Clinical significance of PNO1 as a novel biomarker and therapeutic target of hepatocellular carcinoma.

The RNA-binding protein PNO1 plays an essential role in ribosome biogenesis. Recent studies have shown that it is involved in tumorigenesis; however, its role in hepatocellular carcinoma (HCC) is not well understood. The purpose of this study was to examine whether PNO1 can be used as a biomarker of HCC and also examine the therapeutic potential of PNO1 knockout for the treatment of HCC. PNO1 expression was upregulated in HCC and associated with poor prognosis. PNO1 expression was positively associated with tumour stage, lymph node metastasis and poor survival. PNO1 expression was significantly higher in HCC compared to that in fibrolamellar carcinoma or normal tissues. Furthermore, HCC tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53. PNO1 knockout suppressed cell viability, colony formation and EMT of HCC cells. Since activation of Notch signalling pathway promotes HCC, we measured the effects of PNO1 knockout on the components of Notch pathway and its targets. PNO1 knockout suppressed Notch signalling by modulating the expression of Notch ligands and their receptors, and downstream targets. PNO1 knockout also inhibited genes involved in surface adhesion, cell cycle, inflammation and chemotaxis. PNO1 knockout also inhibited colony and spheroid formation, cell migration and invasion, and markers of stem cells, pluripotency and EMT in CSCs. Overall, our data suggest that PNO1 can be used as a diagnostic and prognostic biomarker of HCC, and knockout of PNO1 by CRISPR/Cas9 can be beneficial for the management of HCC by targeting CSCs.

The people behind the papers - Julie Klepstad and Luciano Marcon.

The patterning of somites is coordinated by presomitic mesoderm cells through synchronised oscillations of Notch signalling, creating sequential waves of gene expression that propagate from the posterior to the anterior end of the tissue. In a new study, Klepstad and Marcon propose a new theoretical framework that recapitulates the dynamics of mouse somitogenesis observed in vivo and in vitro. To learn more about the story behind the paper, we caught up with first author Julie Klepstad and corresponding author Luciano Marcon, Principal Investigator at the Andalusian Center for Developmental Biology.

Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind.

Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.

To correctly give rise to future tissues, cells in an embryo must receive and respond to the right signals, at the right time, in the right way. This involves genes being switched on quickly, with cells often ensuring that a range of molecular actors physically come together at 'transcription hubs' in the nucleus ­ the compartment that houses genetic information. These hubs are thought to foster a microenvironment that facilitates the assembly of the machinery that will activate and copy the required genes into messenger RNA molecules. The resulting 'mRNAs' act as templates for producing the corresponding proteins, allowing cells to adequately respond to signals. For example, the activation at the cell surface of a molecule called Notch triggers a series of events that lead to important developmental genes being transcribed within minutes. This process involves a dedicated group of proteins, known as Notch nuclear complexes, quickly getting together in the nucleus and interacting with the transcriptional machinery. How they do this efficiently at the right gene locations is, however, still poorly understood. In particular, it remained unclear whether Notch nuclear complexes participate in the formation of transcription hubs, as well as how these influence mRNA production and the way cells 'remember' having been exposed to Notch activity. To investigate these questions, DeHaro-Arbona et al. genetically engineered fruit flies so that their Notch nuclear complexes and Notch target genes both carried visible tags that could be tracked in living cells in real time. Microscopy imaging of fly tissues revealed that, due to their characteristics, Notch complexes clustered with the transcription machinery and formed transcription hubs near their target genes. All cells exposed to Notch exhibited these hubs, but only a third produced the mRNAs associated with Notch target genes; adding a second signal (an insect hormone) significantly increased the proportion. This illustrates how 'chance' and collaboration influence the way the organism responds to Notch signalling. Finally, the experiments revealed that the hubs persisted for at least a day after removing the Notch signal. This 'molecular memory' led to cells responding faster when presented with Notch activity again. The work by DeHaro-Arbona sheds light on how individual cells respond to Notch signalling, and the factors that influence the activation of its target genes. This knowledge may prove useful when trying to better understand diseases in which this pathway is implicated, such as cancer.

