GoT-Splice protocol for multi-omics profiling of gene expression, cell-surface proteins, mutational status, and RNA splicing in human cells.

Studying RNA splicing factor mutations is challenging due to difficulties in distinguishing wild-type and mutant cells within complex human tissues and inaccuracies associated with reconstructing splicing signals from short-read sequencing data. Here, we present Genotyping of Transcriptomes (GoT)-Splice, a protocol that overcomes these limitations by combining GoT with enhanced long-read single-cell transcriptome and cell-surface proteomics profiling. We describe steps for long-read library preparation and analysis, followed by cDNA re-amplification, enrichment of mutation of interest, sample indexing, and GoT library preparation. For complete details on the use and execution of this protocol, please refer to Cortés-López et al.1.

SON is an essential m6A target for hematopoietic stem cell fate.

Stem cells regulate their self-renewal and differentiation fate outcomes through both symmetric and asymmetric divisions. m6A RNA methylation controls symmetric commitment and inflammation of hematopoietic stem cells (HSCs) through unknown mechanisms. Here, we demonstrate that the nuclear speckle protein SON is an essential m6A target required for murine HSC self-renewal, symmetric commitment, and inflammation control. Global profiling of m6A identified that m6A mRNA methylation of Son increases during HSC commitment. Upon m6A depletion, Son mRNA increases, but its protein is depleted. Reintroduction of SON rescues defects in HSC symmetric commitment divisions and engraftment. Conversely, Son deletion results in a loss of HSC fitness, while overexpression of SON improves mouse and human HSC engraftment potential by increasing quiescence. Mechanistically, we found that SON rescues MYC and suppresses the METTL3-HSC inflammatory gene expression program, including CCL5, through transcriptional regulation. Thus, our findings define a m6A-SON-CCL5 axis that controls inflammation and HSC fate.

Single-cell multi-omics defines the cell-type-specific impact of splicing aberrations in human hematopoietic clonal outgrowths.

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1. SF3B1mut cells were enriched in the megakaryocytic-erythroid lineage, with expansion of SF3B1mut erythroid progenitor cells. We uncovered distinct cryptic 3' splice site usage in different progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid bias and cell-type-specific cryptic 3' splice site usage in SF3B1mut cells precede overt MDS. Collectively, GoT-Splice defines the cell-type-specific impact of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples.

Analysis of RBP expression and binding sites identifies PTBP1 as a regulator of CD19 expression in B-ALL.

Despite massive improvements in the treatment of B-ALL through CART-19 immunotherapy, a large number of patients suffer a relapse due to loss of the targeted epitope. Mutations in the CD19 locus and aberrant splicing events are known to account for the absence of surface antigen. However, early molecular determinants suggesting therapy resistance as well as the time point when first signs of epitope loss appear to be detectable are not enlightened so far. By deep sequencing of the CD19 locus, we identified a blast-specific 2-nucleotide deletion in intron 2 that exists in 35% of B-ALL samples at initial diagnosis. This deletion overlaps with the binding site of RNA binding proteins (RBPs) including PTBP1 and might thereby affect CD19 splicing. Moreover, we could identify a number of other RBPs that are predicted to bind to the CD19 locus being deregulated in leukemic blasts, including NONO. Their expression is highly heterogeneous across B-ALL molecular subtypes as shown by analyzing 706 B-ALL samples accessed via the St. Jude Cloud. Mechanistically, we show that downregulation of PTBP1, but not of NONO, in 697 cells reduces CD19 total protein by increasing intron 2 retention. Isoform analysis in patient samples revealed that blasts, at diagnosis, express increased amounts of CD19 intron 2 retention compared to normal B cells. Our data suggest that loss of RBP functionality by mutations altering their binding motifs or by deregulated expression might harbor the potential for the disease-associated accumulation of therapy-resistant CD19 isoforms.

High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance.

Following CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to developing CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to pervasive CD19 mis-splicing. Our dataset represents a comprehensive resource for identifying predictive biomarkers for CART-19 therapy.

Effect of Experimental Gestational Diabetes Mellitus on Mechanical Sensitivity, Capsaicin-Induced Pain Behaviors and Hind Paw Glabrous Skin Innervation of Male and Female Mouse Offspring.

