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Scientific Reports Jun 2023The defining biology that distinguishes neutrophil extracellular traps (NETs) from other forms of cell death is unresolved, and techniques which unambiguously identify...
The defining biology that distinguishes neutrophil extracellular traps (NETs) from other forms of cell death is unresolved, and techniques which unambiguously identify NETs remain elusive. Raman scattering measurement provides a holistic overview of cell molecular composition based on characteristic bond vibrations in components such as lipids and proteins. We collected Raman spectra from NETs and freeze/thaw necrotic cells using a custom built high-throughput platform which is able to rapidly measure spectra from single cells. Principal component analysis of Raman spectra from NETs clearly distinguished them from necrotic cells despite their similar morphology, demonstrating their fundamental molecular differences. In contrast, classical techniques used for NET analysis, immunofluorescence microscopy, extracellular DNA, and ELISA, could not differentiate these cells. Additionally, machine learning analysis of Raman spectra indicated subtle differences in lipopolysaccharide (LPS)-induced as opposed to phorbol myristate acetate (PMA)-induced NETs, demonstrating the molecular composition of NETs varies depending on the stimulant used. This study demonstrates the benefits of Raman microscopy in discriminating NETs from other types of cell death and by their pathway of induction.
Topics: Humans; Extracellular Traps; Neutrophils; Tetradecanoylphorbol Acetate; Microscopy, Fluorescence; Necrosis
PubMed: 37344494
DOI: 10.1038/s41598-023-36667-3 -
BioRxiv : the Preprint Server For... Aug 2023Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt...
Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here we show that a macropinocytosis activator, the tumor promoter Phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.
PubMed: 37333286
DOI: 10.1101/2023.06.02.543509 -
International Journal of Molecular... May 2023CD248 (endosialin) belongs to a glycoprotein family that also includes thrombomodulin (CD141), CLEC14A, and CD93 (AA4) stem cell markers. We analyzed the regulated...
CD248 (endosialin) belongs to a glycoprotein family that also includes thrombomodulin (CD141), CLEC14A, and CD93 (AA4) stem cell markers. We analyzed the regulated expression of CD248 in vitro using skin (HFFF) and synovial (FLS) mesenchymal stem cell lines, and in fluid and tissue samples of rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Cells were incubated with either rhVEGF, bFGF, TGF-β1, IL1-β, TNF-α, TGFβ1, IFN-γ, or PMA (Phorbol ester). There was no statistically significant change in membrane expression. A soluble (s) form of cleaved CD248 (sCD248) was detected after cell treatment with IL1-β and PMA. Matrix metalloprotease (MMP) MMP-1 and MMP-3 mRNAs were significantly up-regulated by IL1-β and PMA. A broad MMP inhibitor blocked the release of soluble CD248. In RA synovial tissue, we identified CD90 perivascular MSCs double-stained for CD248 and VEGF. High sCD248 levels were detected in synovial fluid from RA. In culture, subpopulations of CD90 CD14 RA MSCs were either identified as CD248 or CD141 cells but CD93. CD248 is abundantly expressed by inflammatory MSCs and shed in an MMP-dependent manner in response to cytokines and pro-angiogenic growth factors. Both membrane-bound and soluble CD248 (acting as a decoy receptor) may contribute to RA pathogenesis.
Topics: Humans; Lectins, C-Type; Arthritis, Rheumatoid; Synovial Membrane; Cytokines; Mesenchymal Stem Cells; Cells, Cultured; Fibroblasts; Antigens, Neoplasm; Antigens, CD; Cell Adhesion Molecules
PubMed: 37298499
DOI: 10.3390/ijms24119546 -
Frontiers in Cellular Neuroscience 2023Dynamic physiological changes in brain extracellular calcium ([Ca]) occur when high levels of neuronal activity lead to substantial Ca entry ion channels reducing local...
