Animal Cells and Systems最新文献

Xenopus embryo is a useful model for evaluating the adverse effects of any compounds on the cellular processes essential for early development and adult tissue homeostasis. Our chemical library screening with frog embryos identified artesunate (ART) as an inhibitor of the BMP signaling pathway to interfere with the specification of embryonic germ layers. Exposure to ART led to reduction of the anterior-posterior body axis, malformed tail structures and loss of pigment cells in the trunk region of embryos. The severely defective embryos exhibited truncation of posterior structures, resembling the phenotypes of tadpoles depleted of BMPs. Consistent with these morphological deformities, ART exposure inhibited the BMP-dependent transcriptions of target genes and specification of ventral mesoderm. In contrast, the expression of an organizer-specific gene induced by Activin/Nodal signaling remained unchanged in ART-treated cells. ART also enhanced anterior neural differentiation at the expense of epidermal and neural crest cell fates. Unexpectedly, we observed that ART exposure accelerates proteasomal degradation of a BMP transducer Smad1, leading to upregulation of MAP kinase activity. Taken together, these results suggest that ART acts as an inhibitor of BMP signaling pathway, exerting severe adverse effects on the specification of germ layers in vertebrate early development.

Yes-associated protein (YAP), a key co-transcription factor of the Hippo pathway, is a promising drug target for cancer therapy due to its critical role in promoting cell proliferation, survival, and tumor progression when dysregulated. While most Hippo pathway-targeting drugs focus on disrupting TEAD-YAP interactions or modulating the MST or LATS kinase cascade, new approaches are needed to identify small molecules that regulate YAP activity. In this study, we conducted high-throughput screening of FDA-approved drugs to discover potential YAP modulators. Using a NanoBiT-based system, which enables real-time and quantitative measurement of protein interactions, combined with phenotype-based assays in EGFP-YAP-expressing cells, we identified compounds that activate or inhibit YAP function. Among the identified YAP regulators, the microtubule destabilizer vinorelbine promoted YAP nuclear localization and transcriptional activation, while the antipsychotic drug thioridazine enhanced YAP phosphorylation at Ser127, resulting in its cytoplasmic retention and reduced transcriptional activity, effectively suppressing cancer cell growth. These findings demonstrate the potential of FDA-approved drugs in modulating YAP activity and present a novel screening strategy for developing YAP-targeting therapeutics. Furthermore, this approach can be extended to identify modulators of other signaling pathways, advancing drug discovery for a wide range of diseases.

Objective: Fetal growth restriction (FGR) is a serious pregnancy complication associated with an increased risk of perinatal morbidity and mortality. Notably, circular RNAs (circRNAs) significantly influence physiological development and disease pathogenesis. We reported previously that lower circ_0081343 expression is associated with placental trophoblast dysfunction. However, only a few studies have reported the role of circRNAs in FGR in vivo. Therefore, we investigated the effects of circ_0081343 overexpression in the FGR mouse model induced by maternal hypoxia.

Methods: Pregnant C57BL/6 mice were kept under hypoxic conditions (10.5% O2) from gestational days 11-17.5, whereas control mice were kept in normal oxygen conditions throughout the gestation period. The animals were sacrificed on the 18.5th day of gestation for prenatal observation. We recorded the maternal body weight, fetal body weight, crown-rump length, and placental weight. Subsequently, we assessed the expression of autophagy, pyroptosis-related protein, and PI3 K/AKT/HIF-1α pathway molecules in placental tissues using RT-PCR, western blotting, ELISA, and immunohistochemistry analysis.

Results: We observed low mmu_circ_0081343 expression in the placental tissues of the FGR mouse. However, the expression increased following the injection of adenovirus-mmu-circ_0081343. The overexpression of mmu-circ_0081343 alleviated FGR symptoms in the pregnant mice, including increasing fetal body and placental weight and ameliorating histological injury of the placenta. Additionally, overexpression of mmu-circ_0081343 upregulated Beclin1 expression, increased the LC3II/I ratio, and downregulated P62 expression, while suppressing the PI3 K/AKT/HIF-1α pathway.

Conclusions: circ_0081343 alleviated gestational hypoxia-induced placental dysfunction and fetal growth restriction (FGR) by promoting autophagy and inhibiting pyroptosis, potentially through the PI3 K/AKT/HIF-1α pathway.

