Analysis of feeding and eating disorders in 191 children according to psychiatric or gastroenterological recruitment: The PEDIAFED cohort study.

OBJECTIVE: The DSM-5 classification introduced new Feeding and Eating Disorders (FED) diagnostic categories, notably Avoidant and Restrictive Food Intake Disorder (ARFID), which, like other FED, can present psychiatric and gastrointestinal symptoms. However, paediatric clinical research that focuses on children below the age of 12 years remains scarce. The aim of this study was first to investigate the clinical features of FED in a cohort of children, second to compare them according to their recruitment (gastroenterology or psychiatry unit). METHOD: This non-interventional retrospective cohort study analysed 191 patients in a French paediatric tertiary care centre (gastroenterology n = 100, psychiatry n = 91). The main outcome variables were clinical data (type of FED, BMI, nutritional support, chronic diseases, psychiatric comorbidities, sensory, sleep, language disorders, gastrointestinal complaints, adverse life events, family history). The outcome was defined by a Clinical Global Impression of Change-score. RESULTS: FED diagnoses were ARFID (n = 100), Unspecified FED (UFED, n = 57), anorexia nervosa (AN, n = 33) and one pica/rumination. Mean follow-up was 3.28 years (SD 1.91). ARFID was associated with selective and sensory disorders (p < 0.001); they had more anxiety disorders than patients with UFED (p < 0.001). Patients with UFED had more chewing difficulties, language disorder (p < 0.001), and more FED related to chronic disease (p < 0.05) than patients with ARFID and AN. Patients with AN were female, underweight, referred exclusively to the psychiatrist, and had more depression than patients with ARFID and UFED (p < 0.001). The gastroenterology cohort included more UFED, while the psychiatry cohort included more psychiatric comorbidities (p < 0.001). A worse clinical outcome was associated with ARFID, a younger age at onset (p < 0.001), selective/sensory disorders and nutritional support (p < 0.05). CONCLUSION: ARFID and UFED children were diagnosed either by gastroenterologists or psychiatrists. Due to frequently associated somatic and psychiatric comorbidities, children with FED should benefit from a multidisciplinary assessment and care.

A longitudinal single-cell atlas of treatment response in pediatric AML.

Pediatric acute myeloid leukemia (pAML) is characterized by heterogeneous cellular composition, driver alterations and prognosis. Characterization of this heterogeneity and how it affects treatment response remains understudied in pediatric patients. We used single-cell RNA sequencing and single-cell ATAC sequencing to profile 28 patients representing different pAML subtypes at diagnosis, remission and relapse. At diagnosis, cellular composition differed between genetic subgroups. Upon relapse, cellular hierarchies transitioned toward a more primitive state regardless of subtype. Primitive cells in the relapsed tumor were distinct compared to cells at diagnosis, with under-representation of myeloid transcriptional programs and over-representation of other lineage programs. In some patients, this was accompanied by the appearance of a B-lymphoid-like hierarchy. Our data thus reveal the emergence of apparent subtype-specific plasticity upon treatment and inform on potentially targetable processes.

Multi-Omic Analysis of CIC's Functional Networks Reveals Novel Interaction Partners and a Potential Role in Mitotic Fidelity.

CIC encodes a transcriptional repressor and MAPK signalling effector that is inactivated by loss-of-function mutations in several cancer types, consistent with a role as a tumour suppressor. Here, we used bioinformatic, genomic, and proteomic approaches to investigate CIC's interaction networks. We observed both previously identified and novel candidate interactions between CIC and SWI/SNF complex members, as well as novel interactions between CIC and cell cycle regulators and RNA processing factors. We found that CIC loss is associated with an increased frequency of mitotic defects in human cell lines and an in vivo mouse model and with dysregulated expression of mitotic regulators. We also observed aberrant splicing in CIC-deficient cell lines, predominantly at 3' and 5' untranslated regions of genes, including genes involved in MAPK signalling, DNA repair, and cell cycle regulation. Our study thus characterises the complexity of CIC's functional network and describes the effect of its loss on cell cycle regulation, mitotic integrity, and transcriptional splicing, thereby expanding our understanding of CIC's potential roles in cancer. In addition, our work exemplifies how multi-omic, network-based analyses can be used to uncover novel insights into the interconnected functions of pleiotropic genes/proteins across cellular contexts.

Oral exploration and food selectivity: A case-control study conducted in a multidisciplinary outpatient setting.

