RESUMO
Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.
Assuntos
Anatomia Artística , Atlas como Assunto , Desenvolvimento Embrionário , Endoderma/embriologia , Modelos Biológicos , Organoides/embriologia , Fator de Transcrição CDX2/metabolismo , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , Feminino , Gastrulação , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Intestinos/embriologia , Masculino , Mesoderma/embriologia , Pessoa de Meia-Idade , Neuregulina-1/metabolismo , Especificidade de Órgãos , Células-Tronco Pluripotentes/citologiaRESUMO
For shade-intolerant plants, changes in light quality through competition from neighbors trigger shade avoidance syndrome (SAS): a series of morphological and physiological adaptations that are ultimately detrimental to plant health and crop yield. Phytochrome-interacting factor 7 (PIF7) is a major transcriptional regulator of SAS in Arabidopsis; however, how it regulates gene expression is not fully understood. Here, we show that PIF7 directly interacts with the histone chaperone anti-silencing factor 1 (ASF1). The ASF1-deprived asf1ab mutant showed defective shade-induced hypocotyl elongation. Histone regulator homolog A (HIRA), which mediates deposition of the H3.3 variant into chromatin, is also involved in SAS. RNA/ChIP-sequencing analyses identified the role of ASF1 in the direct regulation of a subset of PIF7 target genes. Furthermore, shade-elicited gene activation is accompanied by H3.3 enrichment, which is mediated by the PIF7-ASF1-HIRA regulatory module. Collectively, our data reveal that PIF7 recruits ASF1-HIRA to increase H3.3 incorporation into chromatin to promote gene transcription, thus enabling plants to effectively respond to environmental shade.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fator VII/genética , Fitocromo/genética , Cromatina/metabolismo , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Proteínas de Ligação a DNA/metabolismoRESUMO
Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Fator Nuclear 1 de Tireoide/metabolismo , Células Epiteliais Alveolares , Diferenciação Celular , Humanos , Pulmão , OrganoidesRESUMO
Many esophageal diseases can arise during development or throughout life. Therefore, well-characterized in vitro models and detailed methods are essential for studying human esophageal development, homeostasis and disease. Here, we (1) create an atlas of the cell types observed in the normal adult human esophagus; (2) establish an ancestrally diverse biobank of in vitro esophagus tissue to interrogate homeostasis and injury; and (3) benchmark in vitro models using the adult human esophagus atlas. We created a single-cell RNA sequencing reference atlas using fresh adult esophagus biopsies and a continuously expanding biobank of patient-derived in vitro cultures (n=55 lines). We identify and validate several transcriptionally distinct cell classes in the native human adult esophagus, with four populations belonging to the epithelial layer, including basal, epibasal, early differentiating and terminally differentiated luminal cells. Benchmarking in vitro esophagus cultures to the in vivo reference using single-cell RNA sequencing shows that the basal stem cells are robustly maintained in vitro, and the diversity of epithelial cell types in culture is dependent on cell density. We also demonstrate that cultures can be grown in 2D or as 3D organoids, and these methods can be employed for modeling the complete epithelial layers, thereby enabling in vitro modeling of the human adult esophagus.
