H3K4me1 facilitates promoter-enhancer interactions and gene activation during embryonic stem cell differentiation.

Histone H3 lysine 4 mono-methylation (H3K4me1) marks poised or active enhancers. KMT2C (MLL3) and KMT2D (MLL4) catalyze H3K4me1, but their histone methyltransferase activities are largely dispensable for transcription during early embryogenesis in mammals. To better understand the role of H3K4me1 in enhancer function, we analyze dynamic enhancer-promoter (E-P) interactions and gene expression during neural differentiation of the mouse embryonic stem cells. We found that KMT2C/D catalytic activities were only required for H3K4me1 and E-P contacts at a subset of candidate enhancers, induced upon neural differentiation. By contrast, a majority of enhancers retained H3K4me1 in KMT2C/D catalytic mutant cells. Surprisingly, H3K4me1 signals at these KMT2C/D-independent sites were reduced after acute depletion of KMT2B, resulting in aggravated transcriptional defects. Our observations therefore implicate KMT2B in the catalysis of H3K4me1 at enhancers and provide additional support for an active role of H3K4me1 in enhancer-promoter interactions and transcription in mammalian cells.

Spatially organized cellular communities form the developing human heart.

The heart, which is the first organ to develop, is highly dependent on its form to function1,2. However, how diverse cardiac cell types spatially coordinate to create the complex morphological structures that are crucial for heart function remains unclear. Here we integrated single-cell RNA-sequencing with high-resolution multiplexed error-robust fluorescence in situ hybridization to resolve the identity of the cardiac cell types that develop the human heart. This approach also provided a spatial mapping of individual cells that enables illumination of their organization into cellular communities that form distinct cardiac structures. We discovered that many of these cardiac cell types further specified into subpopulations exclusive to specific communities, which support their specialization according to the cellular ecosystem and anatomical region. In particular, ventricular cardiomyocyte subpopulations displayed an unexpected complex laminar organization across the ventricular wall and formed, with other cell subpopulations, several cellular communities. Interrogating cell-cell interactions within these communities using in vivo conditional genetic mouse models and in vitro human pluripotent stem cell systems revealed multicellular signalling pathways that orchestrate the spatial organization of cardiac cell subpopulations during ventricular wall morphogenesis. These detailed findings into the cellular social interactions and specialization of cardiac cell types constructing and remodelling the human heart offer new insights into structural heart diseases and the engineering of complex multicellular tissues for human heart repair.

A fast, scalable and versatile tool for analysis of single-cell omics data.

Single-cell omics technologies have revolutionized the study of gene regulation in complex tissues. A major computational challenge in analyzing these datasets is to project the large-scale and high-dimensional data into low-dimensional space while retaining the relative relationships between cells. This low dimension embedding is necessary to decompose cellular heterogeneity and reconstruct cell-type-specific gene regulatory programs. Traditional dimensionality reduction techniques, however, face challenges in computational efficiency and in comprehensively addressing cellular diversity across varied molecular modalities. Here we introduce a nonlinear dimensionality reduction algorithm, embodied in the Python package SnapATAC2, which not only achieves a more precise capture of single-cell omics data heterogeneities but also ensures efficient runtime and memory usage, scaling linearly with the number of cells. Our algorithm demonstrates exceptional performance, scalability and versatility across diverse single-cell omics datasets, including single-cell assay for transposase-accessible chromatin using sequencing, single-cell RNA sequencing, single-cell Hi-C and single-cell multi-omics datasets, underscoring its utility in advancing single-cell analysis.

Robust Ionics Reinforced Fiber As Implantable Sensor for Early Operando Monitoring Cell Thermal Safety of Commercial Lithium-Ion Batteries.

