Neurogenin Research Articles

The SWI/SNF chromatin remodelling complex regulates pancreatic endocrine cell expansion and differentiation in mice in vivo.

Strategies to augment functional beta cell mass include directed differentiation of stem cells towards a beta cell fate, which requires extensive knowledge of transcriptional programs governing endocrine progenitor cell differentiation in vivo. We aimed to study the contributions of the Brahma-related gene-1 (BRG1) and Brahma (BRM) ATPase subunits of the SWI/SNF chromatin remodelling complex to endocrine cell development. We generated mice with endocrine progenitor-specific Neurog3-Cre BRG1 removal in the presence of heterozygous (Brg1Δendo;Brm+/-) or homozygous (double knockout: DKOΔendo) BRM deficiency. Whole-body metabolic phenotyping, islet function characterisation, islet quantitative PCR and histological characterisation were performed on animals and tissues postnatally. To test the mechanistic actions of SWI/SNF in controlling gene expression during endocrine cell development, single-cell RNA-seq was performed on flow-sorted endocrine-committed cells from embryonic day 15.5 control and mutant embryos. Brg1Δendo;Brm+/- mice exhibit severe glucose intolerance, hyperglycaemia and hypoinsulinaemia, resulting, in part, from reduced islet number; diminished alpha, beta and delta cell mass; compromised islet insulin secretion; and altered islet gene expression programs, including reductions in MAFA and urocortin 3 (UCN3). DKOΔendo mice were not recovered at weaning; however, postnatal day 6 DKOΔendo mice were severely hyperglycaemic with reduced serum insulin levels and beta cell area. Single-cell RNA-seq of embryonic day 15.5 lineage-labelled cells revealed endocrine progenitor, alpha and beta cell populations from SWI/SNF mutants have reduced expression of Mafa, Gcg, Ins1 and Ins2, suggesting limited differentiation capacity. Reduced Neurog3 transcripts were discovered in DKOΔendo endocrine progenitor clusters, and the proliferative capacity of neurogenin 3 (NEUROG3)+ cells was reduced in Brg1Δendo;Brm+/- and DKOΔendo mutants. Loss of BRG1 from developing endocrine progenitor cells has a severe postnatal impact on glucose homeostasis, and loss of both subunits impedes animal survival, with both groups exhibiting alterations in hormone transcripts embryonically. Taken together, these data highlight the critical role SWI/SNF plays in governing gene expression programs essential for endocrine cell development and expansion. Raw and processed data for scRNA-seq have been deposited into the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE248369.

Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ∼0.9 in as little as six-weeks with a mean firing rate of ∼13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of blood-brain barrier breakdown by using human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

Mechanism of electroacupuncture regulating nuclear factor-κB pathway to improve the dedifferentiation of pancreatic β-cells in rats with T2DM