Assuntos
Esôfago , Organoides , Adulto , Humanos , Células-Tronco , Células Epiteliais/metabolismo , Diferenciação CelularRESUMO
Shade avoidance syndrome (SAS) commonly occurs in plants experiencing vegetative shade, triggering a series of morphological and physiological changes for the plants to reach more light. A number of positive regulators, such as PHYTOCHROME-INTERACTING 7 (PIF7), and negative regulators, such as PHYTOCHROMES, are known to ensure appropriate SAS. Here, we identify 211 shade-regulated long non-coding RNAs (lncRNAs) in Arabidopsis. We further characterize PUAR (PHYA UTR Antisense RNA), a lncRNA produced from the intron of the 5' UTR of the PHYTOCHROME A (PHYA) locus. PUAR is induced by shade and promotes shade-induced hypocotyl elongation. PUAR physically associates with PIF7 and represses the shade-mediated induction of PHYA by blocking the binding of PIF7 to the 5' UTR of PHYA. Our findings highlight a role for lncRNAs in SAS and provide insight into the mechanism of PUAR in regulating PHYA gene expression and SAS.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , RNA Longo não Codificante , Regiões 5' não Traduzidas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Hipocótilo/metabolismo , Luz , Fitocromo/genética , Fitocromo/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Infection of the human gut by Salmonella enterica Typhimurium (STM) results in a localized inflammatory disease that is not mimicked in murine infections. To determine mechanisms by which neutrophils, as early responders to bacterial challenge, direct inflammatory programming of human intestinal epithelium, we established a multi-component human intestinal organoid (HIO) model of STM infection. HIOs were micro-injected with STM and seeded with primary human polymorphonuclear leukocytes (PMN-HIOs). PMNs did not significantly alter luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed TUNEL+ epithelial cells that was driven by PMN Caspase-1 activity. Inhibition of Caspases-1, -3 or -4 abrogated epithelial cell death and extrusion in the infected PMN-HIOs but only Caspase-1 inhibition significantly increased bacterial burden in the PMN-HIO epithelium. Thus, PMNs promote cell death in human intestinal epithelial cells through multiple caspases as a protective response to infection. IL-1ß was necessary and sufficient to induce cell shedding in the infected HIOs. These data support a critical innate immune function for human neutrophils in amplifying cell death and extrusion of human epithelial cells from the Salmonella-infected intestinal monolayer.
Assuntos
Neutrófilos , Infecções por Salmonella , Animais , Humanos , Camundongos , Caspases/metabolismo , Células Epiteliais , Mucosa Intestinal/microbiologia , Infecções por Salmonella/metabolismo , Salmonella typhimuriumRESUMO
OBJECTIVE: The aim was to determine the relationship between low handgrip strength (HGS) only, asymmetric HGS only, and low HGS combined with asymmetric HGS and low muscle mass in the West China Health and Aging Trends Study (WCHAT) data. STUDY DESIGN: Individuals aged at least 50 years old were included in this cross-sectional study using WCHAT data. Demographic characteristics, such as age, marital status, education level, ethnicity, and drinking and smoking history, as well as chronic diseases, were recorded for all participants. The HGS of both hands was tested three times using a grip dynanometer with the participant in a standing position with arms extended, before recording the maximum value for both hands. The maximum value referred to values < 28 kg and < 18 kg for males and females, respectively. HGS ratios (non-dominant HGS/dominant HGS) of < 0.90 or > 1.10 suggest asymmetric HGS. The subjects were then allocated to the low HGS, asymmetrical HGS, and combined low and asymmetrical HGS (BOTH group) groups, and those with neither low nor asymmetric HGS (the normal group). The InBody 770 instrument was used for the analysis of muscle mass, with low muscle mass defined as a skeletal muscle mass index (SMI) of < 7.0 kg/m2 or < 5.7 kg/m2 for males and females, respectively. The associations between the different HGS groups and low muscle mass were assessed by logistic regression analysis. RESULTS: The study included 1748 subjects, of whom 1272 (72.77%) were over the age of 60 years. The numbers of Han, Tibetan, and Qiang were 885 (50.63%), 217 (12.41%), and 579 (33.12%), respectively. A total of 465 individuals (26.60%) were classified as having low muscle mass, while 228 (13.04%), 536 (30.66%), and 125 (7.15%) participants were allocated to the low HGS, asymmetric HGS, and BOTH groups, respectively. The average SMI differed significantly between the normal group and the other groups (normal group vs. asymmetric HGS group vs. low HGS group vs. BOTH group: 6.627 kg/m2 vs. 6.633 kg/m2 vs. 6.492 kg/m2 vs. 5.995 kg/m2, respectively, P < 0.05). In addition, the prevalence of low muscle mass in the normal, asymmetric HGS, low HGS, and BOTH groups increased sequentially, with significant differences (normal group vs. asymmetric HGS group vs. low HGS group vs. BOTH group: 21.5% vs. 22.4% vs. 39.5% vs. 56%, respectively, P = 0.001). Further logistic regression analysis showed that the presence of low HGS (OR = 1.7, 95%CI: 1.203-2.402) and both low and asymmetric HGS (OR = 3.378, 95%CI: 2.173-5.252) were predictive of low muscle mass, with the chance being higher for the latter condition. CONCLUSION: The findings suggest that although asymmetrical HGS itself does not increase the chances of low muscle mass. When low HGS and a combination of both features (low HGS combined with asymmetric HGS) is present in subjects, the chance of low muscle mass increases.