INTRODUCTION
Dynamic physiological changes in brain extracellular calcium ([Ca]) occur when high levels of neuronal activity lead to substantial Ca entry ion channels reducing local [Ca]. Perturbations of the extracellular microenvironment that increase [Ca] are commonly used to study how [Ca] regulates neuronal activity. At excitatory synapses, the Ca-sensing receptor (CaSR) and other G-protein coupled receptors link [Ca] and spontaneous glutamate release. Phospholipase C (PLC) is activated by G-proteins and is hypothesized to mediate this process.
METHODS
Patch-clamping cultured neocortical neurons, we tested how spontaneous glutamate release was affected by [Ca] and inhibition of PLC activity. We used hypertonic sucrose (HS) to evaluate the readily releasable pool (RRP) and test if it was affected by inhibition of PLC activity.
RESULTS
Spontaneous glutamate release substantially increased with [Ca], and inhibition of PLC activity, with U73122, abolished this effect. PLC-β1 is an abundant isoform in the neocortex, however, [Ca]-dependent spontaneous release was unchanged in PLC-β1 null mutants (). U73122 completely suppressed this response in neurons, indicating that this residual [Ca]-sensitivity may be mediated by other PLC isoforms. The RRP size was substantially reduced after incubation in U73122, but not U73343. Phorbol esters increased RRP size after PLC inhibition.
DISCUSSION
Together these data point to a strong role for PLC in mediating changes in spontaneous release elicited by [Ca] and other extracellular cues, possibly by modifying the size of the RRP.
PubMed: 37260580
DOI: 10.3389/fncel.2023.1193485 -
Heliyon Jun 2023Anti-inflammatory effects of caffeic acid derivatives have been widely reported. However, the effect of caffeic acid methyl ester (CAME) on the anti-allergic effect in...
Anti-inflammatory effects of caffeic acid derivatives have been widely reported. However, the effect of caffeic acid methyl ester (CAME) on the anti-allergic effect in mast cells has not been elucidated. The present study was aimed to investigate the anti-allergic properties of CAME and its underlying mechanism. Rat basophilic leukemia (RBL-2H3) cells were incubated withphorbol-12-myristate-13-acetate (PMA) and a calcium ionophore, A23187 to induce mast cell activation. Anti-allergic effect of CAME was examined by measuring cytokine, histamine and β-hexosaminidase release. Western blotting was conducted to determine cyclooxygenase-2 (COX-2) expression, Mitogen-activated protein kinases (MAPKs) activation and nuclear factor-κB (NF-κB) translocation. CAME significantly suppressed PMA/A23187-induced TNF-α secretion, and β-hexosaminidase and histamine release in a concentration-dependent manner. Furthermore, CAME significantly attenuated PMA/A23187-induced COX-2 expression and nuclear translocation of NF-κB. CAME significantly suppressed PMA/A23187-induced increased phosphorylation of p38, ERK and JNK RBL-2H3 cells. The results demonstrate that CAME significantly attenuates anti-allergic action by suppressing degranulation of mast cells through the suppression of MAPKs/NF-κB signaling pathway in RBL-2H3 cells.
PubMed: 37255982
DOI: 10.1016/j.heliyon.2023.e16529 -
Cell Reports Jun 2023Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic...
Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic homeostatic potentiation (PHP) of NT release stabilizes transmission over minutes. Despite different timescales of STF and PHP, our analysis of Drosophila neuromuscular junctions reveals functional overlap and shared molecular dependence on the release-site protein Unc13A. Mutating Unc13A's calmodulin binding domain (CaM-domain) increases baseline transmission while blocking STF and PHP. Mathematical modeling suggests that Ca/calmodulin/Unc13A interaction plastically stabilizes vesicle priming at release sites and that CaM-domain mutation causes constitutive stabilization, thereby blocking plasticity. Labeling the functionally essential Unc13A MUN domain reveals higher STED microscopy signals closer to release sites following CaM-domain mutation. Acute phorbol ester treatment similarly enhances NT release and blocks STF/PHP in synapses expressing wild-type Unc13A, while CaM-domain mutation occludes this, indicating common downstream effects. Thus, Unc13A regulatory domains integrate signals across timescales to switch release-site participation for synaptic plasticity.
