EPAS1, a hypoxia- and ferroptosis-related gene, promotes malignant behaviour of cervical cancer by ceRNA and super-enhancer.

Hypoxia and Ferroptosis are associated with the malignant behaviour of cervical cancer. Endothelial PAS domain-containing protein 1 (EPAS1) contributes to the progression of cervical cancer. EPAS1 plays important roles in hypoxia and ferroptosis. Using the GEO dataset, machine-learning algorithms were used to screen for hypoxia- and ferroptosis-related genes (HFRGs) in cervical cancer. EPAS1 was identified as the hub gene. qPCR and WB were used to investigate the expression of EPAS1 in normal and cervical cancer tissues. The proliferation, invasion and migration of EPAS1 cells in HeLa and SiHa cell lines were detected using CCK8, transwell and wound healing assays, respectively. Apoptosis was detected by flow cytometry. A dual-luciferase assay was used to analyse the MALAT1-miR-182-5P-EPAS1 mRNA axis and core promoter elements of the super-enhancer. EPAS1 was significantly overexpressed in cervical cancer tissues. EPAS1 could increase the proliferation, invasion, migration of HeLa and SiHa cells and reduce the apoptosis of HeLa and SiHa cell. According to the double-luciferase assay, EPAS1 expression was regulated by the MALAT1-Mir-182-5p-EPAS1 mRNA axis. EPAS1 is associated with super-enhancers. Double-luciferase assay showed that the core elements of the super-enhancer were E1 and E3. EPAS1, an HFRG, is significantly overexpressed in cervical cancer. EPAS1 promotes malignant behaviour of cervical cancer cells. EPAS1 expression is regulated by super-enhancers and the MALAT1-miR-182-5P- EPAS1 mRNA axis. EPAS1 may be a target for the diagnosis and treatment of cervical cancer.

Microbiota-derived I3A protects the intestine against radiation injury by activating AhR/IL-10/Wnt signaling and enhancing the abundance of probiotics.

The intestine is prone to radiation damage in patients undergoing radiotherapy for pelvic tumors. However, there are currently no effective drugs available for the prevention or treatment of radiation-induced enteropathy (RIE). In this study, we aimed at investigating the impact of indole-3-carboxaldehyde (I3A) derived from the intestinal microbiota on RIE. Intestinal organoids were isolated and cultivated for screening radioprotective tryptophan metabolites. A RIE model was established using 13 Gy whole-abdominal irradiation in male C57BL/6J mice. After oral administration of I3A, its radioprotective ability was assessed through the observation of survival rates, clinical scores, and pathological analysis. Intestinal stem cell survival and changes in the intestinal barrier were observed through immunofluorescence and immunohistochemistry. Subsequently, the radioprotective mechanisms of I3A was investigated through 16S rRNA and transcriptome sequencing, respectively. Finally, human colon cancer cells and organoids were cultured to assess the influence of I3A on tumor radiotherapy. I3A exhibited the most potent radioprotective effect on intestinal organoids. Oral administration of I3A treatment significantly increased the survival rate in irradiated mice, improved clinical and histological scores, mitigated mucosal damage, enhanced the proliferation and differentiation of Lgr5+ intestinal stem cells, and maintained intestinal barrier integrity. Furthermore, I3A enhanced the abundance of probiotics, and activated the AhR/IL-10/Wnt signaling pathway to promote intestinal epithelial proliferation. As a crucial tryptophan metabolite, I3A promotes intestinal epithelial cell proliferation through the AhR/IL-10/Wnt signaling pathway and upregulates the abundance of probiotics to treat RIE. Microbiota-derived I3A demonstrates potential clinical application value for the treatment of RIE.

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

Prognostic Implications of Small Cell Lung Cancer Transcriptional Subtyping for CNS Metastases.