Jag1/2 maintain esophageal homeostasis and suppress foregut tumorigenesis by restricting the basal progenitor cell pool.

Basal progenitor cells are crucial for maintaining foregut (the esophagus and forestomach) homeostasis. When their function is dysregulated, it can promote inflammation and tumorigenesis. However, the mechanisms underlying these processes remain largely unclear. Here, we employ genetic mouse models to reveal that Jag1/2 regulate esophageal homeostasis and foregut tumorigenesis by modulating the function of basal progenitor cells. Deletion of Jag1/2 in mice disrupts esophageal and forestomach epithelial homeostasis. Mechanistically, Jag1/2 deficiency impairs activation of Notch signaling, leading to reduced squamous epithelial differentiation and expansion of basal progenitor cells. Moreover, Jag1/2 deficiency exacerbates the deoxycholic acid (DCA)-induced squamous epithelial injury and accelerates the initiation of squamous cell carcinoma (SCC) in the forestomach. Importantly, expression levels of JAG1/2 are lower in the early stages of human esophageal squamous cell carcinoma (ESCC) carcinogenesis. Collectively, our study demonstrates that Jag1/2 are important for maintaining esophageal and forestomach homeostasis and the onset of foregut SCC.

A Notch toward Progenitor D(G)FRentiation: Defining a NOTCH-PDGFR axis in brown adipogenesis.

Brown adipocytes are found in several fat depots, however, the origins and contributions of different lineages of adipogenic progenitor cells (APCs) to these depots are unclear. In this issue of Developmental Cell, Shi et al. show that platelet-derived growth factor receptor ß (PDGFRß)-lineage and T-box transcription factor 18 (TBX18)-lineage APCs differentially contribute to brown adipogenesis across these depots.

The First Defined Null Allele of the Notch Regulator, a Suppressor of Deltex: Uncovering Its Novel Roles in Drosophila melanogaster Oogenesis.

Suppressor of deltex (Su(dx)) is a Drosophila melanogaster member of the NEDD4 family of the HECT domain E3 ubiquitin ligases. Su(dx) acts as a regulator of Notch endocytic trafficking, promoting Notch lysosomal degradation and the down-regulation of both ligand-dependent and ligand-independent signalling, the latter involving trafficking through the endocytic pathway and activation of the endo/lysosomal membrane. Mutations of Su(dx) result in developmental phenotypes in the Drosophila wing that reflect increased Notch signalling, leading to gaps in the specification of the wing veins, and Su(dx) functions to provide the developmental robustness of Notch activity to environmental temperature shifts. The full developmental functions of Su(dx) are unclear; however, this is due to a lack of a clearly defined null allele. Here we report the first defined null mutation of Su(dx), generated by P-element excision, which removes the complete open reading frame. We show that the mutation is recessive-viable, with the Notch gain of function phenotypes affecting wing vein and leg development. We further uncover new roles for Su(dx) in Drosophila oogenesis, where it regulates interfollicular stalk formation, egg chamber separation and germline cyst enwrapment by the follicle stem cells. Interestingly, while the null allele exhibited a gain in Notch activity during oogenesis, the previously described Su(dx)SP allele, which carries a seven amino acid in-frame deletion, displayed a Notch loss of function phenotypes and an increase in follicle stem cell turnover. This is despite both alleles displaying similar Notch gain of function in wing development. We attribute this unexpected context-dependent outcome of Su(dx)sp being due to the partial retention of function by the intact C2 and WW domain regions of the protein. Our results extend our understanding of the developmental role of Su(dx) in the tissue renewal and homeostasis of the Drosophila ovary and illustrate the importance of examining an allelic series of mutations to fully understand developmental functions.

Linarin Ameliorates Restenosis After Vascular Injury in Type 2 Diabetes Mellitus via Regulating ADAM10-Mediated Notch Signaling Pathway.