To observe the effects of electroacupuncture (EA) on the expression of serum interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and the pancreatic nuclear factor-κB (NF-κB) pathway in type 2 diabetes mellitus (T2DM) rats, and to explore the possible mechanism by which EA improving the dedifferentiation of pancreatic β-cells in the treatment of T2DM. Among 18 SPF-grade male Wistar rats, 6 rats were randomly selected as the control group, and the remaining 12 rats were fed with high-sugar and high-fat diet combined with intraperitoneal injection of 2% streptozotocin solution (35 mg/kg) to establish T2DM model. After successful modeling, the 12 rats were randomly divided into a model group and an EA group, with 6 rats in each group. The EA group received EA at bilateral "Zusanli" (ST 36), "Sanyinjiao" (SP 6), "Weiwanxiashu" (EX-B 3), and "Pishu" (BL 20), with continuous wave, frequency of 15 Hz, current intensity of 2 mA, for 20 min each time, once a day, 6 times a week, for a total of 6 weeks. Fasting blood glucose (FBG) levels were measured before modeling and before and after intervention. After intervention, ELISA was used to detect the serum fasting insulin (FINS), IL-1β and TNF-α levels, and the β-cell function index (HOMA-β) and insulin resistance index (HOMA-IR) were calculated; HE staining was used to observe the morphology of the pancreatic islets; Western blot was used to detect the protein expression of pancreatic forkhead box protein O1 (FoxO1), pancreatic and duodenal homeobox 1 (PDX-1), neurogenin 3 (NGN3), and NF-κB p65. After intervention, the FBG in the model group was higher than that in the control group (P<0.01), and the FBG in the EA group was lower than that in the model group (P<0.01). Compared with the control group, the model group had increased levels of serum FINS, IL-1β, TNF-α, and HOMA-IR (P<0.01), and decreased HOMA-β (P<0.01), reduced protein expression of pancreatic FoxO1 and PDX-1 (P<0.01), and increased protein expression of pancreatic NGN3 and NF-κB p65 (P<0.01, P<0.05). Compared with the model group, the EA group had lower serum FINS, IL-1β, TNF-α levels, and HOMA-IR (P<0.01), higher HOMA-β (P<0.05), increased protein expression of pancreatic FoxO1 and PDX-1 (P<0.01, P<0.05), and decreased protein expression of pancreatic NGN3 and NF-κB p65 (P<0.01, P<0.05). The control group's pancreatic islets showed no obvious abnormalities; the model group's pancreatic islets were irregular in shape and had unclear boundaries with the surrounding area, with immune cell infiltration, reduced β-cell nuclei, disordered arrangement of islet cells, and increased intercellular spaces; the EA group showed improvements in islet morphology, immune cell infiltration, β-cell nuclei count, and the arrangement and spacing of islet cells approaching normal. EA could lower the blood glucose levels in T2DM rats, alleviate chronic inflammatory responses in the islets, and improve the dedifferentiation of pancreatic β-cells, which may be related to the inhibition of pancreatic NF-κB pathway expression.

Generation of three isogenic gene-edited Huntington’s disease human embryonic stem cell lines with DOX-inducible NGN2 expression cassette in the AAVS1 safe locus

Neurogenin 2 (NGN2), a neuronal transcription factor, can expedite differentiation of stem cells into mature glutamatergic neurons. We have utilized an allelic series of previously published and characterized isogenic Huntington's disease (IsoHD) human embryonic stem cell lines (Ooi et al., 2019), carrying different CAG repeat lengths in the first exon of the huntingtin gene. These IsoHDs were modified using CRISPR/Cas9 to insert NGN2 under the TET-ON doxycycline inducible promoter. The resulting IsoHD-NGN2 cell lines retained pluripotency in the absence of doxycycline (DOX), and via addition of DOX to the culturing media differentiation to neurons was achieved within 14 days.

Sex-Dependent Variations in Hypothalamic Fatty Acid Profile and Neuropeptides in Offspring Exposed to Maternal Obesity and High-Fat Diet.

Maternal obesity and/or high-fat diet (HF) consumption can disrupt appetite regulation in their offspring, contributing to transgenerational obesity and metabolic diseases. As fatty acids (FAs) play a role in appetite regulation, we investigated the maternal and fetal levels of FAs as potential contributors to programmed hyperphagia observed in the offspring of obese dams. Female mice were fed either a control diet (CT) or HF prior to mating, and fetal and maternal blood and tissues were collected at 19 days of gestation. Elevated levels of linoleic acid were observed in the serum of HF dams as well as in the serum of their fetuses. An increased concentration of eicosadienoic acid was also detected in the hypothalamus of female HF-O fetuses. HF-O male fetuses showed increased hypothalamic neuropeptide Y (Npy) gene expression, while HF-O female fetuses showed decreased hypothalamic pro-opiomelanocortin (POMC) protein content. Both male and female fetuses exhibited reduced hypothalamic neurogenin 3 (NGN-3) gene expression. In vitro experiments confirmed that LA contributed to the decreased gene expression of Pomc and Ngn-3 in neuronal cells. During lactation, HF female offspring consumed more milk and had a higher body weight compared to CT. In summary, this study demonstrated that exposure to HF prior to and during gestation alters the FA composition in maternal serum and fetal serum and hypothalamus, particularly increasing n-6, which may play a role in the switch from POMC to NPY neurons, leading to increased weight gain in the offspring during lactation.

