Response Element Research Articles

Glial 'omics in ischemia: Acute stroke and chronic cerebral small vessel disease.

Vascular injury and pathologies underlie common diseases including ischemic stroke and cerebral small vessel disease (CSVD). Prior work has identified a key role for glial cells, including microglia, in the multifaceted and temporally evolving neuroimmune response to both stroke and CSVD. Transcriptional profiling has led to important advances including identification of distinct gene expression signatures in ischemia-exposed, flow cytometrically sorted microglia and more recently single cell RNA sequencing-identified microglial subpopulations or clusters. There is a reassuring degree of overlap in the results from these two distinct methodologies with both identifying a proliferative and a separate type I interferon responsive microglial element. Similar patterns were later seen using multimodal and spatial transcriptomal profiling in ischemia-exposed microglia and astrocytes. Methodological advances including enrichment of specific neuroanatomic/functional regions (such as the neurovascular unit) prior to single cell RNA sequencing has led to identification of novel cellular subtypes and generation of new credible hypotheses as to cellular function based on the enhanced cell sub-type specific gene expression patterns. A ribosomal tagging strategy focusing on the cellular translatome analyses carried out in the acute phases post stroke has revealed distinct inflammation-regulating roles for microglia and astrocytes in this setting. Early spatial transcriptomics experiments using cerebral ischemia models have identified regionally distinct microglial cell clusters in ischemic core versus penumbra. There is great potential for combination of these methods for multi-omics approaches to further elucidate glial responses in the context of both acute ischemic stroke and chronic CSVD.

Multi-Responsive Polymer Nanoparticles: A Versatile Platform for Double-Security Anticounterfeiting and Smart Food Packaging.

Potential applications of colloidal polymer nanoparticles in the preparation of smart inks are investigated by physical incorporation of the oxazolidine molecules. Precise adjusting the polymer chain flexibility and polarity is achieved by controlling the ratio of methyl methacrylate and butyl acrylate monomers in the polymerization reaction. In addition, nanofibrous indicators of acid-base vapors are prepared from the latex nanoparticles. This can be beneficial for creating materials that sense and respond to environmental changes, such as humidity or moisture and acidity. Thermochromic inks are prepared by microencapsulating crystal violet lactone dye (CVL) in polymer matrices to prevent their release into the aqueous media. Combining two distinct systems with varying triggers, such as light and temperature, provides an effective strategy for double-encryption anticounterfeiting and crack and scratch detection and indication applications. Preparing labels impregnated with double-responsive inks, a novel approach is developed for food spoilage detection and preservation indication. Labels are manufactured using polymer nanoparticles, which contain photoluminescent oxazolidine molecules, as well as a trinary mixture of CVL within core-shell latex particles as the thermochromic dye. The combination of these two responsive elements transforms traditional packaging into a dynamic and interactive sentinel for the food it holds.

Decoding Critical Targets and Signaling Pathways in EBV-Mediated Diseases Using Large Language Models.

Epstein-Barr virus (EBV), a member of the gamma herpesvirus, is the first identified human oncovirus and is associated with various malignancies. Understanding the intricate interactions between EBV antigens and cellular pathways is crucial to unraveling the molecular mechanisms in EBV-mediated diseases. However, fully elucidating EBV-host interactions and the associated pathogenesis remains a significant challenge. In this study, we employed large language models (LLMs) to screen 36,105 EBV-relevant scientific publications and summarize the current literature landscape on various EBV-associated diseases like Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), nasopharyngeal carcinoma (NPC), and so on. LLM-generated data indicate that the most-studied EBV-associated pathways are enriched in immune response, apoptosis, cell growth, and replication. The analyses of protein-protein interactions (PPIs) reveal three principal EBV-related protein clusters: TP53-centered apoptotic factors, EBV-associated transcription factors, and immune response elements. Utilizing our dataset and public databases, we demonstrated that BLLF3-targeted TLR2-associated factors are effective diagnostic markers for DLBCL. Next, we confirmed the co-expression of LMP1-targeted calcium pathway factors in BL. Finally, we demonstrated the correlation and co-expression of LMP1-induced PARP1, HIF1A, HK2, and key glycolysis-related factors, further suggesting that LMP1 actively regulates the glycolysis pathway. Therefore, our study presents a comprehensive functional encyclopedia of the interactions between EBV antigens and host signaling pathways across various EBV-associated diseases, providing valuable insights for the development of therapeutic strategies.