PURPOSE: Small cell lung cancer (SCLC) often metastasizes to the brain and has poor prognosis. SCLC subtypes distinguished by expressing transcriptional factors ASCL1 or NEUROD1 have been identified. This study investigates the impact of transcription factor-defined SCLC subtype on incidence and outcomes of brain metastases (BMs). METHODS: Patients with SCLC with ASCL1 (A) and NEUROD1 (N) immunohistochemical expression status were identified and classified: (1) A+/N-, (2) A+/N+, (3) A-/N+, and (4) A-/N-. Cumulative incidence competing risk analyses were used to assess incidence of CNS progression. Cox proportional hazards models were used for multivariable analyses of overall survival (OS) and CNS progression-free survival (CNS-PFS). RESULTS: Of 164 patients, most were either A+/N- or A+/N+ (n = 62, n = 63, respectively). BMs were present at diagnosis in 24 patients (15%). Among them, the 12-month cumulative incidence of subsequent CNS progression was numerically highest for A+/N- (50% [95% CI, 10.5 to 74.7]; P = .47). Among those BM-free at diagnosis, the 12-month cumulative incidence of CNS progression was numerically the highest for A+/N- (16% [95% CI, 7.5 to 27.9]) and A-/N+ (9.1% [95% CI, 0.0 to 34.8]; P = .20). Both subtypes, A+/N- and A-/N+, had worse OS compared with A+/N+ (A+/N-: hazard ratio [HR], 1.62 [95% CI, 1.01 to 2.51]; P < .05; A-/N+: HR, 3.02 [95% CI, 1.35 to 6.76]; P = .007). Excellent response rates (28, 65% CR/PR) across subtypes were seen in patients who had CNS-directed radiotherapy versus systemic therapy alone (9, 36% CR/PR). CONCLUSION: To our knowledge, this report is the first to investigate CNS-specific outcomes based on transcription factor subtypes in patients with SCLC. BM-free patients at diagnosis with A+/N- or A-/N+ subtypes had worse outcomes compared with those with transcriptional factor coexpression. Further investigation into the mechanisms and implications of SCLC subtyping on CNS-specific outcomes is warranted to ultimately guide personalized care.

Identification of molecular subtypes based on chromatin regulator-related genes and experimental verification of the role of ASCL1 in conferring chemotherapy resistance to breast cancer.

Objective: The aim of this study was to identify the molecular subtypes of breast cancer based on chromatin regulator-related genes. Methods: The RNA sequencing data of The Cancer Genome Atlas-Breast Cancer cohort were obtained from the official website, while the single-cell data were downloaded from the Gene Expression Omnibus database (GSE176078). Validation was performed using the Molecular Taxonomy of Breast Cancer International Consortium dataset. Furthermore, the immune characteristics, tumor stemness, heterogeneity, and clinical characteristics of these molecular subtypes were analyzed. The correlation between chromatin regulators and chemotherapy resistance was examined in vitro using the quantitative real-time polymerase chain reaction (qRT-PCR) and Cell Counting Kit-8 (CCK8) assays. Results: This study identified three stable molecular subtypes with different prognostic and pathological features. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction analyses revealed that the differentially expressed genes were associated with disease processes, such as mitotic nuclear division, chromosome segregation, condensed chromosome, and specific chromosome region. The T stage and subtypes were correlated with the clinical features. Tumor heterogeneity (mutant-allele tumor heterogeneity, tumor mutational burden, purity, and homologous recombination deficiency) and tumor stemness (RNA expression-based stemness score, epigenetically regulated RNA expression-based stemness score, DNA methylation-based stemness score, and epigenetically regulated DNA methylation-based stemness score) significantly varied between the three subtypes. Furthermore, Western blotting, qRT-PCR, and CCK8 assays demonstrated that the expression of ASCL1 was positively correlated with chemotherapy resistance in breast cancer. Conclusion: This study identified the subtypes of breast cancer based on chromatin regulators and analyzed their clinical features, gene mutation status, immunophenotype, and drug sensitivity. The results of this study provide effective strategies for assessing clinical prognosis and developing personalized treatment strategies.

A knock-in allele of Hand2 expressing Dre recombinase.

HAND2 is a basic helix-loop-helix transcription factor with diverse functions during development. To facilitate the investigation of genetic and functional diversity among Hand2-expressing cells in the mouse, we have generated Hand2Dre, a knock-in allele expressing Dre recombinase. To avoid disrupting Hand2 function, the Dre cDNA is inserted at the 3' end of the Hand2 coding sequence following a viral 2A peptide. Hand2Dre homozygotes can therefore be used in complex crosses to increase the proportion of useful genotypes among offspring. Dre expression in mid-gestation Hand2Dre embryos is indistinguishable from wild-type Hand2 expression, and HandDre efficiently recombines rox target sites in vivo. In combination with existing Cre and Flp mouse lines, Hand2Dre will therefore extend the ability to perform genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by developmental expression of Hand2.