Directed Differentiation of Neurons from Human iPSCs for Modeling Neurological Disorders.

Human-induced pluripotent stem cell (hiPSC) technology has enabledcomprehensive human cell-based disease modeling in vitro. Due to limited accessibility of primary human neurons as well as species-specific divergence between human and rodent brain tissues, hiPSC-derived neurons have become a popular tool for studying neuronal biology in a dish. Here, we provide methods for transcription factor-driven directed differentiation of neurons from hiPSCs via a neural progenitor cell (NPC) intermediate. Doxycycline-inducible expression of neuron fate-determining transcription factors neurogenin 2 (NGN2) and achaete-scute homolog 1 (ASCL1) enables rapid and controllable differentiation of human neurons for disease modeling applications. The provided method is also designed to improve the reproducibility of human neuron differentiation by reducing the batch-to-batch variation of NPC differentiation and lentiviral transduction.

Integrating single-cell imaging and RNA sequencing datasets links differentiation and morphogenetic dynamics of human pancreatic endocrine progenitors.

Basic helix-loop-helix genes, particularly proneural genes, are well-described triggers of cell differentiation, yet information on their dynamics is limited, notably in human development. Here, we focus on Neurogenin 3 (NEUROG3), which is crucial for pancreatic endocrine lineage initiation. By monitoring both NEUROG3 gene expression and protein in single cells using a knockin dual reporter in 2D and 3D models of human pancreas development, we show an approximately 2-fold slower expression of human NEUROG3 than that of the mouse. We observe heterogeneous peak levels of NEUROG3 expression and reveal through long-term live imaging that both low and high NEUROG3 peak levels can trigger differentiation into hormone-expressing cells. Based on fluorescence intensity, we statistically integrate single-cell transcriptome with dynamic behaviors of live cells and propose a data-mapping methodology applicable to other contexts. Using this methodology, we identify a role for KLK12 in motility at the onset of NEUROG3 expression.

Arginine 65 methylation of Neurogenin 3 by PRMT1 is required for pancreatic endocrine development of hESCs

Neurogenin 3 (NGN3) is a key transcription factor in the cell fate determination of endocrine progenitors (EPs) in the developing pancreas. Previous studies have shown that the stability and activity of NGN3 are regulated by phosphorylation. However, the role of NGN3 methylation is poorly understood. Here, we report that protein arginine methyltransferase-1 (PRMT1)-mediated arginine 65 methylation of NGN3 is required for the pancreatic endocrine development of human embryonic stem cells (hESCs) in vitro. We found that inducible PRMT1-knockout (P-iKO) hESCs did not differentiate from EPs into endocrine cells (ECs) in the presence of doxycycline. Loss of PRMT1 caused NGN3 accumulation in the cytoplasm of EPs and decreased the transcriptional activity of NGN3. We found that PRMT1 specifically methylates NGN3 arginine 65 and that this modification is a prerequisite for ubiquitin-mediated degradation. Our findings demonstrate that arginine 65 methylation of NGN3 is a key molecular switch in hESCs permitting their differentiation into pancreatic ECs.

