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Chemistry (Weinheim An Der Bergstrasse,... Jun 2024Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of...
Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC click reaction of ABNO-H-PEG4-N3 with 5-ethynyl-dUMP or -dUTP. The modified triphosphate was used as substrate for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The keto-ABNO radical reacted with tryptophan (Trp) and Trp-containing peptides to form a stable 3-fused hexahydropyrrolo-indole conjugates. Similarly modified ABNO-H-linked nucleotides reacted with Trp-containing peptides to form a stable conjugate in the presence but surprisingly even in the absence of NaNO2 (presumably through activation by O2). The reactive ABNO-H-modified DNA probe was used for bioconjugations and crosslinking with Trp-containing peptides or proteins.
PubMed: 38924659
DOI: 10.1002/chem.202402151 -
Biomolecules & Biomedicine Jun 2024Knee osteoarthritis (KOA) is one of the most common degenerative joint diseases in the elderly worldwide. The primary lesion in patients with KOA is the degeneration of...
Knee osteoarthritis (KOA) is one of the most common degenerative joint diseases in the elderly worldwide. The primary lesion in patients with KOA is the degeneration of articular cartilage. This study aimed to observe the biological effects of cyclic negative pressure on C28/I2 chondrocytes and to elucidate the underlying molecular mechanisms. We designed a bi-directional intelligent micro-pressure control device for cyclic negative pressure intervention on C28/I2 chondrocytes. Chondrocyte vitality and proliferation were assessed using Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays. The extracellular matrix was analyzed using real-time fluorescence quantitative polymerase chain reaction (PCR) and western blot, while the molecular mechanism of the chondrocyte response to cyclic negative pressure was explored through mRNA sequencing. Experimental data demonstrated that cyclic negative pressure promoted chondrocyte proliferation and upregulated the expression of chondrocyte-specific protein, namely the collagen type II alpha 1 chain (COL2A1) protein, and the transcription factor SRY-box transcription factor 9 (SOX9). Additionally, RNA sequencing analysis revealed that the gene levels of insulin-like growth factor 2 (IGF-2) and early growth response 1 (EGR-1) were significantly elevated in the cyclic negative pressure group. This study demonstrates that cyclic negative pressure stimulates the proliferation of C28/I2 chondrocytes by promoting the expression of EGR-1 and IGF-2. This new discovery may provide novel insights into cartilage health and KOA prevention.
PubMed: 38912889
DOI: 10.17305/bb.2024.10487 -
Journal of Experimental Orthopaedics Jul 2024To investigate the level of cellular senescence in stem cells derived from microfragmented abdominal adipose tissue harvested from patients with knee osteoarthritis (OA).
Microfragmented abdominal adipose tissue-derived stem cells from knee osteoarthritis patients aged 29-65 years demonstrate in vitro stemness and low levels of cellular senescence.
PURPOSE
To investigate the level of cellular senescence in stem cells derived from microfragmented abdominal adipose tissue harvested from patients with knee osteoarthritis (OA).
METHODS
Stem cells harvested from microfragmented abdominal adipose tissue from 20 patients with knee OA, aged 29-65 years (mean = 49.8, SD = 9.58), were analysed as a function of patient age and compared with control cells exhibiting signs of cellular senescence. Steady-state mRNA levels of a panel of genes associated with senescence were measured by qPCR. Intracellular senescence-associated proteins p16 and p21, and senescence-associated β-galactosidase activity were measured by flow cytometry. Cellular proliferation was assessed using a 5-ethynyl-2'-deoxyuridine proliferation assay. Stemness was assessed by stem cell surface markers using flow cytometry and the capacity to undergo adipogenic and osteogenic differentiation in vitro.
RESULTS
No correlation was found between cellular senescence levels of the microfragmented adipose tissue-derived stem cells and patient age for any of the standard assays used to quantify senescence. The level of cellular senescence was generally low across all senescence-associated assays compared to the positive senescence control. Stemness was verified for all samples. An increased capacity to undergo adipogenic differentiation was shown with increasing patient age ( = 0.02). No effect of patient age was found for osteogenic differentiation.
