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Journal of Experimental & Clinical... Oct 2022Ferroptosis is a novel form of iron-dependent cell death and participates in the malignant progression of glioblastoma (GBM). Although circular RNAs (circRNAs) are found...
BACKGROUND
Ferroptosis is a novel form of iron-dependent cell death and participates in the malignant progression of glioblastoma (GBM). Although circular RNAs (circRNAs) are found to play key roles in ferroptosis via several mechanisms, including regulating iron metabolism, glutathione metabolism, lipid peroxidation and mitochondrial-related proteins, there are many novel circRNAs regulating ferroptosis need to be found, and they may become a new molecular treatment target in GBM.
METHODS
The expression levels of circLRFN5, PRRX2 and GCH1 were detected by qPCR, western blotting, and immunohistochemistry. Lentiviral-based infections were used to overexpress or knockdown these molecules in glioma stem cells (GSCs). The biological functions of these molecules on GSCs were detected by MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay, transwell, neurosphere formation assays, Extreme Limiting Dilution Analysis (ELDA) and xenograft experiments. The content of ferroptosis levels in GSCs was detected by BODIPY 581/591 C11 assay, glutathione (GSH) assay and malondialdehyde (MDA) assay. The regulating mechanisms among these molecules were studied by RNA immunoprecipitation assay, RNA pull-down assay, ubiquitination assay, dual-luciferase reporter assay and chromatin immunoprecipitation assay.
RESULTS
We found a novel circRNA circLRFN5 is downregulated in GBM and associated with GBM patients' poor prognosis. CircLRFN5 overexpression inhibits the cell viabilities, proliferation, neurospheres formation, stemness and tumorigenesis of GSCs via inducing ferroptosis. Mechanistically, circLRFN5 binds to PRRX2 protein and promotes its degradation via a ubiquitin-mediated proteasomal pathway. PRRX2 can transcriptionally upregulate GCH1 expression in GSCs, which is a ferroptosis suppressor via generating the antioxidant tetrahydrobiopterin (BH4).
CONCLUSIONS
Our study found circLRFN5 as a tumor-suppressive circRNA and identified its role in the progression of ferroptosis and GBM. CircLRFN5 can be used as a potential GBM biomarker and become a target for molecular therapies or ferroptosis-dependent therapy in GBM.
Topics: Humans; Antioxidants; Biomarkers; Brain Neoplasms; Deoxyuridine; Ferroptosis; Glioblastoma; Glioma; Glutathione; Homeodomain Proteins; Iron; Malondialdehyde; RNA, Circular; Ubiquitins; GTP Cyclohydrolase
PubMed: 36266731
DOI: 10.1186/s13046-022-02518-8 -
Science (New York, N.Y.) Aug 2016Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult...
Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by 5-ethynyl-2'-deoxyuridine (EdU) to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. We also apply Div-Seq to identify and profile rare newborn neurons in the adult spinal cord, a noncanonical neurogenic region. sNuc-Seq and Div-Seq open the way for unbiased analysis of diverse complex tissues.
Topics: Animals; Cell Division; Cell Nucleus; Deoxyuridine; Hippocampus; Isotope Labeling; Mice; Neurogenesis; Neurons; Sequence Analysis, RNA; Single-Cell Analysis; Spinal Cord; Transcription, Genetic; Transcriptome
PubMed: 27471252
DOI: 10.1126/science.aad7038 -
Chinese Medical Journal Dec 2023Posttraumatic stress disorder (PTSD) and depression are highly comorbid. Psilocybin exerts substantial therapeutic effects on depression by promoting neuroplasticity....
BACKGROUND
Posttraumatic stress disorder (PTSD) and depression are highly comorbid. Psilocybin exerts substantial therapeutic effects on depression by promoting neuroplasticity. Fear extinction is a key process in the mechanism of first-line exposure-based therapies for PTSD. We hypothesized that psilocybin would facilitate fear extinction by promoting hippocampal neuroplasticity.
