A brain-enriched circular RNA controls excitatory neurotransmission and restricts sensitivity to aversive stimuli.

Circular RNAs (circRNAs) are a large class of noncoding RNAs. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains unexplored, partly because of the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, an abundant circRNA derived from the Tulp4 gene that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof of principle for the regulatory role of circRNAs in neural processes.

A promoter-dependent upstream activator augments CFTR expression in diverse epithelial cell types.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an anion-selective channel found in epithelial cell membranes. Mutations in CFTR cause cystic fibrosis (CF), an inherited disorder that impairs epithelial function in multiple organs. Most men with CF are infertile due to loss of intact genital ducts. Here we investigated a novel epididymis-selective cis-regulatory element (CRE), located within a peak of open chromatin at -9.5 kb 5' to the CFTR gene promoter. Activation of the -9.5 kb CRE alone by CRISPRa had no impact on CFTR gene expression. However, CRISPRa co-activation of the -9.5 kb CRE and the CFTR gene promoter in epididymis cells significantly augmented CFTR mRNA and protein expression when compared to promoter activation alone. This increase was accompanied by enhanced chromatin accessibility at both sites. Furthermore, the combined CRISPRa strategy activated CFTR expression in other epithelial cells that lack open chromatin at the -9.5 kb site and in which the locus is normally inactive. However, the -9.5 kb CRE does not function as a classical enhancer of the CFTR promoter in transient reporter gene assays. These data provide a novel mechanism for activating/augmenting CFTR expression, which may have therapeutic utility for mutations that perturb CFTR transcription.

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35 kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10 kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35 kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

Dual SMAD inhibition enhances the longevity of human epididymis epithelial cells.

Primary human epididymis epithelial (HEE) cells are valuable reagents for functional studies on the human epididymis. We used them previously to determine the transcriptional networks that establish cell identity along the length of the epididymis from caput, corpus, and cauda. These studies on HEE cells and organoids derived from them revealed important cellular properties. However, similar to other primary cells, HEE cells undergo replicative senescence and de-differentiation in culture. A cocktail of small molecules was shown elsewhere to extend longevity of epithelial basal cells. The components included transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) antagonists, WNT agonist, and Rho-associated and coiled-coil containing protein kinase (ROCK) inhibitor (ROCKi), which together prevented the senescence-related upregulation of TGF-ß signaling pathway members. Here, we treat HEE cells with the same cocktail and observed enhanced replicative potential and prolonged expression of markers of HEE differentiation. This treatment expands the differentiated HEE cell population available from individual epididymis tissue samples that can be used for molecular, cellular, and functional studies.

Perioperative Care of Children with Severe Neurological Impairment and Neuromuscular Scoliosis-A Practical Pathway to Optimize Peri-Operative Health and Guide Decision Making.

Neuromuscular scoliosis is a common feature in children with severe neurological impairment (SNI), including those with severe cerebral palsy. Surgical correction of scoliosis is the mainstay of treatment. This group of patients also have associated medical complexity. The complication rates post-surgery are high, although, for many, they are worth the risk. There are currently no published practice guidelines or care pathways for children with SNI who are undergoing scoliosis corrective surgery. In response to the high uptake of this surgery, coupled with the expected complication rates, our hospital established a perioperative clinic. The purpose of this paper is to describe our perioperative approach. This clinic has developed into a service beyond perioperative care and, with the collaborative meeting, enables shared decision-making to identify the right candidate for surgery. The process involves surgical expertise, understanding the family and child at the centre, and optimisation of medical care pre- and post-surgery. In this paper, we describe the process in a step-by-step manner. We provide clinical vignettes, as well as the proformas that we use, and we highlight the benefits of the team-based process.