CONCLUSIONS
Autologous microfragmented adipose tissue-derived stem cells may be used in clinical trials of knee OA of patients aged 29-65 years, at least until passage 4, as they show stemness potential and negligible senescence in vitro.
LEVEL OF EVIDENCE
Not applicable.
PubMed: 38911188
DOI: 10.1002/jeo2.12056 -
Archives of Oral Biology Sep 2024This study was designed to investigate the biological role and the reaction mechanism of Tweety family member 3 (TTYH3) in oral squamous cell carcinoma (OSCC).
OBJECTIVE
This study was designed to investigate the biological role and the reaction mechanism of Tweety family member 3 (TTYH3) in oral squamous cell carcinoma (OSCC).
DESIGN
The mRNA and protein expressions of TTYH3 were assessed with RT-qPCR and western blot. After silencing TTYH3 expression, the proliferation of OSCC cells were detected using cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining and colony formation assay. Cell migration and invasion were detected using wound healing and transwell. Gelatin zymography protease assay was used to detect matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-2 (MMP9) activity and western blot was used to detect the expressions of proteins associated with proliferation and epithelial-mesenchymal transition (EMT). The mRNA expression of TTYH3 in THP-1-derived macrophage was detected using real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). The number of CD86-positive cells and CD206-positive cells was detected using immunofluorescence assay. RT-qPCR was used to detect the expressions of M2 markers arginase 1 (ARG1), chitinase-like 3 (YM1) and mannose receptor C-type 1 (MRC1).
RESULTS
In this study, it was found that TTYH3 expression was upregulated in OSCC cell lines and TTYH3 knockdown could inhibit the proliferation, migration, invasion and EMT process in OSCC via suppressing M2 polarization of tumor-associated macrophages.
CONCLUSIONS
Collectively, TTYH3 facilitated the progression of OSCC through the regulation of tumor-associated macrophages polarization.
Topics: Humans; Mouth Neoplasms; Carcinoma, Squamous Cell; Cell Proliferation; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Tumor-Associated Macrophages; Disease Progression; Matrix Metalloproteinase 9; Matrix Metalloproteinase 2; Blotting, Western; Real-Time Polymerase Chain Reaction
PubMed: 38908074
DOI: 10.1016/j.archoralbio.2024.106028 -
Biomedicine & Pharmacotherapy =... Jun 2024Myocardial fibrosis is a pathological, physiological change that results from alterations, such as inflammation and metabolic dysfunction, after myocardial infarction...
Caffeic acid mitigates myocardial fibrosis and improves heart function in post-myocardial infarction by inhibiting transforming growth factor-β receptor 1 signaling pathways.
Myocardial fibrosis is a pathological, physiological change that results from alterations, such as inflammation and metabolic dysfunction, after myocardial infarction (MI). Excessive fibrosis can cause cardiac dysfunction, ventricular remodeling, and heart failure. Caffeic acid (CA), a natural polyphenolic acid in various foods, has cardioprotective effects. This study aimed to explore whether CA exerts a cardioprotective effect to inhibit myocardial fibrosis post-MI and elucidate the underlying mechanisms. Histological observations indicated that CA ameliorated ventricular remodeling induced by left anterior descending coronary artery ligation in MI mice and partially restored cardiac function. CA selectively targeted transforming growth factor-β receptor 1 (TGFBR1) and inhibited TGFBR1-Smad2/3 signaling, reducing collagen deposition in the infarcted area of MI mice hearts. Furthermore, cell counting (CCK-8) assay, 5-ethynyl-2'-deoxyuridine assay, and western blotting revealed that CA dose-dependently decreased the proliferation, collagen synthesis, and activation of the TGFBR1-Smad2/3 pathway in primary cardiac fibroblasts (CFs) stimulated by TGF-β1 in vitro. Notably, TGFBR1 overexpression in CFs partially counteracted the inhibitory effects of CA. These findings suggest that CA effectively mitigates myocardial fibrosis and enhances cardiac function following MI and that this effect may be associated with the direct targeting of TGFBR1 by CA.