PURPOSE: Gestational diabetes mellitus (GDM) induces cardiovascular and metabolic disturbances in offspring. However, the effects of GDM in pain processing in offspring and whether male and female offspring are equally affected is not well known. Thus, we determined: i) whether GDM in mice affects offspring hindpaw mechanical sensitivity, capsaicin-induced spontaneous pain-like behaviors, and epidermal nerve fiber density (ENFD); and ii) whether there is sexual dimorphism in these parameters in offspring from GDM dams. METHODS: GDM was induced in pregnant ICR mice via i.p. streptozotocin (STZ). Then, glucose levels from dams and offspring were determined. Male and female offspring 2-3 months of age were evaluated for: a) baseline mechanical sensitivity of the hind paw by using von Frey filaments; b) number of flinches and time spent guarding induced by intraplantar capsaicin (0.1%); and c) density of PGP-9.5 and CGRP axons in the epidermis from the hind paw glabrous skin. RESULTS: Prepartum levels of glucose in STZ-treated dams were significantly increased compared to vehicle-treated dams; however, GDM or vehicle offspring displayed normal and similar blood glucose levels. Male and female GDM offspring showed significantly greater mechanical sensitivity and capsaicin-induced pain behaviors compared to vehicle offspring. Male GDM offspring displayed a slightly more intense nociceptive phenotype in the capsaicin test. PGP-9.5 and CGRP ENFD in hind paw glabrous skin were greater in male and female GDM offspring versus their controls. Sexual dimorphism was generally not observed in GDM offspring in most of the studied parameters. CONCLUSION: These results suggest GDM induced greater pain-like behaviors in adult offspring regardless of sex along with an increased ENFD of PGP-9.5 and CGRP in the hind paw glabrous skin. We show that GDM peripheral neuropathy differs from diabetic peripheral neuropathy acquired in adulthood and set the foundation to further study this in human babies exposed to GDM.

Direct long-read RNA sequencing identifies a subset of questionable exitrons likely arising from reverse transcription artifacts.

Resistance to CD19-directed immunotherapies in lymphoblastic leukemia has been attributed, among other factors, to several aberrant CD19 pre-mRNA splicing events, including recently reported excision of a cryptic intron embedded within CD19 exon 2. While "exitrons" are known to exist in hundreds of human transcripts, we discovered, using reporter assays and direct long-read RNA sequencing (dRNA-seq), that the CD19 exitron is an artifact of reverse transcription. Extending our analysis to publicly available datasets, we identified dozens of questionable exitrons, dubbed "falsitrons," that appear only in cDNA-seq, but never in dRNA-seq. Our results highlight the importance of dRNA-seq for transcript isoform validation.

Análisis comparativo de los modelos estéticos corporales de una universidad pública y una privada / Comparative analysis of aesthetics body models of a public and private university

Introducción: El modelo estético corporal es un conjunto de pautas fuertemente influyentes sobre las características físicas corporales, regidas principalmente por el culto al atractivo físico con el exaltamiento a la delgadez del cuerpo. Objetivo: Comparar la influencia del modelo estético corporal que contribuye al adelgazamiento entre estudiantes de una universidad privada y una pública. Métodos: Estudio descriptivo, transversal, no probabilístico aplicado a mujeres estudiantes de nutrición entre 18 y 24 años de edad. Se utilizó el CIMEC-40 como instrumento de medición de la interiorización del modelo estético corporal con un punto de corte = 23-24. Se utilizó la prueba t de muestras independientes para determinar igualdad de medias. También se aplicaron los análisis: de confiabilidad, exploratorio, factorial confirmatoria y de varianza multivariado. En todas las pruebas se utilizó un nivel de significancia = 0,05. Resultados: El instrumento evidenció una alta confiabilidad (alfa de Cronbach = 0.91). Se encontró que 69 % (99/143) de las estudiantes de la universidad privada y 50% (66/131) de la universidad pública sobrepasaron el punto de corte establecido; existiendo una diferencia significativa entre ambos (valor p = 0.000083). El análisis factorial constató la validez de cuatro factores de medición: 1) malestar por la imagen corporal, 2) influencia de publicidad 3) in-fluencia de los mensajes verbales y 4) influencias de los modelos sociales. Conclusiones: Las estudiantes de la universidad privada son más sensibles a las influencias del modelo estético corporal que contribuyen al adelgazamiento y con ello la probabilidad de incorporar prácticas habituales alimentarias de riesgo. (AU)