Knockout of the Carbohydrate Responsive Element Binding Protein Enhances Proliferation and Tumorigenesis in Renal Tubules of Mice.

Glycogen-storing so-called clear cell kidney tubules (CCTs), precursor lesions of renal cell carcinoma, have been described in diabetic rats and in humans. The lesions show upregulation of the Akt/mTOR-pathway and the related transcription factor carbohydrate responsive element binding protein (ChREBP), which is supposedly pro-oncogenic. We investigated the effect of ChREBP-knockout on nephrocarcinogenesis in streptozotocin-induced diabetic and normoglycemic mice. Diabetic, but not non-diabetic mice, showed CCTs at 3, 6 and 12 months of age. Glycogenosis was confirmed by periodic acid schiff reaction and transmission electron microscopy. CCTs in ChREBP-knockout mice consisted of larger cells and occurred more frequently compared to wildtype mice. Progression towards kidney tumors was observed in both diabetic groups but occurred earlier in ChREBP-knockout mice. Proliferative activity assessed by BrdU-labeling was lower in 1-week-old but higher in 12-month-old diabetic ChREBP-knockout mice. Surprisingly, renal neoplasms occurred spontaneously in non-diabetic ChREBP-knockout, but not non-diabetic wildtype mice, indicating an unexpected tumor-suppressive function of ChREBP. Immunohistochemistry showed upregulated glycolysis and lipogenesis, along with activated Akt/mTOR-signaling in tumors of ChREBP-knockout groups. Immunohistochemistry of human clear cell renal cell carcinomas revealed reduced ChREBP expression compared to normal kidney tissue. However, the molecular mechanisms by which loss of ChREBP might facilitate tumorigenesis require further investigation.

Genome-Wide Identification of COMT Gene Family in Maize and its Function in Response to Light.

Maize is a major crop, feed, and industrial material. Caffeic acid-O-methyltransferase (COMT) is a methylase closely associated with lignin biosynthesis and plant growth and resistance. In this study, we identified the COMT gene (ZmCOMT) family in maize and further analyzed its phylogenetic evolution, subcellular localization, and its function in response to light. Thirty-one ZmCOMT genes were identified in the maize genome, which were distributed across eight chromosomes and mainly clustered on chromosome 4. Most ZmCOMT proteins were predicted to localize in the cytoplasm. Ten different conserved motifs were present in most ZmCOMT proteins, and motif1, motif6, and motif7 were highly conserved and present in all ZmCOMT proteins. The photoresponsivity elements were conserved among all members, and ZmCOMT22 and ZmCOMT10 genes responsive to light. This result suggests a potential function for these two genes in lignin biosynthesis which a previous study had linked to light regulation. Jasmonic acid responsive and abscisic acid cis-acting elements were present in the promoter regions of family members, thus the family may be regulated by hormone signaling pathways of maize. In summary, ZmCOMT genes are ancient, and the highly conserved motifs may be significant in survival and evolution of maize. Furthermore, light may influence lignin biosynthesis and photosynthesis through ZmCOMT genes. This research provided theoretical basis for lignin biosynthesis of maize and the potential value of ZmCOMT22 and ZmCOMT10 genes to enhance plant photosynthesis for facing global warming.

Activation of estrogen-related receptor γ by calcium and cadmium.