Commercial batteries have been largely applied in mobile electronics, electric vehicles, and scalable energy storage systems. However, thermal runaway of batteries still obstructs the reliability of electric equipment. Considering this, building upon recent investigations of energy thermal safety, commercially available organogel fiber-based implantable sensors have been developed through 3D printing technology for first operando implantable monitoring of cell temperature. The printed fibers present excellent reliability and superelasticity because of internal supramolecular cross-linking. High temperature sensitivity (-39.84% °C-1/-1.557% °C-1) within a wide range (-15 to 80 °C) is achieved, and the corresponding mechanism is clarified based on in situ temperature-dependent Raman technology. Furthermore, taking the pouch cell as an example, combined with finite element analysis, the real-time observation system of cell temperature is successfully demonstrated through an implanted sensor with wireless Bluetooth transmission. This enlightening approach paves the way for achieving safety monitoring and smart warnings for various electric equipment.

Role of H3K4 monomethylation in gene regulation.

Methylation of histone H3 on the lysine-4 residue (H3K4me) is found throughout the eukaryotic domain, and its initial discovery as a conserved epigenetic mark of active transcription from yeast to mammalian cells has contributed to the histone code hypothesis. However, recent studies have raised questions on whether the different forms of H3K4me play a direct role in gene regulation or are simply by-products of the transcription process. Here, we review the often-conflicting experimental evidence, focusing on the monomethylation of lysine 4 on histone H3 that has been linked to the transcriptional state of enhancers in metazoans. We suggest that this epigenetic mark acts in a context-dependent manner to directly facilitate the transcriptional output of the genome and the establishment of cellular identity.

First International Conference on Unconventional Animal Models of Alzheimer's Disease and Aging.

The first International Conference on Unconventional Animal Models of Alzheimer's Disease and Aging (UAMAA) took place on December 13-16, 2023, in Santiago, Chile. The Alzheimer's disease (AD) research field is currently in search for new and unconventional models that could hold greater translational potential than transgenic mouse models. Thus this UAMAA conference is timely and significant. The event consisted of 6 sessions with talks from 28 world-class scientists from all over the world. These animal models of interest include the degu (Octodon degu), the dog (Canis familiaris), and certain species of nonhuman primates that may better recapitulate neuropathology and cognitive impairments in human AD. Our conference has provided a formal forum to discuss and highlight new research directions, alternative animal models, and innovative approaches for the AD and aging research field.

Vapor-induced phase-separation-enabled versatile direct ink writing.

Versatile printing of polymers, metals, and composites always calls for simple, economic approaches. Here we present an approach to three-dimensional (3D) printing of polymeric, metallic, and composite materials at room conditions, based on the polymeric vapor-induced phase separation (VIPS) process. During VIPS 3D printing (VIPS-3DP), a dissolved polymer-based ink is deposited in an environment where nebulized non-solvent is present, inducing the low-volatility solvent to be extracted from the filament in a controllable manner due to its higher chemical affinity with the non-solvent used. The polymeric phase is hardened in situ as a result of the induced phase separation process. The low volatility of the solvent enables its reclamation after the printing process, significantly reducing its environmental footprint. We first demonstrate the use of VIPS-3DP for polymer printing, showcasing its potential in printing intricate structures. We further extend VIPS-3DP to the deposition of polymer-based metallic inks or composite powder-laden polymeric inks, which become metallic parts or composites after a thermal cycle is applied. Furthermore, spatially tunable porous structures and functionally graded parts are printed by using the printing path to set the inter-filament porosity as well as an inorganic space-holder as an intra-filament porogen.

Droplet Hi-C for Fast and Scalable Profiling of Chromatin Architecture in Single Cells.

Comprehensive analysis of chromatin architecture is crucial for understanding the gene regulatory programs during development and in disease pathogenesis, yet current methods often inadequately address the unique challenges presented by analysis of heterogeneous tissue samples. Here, we introduce Droplet Hi-C, which employs a commercial microfluidic device for high-throughput, single-cell chromatin conformation profiling in droplets. Using Droplet Hi-C, we mapped the chromatin architecture at single-cell resolution from the mouse cortex and analyzed gene regulatory programs in major cortical cell types. Additionally, we used this technique to detect copy number variation (CNV), structural variations (SVs) and extrachromosomal DNA (ecDNA) in cancer cells, revealing clonal dynamics and other oncogenic events during treatment. We further refined this technique to allow for joint profiling of chromatin architecture and transcriptome in single cells, facilitating a more comprehensive exploration of the links between chromatin architecture and gene expression in both normal tissues and tumors. Thus, Droplet Hi-C not only addresses critical gaps in chromatin analysis of heterogeneous tissues but also emerges as a versatile tool enhancing our understanding of gene regulation in health and disease.

