Generation and characterization of Chd7-iCreERT2-tdTomato mice.

Heterozygous mutation of CHD7 gene causes a severe developmental disorder called CHARGE syndrome. In order to further explore the expression and function of Chd7 in vivo, we generated a Chd7-P2A-iCreERT2-P2A-tdTomato (in short, Chd7-CT-tdT) knockin mouse line using the CRISPR/Cas9 technology. The specificity and efficiency of two knockin genetic elements were validated. The Chd7-CT-tdT reporter gene could accurately reflect both the dynamic expression pattern of endogenous Chd7 during neurodevelopment and cell-type specific expression in the brain and eye. The recombination efficiency of Chd7-CT-tdT in postnatal cerebellum is very high. Moreover, lineage tracing experiment showed that Chd7 is expressed in intestinal stem cells. In summary, the newly constructed Chd7-CT-tdT mouse line provide a useful tool to study the function of Chd7.

A Novel Fusion of IL-10 Engineered to Traffic across Intestinal Epithelium to Treat Colitis.

IL-10 is a potent anti-inflammatory cytokine capable of suppressing a number of proinflammatory signals associated with intestinal inflammatory diseases, such as ulcerative colitis and Crohn's disease. Clinical use of human IL-10 (hIL-10) has been limited by anemia and thrombocytopenia following systemic injection, side effects that might be eliminated by a gut-restricted distribution. We have identified a transcytosis pathway used by cholix, an exotoxin secreted by nonpandemic forms of the intestinal pathogen Vibrio cholerae A nontoxic fragment of the first 386 aa of cholix was genetically fused to hIL-10 to produce recombinant AMT-101. In vitro and in vivo characterization of AMT-101 showed it to efficiently cross healthy human intestinal epithelium (SMI-100) by a vesicular transcytosis process, activate hIL-10 receptors in an engineered U2OS osteosarcoma cell line, and increase cellular phospho-STAT3 levels in J774.2 mouse macrophage cells. AMT-101 was taken up by inflamed intestinal mucosa and activated pSTAT3 in the lamina propria with limited systemic distribution. AMT-101 administered to healthy mice by oral gavage or to cynomolgus monkeys (nonhuman primates) by colonic spray increased circulating levels of IL-1R antagonist (IL-1Ra). Oral gavage of AMT-101 in two mouse models of induced colitis prevented associated pathological events and plasma cytokine changes. Overall, these studies suggest that AMT-101 can efficiently overcome the epithelial barrier to focus biologically active IL-10 to the intestinal lamina propria.

Risk stratification by long non-coding RNAs profiling in COVID-19 patients.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic worldwide. Long non-coding RNAs (lncRNAs) are a subclass of endogenous, non-protein-coding RNA, which lacks an open reading frame and is more than 200 nucleotides in length. However, the functions for lncRNAs in COVID-19 have not been unravelled. The present study aimed at identifying the related lncRNAs based on RNA sequencing of peripheral blood mononuclear cells from patients with SARS-CoV-2 infection as well as health individuals. Overall, 17 severe, 12 non-severe patients and 10 healthy controls were enrolled in this study. Firstly, we reported some altered lncRNAs between severe, non-severe COVID-19 patients and healthy controls. Next, we developed a 7-lncRNA panel with a good differential ability between severe and non-severe COVID-19 patients using least absolute shrinkage and selection operator regression. Finally, we observed that COVID-19 is a heterogeneous disease among which severe COVID-19 patients have two subtypes with similar risk score and immune score based on lncRNA panel using iCluster algorithm. As the roles of lncRNAs in COVID-19 have not yet been fully identified and understood, our analysis should provide valuable resource and information for the future studies.

The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions.

rRNA genes (rDNA) exist in two distinct epigenetic states, active promoters being unmethylated and marked by euchromatic histone modifications, whereas silent ones are methylated and exhibit heterochromatic features. Here we show that the nucleosome remodeling and deacetylation (NuRD) complex establishes a specific chromatin structure at rRNA genes that are poised for transcription activation. The promoter of poised rRNA genes is unmethylated, associated with components of the preinitiation complex, marked by bivalent histone modifications and covered by a nucleosome in the "off" position, which is refractory to transcription initiation. Repression of rDNA transcription in growth-arrested and differentiated cells correlates with elevated association of NuRD and increased levels of poised rRNA genes. Reactivation of transcription requires resetting the promoter-bound nucleosome into the "on" position by the DNA-dependent ATPase CSB (Cockayne syndrome protein B). The results uncover a unique mechanism by which ATP-dependent chromatin remodeling complexes with opposing activities establish a specific chromatin state and regulate transcription.