Estrogen-related receptor γ (ERRγ) is a metabolic regulator with no identified physiological ligands. This study investigates whether calcium is an ERRγ ligand that mediates the effects of glucagon and whether cadmium, which mimics the effects of calcium, disrupts metabolism through ERRγ. HepG2, MCF-7, and HEK293T transfected with ERRγ were treated with glucagon, calcium, cadmium, ERRγ agonist, or ERRγ inhibitor. Cells were then collected for in vitro assays including real-time qPCR, Western blot, ChIP, immunofluorescence, mutational analysis, or gene set enrichment analysis. Molecular dynamics simulations were performed to study mutation sites. In HepG2 cells, treatment with glucagon, calcium, or cadmium re-localized ERRγ to the cell nucleus, recruited ERRγ to estrogen-related response elements, induced the expression of ERRγ-regulated genes, and increased extracellular glucose that was blocked by an ERRγ antagonist. In MCF-7 cells and HEK293T cells transfected with ERRγ, similar treatments induced the expression of metabolic genes. Mutational analysis identified S303, T429, and E452 in the ligand-binding domain as potential interaction sites. Molecular dynamics simulations showed that calcium induced changes in ERRγ similar to ERRγ agonist. The results suggest that calcium is a potential ligand of ERRγ that mediates the effects of glucagon and cadmium disrupts metabolism through ERRγ.

Role of BIX01294 in the intracranial inhibition of H3K9 methylation lessens neuronal loss in vascular dementia model.

Dementia develops as a result of multiple factors, including cerebrovascular disease which is called vascular dementia (VD). Histone-3 lysine-9 dimethylation (H3K9me2) broadly increases during VD and inhibits neuroprotective gene expressions. So, we aimed to determine how H3K9me2 inhibitor (BIX01294) affects neuronal damage in VD. An in vivo model of VD was used followed by BIX01294 treatment. Behavioral tests, hematoxylin, and eosin (H&E), Congo red, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were carried out. Hippocampal phosphorylated cyclic-AMP responsive element binding protein (p-CREB), c-fos, brain-derived neurotrophic factor (BDNF), and H3K9me2, were detected by western blot analysis technique. Neurological deficit and anxiety-related behavior significantly reduced in the treatment group compared to the VD group (p < 0.05). BIX01294 improved spatial and passive avoidance memory (p < 0.01 and p < 0.05, respectively) compared to the VD group. Treatment with BIX01294 restored the level of p-CREB/CREB ratio (p < 0.05), cfos (p < 0.01), BDNF (p < 0.01), and suppressed H3K9me2 (p < 0.001) when compared to the VD group. BIX01294 microinjection reduced the apoptosis level in TUNEL staining (p < 0.05), and raised neural cell count in H&E staining (p < 0.01); amyloid beta accumulation significantly decreased in the treatment group (p < 0.05) compared to the VD group. In conclusion, long-term treatment with a low dose of BIX01294 can prevent the progression of neuronal loss in VD model by raising the expression of neurotrophic factors, and reducing the apoptosis level.

The effects of chronic desipramine treatment on neurotrophin-3 in the brain of mice with selective depletion of CREB and CREM in noradrenergic neurons

The disturbances in neurotrophic support are thought to be one of the main causes of depression, which depend not only on the neurotrophins themselves but also on the molecules regulating their synthesis and effector functions. One such molecule is cAMP responsive element binding protein (CREB), which role in depression and antidepressant drugs mechanism of action has been extensively studied. However, CREB’s effects vary depending on brain structure, necessitating specific transgenic models for studying its function. Moreover, deletion of CREB enhances cAMP response element modulator (CREM) expression, suspected to compensate for CREB in its absence.Previously, mice lacking CREB in noradrenergic neurons and CREM (Creb1DbhCreCrem-/-) showed to be insensitive to acute desipramine, whereas mice lacking only CREB (Creb1DbhCre) showed similar effects as wild type animals (w/t). As neurotrophic changes require chronic antidepressant treatment, in current study mice (w/t, Creb1DbhCre and Creb1DbhCreCrem-/-; both males and females) were given desipramine for 21 days, to assess the effects of the drug on CREB, neurotrophins and their receptors in the hippocampus and prefrontal cortex.Interestingly, desipramine had no effect on CREB in neither of studied groups. However, both male and female mice lacking CREB and CREM displayed alterations in neurotrophin-3 (NTF3) expression or protein levels, modulated by desipramine. These findings suggest NTF3 is connected with inhibited response to acute and probably chronic desipramine administration in Creb1DbhCreCrem–/– mice, although in w/t chronic desipramine had no effect on NTF3. Nevertheless, our findings give insight into the role of non-BDNF neurotrophins in the mechanism of antidepressant drugs.