Vascular lesions frequently arise as complication in patients diagnosed with diabetes mellitus (DM). Presently, percutaneous coronary intervention (PCI) and antithrombotic therapy serve as primary treatments. However, in-stent restenosis persists as a challenging clinical issue following PCI, lacking sustained and effective treatment. Linarin (LN) exhibits diverse pharmacological activities and is regarded as a potential drug for treating various diseases, including DM. But its specific role in restenosis after vascular injury in DM patients remains unclear. A rat model of diabetes-related restenosis was established to evaluate the role of LN on neointimal hyperplasia. Vascular smooth muscle cells (VSMCs) stimulated by high glucose (HG, 30 mM) underwent LN treatment. Additionally, an overexpression plasmid of A disintegrin and metalloproteinases (ADAM10) was constructed to transfect VSMCs. We employed CCK-8, Brdu, wound-healing scratch, and transwell migration assays to evaluate the proliferation and migration of VSMCs. Furthermore, western blot and immunofluorescence assays were utilized to investigate the expressions of ADAM10 and the downstream Notch signaling pathway in vivo and in vitro models. LN notably alleviated intimal hyperplasia after vascular injury in DM rats and reduced the protein expression of ADAM10, alongside its downstream Notch1 signaling pathway-related proteins (Notch1, NICD and Hes1) in rat carotid artery tissues. LN effectively suppressed the proliferation and migration of VSMCs induced by HG, downregulating the protein expression of ADAM10, Notch1, NICD and Hes1. Moreover, our findings indicated that ADAM10 overexpression significantly reversed LN's effects on proliferation, migration, and the expression of Notch1 signaling pathway-related proteins in HG-treated VSMCs. LN demonstrates potential therapeutic efficacy in addressing restenosis after diabetic-related vascular injury, with the ADAM10 mediated Notch signaling pathway playing a pivotal role.

The Clock and Wavefront Self-Organizing model recreates the dynamics of mouse somitogenesis in vivo and in vitro.

During mouse development, presomitic mesoderm cells synchronize Wnt and Notch oscillations, creating sequential phase waves that pattern somites. Traditional somitogenesis models attribute phase waves to a global modulation of the oscillation frequency. However, increasing evidence suggests that they could arise in a self-organizing manner. Here, we introduce the Sevilletor, a novel reaction-diffusion system that serves as a framework to compare different somitogenesis patterning hypotheses. Using this framework, we propose the Clock and Wavefront Self-Organizing model that considers an excitable self-organizing region where phase waves form independent of global frequency gradients. The model recapitulates the change in relative phase of Wnt and Notch observed during mouse somitogenesis and provides a theoretical basis for understanding the excitability of mouse presomitic mesoderm cells in vitro.

Notch signaling pathway in cancer: from mechanistic insights to targeted therapies.

Notch signaling, renowned for its role in regulating cell fate, organ development, and tissue homeostasis across metazoans, is highly conserved throughout evolution. The Notch receptor and its ligands are transmembrane proteins containing epidermal growth factor-like repeat sequences, typically necessitating receptor-ligand interaction to initiate classical Notch signaling transduction. Accumulating evidence indicates that the Notch signaling pathway serves as both an oncogenic factor and a tumor suppressor in various cancer types. Dysregulation of this pathway promotes epithelial-mesenchymal transition and angiogenesis in malignancies, closely linked to cancer proliferation, invasion, and metastasis. Furthermore, the Notch signaling pathway contributes to maintaining stem-like properties in cancer cells, thereby enhancing cancer invasiveness. The regulatory role of the Notch signaling pathway in cancer metabolic reprogramming and the tumor microenvironment suggests its pivotal involvement in balancing oncogenic and tumor suppressive effects. Moreover, the Notch signaling pathway is implicated in conferring chemoresistance to tumor cells. Therefore, a comprehensive understanding of these biological processes is crucial for developing innovative therapeutic strategies targeting Notch signaling. This review focuses on the research progress of the Notch signaling pathway in cancers, providing in-depth insights into the potential mechanisms of Notch signaling regulation in the occurrence and progression of cancer. Additionally, the review summarizes pharmaceutical clinical trials targeting Notch signaling for cancer therapy, aiming to offer new insights into therapeutic strategies for human malignancies.

