A cryo-fixation protocol to study the structure of the synaptonemal complex.

Genetic variability in sexually reproducing organisms results from an exchange of genetic material between homologous chromosomes. The genetic exchange mechanism is dependent on the synaptonemal complex (SC), a protein structure localized between the homologous chromosomes. The current structural models of the mammalian SC are based on electron microscopy, superresolution, and expansion microscopy studies using chemical fixatives and sample dehydration of gonads, which are methodologies known to produce structural artifacts. To further analyze the structure of the SC, without chemical fixation, we have adapted a cryo-fixation method for electron microscopy where pachytene cells are isolated from mouse testis by FACS, followed by cryo-fixation, cryo-substitution, and electron tomography. In parallel, we performed conventional chemical fixation and electron tomography on mouse seminiferous tubules to compare the SC structure obtained with the two fixation methods. We found several differences in the structure and organization of the SC in cryo-fixed samples when compared to chemically preserved samples. We found the central region of the SC to be wider and the transverse filaments to be more densely packed in the central region of the SC.

Synaptonemal complex formation produces a particular arrangement of the lateral element-associated DNA.

During meiosis, homologous chromosomes exchange genetic material. This exchange or meiotic recombination is mediated by a proteinaceous scaffold known as the Synaptonemal complex (SC). Any defects in its formation produce failures in meiotic recombination, chromosome segregation and meiosis completion. It has been proposed that DNA repair events that will be resolved by crossover between homologous chromosomes are predetermined by the SC. Hence, structural analysis of the organization of the DNA in the SC could shed light on the process of crossover interference. In this work, we employed an ultrastructural DNA staining technique on mouse testis and followed nuclei of pachytene cells. We observed structures organized similarly to the SCs stained with conventional techniques. These structures, presumably the DNA in the SCs, are delineating the edges of both lateral elements and no staining was observed between them. DNA in the LEs resembles two parallel tracks. However, a bubble-like staining pattern in certain regions of the SC was observed. Furthermore, this staining pattern is found in SCs formed between non-homologous chromosomes, in SCs formed between sister chromatids and in SCs without lateral elements, suggesting that this particular organization of the DNA is determined by the synapsis of the chromosomes despite their lack of homology or the presence of partially formed SCs.

The Transcriptomic Landscape of Pediatric Astrocytoma.

Central nervous system tumors are the most common solid neoplasia during childhood and represent one of the leading causes of cancer-related mortality. Tumors arising from astrocytic cells (astrocytomas) are the most frequently diagnosed, and according to their histological and pathological characteristics, they are classified into four categories. However, an additional layer of molecular classification considering the DNA sequence of the tumorigenesis-associated genes IDH1/2 and H3F3A has recently been incorporated into the classification guidelines. Although mutations in H3F3A are found exclusively in a subtype of grade IV pediatric astrocytoma, mutations in IDH1/2 genes are very rare in children under 14 years of age. The transcriptomic profiles of astrocytoma in adults and children have been extensively studied. However, there is scarce information on these profiles in pediatric populations considering the status of tumorigenesis-associated genes. Therefore, here we report the transcriptomic landscape of the four grades of pediatric astrocytoma by RNA sequencing. We found several well-documented biological functions associated with the misregulated genes in the four grades of astrocytoma, as well as additional biological pathways. Among the four grades of astrocytoma, we found shared misregulated genes that could have implications in tumorigenesis. Finally, we identified a transcriptional signature for almost all grades of astrocytoma that could be used as a transcription-based identification method.

The width of the lateral element of the synaptonemal complex is determined by a multilayered organization of its components.

The synaptonemal complex (SC) is a proteinaceous structure that holds the homologous chromosomes in close proximity while they exchange genetic material in a process known as meiotic recombination. This meiotic recombination leads to genetic variability in sexually reproducing organisms. The ultrastructure of the SC is studied by electron microscopy and it is observed as a tripartite structure. Two lateral elements (LE) separated by a central region (CR) confer its classical tripartite organization. The LEs are the anchoring platform for the replicated homologous chromosomes to properly exchange genetic material with one another. An accurate assembly of the LE is indispensable for the proper completion of meiosis. Ultrastructural studies suggested that the LE is organized as a multilayered unit. However, no validation of this model has been previously provided. In this ultrastructural study, by using mice with different genetic backgrounds that affect the LE width, we provide further evidence that support a multilayered organization of the LE. Additionally, we provide data suggesting additional roles of the different cohesin complex components in the structure of the LEs of the SC.