Topics: Animals; Drosophila Proteins; Calmodulin; Presynaptic Terminals; Drosophila; Synaptic Transmission; Synapses; Neuronal Plasticity
PubMed: 37243591
DOI: 10.1016/j.celrep.2023.112541 -
Biochemistry and Biophysics Reports Jul 2023Many chronic inflammatory diseases, such as autoimmune inflammation, are associated with M1 macrophages, and the key to their treatment is blocking inflammation....
Many chronic inflammatory diseases, such as autoimmune inflammation, are associated with M1 macrophages, and the key to their treatment is blocking inflammation. Oxymatrine (OMT), a traditional Chinese medicine, has a marked anti-inflammatory effect. However, its anti-inflammatory target and mechanism in M1 cells remain unclear, which limits its clinical application. In this study, we investigated the anti-inflammatory effects of oxymatrine (OMT) on the M1 inflammatory response. We also determined the relationship between OMT treatment and the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway with OMT treatment. To this end, we induced the differentiation of human peripheral blood monocytes (THP-1) into M1 cells. THP-1 cells were induced with a phorbol ester (phorbol-12-myristate-13-acetate (PMA)) and differentiated into naïve M0 macrophages. M0 cells were induced into M1 cells using lipopolysaccharide (LPS). The experimental groups were divided into the M0 macrophage group (NC), M1 inflammatory response group (LPS group), and M1 group treated with different concentrations of OMT (LPS + OMT-L, LPS + OMT-M, LPS + OMT-H). The cells in the OMT-treated groups were treated with OMT for 6 h, followed by LPS for 24 h, and the LPS group was treated with LPS only. The resulting supernatants and cells were collected. The secretion levels of NO were detected by the Griess method and the secretion levels of TNF-α and IL-1β in the supernatants were detected by the ELISA method. The secretion levels of these inflammatory factors were reduced in every OMT-treated group compared to the LPS group (P < 0.01), and the most significant reductions were found in the OMT-H group (P < 0.0001). By western blotting, the protein expression levels of TLR4, NF-κB, NLRP3, and Caspase-1 were all found to be downregulated in the cells of OMT-treated groups compared to the LPS group (P < 0.0001). changes in NLRP3 expression were observed using immunofluorescence. The fluorescence intensity of NLRP3 in M1 cells was weaker in all OMT intervention groups than in the LPS group (P < 0.001). In conclusion, OMT has significant anti-inflammatory effects on the M1 inflammatory responses, and the TLR4/NF-κB/NLRP3 pathway was blocked proportional to the concentration of OMT.
PubMed: 37215292
DOI: 10.1016/j.bbrep.2023.101482 -
Biomedicine & Pharmacotherapy =... Jul 2023Research on transient receptor potential vanilloid-4 (TRPV4) can provide a promising potential therapeutic target in the development of novel medicines for lung... (Review)
Review
Research on transient receptor potential vanilloid-4 (TRPV4) can provide a promising potential therapeutic target in the development of novel medicines for lung disorders. TRPV4 expresses in lung tissue and plays an important role in the maintenance of respiratory homeostatic function. TRPV4 is upregulated in life-threatening respiratory diseases like pulmonary hypertension, asthma, cystic fibrosis, and chronic obstructive pulmonary diseases. TRPV4 is linked to several proteins that have physiological functions and are sensitive to a wide variety of stimuli, such as mechanical stimulation, changes in temperature, and hypotonicity, and responds to a variety of proteins and lipid mediators, including anandamide (AA), the arachidonic acid metabolite, 5,6-epoxyeicosatrienoic acid (5,6-EET), a plant dimeric diterpenoid called bisandrographolide A (BAA), and the phorbol ester 4-alpha-phorbol-12,13-didecanoate (4α-PDD). This study focused on relevant research evidence of TRPV4 in lung disorders and its agonist and antagonist effects. TRPV4 can be a possible target of discovered molecules that exerts high therapeutic potential in the treatment of respiratory diseases by inhibiting TRPV4.