De la presencialidad a la virtualidad en tiempos de pandemia: la experiencia del Hospital Italiano de Buenos Aires / From face-to-face to virtuality in pandemic times: the experience of the Hospital Italiano de Buenos Aires

El 11 de marzo de 2020, la Organización Mundial de la Salud (OMS) declaró el COVID-19 como pandemia, afectando drásticamente la atención de la salud. A nivel global se adoptaron medidas como el distanciamiento social y la cuarentena. Ello representó un enorme desafío para los Sistemas de Información en Salud (SIS), que rápidamente debieron adaptarse, frente a una razón ineludible para abrazar por completo la transformación digital. Surge la necesidad de explorar las tecnologías digitales utilizadas durante la pandemia y considerarlas para su uso continuado en el tiempo o cíclicamente en caso de brotes recurrentes. Las herramientas informáticas se han utilizado para la prestación de servicios de telemedicina, monitorización remota de pacientes, comunicación digital entre líderes políticos y autoridades científicas, monitorización de datos para analizar la propagación y evolución del COVID-19, etc. Los países y organizaciones han impulsado el uso de soluciones tecnológicas con distintas limitaciones. El Hospital Italiano de Buenos Aires posee una trayectoria de más de 20 años en implementaciones e innovaciones tecnológicas; sin embargo, la pandemia impulsó una serie de adaptaciones en su SIS. El objetivo de este trabajo fue describir dicho proceso de adaptación digital desde marzo a diciembre de 2020, e identificar los principales resultados utilizando un modelo sociotécnico. Se empleó el modelo de Sittig que incluye 8 dimensiones: Infraestructura, Contenido clínico, Interfaz Humano-computadora, Personas, Comunicación y procesos, Regulaciones, Características organizacionales y Políticas internas y Medición y monitorización. (AU)

On March 11, 2020, the World Health Organization (WHO) declared COVID-19 a pandemic, dramatically affecting health care. Measures such as social distancing and quarantine were adopted globally. This new context represented a huge challenge for Health Information Systems (HIS) that had to adapt quickly, facing an inescapable reason to fully embrace the digital transformation. There is a need to explore the digital technologies used during the pandemic and consider them for continued use over time or cyclically in the event of recurring outbreaks. Digital tools have been used for the provision of telemedicine services, remote patient monitoring, digital communication between political leaders and scientific authorities, data monitoring to analyze the spread and evolution of COVID-19, etc. Countries and organizations have promoted the use of technological solutions with different limitations. The Hospital Italiano de Buenos Aires has a history of more than 20 years in technological implementations and innovations, however, the pandemic prompted a series of adaptations in its SIS. The objective of this work was to describe said digital adaptation process from March to December 2020, and to identify the main results using a sociotechnical model. Sittig´model was used, which includes 8 dimensions: Infrastructure, Clinical Content, Human-Computer Interface, People, Communication and Processes, Regulations, Organizational Characteristics and Internal Policies, and Measurement and Monitoring. (AU)

Random changepoint segmented regression with smooth transition.

We consider random changepoint segmented regression models to analyse data from a study conducted to verify whether treatment with stem cells may delay the onset of a symptom of amyotrophic lateral sclerosis in genetically modified mice. The proposed models capture the biological aspects of the data, accommodating a smooth transition between the periods with and without symptoms. An additional changepoint is considered to avoid negative predicted responses. Given the nonlinear nature of the model, we propose an algorithm to estimate the fixed parameters and to predict the random effects by fitting linear mixed models iteratively via standard software. We compare the variances obtained in the final step with bootstrapped and robust ones.

An epilepsy-associated ACTL6B variant captures neuronal hyperexcitability in a human induced pluripotent stem cell model.

