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Methods in Molecular Biology (Clifton,... 2023Primary cultures of bovine chromaffin cells are considered a good model to evaluate potential neuroprotective compounds for two major reasons: (i) they share many common...
Primary cultures of bovine chromaffin cells are considered a good model to evaluate potential neuroprotective compounds for two major reasons: (i) they share many common features to neurons as they synthesize, store, and release neurotransmitters; they are excitable cells that express voltage-dependent calcium, potassium, and sodium channels; they express different neuronal receptor subtypes; and (ii) they can be easily cultured in high quantities from adult animals; as adult para-neurons, they can be used to reproduce different neurodegenerative-like cytotoxicity models. In this chapter, we describe protocols to mimic calcium overload (veratridine and thapsigargin) and oxidative stress (rotenone plus oligomycin-A and 6-hydroxydopamine) to evaluate potential neuroprotective compounds.
Topics: Animals; Calcium; Cattle; Cells, Cultured; Chromaffin Cells; Neuroprotective Agents; Neurotransmitter Agents; Oligomycins; Oxidopamine; Potassium; Rotenone; Sodium Channels; Thapsigargin; Veratridine
PubMed: 36205906
DOI: 10.1007/978-1-0716-2671-9_24 -
International Journal of Experimental... Dec 2023This study aimed to investigate the effects of mitochondrial homeostasis on lipopolysaccharide (LPS)-induced endothelial cell barrier function and the mechanisms that...
This study aimed to investigate the effects of mitochondrial homeostasis on lipopolysaccharide (LPS)-induced endothelial cell barrier function and the mechanisms that underlie these effects. Cells were treated with LPS or oligomycin (mitochondrial adenosine triphosphate synthase inhibitor) and the mitochondrial morphology, mitochondrial reactive oxygen species (mtROS), and mitochondrial membrane potential (ΔΨm) were evaluated. Moreover, the shedding of glycocalyx-heparan sulphate (HS), the levels of HS-specific degrading enzyme heparanase (HPA), and the expression of occludin and zonula occludens (ZO-1) of Tight Junctions (TJ)s, which are mediated by myosin light chain phosphorylation (p-MLC), were assessed. Examining the changes in mitochondrial homeostasis showed that adding heparinase III, which is an exogenous HPA, can destroy the integrity of glycocalyx. LPS simultaneously increased mitochondrial swelling, mtROS, and ΔΨm. Without oligomycin effects, HS, HPA levels, and p-MLC were found to be elevated, and the destruction of occludin and ZO-1 increased. Heparinase III not only damaged the glycocalyx by increasing HS shedding but also increased mitochondrial swelling and mtROS and decreased ΔΨm. Mitochondrial homeostasis is involved in LPS-induced endothelial cell barrier dysfunction by aggravating HPA and p-MLC levels. In turn, the integrated glycocalyx protects mitochondrial homeostasis.
Topics: Lipopolysaccharides; Occludin; Endothelial Cells; Tight Junctions; Oligomycins
PubMed: 37828780
DOI: 10.1111/iep.12495 -
Scientific Reports Jul 2020Mesenchymal stem cell (MSC)-based therapy is being increasingly considered a powerful opportunity for several disorders based on MSC immunoregulatory properties....
Mesenchymal stem cell (MSC)-based therapy is being increasingly considered a powerful opportunity for several disorders based on MSC immunoregulatory properties. Nonetheless, MSC are versatile and plastic cells that require an efficient control of their features and functions for their optimal use in clinic. Recently, we have shown that PPARβ/δ is pivotal for MSC immunoregulatory and therapeutic functions. However, the role of PPARβ/δ on MSC metabolic activity and the relevance of PPARβ/δ metabolic control on MSC immunosuppressive properties have never been addressed. Here, we demonstrate that PPARβ/δ deficiency forces MSC metabolic adaptation increasing their glycolytic activity required for their immunoregulatory functions on Th1 and Th17 cells. Additionally, we show that the inhibition of the mitochondrial production of ATP in MSC expressing PPARβ/δ, promotes their metabolic switch towards aerobic glycolysis to stably enhance their immunosuppressive capacities significantly. Altogether, these data demonstrate that PPARβ/δ governs the immunoregulatory potential of MSC by dictating their metabolic reprogramming and pave the way for enhancing MSC immunoregulatory properties and counteracting their versatility.
