Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs.

Transient delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) into the central nervous system (CNS) for therapeutic genome editing could avoid limitations of viral vector-based delivery including cargo capacity, immunogenicity, and cost. Here, we tested the ability of cell-penetrant Cas9 RNPs to edit the mouse striatum when introduced using a convection-enhanced delivery system. These transient Cas9 RNPs showed comparable editing of neurons and reduced adaptive immune responses relative to one formulation of Cas9 delivered using AAV serotype 9. The production of ultra-low endotoxin Cas9 protein manufactured at scale further improved innate immunity. We conclude that injection-based delivery of minimally immunogenic CRISPR genome editing RNPs into the CNS provides a valuable alternative to virus-mediated genome editing.

The Complete Chloroplast Genome of Plukenetia volubilis Provides Insights Into the Organelle Inheritance.

Plukenetia volubilis L. (Malpighiales: Euphorbiaceae), also known as Sacha inchi, is considered a promising crop due to its high seed content of unsaturated fatty acids (UFAs), all of them highly valuable for food and cosmetic industries, but the genetic basis of oil biosynthesis of this non-model plant is still insufficient. Here, we sequenced the total DNA of Sacha inchi by using Illumina and Nanopore technologies and approached a de novo reconstruction of the whole nucleotide sequence and the organization of its 164,111 bp length of the chloroplast genome, displaying two copies of an inverted repeat sequence [inverted repeat A (IRA) and inverted repeat B (IRB)] of 28,209 bp, each one separating a small single copy (SSC) region of 17,860 bp and a large single copy (LSC) region of 89,833 bp. We detected two large inversions on the chloroplast genome that were not presented in the previously reported sequence and studied a promising cpDNA marker, useful in phylogenetic approaches. This chloroplast DNA (cpDNA) marker was used on a set of five distinct Colombian cultivars of P. volubilis from different geographical locations to reveal their phylogenetic relationships. Thus, we evaluated if it has enough resolution to genotype cultivars, intending to crossbreed parents and following marker's trace down to the F1 generation. We finally elucidated, by using molecular and cytological methods on cut flower buds, that the inheritance mode of P. volubilis cpDNA is maternally transmitted and proposed that it occurs as long as it is physically excluded during pollen development. This de novo chloroplast genome will provide a valuable resource for studying this promising crop, allowing the determination of the organellar inheritance mechanism of some critical phenotypic traits and enabling the use of genetic engineering in breeding programs to develop new varieties.

Efficient direct shoot organogenesis and genetic stability in micropropagated sacha inchi (Plukenetia volubilis L.).

OBJECTIVE: It is necessary to improve biotech platforms based on in vitro cell tissue culture to support sacha inchi (Plukenetia volubilis L.) research programs and draw on the nutritional value of the high polyunsaturated fatty acid content of its oilseed. Here, we developed a rapid and efficient method for induction and direct in vitro shoot development for this species. RESULTS: Shoots were generated from hypocotyl explants. The highest organogenic response was obtained in woody plant medium supplemented with 1 mg/L thidiazuron and 0.5 mg/L zeatin supplemented with L-glutamine, adenine hemisulfate, and L-arginine. Shoots obtained using this medium were transferred and subcultivated with different concentrations of indole-3-butyric acid and 1-naphthylacetic acid for rooting. For the first time, a histological analysis was performed supporting direct organogenic development in this species. The plantlets obtained were transferred ex vitro with a survival percentage of 80%. The genetic stability of the plants recovered was confirmed by randomly amplified polymorphic DNA analysis. All results indicate that it would be possible to stimulate direct shoot formation from hypocotyls to support the sustainable use of this species.

[National consensus on the modified Atkins diet]. / Consenso nacional de dieta Atkins modificada.