METHODS
First, we assessed the effects of psilocybin on percentage of freezing time in an auditory cued fear conditioning (FC) and fear extinction paradigm in mice. Psilocybin was administered 30 min before extinction training. Fear extinction testing was performed on the first day; fear extinction retrieval and fear renewal were tested on the sixth and seventh days, respectively. Furthermore, we verified the effect of psilocybin on hippocampal neuroplasticity using Golgi staining for the dendritic complexity and spine density, Western blotting for the protein levels of brain derived neurotrophic factor (BDNF) and mechanistic target of rapamycin (mTOR), and immunofluorescence staining for the numbers of doublecortin (DCX)- and bromodeoxyuridine (BrdU)-positive cells.
RESULTS
A single dose of psilocybin (2.5 mg/kg, i.p.) reduced the increase in the percentage of freezing time induced by FC at 24 h, 6th day and 7th day after administration. In terms of structural neuroplasticity, psilocybin rescued the decrease in hippocampal dendritic complexity and spine density induced by FC; in terms of neuroplasticity related proteins, psilocybin rescued the decrease in the protein levels of hippocampal BDNF and mTOR induced by FC; in terms of neurogenesis, psilocybin rescued the decrease in the numbers of DCX- and BrdU-positive cells in the hippocampal dentate gyrus induced by FC.
CONCLUSIONS
A single dose of psilocybin facilitated rapid and sustained fear extinction; this effect might be partially mediated by the promotion of hippocampal neuroplasticity. This study indicates that psilocybin may be a useful adjunct to exposure-based therapies for PTSD and other mental disorders characterized by failure of fear extinction.
Topics: Humans; Mice; Animals; Psilocybin; Fear; Extinction, Psychological; Brain-Derived Neurotrophic Factor; Bromodeoxyuridine; Hippocampus; Neuronal Plasticity; TOR Serine-Threonine Kinases
PubMed: 37000971
DOI: 10.1097/CM9.0000000000002647 -
JAMA Oncology Jan 2020Unresectable intrahepatic cholangiocarcinoma (IHC) carries a poor prognosis, with a median overall survival (OS) of 11 months. Hepatic arterial infusion (HAI) of...
Assessment of Hepatic Arterial Infusion of Floxuridine in Combination With Systemic Gemcitabine and Oxaliplatin in Patients With Unresectable Intrahepatic Cholangiocarcinoma: A Phase 2 Clinical Trial.
IMPORTANCE
Unresectable intrahepatic cholangiocarcinoma (IHC) carries a poor prognosis, with a median overall survival (OS) of 11 months. Hepatic arterial infusion (HAI) of high-dose chemotherapy may have potential benefit in these patients.
OBJECTIVE
To evaluate clinical outcomes when HAI chemotherapy is combined with systemic chemotherapy in patients with unresectable IHC.
DESIGN, SETTING, AND PARTICIPANTS
A single-institution, phase 2 clinical trial including 38 patients was conducted with HAI floxuridine plus systemic gemcitabine and oxaliplatin in patients with unresectable IHC at Memorial Sloan Kettering Cancer Center between May 20, 2013, and June 27, 2019. A confirmatory phase 1/2 study using the same therapy was conducted during the same time period at Washington University in St Louis. Patients with histologically confirmed, unresectable IHC were eligible. Resectable metastatic disease to regional lymph nodes and prior systemic therapy were permitted. Patients with distant metastatic disease were excluded.
INTERVENTIONS
Hepatic arterial infusion of floxuridine and systemic administration of gemcitabine and oxaliplatin.
MAIN OUTCOMES AND MEASURES
The primary outcome was progression-free survival (PFS) of 80% at 6 months.