Introduction: The body aesthetic model is a set of strongly influential guidelines of the physical body characteristics, governed mainly by the cult of physical attractiveness with the exaltation of the body thinness. Objective: To compare the influence that have an aesthetic body model that contributes to weight loss between students of a private and public university. Methods: Descriptive, cross-sectional, non-probabilistic study applied to female nutrition students between 18 and 24 years old. The CIMEC-40 was used as an instrument to measure the internalization of the body aesthetic model with a cutoff point = 23-24. The independent samples t-test was used to determine equality of means. Reliability, exploratory, confirmatory factorial and multivariate variance analyzes were also applied at this method. A significance level = 0.05 was used in all tests. Results: The instrument showed high reliability (Cronbach’s alpha = 0.91). It was found that 69% (99/143) of all students from the private university and 50% (66/131) from the public university exceeded the established cut-off point, there being a significant difference between the two (p-value = 0.000083). The factorial analysis verified that the validity of four measurement factors: 1) body image discomfort, 2) advertising influence, 3) influence of verbal messages and 4) influences of social models. Conclusions: The students of the private university studied are more sensitive to the influences of the aesthetic body model that contribute to the weight loss and with it the probability of incorporating risky eating habits. (AU)

Utility of a Fluid Library with Samples of Humans, Reservoirs and Vectors Collected in Filter Paper, for Retrospective Diagnosis of American Trypanosomiasis in Endemic Areas of Venezuela.

INTRODUCTION: We define a fluid library as a library of samples of different biological fluids (from humans, animals or vectors) collected and properly stored on filter paper, which allows retrospective studies, especially of diagnosis or detection of infectious agents in these samples, using different techniques. The objective of this work was the retrospective diagnosis of American trypanosomiasis by PCR in a Venezuelan endemic area using a fluid library. METHODS: A fluid library with samples that had been collected on filter paper, 5 years ago, was used for the detection of Trypanosoma cruzi DNA. 165 blood samples of humans, 30 samples of 25 animals (Didelphis marsupialis, Canis familiaris, Equus asinus and Felis catus) and 8 samples of vectors from endemic areas of Anzoátegui state, were analysed by PCR. RESULTS: The results revealed that 16.4% of the humans samples were positive, 11.1% of those detected positive were children younger than 10 years old, and 26.72% young people aged 11-20 years, suggesting that T. cruzi infection has been active for the past two decades. 56% of the animal samples showed amplification; Didelphis marsupialis 66%, Canis familiaris 54.5%, Equus asinus 50%, and Felis catus 33.3%. On the other hand, positivity (50%) was detected in the studied vectors, of which the 3 most important species in Venezuela (Rhodnius prolixus, Triatoma maculata and Panstrongylus geniculatus) were involved. CONCLUSIONS: The PCR using a fluid library allowed the detection of T. cruzi DNA in old samples from the three host of the epidemiological chain, suggesting that retrospective diagnosis can be made through this strategy and demonstrate that there has been active transmission, which helps to clarify the epidemiological situation in areas where there are no previous reports.

Alfabetización Informacional: enseñanza y desarrollo de su competencia en la educación básica / Information Literacy: teaching and development of its competence in elementary school

Resumen Este artículo presenta los resultados de una investigación sobre la enseñanza de Alfabetización Informacional (ALFIN) en la educación básica, cuyo objetivo fue determinar las mejores prácticas para su implementación e identificar los temas a incorporar en una iniciativa de esta naturaleza, así como las formas de afrontar la problemática de la saturación de la información, de modo que se pretende que cada estudiante aprenda a buscar, a encontrar, a seleccionar, a evaluar y a comunicar información, para adquirir conocimientos, habilidades y actitudes. La metodología del estudio se basó en el análisis de experiencias usando un enfoque cualitativo, donde se concluye que ALFIN se implementó exitosamente por medio del uso del juego, aplicaciones digitales para su enseñanza, cursos específicos en sobre el tema, juegos de mesa, la técnica booleana de búsqueda de información, la lectoescritura, enseñanza por medio del dibujo e instrumentos para la evaluación y utilización de recursos educativos. El logro principal fue contribuir con el desarrollo de las competencias que necesita el estudiantado para desempeñarse con éxito en la escuela y para lograr la capacidad del aprendizaje permanente.