Background: Pediatric feeding disorders (PFDs) are common, and their great phenotypic variability reflects the breadth of the associated nosological profiles. PFDs should be assessed and managed by multidisciplinary teams. Our study aimed to describe clinical signs of feeding difficulties in a group of PFD patients assessed by such a team, and to compare them with children in a control group. Methods: In this case-control study, case group patients 1 to 6 years old were consecutively recruited through the multidisciplinary unit for the treatment of pediatric feeding difficulties based at Robert Debré Teaching Hospital in Paris, France. Children with an encephalopathy, severe neurometabolic disorder, or genetic syndrome (suspected or confirmed) were excluded. Members of the control group, consisting of children with no feeding difficulties (i.e., Montreal Children's Hospital Feeding Scale scores below 60) or severe chronic diseases, were recruited from a day care center and 2 kindergartens. Data from medical histories and clinical examination related to mealtime practices, oral motor skills, neurodevelopment, sensory processing, and any functional gastrointestinal disorders (FGIDs) were recorded and compared between groups. Results: In all, 244 PFD cases were compared with 109 controls (mean ages: cases, 3.42 [±1.47]; controls, 3.32 [±1.17]; P = 0.55). Use of distractions during meals was much more among PFD children (cases, 77.46%; controls, 5.5%; P < 0.001), as was conflict during meals. While the groups did not differ in their members' hand-mouth coordination or ability to grab objects, cases began exploring their environments later; mouthing, especially, was less common in the case group (cases, n = 80 [32.92%]; controls, n = 102 [94.44%]; P < 0.001). FGIDs and signs of visual, olfactory, tactile, and oral hypersensitivity were significantly more frequent among cases. Conclusion: Initial clinical assessments showed that, in the children with PFDs, normal stages of environmental exploration were altered, and that this was often associated with signs of sensory hypersensitivity and digestive discomfort.

Exploration of how primary care models influence job satisfaction among primary care providers during the COVID-19 pandemic in New Brunswick: a descriptive and comparative study.

BACKGROUND: The COVID-19 pandemic has highlighted human resource gaps and physician shortages in healthcare systems in New Brunswick (NB), as evidenced by multiple healthcare service interruptions. In addition, the New Brunswick Health Council gathered data from citizens on the type of primary care models (i.e. physicians in solo practice, physicians in collaborative practice, and collaborative practice with physicians and nurse practitioners) they use as their usual place of care. To add to their survey's findings, our study aims to see how these different primary care models were associated with job satisfaction as reported by primary care providers. METHODS: In total, 120 primary care providers responded to an online survey about their primary care models and job satisfaction levels. We used IBM's "SPSS Statistics" software to run Chi-square and Fisher's exact tests to compare job satisfaction levels between variable groups to determine if there were statistically significant variations. RESULTS: Overall, 77% of participants declared being satisfied at work. The reported job satisfaction levels did not appear to be influenced by the primary care model. Participants reported similar job satisfaction levels regardless of if they practiced alone or in collaboration. Although 50% of primary care providers reported having symptoms of burnout and experienced a decline in job satisfaction during the COVID-19 pandemic, the primary care model was not associated with these experiences. Therefore, participants who reported burnout or a decline in job satisfaction were similar in all primary care models. Our study's results suggest that the autonomy to choose a preferred model was important, since 45.8% of participants reported choosing their primary care models, based on preference. Proximity to family and friends and balancing work and family emerged as critical factors that influence choosing a job and staying in that job. CONCLUSION: Primary care providers' staffing recruitment and retention strategies should include the factors reported as determinants in our study. Primary care models do not appear to influence job satisfaction levels, although having the autonomy to choose a preferred model was reported as highly important. Consequently, it may be counterproductive to impose specific primary care models if one aims to prioritize primary care providers' job satisfaction and wellness.

Pharmacist-led interventions during transitions of care of older adults admitted to short term geriatric units: Current practices and perceived barriers.