Reactive Oxygen Species-Responsive Delivery of a Notch Inhibitor to Alleviate Nonalcoholic Steatohepatitis by Inhibiting Hepatic de Novo Lipogenesis and Inflammation.

With the increased prevalence of nonalcoholic steatohepatitis (NASH) in the world, effective pharmacotherapy in clinical practice is still lacking. Previous studies have shown that dibenzazepine (DBZ), a Notch inhibitor, could alleviate NASH development in a mouse model. However, low bioavailability, poor water solubility, and extrahepatic side effects restrict its clinical application. To overcome these barriers, we developed a reactive oxygen species (ROS)-sensitive nanoparticle based on the conjugation of bilirubin to poly(ethylene glycol) (PEG) chains, taking into account the overaccumulation of hepatic ROS in the pathologic state of nonalcoholic steatohepatitis (NASH). The PEGylated bilirubin can self-assemble into nanoparticles in an aqueous solution and encapsulate insoluble DBZ into its hydrophobic cavity. DBZ nanoparticles (DBZ Nps) had good stability, rapidly released DBZ in response to H2O2, and effectively scavenged intracellular ROS of hepatocytes. After systemic administration, DBZ Nps could accumulate in the liver of the NASH mice, extend persistence in circulation, and improve the bioavailability of DBZ. Furthermore, DBZ Nps significantly improved glucose intolerance, relieved hepatic lipid accumulation and inflammation, and ameliorated NASH-induced liver fibrosis. Additionally, DBZ Nps had no significant extrahepatic side effects. Taken together, our results highlight the potential of the ROS-sensitive DBZ nanoparticle as a promising therapeutic strategy for NASH.

Reduction of specific enterocytes from loss of intestinal LGR4 improves lipid metabolism in mice.

Whether intestinal Leucine-rich repeat containing G-protein-coupled receptor 4 (LGR4) impacts nutrition absorption and energy homeostasis remains unknown. Here, we report that deficiency of Lgr4 (Lgr4iKO) in intestinal epithelium decreased the proportion of enterocytes selective for long-chain fatty acid absorption, leading to reduction in lipid absorption and subsequent improvement in lipid and glucose metabolism. Single-cell RNA sequencing demonstrates the heterogeneity of absorptive enterocytes, with a decrease in enterocytes selective for long-chain fatty acid-absorption and an increase in enterocytes selective for carbohydrate absorption in Lgr4iKO mice. Activation of Notch signaling and concurrent inhibition of Wnt signaling are observed in the transgenes. Associated with these alterations is the substantial reduction in lipid absorption. Decrement in lipid absorption renders Lgr4iKO mice resistant to high fat diet-induced obesity relevant to wild type littermates. Our study thus suggests that targeting intestinal LGR4 is a potential strategy for the intervention of obesity and liver steatosis.

Polystyrene nanoplastics exert cardiotoxicity through the Notch and Wnt pathways in zebrafish (Danio rerio).

The ubiquity of micro(nano)plastics has raised significant concerns among people. Their accumulation in the cardiovascular system necessitates attention to their cardiotoxicity. However, research on the cardiotoxicity of micro(nano)plastics remains scarce. Our study exposed zebrafish embryos to four different concentrations (0, 1, 10, 100 µg/mL) of polystyrene nanoplastics (PSNPs) for a period of 7 days. The results indicated that PSNPs noticeably decreased the hatching and survival rates of zebrafish and also induced cardiac developmental abnormalities. The mRNA level analysis revealed significant upregulations of heart development-related genes nkx2.5, cmlc-2, and myh-7 in response to PSNPs. Additionally, PSNPs significantly up-regulated the mRNA level associated with the Notch signaling pathway (notch-1a, jag-1a, and her-7) while remarkably suppressing the expression of the Wnt signaling pathway gene (wnt-3a). Further research showed that PSNPs significantly increased the expression of endoplasmic reticulum stress genes atf-6 and chop, while noticeably inhibiting mitochondrial copy numbers. Moreover, PSNPs were found to decrease calcium ion level and superoxide dismutase (SOD) activity in zebrafish larvae. Additionally, prolonged exposure to PSNPs for 7 days exacerbated abnormalities in various indicators compared to a 4-day exposure. In conclusion, our study demonstrates that PSNPs induce oxidative stress in zebrafish larvae, thereby activating endoplasmic reticulum stress and inhibiting mitochondrial activity, ultimately disrupting the Notch and Wnt signaling pathways. These disruptions result in abnormalities in cardiac developmental genes, ultimately leading to cardiac developmental abnormalities in zebrafish. The present research contributes to a novel understanding of the cardiotoxicity of PSNPs.