ACTL6B is a component of the neuronal BRG1/brm-associated factor (nBAF) complex, which is required for chromatin remodeling in postmitotic neurons. We recently reported biallelic pathogenic variants in ACTL6B in patients diagnosed with early infantile epileptic encephalopathy, subtype 76 (EIEE-76), presenting with severe, global developmental delay, epileptic encephalopathy, cerebral atrophy, and abnormal central nervous system myelination. However, the pathophysiological mechanisms underlying their phenotype is unknown. Here, we investigate the molecular pathogenesis of ACTL6B p.(Val421_Cys425del) using in silico 3D protein modeling predictions and patient-specific induced pluripotent stem cell-derived neurons. We found neurons derived from EIEE-76 patients showed impaired accumulation of ACTL6B compared to unaffected relatives, caused by reduced protein stability. Furthermore, EIEE-76 patient-derived neurons had dysregulated nBAF target gene expression, including genes important for neuronal development and disease. Multielectrode array system analysis unveiled elevated electrophysiological activity of EIEE-76 patients-derived neurons, consistent with the patient phenotype. Taken together, our findings validate a new model for EIEE-76 and reveal how reduced ACTL6B expression affects neuronal function.

Pain in children with dyskinetic and mixed dyskinetic/spastic cerebral palsy.

AIM: To evaluate pain prevalence and characteristics in children and adolescents with predominant dyskinetic and mixed (dyskinetic/spastic) cerebral palsy (CP) motor types. METHOD: Seventy-five participants with a diagnosis of CP and confirmed dyskinetic or mixed (dyskinetic/spastic) motor type took part in a multisite cross-sectional study. The primary outcome was carer-reported pain prevalence (preceding 2wks) measured using the Health Utilities Index-3. Secondary outcomes were chronicity, intensity, body locations, quality of life, and activity impact. RESULTS: Mean participant age was 10 years 11 months (SD 4y 2mo, range 5-18y). There were 44 males and 31 females and 37 (49%) had predominant dyskinetic CP. Pain was prevalent in 85% and it was chronic in 77% of participants. Fifty-two per cent experienced moderate-to-high carer-reported pain intensity, which was significantly associated with predominant dyskinetic motor types (p=0.008). Pain occurred at multiple body locations (5 out of 21), with significantly increased numbers of locations at higher Gross Motor Function Classification System levels (p=0.02). Face, jaw, and temple pain was significantly associated with predominant dyskinetic motor types (p=0.005). Poorer carer proxy-reported quality of life was detected in those with chronic pain compared to those without (p=0.03); however, chronic pain did not affect quality of life for self-reporting participants. INTERPRETATION: Pain was highly prevalent in children and adolescents with predominant dyskinetic and mixed (dyskinetic/spastic) motor types, highlighting a population in need of lifespan pain management. WHAT THIS PAPER ADDS: Chronic pain prevalence in children and adolescents with predominant dyskinetic and mixed (dyskinetic/spastic) motor types is high. Pain occurs across multiple body locations in predominant dyskinetic and mixed (dyskinetic/spastic) motor types. Less recognized locations of pain include the face, jaw, and temple for predominant dyskinetic motor types.

The lived experience of chronic pain and dyskinesia in children and adolescents with cerebral palsy.

BACKGROUND: To explore the lived experience of chronic pain and dyskinesia in children and adolescents with cerebral palsy. METHODS: A convergent parallel mixed methods design was undertaken. First, a quantitative cross-sectional study of participants able to self-report their quality of life was undertaken. This study characterised pain chronicity, intensity, body locations, and quality of life. Second, semi-structured interviews were undertaken with a subset of children and adolescents experiencing chronic pain. RESULTS: Twenty-five children and adolescents took part in the cross-sectional study, 23 of whom experienced chronic pain and 13 of moderate intensity. Pain was often located in multiple bodily regions (6/21), with no trends in quality of life outcomes detected. Eight participated in semi-structured interviews, which identified three key themes including 'lives embedded with dyskinesia', 'real world challenges of chronic pain', and 'still learning strategies to manage their pain and dyskinesia'. CONCLUSIONS: A high proportion of children and adolescents with cerebral palsy and dyskinesia who were able to self-report experienced chronic pain. The physical and emotional impacts of living with chronic pain and dyskinesia existed along a spectrum, from those with lesser to greater extent of their impacts. Children and adolescents may benefit from targeted chronic pain education and management within bio-psychosocial models.