Tripartite motif (TRIM) family proteins are increasingly recognized as important regulators of autophagy under various physiological and pathological conditions. TRIM22 has been previously shown to mediate autophagosome-lysosome fusion, but its potential role in earlier stages of autophagy remained unexplored. In this study, we investigated the function of TRIM22 in autophagy initiation. Overexpression of TRIM22 increased LC3-II levels and enhanced autophagic flux without affecting mTOR and AMPK activity. We found that TRIM22 interacts with components of both the ULK1 complex and the class III PI3K complex through distinct domains, recruiting them into punctate structures that represent autophagosome formation sites. Domain mapping revealed that the SPRY domain mediates interactions with ATG13 and FIP200, while the N-terminal region interacts with ULK1 and ATG101. The B-box domain of TRIM22 was identified as crucial for its interaction with Beclin-1, a key component of the class III PI3K complex. Deletion of this domain impaired the ability of TRIM22 to assemble the class III PI3K complex and induce autophagic flux. Interestingly, competitive binding assays revealed that Beclin-1 and PLEKHM1 bind to the same region of TRIM22, suggesting a mechanism for coordinating different stages of autophagy. The Alzheimer's disease-associated TRIM22 variant R321K maintained autophagy initiation function in both cell lines and primary neurons. These findings demonstrate that TRIM22 acts as a scaffold protein to promote autophagy initiation, in addition to its previously described role in autophagosome-lysosome fusion. Our study provides new insights into the molecular mechanisms by which TRIM proteins regulate multiple stages of the autophagy process.

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by social interaction deficits and repetitive behaviors. While precise causes of ASD remain elusive, growing evidence highlights that an imbalance in excitatory and inhibitory (E/I) signaling is a pivotal factor in ASD development and modulation. Balanced E/I neurotransmission is critical for circuit formation, synaptic plasticity, and developmental timing. However, key questions persist, including the critical perturbation window, neurological and neurodevelopmental effects, and clinical implications of E/I imbalance. This study investigated early-life modulation of the GABAergic system's impact on E/I balance and ASD-like behaviors in mice. Mice were treated with bicuculline, a GABAA receptor antagonist, from postnatal days 7-11, and behavioral tests were conducted during adolescence. Results revealed deficits in social interaction in both male and female mice and increased repetitive behaviors in bicuculline-treated male mice. Electrophysiological recordings in the mPFC indicated reduced resting membrane potential, heightened neuronal excitability, and a shift in the E/I ratio. In the hippocampus, recordings displayed enhanced LTP and altered synaptic plasticity. DEG analysis of the PFC in bicuculline-treated mice unveiled aberrant gene profiles related to the regulation of synaptic function. Clinical significance and underlying mechanisms of abnormal brain activity, neurodevelopment, and ASD-related behaviors prompted by neonatal bicuculline treatment require further investigation. Nevertheless, these results suggest that GABAergic signaling disruption during the neonatal period might contribute to ASD-related brain pathophysiological changes.

Endocrine-disrupting chemicals (EDCs) are exogenous compounds that interact with the estrogen receptor (ER), thereby disrupting estrogen-mediated signaling. In a previous study, we employed a bioluminescence resonance energy transfer (BRET) system to assess ER dimerization for detecting EDCs. To further determine whether the BRET assay could be used independently to identify EDCs, we investigated ER-EDC interactions before and after dimerization. Results from isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) revealed that ER dimerization can be mediated by EDCs. Consequently, the BRET assay proved effective in detecting dimerization and clarifying its relevance to EDC-induced signaling disruption. Additionally, to examine EDC-induced transcriptional changes, we performed chromatin immunoprecipitation sequencing (ChIP-seq), followed by gene ontology (GO) analysis. These analyses demonstrated that EDCs affect various signaling pathways, including those involved in antibody-dependent cytotoxicity, bone morphogenetic protein (BMP) signaling in cardiac induction, and hepatocyte growth factor receptor signaling. Overall, this study elucidates the molecular mechanisms by which EDCs influence ER dimerization and signaling. These findings highlight the utility of the BRET-based assay for EDC detection and contribute to a deeper understanding of the systemic effects of EDCs on endocrine disruption.