NPAS2, transcriptionally activated by ARRB1, promotes the malignant behaviours of lung adenocarcinoma cells and regulates the reprogramming of glucose metabolism.

Lung adenocarcinoma (LUAD) is a serious threat to public health and is accompanied by increased morbidity and mortality worldwide. Neuronal PAS domain protein2 (NPAS2) has been confirmed as an oncogene in LUAD; however, little is known about its molecular mechanism. Here, the expression level of NPAS2 was detected in LUAD cell lines and 16HBE cells. Gain- and loss-of-function experiments were performed. Cell Counting Kit-8, colony formation, flow cytometry, wound-healing and Transwell assays were conducted to assess cell proliferation, apoptosis, migration and invasion, respectively. Reprogramming of glucose metabolism was evaluated via oxygen consumption rate (OCR), complexes activities, lactic production and glucose consumption. The expression of critical proteins was examined by western blot. We demonstrated aberrant upregulation of NPAS2 and ß-arrestin-1 (ARRB1) in LUAD cell lines. ARRB1 was found to be a critical transcription factor of NPAS2 with binding sites within the promoter region of NPAS2, thereby causing its transcriptional activation. Functional experiments revealed that NPAS2 depletion significantly inhibited the malignant behaviours of A549 cells by suppressing cell proliferation, migration, invasion and epithelial-mesenchymal transition and promoting cell apoptosis. Meanwhile, NPAS2 depletion increased OCR and activities of complexes (I, II, III and V), and reduced lactic acid production and glucose uptake in A549 cells, indicating that NPAS2 depletion inhibited aerobic glycolysis, accompanied by reduced expression of glycolytic enzymes. However, the changes caused by NPAS2 knockdown were partly restored by ARRB1 overexpression. In conclusion, our study suggests that ARRB1 could transcriptionally activate NPAS2, facilitating malignant activities and glycolysis, and ultimately promoting the progression of LUAD, proving a novel therapeutic strategy for the treatment of LUAD.

[Astragali Radix-Curcumae Rhizoma inhibits colon cancer progression and enhances 5-FU efficacy by regulating hypoxia-inducible factors and tumor stem cells].

The animal and cell models were used in this study to investigate the mechanism of Astragali Radix-Curcumae Rhizoma(HQEZ) in inhibiting colon cancer progression and enhancing the efficacy of 5-fluorouracil(5-FU) by regulating hypoxia-inducible factors and tumor stem cells. The animal model was established by subcutaneous transplantation of colon cancer HCT116 cells in nude mice, and 24 successfully modeled mice were randomized into model, 5-FU, HQEZ, and 5-FU+HQEZ groups. The tumor volume was measured every two days. Western blot was employed to measure the protein levels of epidermal growth factor receptor(EGFR), dihydropyrimidine dehydrogenase(DPYD), and thymidylate synthase(TYMS), the key targets of the hypoxic core region, as well as the hypoxia-inducible factors HIF-1α and HIF-2α and the cancer stem cell surface marker CD133 and SRY-box transcription factor 2(SOX2). The results of animal experiments showed that HQEZ slowed down the tumor growth and significantly increased the tumor inhibition rate of 5-FU. Compared with the model group, HQEZ significantly down-regulated the protein levels of EGFR and DPYD, and 5-FU+HQEZ significantly down-regulated the protein levels of EGFR and TYMS in tumors. Compared with the model group, HQEZ significantly down-regulated the protein levels of HIF-1α, HIF-2α, SOX2, and CD133 in the hypoxic core region. Compared with the 5-FU group, 5-FU+HQEZ lowered the protein levels of HIF-1α, HIF-2α, and SOX2. The cell experiments showed that the protein le-vels of HIF-1α and HIF-2α in HCT116 cells elevated significantly after low oxygen treatment. Compared with 5-FU(1.38 µmol·L~(-1)) alone, HQEZ(40 mg·mL~(-1)) and 5-FU+HQEZ significantly down-regulated the protein levels of HIF-1α, HIF-2α, and TYMS. In conclusion, HQEZ can inhibit the expression of hypoxia-responsive molecules in colon cancer cells and reduce the properties of cancer stem cells, thereby enhancing the therapeutic effect of 5-FU on colon cancer.

Spatio-temporal dynamics of early somite segmentation in the chicken embryo.

