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Scientific Reports Sep 2023Stimulation of glucose uptake in response to ischemic metabolic stress is important for cardiomyocyte function and survival. Chronic exposure of cardiomyocytes to fatty...
Stimulation of glucose uptake in response to ischemic metabolic stress is important for cardiomyocyte function and survival. Chronic exposure of cardiomyocytes to fatty acids (FA) impairs the stimulation of glucose uptake, whereas induction of lipid droplets (LD) is associated with preserved glucose uptake. However, the mechanisms by which LD induction prevents glucose uptake impairment remain elusive. We induced LD with either tetradecanoyl phorbol acetate (TPA) or 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Triacylglycerol biosynthesis enzymes were inhibited in cardiomyocytes exposed to FA ± LD inducers, either upstream (glycerol-3-phosphate acyltransferases; GPAT) or downstream (diacylglycerol acyltransferases; DGAT) of the diacylglycerol step. Although both inhibitions reduced LD formation in cardiomyocytes treated with FA and LD inducers, only DGAT inhibition impaired metabolic stress-stimulated glucose uptake. DGAT inhibition in FA plus TPA-treated cardiomyocytes reduced triacylglycerol but not diacylglycerol content, thus increasing the diacylglycerol/triacylglycerol ratio. In cardiomyocytes exposed to FA alone, GPAT inhibition reduced diacylglycerol but not triacylglycerol, thus decreasing the diacylglycerol/triacylglycerol ratio, prevented PKCδ activation and improved metabolic stress-stimulated glucose uptake. Changes in AMP-activated Protein Kinase activity failed to explain variations in metabolic stress-stimulated glucose uptake. Thus, LD formation regulates metabolic stress-stimulated glucose uptake in a manner best reflected by the diacylglycerol/triacylglycerol ratio.
Topics: Myocytes, Cardiac; Biological Transport; Diacylglycerol O-Acyltransferase; Fatty Acids; Tetradecanoylphorbol Acetate; Glucose
PubMed: 37684349
DOI: 10.1038/s41598-023-42072-7 -
Cells Sep 2023Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the...
Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.
Topics: Neutrophils; Calcimycin; Microscopy, Atomic Force; Extracellular Traps; Cell Nucleus; Tetradecanoylphorbol Acetate
PubMed: 37681931
DOI: 10.3390/cells12172199 -
BMC Complementary Medicine and Therapies Sep 2023Allergy is an inflammatory disorder affecting around 20% of the global population. The adverse effects of current conventional treatments give rise to the increased...
BACKGROUND
Allergy is an inflammatory disorder affecting around 20% of the global population. The adverse effects of current conventional treatments give rise to the increased popularity of using natural food products as complementary and alternative medicine against allergic diseases. Stingless bee honey, commonly known as Kelulut honey (KH) in Malaysia, has been used locally as a traditional remedy to relieve cough and asthma. This study evaluated the anti-allergic potential of KH collected from four different botanical sources on phorbol ester 12-myristate-3-acetate and calcium ionophore-activated human mast cells.
METHODS
The present study examined the inhibitory effects of all collected honey on the release of selected inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-4, IL-6, IL-8, histamine, and β-hexosaminidase in an activated HMC. Besides that, all honey's total phenolic content (TPC) was also examined, followed by using liquid chromatography with tandem mass spectrometry (LC-MS/MS) to identify the phytochemicals in the honey. Further examination of the identified phytochemicals on their potential interaction with selected signaling molecules in an activated mast cell was conducted using computational methods.
RESULTS
The results indicated that there were significant inhibitory effects on all selected inflammatory mediators' release by KH sourced from bamboo (BH) and rubber tree (RH) at 0.5% and 1%, but not KH sourced from mango (AH) and noni (EH). BH and RH were found to have higher TPC values and were rich in their phytochemical profiles based on the LC-MS/MS results. Computational studies were employed to determine the possible molecular target of KH through molecular docking using HADDOCK and PRODIGY web servers.