Puerarin alleviates symptoms of preeclampsia through the repression of trophoblast ferroptosis via the CREB/HO-1 pathway

IntroductionPreeclampsia (PE) is a pregnancy-associated complication characterised by new-onset hypertension and proteinuria. This study explored the therapeutic potential of puerarin (Pue) in PE and investigated the underlying mechanism, with a focus on placental ferroptosis. MethodsUsing an NG-nitro-L-arginine methyl ester (L-NAME)-induced PE mouse model, we assessed the effects of Pue on PE phenotypes and placental ferroptosis. Antioxidative and anti-ferroptotic effects of Pue were studied in three ferroptotic cell models (hypoxia/reperfusion, cobalt chloride, and erastin). The regulation of Pue on cAMP response element binding protein (CREB) and heme oxygenase-1 (HO-1) was evaluated through gain- and loss-of-function assays. Luciferase assays were used to elucidate the effect of Flag-CREB on Hmox1 promoter fragments. CREB/HO-1 modulation by Pue was validated in mouse placentas with PE. ResultsPue significantly alleviated maternal hypertension, proteinuria, fetal growth restriction, and placental damage in PE mice. This was associated with an upregulation of the anti-ferroptosis system (glutathione peroxidase 4 [GPX4], cys2/glutamate antiporter [SLC7A11], and glutathione [GSH]) and repression of reactive oxygen species (ROS) and malondialdehyde (MDA) in trophoblasts. Pue reduced HO-1 and CREB, and HO-1 deficiency upregulated GPX4 and SLC7A11. Manipulation of CREB expression led to changes in HO-1/GPX4; whereas, the regulation reversed by Pue administration. Flag-CREB enhanced luciferase activity on the full length Hmox1 promoter (−2000/+78), which contains three CREB1 binding sites (S1–S3). In contrast, no increase in luciferase activity was observed with promoter fragments (−850/+78) and (−550/+78), which contain only the CREB1 binding sites S2 and S3, respectively. DiscussionPue ameliorated PE-like symptoms in mice by repressing trophoblast ferroptosis via inhibition of CREB signalling and affecting the Homx1 promoter.

Disruption of canonical AHR-mediated induction of hepatocyte PKM2 expression compromises antioxidant defenses and increases TCDD-induced hepatotoxicity

Metabolic reprogramming by the pyruvate kinase M2 isoform is associated with cell proliferation and reactive oxygen species (ROS) defenses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant that induces ROS and hepatotoxicity, dose-dependently induces pyruvate kinase muscle isoform M2 (PKM2) in the liver. To further investigate its role in combating TCDD hepatotoxicity, a PkmΔDRE mouse was constructed lacking the dioxin response element mediating aryl hydrocarbon receptor (AHR) induction. TCDD failed to induce hepatic PKM2 in PkmΔDRE mice and in primary hepatocytes isolated from an AHR knockout model (AHRV375Afl/flAlb-CreERT2), demonstrating induction is AHR dependent. Both wild-type (WT) and PkmΔDRE mice exhibited dose-dependent increases in liver weight after treatment with TCDD every 4 days for 28 days. Glutathione (GSH) levels increased in WT mice while oxidized glutathione (GSSG) levels increased in both models with a 24-fold decrease in the GSH/GSSG ratio in PkmΔDRE mice suggesting lower antioxidant and recycling capacity. Moreover, TCDD-induced fibrosis was more severe in PkmΔDRE mice while PkmΔDRE hepatocytes exhibited greater cytotoxicity following co-treatment with TCDD and hydrogen peroxide. TCDD also induced PKM2 in human HepaRG™ cells with AHR enrichment at a conserved DRE core within the locus. These results suggest AHR-mediated PKM2 induction is a novel antioxidant response to TCDD.