During vertebrate embryo development, the body is progressively segmented along the anterior-posterior (A-P) axis early in development. The rate of somite formation is controlled by the somitogenesis embryo clock (EC), which was first described as gene expression oscillations of hairy1 (hes4) in the presomitic mesoderm of chick embryos with 15-20 somites. Here, the EC displays the same periodicity as somite formation, 90 min, whereas the posterior-most somites (44-52) only arise every 150 minutes, matched by a corresponding slower pace of the EC. Evidence suggests that the rostral-most somites are formed faster, however, their periodicity and the EC expression dynamics in these early stages are unknown. In this study, we used time-lapse imaging of chicken embryos from primitive streak to somitogenesis stages with high temporal resolution (3-minute intervals). We measured the length between the anterior-most and the last formed somitic clefts in each captured frame and developed a simple algorithm to automatically infer both the length and time of formation of each somite. We found that the occipital somites (up to somite 5) form at an average rate of 75 minutes, while somites 6 onwards are formed approximately every 90 minutes. We also assessed the expression dynamics of hairy1 using half-embryo explants cultured for different periods of time. This showed that EC hairy1 expression is highly dynamic prior to somitogenesis and assumes a clear oscillatory behaviour as the first somites are formed. Importantly, using ex ovo culture and live-imaging techniques, we showed that the hairy1 expression pattern recapitulates with the formation of each new pair of somites, indicating that somite segmentation is coupled with EC oscillations since the onset of somitogenesis.

Characterization and expression analysis of GLABRA3 (GL3) genes in cotton: insights into trichome development and hormonal regulation.

BACKGROUND: GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) genes encode a typical helix-loop-helix (bHLH) transcription factors that primarily regulate trichome branching and root hair development, DNA endoreduplication, trichoblast size, and stomatal formation. The functions of GL3 genes in cotton crop have been poorly characterized. In this study, we performed comprehensive genome-wide scans for GL3 and EGL3 homologs to enhance our comprehension of their potential roles in trichome and fiber development in cotton crop. METHODS AND RESULTS: Our findings paraded that Gossypium hirsutum and G. barbadense have 6 GL3s each, unevenly distributed on 4 chromosomes whereas, G. arboreum, and G. raimondii have 3 GL3s each, unevenly distributed on 2 chromosomes. Gh_A08G2088 and Gb_A09G2187, despite having the same bHLH domain as the other GL3 genes, were excluded due to remarkable short sequences and limited number of motifs, indicating a lack of potential functional activity. The phylogenetic analysis categorized remaining 16 GL3s into three subfamilies (Group I-III) closely related to A. thaliana. The 16 GL3s have complete bHLH domain, encompassing 590-631 amino acids, with molecular weights (MWs) ranging from 65.92 to 71.36 kDa. Within each subfamily GL3s depicted shared similar gene structures and motifs, indicating conserved characteristics within respective groups. Promoter region analysis revealed 27 cis-acting elements, these elements were responsive to salicylic acid, abscisic acid (ABA), methyl jasmonate (MeJA), and gibberellin. The expression of GL3 genes was analyzed across 12 tissues in both G. barbadense and G. hirsutum using the publicly available RNA-seq data. Among GL3s, Gb_D11G0219, Gb_D11G0214, and Gb_D08G2182, were identified as relatively highly expressed across different tissues, consequently selected for hormone treatment and expression validation in G. barbadense. RT-qPCR results demonstrated significant alterations in the expression levels of Gb_D11G0219 and Gb_D11G0214 following MeJA, GA, and ABA treatment. Subcellular localization prediction revealed that most GL3 proteins were predominantly expressed in the nucleus, while a few were localized in the cytoplasm and chloroplasts. CONCLUSIONS: In summary, this study lays the foundation for subsequent functional validation of GL3 genes by identifying hormonal regulation patterns and probable sites of action in cotton trichome formation and fiber development. The results stipulate a rationale to elucidate the roles and regulatory mechanisms of GL3 genes in the intricate process of cotton fibre and trichome development.

Characterization of PIF4 Phosphorylation by SPA1.

The PHYTOCHROME INTERACTING FACTORs (PIFs) play pivotal roles in regulating thermo- and photo-morphogenesis in Arabidopsis. One of the main hubs in thermomorphogenesis is PIF4, which regulates plant development under high ambient temperature along with other PIFs. PIF4 enhances its own transcription and PIF4 protein is stabilized under high ambient temperature. However, the mechanisms of thermo-stabilization of PIF4 are less understood. Recently, it was shown that SUPPRESSOR OF PHYA-105 1 (SPA1) can function as a serine/threonine kinase to phosphorylate PIF4 in vitro, and the phosphorylated form of PIF4 is more stable under high ambient temperature conditions. In this chapter, we describe the in vitro kinase assay of PIF4 by SPA1. In principle, this protocol can be applied for other putative substrates and kinases.