PubMed: 38906025
DOI: 10.1016/j.biopha.2024.117012 -
Biology Direct Jun 2024Long noncoding RNAs (lncRNAs) are implicated in the initiation and progression of diffuse large B-cell lymphoma (DLBCL). Small nucleolar RNA host gene 20 (SNHG20) has...
BACKGROUND
Long noncoding RNAs (lncRNAs) are implicated in the initiation and progression of diffuse large B-cell lymphoma (DLBCL). Small nucleolar RNA host gene 20 (SNHG20) has been recognized as a critical lncRNA in multiple human cancers. However, the role of SNHG20 and its underlying mechanism in DLBCL are still unclear.
METHODS
The expression levels of SNHG20, c-MYC, β-catenin, and ubiquitin-specific peptidase 14 (USP14) were measured by reverse transcription-quantitative polymerase chain reaction (RT‒qPCR) and immunoblotting. Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) incorporation, and flow cytometry assays were used to assess the proliferation and apoptosis of DLBCL cells. The transcriptional regulation of SNHG20 by c-MYC was confirmed by a luciferase reporter assay and RNA immunoprecipitation. The interaction between USP14 and β-catenin was demonstrated using coimmunoprecipitation. A subcutaneous xenograft model was constructed to determine the role of SNHG20 in vivo.
RESULTS
In the present study, we found that SNHG20 expression was upregulated in DLBCL cell lines and tissues compared to their normal counterparts. SNHG20 knockdown prominently reduced the proliferation and induced the apoptosis of U2932 and OCI-LY3 cells. However, SNHG20 overexpression increased the proliferation and apoptosis resistance of DLBCL cells. Mechanistically, the expression of SNHG20 was positively regulated by c-MYC in DLBCL cells. C-MYC directly bound to the promoter of SNHG20 to activate its transcription. SNHG20 was expressed mainly in the cytosol in DLBCL cells. SNHG20 silencing did not impact USP14 expression but markedly decreased the level of β-catenin, the substrate of USP14, in DLBCL cells. USP14 overexpression increased the β-catenin level, and this increase was attenuated by SNHG20 knockdown. Treatment with the proteasome inhibitor MG132 abolished SNHG20 knockdown-induced β-catenin downregulation. Moreover, SNHG20 silencing reduced the half-life but increased the ubiquitination of β-catenin in DLBCL cells. SNHG20 knockdown weakened the interaction between both endogenous and exogenous USP14 and β-catenin. In turn, SNHG20 overexpression increased the c-MYC level, and this increase was attenuated by β-catenin knockdown. Importantly, β-catenin knockdown attenuated the SNHG20-mediated increase in DLBCL cell proliferation in vitro and tumour growth in vivo.
CONCLUSIONS
Taken together, our results suggested that c-MYC-activated SNHG20 accelerated the proliferation and increased the apoptosis resistance of DLBCL cells via USP14-mediated deubiquitination of β-catenin. The c-MYC/SNHG20 positive feedback loop may be a new target for anti-DLBCL treatment.
Topics: RNA, Long Noncoding; Humans; beta Catenin; Cell Proliferation; Lymphoma, Large B-Cell, Diffuse; Proto-Oncogene Proteins c-myc; Cell Line, Tumor; Animals; Mice; Ubiquitination; Ubiquitin Thiolesterase; Gene Expression Regulation, Neoplastic; Apoptosis; Mice, Nude
PubMed: 38886753
DOI: 10.1186/s13062-024-00488-9 -
Nature Methods Jun 2024In a human cell, thousands of replication forks simultaneously coordinate duplication of the entire genome. The rate at which this process occurs might depend on the...