The Role of Retinoic-Acid-Related Orphan Receptor (RORs) in Cellular Homeostasis.

Retinoic-acid-related orphan receptors (RORs) are transcription factors belonging to the nuclear receptor subfamily consisting of RORα, RORβ, and RORγ. By binding to the ROR response elements (ROREs) on target gene promoters, RORs regulate a wide variety of cellular processes, including autophagy, mitophagy, oxidative stress, and inflammation. The regulatory roles of RORs are observed in cardiac cells, hepatocytes, pulmonary epithelial cells, renal cells, immune cells, and cancer cells. A growing body of clinical and experimental evidence suggests that ROR expression levels are markedly reduced under different pathological and stress conditions, suggesting that RORs may play a critical role in the pathogenesis of a variety of disease states, including myocardial infarction, immune disorders, cancer, and metabolic syndrome. Reductions in RORs are also associated with inhibition of autophagy, increased reactive oxygen species (ROS), and increased cell death, underscoring the importance of RORs in the regulation of these processes. Herein, we highlight the relationship between RORs and homeostatic processes that influence cell viability. Understanding how these intricate processes are governed at the cellular level is of high scientific and clinical importance to develop new therapeutic strategies that modulate ROR expression and disease progression.

Hypoxia Reduces Mouse Urine Output via HIF1α-Mediated Upregulation of Renal AQP1

Introduction: Patients with acute mountain sickness (AMS) due to hypoxia at high altitudes often exhibit abnormal water metabolism. Hypoxia-inducible factors (HIFs) are major regulators of adaptive responses to hypoxia. As transcription factors, HIFs are involved in the regulation of erythropoiesis, iron metabolism, angiogenesis, energy metabolism, and cell survival by promoting the transcriptional expression of hundreds of target genes. Roxadustat, a novel drug for the treatment of anemia associated with chronic kidney disease (CKD), acts by inhibiting the degradation of HIFs to increase their protein levels. However, the clinical use of roxadustat is frequently associated with peripheral edema, suggesting the involvement of HIFs in regulating the body’s water balance possibly by modulating water reabsorption in the kidney. Methods: We first evaluated the effect of hypoxia (8% O2) on mouse urine output. We then performed in vitro experiments using hypoxia (1% O2) and roxadustat on mouse primary proximal tubular cells (mPTCs). The quantitative polymerase chain reaction, Western blot, and immunofluorescence were used to assess AQP1 mRNA and protein expression levels. Luciferase, Chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA) were used to investigate the transcriptional regulation of AQP1 by HIF1α. Results: We found that mice exposed to hypoxia (8% O2) had significantly reduced urine volume compared to mice exposed to normoxia (21% O2). Hypoxia significantly elevated AQP1 expression at both mRNA and protein levels. In vitro experiments using mouse primary cultured proximal tubular cells (mPTCs) revealed that both hypoxia and roxadustat increased AQP1 expression. Mechanistically, overexpression of HIF1α, but not HIF2α, markedly increased AQP1 protein expression. Furthermore, the upregulation of AQP1 by hypoxia and roxadustat can be blocked by the HIF1α inhibitor PX-478 in mPTCs. Finally, we found that the AQP1 gene promoter contains a putative hypoxia response element and confirmed that AQP1 is a target gene of HIF1α using Luciferase reporter, ChIP, and EMSA assays. Conclusion: This study demonstrates that hypoxia can reduce the urine volume of mice via upregulating AQP1 expression by HIF1α in the proximal tubular epithelial cells. Our findings also suggest a potential mechanism involved in water metabolism disorders in patients with AMS and in patients with CKD receiving roxadustat treatment.

Morphine-induced hyperalgesia impacts small extracellular vesicle miRNA composition and function.