GST Pull-Down Assay to Study PIF4 Binding In Vitro.

The phytochrome-interacting factor 4 (PIF4) is a well-known transcription factor that plays a pivotal role in plant thermomorphogenesis, coordinating growth and development in response to temperature changes. As PIF4 functions by forming complexes with other proteins, determining its interacting partners is essential for understanding its diverse roles in plant thermal responses. The GST (glutathione-S-transferase) pull-down assay is a widely used biochemical technique that enables the investigation of protein-protein interactions in vitro. It is particularly useful for studying transient or weak interactions between proteins. In this chapter, we describe the GST pull-down approach to detect the interaction between PIF4 and a known or suspected interacting protein. We provide detailed step-by-step descriptions of the assay procedures, from the preparation of recombinant GST-PIF4 fusion protein to the binding and elution of interacting partners. Additionally, we provide guidelines for data interpretation, quantification, and statistical analysis to ensure robust and reliable results.

Assessing the Function of CBF1 in Modulating the Interaction Between Phytochrome B and PIF4.

Phytochromes are red (R) and far-red (FR) light photoreceptors in plants. Upon light exposure, photoactivated phytochromes translocate into the nucleus, where they interact with their partner proteins to transduce light signals. The yeast two-hybrid (Y2H) system is a powerful technique for rapidly identifying and verifying protein-protein interactions, and PHYTOCHROME-INTERACTING FACTOR3 (PIF3), the founding member of the PIF proteins, was initially identified in a Y2H screen for phytochrome B (phyB)-interacting proteins. Recently, we developed a yeast three-hybrid (Y3H) system by introducing an additional vector into this Y2H system, and thus a new regulator could be co-expressed and its role in modulating the interactions between phytochromes and their signaling partners could be examined. By employing this Y3H system, we recently showed that both MYB30 and CBF1, two negative regulators of seedlings photomorphogenesis, act to inhibit the interactions between phyB and PIF4/PIF5. In this chapter, we will use the CBF1-phyB-PIF4 module as an example and describe the detailed procedure for performing this Y3H assay. It will be intriguing and exciting to explore the potential usage of this Y3H system in future research.

Photobody formation spatially segregates two opposing phytochrome B signaling actions of PIF5 degradation and stabilization.

Photoactivation of the plant photoreceptor and thermosensor phytochrome B (PHYB) triggers its condensation into subnuclear membraneless organelles named photobodies (PBs). However, the function of PBs in PHYB signaling remains frustratingly elusive. Here, we found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) to PBs. Surprisingly, PHYB exerts opposing roles in degrading and stabilizing PIF5. Perturbing PB size by overproducing PHYB provoked a biphasic PIF5 response: while a moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Conversely, reducing PB size by dim light, which enhanced PB dynamics and nucleoplasmic PHYB and PIF5, switched the balance towards PIF5 degradation. Together, these results reveal that PB formation spatially segregates two antagonistic PHYB signaling actions - PIF5 stabilization in PBs and PIF5 degradation in the surrounding nucleoplasm - which could enable an environmentally sensitive, counterbalancing mechanism to titrate nucleoplasmic PIF5 and environmental responses.

DEC1 is involved in circadian rhythm disruption-exacerbated pulmonary fibrosis.

BACKGROUND: The alveolar epithelial type II cell (AT2) and its senescence play a pivotal role in alveolar damage and pulmonary fibrosis. Cell circadian rhythm is strongly associated with cell senescence. Differentiated embryonic chondrocyte expressed gene 1 (DEC1) is a very important circadian clock gene. However, the role of DEC1 in AT2 senescence and pulmonary fibrosis was still unclear. RESULTS: In this study, a circadian disruption model of light intervention was used. It was found that circadian disruption exacerbated pulmonary fibrosis in mice. To understand the underlying mechanism, DEC1 levels were investigated. Results showed that DEC1 levels increased in lung tissues of IPF patients and in bleomycin-induced mouse fibrotic lungs. In vitro study revealed that bleomycin and TGF-ß1 increased the expressions of DEC1, collagen-I, and fibronectin in AT2 cells. Inhibition of DEC1 mitigated bleomycin-induced fibrotic changes in vitro and in vivo. After that, cell senescence was observed in bleomycin-treated AT2 cells and mouse models, but these were prevented by DEC1 inhibition. At last, p21 was confirmed having circadian rhythm followed DEC1 in normal conditions. But bleomycin disrupted the circadian rhythm and increased DEC1 which promoted p21 expression, increased p21 mediated AT2 senescence and pulmonary fibrosis. CONCLUSIONS: Taken together, circadian clock protein DEC1 mediated pulmonary fibrosis via p21 and cell senescence in alveolar epithelial type II cells.