Topics: Animals; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Cell Proliferation; Gene Silencing; Glycolysis; Immunosuppression Therapy; Mesenchymal Stem Cells; Mice; Oligomycins; PPAR-beta; Receptors, Cytoplasmic and Nuclear; Th1 Cells; Th17 Cells
PubMed: 32651456
DOI: 10.1038/s41598-020-68347-x -
MSphere Aug 2023is a prominent opportunistic fungal pathogen of humans. The increasing incidence of infections is attributed to both innate and acquired resistance to antifungals....
is a prominent opportunistic fungal pathogen of humans. The increasing incidence of infections is attributed to both innate and acquired resistance to antifungals. Previous studies suggest the transcription factor Pdr1 and several target genes encoding ABC transporters are critical elements of pleiotropic defense against azoles and other antifungals. This study utilizes transposon insertion profiling to investigate Pdr1-independent and Pdr1-dependent mechanisms that alter susceptibility to the frontline antifungal fluconazole. Several new genes were found to alter fluconazole susceptibility independent of Pdr1 (, , , , ). A bZIP transcription repressor of mitochondrial function () positively regulated Pdr1 while hundreds of genes encoding mitochondrial proteins were confirmed as negative regulators of Pdr1. The antibiotic oligomycin activated Pdr1 and antagonized fluconazole efficacy likely by interfering with mitochondrial processes in . Unexpectedly, disruption of many 60S ribosomal proteins also activated Pdr1, thus mimicking the effects of the mRNA translation inhibitors. Cycloheximide failed to fully activate Pdr1 in a cycloheximide-resistant Rpl28-Q38E mutant. Similarly, fluconazole failed to fully activate Pdr1 in a strain expressing a low-affinity variant of Erg11. Fluconazole activated Pdr1 with very slow kinetics that correlated with the delayed onset of cellular stress. These findings are inconsistent with the idea that Pdr1 directly senses xenobiotics and support an alternative hypothesis where Pdr1 senses cellular stresses that arise only after engagement of xenobiotics with their targets. IMPORTANCE is an opportunistic pathogenic yeast that causes discomfort and death. Its incidence has been increasing because of natural defenses to our common antifungal medications. This study explores the entire genome for impacts on resistance to fluconazole. We find several new and unexpected genes can impact susceptibility to fluconazole. Several antibiotics can also alter the efficacy of fluconazole. Most importantly, we find that Pdr1-a key determinant of fluconazole resistance-is not regulated directly through binding of fluconazole and instead is regulated indirectly by sensing the cellular stresses caused by fluconazole blockage of sterol biosynthesis. This new understanding of drug resistance mechanisms could improve the outcomes of current antifungals and accelerate the development of novel therapeutics.
Topics: Humans; Antifungal Agents; Candida glabrata; Cycloheximide; Drug Resistance, Fungal; Fluconazole; Fungal Proteins; Transcription Factors; Xenobiotics
PubMed: 37358297
DOI: 10.1128/msphere.00254-23 -
Experimental & Molecular Medicine Nov 2022Transarterial chemoembolization (TACE) is the first-line treatment for unresectable intermediate-stage hepatocellular carcinoma (HCC). It is of high clinical...
Transarterial chemoembolization (TACE) is the first-line treatment for unresectable intermediate-stage hepatocellular carcinoma (HCC). It is of high clinical significance to explore the synergistic effect of TACE with antiangiogenic inhibitors and the molecular mechanisms involved. This study determined that glucose, but not other analyzed nutrients, offered significant protection against cell death induced by sorafenib, as indicated by glucose deprivation sensitizing cells to sorafenib-induced cell death. Next, this synergistic effect was found to be specific to sorafenib, not to lenvatinib or the chemotherapeutic drugs cisplatin and doxorubicin. Mechanistically, sorafenib-induced mitophagy, as indicated by PINK1 accumulation, increased the phospho-poly-ubiquitination modification, accelerated mitochondrial membrane protein and mitochondrial DNA degradation, and increased the amount of mitochondrion-localized mKeima-Red engulfed by lysosomes. Among several E3 ubiquitin ligases tested, SIAH1 was found to be essential for inducing mitophagy; that is, SIAH1 silencing markedly repressed mitophagy and sensitized cells to sorafenib-induced death. Notably, the combined treatment of glucose restriction and sorafenib abolished ATP generation and mitophagy, which led to a high cell death rate. Oligomycin and antimycin, inhibitors of electron transport chain complexes, mimicked the synergistic effect of sorafenib with glucose restriction to promote cell death mediated via mitophagy inhibition. Finally, inhibition of the glucose transporter by canagliflozin (a clinically available drug used for type-II diabetes) effectively synergized with sorafenib to induce HCC cell death in vitro and to inhibit xenograft tumor growth in vivo. This study demonstrates that simultaneous treatment with sorafenib and glucose restriction is an effective approach to treat HCC, suggesting a promising combination strategy such as transarterial sorafenib-embolization (TASE) for the treatment of unresectable HCC.