NGN2-based neuronal programming of hiPSCs in an automated microfluidic platform

The generation of induced pluripotent stem cells (iPSCs) via somatic cell reprogramming allowed to have an unlimited in vitro source of patient-specific cells.This achievement has introduced a new revolutionary way to create human in vitro models and to study human diseases starting from patient's own cells, especially important for inaccessible tissues like the brain.Recently, lab-on-a-chip technology has opened new reliable alternatives to conventional in vitro models able to replicate key aspects of human physiology, thanks to the intrinsic high surface-area-to-volume ratio, which allows fine control of the cellular microenvironment.The development of automated microfluidic platforms allowed the implementation of this technology to perform high-throughput, standardized and parallelized assays, suitable for drug screenings and developing new therapeutic approaches in a cost-effective way.However, the major challenges in the broad application of automated lab-on-a-chip in biological research are the lack of production robustness and ease of use of the devices.Here, we present an automated microfluidic platform able to host the rapid conversion of human iPSCs (hiPSCs) into neurons via viral-mediated overexpression of Neurogenin 2 (NGN2) in a user-friendly manner.The design of the platform, built with multilayer soft-lithography techniques, shows easiness in the fabrication and assembly thanks to the simple geometry and experimental reproducibility at the same time.All operations are managed automatically, from the cell seeding, medium change, doxycycline-mediated neuronal induction, selection of the genetically engineered cells, and analysis of the output of differentiation, including immunofluorescence assay.Our results show a high-throughput, efficient and homogenous conversion of hiPSCs into neurons in 10 days, characterized by the expression of the mature neuronal marker MAP2 and calcium signaling.The neurons-on-chip model here described represents a fully automated loop system able to address the challenges in the field of neurological diseases modelling in vitro and improve current preclinical models.

USP7 controls NGN3 stability and pancreatic endocrine lineage development

Understanding the factors and mechanisms involved in beta-cell development will guide therapeutic efforts to generate fully functional beta cells for diabetes. Neurogenin 3 (NGN3) is the key transcription factor that marks endocrine progenitors and drives beta-cell differentiation. Here we screen for binding partners of NGN3 and identify the deubiquitylating enzyme USP7 as a key regulator of NGN3 stability. Mechanistically, USP7 interacts with, deubiquitinates and stabilizes NGN3. In vivo, conditional knockout of Usp7 in the mouse embryonic pancreas causes a dramatic reduction in islet formation and hyperglycemia in adult mice, due to impaired NGN3-mediated endocrine specification during pancreatic development. Furthermore, pharmacological inhibition of USP7 during endocrine specification in human iPSC models of beta-cell differentiation decreases NGN3 expressing progenitor cell numbers and impairs beta cell differentiation. Thus, the USP7-NGN3 axis is an essential mechanism for driving endocrine development and beta-cell differentiation, which can be therapeutically exploited.

A novel case of two siblings harbouring homozygous variant in the NEUROG1 gene with autism as an additional phenotype: a case report

IntroductionNEUROG1 gene is yet to be associated with a set of human phenotypes in the OMIM database. Three cases have previously been diagnosed with cranial dysinnervation due to biallelic variants in the NEUROG1 gene. This is the fourth and a novel report of a sibling pair harboring a homozygous variant in the NEUROG1 gene with autism as an additional phenotype. A brief review of the literature in conjunction with a genotype–phenotype correlation has been described. A potential hypothesis for the presence of the autistic phenotype in the present case has also been elucidated.Case presentationA female aged 6 years and 9 months born to endogamous and phenotypically healthy parents was diagnosed with global developmental delay, autism spectrum disorder, hearing loss, corneal opacity and no eye blinking. Her MRI of the brain revealed mild peritrigonal white matter hyperintensity, and MRI and CT scan of the temporal bones showed abnormal cranial nerves. The proband’s younger sister, aged 4-years, was similarly affected. Whole exome sequencing was performed in the proband, which revealed a novel homozygous, likely pathogenic, truncating frameshift variant, c.228_231dup (p.Thr78ProfsTer122) in exon 1 of the NEUROG1 gene (ENST00000314744.4). Segregation analysis by Sanger sequencing showed the proband and her younger sister to be homozygotes and their parents to be heterozygous carriers.ConclusionThis is the fourth report across the globe with a variant identified in the NEUROG1 gene to be associated with cranial dysinnervation phenotype. An additional phenotype of autism in two female siblings was a novel observation. We provide a hypothetical framework which could explain the pleiotropic effect of a dysfunctional NEUROG1 protein leading to autism and posit it as a candidate for diagnosis of autism spectrum disorder with congenital cranial dysinnervation disorder.