Background: During transitions of care, older adults are at risk of adverse drug events which could lead to avoidable hospital visits. Pharmacists are increasingly involved in care teams at various stages of the continuum of care. The types and frequency of clinical interventions performed by pharmacists in the geriatric practice setting remain poorly documented. Objectives: This study aimed to describe the current integration of pharmacist interventions during transitions of care of older adults admitted in short-term geriatric units (STGUs) and to explore barriers and facilitators to their implementation in clinical practice. The secondary objective was to explore associations between certain patient characteristics and pharmacist-led interventions during transitional care. Methods: A mixed methods study was conducted with pharmacists practicing in STGUs in the Montreal area, Canada. The application of 8 pharmaceutical interventions was assessed using a self-administered questionnaire, along with as a retrospective chart review. Four semi-structured group interviews were conducted in order to identify perceived barriers and facilitators. Results: Thirteen pharmacists participated in the study. In the questionnaire, medication reconciliation on admission and at discharge was reported as being performed at least half the time by 12 (92%) and 7 (54%) pharmacists, respectively. The retrospective chart review revealed that these interventions were documented in 95 (98%) and 25 (26%) files, respectively. While 35% of patients had a documented pharmaceutical care plan on admission, none was documented at discharge. Several barriers to implementing clinical interventions were identified such as lack of time, technical support, communication and standardization. Conclusions: Pharmacists are involved at different periods of transitional care; however, certain barriers should be addressed in order to expand their role in discharge planning. Providing guidelines on what is expected at discharge and post-discharge, and having a practice focused on delegation and collaboration would help pharmacists increase their role throughout the transition of care of older adults.

Single-cell landscapes of primary glioblastomas and matched explants and cell lines show variable retention of inter- and intratumor heterogeneity.

Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive inter- and intratumor heterogeneity. Patient-derived models, such as organoids and explants, have recently emerged as useful models to study such heterogeneity, although the extent to which they can recapitulate GBM genomic features remains unclear. Here, we analyze bulk exome and single-cell genome and transcriptome profiles of 12 IDH wild-type GBMs, including two recurrent tumors, and of patient-derived explants (PDEs) and gliomasphere (GS) lines derived from these tumors. We find that PDEs are genetically similar to, and variably retain gene expression characteristics of, their parent tumors. Notably, PDEs appear to exhibit similar levels of transcriptional heterogeneity compared with their parent tumors, whereas GS lines tend to be enriched for cells in a more uniform transcriptional state. The approaches and datasets introduced here will provide a valuable resource to help guide experiments using GBM-derived models, especially in the context of studying cellular heterogeneity.

Weaning children from prolonged enteral nutrition: A position paper.

Enteral nutrition (EN) allows adequate nutritional intake in children for whom oral intake is impossible, insufficient or unsafe. With maturation and health improvements, most children ameliorate oral skills and become able to eat orally, therefore weaning from EN becomes a therapeutic goal. No recommendations currently exist on tube weaning, and practices vary widely between centres. With this report, the French Network of Rare Digestive Diseases (FIMATHO) and the French-Speaking Group of Paediatric Hepatology, Gastroenterology and Nutrition (GFHGNP) aim to develop uniform clinical practice recommendations for weaning children from EN. A multidisciplinary working group (WG) encompassing paediatricians, paediatric gastroenterologists, speech-language therapists, psychologists, dietitians and occupational therapists, was formed in June 2018. A systematic literature search was performed on those published from January 1, 1998, to April 30, 2020, using MEDLINE. After several rounds of e-discussions, relevant items for paediatric tube weaning were identified, and recommendations were developed, discussed and finalized. The WG members voted on each recommendation using a nominal voting technique. Expert opinion was applied to support the recommendations where no high-quality studies were available.

Integrative multi-omic analysis reveals neurodevelopmental gene dysregulation in CIC-knockout and IDH1-mutant cells.

Capicua (CIC)'s transcriptional repressor function is implicated in neurodevelopment and in oligodendroglioma (ODG) aetiology. However, CIC's role in these contexts remains obscure, primarily from our currently limited knowledge regarding its biological functions. Moreover, CIC mutations in ODG invariably co-occur with a neomorphic IDH1/2 mutation, yet the functional relationship between these two genetic events is unknown. Here, we analysed models derived from an E6/E7/hTERT-immortalized (i.e. p53- and RB-deficient) normal human astrocyte cell line. To examine the consequences of CIC loss, we compared transcriptomic and epigenomic profiles between CIC wild-type and knockout cell lines, with and without mutant IDH1 expression. Our analyses revealed dysregulation of neurodevelopmental genes in association with CIC loss. CIC ChIP-seq was also performed to expand upon the currently limited ensemble of known CIC target genes. Among the newly identified direct CIC target genes were EPHA2 and ID1, whose functions are linked to neurodevelopment and the tumourigenicity of in vivo glioma tumour models. NFIA, a known mediator of gliogenesis, was discovered to be uniquely overexpressed in CIC-knockout cells expressing mutant IDH1-R132H protein. These results identify neurodevelopment and specific genes within this context as candidate targets through which CIC alterations may contribute to the progression of IDH-mutant gliomas. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Ceftazidime-Induced Neurotoxicity in an 80-Year-Old Female With Renal Dysfunction: A Case Report.