Paraptosis-like cell death in Wistar rat granulosa cells.

Follicular atresia, a common process present in all mammals, involves apoptotic and autophagic cell death. However, the participation of paraptosis, a type of caspase-independent cell death, during follicular atresia is unknown. This study found swollen endoplasmic reticulum in the granulosa cells of adult Wistar rats. Calnexin was used as a marker of the endoplasmic reticulum at the ultrastructural and optical levels. The cells with swelling of the endoplasmic reticulum were negative to the TUNEL assay and active caspase-3 immunodetection, indicating that this swelling is not part of any apoptotic or autophagic process. Additionally, immunodetection of the CHOP protein was used as a marker of endoplasmic reticulum stress, and this confirmed the presence of the paraptosis process. These data suggest that paraptosis-like cell death is associated with the death of granulosa cells during follicular atresia in adult Wistar rats.

Brain-computer interface technologies: from signal to action.

Here, we present a state-of-the-art review of the research performed on the brain-computer interface (BCI) technologies with a focus on signal processing approaches. BCI can be divided into three main components: signal acquisition, signal processing, and effector device. The signal acquisition component is generally divided into two categories: noninvasive and invasive. For noninvasive, this review focuses on electroencephalogram. For the invasive, the review includes electrocorticography, local field potentials, multiple-unit activity, and single-unit action potentials. Signal processing techniques reviewed are divided into time-frequency methods such as Fourier transform, autoregressive models, wavelets, and Kalman filter and spatiotemporal techniques such as Laplacian filter and common spatial patterns. Additionally, various signal feature classification algorithms are discussed such as linear discriminant analysis, support vector machines, artificial neural networks, and Bayesian classifiers. The article ends with a discussion of challenges facing BCI and concluding remarks on the future of the technology.

Agaricus macrochlamys, a New Species from the (Sub)tropical Cloud Forests of North America and the Caribbean, and Agaricus fiardii, a New Synonym of Agaricus subrufescens.

Agaricus is a genus of fungi in the family Agaricaceae, with several highly priced edible and medicinal species. Here we describe Agaricus macrochlamys, a new species, in A. sect. Arvenses, sympatric and morphologically cryptic with the edible and medicinally cultivated mushroom, A. subrufescens. Phylogenetic analyses showed that A. macrochlamys is closely related to A. subrufescens, and that A. fiardii is a new synonym of A. subrufescens. Despite being morphologically cryptic, A. macrochlamys can be distinguished from A. subrufescens by several ITS and tef1α species-specific markers and a 4-bp insertion in the tef1α sequence. Furthermore, A. subrufescens is a cosmopolitan species, while A. macrochlamys distribution is so far restricted to Mexico, the Dominican Republic, and the United States.

Synaptonemal complex stability depends on repressive histone marks of the lateral element-associated repeat sequences.

The synaptonemal complex (SC) is the central key structure for meiosis in organisms undergoing sexual reproduction. During meiotic prophase I, homologous chromosomes exchange genetic information at the time they are attached to the lateral elements by specific DNA sequences. Most of these sequences, so far identified, consist of repeat DNA, which are subject to chromatin structural changes during meiotic prophase I. In this work, we addressed the effect of altering the chromatin structure of repeat DNA sequences mediating anchorage to the lateral elements of the SC. Administration of the histone deacetylase inhibitor trichostatin A into live rats caused death of cells in the pachytene stage as well as changes in histone marks along the synaptonemal complex. The most notable effect was partial loss of histone H3 lysine 27 trimethylation. Our work describes the epigenetic landscape of lateral element-associated chromatin and reveals a critical role of histone marks in synaptonemal complex integrity.

Desarmillaria caespitosa, a North American vicariant of D. tabescens.

Desarmillaria caespitosa, a North American vicariant species of European D. tabescens, is redescribed in detail based on recent collections from the USA and Mexico. This species is characterized by morphological features and multilocus phylogenetic analyses using portions of nuc rDNA 28S (28S), translation elongation factor 1-alpha (tef1), the second largest subunit of RNA polymerase II (rpb2), actin (act), and glyceraldehyde-3-phosphate dehydrogenase (gpd). A neotype of D. caespitosa is designated here. Morphological and genetic differences between D. caespitosa and D. tabescens were identified. Morphologically, D. caespitosa differs from D. tabescens by having wider basidiospores, narrower cheilocystidia, which are often irregular or mixed (regular, irregular, or coralloid), and narrower caulocystidia. Phylogenetic analyses of five independent gene regions show that D. caespitosa and D. tabescens are separated by nodes with strong support. The new combination, D. caespitosa, is proposed.