Triptolide, a Cancer Cell Proliferation Inhibitor, Causes Zebrafish Muscle Defects by Regulating Notch and STAT3 Signaling Pathways.

Triptolide is a natural compound in herbal remedies with anti-inflammatory and anti-proliferative properties. We studied its effects on critical signaling processes within the cell, including Notch1 and STAT3 signaling. Our research showed that triptolide reduces cancer cell proliferation by decreasing the expression of downstream targets of these signals. The levels of each signal-related protein and mRNA were analyzed using Western blot and qPCR methods. Interestingly, inhibiting one signal with a single inhibitor alone did not significantly reduce cancer cell proliferation. Instead, MTT assays showed that the simultaneous inhibition of Notch1 and STAT3 signaling reduced cell proliferation. The effect of triptolide was similar to a combination treatment with inhibitors for both signals. When we conducted a study on the impact of triptolide on zebrafish larvae, we found that it inhibited muscle development and interfered with muscle cell proliferation, as evidenced by differences in the staining of myosin heavy chain and F-actin proteins in confocal fluorescence microscopy. Additionally, we noticed that inhibiting a single type of signaling did not lead to any significant muscle defects. This implies that triptolide obstructs multiple signals simultaneously, including Notch1 and STAT3, during muscle development. Chemotherapy is commonly used to treat cancer, but it may cause muscle loss due to drug-related adverse reactions or other complex mechanisms. Our study suggests that anticancer agents like triptolide, inhibiting essential signaling pathways including Notch1 and STAT3 signaling, may cause muscle atrophy through anti-proliferative activity.

Notch Signaling: An Emerging Paradigm in the Pathogenesis of Reproductive Disorders and Diverse Pathological Conditions.

The highly conserved Notch pathway, a pillar of juxtacrine signaling, orchestrates intricate intercellular communication, governing diverse developmental and homeostatic processes through a tightly regulated cascade of proteolytic cleavages. This pathway, culminating in the migration of the Notch intracellular domain (NICD) to the nucleus and the subsequent activation of downstream target genes, exerts a profound influence on a plethora of molecular processes, including cell cycle progression, lineage specification, cell-cell adhesion, and fate determination. Accumulating evidence underscores the pivotal role of Notch dysregulation, encompassing both gain and loss-of-function mutations, in the pathogenesis of numerous human diseases. This review delves deep into the multifaceted roles of Notch signaling in cellular dynamics, encompassing proliferation, differentiation, polarity maintenance, epithelial-mesenchymal transition (EMT), tissue regeneration/remodeling, and its intricate interplay with other signaling pathways. We then focus on the emerging landscape of Notch aberrations in gynecological pathologies predisposing individuals to infertility. By highlighting the exquisite conservation of Notch signaling in Drosophila and its power as a model organism, we pave the way for further dissection of disease mechanisms and potential therapeutic interventions through targeted modulation of this master regulatory pathway.

SNP and Structural Study of the Notch Superfamily Provides Insights and Novel Pharmacological Targets against the CADASIL Syndrome and Neurodegenerative Diseases.