Sensory Input, Sex, and Function Shape Hypothalamic Cell Type Development.

Mammalian behavior and physiology undergo dramatic changes in early life. Young animals rely on conspecifics to meet their homeostatic needs, until weaning and puberty initiate nutritional independence and sex-specific social interactions, respectively. How neuronal populations regulating homeostatic functions and social behaviors develop and mature during these transitions remains unclear. We used paired transcriptomic and chromatin accessibility profiling to examine the developmental trajectories of neuronal populations in the hypothalamic preoptic region, where cell types with key roles in physiological and behavioral control have been identified1-6. These data reveal a remarkable diversity of developmental trajectories shaped by the sex of the animal, and the location and behavioral or physiological function of the corresponding cell types. We identify key stages of preoptic development, including the perinatal emergence of sex differences, postnatal maturation and subsequent refinement of signaling networks, and nonlinear transcriptional changes accelerating at the time of weaning and puberty. We assessed preoptic development in various sensory mutants and find a major role for vomeronasal sensing in the timing of preoptic cell type maturation. These results provide novel insights into the development of neurons controlling homeostatic functions and social behaviors and lay ground for examining the dynamics of these functions in early life.

BORIS/CTCFL epigenetically reprograms clustered CTCF binding sites into alternative transcriptional start sites.

BACKGROUND: Pervasive usage of alternative promoters leads to the deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters. RESULTS: Here we describe how alternative cancer-testis-specific transcription is activated. We show that intergenic and intronic CTCF binding sites, which are transcriptionally inert in normal somatic cells, could be epigenetically reprogrammed into active de novo promoters in germ and cancer cells. BORIS/CTCFL, the testis-specific paralog of the ubiquitously expressed CTCF, triggers the epigenetic reprogramming of CTCF sites into units of active transcription. BORIS binding initiates the recruitment of the chromatin remodeling factor, SRCAP, followed by the replacement of H2A histone with H2A.Z, resulting in a more relaxed chromatin state in the nucleosomes flanking the CTCF binding sites. The relaxation of chromatin around CTCF binding sites facilitates the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the epigenetically reprogrammed CTCF binding sites can drive the expression of cancer-testis genes, long noncoding RNAs, retro-pseudogenes, and dormant transposable elements. CONCLUSIONS: Thus, BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.

RUNX1 C-terminal mutations impair blood cell differentiation by perturbing specific enhancer-promoter networks.

ABSTRACT: The transcription factor RUNX1 is a master regulator of hematopoiesis and is frequently mutated in myeloid malignancies. Mutations in its runt homology domain (RHD) frequently disrupt DNA binding and result in loss of RUNX1 function. However, it is not clearly understood how other RUNX1 mutations contribute to disease development. Here, we characterized RUNX1 mutations outside of the RHD. Our analysis of the patient data sets revealed that mutations within the C-terminus frequently occur in hematopoietic disorders. Remarkably, most of these mutations were nonsense or frameshift mutations and were predicted to be exempt from nonsense-mediated messenger RNA decay. Therefore, this class of mutation is projected to produce DNA-binding proteins that contribute to the pathogenesis in a distinct manner. To model this, we introduced the RUNX1R320∗ mutation into the endogenous gene locus and demonstrated the production of RUNX1R320∗ protein. Expression of RUNX1R320∗ resulted in the disruption of RUNX1 regulated processes such as megakaryocytic differentiation, through a transcriptional signature different from RUNX1 depletion. To understand the underlying mechanisms, we used Global RNA Interactions with DNA by deep sequencing (GRID-seq) to examine enhancer-promoter connections. We identified widespread alterations in the enhancer-promoter networks within RUNX1 mutant cells. Additionally, we uncovered enrichment of RUNX1R320∗ and FOXK2 binding at the MYC super enhancer locus, significantly upregulating MYC transcription and signaling pathways. Together, our study demonstrated that most RUNX1 mutations outside the DNA-binding domain are not subject to nonsense-mediated decay, producing protein products that act in concert with additional cofactors to dysregulate hematopoiesis through mechanisms distinct from those induced by RUNX1 depletion.