Neurological toxicity is a relatively rare adverse reaction reported in elderly patients treated with cephalosporins. We present a case of ceftazidime-induced encephalopathy in the context of acute kidney injury in an 80-year-old female treated for a Pseudomonas aeruginosa prosthetic joint infection. During the course of treatment, the patient developed sudden confusion and disorientation. The patient's mental state progressively worsened, eventually leading to intubation and admission to the intensive care unit. As imaging and laboratory analyses revealed no alternative causes explaining the patient's symptoms, ceftazidime was stopped under the suspicion of drug-induced neurotoxicity. Shortly after ceftazidime discontinuation, the patient's condition drastically improved and returned to baseline within 5 days. This case reveals the potential severity of cephalosporin-induced neurotoxicity in elderly patients and highlights the importance of quickly detecting such adverse events in order to prevent dire outcomes.

A Scalable Strand-Specific Protocol Enabling Full-Length Total RNA Sequencing From Single Cells.

RNA sequencing (RNAseq) has been widely used to generate bulk gene expression measurements collected from pools of cells. Only relatively recently have single-cell RNAseq (scRNAseq) methods provided opportunities for gene expression analyses at the single-cell level, allowing researchers to study heterogeneous mixtures of cells at unprecedented resolution. Tumors tend to be composed of heterogeneous cellular mixtures and are frequently the subjects of such analyses. Extensive method developments have led to several protocols for scRNAseq but, owing to the small amounts of RNA in single cells, technical constraints have required compromises. For example, the majority of scRNAseq methods are limited to sequencing only the 3' or 5' termini of transcripts. Other protocols that facilitate full-length transcript profiling tend to capture only polyadenylated mRNAs and are generally limited to processing only 96 cells at a time. Here, we address these limitations and present a novel protocol that allows for the high-throughput sequencing of full-length, total RNA at single-cell resolution. We demonstrate that our method produced strand-specific sequencing data for both polyadenylated and non-polyadenylated transcripts, enabled the profiling of transcript regions beyond only transcript termini, and yielded data rich enough to allow identification of cell types from heterogeneous biological samples.

Impact of pharmacist interventions during transition of care in older adults to reduce the use of healthcare services: A scoping review.

BACKGROUND: Current literature has shown increasing risk of error in transition of care between different healthcare settings, especially in the older population. Moreover, drug-related hospital readmission has been reported due to lack of appropriate communication. However, the literature is not clear about the impact of pharmacist interventions during transition of care of older adults on the reduction in use of healthcare services. OBJECTIVE: The goal of the scoping review was to describe the impact of pharmacist interventions during transitions of care for older adults on the use of healthcare services. METHODS: MEDLINE was searched for randomized controlled trials and controlled studies that analyzed pharmacist interventions during transition of care of older adults with regard to use of healthcare services. Four reviewers, grouped in pairs, independently screened all references published from 1990 to 2019 and extracted and analyzed the data. A pharmaceutical model of 8 pharmacist-led interventions was adapted from literature to compare the included studies. RESULTS: There were 1527 publications screened, 17 of which met inclusion criteria. Pharmacist-led interventions decreased the use of healthcare services in 11 of these studies. The majority of studies were of very good or good quality based on Mixed Methods Appraisal Tool. Pharmacist were implicated at all times during the transition of care process (i.e. admission/during stay, discharge and post-discharge) in 4 of the effective studies, whereas none did in the not effective studies. More interventions were accomplished by pharmacists in studies with positive outcomes. CONCLUSION: By diversifying their interventions at different moments throughout transition of care, pharmacists can reduce the use of healthcare services for older adults during transition of care. This scoping review also shows the need to better understand key components of post-discharge interventions and to have a dynamic pharmaceutical model accepted by the scientific community.

Transcriptomic analysis of CIC and ATXN1L reveal a functional relationship exploited by cancer.