Evaluation of the melting temperature of TaqMan probes as a genotyping method for IDH1, IDH2, and H3F3A in pediatric astrocytomas.

Background: Astrocytomas are cancer tumors of the central nervous system and represent the most common type of solid tumors during human childhood. In 2016, the World Health Organization established a molecular classification system to regroup tumor entities to achieve a more accurate diagnosis and a better clinical decision-making and selection of treatment in patients with these types of tumors. Methods: We evaluated a genotyping assay for rapid and cost-effective mutation detection in astrocytomas using TaqMan probes in an asymmetric polymerase chain reaction (PCR) assay. Results: Four diffuse astrocytomas (Grade II), three anaplastic astrocytomas (Grade III), and four glioblastomas (Grade IV) were sequenced, and all of them displayed the wild-type (WT) sequence. We tried to set up this melting analysis for the genotyping of pediatric astrocytomas by identifying the specific melting temperatures of the TaqMan probes due to the presence of the WT sequences in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and H3.3 histone A genes (H3F3A). We used an IDH1-TaqMan probe to identify the WT status of IDH1 in two different WT deoxyribonucleic acid (DNA) templates (pilocytic and diffuse astrocytoma) and obtained four melting temperature values ranged from 65.6 to 92.2°C. Furthermore, only four out of 29 reactions displayed amplification of the DNA template. Sanger sequencing was faster and more reliable to detect the gene status in all the sequenced samples. Conclusions: We conclude that conventional Sanger sequencing remains the gold standard for the genotyping of pediatric astrocytomas.

Introducción: Los astrocitomas son un tipo de cáncer que afecta al sistema nervioso central y representan el tumor sólido más común durante la infancia. En el año 2016, la Organización Mundial de la Salud estableció un sistema de clasificación molecular para reagrupar tumores con identidades genéticas similares y lograr un diagnóstico más preciso, lo que lleva a tomar las decisiones clínicas idóneas al elegir el tratamiento de pacientes con este tipo de tumores. Métodos: Se evaluó un protocolo que involucra el uso de sondas TaqMan en un ensayo de reacción en cadena de la polimerasa asimétrica para la detección de mutaciones en astrocitomas. Se secuenciaron cuatro astrocitomas difusos (Grado II), tres astrocitomas anaplásicos (Grado III) y cuatro glioblastomas (Grado IV). Se intentó establecer las condiciones del análisis para la genotipificación de los astrocitomas pediátricos mediante la identificación de las temperaturas de disociación específicas de las sondas TaqMan producidas por la prescencia de las secuancias WT en los genes isocitrato deshidrogenasa 1 y 2 (IDH1, IDH2) y H3.3 histona A (H3F3A). Resultados: Los astrocitomas mostraron la secuencia wild type (WT) (silvestre) de los genes. Se utilizó una sonda TaqMan IDH1 para identificar el estado de este gen en dos templados WT de DNA (astrocitoma pilocítico y difuso) y se obtuvieron cuatro valores de temperatura de disociación (65.6-92.2 °C). Solo cuatro de las 29 reacciones mostraron amplificación de DNA. La secuenciación de Sanger fue más rápida y confiable para detectar el estado de los genes en todas las muestras. Conclusiones: La secuenciación de Sanger sigue siendo la técnica más práctica para la genotipificación de astrocitomas pediátricos.

Histochemical Study of the Emergence of Apoptosis and Altered SYCP3 Protein Distribution During the First Spermatogenic Wave in Wistar Rats.