Blood transfusion is a critical, lifesaving medical procedure for dogs. However, the limited availability of blood donors and ethical concerns highlight the need for alternative solutions, such as in vitro-produced red blood cells (RBCs), which remain unexplored in canines. This study aimed to produce canine erythrocytes in vitro from peripheral blood (PB) mononuclear cells (MNCs), optimize culture conditions using either human or canine reagents, and identify relevant cell markers. Results indicated that canine erythropoiesis can be induced by human or canine cytokines, producing RBCs within approximately 20 days. Although cell numbers decreased during the first seven days, immature erythroid cells proliferated, reaching peak expansion and RBC production by day 17. Despite the smaller cell size of the cultured RBCs than that of humans, the morphology at each stage of erythroid maturation was analogous to that of human erythropoiesis. Furthermore, the expression patterns of canine alpha hemoglobin stabilizing protein and erythropoietin receptor mirrored those observed in human erythropoiesis. Oxygen-hemoglobin (oxygen-Hb) association and dissociation curves of cultured RBCs closely resembled those of native canine RBCs, indicating an appropriate oxygen-carrying capacity. This study presents the first evidence of successful in vitro production of canine RBCs, offering a promising tool for research and potential therapeutic applications.

Additional sex comb-like 1 (ASXL1) is a chromatin-associated factor essential for transcriptional regulation. De novo truncating mutations in the ASXL1 gene are linked to Bohring-Opitz syndrome, a developmental disorder characterized by microcephaly; however, the role of Asxl1 in brain development remains unclear. In this study, we demonstrate that Asxl1 deletion in mice induces microcephaly, primarily caused by a reduction in the size and number of cortical neurons. Asxl1 ablation disrupts neural stem cell (NSC) maintenance, as evidenced by decreased proliferation and increased apoptosis. Transcriptomic analysis of Asxl1-deficient NSCs revealed 4,635 differentially expressed genes, including 2,262 upregulated and 2,373 downregulated genes. Gene ontology analysis indicated that Asxl1 regulates NSC survival through the histone methyltransferase Ezh2, a core component of the Polycomb Repressive Complex 2 (PRC2). Inhibition of H3K27me3 using GSK343 significantly reduced the viability of wild-type NSCs, but had a markedly diminished effect on Asxl1-deficient NSCs. Furthermore, Ezh2 target genes associated with apoptosis, such as Epha7 and Osr1, were upregulated in wild-type NSCs following GSK343 treatment but not significantly affected in Asxl1-deficient NSCs. These findings establish Asxl1 as a critical regulator of NSC survival and neurogenesis via Ezh2-mediated chromatin modification and provide insights into the mechanisms underlying microcephaly in developmental disorders.

Polypterus senegalus is a fish species characterized by primitive traits and unique physiological-anatomical features, representing a transitional stage toward terrestrial vertebrates. Diverging from other ray-finned fishes approximately 420 million years ago, it serves as a key model for vertebrate evolutionary studies. In this study, we identified 19,295 unique P. senegalus transcripts using Pacific Biosciences Iso-Seq, with most classified as novel exon combinations, intron retentions, or novel start and stop codons. This high-quality, full-length transcriptome revealed previously unreported isoforms and investigated their functional roles to enhance our understanding of the genomic complexity and evolutionary traits of the species. Notably, these isoforms were identified in genes associated with Polypterus's distinctive features, including limb flexibility (ACTN3B, MYBPC1), lung function (FAM13A, ERBB2, TCF7), and circulation (FBN1, MYH11B, MEF2CB). These findings offer molecular insights into early vertebrate terrestrial adaptation and the functional integration of key tissues in extant species.

Epigenetics and epigenomics are captivating fields of molecular biology, dedicated to the exploration of heritable alterations in gene expression and cellular phenotypes, which transpire devoid of any discernible modifications to the fundamental DNA sequence. This intricate regulatory apparatus encompasses multiple mechanisms, prominently featuring DNA methylation, histone modifications, and the involvement of non-coding RNA molecules in pivotal roles. To achieve a comprehensive grasp of these diverse mechanisms, it is imperative to conduct research employing animal models as proxies for human studies. Since experimental animal models like mice and rats struggle to replicate the diverse environmental conditions experienced by humans, this review focuses on comparing common epigenetic alterations in naturally occurring tumors in canine models, which share the human environment, with those in humans. Through this, we emphasize the importance of an epigenetic regulation in the comparative medical approach to a deeper understanding of cancers and further development of cancer treatments. Additionally, we elucidate epigenetic modifications pertinent to specific developmental stages, the ageing process, and the progression of various diseases.