INTRODUCTION: Epilepsy is a chronic disease that affects 0.5-1% of the population. One third of the patients become refractory to antiepileptic drugs. Among the non-pharmacological treatments available, the modified Atkins diet is an effective treatment used since 2003 as another alternative for children and adults with refractory epilepsy. DEVELOPMENT: The Ketogenic Diet National Committee, which depends on the Argentine Society of Pediatric Neurology, elaborated this consensus on the modified Atkins diet, basing itself on a review of the literature and on their clinical experience. This consensus in Spanish explains the different aspects to be taken into account regarding the modified Atkins diet, patient selection, implementation, different controls and adverse effects. Unlike the classic ketogenic diet, the modified Atkins diet is initiated without fasting or hospital stay, nor does it require protein, calorie or fluid restriction, thus improving patient palatability and consequently patient tolerability. CONCLUSIONS: The modified Atkins diet is a useful treatment for patients with intractable epilepsy. The publication of this consensus offers the possibility for new centers to get oriented regarding this diet implementation.

TITLE: Consenso nacional de dieta Atkins modificada.Introduccion. La epilepsia es una enfermedad cronica que afecta al 0,5-1% de la poblacion, y un tercio de los pacientes evoluciona hacia una forma refractaria a los farmacos antiepilepticos. Dentro de los tratamientos no farmacologicos disponibles, la dieta cetogenica Atkins modificada es un tratamiento efectivo utilizado desde 2003 como otra alternativa en niños y adultos con epilepsia refractaria. Desarrollo. El Comite Nacional de Dieta Cetogenica, dependiente de la Sociedad Argentina de Neurologia Infantil, elaboro este consenso sobre dieta Atkins modificada basandose en una revision de la bibliografia y en su experiencia clinica. Este consenso explica los distintos aspectos que hay que tener en cuenta sobre la dieta Atkins modificada, eleccion de pacientes, forma de implementacion, diversos controles y efectos adversos. A diferencia de la dieta cetogenica clasica, se inicia sin ayuno ni hospitalizacion, y no hay restriccion proteica, calorica o hidrica, por lo que mejora la palatabilidad y, consecuentemente, la tolerabilidad. Conclusiones. La dieta Atkins modificada es un tratamiento util para pacientes con epilepsia intratable. La publicacion de este consenso ofrece la posibilidad de orientar a nuevos centros en su implementacion.

Consenso nacional de dieta Atkins modificada / National consensus on the modified Atkins diet

Introducción. La epilepsia es una enfermedad crónica que afecta al 0,5-1% de la población, y un tercio de los pacientes evoluciona hacia una forma refractaria a los fármacos antiepilépticos. Dentro de los tratamientos no farmacológicos disponibles, la dieta cetogénica Atkins modificada es un tratamiento efectivo utilizado desde 2003 como otra alternativa en niños y adultos con epilepsia refractaria. Desarrollo. El Comité Nacional de Dieta Cetogénica, dependiente de la Sociedad Argentina de Neurología Infantil, elaboró este consenso sobre dieta Atkins modificada basándose en una revisión de la bibliografía y en su experiencia clínica. Este consenso explica los distintos aspectos que hay que tener en cuenta sobre la dieta Atkins modificada, elección de pacientes, forma de implementación, diversos controles y efectos adversos. A diferencia de la dieta cetogénica clásica, se inicia sin ayuno ni hospitalización, y no hay restricción proteica, calórica o hídrica, por lo que mejora la palatabilidad y, consecuentemente, la tolerabilidad. Conclusiones. La dieta Atkins modificada es un tratamiento útil para pacientes con epilepsia intratable. La publicación de este consenso ofrece la posibilidad de orientar a nuevos centros en su implementación (AU)

Introduction. Epilepsy is a chronic disease that affects 0.5-1% of the population. One third of the patients become refractory to antiepileptic drugs. Among the non-pharmacological treatments available, the modified Atkins diet is an effective treatment used since 2003 as another alternative for children and adults with refractory epilepsy. Development. The Ketogenic Diet National Committee, which depends on the Argentine Society of Pediatric Neurology, elaborated this consensus on the modified Atkins diet, basing itself on a review of the literature and on their clinical experience. This consensus in Spanish explains the different aspects to be taken into account regarding the modified Atkins diet, patient selection, implementation, different controls and adverse effects. Unlike the classic ketogenic diet, the modified Atkins diet is initiated without fasting or hospital stay, nor does it require protein, calorie or fluid restriction, thus improving patient palatability and consequently patient tolerability. Conclusions. The modified Atkins diet is a useful treatment for patients with intractable epilepsy. The publication of this consensus offers the possibility for new centers to get oriented regarding this diet implementation (AU)

Recommendations for the clinical management of children with refractory epilepsy receiving the ketogenic diet.