In a human cell, thousands of replication forks simultaneously coordinate duplication of the entire genome. The rate at which this process occurs might depend on the epigenetic state of the genome and vary between, or even within, cell types. To accurately measure DNA replication speeds, we developed single-cell 5-ethynyl-2'-deoxyuridine sequencing to detect nascent replicated DNA. We observed that the DNA replication speed is not constant but increases during S phase of the cell cycle. Using genetic and pharmacological perturbations we were able to alter this acceleration of replication and conclude that DNA damage inflicted by the process of transcription limits the speed of replication during early S phase. In late S phase, during which less-transcribed regions replicate, replication accelerates and approaches its maximum speed.
PubMed: 38886577
DOI: 10.1038/s41592-024-02308-4 -
BioRxiv : the Preprint Server For... Apr 2024DNA damage and cytoplasmic DNA induce type-1 interferon (IFN-1) and potentiate responses to immune checkpoint inhibitors. Our prior work found that inhibitors of the DNA...
UNLABELLED
DNA damage and cytoplasmic DNA induce type-1 interferon (IFN-1) and potentiate responses to immune checkpoint inhibitors. Our prior work found that inhibitors of the DNA damage response kinase ATR (ATRi) induce IFN-1 and deoxyuridine (dU) incorporation by DNA polymerases, akin to antimetabolites. Whether and how dU incorporation is required for ATRi-induced IFN-1 signaling is not known. Here, we show that ATRi-dependent IFN-1 responses require uracil DNA glycosylase (UNG)-initiated base excision repair and STING. Quantitative analyses of nine distinct nucleosides reveals that ATRi induce dU incorporation more rapidly in UNG wild-type than knockout cells, and that induction of IFN-1 is associated with futile cycles of repair. While ATRi induce similar numbers of micronuclei in UNG wild-type and knockout cells, dU containing micronuclei and cytoplasmic DNA are increased in knockout cells. Surprisingly, DNA fragments containing dU block STING-dependent induction of IFN-1, MHC-1, and PD-L1. Furthermore, UNG knockout sensitizes cells to IFN-γ , and potentiates responses to anti-PD-L1 in resistant tumors . These data demonstrate an unexpected and specific role for dU-rich DNA in suppressing STING-dependent IFN-1 responses, and show that UNG-deficient tumors have a heightened response to immune checkpoint inhibitors.
STATEMENT OF SIGNIFICANCE
Antimetabolites disrupt nucleotide pools and increase dU incorporation by DNA polymerases. We show that unrepaired dU potentiates responses to checkpoint inhibitors in mouse models of cancer. Patients with low tumor UNG may respond to antimetabolites combined with checkpoint inhibitors, and patients with high tumor UNG may respond to UNG inhibitors combined with checkpoint inhibitors.
PubMed: 38883769
DOI: 10.1101/2024.04.04.588079 -
Cell Biochemistry and Biophysics Jun 2024Prostate cancer (PCa) remains a leading cause of cancer-related incidence and mortality in men. Disruptions in amino acid (AA) metabolism contribute to the disease...