Assuntos
Envelhecimento , Força da Mão , Músculo Esquelético , Humanos , Masculino , Estudos Transversais , Feminino , Força da Mão/fisiologia , China/epidemiologia , Idoso , Pessoa de Meia-Idade , Envelhecimento/fisiologia , Músculo Esquelético/fisiologia , Sarcopenia/epidemiologia , Sarcopenia/fisiopatologia , Sarcopenia/diagnóstico , Idoso de 80 Anos ou maisRESUMO
BACKGROUND: MicroRNAs (miRNAs) are important post-transcriptional gene regulators controlling cellular lineage specification and differentiation during embryonic development, including the gastrointestinal system. However, miRNA-mediated regulatory mechanisms involved in early embryonic development of human small intestine (SI) remains underexplored. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage. RESULTS: We leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Small RNA-seq profiling revealed temporal dynamics of miRNA signatures across different developmental events of the model, including definitive endoderm formation, SI lineage specification and SI regional patterning. Our multi-omic, integrative analyses showed further that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut master marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development. CONCLUSION: The present study uncovers a novel facet of miRNAs in regulating prenatal SI development. We leveraged multi-omic, systems biology approaches to discover candidate miRNA regulators associated with early SI developmental events in a human organoid model. In this study, we highlighted miRNA-mediated post-transcriptional regulation relevant to the event of SI lineage specification. The candidate miRNA regulators that we identified for the other stages of SI development also warrant detailed characterization in the future.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs , Humanos , Animais , Camundongos , Diferenciação Celular/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Intestino Delgado/metabolismo , Organoides/metabolismoRESUMO
BACKGROUND & AIMS: Drug-induced liver injury (DILI), both intrinsic and idiosyncratic, causes frequent morbidity, mortality, clinical trial failures and post-approval withdrawal. This suggests an unmet need for improved in vitro models for DILI risk prediction that can account for diverse host genetics and other clinical factors. In this study, we evaluated the utility of human liver organoids (HLOs) for high-throughput DILI risk prediction and in an organ-on-chip system. METHODS: HLOs were derived from three separate iPSC lines and benchmarked on two platforms for their ability to model in vitro liver function and identify hepatotoxic compounds using biochemical assays for albumin, ALT, AST, microscopy-based morphological profiling, and single-cell transcriptomics: i) HLOs dispersed in 384-well-formatted plates and exposed to a library of compounds; ii) HLOs adapted to a liver-on-chip system. RESULTS: Dispersed HLOs derived from the three iPSC lines had similar DILI predictive capacity as intact HLOs in a high-throughput screening format, allowing for measurable IC50 values of compound cytotoxicity. Distinct morphological differences were observed in cells treated with drugs exerting differing mechanisms of toxicity. On-chip HLOs significantly increased albumin production, CYP450 expression, and ALT/AST release when treated with known hepatoxic drugs compared to dispersed HLOs and primary human hepatocytes. On-chip HLOs were able to predict the synergistic hepatotoxicity of tenofovir-inarigivir and displayed steatosis and mitochondrial perturbation, via phenotypic and transcriptomic analysis, on exposure to fialuridine and acetaminophen, respectively. CONCLUSIONS: The high-throughput and liver-on-chip systems exhibit enhanced in vivo-like functions and demonstrate the potential utility of these platforms for DILI risk assessment. Tenofovir-inarigivr-associated hepatotoxicity was observed and correlates with the clinical manifestation of DILI observed in patients. IMPACT AND IMPLICATIONS: Idiosyncratic (spontaneous, patient-specific) drug-induced liver injury (DILI) is difficult to study due to the lack of liver models that function as human liver tissue and are adaptable for large-scale drug screening. Human liver organoids grown from patient stem cells respond to known DILI-causing drugs in both a high-throughput and on a physiological "chip" culture system. These platforms show promise for researchers in their use as predictive models for novel drugs before entering clinical trials and as a potential in vitro diagnostic tool. Our findings support further development of patient-derived liver organoid lines and their use in the context of DILI research.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Fígado/metabolismo , Hepatócitos/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Organoides , AlbuminasRESUMO