CONCLUSIONS
In short, the results indicated that KH possesses anti-allergic effects towards an activated HMC, possibly by targeting downstream MAPKs. However, their anti-allergic effects may vary according to their botanical sources. Nevertheless, the present study has provided insight into the potential application of stingless bee honey as a complementary and alternative medicine to treat various allergic diseases.
Topics: Humans; Bees; Animals; Honey; Anti-Allergic Agents; Mast Cells; Cell Degranulation; Chromatography, Liquid; Molecular Docking Simulation; Tandem Mass Spectrometry; Hypersensitivity
PubMed: 37667314
DOI: 10.1186/s12906-023-04129-y -
Biochemistry Sep 2023Munc13-1 is a key protein necessary for vesicle fusion and neurotransmitter release in the brain. Diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma...
Munc13-1 is a key protein necessary for vesicle fusion and neurotransmitter release in the brain. Diacylglycerol (DAG)/phorbol ester binds to its C1 domain in the plasma membrane and activates it. The C1 domain of Munc13-1 and protein kinase C (PKC) are homologous in terms of sequence and structure. In order to identify small-molecule modulators of Munc13-1 targeting the C1 domain, we studied the effect of three DAG-lactones, ()-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (JH-131e-153), ()-(2-(hydroxymethyl)-4-(3-isobutyl-5-methylhexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (AJH-836), and ()-(2-(hydroxymethyl)-4-(4-nitrobenzylidene)-5-oxotetrahydrofuran-2-yl)methyl 4-(dimethylamino)benzoate (130C037), on Munc13-1 activation using the ligand-induced membrane translocation assay. JH-131e-153 showed higher activation than AJH-836, and 130C037 was not able to activate Munc13-1. To understand the role of the ligand-binding site residues in the activation process, three alanine mutants were generated. For AJH-836, the order of activation was wild-type (WT) Munc13-1 > R592A > W588A > I590A. For JH-131e-153, the order of activation was WT > I590 ≈ R592A ≈ W588A. Overall, the isomer of DAG-lactones showed higher potency than the isomer and Trp-588, Ile-590, and Arg-592 were important for its binding. When comparing the activation of Munc13-1 and PKC, the order of activation for JH-131e-153 was PKCα > Munc13-1 > PKCε and for AJH-836, the order of activation was PKCε > PKCα > Munc13-1. Molecular docking supported higher binding of JH-131e-153 than AJH-836 with the Munc13-1 C1 domain. Our results suggest that DAG-lactones have the potential to modulate neuronal processes via Munc13-1 and can be further developed for therapeutic intervention for neurodegenerative diseases.
Topics: Protein Kinase C-alpha; Diglycerides; Ligands; Molecular Docking Simulation; Protein Kinase C; Lactones
PubMed: 37651159
DOI: 10.1021/acs.biochem.3c00375 -
Frontiers in Immunology 2023The infusion of -generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including...
INTRODUCTION
The infusion of -generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including graft-versus-host disease.
METHODS
Previously, we developed a protocol for the generation of a novel population of regulatory B cells, which are characterized by secretion of enzymatically active granzyme B (). This protocol uses recombinant interleukin 21 (IL-21) and goat-derived F(ab)'2 fragments against the human B cell receptor (anti-BCR). Generally, the use of xenogeneic material for the manufacturing of advanced therapy medicinal products should be avoided to prevent adverse immune reactions as well as potential transmission of so far unknown diseases.
RESULTS
In the present work we demonstrated that phorbol-12-myristate-13-acetate (PMA/TPA), a phorbol ester with a particular analogy to the second messenger diacylglycerol (DAG), is a potent enhancer of IL-21-induced differentiation of pre-activated B cells into . The percentage of after stimulation of pre-activated B cells with IL-21 and PMA/TPA was not significantly lower compared to stimulation with IL-21 and anti-BCR.
DISCUSSION
Given that PMA/TPA has already undergone encouraging clinical testing in patients with certain haematological diseases, our results suggest that PMA/TPA may be a safe and feasible alternative for manufacturing of .
Topics: Humans; B-Lymphocytes, Regulatory; Granzymes; Tetradecanoylphorbol Acetate
PubMed: 37588597
DOI: 10.3389/fimmu.2023.1194880 -
Lab on a Chip Sep 2023Neutrophils are the most abundant circulating white blood cells and one of their critical functions to eliminate pathogenic threats includes the release of extracellular...