Topics: Humans; Carcinoma, Hepatocellular; Sorafenib; Chemoembolization, Therapeutic; Liver Neoplasms; Mitophagy; Glucose; Niacinamide; Antineoplastic Agents
PubMed: 36385558
DOI: 10.1038/s12276-022-00878-x -
PloS One 2022Measurement of oxygen consumption of cultured cells is widely used for diagnosis of mitochondrial diseases, drug testing, biotechnology, and toxicology. Fibroblasts are...
Measurement of oxygen consumption of cultured cells is widely used for diagnosis of mitochondrial diseases, drug testing, biotechnology, and toxicology. Fibroblasts are cultured in monolayers, but physiological measurements are carried out in suspended or attached cells. We address the question whether respiration differs in attached versus suspended cells using multiwell respirometry (Agilent Seahorse XF24) and high-resolution respirometry (Oroboros O2k), respectively. Respiration of human dermal fibroblasts measured in culture medium was baseline-corrected for residual oxygen consumption and expressed as oxygen flow per cell. No differences were observed between attached and suspended cells in ROUTINE respiration of living cells and LEAK respiration obtained after inhibition of ATP synthase by oligomycin. The electron transfer capacity was higher in the O2k than in the XF24. This could be explained by a limitation to two uncoupler titrations in the XF24 which led to an underestimation compared to multiple titration steps in the O2k. A quantitative evaluation of respiration measured via different platforms revealed that short-term suspension of fibroblasts did not affect respiratory activity and coupling control. Evaluation of results obtained by different platforms provides a test for reproducibility beyond repeatability. Repeatability and reproducibility are required for building a validated respirometric database.
Topics: Cell Respiration; Fibroblasts; Humans; Oxidative Phosphorylation; Oxygen Consumption; Reproducibility of Results
PubMed: 35239701
DOI: 10.1371/journal.pone.0264496 -
Phytomedicine : International Journal... May 2021Starting December 2019, mankind faced an unprecedented enemy, the COVID-19 virus. The world convened in international efforts, experiences and technologies in order to... (Review)
Review
BACKGROUND
Starting December 2019, mankind faced an unprecedented enemy, the COVID-19 virus. The world convened in international efforts, experiences and technologies in order to fight the emerging pandemic. Isolation, hygiene measure, diagnosis, and treatment are the most efficient ways of prevention and intervention nowadays. The health organizations and global care systems screened the available resources and offered recommendations of approved and proposed medications. However, the search for a specific selective therapy or vaccine against COVID-19 remains a challenge.
METHODS
A literature search was performed for the screening of natural and derived bio-active compounds which showed potent antiviral activity against coronaviruses using published articles, patents, clinical trials website (https://clinicaltrials.gov/) and web databases (PubMed, SCI Finder, Science Direct, and Google Scholar).
RESULTS
Through the screening for natural products with antiviral activities against different types of the human coronavirus, extracts of Lycoris radiata (L'Hér.), Gentiana scabra Bunge, Dioscorea batatas Decne., Cassia tora L., Taxillus chinensis (DC.), Cibotium barometz L. and Echinacea purpurea L. showed a promising effect against SARS-CoV. Out of the listed compound Lycorine, emetine dihydrochloride hydrate, pristimerin, harmine, conessine, berbamine, 4`-hydroxychalcone, papaverine, mycophenolic acid, mycophenolate mofetil, monensin sodium, cycloheximide, oligomycin and valinomycin show potent activity against human coronaviruses. Additionally, it is worth noting that some compounds have already moved into clinical trials for their activity against COVID-19 including fingolimod, methylprednisolone, chloroquine, tetrandrine and tocilizumab.
CONCLUSION
Natural compounds and their derivatives could be used for developing potent therapeutics with significant activity against SARS-COV-2, providing a promising frontline in the fighting against COVID-19.
Topics: Antiviral Agents; Biological Products; COVID-19 Vaccines; Clinical Trials as Topic; Drug Evaluation, Preclinical; Humans; Molecular Structure; Pandemics; Plant Preparations; SARS-CoV-2; COVID-19 Drug Treatment
PubMed: 33067112
DOI: 10.1016/j.phymed.2020.153311 -
Frontiers in Immunology 2022The protozoan parasite is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous...
The protozoan parasite is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis-an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)-is elicited by stages. So far, the metabolic requirements of -triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) in sporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studied chemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminished PMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved in sporozoite-induced NETosis.