Aberrations in Capicua (CIC) have recently been implicated as a negative prognostic factor in a multitude of cancer types through activation of the MAPK signalling cascade and derepression of oncogenic ETS transcription factors. The Ataxin-family protein ATXN1L has previously been reported to interact with CIC in developmental and disease contexts to facilitate the repression of CIC target genes. To further investigate this relationship, we performed functional in vitro studies utilizing ATXN1LKO and CICKO human cell lines and characterized a reciprocal functional relationship between CIC and ATXN1L. Transcriptomic interrogation of the CIC-ATXN1-ATXN1L axis in low-grade glioma, prostate adenocarcinoma and stomach adenocarcinoma TCGA cohorts revealed context-dependent convergence of gene sets and pathways related to mitotic cell cycle and division. This study highlights the CIC-ATXN1-ATXN1L axis as a more potent regulator of the cell cycle than previously appreciated.

N-Acetylglucosamine-1-Phosphate Transferase, WecA, as a Validated Drug Target in Mycobacterium tuberculosis.

The mycobacterial phosphoglycosyltransferase WecA, which initiates arabinogalactan biosynthesis in Mycobacterium tuberculosis, has been proposed as a target of the caprazamycin derivative CPZEN-45, a preclinical drug candidate for the treatment of tuberculosis. In this report, we describe the functional characterization of mycobacterial WecA and confirm the essentiality of its encoding gene in M. tuberculosis by demonstrating that the transcriptional silencing of wecA is bactericidal in vitro and in macrophages. Silencing wecA also conferred hypersensitivity of M. tuberculosis to the drug tunicamycin, confirming its target selectivity for WecA in whole cells. Simple radiometric assays performed with mycobacterial membranes and commercially available substrates allowed chemical validation of other putative WecA inhibitors and resolved their selectivity toward WecA versus another attractive cell wall target, translocase I, which catalyzes the first membrane step in the biosynthesis of peptidoglycan. These assays and the mutant strain described herein will be useful for identifying potential antitubercular leads by screening chemical libraries for novel WecA inhibitors.

Comparative transcriptome analysis of isogenic cell line models and primary cancers links capicua (CIC) loss to activation of the MAPK signalling cascade.

CIC encodes a transcriptional repressor, capicua (CIC), whose disrupted activity appears to be involved in several cancer types, including type I low-grade gliomas (LGGs) and stomach adenocarcinomas (STADs). To explore human CIC's transcriptional network in an isogenic background, we developed novel isogenic CIC knockout cell lines as model systems, and used these in transcriptome analyses to study the consequences of CIC loss. We also compared our results with analyses of transcriptome data from TCGA for type I LGGs and STADs. We identified 39 candidate targets of CIC transcriptional regulation, and confirmed seven of these as direct targets. We showed that, although many CIC targets appear to be context-specific, the effects of CIC loss converge on the dysregulation of similar biological processes in different cancer types. For example, we found that CIC deficiency was associated with disruptions in the expression of genes involved in cell-cell adhesion, and in the development of several cell and tissue types. We also showed that loss of CIC leads to overexpression of downstream members of the mitogen-activated protein kinase (MAPK) signalling cascade, indicating that CIC deficiency may present a novel mechanism for activation of this oncogenic pathway. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Micrococcin P1 - A bactericidal thiopeptide active against Mycobacterium tuberculosis.

The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains. In addition, in the case of TB, which claimed 1.5 million lives in 2014, the treatment of the least complicated, drug sensitive cases is lengthy and disagreeable. Therefore, new drugs with novel targets are urgently needed to control resistant Mycobacterium tuberculosis strains. In this manuscript we report the characterization of the thiopeptide micrococcin P1 as an anti-tubercular agent. Our biochemical experiments show that this antibiotic inhibits the elongation step of protein synthesis in mycobacteria. We have further identified micrococcin resistant mutations in the ribosomal protein L11 (RplK); the mutations were located in the proline loop at the N-terminus. Reintroduction of the mutations into a clean genetic background, confirmed that they conferred resistance, while introduction of the wild type RplK allele into resistant strains re-established sensitivity. We also identified a mutation in the 23S rRNA gene. These data, in good agreement with previous structural studies suggest that also in M. tuberculosis micrococcin P1 functions by binding to the cleft between the 23S rRNA and the L11 protein loop, thus interfering with the binding of elongation factors Tu and G (EF-Tu and EF-G) and inhibiting protein translocation.

Effect of O6-Substituted Guanine Analogs on O6-methylguanine DNA-methyltransferase Expression and Glioblastoma Cells Viability.