Disruption of DDX53 coding sequence has limited impact on iPSC-derived human NGN2 neurons

BackgroundThe X-linked PTCHD1 locus is strongly associated with autism spectrum disorder (ASD). Males who carry chromosome microdeletions of PTCHD1 antisense long non-coding RNA (PTCHD1-AS)/DEAD-box helicase 53 (DDX53) have ASD, or a sub-clinical form called Broader Autism Phenotype. If the deletion extends beyond PTCHD1-AS/DDX53 to the next gene, PTCHD1, which is protein-coding, the individuals typically have ASD and intellectual disability (ID). Three male siblings with a 90 kb deletion that affects only PTCHD1-AS (and not including DDX53) have ASD. We performed a functional analysis of DDX53 to examine its role in NGN2 neurons.MethodsWe used the clustered regularly interspaced short palindromic repeats (CRISPR) gene editing strategy to knock out DDX53 protein by inserting 3 termination codons (3TCs) into two different induced pluripotent stem cell (iPSC) lines. DDX53 CRISPR-edited iPSCs were differentiated into cortical excitatory neurons by Neurogenin 2 (NGN-2) directed differentiation. The functional differences of DDX53-3TC neurons compared to isogenic control neurons with molecular and electrophysiological approaches were assessed.ResultsIsogenic iPSC-derived control neurons exhibited low levels of DDX53 transcripts. Transcriptional analysis revealed the generation of excitatory cortical neurons and DDX53 protein was not detected in iPSC-derived control neurons by western blot. Control lines and DDX53-3TC neurons were active in the multi-electrode array, but no overt electrophysiological phenotype in either isogenic line was observed.ConclusionDDX53-3TC mutation does not alter NGN2 neuronal function in these experiments, suggesting that synaptic deficits causing ASD are unlikely in this cell type.

Novel Variants and Phenotypes in NEUROG3-Associated Syndrome.

Biallelic pathogenic variants in the NEUROG3 gene cause malabsorptive diarrhea, insulin-dependent diabetes mellitus (IDDM), and rarely hypogonadotropic hypogonadism. With only 17 reported cases, the clinical and mutational spectra of this disease are far from complete. To identify the underlying genetic etiology in 3 unrelated Thai patients who presented with early-onset malabsorptive diarrhea, endocrine abnormalities, and renal defects and to determine the pathogenicity of the newly identified pathogenic variants using luciferase reporter assays and western blot. Three unrelated patients with congenital diarrhea were recruited. Detailed clinical and endocrinological features were obtained. Exome sequencing was performed to identify mutations and in vitro functional experiments including luciferase reporter assay were studied to validate their pathogenicity. In addition to malabsorptive diarrhea due to enteric anendocrinosis, IDDM, short stature, and delayed puberty, our patients also exhibited pituitary gland hypoplasia with multiple pituitary hormone deficiencies (Patient 1, 2, 3) and proximal renal tubulopathy (Patient 2, 3) that have not previously reported. Exome sequencing revealed that Patient 1 was homozygous for c.371C > G (p.Thr124Arg) while the other 2 patients were homozygous for c.284G > C (p.Arg95Pro) in NEUROG3. Both variants have never been previously reported. Luciferase reporter assay demonstrated that these 2 variants impaired transcriptional activity of NEUROG3. This study reported pituitary gland hypoplasia with multiple pituitary hormone deficiencies and proximal renal tubulopathy and 2 newly identified NEUROG3 loss-of-function variants in the patients with NEUROG3-associated syndrome.