The Antioxidant Drug Edaravone Binds to the Aryl Hydrocarbon Receptor (AHR) and Promotes the Downstream Signaling Pathway Activation.

A considerable effort has been spent in the past decades to develop targeted therapies for the treatment of demyelinating diseases, such as multiple sclerosis (MS). Among drugs with free radical scavenging activity and oligodendrocyte protecting effects, Edaravone (Radicava) has recently received increasing attention because of being able to enhance remyelination in experimental in vitro and in vivo disease models. While its beneficial effects are greatly supported by experimental evidence, there is a current paucity of information regarding its mechanism of action and main molecular targets. By using high-throughput RNA-seq and biochemical experiments in murine oligodendrocyte progenitors and SH-SY5Y neuroblastoma cells combined with molecular docking and molecular dynamics simulation, we here provide evidence that Edaravone triggers the activation of aryl hydrocarbon receptor (AHR) signaling by eliciting AHR nuclear translocation and the transcriptional-mediated induction of key cytoprotective gene expression. We also show that an Edaravone-dependent AHR signaling transduction occurs in the zebrafish experimental model, associated with a downstream upregulation of the NRF2 signaling pathway. We finally demonstrate that its rapid cytoprotective and antioxidant actions boost increased expression of the promyelinating Olig2 protein as well as of an Olig2:GFP transgene in vivo. We therefore shed light on a still undescribed potential mechanism of action for this drug, providing further support to its therapeutic potential in the context of debilitating demyelinating conditions.

Cell type-dependent response to benzo(a)pyrene exposure of human placental cell lines under normoxic, hypoxic, and pro-inflammatory conditions.

Benzo(a)pyrene (BaP) can be detected in the human placenta. However, little is known about the effects of BaP exposure on different placental cells under various conditions. In this study, we aimed to investigate the effects of BaP on mitochondrial function, pyrin domain-containing protein 3 (NLRP3) inflammasome, and apoptosis in three human trophoblast cell lines under normoxia, hypoxia, and inflammatory conditions. JEG-3, BeWo, and HTR-8/SVneo cell lines were exposed to BaP under normoxia, hypoxia, or inflammatory conditions for 24 h. After treatment, we evaluated cell viability, apoptosis, aryl hydrocarbon receptor (AhR) protein and cytochrome P450 (CYP) gene expression, mitochondrial function, including mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨm), intracellular adenosine triphosphate (iATP), and extracellular ATP (eATP), nitric oxide (NO), NLPR3 inflammasome proteins, and interleukin (IL)-1ß. We found that BaP upregulated the expression of AhR or CYP genes to varying degrees in all three cell lines. Exposure to BaP alone increased ΔΨm in all cell lines but decreased NO in BeWo and HTR-8/SVneo, iATP in HTR-8/SVneo, and cell viability in JEG-3, without affecting apoptosis. Under hypoxic conditions, BaP did not increase the expression of AhR and CYP genes in JEG-3 cells but increased CYP gene expression in two others. Pro-inflammatory conditions did not affect the response of the 3 cell lines to BaP with respect to the expression of CYP genes and changes in the mitochondrial function and NLRP3 inflammasome proteins. In addition, in HTR-8/SVneo cells, BaP increased IL-1ß secretion in the presence of hypoxia and poly(I:C). In conclusion, our results showed that BaP affected mitochondrial function in trophoblast cell lines by increasing ΔΨm. This increased ΔΨm may have rescued the trophoblast cells from activation of the NLRP3 inflammasome and apoptosis after BaP treatment. We also observed that different human trophoblast cell lines had cell type-dependent responses to BaP exposure under normoxia, hypoxia, or pro-inflammatory conditions.

Electrophysiological Properties of Proprioception-Related Neurons in the Intermediate Thoracolumbar Spinal Cord.