A spectrum of AKT3 activating mutations cause focal malformations of cortical development (FMCDs) in cortical organoids.

Focal malformations of cortical development (FMCDs) are brain disorders mainly caused by hyperactive mTOR signaling due to both inactivating and activating mutations of genes in the PI3K-AKT-mTOR pathway. Among them, mosaic and somatic activating mutations of the mTOR pathway activators are more frequently linked to severe form of FMCDs. A human stem cell-based FMCDs model to study these activating mutations is still lacking. Herein, we genetically engineer human embryonic stem cell lines carrying these activating mutations to generate cortical organoids. Mosaic and somatic expression of AKT3 activating mutations in cortical organoids mimicking the disease presentation with overproliferation and the formation of dysmorphic neurons. In parallel comparison of various AKT3 activating mutations reveals that stronger mutation is associated with more severe neuronal migratory and overgrowth defects. Together, we have established a feasible human stem cell-based model for FMCDs that could help to better understand pathogenic mechanism and develop novel therapeutic strategy.

A single-cell atlas reveals the heterogeneity of meningeal immunity in a mouse model of Methyl CpG binding protein 2 deficiency.

Methyl CpG binding protein 2 (MeCP2) is a DNA methylation reader protein. Mutations in MeCP2 are the major cause of Rett syndrome (RTT). Increasing evidence has shown that dysregulated immunity and chronic subclinical inflammation are linked to MeCP2 deficiency and contribute to RTT development and deterioration. The meninges surrounding the central nervous system (CNS) contain a wide repertoire of immune cells that participate in immune surveillance within the CNS and influence various brain functions; however, the characterization and role of meningeal immunity in CNS with MeCP2 deficiency remain poorly addressed. Here, we used single-cell sequencing to profile Mecp2-deficient meningeal immune cells from the dura mater, which has been reported to contain the most meningeal immune cells during homeostasis. Data showed that the meninges of Mecp2-null mice contained the same diverse immune cell populations as control mice and showed an up-regulation of immune-related processes. B cell populations were greater in Mecp2-null mice than in control mice, and the expression of genes encoding for immunoglobulins was remarkably higher. Mecp2-deficient meninges also contained more cytotoxic CD8+ T cells than control meninges. With increased interferon-γ transcription in T and natural killer cells, meningeal macrophages showed decreased suppression and increased activity in Mecp2-deficienct mice. Together, these findings provide novel insights into meningeal immunity, which is a less studied aspect of neuroimmune interactions in Mecp2-mutated diseases, and offer an essential resource for comparative analyses and data exploration to better understand the functional role of meningeal immunity in RTT.

CHD7 in oocytes is essential for female fertility.

Background: Chromodomain helicase DNA-binding protein 7 (CHD7), which is associated with CHARGE (Coloboma, Heart defect, Atresia choanae, Restricted growth, Genital hypoplasia and Ear abnormality) syndrome is an important regulator in many vital developmental processes. However, its role during oocyte development remains unknown. Methods: We screened the Gene Expression Omnibus (GEO) database for expression levels of CHD7 during folliculogenesis. We generated a conditional knockout (cKO) mouse strain with oocyte-specific deletion of CHD7 (Gdf9-Cre:Chd7f/f ) using the Cre-loxP approach. Evaluation of follicle numbers and reproductive ability was then conducted. In addition, granulosa cell (GC) apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and cleaved caspase-3, using immunohistochemistry (IHC) and immunofluorescence (IF). GC proliferation was measured by Ki67 staining as evaluated by IHC. Results: In our study, we demonstrated that CHD7 has high expression throughout all developmental stages of the oocyte. We found that deletion of Chd7 in oocytes can cause infertility or sub-fertility in female mice and is associated with decreased follicle numbers at all stages. In addition, we found that GC apoptosis was significantly higher in cKO mice. Conclusions: To our knowledge, our study has been the first to show that CHD7 plays a specific role during oogenesis. Our findings provide new insights into CHD7-related infertility.