Morphine and other synthetic opioids are widely prescribed to treat pain. Prolonged morphine exposure can paradoxically enhance pain sensitivity in humans and nociceptive behavior in rodents. To better understand the molecular mechanisms underlying opioid-induced hyperalgesia, we investigated changes in miRNA composition of small extracellular vesicles (sEVs) from the serum of mice after a morphine treatment paradigm that induces hyperalgesia. We observed significant differential expression of 18 miRNAs in sEVs from morphine-treated mice of both sexes compared to controls. Several of these miRNAs were bioinformatically predicted to regulate cyclic AMP response element binding protein (CREB), a well-characterized transcription factor implicated in pain and drug addiction. We confirmed the binding and repression of Creb mRNA by miR-155 and miR-10a. We tested if serum-derived sEVs from morphine-treated mice could elicit nociceptive behavior in naïve recipient mice. Intrathecal injection of 1 μg sEVs did not significantly impact basal mechanical and thermal threshold in naïve recipient mice. However, prophylactic 1 μg sEV administration in recipient mice resulted in faster resolution of complete Freund's adjuvant-induced mechanical and thermal inflammatory hypersensitivity. Other behaviors assayed following administration of these sEVs were not impacted including sEV conditioned place preference and locomotor sensitization. These results indicate that morphine regulation of serum sEV composition can contribute to analgesia and suggest a potential for sEVs to be a non-opioid therapeutic intervention strategy to treat pain.

Novel bioassays based on 3D-printed device for sensing of hypoxia and p53 pathway in 3D cell models.

Cell-based assays are widely exploited for drug screening and biosensing, providing useful information about bioactivity of target analytes and complex biological samples. It is well recognized that 3D cell models are required to achieve highly valuable information, also from the perspective of replacing animal models. However, bioassays relying on 3D cell models are generally highly demanding in terms of facilities, equipment, and skilled personnel requirements. To reduce cost, increase sustainability, and provide a flexible 3D cell-based platform for bioassays, we here report a novel approach based on a 3D-printed microtissue device. To assess the suitability of this strategy for reporter gene technology, we selected to monitor two molecular pathways which were of interest in several applications, hypoxia signaling and the p53 pathway. The investigation of such pathways is highly relevant in fields spanning from drug screening to bioactivity monitoring for industrial by-product valorization. Microtissues of human hepatocarcinoma (HepG2) and human embryonic kidney (Hek293T) cell lines were obtained with a low-cost and sustainable chip platform and bioassays were developed to monitor the hypoxia-inducible factors (HIFs) and the p53 tumor suppressor pathway. HepG2 and Hek293T 3D cell models were genetically engineered to express the Luc2P from Photinus pyralis firefly either under the regulation of p53 or HIF response elements. The bioassays allowed quantitative assessment of hypoxia and tumoral activity with 1,10-phenanthroline for HIF and with doxorubicin for p53 pathway activation, respectively, showing good potential for applications of this sustainable and low-cost 3D-printed microfluidic platform for bioactivity analyses, drug screening, and precision medicine.

Sargassum horneri fucoidan oligosaccharide: Purification, characterization, and antioxidant effects targeting the MAPK and KEAP1-NRF2 signaling pathways

The current research looked into the inhibitory effect of a purified component of Sargassum horneri fucoidan oligosaccharide (F2) with a sulfate content of 16.35 %, which showed substantial protective effects on H2O2-induced oxidative damage in RAW 264.7 cells and zebrafish. The chemical and structural characteristics were examined by GC, FTIR, MS, and NMR spectroscopy, revealing that F2 was composed of not only a mixed oligosaccharide with 1–4 degrees of polymerization but also had a main chain consisting of (1 → 3) linked α-L-Fucp and (1 → 3,4) linked α-L-Fucp, sulfated groups mainly at C2 position. It was found that F2 could activate the MAPK and Keap1-Nrf2 signaling pathways through the dissociation of the Keap1-Nrf2 complex in RAW 264.7 cells. Then Nrf2 entered the cell and bound to the antioxidant response element (ARE), resulting in activating the expression of downstream antioxidant genes. F2 was also found to have a strong protective effect on oxidative stress in zebrafish induced by H2O2, which can reduce the heart rate of zebrafish and prevent the generation of intracellular ROS and cell death. Therefore, S. horneri fucoidan oligosaccharide (F2) could be a potential ingredient for functional foods, cosmetics, and pharmaceuticals because of its antioxidant activity.