Neutrophils are the most abundant circulating white blood cells and one of their critical functions to eliminate pathogenic threats includes the release of extracellular DNA, also known as neutrophil extracellular traps (NETs), which is dysregulated in many diseases including cancer, type 2 diabetes mellitus and infectious diseases. Currently, conventional methods to quantify the NET formation (NETosis) rely on fluorescence antibody-based NET labelling or circulating NET-associated protein detection by ELISA, which are expensive, laborious, and time-consuming. In this work, we employed a novel "virtual staining" using deep convolutional neural networks (CNNs) to facilitate label-free quantification of NETs trapped in a micropillar array in a microfluidic device. Virtual staining is constructed to establish relations between morphological features in phase contrast images and fluorescence features in Sytox-green (DNA dye) images. We first investigated the effect of different learning rates on model training and optimized the learning rate to achieve the best model which can provide outputs close to Sytox green staining based on various reconstruction metrics (, structural similarity (SSIM) and pixel-wise error (MAE, MSE)). The virtual staining of different NET concentrations was investigated which showed a linear correlation with fluorescent staining. As a proof of concept for clinical testing, the model was used to characterize purified neutrophils treated with NETosis inducers, including lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore (CaI), and successfully detected different NET profiles for different treatments. Collectively, these results demonstrated the potential of using deep learning for enhanced label-free image analysis of NETs for clinical research, drug discovery and point-of-care testing of diseases.
Topics: Humans; Extracellular Traps; Microfluidics; Diabetes Mellitus, Type 2; Neutrophils; Tetradecanoylphorbol Acetate; DNA
PubMed: 37584074
DOI: 10.1039/d3lc00398a -
Journal of Virology Aug 2023Transcription of the human papillomavirus (HPV) oncogenes, and , is regulated by the long control region (LCR) of the viral genome. Although various transcription...
Transcription of the human papillomavirus (HPV) oncogenes, and , is regulated by the long control region (LCR) of the viral genome. Although various transcription factors have been reported to bind to the LCR, little is known about the transcriptional cofactors that modulate HPV oncogene expression in association with these transcription factors. Here, we performed DNA-pulldown purification of nuclear proteins in cervical cancer cells, followed by proteomic analyses to identify transcriptional cofactors that bind to the HPV16 LCR via the transcription factor TEAD1. We detected the proinflammatory cytokine S100A9 that localized to the nucleus of cervical cancer cells and associated with the LCR via direct interaction with TEAD1. Nuclear S100A9 levels and its association with the LCR were increased in cervical cancer cells by treatment with a proinflammatory phorbol ester. Knockdown of S100A9 decreased HPV oncogene expression and reduced the growth of cervical cancer cells and their susceptibility to cisplatin, whereas forced nuclear expression of S100A9 using nuclear localization signals exerted opposite effects. Thus, we conclude that nuclear S100A9 binds to the HPV LCR via TEAD1 and enhances viral oncogene expression by acting as a transcriptional coactivator. IMPORTANCE Human papillomavirus (HPV) infection is the primary cause of cervical cancer, and the viral oncogenes and play crucial roles in carcinogenesis. Although cervical inflammation contributes to the development of cervical cancer, the molecular mechanisms underlying the role of these inflammatory responses in HPV carcinogenesis are not fully understood. Our study shows that S100A9, a proinflammatory cytokine, is induced in the nucleus of cervical cancer cells by inflammatory stimuli, and it enhances HPV oncogene expression by acting as a transcriptional coactivator of TEAD1. These findings provide new molecular insights into the relationship between inflammation and viral carcinogenesis.