RESULTS
For the phase 2 clinical trial at Memorial Sloan Kettering Cancer Center, 42 patients with unresectable IHC were included and, of these, 38 patients were treated (13 [34%] men; median [range] age at diagnosis, 64 [39-81] years). The median follow-up was 30.5 months. Twenty-two patients (58%) achieved a partial radiographic response, and 32 patients (84%) achieved disease control at 6 months. Four patients had sufficient response to undergo resection, and 1 patient had a complete pathologic response. The median PFS was 11.8 months (1-sided 90% CI, 11.1) with a 6-month PFS rate of 84.1% (90% CI, 74.8%-infinity), thereby meeting the primary end point (6-month PFS rate, 80%). The median OS was 25.0 months (95% CI, 20.6-not reached), and the 1-year OS rate was 89.5% (95% CI, 80.2%-99.8%). Patients with resectable regional lymph nodes (18 [47%]) showed no difference in OS compared with patients with node-negative disease (24-month OS: lymph node negative: 60%; 95% CI, 40%-91% vs lymph node positive: 50%; 95% CI, 30%-83%; P = .66). Four patients (11%) had grade 4 toxic effects requiring removal from the study (1 portal hypertension, 2 gastroduodenal artery aneurysms, 1 infection in the pump pocket). Subgroup analysis showed significant improvement in survival in patients with IDH1/2 mutated tumors (2-year OS, 90%; 95% CI, 73%-99%) vs wild-type (2-year OS, 33%; 95% CI, 18%-63%) (P = .01). In the Washington University in St Louis confirmatory cohort, 9 patients (90%) achieved disease control at 6 months; the most common grade 3 toxic effect was elevated results of liver function tests, and median PFS was 12.8 months (1-sided 90% CI, 6.4).
CONCLUSIONS AND RELEVANCE
Hepatic arterial infusion plus systemic chemotherapy appears to be highly active and tolerable in patients with unresectable IHC; further evaluation is warranted.
Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Bile Duct Neoplasms; Bile Ducts, Intrahepatic; Cholangiocarcinoma; Colorectal Neoplasms; Deoxycytidine; Female; Floxuridine; Humans; Liver Neoplasms; Male; Middle Aged; Oxaliplatin; Treatment Outcome; Gemcitabine
PubMed: 31670750
DOI: 10.1001/jamaoncol.2019.3718 -
Antiviral Chemistry & Chemotherapy 2023Brivudin, (()-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) can be considered the gold standard for the treatment of varicella-zoster virus (VZV) infections, such as herpes... (Review)
Review
Brivudin, (()-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) can be considered the gold standard for the treatment of varicella-zoster virus (VZV) infections, such as herpes zoster (shingles). It is available for clinical use in most European countries (except for the UK) and over the whole world (except for the US and Canada). Besides VZV its activity spectrum also includes various other herpesviruses, such as herpes simplex virus type 1 (HSV-1). Its activity against VZV and HSV-1 depends on phosphorylation by the virus-encoded thymidine kinase (TK). In its active form (BVDU TP or BVDU 5'-triphosphate), it can act as both substrate and inhibitor of the viral (i.e., HSV-1) DNA polymerase. It has proven to be effective against herpes zoster, including post-herpetic neuralgia (PHN). It is contra-indicated in patients concomitantly treated by 5-fluorouracil (FU), since its degradation product, ()-5-(2-bromovinyl)uracil, is inhibitory to the catabolism of FU, which may enhance the toxicity of the latter. A new compound, the bicyclic nucleoside analogue (BCNA) Cf-1743, has been described, which is a more potent inhibitor of VZV replication than BVDU and which does not interfere with the catabolism of FU. It is applicable orally, as its 5'-valine ester FV-100 (Fermavir), but has not (yet) been marketed for clinical use.