Novel compound heterozygous mutation in STAMBP causes a neurodevelopmental disorder by disrupting cortical proliferation.

Background: Mutations in the STAMBP gene, which encodes a deubiquitinating isopeptidase called STAM-binding protein, are related to global developmental delay, microcephaly, and capillary malformation. Owing to the limited number of reported cases, the functional and phenotypic characteristics of STAMBP variants require further elucidation. Materials and methods: Whole exome sequencing was performed on a patient presenting with a neurodevelopmental disorder. Novel compound heterozygous mutations in STAMBP [c.843_844del (p.C282Wfs*11) and c.920G > A (p.G307E)] were identified and validated using Sanger sequencing. A 3D human cortical organoid model was used to investigate the function of STAMBP and the pathogenicity of the novel mutation (c.920G > A, p.G307E). Results: The patient was presented with global developmental delay, autism spectrum disorder, microcephaly, epilepsy, and dysmorphic facial features but without apparent capillary malformation on the skin and organs. Cortical organoids with STAMBP knockout (KO) showed significantly lower proliferation of neural stem cells (NSCs), leading to smaller organoids that are characteristic of microcephaly. Furthermore, STAMBP disruption did not affect apoptosis in early cortical organoids. After re-expressing wild-type STAMBP, STAMBP G307E , and STAMBP T313I (a known pathogenic mutation) within STAMBP KO organoids, only STAMBP WT rescued the impaired proliferation of STAMBP deficient organoids, but not STAMBP G307E and STAMBP T313I . Conclusion: Our findings demonstrate that the clinical phenotype of STAMBP mutations is highly variable, and patients with different STAMBP mutations show differences in the severity of symptoms. The STAMBP missense mutation identified here is a novel pathogenic mutation that impairs the proliferation of NSCs in human brain development.

Corosolic Acid Attenuates Hepatic Lipid Accumulation and Inflammatory Response via AMPK/SREBPs and NF-κB/MAPK Signaling Pathways.

Corosolic acid (CA) is the main active component of Lagetstroemia speciosa and has been known to serve as several different pharmacological effects, such as antidiabetic, anti-oxidant, and anticancer effects. In this study, effects of CA on the hepatic lipid accumulation were examined using HepG2 cells and tyloxapol (TY)-induced hyperlipidemia ICR mice. CA significantly inhibited hepatic lipid accumulation via inhibition of SREBPs, and its target genes FAS, SCD1, and HMGCR transcription in HepG2 cells. These effects were mediated through activation of AMPK, and these effects were all abolished in the presence of compound C (CC, an AMPK inhibitor). In addition, CA clearly alleviated serum ALT, AST, TG, TC, low-density lipoprotein cholesterol (LDL-C), and increased high-density lipoprotein cholesterol (HDL-C) levels, and obviously attenuated TY-induced liver steatosis and inflammation. Moreover, CA significantly upregulated AMPK, ACC, LKB1 phosphorylation, and significantly inhibited lipin1, SREBPs, TNF-α, F4/80, caspase-1 expression, NF-κB translocation, and MAPK activation in TY-induced hyperlipidemia mice. Our results suggest that CA is a potent antihyperlipidemia and antihepatic steatosis agent and the mechanism involved both lipogenesis and cholesterol synthesis and inflammation response inhibition via AMPK/SREBPs and NF-κB/MAPK signaling pathways.

Cholix protein domain I functions as a carrier element for efficient apical to basal epithelial transcytosis.