Apoptosis is a type of cell death responsible for maintaining tissue homeostasis that can occur in male gonads. The morphological and biochemical characteristics of apoptosis include cellular contraction, caspase activation, and DNA fragmentation. Dynamic processes of cell renewal and differentiation occur inside the seminiferous tubules, which are regulated by mitosis and meiosis, respectively. During meiosis, recombination is caused by assembly of the synaptonemal complex, which involves the participation of constitutive proteins, such as synaptonemal complex protein-3 (SYCP3). The present study evaluated germinal cell death in immature male rats and the distribution of the SYCP3 protein. Our results indicate that as germinal cells progress to the second meiotic stage, significant numbers of them are eliminated by apoptosis. We determined that the SYCP3 protein is not always incorporated into the structure of the synaptonemal complex but rather forms a nuclear cumulus near the inner nuclear membrane, causing many of these cells to undergo apoptosis. We propose that both the excess of the SYCP3 protein and its accumulation during the first meiotic division could contribute to the cell death of primary spermatocytes during the first spermatogenic wave in prepubertal Wistar rats. Anat Rec, 302:2082-2092, 2019. © 2019 American Association for Anatomy.

Artificial covalent linkage of bacterial acyl carrier proteins for fatty acid production.

Acyl carrier proteins (ACPs) are essential to the production of fatty acids. In some species of marine bacteria, ACPs are arranged into tandem repeats joined by peptide linkers, an arrangement that results in high fatty acid yields. By contrast, Escherichia coli, a relatively low producer of fatty acids, uses a single-domain ACP. In this work, we have engineered the native E. coli ACP into tandem di- and tri-domain constructs joined by a naturally occurring peptide linker from the PUFA synthase of Photobacterium profundum. The size of these tandem fused ACPs was determined by size exclusion chromatography to be higher (21 kDa, 36 kDa and 141 kDa) than expected based on the amino acid sequence (12 kDa, 24 kDa and 37 kDa, respectively) suggesting the formation of a flexible extended conformation. Structural studies using small-angle X-ray scattering (SAXS), confirmed this conformational flexibility. The thermal stability for the di- and tri-domain constructs was similar to that of the unfused ACP, indicating a lack of interaction between domains. Lastly, E. coli cultures harboring tandem ACPs produced up to 1.6 times more fatty acids than wild-type ACP, demonstrating the viability of ACP fusion as a method to enhance fatty acid yield in bacteria.

Primary hepatic carcinoid tumor metastasizing to eyelid: A case report.

PURPOSE: We report a rare case of a metastatic carcinoid tumor to the right lower lid masquerading as a chalazion. OBSERVATIONS: A 78-year-old Hispanic woman who presented with a 3-month history of a non-resolving chalazion on the right lower lid despite aggressive medical treatment. The patient had a history of noninfectious anterior uveitis and primary hepatic carcinoid tumor that was incidentally diagnosed during the initial uveitis work-up. The right lower eyelid lesion was biopsied and histological and immunopathological analysis revealed a well differentiated neuroendocrine tumor consistent with a carcinoid tumor. CONCLUSIONAND IMPORTANCE: Neuroendocrine tumors should be considered as part of the differential diagnosis of focal, vascularized eyelid masses. To the authors best knowledge this is the first reported case of primary hepatic carcinoid tumor with metastasis to the eyelids. We also highlight the importance of pursuing a histopathologic diagnosis, in the setting of a non-resolving or recurrent chalazion.

[Speech perception development in children with dyslexia]. / Desarrollo de la percepción del habla en niños con dislexia.

Several studies have indicated that dyslexics show a deficit in speech perception (SP). The main purpose of this research is to determine the development of SP in dyslexics and normal readers paired by grades from 2nd to 6th grade of primary school and to know whether the phonetic contrasts that are relevant for SP change during development, taking into account the individual differences. The achievement of both groups was compared in the phonetic tasks: voicing contrast, place of articulation contrast and manner of articulation contrast. The results showed that the dyslexic performed poorer than the normal readers in SP. In place of articulation contrast, the developmental pattern is similar in both groups but not in voicing and manner of articulation. Manner of articulation has more influence on SP, and its development is higher than the other contrast tasks in both groups.

Armillaria mexicana, a newly described species from Mexico.