Topics: Humans; Antiviral Agents; Bromodeoxyuridine; Herpesvirus 3, Human; Herpes Zoster; Herpesvirus 1, Human; Fluorouracil; Thymidine Kinase
PubMed: 36710501
DOI: 10.1177/20402066231152971 -
International Journal of Molecular... Feb 2023Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases;... (Review)
Review
Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases; the length of these particular cell cycle phases is an important characteristic of cell life. The progression of cells through these phases is a highly orchestrated process governed by endogenous and exogenous factors. For the elucidation of the role of these factors, including pathological aspects, various methods have been developed. Among these methods, those focused on the analysis of the duration of distinct cell cycle phases play important role. The main aim of this review is to guide the readers through the basic methods of the determination of cell cycle phases and estimation of their length, with a focus on the effectiveness and reproducibility of the described methods.
Topics: Bromodeoxyuridine; Reproducibility of Results; Cell Cycle; Cell Division; Cell Proliferation
PubMed: 36835083
DOI: 10.3390/ijms24043674 -
Science (New York, N.Y.) Aug 2021Stochastic fluctuations in gene expression ("noise") are often considered detrimental, but fluctuations can also be exploited for benefit (e.g., dither). We show here...
Stochastic fluctuations in gene expression ("noise") are often considered detrimental, but fluctuations can also be exploited for benefit (e.g., dither). We show here that DNA base excision repair amplifies transcriptional noise to facilitate cellular reprogramming. Specifically, the DNA repair protein Apex1, which recognizes both naturally occurring and unnatural base modifications, amplifies expression noise while homeostatically maintaining mean expression levels. This amplified expression noise originates from shorter-duration, higher-intensity transcriptional bursts generated by Apex1-mediated DNA supercoiling. The remodeling of DNA topology first impedes and then accelerates transcription to maintain mean levels. This mechanism, which we refer to as "discordant transcription through repair" ("DiThR," which is pronounced "dither"), potentiates cellular reprogramming and differentiation. Our study reveals a potential functional role for transcriptional fluctuations mediated by DNA base modifications in embryonic development and disease.
Topics: Animals; Cell Differentiation; Cells, Cultured; Cellular Reprogramming; Computer Simulation; DNA; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Embryonic Stem Cells; Gene Expression; Idoxuridine; Mice; Models, Genetic; Nanog Homeobox Protein; Nucleic Acid Conformation; RNA, Messenger; Single-Cell Analysis; Stochastic Processes; Thymidine Kinase; Transcription, Genetic
PubMed: 34301855
DOI: 10.1126/science.abc6506 -
Cells Jul 2021Human adipose tissue-derived stem cells (hADSCs) are highly suitable for regeneration therapies being easily collected and propagated in vitro. The effects of different...
Human adipose tissue-derived stem cells (hADSCs) are highly suitable for regeneration therapies being easily collected and propagated in vitro. The effects of different external factors and culturing conditions are able to affect hADSC proliferation, senescence, differentiation, and migration, even at the molecular level. In the present paper, we exposed hADSCs to an exhausted medium from the breast cancer cell line (MCF-7) to evaluate whether the soluble factors released by these cells may be able to induce changes in stem cell behavior. In particular, we investigated the expression of stemness-related genes (OCT4; Sox 2; Nanog), the cell-cycle regulators p21 (WAF1/CIP1) p53, epigenetic markers (DNMT1 and Sirt1), and autophagy-related proteins. From our results, we can infer that the exhausted medium from MCF-7 is able to influence the hADSCs behavior increasing the expression of stemness-related genes, cell proliferation, and autophagy. Polyamines detectable in MCF-7 exhausted medium could be related to the higher proliferation capability observed in hADSCs, suggesting direct crosstalk between these molecules and the observed changes in stem cell potency.
Topics: Adipose Tissue; Autophagosomes; Autophagy; Bromodeoxyuridine; Cell Proliferation; Cell Shape; Cell Survival; Culture Media; Cyclin-Dependent Kinase Inhibitor p21; Epigenesis, Genetic; Humans; MCF-7 Cells; Middle Aged; Polyamines; Stem Cells; Tumor Suppressor Protein p53; bcl-2-Associated X Protein
PubMed: 34359925
DOI: 10.3390/cells10071754 -
Cells Jun 2022Tagging proliferating cells with thymidine analogs is an indispensable research tool; however, the issue of the potential in vivo cytotoxicity of these compounds remains...