Cholix (Chx) is expressed by the intestinal pathogen Vibrio cholerae as a single chain of 634 amino acids (~70.7 kDa protein) that folds into three distinct domains, with elements of the second and third domains being involved in accessing the cytoplasm of nonpolarized cells and inciting cell death via ADP-ribosylation of elongation factor 2, respectively. In order to reach nonpolarized cells within the intestinal lamina propria, however, Chx must cross the polarized epithelial barrier in an intact form. Here, we provide invitro and invivo demonstrations that a nontoxic Chx transports across intestinal epithelium via a vesicular trafficking pathway that rapidly achieves vesicular apical to basal (A→B) transcytosis and avoids routing to lysosomes. Specifically, Chx traffics in apical endocytic Rab7+ vesicles and in basal exocytic Rab11+ vesicles with a transition between these domains occurring in the ER-Golgi intermediate compartment (ERGIC) through interactions with the lectin mannose-binding protein 1 (LMAN1) protein that undergoes an intracellular re-distribution that coincides with the re-organization of COPI+ and COPII+ vesicular structures. Truncation studies demonstrated that domain I of Chx alone was sufficient to efficiently complete A→B transcytosis and capable of ferrying genetically conjoined human growth hormone (hGH). These studies provide evidence for a pathophysiological strategy where native Chx exotoxin secreted in the intestinal lumen by nonpandemic V. cholerae can reach nonpolarized cells within the lamina propria in an intact form by using a nondestructive pathway to cross in the intestinal epithelial that appears useful for oral delivery of biopharmaceuticals.One-Sentence Summary: Elements within the first domain of the Cholix exotoxin protein are essential and sufficient for the apical to basal transcytosis of this Vibrio cholerae-derived virulence factor across polarized intestinal epithelial cells.

CRISPR-engineered genome editing for the next generation neurological disease modeling.

Neurological disorders often occur because of failure of proper brain development and/or appropriate maintenance of neuronal circuits. In order to understand roles of causative factors (e.g. genes, cell types) in disease development, generation of solid animal models has been one of straight-forward approaches. Recent next generation sequencing studies on human patient-derived clinical samples have identified various types of recurrent mutations in individual neurological diseases. While these discoveries have prompted us to evaluate impact of mutated genes on these neurological diseases, a feasible but flexible genome editing tool had remained to be developed. An advance of genome editing technology using the clustered regularly interspaced short palindromic repeats (CRISPR) with the CRISPR-associated protein (Cas) offers us a tremendous potential to create a variety of mutations in the cell, leading to "next generation" disease models carrying disease-associated mutations. We will here review recent progress of CRISPR-based brain disease modeling studies and discuss future requirement to tackle current difficulties in usage of these technologies.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken ß-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

Revealing the Hidden Powers that Fuel Adult Neurogenesis.

A defining characteristic of diverse stem cell populations is their distinct metabolic state, although how these states change during adult hippocampal neurogenesis is unclear. Recently in Neuron, Beckervordersandforth et al. (2017) report that adult neurogenesis requires mitochondrial electron transport and oxidative phosphorylation and that disrupting these pathways induces premature aging phenotypes.

Versatile Roles of the Chromatin Remodeler CHD7 during Brain Development and Disease.

CHD7 (Chromo-Helicase-DNA binding protein 7) protein is an ATP-dependent chromatin remodeler. Heterozygous mutation of the CHD7 gene causes a severe congenital disease known as CHARGE syndrome. Most CHARGE syndrome patients have brain structural anomalies, implicating an important role of CHD7 during brain development. In this review, we summarize studies dissecting developmental functions of CHD7 in the brain and discuss pathogenic mechanisms behind neurodevelopmental defects caused by mutation of CHD7. As we discussed, CHD7 protein exhibits a remarkably specific and dynamic expression pattern in the brain. Studies in human and animal models have revealed that CHD7 is involved in multiple developmental lineages and processes in the brain. Mechanistically, CHD7 is essential for neural differentiation due to its transcriptional regulation in progenitor cells.

Chd7 is indispensable for mammalian brain development through activation of a neuronal differentiation programme.

Mutations in chromatin modifier genes are frequently associated with neurodevelopmental diseases. We herein demonstrate that the chromodomain helicase DNA-binding protein 7 (Chd7), frequently associated with CHARGE syndrome, is indispensable for normal cerebellar development. Genetic inactivation of Chd7 in cerebellar granule neuron progenitors leads to cerebellar hypoplasia in mice, due to the impairment of granule neuron differentiation, induction of apoptosis and abnormal localization of Purkinje cells, which closely recapitulates known clinical features in the cerebella of CHARGE patients. Combinatory molecular analyses reveal that Chd7 is required for the maintenance of open chromatin and thus activation of genes essential for granule neuron differentiation. We further demonstrate that both Chd7 and Top2b are necessary for the transcription of a set of long neuronal genes in cerebellar granule neurons. Altogether, our comprehensive analyses reveal a mechanism with chromatin remodellers governing brain development via controlling a core transcriptional programme for cell-specific differentiation.

TR surfaces and conformations required to bind nuclear receptor corepressor.