Computational Analysis of Functional Coding/Noncoding Single Nucleotide Polymorphisms (SNPs/Indels) in Human NEUROG1 gene

Human NEUROG1 gene encodes a very important protein Neurogenin1, which has been demonstrated to have an essential role as a transcription factor in the process of neurogenesis and neural repair. Mutations in NEUROG1 gene have been linked to many congenital diseases and some neurodegenerative diseases. This study used bioinformatics tools to evaluate the effects of mutations along the sequence of the gene. The 3D structural modeling of NEUROG1 was generated through I-TASSER and validated by different software. Genomic data has been retrieved from NCBI, GenBank, and Ensembl databases. Of the 617 SNPs and INDels reported in the dbSNP spanning coding and noncoding regions, only 4 of the 193 SNPs affecting coding regions were predicted to be deleterious with bioinformatics software. Analysis of 3' UTR (3ʹ-untranslated regions) revealed that eight SNPs were found to affect microRNA binding sites either by creating or disturbing them, and other three INDels had no effect This study is a proposed computational analysis for the possible effects of Non- synonymous SNPs on the functionality of NEUROG1 gene and subsequently its protein as an important cofactor in new neurons formation. The results suggested that, NEUROG1 gene can serve as a candidate for gene therapy at genetics and epigenetics levels and/or drug design for neurodegenerative and other neurological diseases.

Cend1 and Neurog2 efficiently reprogram human cortical astrocytes to neural precursor cells and induced-neurons.

Direct reprogramming of glial cells into induced-neurons is a promising strategy for CNS repair after acute injury or neurodegenerative diseases. Grey matter astrocytes, which exhibit features of neural stem cells when activated, are an ideal cell source for direct neuronal conversion. The aim of the study is the investigation of the neuronal reprogramming capacity of CEND1 and/or Neurogenin-2 (NEUROG2) upon their overexpression on primary human adult cortical astrocytes. Our data indicate that adult human cortical astrocytes can be directly reprogrammed by either CEND1 or NEUROG2 to cells with differentiated neuronal morphology, exhibiting long neurites and branched processes. Exploration of gene expression dynamics along the conversion process revealed that neuronal genes are significantly up-regulated while astrocytic genes are down-regulated. Differentiated induced-neurons (iNs) exhibit either GABAergic or glutamatergic/dopaminergic identity upon CEND1 and NEUROG2 overexpression respectively. Co-expression of CEND1 and NEUROG2 in double-transduced cultures induced elevated expression levels of neural progenitor/stem genes and appearance of highly proliferative spheres with neural progenitor cell (NPC) properties in culture.

Protein Production and Purification of a Codon-Optimized Human NGN3 Transcription Factor from E. coli.

Neurogenin 3 (NGN3) transcription factor is vital for the development of endocrine cells of the intestine and pancreas. NGN3 is also critical for the neural precursor cell determination in the neuroectoderm. Additionally, it is one of the vital transcription factors for deriving human β-cells from specialized somatic cells. In the current study, the production and purification of the human NGN3 protein from Escherichia coli (E. coli) is reported. First, the 642bp protein-coding nucleotide sequence of the NGN3 gene was codon-optimized to enable enhanced protein expression in E. coli strain BL21(DE3). The codon-optimized NGN3 sequence was fused in-frame to three different fusion tags to enable cell penetration, nuclear translocation, and affinity purification. The gene insert with the fusion tags was subsequently cloned into an expression vector (pET28a( +)) for heterologous expression in BL21(DE3) cells. A suitable genetic construct and the ideal expression conditions were subsequently identified that produced a soluble form of the recombinant NGN3 fusion protein. This NGN3 fusion protein was purified to homogeneity (purity > 90%) under native conditions, and its secondary structure was retained post-purification. This purified protein, when applied to human cells, did not induce cytotoxicity. Further, the cellular uptake and nuclear translocation of the NGN3 fusion protein was demonstrated followed by its biological activity in PANC-1 cells. Prospectively, this recombinant protein can be utilized for various biological applications to investigate its functionality in cell reprogramming, biological processes, and diseases.

NGN2 induces diverse neuron types from human pluripotency.

SummaryHuman neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.