Tagging proliferating cells with thymidine analogs is an indispensable research tool; however, the issue of the potential in vivo cytotoxicity of these compounds remains unresolved. Here, we address these concerns by examining the effects of BrdU and EdU on adult hippocampal neurogenesis and EdU on the perinatal somatic development of mice. We show that, in a wide range of doses, EdU and BrdU label similar numbers of cells in the dentate gyrus shortly after administration. Furthermore, whereas the administration of EdU does not affect the division and survival of neural progenitor within 48 h after injection, it does affect cell survival, as evaluated 6 weeks later. We also show that a single injection of various doses of EdU on the first postnatal day does not lead to noticeable changes in a panel of morphometric criteria within the first week; however, higher doses of EdU adversely affect the subsequent somatic maturation and brain growth of the mouse pups. Our results indicate the potential caveats in labeling the replicating DNA using thymidine analogs and suggest guidelines for applying this approach.
Topics: Animals; Bromodeoxyuridine; Cell Count; Cell Proliferation; Mice; Neurogenesis; Thymidine
PubMed: 35741018
DOI: 10.3390/cells11121888 -
Cardiovascular Research Sep 2022Intimal hyperplasia is a common feature of vascular remodelling disorders. Accumulation of synthetic smooth muscle cell (SMC)-like cells is the main underlying cause....
AIMS
Intimal hyperplasia is a common feature of vascular remodelling disorders. Accumulation of synthetic smooth muscle cell (SMC)-like cells is the main underlying cause. Current therapeutic approaches including drug-eluting stents are not perfect due to the toxicity on endothelial cells and novel therapeutic strategies are needed. Our preliminary screening for dysregulated cyclic nucleotide phosphodiesterases (PDEs) in growing SMCs revealed the alteration of PDE10A expression. Herein, we investigated the function of PDE10A in SMC proliferation and intimal hyperplasia both in vitro and in vivo.
METHODS AND RESULTS
RT-qPCR, immunoblot, and in situ proximity ligation assay were performed to determine PDE10A expression in synthetic SMCs and injured vessels. We found that PDE10A mRNA and/or protein levels are up-regulated in cultured SMCs upon growth stimulation, as well as in intimal cells in injured mouse femoral arteries. To determine the cellular functions of PDE10A, we focused on its role in SMC proliferation. The anti-mitogenic effects of PDE10A on SMCs were evaluated via cell counting, BrdU incorporation, and flow cytometry. We found that PDE10A deficiency or inhibition arrested the SMC cell cycle at G1-phase with a reduction of cyclin D1. The anti-mitotic effect of PDE10A inhibition was dependent on cGMP-dependent protein kinase Iα (PKGIα), involving C-natriuretic peptide (CNP) and particulate guanylate cyclase natriuretic peptide receptor 2 (NPR2). In addition, the effects of genetic depletion and pharmacological inhibition of PDE10A on neointimal formation were examined in a mouse model of femoral artery wire injury. Both PDE10A knockout and inhibition decreased injury-induced intimal thickening in femoral arteries by at least 50%. Moreover, PDE10A inhibition decreased ex vivo remodelling of cultured human saphenous vein segments.
CONCLUSIONS
Our findings indicate that PDE10A contributes to SMC proliferation and intimal hyperplasia at least partially via antagonizing CNP/NPR2/cGMP/PKG1α signalling and suggest that PDE10A may be a novel drug target for treating vascular occlusive disease.
Topics: Animals; Bromodeoxyuridine; Cell Proliferation; Cells, Cultured; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclin D1; Endothelial Cells; Guanylate Cyclase; Humans; Hyperplasia; Mice; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphoric Diester Hydrolases; RNA, Messenger; Vascular Remodeling; Vascular System Injuries
PubMed: 34550322
DOI: 10.1093/cvr/cvab304