The ketogenic diet, a non-drug treatment with proven effectiveness, has been the most commonly used therapy in the past decade for the management of refractory epilepsy in the pediatric population. Compared to adding a new drug to a pre-existing treatment, the ketogenic diet is highly effective and reduces the number of seizures by 50-90% in approximately 45-60% of children after six months of treatment. For this reason, the Argentine Society of Pediatric Neurology established the Ketogenic Diet Working Group. It is integrated by pediatric dietitians, pediatricians, pediatric neurologists and B.S. in Nutrition, who developed recommendations for the optimal management of patients receiving the classical ketogenic diet based on expert consensus and scientific publications in this field.

La dieta cetogénica constituye el tratamiento no farmacológico de eficacia probada más utilizado en la última década para el manejo de la epilepsia refractaria en la población pediátrica. En comparación con el agregado de un nuevo fármaco a un tratamiento preexistente, esta terapia es altamente efectiva, con una reducción de las crisis de entre un 50% y un 90% en aproximadamente 45%-60% de los niños tras 6 meses de tratamiento. Es por ello por lo que la Sociedad Argentina de Neurología Infantil creó el Grupo de Trabajo de Dieta Cetogénica. Este está conformado por médicos nutricionistas infantiles, pediatras, neurólogos infantiles y licenciados en Nutrición, quienes elaboraron estas recomendaciones para un manejo óptimo de los pacientes que reciben la dieta cetogénica clásica, basándose en el consenso de los expertos y la bibliografía publicada en el tema.

Mitochondrial genome characterization of Tecia solanivora (Lepidoptera: Gelechiidae) and its phylogenetic relationship with other lepidopteran insects.

The complete mitogenome of the potato tuber moth Tecia solanivora (Lepidoptera: Gelechiidae) was sequenced, annotated, characterized and compared with 140 species of the order Lepidoptera. The circular genome is 15,251 bp, containing 37 genes (13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A+T-rich region). The gene arrangement was identical to other lepidopteran mitogenomes but different from the ancestral arrangement found in most insects for the tRNA-Met gene (A+T-region, tRNA-I, tRNA-Q, tRNA-M). The mitogenome of T. solanivora is highly A+T-biased (78.2%) and exhibits negative AT- and GC-skews. All PCGs are initiated by canonical ATN start codons, except for Cytochrome Oxidase subunit 1 (COI), which is initiated by CGA. Most PCGs have a complete typical stop codon (TAA). Only NAD1 has a TAG stop codon and the COII and NAD5 genes have an incomplete stop codon consisting of just a T. The A+T-rich region is 332 bp long and contains common features found in lepidopteran mitogenomes, including the 'ATAGA' motif, a 17 bp poly (T) stretch and a (AT)8 element preceded by the 'ATTTA' motif. Other tandem repeats like (TAA)4 and (TAT)7 were found, as well as (T)6 and (A)10 mononucleotide repeat elements. Finally, this mitogenome has 20 intergenic spacer regions. The phylogenetic relationship of T. solanivora with 28 other lepidopteran families (12 superfamilies) showed that taxonomic classification by morphological features coincides with the inferred phylogeny. Thus, the Gelechiidae family represents a monophyletic group, suggesting that T. solanivora and Pectinophora gossypiella have a recent common ancestor.

Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system.