Extensive folding variability between homologous chromosomes in mammalian cells.

Genetic variation and 3D chromatin structure have major roles in gene regulation. Due to challenges in mapping chromatin conformation with haplotype-specific resolution, the effects of genetic sequence variation on 3D genome structure and gene expression imbalance remain understudied. Here, we applied Genome Architecture Mapping (GAM) to a hybrid mouse embryonic stem cell (mESC) line with high density of single nucleotide polymorphisms (SNPs). GAM resolved haplotype-specific 3D genome structures with high sensitivity, revealing extensive allelic differences in chromatin compartments, topologically associating domains (TADs), long-range enhancer-promoter contacts, and CTCF loops. Architectural differences often coincide with allele-specific differences in gene expression, mediated by Polycomb repression. We show that histone genes are expressed with allelic imbalance in mESCs, are involved in haplotype-specific chromatin contact marked by H3K27me3, and are targets of Polycomb repression through conditional knockouts of Ezh2 or Ring1b. Our work reveals highly distinct 3D folding structures between homologous chromosomes, and highlights their intricate connections with allelic gene expression.

Dynamic enhancer landscapes in human craniofacial development.

The genetic basis of human facial variation and craniofacial birth defects remains poorly understood. Distant-acting transcriptional enhancers control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development. However, a lack of accurate maps of the genomic locations and cell type-resolved activities of craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combine histone modification, chromatin accessibility, and gene expression profiling of human craniofacial development with single-cell analyses of the developing mouse face to define the regulatory landscape of facial development at tissue- and single cell-resolution. We provide temporal activity profiles for 14,000 human developmental craniofacial enhancers. We find that 56% of human craniofacial enhancers share chromatin accessibility in the mouse and we provide cell population- and embryonic stage-resolved predictions of their in vivo activity. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development.

Cell-type-specific 3D-genome organization and transcription regulation in the brain.

3D organization of the genome plays a critical role in regulating gene expression. However, it remains unclear how chromatin organization differs among different cell types in the brain. Here we used genome-scale DNA and RNA imaging to investigate 3D-genome organization in transcriptionally distinct cell types in the primary motor cortex of the mouse brain. We uncovered a wide spectrum of differences in the nuclear architecture and 3D-genome organization among different cell types, ranging from the physical size of the cell nucleus to the active-inactive chromatin compartmentalization and radial positioning of chromatin loci within the nucleus. These cell-type-dependent variations in nuclear architecture and chromatin organization exhibited strong correlation with both total transcriptional activity of the cell and transcriptional regulation of cell-type-specific marker genes. Moreover, we found that the methylated-DNA-binding protein MeCP2 regulates transcription in a divergent manner, depending on the nuclear radial positions of chromatin loci, through modulating active-inactive chromatin compartmentalization.

Design and Realization of Lung Organoid Cultures for COVID-19 Applications.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading globally and threatening public health. Advanced in vitro models that recapitulate the architecture and functioning of specific tissues and organs are in high demand for COVID-19-related pathology studies and drug screening. Three-dimensional (3D) in vitro cultures such as self-assembled and engineered organoid cultures surpass conventional two-dimensional (2D) cultures and animal models with respect to the increased cellular complexity, better human-relevant environment, and reduced cost, thus presenting as promising platforms for understanding viral pathogenesis and developing new therapeutics. This review highlights the recent advances in self-assembled and engineered organoid technologies that are used for COVID-19 studies. The challenges and future perspectives are also discussed.