Neuropeptide Precursor VGF Promotes Liver Metastatic Colonization of Gαq Mutant Uveal Melanoma by Facilitating Tumor Microenvironment via Paracrine Loops.

Uveal melanoma (UM), the predominant primary ocular malignancy, often progresses to liver metastasis with limited therapeutic options. The interplay of the tumor microenvironment, encompassing secreted soluble factors, plays a crucial role in facilitating liver metastasis. In this study, the role is elucidated of the neural growth factor-inducible gene (VGF), a secreted neuropeptide precursor, in Gαq mutant UM. Employing a multiomics approach, encompassing transcriptomic and secretomic analyses, the intricate involvement of VGF in UM progression is unveiled. VGF is upregulated in Gαq mutant UM cells and associated with poor prognosis of UM patients. Targeting VGF significantly suppressed the growth of UM in vitro and in vivo. Further evidence shows that VGF is regulated by Gαq through MAPK/CREB pathway. Mechanistically, CREB modulates VGF expression by directly binding to consensus DNA response elements in the promoters of the VGF gene. Combined inhibition of Gαq and MEK remarkably reduces tumor burden in the UM xenograft model. Notably, VGF triggers liver metastatic colonization of UM and activates the fibrosis of hepatic stellate cells (HSCs), creating a favorable microenvironment, through an autocrine and paracrine loop. Furthermore, VGF directly binds to TGFBR2 and regulates TGF-β-SMAD signaling pathway, thereby regulating genes associated with endothelial-mesenchymal transition (EMT) to promote metastasis. Taken together, these findings identify VGF as a pivotal driver in the progression and metastasis of Gαq mutant UM and confers a promising therapeutic target and strategy for UM patients.

Hypoxia drives estrogen receptor β-mediated cell growth via transcription activation in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor with a poor prognosis. Hypoxia conditions affect multiple cellular processes promoting the adaptation and progression of cancer cells via the activation of hypoxia-inducible factors (HIF) and subsequent transcription activation of their target genes. Preliminary studies have suggested that estrogen receptor β (ERβ) might play a promoting role in the progression of NSCLC. However, the precise mechanisms, particularly its connection to HIF-1α-mediated modulation under hypoxia, remain unclear. Our findings demonstrated that the overexpression of ERβ, not ERα, increased cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. Tissue microarray staining revealed a strong correlation between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in CoCl2-induced hypoxia, 1% O2 incubation, or HIF-1α overexpressing cells. ChIP identified HIF-1α binding to a hypoxia response element in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the tumor growth, thus emphasizing the promising prospects of targeting HIF-1α and ERβ as a therapeutic approach for the treatment of NSCLC. KEY MESSAGES: ERβ, not ERα, increases cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. A strong correlation exists between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in hypoxic cells via binding to HRE in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the NSCLC tumor growth.

Integrated Analysis of Transcriptome and Metabolome Provides Insights into Flavonoid Biosynthesis of Blueberry Leaves in Response to Drought Stress.

Blueberries (Vaccinium spp.) are extremely sensitive to drought stress. Flavonoids are crucial secondary metabolites that possess the ability to withstand drought stress. Therefore, improving the drought resistance of blueberries by increasing the flavonoid content is crucial for the development of the blueberry industry. To explore the underlying molecular mechanism of blueberry in adaptation to drought stress, we performed an integrated analysis of the metabolome and transcriptome of blueberry leaves under drought stress. We found that the most enriched drought-responsive genes are mainly involved in flavonoid biosynthesis and plant hormone signal transduction pathways based on transcriptome data and the main drought-responsive metabolites come from the flavonoid class based on metabolome data. The UDP-glucose flavonoid 3-O-glucosyl transferase (UFGT), flavonol synthase (FLS), and anthocyanidin reductase (ANR-2) genes may be the key genes for the accumulation of anthocyanins, flavonols, and flavans in response to drought stress in blueberry leaves, respectively. Delphinidin 3-glucoside and delphinidin-3-O-glucoside chloride may be the most important drought-responsive flavonoid metabolites. VcMYB1, VcMYBPA1, MYBPA1.2, and MYBPA2.1 might be responsible for drought-induced flavonoid biosynthesis and VcMYB14, MYB14, MYB102, and MYB108 may be responsible for blueberry leaf drought tolerance. ABA responsive elements binding factor (ABF) genes, MYB genes, bHLH genes, and flavonoid biosynthetic genes might form a regulatory network to regulate drought-induced accumulation of flavonoid metabolites in blueberry leaves. Our study provides a useful reference for breeding drought-resistant blueberry varieties.