In the enteric nervous system (ENS), glia outnumber neurons by 4-fold and form an extensive network throughout the gastrointestinal tract. Growing evidence for the essential role of enteric glia in bowel function makes it imperative to understand better their molecular marker expression and how they relate to glia in the rest of the nervous system. We analyzed expression of markers of astrocytes and oligodendrocytes in the ENS and found, unexpectedly, that proteolipid protein 1 (PLP1) is specifically expressed by glia in adult mouse intestine. PLP1 and S100ß are the markers most widely expressed by enteric glia, while glial fibrillary acidic protein expression is more restricted. Marker expression in addition to cellular location and morphology distinguishes a specific subpopulation of intramuscular enteric glia, suggesting that a combinatorial code of molecular markers can be used to identify distinct subtypes. To assess the similarity between enteric and extraenteric glia, we performed RNA sequencing analysis on PLP1-expressing cells in the mouse intestine and compared their gene expression pattern to that of other types of glia. This analysis shows that enteric glia are transcriptionally unique and distinct from other cell types in the nervous system. Enteric glia express many genes characteristic of the myelinating glia, Schwann cells and oligodendrocytes, although there is no evidence of myelination in the murine ENS. GLIA 2015;63:2040-2057.

Consenso nacional sobre dieta cetogénica / National consensus on the ketogenic diet

Introducción. La epilepsia es una enfermedad crónica que afecta al 0,5-1% de la población, mayormente de inicio durante la infancia. Un tercio de los pacientes evoluciona hacia una forma refractaria al tratamiento con fármacos antiepilépticos, lo que plantea al equipo de salud un desafío terapéutico. La dieta cetogénica (DC) es un tratamiento no farmacológico efectivo utilizado como un método alternativo para el tratamiento de la epilepsia refractaria. Objetivos. Es necesario establecer directrices para utilizar la DC adecuadamente y así expandir su conocimiento y utilización en países hispanoparlantes. El Comité Nacional de Dieta Cetogénica, dependiente de la Sociedad Argentina de Neurología Infantil, elaboró este consenso para estandarizar el uso de la DC basándose en la bibliografía publicada y la experiencia clínica. El grupo está formado por neuropediatras, médicos nutricionistas y licenciadas en nutrición de cinco provincias de Argentina pertenecientes a 10 centros que aplican la DC como tratamiento de la epilepsia refractaria. Desarrollo. Se exponen temas tales como la selección del paciente, el asesoramiento a la familia antes del tratamiento, las interacciones de la DC con la medicación anticonvulsionante, los suplementos, el control de efectos adversos y la retirada de dicha dieta. Conclusiones. La DC es un tratamiento útil para los pacientes pediátricos con epilepsia intratable. Es fundamental la educación y colaboración del paciente y la familia. El tratamiento debe llevarlo a cabo un equipo interdisciplinar experimentado, siguiendo un protocolo. La formación de un grupo nacional interdisciplinar, y la publicación de este consenso, ofrece la posibilidad de orientar a nuevos centros en su implantación (AU)

Introduction. Epilepsy is a chronic disease with onset in infancy affecting 0.5-1% of the population. One third of the patients is refractory to antiepileptic drugs and they pose a challenge for the health care team. The ketogenic diet is an effective, non-pharmacological, alternative treatment for the management of refractory epilepsy. Aims. There is a need to establish guidelines for the adequate and increased use of the ketogenic diet in Spanish-speaking countries. The National Committee on the Ketogenic Diet, consisting of paediatric neurologists, clinical nutritionists, and dietitians, of the Argentine Society of Child Neurology has developed this consensus statement to standardize the use of the ketogenic diet based on the literature and clinical experience. Development. Patient selection, pre-treatment family counseling, drug interactions, micronutrient supplementation, adverse effects, and discontinuation of the diet are discussed. Conclusions. The ketogenic diet is an effective treatment for children with refractory epilepsy. Education and collaboration of the patient and their family is essential. The patient should be managed by an experienced multidisciplinary team using a protocol. The formation of a national multidisciplinary team and the publication of this document provide possibilities for new centers to integrate the ketogenic diet into their treatment options (AU)

[National consensus on the ketogenic diet]. / Consenso nacional sobre dieta cetogénica.