Armillaria mexicana (Agaricales, Physalacriaceae) is described as a new species based on morphology, DNA sequence data, and phylogenetic analyses. It clearly differs from previously reported Armillaria species in North, Central, and South America. It is characterized by the absence of fibulae in the basidioma, abundant cheilocystidia, and ellipsoidal, hyaline basidiospores that are apparently smooth under light microscope, but slightly to moderately rugulose under scanning electron microscope. It is differentiated from other Armillaria species by macromorphological characters, including annulus structure, pileus and stipe coloration, and other structures. DNA sequence data (nuc rDNA internal transcribed spacers [ITS1-5.8S-ITS2 = ITS], 28S D-domain, 3' end of 28S intergenic spacer 1, and translation elongation factor 1-α [TEF1]) show that A. mexicana sequences are quite distinct from sequences of analogous Armillaria species in GenBank. In addition, sequences of ITS of the A. mexicana ex-type culture reveal an ITS1 of 1299 bp and an ITS2 of 582 bp, the longest ITS regions reported thus far in fungi. Phylogenetic analysis based on TEF1 sequences place A. mexicana in a well-separated, monophyletic clade basal to the polyphyletic A. mellea complex.

Locus and nature of perceptual phonological deficit in Spanish children with reading disabilities.

The aims of this study were (a) to determine whether Spanish children with reading disabilities (RD) show a speech perception deficit and (b) to explore the locus and nature of this perceptive deficit. A group of 29 children with RD, 41 chronological age-matched controls, and 27 reading ability-matched younger controls were tested on tasks of speech perception. The effect of linguistic unit (word vs. syllable) and type of phonetic contrast (voicing, place and manner of articulation) were analyzed in terms of the number of errors and the response time. The results revealed a speech perception deficit in Spanish children with RD that was independent of the type of phonetic contrast and of linguistic unit.

MUSIC-Expected maximization gaussian mixture methodology for clustering and detection of task-related neuronal firing rates.

Researchers often rely on simple methods to identify involvement of neurons in a particular motor task. The historical approach has been to inspect large groups of neurons and subjectively separate neurons into groups based on the expertise of the investigator. In cases where neuron populations are small it is reasonable to inspect these neuronal recordings and their firing rates carefully to avoid data omissions. In this paper, a new methodology is presented for automatic objective classification of neurons recorded in association with behavioral tasks into groups. By identifying characteristics of neurons in a particular group, the investigator can then identify functional classes of neurons based on their relationship to the task. The methodology is based on integration of a multiple signal classification (MUSIC) algorithm to extract relevant features from the firing rate and an expectation-maximization Gaussian mixture algorithm (EM-GMM) to cluster the extracted features. The methodology is capable of identifying and clustering similar firing rate profiles automatically based on specific signal features. An empirical wavelet transform (EWT) was used to validate the features found in the MUSIC pseudospectrum and the resulting signal features captured by the methodology. Additionally, this methodology was used to inspect behavioral elements of neurons to physiologically validate the model. This methodology was tested using a set of data collected from awake behaving non-human primates.

WHATCH'EM: A Weather-Driven Energy Balance Model for Determining Water Height and Temperature in Container Habitats for Aedes aegypti.

The mosquito virus vector Aedes (Ae.) aegypti exploits a wide range of containers as sites for egg laying and development of the immature life stages, yet the approaches for modeling meteorologically sensitive container water dynamics have been limited. This study introduces the Water Height and Temperature in Container Habitats Energy Model (WHATCH'EM), a state-of-the-science, physically based energy balance model of water height and temperature in containers that may serve as development sites for mosquitoes. The authors employ WHATCH'EM to model container water dynamics in three cities along a climatic gradient in México ranging from sea level, where Ae. aegypti is highly abundant, to ~2100 m, where Ae. aegypti is rarely found. When compared with measurements from a 1-month field experiment in two of these cities during summer 2013, WHATCH'EM realistically simulates the daily mean and range of water temperature for a variety of containers. To examine container dynamics for an entire season, WHATCH'EM is also driven with field-derived meteorological data from May to September 2011 and evaluated for three commonly encountered container types. WHATCH'EM simulates the highly nonlinear manner in which air temperature, humidity, rainfall, clouds, and container characteristics (shape, size, and color) determine water temperature and height. Sunlight exposure, modulated by clouds and shading from nearby objects, plays a first-order role. In general, simulated water temperatures are higher for containers that are larger, darker, and receive more sunlight. WHATCH'EM simulations will be helpful in understanding the limiting meteorological and container-related factors for proliferation of Ae. aegypti and may be useful for informing weather-driven early warning systems for viruses transmitted by Ae. aegypti.

Wavelet methodology to improve single unit isolation in primary motor cortex cells.