Neuronal iron overload contributes to synaptic damage and neuropsychiatric disorders. However, the molecular mechanisms underlying iron deposition in depression remain largely unexplored. Our study aims to investigate how nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) ameliorates hippocampal synaptic dysfunction and reduces brain functional connectivity (FC) associated with excessive iron in depression. We treated mice with chronic unpredictable mild stress (CUMS) with the iron chelator deferoxamine mesylate (DFOM) and a high-iron diet (2.5% carbonyl iron) to examine the role of iron overload in synaptic plasticity. The involvement of Nrf2 in iron metabolism and brain function was assessed using molecular biological techniques and in vivo resting-state functional magnetic resonance imaging (rs-fMRI) through genetic deletion or pharmacologic activation of Nrf2. The results demonstrated a significant correlation between elevated serum iron levels and impaired hippocampal functional connectivity (FC), which contributed to the development of depression-induced CUMS. Iron overload plays a crucial role in CUMS-induced depression and synaptic dysfunction, as evidenced by the therapeutic effects of a high-iron diet and DFOM. The observed iron overload in this study was associated with decreased Nrf2 levels and increased expression of transferrin receptors (TfR). Notably, inhibition of iron accumulation effectively attenuated CUMS-induced synaptic damage mediated by downregulation of brain-derived neurotrophic factor (BDNF). Nrf2-/- mice exhibited compromised FC within the limbic system and the basal ganglia, particularly in the hippocampus, and inhibition of iron accumulation effectively attenuated CUMS-induced synaptic damage mediated by downregulation of brain-derived neurotrophic factor (BDNF). Activation of Nrf2 restored iron homeostasis and reversed vulnerability to depression. Mechanistically, we further identified that Nrf2 deletion promoted iron overload via upregulation of TfR and downregulation of ferritin light chain (FtL), leading to BDNF-mediated synapse damage in the hippocampus. Therefore, our findings unveil a novel role for Nrf2 in regulating iron homeostasis while providing mechanistic insights into poststress susceptibility to depression. Targeting Nrf2-mediated iron metabolism may offer promising strategies for developing more effective antidepressant therapies.
Assuntos
Sobrecarga de Ferro , Ferro , Animais , Camundongos , Fator Neurotrófico Derivado do Encéfalo , Fator 2 Relacionado a NF-E2 , Depressão/etiologia , HipocampoRESUMO
Salmonella enterica represents over 2500 serovars associated with a wide-ranging spectrum of disease; from self-limiting gastroenteritis to invasive infections caused by non-typhoidal serovars (NTS) and typhoidal serovars, respectively. Host factors strongly influence infection outcome as malnourished or immunocompromised individuals can develop invasive infections from NTS, however, comparative analyses of serovar-specific host responses have been constrained by reliance on limited model systems. Here we used human intestinal organoids (HIOs), a three-dimensional "gut-like" in vitro system derived from human embryonic stem cells, to elucidate similarities and differences in host responses to NTS and typhoidal serovars. HIOs discriminated between the two most prevalent NTS, Salmonella enterica serovar Typhimurium (STM) and Salmonella enterica serovar Enteritidis (SE), and typhoidal serovar Salmonella enterica serovar Typhi (ST) in epithelial cell invasion, replication and transcriptional responses. Pro-inflammatory signaling and cytokine output was reduced in ST-infected HIOs compared to NTS infections, consistent with early stages of NTS and typhoidal diseases. While we predicted that ST would induce a distinct transcriptional profile from the NTS strains, more nuanced expression profiles emerged. Notably, pathways involved in cell cycle, metabolism and mitochondrial functions were downregulated in STM-infected HIOs and upregulated in SE-infected HIOs. These results correlated with suppression of cellular proliferation and induction of host cell death in STM-infected HIOs and in contrast, elevated levels of reactive oxygen species production in SE-infected HIOs. Collectively, these results suggest that the HIO model is well suited to reveal host transcriptional programming specific to infection by individual Salmonella serovars, and that individual NTS may provoke unique host epithelial responses during intestinal stages of infection.