Regulation of LAMTOR1 by oxidative stress in retinal pigment epithelium: Implications for age-related macular degeneration pathogenesis

Oxidative stress is a critical pathogenic factor for age-related macular degeneration (AMD). Autophagy serves as a mechanism to counteract oxidative stress. LAMTOR1 regulates mTORC1 activity by recruiting or disassembling it on the lysosome under the addition or deprivation of amino acids. This regulation inhibits or enhances autophagy. Our study investigates whether oxidative stress impacts LAMTOR1, thereby adapting to oxidative conditions. We employed oxidative stressors, menadione (VK3) and 4-hydroxynonenal (4-HNE), and observed a reduction of LAMTOR1 in both human and mouse retinal pigment epithelium (RPE) following short-term (1h) and prolonged exposures (24h). Nrf2 overexpression increased both lamtor1 mRNA and LAMTOR1 protein in the RPE. To determine if Nrf2 regulates lamtor1 transcription, we cloned the deletion mutants of the lamtor1 promoter into a luciferase reporter. Although the promoter contained antioxidant response elements, transcriptional activity depended on the interaction between Nrf2 and the constructs containing the transcriptional start site. Moreover, Nrf2-driven transcription was significantly reduced by an inhibitor of histone acetyltransferase, p300. Correspondingly, Nrf2 overexpression increased levels of acetylated histone 3 and p300. The reduction in LAMTOR1 by 4-HNE was reversed by pepstatin A and NH4Cl which block lysosomal degradation. 4-HNE increased TFEB nuclear translocation which was reversed by LAMTOR1 overexpression. In vivo, LAMTOR1 levels decreased in the photoreceptor and RPE layers of NaIO3-injected mice, compared to PBS-injected controls. In conclusion, oxidative injury reduces LAMTOR1, predominantly through lysosomal degradation although Nrf2-mediated histone acetylation enhances lamtor1 transcription. This study reveals a previously unrecognized regulatory mechanism of lamtor1 by oxidative stress, suggesting a novel role for LAMTOR1 in the pathogenesis of AMD.

Proximal Co-Translation Facilitates Detection of Weak Protein-Protein Interactions

Ubiquitin (Ub) signals are recognized and decoded into cellular responses by Ub-receptors, proteins that tether the Ub-binding domain(s) (UBDs) with response elements. Typically, UBDs bind mono-Ub in highly dynamic and weak affinity manners, presenting challenges in identifying and characterizing their binding interfaces. Here, we report the development of a new approach to facilitate the detection of these weak interactions using split-reporter systems where two interacting proteins are proximally co-translated from a single mRNA. This proximity significantly enhances the readout signals of weak protein–protein interactions (PPIs). We harnessed this system to characterize the ultra-weak UBD and ENTH (Epsin N-terminal Homology) and discovered that the yeast Ent1-ENTH domain contains two Ub-binding patches. One is similar to a previously characterized patch on STAM1(signal-transducing adaptor molecule)-VHS (Vps27, Hrs, and STAM), and the other was predicted by AlphaFold. Using a split-CAT selection system that co-translates Ub and ENTH in combination with mutagenesis, we assessed and confirmed the existence of a novel binding patch around residue F53 on ENTH. Co-translation in the split-CAT system provides an effective tool for studying weak PPIs and offers new insights into Ub-receptor interactions.