BACKGROUND: Glioblastoma multiforme (GBM) is often associated with a poor survival prognostic for patients. The main reason seems to be the acquired or inherent resistance to the chemotherapeutic agent used to treat the tumor, temozolomide (TMZ). To this day, the most recognized pathway of resistance is the DNA Direct Repair pathway by the means of the protein O6- methylguanine DNA-methyltransferase (MGMT). OBJECTIVES: To design and synthesize a series of MGMT inhibitors that can sensitize GBM cells to TMZ. METHODS: Twenty-five O6-alkyl, O6-aryl and O6-substituted-aryl guanine analogs including nine novel compounds were synthesized, characterized, analyzed by molecular docking and tested on the T98G GBM cells viability. RESULTS: Following molecular modeling with MGMT, the newly designed compounds 19, 22, and 24 emerged as the most promising MGMT ligands and displayed modest cytotoxicity. Guanine analog (19), bearing a p-nitrobenzyl moiety, reduced considerably the O6-methylguanine DNAmethyltransferase expression level. When combined with TMZ (1), which is used as first line treatment for brain tumors, compounds 19, 22, and 24 decreased T98G cells proliferation by 32%, 68% and 50%, respectively. TMZ (1) displayed negligible effect on the proliferation of these cells further supporting the notion that this cell model is resistant to this alkylating agent. CONCLUSION: Overall, these results notably highlight a group of MGMT inhibitors that warrants further exploration in the development of therapeutic options to circumvent TMZ resistance in brain tumors.

DNA methylation in adult diffuse gliomas.

Adult diffuse gliomas account for the majority of primary malignant brain tumours, and are in most cases lethal. Current therapies are often only marginally effective, and improved options will almost certainly benefit from further insight into the various processes contributing to gliomagenesis and pathology. While molecular characterization of these tumours classifies them on the basis of genetic alterations and chromosomal abnormalities, DNA methylation patterns are increasingly understood to play a role in glioma pathogenesis. Indeed, a subset of gliomas associated with improved survival is characterized by the glioma CpG island methylator phenotype (G-CIMP), which can be induced by the expression of mutant isocitrate dehydrogenase (IDH1/2). Aberrant methylation of particular genes or regulatory elements, within the context of G-CIMP-positive and/or negative tumours, has also been shown to be associated with differential survival. In this review, we provide an overview of the current knowledge regarding the role of DNA methylation in adult diffuse gliomas. In particular, we discuss IDH mutations and G-CIMP, MGMT promoter methylation, DNA methylation-mediated microRNA regulation and aberrant methylation of specific genes or groups of genes.

Genome-Wide Profiles of Extra-cranial Malignant Rhabdoid Tumors Reveal Heterogeneity and Dysregulated Developmental Pathways.

Malignant rhabdoid tumors (MRTs) are rare lethal tumors of childhood that most commonly occur in the kidney and brain. MRTs are driven by SMARCB1 loss, but the molecular consequences of SMARCB1 loss in extra-cranial tumors have not been comprehensively described and genomic resources for analyses of extra-cranial MRT are limited. To provide such data, we used whole-genome sequencing, whole-genome bisulfite sequencing, whole transcriptome (RNA-seq) and microRNA sequencing (miRNA-seq), and histone modification profiling to characterize extra-cranial MRTs. Our analyses revealed gene expression and methylation subgroups and focused on dysregulated pathways, including those involved in neural crest development.

A simple histological technique to improve immunostaining when using DNA denaturation for BrdU labelling.

The typical immunohistochemistry technique used to reveal 5-bromo-2'-deoxyuridine (BrdU) incorporation requires denaturation of the DNA by heat and acid to permeabilize the cell nucleus. This treatment can damage tissue and reduce the antigenicity of several proteins, which then leads to weak immunostaining and/or false negatives. We show that an overnight post-fixation step following immunohistochemistry for antigens of interest protects immunostaining during the acid/heat denaturation treatment for subsequent BrdU staining. We used this technique to study the differentiation of recently divided oligodendrocyte progenitor cells in NG2CreER:EYFP reporter mice. We used a GFP anti-EYFP antibody to maximize visualization of the EYFP-containing oligodendrocyte progenitor cells, Olig1, and GST-pi to confirm the cell phenotype. Immunostaining for GFP, Olig1, and GST-pi is reduced by DNA denaturation. We found that incorporating a post-fixation step after double immunostaining for GFP/Olig1 and GFP/GST-pi prior to DNA denaturation prevented the fading and false negatives associated with this treatment. This simple addition to BrdU immunohistochemistry protocols extends the range of proteins that can be detected in combination with BrdU, along with the number of antibodies that can be used successfully in the study of cell proliferation.