Proprioception, the sense of limb and body position, is required to produce accurate and precise movements. Proprioceptive sensory neurons transmit muscle length and tension information to the spinal cord. The function of excitatory neurons in the intermediate spinal cord, which receive this proprioceptive information, remains poorly understood. Using genetic labeling strategies and patch-clamp techniques in acute spinal cord preparations in mice, we set out to uncover how two sets of spinal neurons, Clarke's column (CC) and Atoh1-lineage neurons, respond to electrical activity and how their inputs are organized. Both sets of neurons are located in close proximity in laminae V-VII of the thoracolumbar spinal cord and have been described to receive proprioceptive signals. We find that a majority of CC neurons have a tonic-firing type and express a distinctive hyperpolarization-activated current (Ih). Atoh1-lineage neurons, which cluster into two spatially distinct populations, are mostly a fading-firing type and display similar electrophysiological properties to each other, possibly due to their common developmental lineage. Finally, we find that CC neurons respond to stimulation of lumbar dorsal roots, consistent with prior knowledge that CC neurons receive hindlimb proprioceptive information. In contrast, using a combination of electrical stimulation, optogenetic stimulation, and transsynaptic rabies virus tracing, we find that Atoh1-lineage neurons receive heterogeneous, predominantly local thoracic inputs that include parvalbumin-lineage sensory afferents and local interneuron presynaptic inputs. Altogether, we find that CC and Atoh1-lineage neurons have distinct membrane properties and sensory input organization, representing different subcircuit modes of proprioceptive information processing.

Diosgenin improves post-myocardial infarction cardiac function via HAND2-induced angiogenesis.

While diosgenin has been demonstrated effective in various cardiovascular diseases, its specific impact on treating heart attacks remains unclear. Our research revealed that diosgenin significantly improved cardiac function in a myocardial infarction (MI) mouse model, reducing cardiac fibrosis and cell apoptosis while promoting angiogenesis. Mechanistically, diosgenin upregulated the Hand2 expression, promoting the proliferation and migration of endothelial cells under hypoxic conditions. Acting as a transcription factor, HAND2 activated the angiogenesis-related gene Aggf1. Conversely, silencing Hand2 inhibited the diosgenin-induced migration of hypoxic endothelial cells and angiogenesis. In summary, these findings provide new insights into the protective role of diosgenin in MI, validating its effect on angiogenic activity and providing a theoretical basis for clinical treatment strategies.

Circulating-tumour DNA methylation of HAND1 gene: a promising biomarker in early detection of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the significant global health concerns with an increase in cases. Regular screening tests are crucial for early detection as it is often asymptomatic in the initial stages. Liquid biopsies, a non-invasive approach that examines biomarkers in biofluids, offer a promising future in diagnosing and screening cancer. Circulating-tumour DNA (ctDNA) is the genetic material in biofluids released into the circulatory system by cells. ctDNA is a promising marker for monitoring patients since cancer cells display distinct DNA methylation patterns compared to normal cells. The potential of our research to contribute to early detection and improved patient outcomes is significant. AIMS: The primary objective of this research project was to explore the HAND1 methylation levels in plasma ctDNA as a potential biomarker for diagnosing CRC and evaluate the methylation level of the well-established gene SPET9 to compare it with the methylation level of HAND1. MATERIALS AND METHODS: Plasma samples were collected from 30 CRC patients and 15 healthy individuals, with CRC samples obtained pre-treatment. ctDNA was extracted and treated with bisulfite for methylation status assessment. Quantitative methylation-specific PCR (qMS-PCR) was performed for HAND1 and SEPT9, using ß-actin (ACTB gene) as a reference. The study aims to evaluate the potential of these genes as diagnostic biomarkers for CRC, contributing to early detection and improved patient outcomes. RESULTS: Our study yielded significant results: 90% of CRC patients (27 out of 30) had hypermethylation in the SEPT9 gene, and 83% (25 out of 30) exhibited hypermethylation in the HAND1 gene. The methylation levels of both genes were significantly higher in CRC patients than in healthy donors. These findings underscore the potential of SEPT9 and HAND1 methylation as promising biomarkers for diagnosing CRC, potentially leading to early detection and improved patient outcomes. CONCLUSION: These findings highlight the potential of SEPT9 and HAND1 methylation as promising biomarkers for diagnosing CRC. However, further research and validation studies are needed to confirm these findings and to explore their clinical utility in CRC diagnosis and management.