Assuntos
Perfilação da Expressão Gênica , Intestinos/microbiologia , Intestinos/fisiopatologia , Infecções por Salmonella/microbiologia , Infecções por Salmonella/fisiopatologia , Humanos , Organoides , Salmonella enterica , Sorogrupo , TranscriptomaRESUMO
Increasing evidence suggests that insulin resistance in type 2 diabetes mellitus (T2DM) is associated with mitochondrial dysfunction in skeletal muscle, while the underlying molecular mechanisms remain elusive. This study aims to construct a ceRNA regulatory network that is involved in mitochondrial dysfunction of skeletal muscle in T2DM. Based on GEO database analysis, differentially expressed lncRNA and mRNA profiles were identified in skeletal muscle tissues of T2DM. Next, LASSO regression analysis was conducted to predict the key lncRNAs related to T2DM, which was validated by receiver operating characteristic (ROC) analysis. Moreover, the miRNAs related to skeletal muscle in T2DM were identified by WGCNA, followed by construction of gene-gene interaction network and GO and KEGG enrichment analyses. It was found that 12 lncRNAs and 6 miRNAs were related to skeletal muscle in T2DM. Moreover, the lncRNA-miRNA-mRNA ceRNA network involving UCA1, miR-143-3p, and FGF21 was constructed. UCA1, and FGF21 were downregulated, while miR-143-3p was upregulated in skeletal muscle cells (SkMCs) exposed to palmitic acid. Additionally, ectopic expression experiments were performed in SkMCs to confirm the effects of UCA1/miR-143-3p/FGF21 on mitochondrial dysfunction by determining mitochondrial ROS, oxygen consumption rate (OCR), membrane potential, and ATP level. Overexpression of miR-143-3p increased ROS accumulation and reduced the OCR, fluorescence ratio of JC-1, and ATP level, which were reversed by upregulation of UCA1 or FGF21. Collectively, lncRNA UCA1 inhibited mitochondrial dysfunction of skeletal muscle in T2DM by sequestering miR-143-3p away from FGF21, therefore providing a potential therapeutic target for alleviating mitochondrial dysfunction of skeletal muscle in T2DM.
Assuntos
Diabetes Mellitus Tipo 2 , MicroRNAs , RNA Longo não Codificante , Humanos , Trifosfato de Adenosina/metabolismo , Diabetes Mellitus Tipo 2/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Espécies Reativas de Oxigênio/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genéticaRESUMO
Light and temperature are two key environmental signals that share several molecular components that, in turn, regulate plant growth. Darkness and high ambient temperatures promote skoto- and thermomorphogenesis, including stem elongation. Heat shock proteins 90 (HSP90s) facilitate the adaptation of organisms to various adverse environmental stimuli. Here, we showed that HSP90s are required for hypocotyl elongation during both skoto- and thermomorphogenesis. When HSP90s activities are impaired by the knockdown of HSP90s expression or the application of HSP90 inhibitors, the expression levels and protein abundance of PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) markedly decreased. EARLY FLOWERING 3 (ELF3) deficiency was resistant to the inhibition of HSP90s activities. Furthermore, HSP90s interacted with and destabilized ELF3. In the CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) mutant, the changes in endogenous PIF4 and ELF3 protein levels caused by the inhibition of HSP90s activities were abolished. HSP90s enhanced the interaction between COP1 and ELF3, reduced ELF3 functional effects on PIF4 and modulated hypocotyl elongation during skoto- and thermomorphogenesis. Our results indicated that HSP90s participate in light and temperature signalling via the COP1-ELF3-PIF4 module to regulate hypocotyl growth in changing environments.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Hipocótilo/metabolismo , Fitocromo/metabolismo , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismoRESUMO
To investigate the molecular mechanism of communication network factor 1 (CCN1) regulating pentylenetetrazol (PTZ)-induced epileptogenesis, deepen the understanding of epilepsy seizure pathogenesis, and provide new drug action targets for its clinical prevention and treatment. Differentially expressed genes (DEGs) on microarrays GSE47516 and GSE88992 were analyzed online using GEO2R. Pathway enrichment and protein-protein interaction network (PPI) analysis of DEGs were carried out using Metascape. Brain tissue samples of severe traumatic brain injury patients (named Healthy group) and refractory epilepsy patients (named Epilepsy group) were obtained and analyzed by qRT-PCR and immunohistochemistry (IHC) staining. A PTZ-induced epilepsy mouse model was established and verified. Morphological changes of neurons in mouse brain tissue were detected using hematoxylin and eosin (HE) staining. qRT-PCR was conducted to detect the mRNA expressions of apoptosis-associated proteins Bax, Caspase-3 and bcl2. TUNEL staining was performed to detect brain neuron apoptosis. The levels of myocardial enzymology, GSH, MDA and ROS in blood of mouse were detected by biochemical assay. CCN1 expression was increased in epilepsy brain tissue samples. CCN1 decreasing effectively prolongs seizure incubation period and decreases seizure duration. Silencing of CCN1 also reduces neuronal damage and apoptosis, decreases mRNA and protein expression of proapoptotic proteins Bax and Caspase-3, increases mRNA expression of antiapoptotic protein Bcl2. Moreover, decrease of CCN1 decreases myocardial enzymatic indexes CK and CK-MB levels, reduces myocardial tissue hemorrhage, and relieves oxidative stress response in hippocampal and myocardial tissue. CCN1 expression is increased in epileptic samples. CCN1 decreasing protects brain tissue by attenuating oxidative stress and inhibiting neuronal apoptosis triggered by PTZ injection, which probably by regulating Nrf2/HO-1 pathway.
Assuntos
Epilepsia , Pentilenotetrazol , Humanos , Camundongos , Animais , Pentilenotetrazol/efeitos adversos , Caspase 3/metabolismo , Proteína X Associada a bcl-2/metabolismo , Epilepsia/induzido quimicamente , Epilepsia/genética , Epilepsia/tratamento farmacológico , Convulsões/induzido quimicamente , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: Temporal lobe epilepsy (TLE) patients usually suffer from impaired episodic memory (EM), but its underlying electrophysiologic mechanism and impacted cognitive performance are unclear. We aim to investigate the association between episodic memory reserve and physiological measures of memory workload in TLE patients using Event-related potentials (ERP). METHODS: A change detection task with image stimuli assesses visual episodic memory. During the memory encoding and decoding phases, the ERP signals were analyzed from twenty-nine TLE patients (twelve with left TLE patients, seventeen with TLE), and thirty healthy controls. Given that EM is a complex process involving many fundamental cognitive processes, the amplitudes and latencies of EM-related ERP (FN400, late positive potential (LPC), and late posterior negativity (LPN)), and the ERP reflecting the fundamental processes (P100, N100, P200, and P300) were calculated. Then we used a three-by-two factorial design on the ERP metrics for interaction and main effects. The correlation analysis among Wechsler Memory Scales-Chinese Revision (WMS-RC) results, behavioral data, and the ERPs was carried out. RESULTS: The TLE patients performed worse in WMS-RC and the memory task. The increased P200 and decreased P300 amplitudes were observed in the TLE patients, and LPN was abnormal in only LTLE patients. For EM-related components, differences were observed in both the LTLE and RTLE patients: the lack of the FN400 effect, the lack of the reversed LPC effect, and the reduced FN400. No significant inter-group difference was detected for the latencies of all the ERPs. Additionally, there were significant correlations among WMS-RC scores, behaviors, and some ERP amplitudes. CONCLUSIONS: The impaired EM is linked to the increased P200 and decreased P300 amplitudes. LPN seems to be sensitive to left temporal lobe dysfunction. More importantly, the abnormal old or new effects of the FN400 and LPC, and the reduced FN400 amplitude might be associated with the visual EM deficit in the TLE patients. These findings may assist in the deep understanding of the EM disorder and the evaluation of the side effects of antiepileptic drugs.