ABSTRACT This article presents a research on the teaching of Information Literacy (IL) in elementary education, whose objective is to determine the best practices for its implementation and to identify the topics that need to be incorporated into such an initiative, as well as the ways of facing the problem of information saturation; so that students learn to search, retrieve, select, evaluate and communicate information, in order to acquire knowledge, competences and attitudes. The methodology employed was based on the qualitative analysis of experiences, which allowed us to concluded that IL has been successfully implemented through the use of educational games, digital applications for its teaching, specific courses on the subject, board games, Boolean search techniques, teaching through drawing, and instruments for the evaluation and use of educational resources. The main achievement was to contribute to the development of the competences that students need to perform successfully in school and to reach the capacity required for lifelong learning.

Implications of Demographics and Socioeconomic Factors in Breast Cancer Reconstruction.

BACKGROUND: Not all women undergo breast reconstruction despite its vital role in the recovery process. Previous studies have reported that women who are ethnically diverse and of lower socioeconomic status are less likely to undergo breast reconstruction, but the reasons remain unclear. The purpose of this study is to evaluate the demographic characteristics of our patient population and their primary reason for not undergoing breast reconstruction. METHODS: An institutional review board-approved, single-institution study was designed to evaluate all female breast cancer patients of all stages who underwent mastectomy but did not undergo breast reconstruction from 2008 to 2014. Patients were contacted via telephone and asked to participate in a validated, prompted survey. Data regarding their demographic information and primary reason for not undergoing breast reconstruction were collected. RESULTS: Inclusion criteria were met by 181 patients, of which 61% participated in the survey. Overall, the most common reason for not undergoing breast reconstruction (26%) was unwillingness to undergo further procedures. However, the most common reason for patients that identified as Hispanic, Spanish-speaking, high school graduates, or having an annual income less than US $25,000 (P < 0.05) was insufficient information received. CONCLUSIONS: This study demonstrates that ethnicity and socioeconomic factors play a key role in determining why patients forego breast reconstruction. Ethnicity, language, education, income, and employment status are associated with patients not receiving appropriate education regarding their reconstructive options. Breast surgeons with a diverse patient population should ensure that these patients are adequately educated regarding their options, and if perhaps, more of these patients would decide to partake in the reconstruction process.

Influencia de estilos de vida en el estado nutricional de estudiantes universitarios / Influence of Lifestyles on the Nutritional Status of University Students

Resumen Antecedentes: el acceso a la universidad supone un cambio importante en el estilo de vida del estudiante, que puede repercutir en su estado nutricional. Objetivo: buscar una asociación entre estilo de vida y cambios antropométricos en estudiantes universitarios de la Escuela Superior Politécnica del Litoral de Guayaquil, Ecuador en un periodo de tres años. Materiales y métodos: estudio descriptivo de cohorte con una muestra de 336 estudiantes de ambos sexos en quienes se evaluó el estado nutricional por antropometría y se indagó sobre su estilo de vida mediante encuesta previamente validada. Resultados: entre 2014 y 2017 incrementó el exceso de peso (sobrepeso + obesidad) de 25,6 % a 31,9 %, IMC, grasa corporal, circunferencia de cintura, circunferencia de cadera, e índice cintura/cadera (p<0,05). Se asoció el consumo de alcohol con mayor peso (+3,7 kg) e índice cintura/cadera; y, mayor frecuencia de consumo de gaseosas con mayor peso (+5 kg). El consumo de comidas rápidas se asoció con el incremento del índice cintura/cadera (p<0,05). No se encontraron diferencias antropométricas según consumo de cigarrillos, actividad física, consumo de frutas y verduras. Conclusión: en la población universitaria estudiada, el exceso de peso y de grasa corporal está aumentando; el consumo de alcohol, de gaseosas y fumar afectan el estado nutricional.

Abstract Background: Attendance to university is a major change in a student's lifestyle, which may be reflected in their nutritional status. Objective: Seek the association between lifestyle and anthropometric changes in university students of the Polytechnic School of the Litoral in Guayaquil-Ecuador over a period of three years. Materials and Methods: Descriptive cohort study with a sample of 336 students of both sexes, in whom nutritional status was evaluated by anthropometry, and lifestyle was investigated through a previously validated survey. Results: Between 2014 and 2017, excess weight (overweight + obesity) increased from 25.6% to 31.9%, BMI, body fat, waist circumference, hip circumference and waist to hip ratio (p <0.05). Alcohol consumption was associated with greater weight (+3.7 Kg) and waist to hip ratio, and higher frequency of consumption of soft drinks with greater weight (+5 kg). The consumption of fast foods was associated with increase in waist to hip ratio (p <0.005). No anthropometric differences were found associated with cigarette use, physical activity, or consumption of fruits and vegetables. Conclusion: In the university population studied, excess weight and body fat is increasing, while alcohol and soda consumption as well as smoking affect nutritional status.

Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis.

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.

Hipertensión pulmonar asociada al virus de la inmunodeficiencia humana en pediatría: tratamiento con sildenafil. A propósito de un caso / Pulmonary hypertension associated with the human immunodeficiency virus in children: treatment with sildenafil. A case report

La hipertensión pulmonar asociada a la infección por virus de inmunodeficiencia humana es una enfermedad sumamente infrecuente en pediatría, por lo que requiere alta sospecha clínica para llegar a su diagnóstico. Su aparición es de pronóstico desfavorable, pero el diagnóstico precoz y el tratamiento específico pueden mejorar su evolución. Se presenta el caso clínico de un paciente de 15 años con diagnóstico de infección por virus de inmunodeficiencia humana de transmisión vertical, sin tratamiento antirretroviral, con tos y disnea de esfuerzo progresiva asociadas a signos de falla cardíaca derecha en el cual se diagnosticó hipertensión pulmonar grave. Luego de descartarse otras causas, se asumió la hipertensión pulmonar asociada a la infección por virus de inmunodeficiencia humana. Se realizó el tratamiento con sildenafil y presentó buena respuesta.

Pulmonary hypertension associated with human immunodeficiency virus infection is an extremely rare disease in pediatrics; it requires a high clinical suspicion to reach a diagnosis. Its appearance poses an unfavorable prognostic, but early diagnosis and specific treatment can improve outcomes. We report the clinical case of a fifteen-year-old patient diagnosed with human immunodeficiency virus infection of vertical transmission, without antiretroviral treatment, with cough and progressive exertional dyspnea, associated with signs of right heart failure in which severe pulmonary hypertension was diagnosed. After discarding other causes, it was assumed pulmonary hypertension associated with human immunodeficiency virus infection. Treatment was performed with sildenafil with good response.

[Pulmonary hypertension associated with the human immunodeficiency virus in children: treatment with sildenafil. A case report]. / Hipertensión pulmonar asociada al virus de la inmunodeficiencia humana en pediatría: tratamiento con sildenafil. A propósito de un caso.

Pulmonary hypertension associated with human immunodeficiency virus infection is an extremely rare disease in pediatrics; it requires a high clinical suspicion to reach a diagnosis. Its appearance poses an unfavorable prognostic, but early diagnosis and specific treatment can improve outcomes. We report the clinical case of a fifteen-year-old patient diagnosed with human immunodeficiency virus infection of vertical transmission, without antiretroviral treatment, with cough and progressive exertional dyspnea, associated with signs of right heart failure in which severe pulmonary hypertension was diagnosed. After discarding other causes, it was assumed pulmonary hypertension associated with human immunodeficiency virus infection. Treatment was performed with sildenafil with good response.

La hipertensión pulmonar asociada a la infección por virus de inmunodeficiencia humana es una enfermedad sumamente infrecuente en pediatría, por lo que requiere alta sospecha clínica para llegar a su diagnóstico. Su aparición es de pronóstico desfavorable, pero el diagnóstico precoz y el tratamiento específico pueden mejorar su evolución. Se presenta el caso clínico de un paciente de 15 años con diagnóstico de infección por virus de inmunodeficiencia humana de transmisión vertical, sin tratamiento antirretroviral, con tos y disnea de esfuerzo progresiva asociadas a signos de falla cardíaca derecha en el cual se diagnosticó hipertensión pulmonar grave. Luego de descartarse otras causas, se asumió la hipertensión pulmonar asociada a la infección por virus de inmunodeficiencia humana. Se realizó el tratamiento con sildenafil y presentó buena respuesta.

Dexmedetomidine Decreases Postoperative Pain and Narcotic Use in Children Undergoing Alveolar Bone Graft Surgery.