Diagnostic power of DNA methylation markers suggestive of cholangiocarcinoma in ERCP-based brush cytology

Accurate differentiation between cholangiocarcinoma (CCA) and benign biliary stricture is of paramount importance. Biliary brush cytology is a simple and safe diagnostic approach that provides relatively high specificity; however, sensitivity is limited. Previous reports indicated the aberrations of DNA methylation in CCA. This study aimed to investigate the diagnostic performance of the methylation index (MI) of HOXA1 and NEUROG1 gene promoters in CCA. Patients with biliary stricture who underwent ERCP with brush cytology in Siriraj Hospital from September 2016 to December 2019 were prospectively enrolled. The MI of HOXA1 (MI_H) and MI of NEUROG1 (MI_N) were determined by quantitative methylation-specific polymerase chain reaction. The diagnostic power for CCA was tested for MI from both genes and serum carbohydrate antigen 19-9 (CA19-9). Sixty-seven patients were included in the study; 41 patients had a final diagnosis of CCA, and 26 patients were determined to have a benign biliary stricture. The results showed that both MI_H and MI_N had higher sensitivity and accuracy (95.1% and 82.3% and 90.2% and 89.5%, respectively) than brush cytology (61.5% and 78.1%) and CA19-9 (69.4% and 77.8%). The combination of brush cytology, both methylation markers, and CA19-9 increased the sensitivity and accuracy to 97.4% and 91.0%. Methylation markers were positive in 5 of 6 patients with confirmed CCA whose cytology and CA19-9 were negative. DNA methylation increased the sensitivity for the diagnosis of CCA; therefore, the use of DNA methylation is promising for diagnosis of CCA in patients with biliary strictures. A future validation study is warranted to assess its role in clinical practice. (Clinical trial registration number: NCT04568512.).

Effect of NEUROG3 polymorphism rs144643855 on regional spontaneous brain activity in major depressive disorder

PurposeOur previous study identified a significant association between a single nucleotide polymorphism (SNP) located in the neurogenin3 (NEUROG3) gene and post-stroke depression (PSD) in Chinese populations. The present work explores whether polymorphism rs144643855 affects regional brain activity and clinical phenotypes in major depressive disorder (MDD). MethodA total of 182 participants were included: 116 MDD patients and 66 normal controls. All participants underwent resting-state functional magnetic resonance imaging (rs-fMRI) scanning at baseline. Spontaneous brain activity was assessed using amplitude of low-frequency fluctuation (ALFF). The Hamilton Depression Scale-24 (HAMD-24) and Snaith-Hamilton Pleasure Scale (SHAPS) were used to assess participants at baseline. Two-way analysis of covariance (ANCOVA) was used to explore the interaction between diagnostic groups and NEUROG3 rs144643855 on regional brain activity. We performed correlation analysis to further test the association between these interactive brain regions and clinical manifestations of MDD. ResultsGenotype and disease significantly interacted in the left inferior frontal gyrus (IFG-L), right superior frontal gyrus (SFG-R), and left paracentral lobule (PCL-L) (P < 0.05). ALFF values of the IFG-L were found to be significantly associated with anhedonia in MDD patients. ConclusionThese findings suggest a potential relationship between rs144643855 variations and altered frontal brain activity in MDD. NEUROG3 may play an important role in the neuropathophysiology of MDD.

First in place may not be the winner

Neurodevelopment The human brain matures more slowly than the brains of nonhuman primates. A key question is to what extent the timing of neuronal development is determined by the neurons themselves rather than by their setting within the developing brain. Schornig et al. overexpressed the neurogenic transcription factor neurogenin 2 (NGN2) to convert chimpanzee, bonobo, and human pluripotent stem cells (PSCs) into neurons. Functional and transcriptional analyses tracked the development of these cells into excitatory neurons. The authors found that as expression of stem cell markers declined, neurites developed and the cells became synaptically networked. Subsequently, cortical and sensory neurons differentiated. The ape neurons got off to a quicker start than human neurons in terms of both morphology and spontaneous activity. Delays in morphological and functional activity may support the development of greater complexity in the human brain. eLife 10 , e59323 (2021).