Topics: Adenosine Triphosphate; Animals; Cattle; Cattle Diseases; Coccidiosis; Eimeria; Glycolysis; Sporozoites
PubMed: 36032127
DOI: 10.3389/fimmu.2022.842482 -
Journal of Peptide Science : An... Sep 2022Cell-penetrating peptides (CPPs) can aid in intracellular and in vivo drug delivery. However, the mechanisms of CPP-mediated penetration remain unclear, limiting the...
Cell-penetrating peptides (CPPs) can aid in intracellular and in vivo drug delivery. However, the mechanisms of CPP-mediated penetration remain unclear, limiting the development and further application of CPPs. Flow cytometry and laser confocal fluorescence microscopy were performed to detect the effects of different endocytosis inhibitors on the internalization of CC12 and penetratin in ARPE-19 cells. The co-localization of CPPs with the lysosome and macropinosome was detected via an endocytosis tracing experiment. The flow cytometry results showed that chlorpromazine, wortmannin, cytochalasin D, and the ATP inhibitor oligomycin had dose-dependent endocytosis-inhibitory effects on CC12. The laser confocal fluorescence results showed that oligomycin had the most significant inhibitory effect on CC12 uptake; CC12 was co-located with the lysosome, but not with the macropinosome. For penetratin, cytochalasin D and oligomycin had obvious inhibitory effects. The laser confocal fluorescence results indicated that oligomycin had the most significant inhibitory effect on penetratin uptake; the co-localization of penetratin with the lysosome was higher than that with the macropinosome. Cation-independent CC12 and cationic penetratin may be internalized into cells primarily through caveolae and clathrin-mediated endocytosis, and they are typically dependent on ATP. The transport of penetratin could be partly achieved through the direct transmembrane pathway, as the positive charge of penetratin interacts with the negative charge of the cell membrane, and partly through the endocytic pathway.
Topics: Adenosine Triphosphate; Carrier Proteins; Cations; Cell-Penetrating Peptides; Cytochalasin D; Endocytosis; Oligomycins; Transcytosis
PubMed: 35128758
DOI: 10.1002/psc.3408 -
Scientific Reports Aug 2022Mitochondrial dysfunction promotes cancer aggressiveness, metastasis, and resistance to therapy. Similar traits are associated with epithelial mesenchymal transition...
Mitochondrial dysfunction promotes cancer aggressiveness, metastasis, and resistance to therapy. Similar traits are associated with epithelial mesenchymal transition (EMT). We questioned whether mitochondrial dysfunction induces EMT in head and neck cancer (HNC) cell lines. We induced mitochondrial dysfunction in four HNC cell lines with carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial electron transport chain uncoupling agent, and oligomycin, a mitochondrial ATP synthase inhibitor. Extracellular flux analyses and expression of the cystine/glutamate antiporter system xc (xCT) served to confirm mitochondrial dysfunction. Expression of the EMT-related transcription factor SNAI2, the mesenchymal marker vimentin and vimentin/cytokeratin double positivity served to detect EMT. In addition, holotomographic microscopy was used to search for morphological features of EMT. Extracellular flux analysis and xCT expression confirmed that FCCP/oligomycin induced mitochondrial dysfunction in all cell lines. Across the four cell lines, mitochondrial dysfunction resulted in an increase in relative SNAI2 expression from 8.5 ± 0.8 to 12.0 ± 1.1 (mean ± SEM; p = 0.007). This effect was predominantly caused by the CAL 27 cell line (increase from 2.2 ± 0.4 to 5.5 ± 1.0; p < 0.001). Similarly, only in CAL 27 cells vimentin expression increased from 2.2 ± 0.5 × 10 to 33.2 ± 10.2 × 10 (p = 0.002) and vimentin/cytokeratin double positive cells increased from 34.7 ± 5.1 to 67.5 ± 9.8% (p = 0.003), while the other 3 cell lines did not respond with EMT (all p > 0.1). Across all cell lines, FCCP/oligomycin had no effect on EMT characteristics in holotomographic microscopy. Mitochondrial dysfunction induced EMT in 1 of 4 HNC cell lines. Given the heterogeneity of HNC, mitochondrial dysfunction may be sporadically induced by EMT, but EMT does not explain the tumor promoting effects of mitochondrial dysfunction in general.
Topics: Cadherins; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Head and Neck Neoplasms; Humans; Keratins; Mitochondria; Oligomycins; Vimentin
PubMed: 35918485
DOI: 10.1038/s41598-022-16829-5