The evolutionary conserved Notch signaling pathway functions as a mediator of direct cell-cell communication between neighboring cells during development. Notch plays a crucial role in various fundamental biological processes in a wide range of tissues. Accordingly, the aberrant signaling of this pathway underlies multiple genetic pathologies such as developmental syndromes, congenital disorders, neurodegenerative diseases, and cancer. Over the last two decades, significant data have shown that the Notch signaling pathway displays a significant function in the mature brains of vertebrates and invertebrates beyond neuronal development and specification during embryonic development. Neuronal connection, synaptic plasticity, learning, and memory appear to be regulated by this pathway. Specific mutations in human Notch family proteins have been linked to several neurodegenerative diseases including Alzheimer's disease, CADASIL, and ischemic injury. Neurodegenerative diseases are incurable disorders of the central nervous system that cause the progressive degeneration and/or death of brain nerve cells, affecting both mental function and movement (ataxia). There is currently a lot of study being conducted to better understand the molecular mechanisms by which Notch plays an essential role in the mature brain. In this study, an in silico analysis of polymorphisms and mutations in human Notch family members that lead to neurodegenerative diseases was performed in order to investigate the correlations among Notch family proteins and neurodegenerative diseases. Particular emphasis was placed on the study of mutations in the Notch3 protein and the structure analysis of the mutant Notch3 protein that leads to the manifestation of the CADASIL syndrome in order to spot possible conserved mutations and interpret the effect of these mutations in the Notch3 protein structure. Conserved mutations of cysteine residues may be candidate pharmacological targets for the potential therapy of CADASIL syndrome.

Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development.

Hairless (H) encodes the major antagonist in the Notch signaling pathway, which governs cellular differentiation of various tissues in Drosophila. By binding to the Notch signal transducer Suppressor of Hairless (Su(H)), H assembles repressor complexes onto Notch target genes. Using genome engineering, three new H alleles, HFA, HLLAA and HWA were generated and a phenotypic series was established by several parameters, reflecting the residual H-Su(H) binding capacity. Occasionally, homozygous HWA flies develop to adulthood. They were compared with the likewise semi-viable HNN allele affecting H-Su(H) nuclear entry. The H homozygotes were short-lived, sterile and flightless, yet showed largely normal expression of several mitochondrial genes. Typical for H mutants, both HWA and HNN homozygous alleles displayed strong defects in wing venation and mechano-sensory bristle development. Strikingly, however, HWA displayed only a loss of bristles, whereas bristle organs of HNN flies showed a complete shaft-to-socket transformation. Apparently, the impact of HWA is restricted to lateral inhibition, whereas that of HNN also affects the respective cell type specification. Notably, reduction in Su(H) gene dosage only suppressed the HNN bristle phenotype, but amplified that of HWA. We interpret these differences as to the role of H regarding Su(H) stability and availability.

Increased H19/miR-675 Expression in Adult T-Cell Leukemia Is Associated with a Unique Notch Signature Pathway.

The Notch pathway is a key cancer driver and is important in tumor progression. Early research suggested that Notch activity was highly dependent on the expression of the intracellular cleaved domain of Notch-1 (NICD). However, recent insights into Notch signaling reveal the presence of Notch pathway signatures, which may vary depending on different cancer types and tumor microenvironments. Herein, we perform a comprehensive investigation of the Notch signaling pathway in adult T-cell leukemia (ATL) primary patient samples. Using gene arrays, we demonstrate that the Notch pathway is constitutively activated in ATL patient samples. Furthermore, the activation of Notch in ATL cells remains elevated irrespective of the presence of activating mutations in Notch itself or its repressor, FBXW7, and that ATL cells are dependent upon Notch-1 expression for proliferation and survival. We demonstrate that ATL cells exhibit the expression of pivotal Notch-related genes, including notch-1, hes1, c-myc, H19, and hes4, thereby defining a critical Notch signature associated with ATL disease. Finally, we demonstrate that lncRNA H19 is highly expressed in ATL patient samples and ATL cells and contributes to Notch signaling activation. Collectively, our results shed further light on the Notch pathway in ATL leukemia and reveal new therapeutic approaches to inhibit Notch activation in ATL cells.

Novel insight into FCSK-congenital disorder of glycosylation through a CRISPR-generated cell model.