Assuntos
Epilepsia do Lobo Temporal , Memória Episódica , Humanos , Lobo Temporal , Transtornos da Memória/diagnóstico , Potenciais EvocadosRESUMO
OBJECTIVE: This study aimed to explore the relationship between the sarcopenia index (SI) and the risk of pneumonia in hospitalized patients with acute alcohol withdrawal syndrome (AWS). STUDY DESIGN: We have performed a retrospective study of individuals with AWS from a teaching hospital in western China. Patients' data were retrieved from the medicinal record databases. Patients' primary (upon admission) blood serum creatinine (Cr) and cystatin C (CysC) levels were incorporated into the records. Participants were separated into low and high SI cohorts based on the three-quarter digit of SI (SI = serum Cr/serum CysC ratio × 100). The association between SI and the risk of pneumonia in hospitalized patients with AWS was assessed by logistic regression analysis. RESULT: Three hundred and twelve patients with acute AWS were included in this retrospective analysis. Among hospitalized patients with acute AWS, the incidence of pneumonia was 13.78%. The average median age of acute AWS patients with pneumonia was 55.28 (10.65) years, and the mean age of acute AWS individuals without pneumonia was 51.23 (10.08) years. In the univariate analysis, the high SI group (SI > 87.91) had a lower incidence of pneumonia than the low SI group (SI ≤ 87.91) (high SI vs. low SI, 6.41% vs. 16.24%, p = 0.029). Further logistic regression analysis showed that the high SI group demonstrated a poorer risk of pneumonia (OR = 0.353, 95%CI: 0.134-0.932, p = 0.036). After adjusting for possible confounders, the risk of pneumonia remained low in the high SI group (OR = 0.358, 95%CI: 0.132-0.968, p = 0.043). CONCLUSION: Our results showed that SI was linked with the risk of pneumonia in hospitalized individuals with acute AWS. We further suggest that it could be a pneumonia risk factor, especially in medical centers where sarcopenia diagnosis is unavailable.
Assuntos
Alcoolismo , Pneumonia , Sarcopenia , Síndrome de Abstinência a Substâncias , Humanos , Alcoolismo/complicações , Alcoolismo/diagnóstico , Alcoolismo/epidemiologia , Prognóstico , Estudos Retrospectivos , Sarcopenia/diagnóstico , Sarcopenia/epidemiologia , Pneumonia/complicações , Pneumonia/diagnósticoRESUMO
The establishment of the small intestinal (SI) lineage during human embryogenesis ensures functional integrity of the intestine after birth. The chromatin dynamics that drive SI lineage formation and regional patterning in humans are essentially unknown. To fill this knowledge void, we apply a cutting-edge genomic technology to a state-of-the-art human model of early SI development. Specifically, we leverage chromatin run-on sequencing (ChRO-seq) to define the landscape of active promoters, enhancers and gene bodies across distinct stages of directed differentiation of human pluripotent stem cells into SI spheroids with regional specification. Through comprehensive ChRO-seq analysis we identify candidate stage-specific chromatin activity states, novel markers and enhancer hotspots during the directed differentiation. Moreover, we propose a detailed transcriptional network associated with SI lineage formation or regional patterning. Our ChRO-seq analyses uncover a previously undescribed pattern of enhancer activity and transcription at HOX gene loci underlying SI regional patterning. We also validated this unique HOX dynamics by the analysis of single cell RNA-seq data from human fetal SI. Overall, the results lead to a new proposed working model for the regulatory underpinnings of human SI development, thereby adding a novel dimension to the literature that has relied almost exclusively on non-human models.
