An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal epithelial cells.

The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more.

Visualization of intracellular ATP dynamics in different nephron segments under pathophysiological conditions using the kidney slice culture system.

ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here, we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated the ATP synthesis pathway, and analyzed intracellular ATP dynamics using an ex vivo IR-mimicking model and a cisplatin nephropathy model. Proximal tubules (PTs) were found to be strongly dependent on oxidative phosphorylation (OXPHOS) using the inhibitor oligomycin A, whereas podocytes relied on both OXPHOS and glycolysis using phloretin an active transport inhibitor of glucose. We also confirmed that an ex vivo IR-mimicking model could recapitulate ATP dynamics in vivo; ATP recovery in PTs after reoxygenation varied depending on anoxic time length, whereas ATP in distal tubules (DTs) recovered well even after long-term anoxia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor, cimetidine, suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent (Mitochonic Acid 5) delayed the cisplatin-induced ATP decrease in PTs. Thus, our novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.

Oligomycin-producing Streptomyces sp. newly isolated from Swiss soils efficiently protect Arabidopsis thaliana against Botrytis cinerea.

Botrytis cinerea is a necrotrophic phytopathogen able to attack more than 200 different plant species causing strong yield losses worldwide. Many synthetic fungicides have been developed to control this disease, resulting in the rise of fungicide-resistance B. cinerea strains. The aim of this study was to identify Streptomyces strains showing antagonistic activity against B. cinerea to contribute to plant protection in an environmentally friendly way. We isolated 15 Actinomycete strains from 9 different Swiss soils. The culture filtrates of three isolates showing antifungal activity inhibited spore germination and delayed mycelial growth of B. cinerea. Infection experiments showed that Arabidopsis thaliana plants were more resistant to this pathogen after leaf treatment with the Streptomyces filtrates. Bioassay-guided isolation of the active compounds revealed the presence of germicidins A and B as well as of oligomycins A, B, and E. While germicidins were mostly inactive, oligomycin B reduced the mycelial growth of B. cinerea significantly. Moreover, all three oligomycins inhibited this fungus' spore germination, suggesting that these molecules might contribute to the Streptomyces's ability to protect plants against infection by the broad host-pathogen Botrytis cinerea. IMPORTANCE: This study reports the isolation of new Streptomyces strains with strong plant-protective potential mediated by their production of specialized metabolites. Using the broad host range pathogenic fungus Botrytis cinerea, we demonstrate that the cell-free filtrate of selected Streptomyces isolates efficiently inhibits different developmental stages of the fungus, including mycelial growth and the epidemiologically relevant spore germination. Beyond in vitro experiments, the strains and their metabolites also efficiently protected plants against the disease caused by this pathogen. This work further identifies oligomycins as active compounds involved in the observed antifungal activity of the strains. This work shows that we can harness the natural ability of soil-borne microbes and of their metabolites to efficiently fight other microbes responsible for significant crop losses. This opens the way to the development of environmentally friendly health protection measures for crops of agronomical relevance, based on these newly isolated strains or their metabolic extracts containing oligomycins.

Experimental and computational investigation of the kinetic evolution of the glutaminolysis pathway and its interplay with the glycolysis pathway.

Exploring cellular responses necessitates studying real-time metabolic pathway kinetics, considering the adaptable nature of cells. Glycolysis and glutaminolysis are interconnected pathways fundamental to driving cellular metabolism, generating both energy and essential biosynthetic molecules. While prior studies explored glycolysis tracking, this research focuses on monitoring the kinetics of the glutaminolysis pathway by evaluating the effect of glutamine availability on glycolytic kinetics and by investigating the impact of a stimulator (oligomycin) and inhibitor (2DG) on the glycolytic flux in the presence of glutamine. Additionally, we adapted a rate equation model to provide improved understanding of the pathway kinetics. The experimental and simulated results indicate a significant reduction in extracellular lactate production in the presence of glutamine, reflecting a shift from glycolysis towards oxidative phosphorylation, due to the additional contribution of glutamine to energy production through the ETC (electron transport chain), reducing the glycolytic load. Oligomycin, an ETC inhibitor, increases lactate production to the original glycolytic level, despite the presence of glutamine. Nevertheless, its mechanism is influenced by the presence of glutamine, as predicted by the model. Conversely, 2DG notably reduces lactate production, affirming its glycolytic origin. The gradual increase in lactate production under the influence of 2DG implies increased activation of glutaminolysis as an alternative energy source. The model also simulates the varying metabolic responses under varying carbon/modulator concentrations. In conclusion, the kinetic model described here contributes to the understanding of changes in intracellular metabolites and their interrelationships in a way which would be challenging to obtain solely through kinetic assays.

Enhancement of Acetate-Induced Apoptosis of Colorectal Cancer Cells by Cathepsin D Inhibition Depends on Oligomycin A-Sensitive Respiration.