Revictimization as a high-risk factor for development of posttraumatic stress disorder: a systematic review of the literature

Objective: Much research has been published on the role of sexual revictimization in the emergence of mental disorders in adulthood, but findings have sometimes been contradictory. The present systematic review sought to assess the state of the evidence on revictimization as a potential factor for the emergence of posttraumatic stress disorder (PTSD). Methods: Electronic searches were conducted in five databases (MEDLINE/PubMed, Cochrane Library, Campbell Library, PsycINFO, and LILACS), using the terms PTSD, posttraumatic stress disorder, child abuse, and rape. Results: We identified nine articles that established a connection among childhood sexual abuse (CSA), sexual revictimization in adulthood, and development of PTSD. Eight of the nine papers included were classified as having strong methodological quality (grade VI). One was classified as IV, with an average quality-of-evidence rating. The mean methodological quality score of the articles was 5.5, and the quality of evidence was deemed strong. Conclusion: In the included studies, PTSD symptoms were most prevalent in the CSA + adult sexual assault groups, providing further evidence for the revictimization hypothesis.

Revictimization as a high-risk factor for development of posttraumatic stress disorder: a systematic review of the literature.

OBJECTIVE: Much research has been published on the role of sexual revictimization in the emergence of mental disorders in adulthood, but findings have sometimes been contradictory. The present systematic review sought to assess the state of the evidence on revictimization as a potential factor for the emergence of posttraumatic stress disorder (PTSD). METHODS: Electronic searches were conducted in five databases (MEDLINE/PubMed, Cochrane Library, Campbell Library, PsycINFO, and LILACS), using the terms PTSD, posttraumatic stress disorder, child abuse, and rape. RESULTS: We identified nine articles that established a connection among childhood sexual abuse (CSA), sexual revictimization in adulthood, and development of PTSD. Eight of the nine papers included were classified as having strong methodological quality (grade VI). One was classified as IV, with an average quality-of-evidence rating. The mean methodological quality score of the articles was 5.5, and the quality of evidence was deemed strong. CONCLUSION: In the included studies, PTSD symptoms were most prevalent in the CSA + adult sexual assault groups, providing further evidence for the revictimization hypothesis.

Prevalence and characteristics of pain in children and young adults with cerebral palsy: a systematic review.

AIM: The primary aim of this review is to evaluate the evidence for pain prevalence in children and young adults with cerebral palsy. Secondary aims are to identify pain characteristics and types of pain measurement used in this population. METHOD: Ovid MEDLINE, Embase, CINAHL Plus, and PubMed were searched in October 2016 and updated in November 2017. Two authors independently screened studies according to Preferred Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Pain outcomes were categorized within a biopsychosocial pain framework, with pain prevalence extracted for all recall periods and measurement types. RESULTS: One hundred and six publications from 57 studies met inclusion criteria. Pain prevalence varied widely from 14 per cent to 76 per cent and was higher in females, older age groups, and those classified within Gross Motor Function Classification System level V. Pain was most frequent in the lower limbs, back, and abdomen and associated with reduced quality of life or health status. The influence of pain on psychological functioning, interference, and participation was inconclusive. INTERPRETATION: Variation exists in reported pain prevalence because of sampling bias, inconsistent measurement, varying recall periods, and use of different participant age ranges. WHAT THIS PAPER ADDS: Pain prevalence varies from 14 per cent to 76 per cent in children and young adults with cerebral palsy. Pain is more prevalent in females, older age groups, and children in Gross Motor Function Classification System level V.

Genistein Affects Expression of Cytochrome P450 (CYP450) Genes in Hepatocellular Carcinoma (HEPG2/C3A) Cell Line.