OBJECTIVE: Dexmedetomidine is a parenteral agent that combines the benefits of cooperative sedation, anxiolysis, and analgesia without the risks of respiratory depression. Off-label use has been reported in children. We have introduced dexmedetomidine for use in patients having undergone alveolar bone graft (ABG). The objective is to demonstrate the value and safety of postoperative dexmedetomidine infusion in a non-ICU setting following ABG. DESIGN: A retrospective review was performed on patients who underwent ABG by the senior author. Patients were divided into 2 groups: those who received postoperative dexmedetomidine and those who received patient-controlled anesthesia. MAIN OUTCOME MEASURE(S): The primary study outcome measures included patient demographics, adverse events, length of stay, pain scores, and doses of narcotics during admission were collected. RESULTS: Inclusion criteria were met by 54 patients; 39 received dexmedetomidine whereas 15 did not. There were no significant differences between groups in age, gender, and length of stay. The patients who received dexmedetomidine used oral narcotics less often ( P = .01). In addition, more patients reported no pain after surgery ( P = .05) and at the time of discharge if they received dexmedetomidine ( P < .01). There were no reported adverse effects. CONCLUSIONS: Dexmedetomidine provided superior pain control after surgery and at the time of discharge, as well as a significant decrease in the use of oral narcotics. In our institution, it has since replaced the PCA as a postoperative pain control modality. Absent the risk for respiratory depression, dexmedetomidine has demonstrated a safe option for postoperative pain control in our focused group of pediatric patients.

Global accumulation of circRNAs during aging in Caenorhabditis elegans.

BACKGROUND: Circular RNAs (CircRNAs) are a newly appreciated class of RNAs that lack free 5' and 3' ends, are expressed by the thousands in diverse forms of life, and are mostly of enigmatic function. Ostensibly due to their resistance to exonucleases, circRNAs are known to be exceptionally stable. Previous work in Drosophila and mice have shown that circRNAs increase during aging in neural tissues. RESULTS: Here, we examined the global profile of circRNAs in C. elegans during aging by performing ribo-depleted total RNA-seq from the fourth larval stage (L4) through 10-day old adults. Using stringent bioinformatic criteria and experimental validation, we annotated a high-confidence set of 1166 circRNAs, including 575 newly discovered circRNAs. These circRNAs were derived from 797 genes with diverse functions, including genes involved in the determination of lifespan. A massive accumulation of circRNAs during aging was uncovered. Many hundreds of circRNAs were significantly increased among the aging time-points and increases of select circRNAs by over 40-fold during aging were quantified by RT-qPCR. The expression of 459 circRNAs was determined to be distinct from the expression of linear RNAs from the same host genes, demonstrating host gene independence of circRNA age-accumulation. CONCLUSIONS: We attribute the global scale of circRNA age-accumulation to the high composition of post-mitotic cells in adult C. elegans, coupled with the high resistance of circRNAs to decay. These findings suggest that the exceptional stability of circRNAs might explain age-accumulation trends observed from neural tissues of other organisms, which also have a high composition of post-mitotic cells. Given the suitability of C. elegans for aging research, it is now poised as an excellent model system to determine whether there are functional consequences of circRNA accumulation during aging.

Genome-Wide circRNA Profiling from RNA-seq Data.

The genome-wide expression patterns of circular RNAs (circRNAs) are of increasing interest for their potential roles in normal cellular homeostasis, development, and disease. Thousands of circRNAs have been annotated from various species in recent years. Analysis of publically available or user-generated rRNA-depleted total RNA-seq data can be performed to uncover new circRNA expression trends. Here we provide a primer for profiling circRNAs from RNA-seq datasets. The description is tailored for the wet lab scientist with limited or no experience in analyzing RNA-seq data. We begin by describing how to access and interpret circRNA annotations. Next, we cover converting circRNA annotations into junction sequences that are used as scaffolds to align RNA-seq reads. Lastly, we visit quantifying circRNA expression trends from the alignment data.

Loss of adult skeletal muscle stem cells drives age-related neuromuscular junction degeneration.

Neuromuscular junction degeneration is a prominent aspect of sarcopenia, the age-associated loss of skeletal muscle integrity. Previously, we showed that muscle stem cells activate and contribute to mouse neuromuscular junction regeneration in response to denervation (Liu et al., 2015). Here, we examined gene expression profiles and neuromuscular junction integrity in aged mouse muscles, and unexpectedly found limited denervation despite a high level of degenerated neuromuscular junctions. Instead, degenerated neuromuscular junctions were associated with reduced contribution from muscle stem cells. Indeed, muscle stem cell depletion was sufficient to induce neuromuscular junction degeneration at a younger age. Conversely, prevention of muscle stem cell and derived myonuclei loss was associated with attenuation of age-related neuromuscular junction degeneration, muscle atrophy, and the promotion of aged muscle force generation. Our observations demonstrate that deficiencies in muscle stem cell fate and post-synaptic myogenesis provide a cellular basis for age-related neuromuscular junction degeneration and associated skeletal muscle decline.