Topics: Humans; Transient Receptor Potential Channels; TRPV Cation Channels; Phorbol Esters; Hypertension, Pulmonary
PubMed: 37178575
DOI: 10.1016/j.biopha.2023.114861 -
International Journal of Molecular... Apr 2023Five million non-melanoma skin cancers occur globally each year, and it is one of the most common malignant cancers. The dysregulation of the endocannabinoid system,...
Five million non-melanoma skin cancers occur globally each year, and it is one of the most common malignant cancers. The dysregulation of the endocannabinoid system, particularly cannabinoid receptor 2 (CB2), is implicated in skin cancer development, progression, and metastasis. Comparing wildtype (WT) to systemic CB2 knockout (CB2) mice, we performed a spontaneous cancer study in one-year old mice, and subsequently used the multi-stage chemical carcinogenesis model, wherein cancer is initiated by 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). We found that aging CB2 mice have an increased incidence of spontaneous cancerous and precancerous skin lesions compared to their WT counterparts. In the DMBA/TPA model, CB2 developed more and larger papillomas, had decreased spontaneous regression of papillomas, and displayed an altered systemic immune profile, including upregulated CD4+ T cells and dendritic cells, compared to WT mice. Immune cell infiltration in the tumor microenvironment was generally low for both genotypes, although a trend of higher myeloid-derived suppressor cells was observed in the CB2 mice. CB2 expression in carcinogen-exposed skin was significantly higher compared to naïve skin in WT mice, suggesting a role of CB2 on keratinocytes. Taken together, our data show that endogenous CB2 activation plays an anti-tumorigenic role in non-melanoma skin carcinogenesis, potentially via an immune-mediated response involving the alteration of T cells and myeloid cells coupled with the modulation of keratinocyte activity.
Topics: Animals; Mice; 9,10-Dimethyl-1,2-benzanthracene; Carcinogenesis; Carcinogens; Papilloma; Receptors, Cannabinoid; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tumor Microenvironment
PubMed: 37175480
DOI: 10.3390/ijms24097773 -
Carcinogenesis Aug 2023Non-melanoma skin cancer (NMSC) is the most common cancer in the world. Environmental exposure to carcinogens is one of the major causes of NMSC initiation and...
Non-melanoma skin cancer (NMSC) is the most common cancer in the world. Environmental exposure to carcinogens is one of the major causes of NMSC initiation and progression. In the current study, we utilized a two-stage skin carcinogenesis mouse model generated by sequential exposure to cancer-initiating agent benzo[a]pyrene (BaP) and promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), to study epigenetic, transcriptomic and metabolic changes at different stages during the development of NMSC. BaP/TPA caused significant alterations in DNA methylation and gene expression profiles in skin carcinogenesis, as evidenced by DNA-seq and RNA-seq analysis. Correlation analysis between differentially expressed genes and differentially methylated regions found that the mRNA expression of oncogenes leucine rich repeat LGI family member 2 (Lgi2), kallikrein-related peptidase 13 (Klk13) and SRY-Box transcription factor (Sox5) are correlated with the promoter CpG methylation status, indicating BaP/TPA regulates these oncogenes through regulating their promoter methylation at different stages of NMSC. Pathway analysis identified that the modulation of macrophage-stimulating protein-recepteur d'origine nantais and high-mobility group box 1 signaling pathways, superpathway of melatonin degradation, melatonin degradation 1, sirtuin signaling and actin cytoskeleton signaling pathways are associated with the development of NMSC. The metabolomic study showed BaP/TPA regulated cancer-associated metabolisms like pyrimidine and amino acid metabolisms/metabolites and epigenetic-associated metabolites, such as S-adenosylmethionine, methionine and 5-methylcytosine, indicating a critical role in carcinogen-mediated metabolic reprogramming and its consequences on cancer development. Altogether, this study provides novel insights integrating methylomic, transcriptomic and metabolic-signaling pathways that could benefit future skin cancer treatment and interception studies.
Topics: Mice; Animals; Benzo(a)pyrene; Carcinogens, Environmental; Melatonin; Carcinogenesis; Skin Neoplasms; Tetradecanoylphorbol Acetate; Epigenesis, Genetic
PubMed: 37100755
DOI: 10.1093/carcin/bgad024