BACKGROUND: Genistein (5,7-Dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is the most abundant isoflavone in soybean, which has been associated with a lower risk of development of cancer and cardiovascular diseases. Of particular interest regarding cancer preventive properties of flavonoids is their interaction with cytochrome P450 enzymes (CYPs). However, contradictory data report the effect of genistein on expression of СYPs enzymes. OBJECTIVE: The aim of this study was to investigate the effects of genistein on cytochrome P450 (CYP) gene expression levels in human hepatocellular carcinoma (HepG2/C3A) and colon adenocarcinoma (HT29) cells. METHODS: Real-time RT-PCR was used to examine the expression of genes families involved in xenobiotic metabolism, such as CYP1 (CYP1A1, CYP1B1), CYP2 (CYP2E1, CYP2D6), CYP3 (CYP3A4); and of a family involved in the catabolism of the all-trans-retinoic acid (ATRA), CYP26 (CYP26A1, CYP26B1). RESULTS: RT-qPCR data analysis showed that after 12 h of exposure of HepG2/C3A cells to genistein (5 and 50 µM) there was an upregulation of CYP1A1 and CYP1B1 and downregulation of CYP2D6, CYP26A1 and CYP26B1 mRNA levels. There was no change in the mRNA levels of CYP P450 genes in HT29 cells. CONCLUSION: Our results suggest that treatment with genistein in non-toxic concentrations may impact the expression level of CYPs involved in the biotransformation of xenobiotics and drug metabolizing enzymes. Moreover, the downregulation of ATRA metabolism-related genes opens a new research path for the study of genistein as retinoic acid metabolism blocking agent for treating cancer and other pathologies.

Human Adipose-Derived CD146+ Stem Cells Increase Life Span of a Muscular Dystrophy Mouse Model More Efficiently than Mesenchymal Stromal Cells.

Duchenne muscular dystrophy is the most common and severe form of progressive muscular dystrophy. Previous results showed an increased survival in double knockout mice (dko) when treated with adipose-derived CD146+ cells. In this study, we analyzed the effect of CD146+ cells compared to mesenchymal stem/stromal cells (MSCs) derived from the same human adipose sample when injected in the dko mouse model without immunosuppression. Both CD146+ cells and MSCs increased the survival of treated mice when compared to vehicle-injected mice, with a more prominent effect of CD146+ cells than MSCs. Both CD146+ cells and MSCs suppressed peripheral blood mononuclear cell proliferation, indicating immunomodulatory properties. Co-culture experiments showed that MSCs have a more inflammatory profile expression, and angiogenesis assay showed that CD146+ cells can improve blood vessel formation. CD146+ cells can extend survival of muscular dystrophy mice more efficiently than MSCs, possibly due to immunomodulatory and angiogenic properties. Further investigations focusing on exogenous CD146+ cell role in vivo will improve cell therapy understanding and effectiveness.

Physiological and biochemical changes during the loss of desiccation tolerance in germinating Adenanthera pavonina L. seeds.

We investigated the loss of desiccation tolerance (DT) in Adenanthera pavonina seeds during germination. Seeds were subjected to imbibition for 0, 24, 36, 48, 60 and 81 h, then dried to their initial moisture content (13%), rehydrated and evaluated for survival (resumption of growth and development of normal seedlings) and membrane system integrity (electrolyte leakage). Embryonic axes of seeds subjected only to imbibition during the same early time periods were used to investigate the electrophoretic patterns of heat-stable proteins and the relative nuclear DNA content. In A. pavonina seeds, DT remained unchanged until 36 h of imbibition (resulting in germination and 82% normal seedlings), after which it was progressively lost, and seeds with a protruded radicle length of 1 mm did not withstand dehydration. The loss of desiccation tolerance could not be related to either membrane damage caused by drying or the resumption of the cell cycle during germination. However, the decrease in heat-stable protein contents observed throughout germination may be related to the loss of DT in A. pavonina seeds.