Assuntos
Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/metabolismo , Intestino Delgado/embriologia , Modelos Biológicos , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Elementos Facilitadores Genéticos , Genes Homeobox , Células-Tronco Embrionárias Humanas/citologia , Humanos , Intestino Delgado/metabolismo , Camundongos , Organoides , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única , Transcrição GênicaRESUMO
Photomorphogenesis is a critical developmental process bridging light-regulated transcriptional reprogramming with morphological changes in organisms. Strikingly, the chromatin-based transcriptional control of photomorphogenesis remains poorly understood. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog of ATP-dependent chromatin-remodeling factor AtINO80 represses plant photomorphogenesis. Loss of AtINO80 inhibited hypocotyl cell elongation and caused anthocyanin accumulation. Both light-induced genes and dark-induced genes were affected in the atino80 mutant. Genome-wide occupancy of the H2A.Z histone variant and levels of histone H3 were reduced in atino80 In particular, AtINO80 bound the gene body of ELONGATED HYPOCOTYL 5 (HY5), resulting in lower chromatin incorporations of H2A.Z and H3 at HY5 in atino80 Genetic analysis revealed that AtINO80 acts in a phytochrome B- and HY5-dependent manner in the regulation of photomorphogenesis. Together, our study elucidates a mechanism wherein AtINO80 modulates nucleosome density and H2A.Z incorporation and represses the transcription of light-related genes, such as HY5, to fine tune plant photomorphogenesis.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Histonas/metabolismo , Luz , Morfogênese/efeitos da radiação , Nucleossomos/metabolismo , Adenosina Trifosfatases/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Escuridão , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Histonas/genética , Mutação/genética , Transcriptoma/genéticaRESUMO
Introduction: This study attempted to investigate the potential of a risk model constructed for regulatory T cells (Tregs) and their related genes in predicting gastric cancer (GC) prognosis. Material and methods: We used flow cytometry to detect the content of CD4+CD25+ Tregs. After detecting expression of five Treg-related genes by quantitative real-time polymerase chain reaction (qRT-PCR), Pearson analysis was employed to analyze the correlation between Tregs and related gene expression. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation and transwell assays were used to detect the effects of a disintegrin and metalloproteinase with thrombospondin motifs 12 (ADAMTS12) on cell functions. A prognostic risk model was built after Cox regression analysis. The Kaplan-Meier method was employed to assess how Tregs, 5-gene risk scores and expression of 5 genes were correlated with the survival time. Results: A significantly increased content of Tregs was found in GC tissues (p < 0.05). 5 Treg- related genes were significantly up-regulated in GC with a positive correlation with the content of Tregs (p < 0.05). Overexpression of ADAMTS12 significantly enhanced the viability, proliferation, migration and invasion of tumor cells. Kaplan-Meier analysis demonstrated poor overall survival and disease-free survival in the high-risk group. The results of survival analysis of Treg content and related gene expression were consistent with those of Cox analysis. Conclusions: The risk model constructed based on five Treg-related genes can enable effective prediction in the prognosis of GC patients.
RESUMO
Concurrent hearing and genetic screening of newborns is expected to play important roles not only in early detection and diagnosis of congenital deafness, which triggers intervention, but also in predicting late-onset and progressive hearing loss and identifying individuals who are at risk of drug-induced HL. Concurrent hearing and genetic screening in the whole newborn population in Beijing was launched in January 2012. This study included 180,469 infants born in Beijing between April 2013 and March 2014, with last follow-up on February 24, 2018. Hearing screening was performed using transiently evoked otoacoustic emission (TEOAE) and automated auditory brainstem response (AABR). For genetic testing, dried blood spots were collected and nine variants in four genes, GJB2, SLC26A4, mtDNA 12S rRNA, and GJB3, were screened using a DNA microarray platform. Of the 180,469 infants, 1,915 (1.061%) were referred bilaterally or unilaterally for hearing screening; 8,136 (4.508%) were positive for genetic screening (heterozygote, homozygote, or compound heterozygote and mtDNA homoplasmy or heteroplasmy), among whom 7,896 (4.375%) passed hearing screening. Forty (0.022%) infants carried two variants in GJB2 or SLC26A4 (homozygote or compound heterozygote) and 10 of those infants passed newborn hearing screening. In total, 409 (0.227%) infants carried the mtDNA 12S rRNA variant (m.1555A>G or m.1494C>T), and 405 of them passed newborn hearing screening. In this cohort study, 25% of infants with pathogenic combinations of GJB2 or SLC26A4 variants and 99% of infants with an m.1555A>G or m.1494C>T variant passed routine newborn hearing screening, indicating that concurrent screening provides a more comprehensive approach for management of congenital deafness and prevention of ototoxicity.