INTRODUCTION: Epilepsy is a chronic disease with onset in infancy affecting 0.5-1% of the population. One third of the patients is refractory to antiepileptic drugs and they pose a challenge for the health care team. The ketogenic diet is an effective, non-pharmacological, alternative treatment for the management of refractory epilepsy. AIMS: There is a need to establish guidelines for the adequate and increased use of the ketogenic diet in Spanish-speaking countries. The National Committee on the Ketogenic Diet, consisting of paediatric neurologists, clinical nutritionists, and dietitians, of the Argentine Society of Child Neurology has developed this consensus statement to standardize the use of the ketogenic diet based on the literature and clinical experience. DEVELOPMENT: Patient selection, pre-treatment family counseling, drug interactions, micronutrient supplementation, adverse effects, and discontinuation of the diet are discussed. CONCLUSIONS: The ketogenic diet is an effective treatment for children with refractory epilepsy. Education and collaboration of the patient and their family is essential. The patient should be managed by an experienced multidisciplinary team using a protocol. The formation of a national multidisciplinary team and the publication of this document provide possibilities for new centers to integrate the ketogenic diet into their treatment options.

TITLE: Consenso nacional sobre dieta cetogenica.Introduccion. La epilepsia es una enfermedad cronica que afecta al 0,5-1% de la poblacion, mayormente de inicio durante la infancia. Un tercio de los pacientes evoluciona hacia una forma refractaria al tratamiento con farmacos antiepilepticos, lo que plantea al equipo de salud un desafio terapeutico. La dieta cetogenica (DC) es un tratamiento no farmacologico efectivo utilizado como un metodo alternativo para el tratamiento de la epilepsia refractaria. Objetivos. Es necesario establecer directrices para utilizar la DC adecuadamente y asi expandir su conocimiento y utilizacion en paises hispanoparlantes. El Comite Nacional de Dieta Cetogenica, dependiente de la Sociedad Argentina de Neurologia Infantil, elaboro este consenso para estandarizar el uso de la DC basandose en la bibliografia publicada y la experiencia clinica. El grupo esta formado por neuropediatras, medicos nutricionistas y licenciadas en nutricion de cinco provincias de Argentina pertenecientes a 10 centros que aplican la DC como tratamiento de la epilepsia refractaria. Desarrollo. Se exponen temas tales como la seleccion del paciente, el asesoramiento a la familia antes del tratamiento, las interacciones de la DC con la medicacion anticonvulsionante, los suplementos, el control de efectos adversos y la retirada de dicha dieta. Conclusiones. La DC es un tratamiento util para los pacientes pediatricos con epilepsia intratable. Es fundamental la educacion y colaboracion del paciente y la familia. El tratamiento debe llevarlo a cabo un equipo interdisciplinar experimentado, siguiendo un protocolo. La formacion de un grupo nacional interdisciplinar, y la publicacion de este consenso, ofrece la posibilidad de orientar a nuevos centros en su implantacion.

Long-term follow-up of the ketogenic diet for refractory epilepsy: multicenter Argentinean experience in 216 pediatric patients.

PURPOSE: In this Argentinean retrospective, collaborative, multicenter study, we examine the efficacy and tolerability of the ketogenic diet (KD) for different epilepsy syndromes. MATERIALS AND METHODS: we evaluated the clinical records of 216 patients started on the KD between March 1, 1990 and December 31, 2010. RESULTS: One hundred forty of the initial patients (65%) remained on the diet at the end of the study period. Twenty-nine patients (20.5%) became seizure free and 50 children (36%) had a 75-99% decrease in seizures. Thus, 56.5% of the patients had a seizure control of more than 75%. The best results were found in patients with epilepsy with myoclonic-astatic seizures, Lennox-Gastaut syndrome, and West syndrome. Good results were also found in patients with Dravet syndrome, in those with symptomatic focal epilepsy secondary to malformations of cortical development, and in patients with tuberous sclerosis. Seizures were significantly reduced in four patients with fever-induced refractory epileptic encephalopathy in school-age children and in two patients with epileptic encephalopathy with continuous spikes and waves during slow sleep. The median period of follow-up after discontinuation of the diet was 6 years. Twenty patients who had become seizure free discontinued the diet, but seizures recurred in five (25%). Of 40 patients with a seizure reduction of more than 50% who discontinued the diet, 10 presented with recurrent seizures. CONCLUSION: The ketogenic diet is a good option in the treatment of refractory epilepsy. After discontinuing the diet, seizures recurrence occurred in few patients.