Antitussive and expectorant properties of growing and fallen leaves of loquat (Eriobotrya japonica)

ABSTRACT Folium Eriobotryae, the dried leaves of loquat (Eriobotrya japonica, (Thunb.) Lindl., Rosaceae), is a traditional Chinese medicine used to treat cough with phlegm in China. Fallen and growing loquat leaves were tested for their effect on coughing and expectoration in mice. HPLC-ELSD and HPLC-MS analyses of aqueous and ethanol extracts of fallen or growing leaves were used to identify the chemical components responsible for this effect. Both the aqueous and ethanol extracts of growing and fallen leaves of loquat contained antitussive and expectorant activities. Moreover, an aqueous extract of growing loquat leaves with a higher flavonoid content displayed a stronger expectorant activity while the ethanol extract of fallen loquat leaves that contained a higher content of triterpenoid acids induced a stronger antitussive activity.

Mucoadhesive buccal films of tramadol for effective pain management / Filmes bucais mucoadesivos de tramadol para o controle eficaz da dor

Abstract Background and objectives: Tramadol hydrochloride is a centrally-acting synthetic opioid analgesic binding to specific opioid receptors. It is used in the management of chronic pain and is recommended as first line drug in the treatment of postoperative or orthopedic injury induced acute pain. The present work is designed to prepare and evaluate mucoadhesive buccal film of tramadol hydrochloride as a novel form of prolonged analgesia for patients with orthopedic injuries. Methods: Buccal films of tramadol hydrochloride were prepared by solvent casting method. The prepared films were evaluated for the various evaluation parameters like thickness, surface pH, weight uniformity, content uniformity, folding endurance, swelling index, in vitro drug release study, in vitro test for mucoadhesion and in vivo studies (primary mucosal irritancy test and analgesic activity). Results: All the formulations exhibited good results for physicochemical characterizations. In in vitro drug release study the films exhibited controlled release more than 12 hours. The formulation BFT2 (containing chitosan and PVP K-90) showed no irritant effect on buccal mucosa and elicit the significant in vivo analgesic activity with 57.14% analgesia against that of standard (61.04%). It was concluded that the mucoadhesive films of tramadol hydrochloride can be effectively used to alleviate the severe pain of orthopedic injuries with prompt onset and prolonged action.

Resumo Justificativa e objetivos: O cloridrato de tramadol é um analgésico opioide de ação central que se liga a receptores opioides específicos. É usado no tratamento de dor crônica e recomendado como fármaco de primeira linha para o tratamento no pós-operatório ou em dor aguda induzida por lesão ortopédica. O presente estudo visa a preparar e avaliar o filme bucal mucoadesivo de cloridrato de tramadol como uma nova forma de analgesia prolongada para pacientes com lesões ortopédicas. Método: Filmes bucais de cloridrato de tramadol foram preparados pelo método de evaporação de solvente. Os filmes preparados foram avaliados para os vários parâmetros de avaliação, como espessura, pH da superfície, uniformidade do peso, uniformidade do conteúdo, resistência a dobras, índice de intumescimento, estudo de liberação da droga in vitro, teste in vitro para mucoadesão e estudos in vivo (teste de irritação da mucosa primária e atividade analgésica). Resultados: Todas as formulações apresentaram bons resultados para caracterizações físico-químicas. Em estudo de libertação de droga in vitro, os filmes exibiram liberação controlada por mais de 12 horas. A formulação de BFT2 (com quitosana e PVP K-90) não mostrou efeito irritante sobre a mucosa bucal e provocou uma atividade analgésica significativa in vivo com 57,14% de analgesia versus a do padrão (61,04%). Concluiu-se que os filmes mucoadesivos de cloridrato de tramadol podem ser usados eficazmente para aliviar a dor intensa de lesões ortopédicas com início rápido e ação prolongada.