The proper isolation of action potentials recorded extracellularly from neural tissue is an active area of research in the fields of neuroscience and biomedical signal processing. This paper presents an isolation methodology for neural recordings using the wavelet transform (WT), a statistical thresholding scheme, and the principal component analysis (PCA) algorithm. The effectiveness of five different mother wavelets was investigated: biorthogonal, Daubachies, discrete Meyer, symmetric, and Coifman; along with three different wavelet coefficient thresholding schemes: fixed form threshold, Stein's unbiased estimate of risk, and minimax; and two different thresholding rules: soft and hard thresholding. The signal quality was evaluated using three different statistical measures: mean-squared error, root-mean squared, and signal to noise ratio. The clustering quality was evaluated using two different statistical measures: isolation distance, and L-ratio. This research shows that the selection of the mother wavelet has a strong influence on the clustering and isolation of single unit neural activity, with the Daubachies 4 wavelet and minimax thresholding scheme performing the best.

Evaluation of the melting temperature of TaqMan probes as a genotyping method for IDH1, IDH2, and H3F3A in pediatric astrocytomas / Evaluación del método de temperatura de disociación de sondas TaqMan para la genotipificación de IDH1, IDH2 y H3F3A en astrocitomas pediátricos

Abstract Background: Astrocytomas are cancer tumors of the central nervous system and represent the most common type of solid tumors during human childhood. In 2016, the World Health Organization established a molecular classification system to regroup tumor entities to achieve a more accurate diagnosis and a better clinical decision-making and selection of treatment in patients with these types of tumors. Methods: We evaluated a genotyping assay for rapid and cost-effective mutation detection in astrocytomas using TaqMan probes in an asymmetric polymerase chain reaction (PCR) assay. Results: Four diffuse astrocytomas (Grade II), three anaplastic astrocytomas (Grade III), and four glioblastomas (Grade IV) were sequenced, and all of them displayed the wild-type (WT) sequence. We tried to set up this melting analysis for the genotyping of pediatric astrocytomas by identifying the specific melting temperatures of the TaqMan probes due to the presence of the WT sequences in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and H3.3 histone A genes (H3F3A). We used an IDH1-TaqMan probe to identify the WT status of IDH1 in two different WT deoxyribonucleic acid (DNA) templates (pilocytic and diffuse astrocytoma) and obtained four melting temperature values ranged from 65.6 to 92.2°C. Furthermore, only four out of 29 reactions displayed amplification of the DNA template. Sanger sequencing was faster and more reliable to detect the gene status in all the sequenced samples. Conclusions: We conclude that conventional Sanger sequencing remains the gold standard for the genotyping of pediatric astrocytomas.

Resumen Introducción: Los astrocitomas son un tipo de cáncer que afecta al sistema nervioso central y representan el tumor sólido más común durante la infancia. En el año 2016, la Organización Mundial de la Salud estableció un sistema de clasificación molecular para reagrupar tumores con identidades genéticas similares y lograr un diagnóstico más preciso, lo que lleva a tomar las decisiones clínicas idóneas al elegir el tratamiento de pacientes con este tipo de tumores. Métodos: Se evaluó un protocolo que involucra el uso de sondas TaqMan en un ensayo de reacción en cadena de la polimerasa asimétrica para la detección de mutaciones en astrocitomas. Se secuenciaron cuatro astrocitomas difusos (Grado II), tres astrocitomas anaplásicos (Grado III) y cuatro glioblastomas (Grado IV). Se intentó establecer las condiciones del análisis para la genotipificación de los astrocitomas pediátricos mediante la identificación de las temperaturas de disociación específicas de las sondas TaqMan producidas por la prescencia de las secuancias WT en los genes isocitrato deshidrogenasa 1 y 2 (IDH1, IDH2) y H3.3 histona A (H3F3A). Resultados: Los astrocitomas mostraron la secuencia wild type (WT) (silvestre) de los genes. Se utilizó una sonda TaqMan IDH1 para identificar el estado de este gen en dos templados WT de DNA (astrocitoma pilocítico y difuso) y se obtuvieron cuatro valores de temperatura de disociación (65.6-92.2 °C). Solo cuatro de las 29 reacciones mostraron amplificación de DNA. La secuenciación de Sanger fue más rápida y confiable para detectar el estado de los genes en todas las muestras. Conclusiones: La secuenciación de Sanger sigue siendo la técnica más práctica para la genotipificación de astrocitomas pediátricos.