DNA slip-outs cause RNA polymerase II arrest in vitro: potential implications for genetic instability.

The abnormal number of repeats found in triplet repeat diseases arises from 'repeat instability', in which the repetitive section of DNA is subject to a change in copy number. Recent studies implicate transcription in a mechanism for repeat instability proposed to involve RNA polymerase II (RNAPII) arrest caused by a CTG slip-out, triggering transcription-coupled repair (TCR), futile cycles of which may lead to repeat expansion or contraction. In the present study, we use defined DNA constructs to directly test whether the structures formed by CAG and CTG repeat slip-outs can cause transcription arrest in vitro. We found that a slip-out of (CAG)(20) or (CTG)(20) repeats on either strand causes RNAPII arrest in HeLa cell nuclear extracts. Perfect hairpins and loops on either strand also cause RNAPII arrest. These findings are consistent with a transcription-induced repeat instability model in which transcription arrest in mammalian cells may initiate a 'gratuitous' TCR event leading to a change in repeat copy number. An understanding of the underlying mechanism of repeat instability could lead to intervention to slow down expansion and delay the onset of many neurodegenerative diseases in which triplet repeat expansion is implicated.

Monitoring child development in the IMCI Context

[Preface]. If child survival is one of the most important unfinished challenges bequeathed to us by the twentieth century, then guaranteeing healthy growth and development for all children, a goal that is already being addressed in the twenty-first century, is key to meeting this challenge. The progress that has been made, especially in recent decades, in the prevention of diseases and the effective treatment of many of them, has had a major impact on the overall living conditions of people throughout the world, including the Region of the Americas. Life expectancy has increased in the last twenty years, and an important part of this increase has been due to the countries’ efforts to reduce infant mortality. The number of deaths in children under 5 years of age was drastically reduced between the early 1980s and the end of the 1990s, and the goal to reduce the 1990 figure by one-third was attained in 2000... The present manual is intended to provide health professionals—from those involved in academic training to personnel who work directly in child health care—with the tools, integrated into IMCI and placed within the population’s reach, to make effective interventions for the improvement of child development. These conditions will undoubtedly hasten achievement of the Millennium Development Goals for 2015 to which all the countries are committed. By ensuring that all children and their families have the opportunity for healthy growth and development throughout childhood, they not only improve child survival but also make a difference in the lives of those who survive.

Síndrome de West secundario a cirugía cardíaca / West syndrome secondary to cardiac surgery

El síndrome de West puede presentarse como complicación de la cirugía cardíaca con cardioplejía y circulación extracorpórea. Presentamos una niña con diagnóstico de cardiopatía congénita que luego de la intervención quirúrgica, sin haber presentado signos de encefalopatía en el posquirúrgico inmediato, comenzó con crisis de espasmos infantiles e hipsarritmia, constituyendo un síndrome de West

Síndrome de West secundario a cirugía cardíaca / West syndrome secondary to cardiac surgery

El síndrome de West puede presentarse como complicación de la cirugía cardíaca con cardioplejía y circulación extracorpórea. Presentamos una niña con diagnóstico de cardiopatía congénita que luego de la intervención quirúrgica, sin haber presentado signos de encefalopatía en el posquirúrgico inmediato, comenzó con crisis de espasmos infantiles e hipsarritmia, constituyendo un síndrome de West (AU)