Residues of the TR that are critical for binding the nuclear receptor corepressor (N-CoR) were identified by testing more than 100 separate mutations of the full-length human TRbeta that scan the surface of its ligand binding domain. The primary inferred interaction surface overlaps the surface described for binding of p160 coactivators, but differs by extending to a novel site underneath which helix 12 rests in the liganded TR, rather than including residues of helix 12. Nonconservative mutations of this surface diminished binding similarly to three isolated N-CoR receptor interaction domains (RIDs), but conservative mutations affected binding variably, consistent with a role for this surface in RID selectivity. The commonality of this surface in binding N-CoR was confirmed for the RXRs and ERs. Deletion of helix 12 increased N-CoR binding by the TR modestly, and by the RXR and ER to a much greater extent, indicating a competition between this helix and the corepressor that regulates the extent of corepressor binding by nuclear receptors. When helix 12 was deleted, N-CoR binding by the ER was stimulated by tamoxifen, and binding by the TR was stimulated by Triac, indicating that helix 12 is not the only feature that regulates corepressor binding. Two additional mutationsensitive surfaces were found alongside helix 1, near the previously described CoR box, and above helix 11, nearby but separate from residues that help link receptor in dimers. Based on effects of selected mutations on T(3) and coactivator binding, and on results of combined mutations of the three sites on corepressor binding, we propose that the second and third surfaces stabilize TR unliganded conformation(s) required for efficient N-CoR binding. In transfection assays mutations of all three surfaces impaired the corepressor-mediated functions of unliganded TR repression or activation. These detailed mapping results suggest approaches for selective modulation of corepressor interaction that include the shape of the molecular binding surface, the competitive occupancy by helix 12, pharmacological stimulation, and specific conformational stabilization.

[Observation and analysis on mutation of routine STR locus].

OBJECTIVE: To observe and analyze the characteristic of mutation at STR locus. METHODS: 27 mutant genes observed in 1211 paternity testing cases were checked by PAGE-silver stained and PowerPlex 16 System Kit and validated by sequencing. RESULTS: Mutant genes locate on 15 loci. The pattern of mutation was accord with stepwise mutation model. The mutation ratio of male-to-female was 8:1 and correlated to the age of father. CONCLUSION: Mutation rate is correlated to the geometric mean of the number of homogeneous repeats of locus. The higher the mean, the higher the mutation rate. These loci are not so appropriate for use in paternity testing.

Structure-based design and synthesis of a thyroid hormone receptor (TR) antagonist.

Antagonists have been developed for several nuclear receptors but not for others, including TRs. TR antagonists may have significant clinical utility for treating hormone excess states and other conditions. A structure derived "extension hypothesis" was applied to synthesize a TR antagonist. The principal design feature was to attach an extension group to a TR agonist whose structure would perturb formation of the TR coactivator-binding surface. The compound, 3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid, has no (TRalpha) or very weak partial (TRbeta) TR agonist activity and blocks TR binding of T3, formation of the coactivator-binding surface, and both a positive T3 response on a thyroid hormone response element and a negative T3 response on the TSHbeta promoter in cultured cells. The results suggest that 3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid is a TR antagonist for thyroid hormone response element-mediated responses, this approach can be used more generally to generate nuclear receptor antagonists, and this compound or analogues may have medical and research utility.

The chromatin remodeler CHD7 regulates adult neurogenesis via activation of SoxC transcription factors.

Chromatin factors that regulate neurogenesis in the central nervous system remain to be explored. Here, we demonstrate that the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7), a protein frequently mutated in human CHARGE syndrome, is a master regulator of neurogenesis in mammalian brain. CHD7 is selectively expressed in actively dividing neural stem cells (NSCs) and progenitors. Genetic inactivation of CHD7 in NSCs leads to a reduction of neuronal differentiation and aberrant dendritic development of newborn neurons. Strikingly, physical exercise can rescue the CHD7 mutant phenotype in the adult hippocampal dentate gyrus. We further show that in NSCs, CHD7 stimulates the expression of Sox4 and Sox11 genes via remodeling their promoters to an open chromatin state. Our study demonstrates an essential role of CHD7 in activation of the neuronal differentiation program in NSCs, thus providing insights into epigenetic regulation of stem cell differentiation and molecular mechanism of human CHARGE syndrome.