Topics: Female; Humans; Carcinogenesis; Human Papillomavirus Viruses; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Papillomavirus Infections; Proteomics; TEA Domain Transcription Factors; Transcription Factors; Uterine Cervical Neoplasms; Calgranulin B
PubMed: 37578237
DOI: 10.1128/jvi.00815-23 -
The Journal of Investigative Dermatology Jan 2024
Topics: Animals; Mice; Remission, Spontaneous; Carcinogens; 9,10-Dimethyl-1,2-benzanthracene; Skin Diseases; Skin Neoplasms; Tetradecanoylphorbol Acetate; Skin
PubMed: 37543244
DOI: 10.1016/j.jid.2023.07.004 -
Scientific Reports Jul 2023Jatropha curcas is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed,...
Jatropha curcas is an oilseed crop with biorefinery applications. Whilst cake generated following oil extraction offers potential as a protein source for animal feed, inactivation of toxic phorbol esters present in the material is necessary. Pleurotus pulmonarius is a detoxifying agent for jatropha cake with additional potential as animal feed, edible mushroom and for enzyme production. For the characterization of fungal genes involved in phorbol ester degradation, together with other industrial applications, reverse transcription-quantitative PCR (RT-qPCR) is a tool that enables accurate quantification of gene expression. For this, reliable analysis requires reference genes for normalization of mRNA levels validated under conditions employed for target genes. The stability of potential reference genes β-TUB, ACTIN, GAPDH, PHOS, EF1α, TRPHO, LAC, MNP3, MYP and VP were evaluated following growth of P. pulmonarius on toxic, non-toxic jatropha cake and a combined treatment, respectively. NormFinder and geNorm algorithms for expression stability analysis identified PHOS, EF1α and MNP3 as appropriate for normalizing gene expression. Reference gene combinations contrasting in ranking were compared following normalization of relative expression of the CHU_2040 gene, encoding an esterase enzyme potentially involved in phorbol ester degradation. The reference genes for P. pulmonarius will facilitate the elucidation of mechanisms involved in detoxification of phorbol esters as well as analysis of target genes for application in biorefinery models.
Topics: Animals; Reverse Transcription; Pleurotus; Agaricales; Animal Feed; Jatropha
PubMed: 37516784
DOI: 10.1038/s41598-023-39115-4 -
European Journal of Pharmacology Sep 2023Previous studies have demonstrated the role of γ-aminobutyric acid type B (GABA) receptors in skin-related conditions and pain. However, most studies have focused on...
Previous studies have demonstrated the role of γ-aminobutyric acid type B (GABA) receptors in skin-related conditions and pain. However, most studies have focused on the main effects of GABA on the central nervous system. Therefore, this study has aimed to determine the potential topical anti-inflammatory and anti-proliferative effects of baclofen cream in an inflammatory skin disease model. The effects of the baclofen cream were evaluated using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears. Histological and immunohistochemical evaluations were performed using an ear oedema assay. The effect of baclofen on keratinocyte proliferation was assessed in PAM212, the murine keratinocyte cell line. The results demonstrate that a single topical application of 5% baclofen, 7.5% baclofen, and 1% dexamethasone each inhibited acute TPA-induced ear oedema (58.94 ± 6.14%, 47.73 ± 11.26%, and 87.33 ± 4.59%, respectively). These results were confirmed by histological analysis. In the chronic model, baclofen (5%) and dexamethasone (1%) each inhibited ear oedema and the maximum inhibitory effect was reached at the end of the experiment (9 day of TPA application) with a percentage inhibition of 54.60 ± 6.15% for baclofen and 71.68 ± 3.45% for dexamethasone, when compared to the vehicle. These results were confirmed by histological analysis. Baclofen and dexamethasone also reduced proliferating cell nuclear antigen expression by 62.01 ± 6.65% and 70.42 ± 6.11%, respectively. However, baclofen did not inhibit keratinocyte proliferation in PAM212 cells. In conclusion, these results demonstrate that baclofen exhibits notable topical antiproliferative and anti-inflammatory properties and could be a potential therapeutic alternative for treating inflammatory and proliferative skin diseases.
Topics: Animals; Mice; Baclofen; GABA-B Receptor Agonists; Dermatitis; Skin Diseases; Anti-Inflammatory Agents; Inflammation; Dexamethasone; Edema; Tetradecanoylphorbol Acetate
PubMed: 37479017
DOI: 10.1016/j.ejphar.2023.175910