Prostate cancer (PCa) remains a leading cause of cancer-related incidence and mortality in men. Disruptions in amino acid (AA) metabolism contribute to the disease progression, with brucine, a glycine antagonist, exhibiting antitumor effects. This study explores the antitumor impact of brucine on PCa and investigates its mechanisms in regulating AA metabolic pathways. The study employed the PCa cell line DU-145, characterized by high sarcosine (Sar) levels, for various assays including Cell Counting Kit-8 (CCK8), wound healing, Transwell, 5-Ethynyl-2'-deoxyuridine (EDU), TdT mediated dUTP Nick End Labeling (TUNEL), flow cytometry, Western blot, and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Network pharmacological analysis determined the anticancer mechanisms of brucine. Sar levels in DU-145 cells were significantly higher than in normal prostatic epithelial cells RWPE-1. Treatment with brucine resulted in a marked decrease in cell viability, proliferation, invasion, and migration, while promoting apoptosis in a dose-dependent manner. Sar levels decreased with increasing brucine concentration. Network pharmacology analysis linked brucine's anticancer effect to the AA metabolism and glycine N-methyltransferase (GNMT) pathways. GNMT expression in prostate cancer tissues and The Cancer Genome Atlas database was significantly elevated compared to controls. Treatment with brucine led to downregulation of GNMT expression in DU-145 cells without significant effect on sarcosine dehydrogenase (SARDH). Addition of recombinant GNMT partially reversed the inhibitory effects of brucine on DU-145 cells. Treatment with brucine downregulates GNMT expression in DU-145 cells, reducing Sar accumulation and inhibiting tumor progression. These findings provide new insights into the antitumor mechanisms of brucine in PCa.
PubMed: 38877335
DOI: 10.1007/s12013-024-01348-z -
Autophagy Jul 2024Thoracic aortic dissection (TAD) is a severe disease, characterized by numerous apoptotic vascular smooth muscle cells (VSMCs). EDIL3/Del-1 is a secreted protein...
Thoracic aortic dissection (TAD) is a severe disease, characterized by numerous apoptotic vascular smooth muscle cells (VSMCs). EDIL3/Del-1 is a secreted protein involved in macrophage efferocytosis in acute inflammation. Here, we aimed to investigate whether EDIL3 promoted the internalization and degradation of apoptotic VSMCs during TAD. The levels of EDIL3 were decreased in the serum and aortic tissue from TAD mice. Global knockout () mice and bone marrow chimeric mice exhibited a considerable exacerbation in β-aminopropionitrile monofumarate (BAPN)-induced TAD, accompanied with increased apoptotic VSMCs accumulating in the damaged aortic tissue. Two types of phagocytes, RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were used for in vitro efferocytosis assay. -deficient phagocytes exhibited inefficient internalization and degradation of apoptotic VSMCs. Instead, EDIL3 promoted the internalization phase through interacting with phosphatidylserine (PtdSer) on apoptotic VSMCs and binding to the macrophage ITGAV/α-ITGB3/β integrin. In addition, EDIL3 accelerated the degradation phase through activating LC3-associated phagocytosis (LAP). Mechanically, following the engulfment, EDIL3 enhanced the activity of SMPD1/acid sphingomyelinase in the phagosome through blocking ITGAV-ITGB3 integrin, which facilitates phagosomal reactive oxygen species (ROS) production by NAPDH oxidase CYBB/NOX2. Furthermore, exogenous EDIL3 supplementation alleviated BAPN-induced TAD and promoted apoptotic cell clearance. EDIL3 may be a novel factor for the prevention and treatment of TAD. BAPN: β-aminopropionitrile monofumarate; BMDM: bone marrow-derived macrophage; C12FDG: 5-dodecanoylaminofluorescein-di-β-D-galactopyranoside; CTRL: control; CYBB/NOX2: cytochrome b-245, beta polypeptide; DCFH-DA: 2',7'-dichlorofluorescin diacetate; EDIL3/Del-1: EGF-like repeats and discoidin I-like domains 3; EdU: 5-ethynyl-2'-deoxyuridine; EVG: elastic van Gieson; H&E: hematoxylin and eosin; IL: interleukin; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NAC: N-acetylcysteine; PtdSer: phosphatidylserine; rEDIL3: recombinant EDIL3; ROS: reactive oxygen species; SMPD1: sphingomyelin phosphodiesterase 1; TAD: thoracic aortic dissection; TEM: transmission electron microscopy; VSMC: vascular smooth muscle cell; WT: wild-type.
PubMed: 38873925
DOI: 10.1080/15548627.2024.2367191