BACKGROUND: FCSK-congenital disorder of glycosylation (FCSK-CDG) is a recently discovered rare autosomal recessive genetic disorder with defective fucosylation due to mutations in the fucokinase encoding gene, FCSK. Despite the essential role of fucokinase in the fucose salvage pathway and severe multisystem manifestations of FCSK-CDG patients, it is not elucidated which cells or which types of fucosylation are affected by its deficiency. METHODS: In this study, CRISPR/Cas9 was employed to construct an FCSK-CDG cell model and explore the molecular mechanisms of the disease by lectin flow cytometry and real-time PCR analyses. RESULTS: Comparison of cellular fucosylation by lectin flow cytometry in the created CRISPR/Cas9 FCSK knockout and the same unedited cell lines showed no significant change in the amount of cell surface fucosylated glycans, which is consistent with the only documented previous study on different cell types. It suggests a probable effect of this disease on secretory glycoproteins. Investigating O-fucosylation by analysis of the NOTCH3 gene expression as a potential target revealed a significant decrease in the FCSK knockout cells compared with the same unedited ones, proving the effect of fucokinase deficiency on EGF-like repeats O-fucosylation. CONCLUSION: This study expands insight into the FCSK-CDG molecular mechanism; to the best of our knowledge, it is the first research conducted to reveal a gene whose expression level alters due to this disease.

Endothelial HIF2α suppresses retinal angiogenesis in neonatal mice by upregulating NOTCH signaling.

Prolyl hydroxylase domain (PHD) proteins are oxygen sensors that use intracellular oxygen as a substrate to hydroxylate hypoxia-inducible factor (HIF) α proteins, routing them for polyubiquitylation and proteasomal degradation. Typically, HIFα accumulation in hypoxic or PHD-deficient tissues leads to upregulated angiogenesis. Here, we report unexpected retinal phenotypes associated with endothelial cell (EC)-specific gene targeting of Phd2 (Egln1) and Hif2alpha (Epas1). EC-specific Phd2 disruption suppressed retinal angiogenesis, despite HIFα accumulation and VEGFA upregulation. Suppressed retinal angiogenesis was observed both in development and in the oxygen-induced retinopathy (OIR) model. On the other hand, EC-specific deletion of Hif1alpha (Hif1a), Hif2alpha, or both did not affect retinal vascular morphogenesis. Strikingly, retinal angiogenesis appeared normal in mice double-deficient for endothelial PHD2 and HIF2α. In PHD2-deficient retinal vasculature, delta-like 4 (DLL4, a NOTCH ligand) and HEY2 (a NOTCH target) were upregulated by HIF2α-dependent mechanisms. Inhibition of NOTCH signaling by a chemical inhibitor or DLL4 antibody partially rescued retinal angiogenesis. Taken together, our data demonstrate that HIF2α accumulation in retinal ECs inhibits rather than stimulates retinal angiogenesis, in part by upregulating DLL4 expression and NOTCH signaling.

Prognostic value of CD8+T cells related genes and exhaustion regulation of Notch signaling pathway in hepatocellular carcinoma.

Immunotherapy has emerged as the primary treatment modality for patients with advanced Hepatocellular carcinoma (HCC). However, its clinical efficacy remains limited, benefiting only a subset of patients, while most exhibit immune tolerance and face a grim prognosis. The infiltration of immune cells plays a pivotal role in tumor initiation and progression. In this study, we conducted an analysis of immune cell infiltration patterns in HCC patients and observed a substantial proportion of CD8+T cells. Leveraging the weighted gene co-expression network analysis (WGCNA), we identified 235 genes associated with CD8+T cell and constructed a risk prediction model. In this model, HCC patients were stratified into a high-risk and low-risk group. Patients in the high-risk group exhibited a lower survival rate, predominantly presented with intermediate to advanced stages of cancer, displayed compromised immune function, showed limited responsiveness to immunotherapy, and demonstrated elevated expression levels of the Notch signaling pathway. Further examination of clinical samples demonstrated an upregulation of the Notch1+CD8+T cell exhaustion phenotype accompanied by impaired cytotoxicity and cytokine secretion functions that worsened with increasing Notch activation levels. Our study not only presents a prognostic model but also highlights the crucial involvement of the Notch pathway in CD8+T cell exhaustion-a potential target for future immunotherapeutic interventions.