Colorectal cancer (CRC) is a leading cause of death worldwide. Conventional therapies are available with varying effectiveness. Acetate, a short-chain fatty acid produced by human intestinal bacteria, triggers mitochondria-mediated apoptosis preferentially in CRC but not in normal colonocytes, which has spurred an interest in its use for CRC prevention/therapy. We previously uncovered that acetate-induced mitochondrial-mediated apoptosis in CRC cells is significantly enhanced by the inhibition of the lysosomal protease cathepsin D (CatD), which indicates both mitochondria and the lysosome are involved in the regulation of acetate-induced apoptosis. Herein, we sought to determine whether mitochondrial function affects CatD apoptotic function. We found that enhancement of acetate-induced apoptosis by CatD inhibition depends on oligomycin A-sensitive respiration. Mechanistically, the potentiating effect is associated with an increase in cellular and mitochondrial superoxide anion accumulation and mitochondrial mass. Our results provide novel clues into the regulation of CatD function and the effect of tumor heterogeneity in the outcome of combined treatment using acetate and CatD inhibitors.

Structures, Biosynthesis, and Bioactivity of Oligomycins from the Marine-Derived Streptomyces sp. FXY-T5.

Oligomycins are potent antifungal and antitumor agents. Mass spectrometry (MS)- and nuclear magnetic resonance (NMR)-based metabolomic fingerprinting analysis of marine-derived actinomycetes in our in-house library provided an oligomycin-producing strain, Streptomyces sp. FXY-T5. Chemical investigation led to the discovery of five new oligomycins, 24-lumooligomycin B (1), 4-lumooligomycin B (2), 6-lumooligomycin B (3), 40-homooligomycin B (4), and 15-hydroxy-oligomycin B (5), together with seven biosynthetically related known derivatives. Their structures were assigned by MS, NMR, electronic circular dichroism (ECD), and single-crystal X-ray diffraction analyses. The biosynthesis pathway of oligomycins was first proposed based on the analysis of a type I modular polyketide synthase (PKS) system and targeted gene disruption. As expected, the isolated oligomycins showed significant antiagricultural fungal pathogen activity and antiproliferative properties from which the possible structure-activity relationships were first suggested. More importantly, oligomycins induced significant G1-phase cell cycle arrest on cancer cells and significantly attenuated their Cyclin D1 and PCNA expression through a ß-catenin signaling pathway.

In vitro reduction of bovine oocyte ATP production with oligomycin affects embryo epigenome.

In brief: Epigenetic programming is a crucial process during early embryo development that can have a significant impact on the results of assisted reproductive technology (ART) and offspring health. Here we show evidence using a bovine in vitro experiment that embryo epigenetic programing is dependent on oocyte mitochondrial bioenergetic activity during maturation. Abstract: This study investigated if oocyte and early embryo epigenetic programming are dependent on oocyte mitochondrial ATP production. A bovine in vitro experiment was performed in which oocyte mitochondrial ATP production was reduced using 5 nmol/L oligomycin A (OM; ATP synthase inhibitor) during in vitro maturation (IVM) compared to control (CONT). OM exposure significantly reduced mitochondrial ATP production rate in MII oocytes (34.6% reduction, P = 0.018) and significantly decreased embryo cleavage rate at 48 h post insemination (7.6% reduction, P = 0.031). Compared to CONT, global DNA methylation (5mC) levels were decreased in OM-exposed MII oocytes (9.8% reduction, P = 0.019) while global histone methylation (H3K9me2) was increased (9.4% increase, P = 0.024). In zygotes, OM exposure during IVM increased 5mC (22.3% increase, P < 0.001) and histone acetylation (H3K9ac, 17.3% increase, P = 0.023) levels, while H3K9me2 levels were not affected. In morulae, 5mC levels were increased (10.3% increase, P = 0.041) after OM exposure compared to CONT, while there was no significant difference in H3K9ac and H3K9me2 levels. These epigenetic alterations were not associated with any persistent effects on embryo mitochondrial ATP production rate or mitochondrial membrane potential (assessed at the four-cell stage). Also, epigenetic regulatory genes were not differentially expressed in OM-exposed zygotes or morulae. Finally, apoptotic cell index in blastocysts was increased after OM exposure during oocyte maturation (41.1% increase, P < 0.001). We conclude that oocyte and early embryo epigenetic programming are dependent on mitochondrial ATP production during IVM.

Synergistic lethality in chronic myeloid leukemia - targeting oxidative phosphorylation and unfolded protein response effectively complements tyrosine kinase inhibitor treatment.

Chronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), targeting the BCR::ABL1 oncoprotein. Still, resistance to therapy, relapse after treatment discontinuation, and side effects remain significant issues of long-term TKI treatment. Preliminary studies have shown that targeting oxidative phosphorylation (oxPhos) and the unfolded protein response (UPR) are promising therapeutic approaches to complement CML treatment. Here, we tested the efficacy of different TKIs, combined with the ATP synthase inhibitor oligomycin and the ER stress inducer thapsigargin in the CML cell lines K562, BV173, and KU812 and found a significant increase in cell death. Both, oligomycin and thapsigargin, triggered the upregulation of the UPR proteins ATF4 and CHOP, which was inhibited by imatinib. We observed comparable effects on cell death when combining TKIs with the ATP synthase inhibitor 8-chloroadenosine (8-Cl-Ado) as a potentially clinically applicable therapeutic agent. Stress-related apoptosis was triggered via a caspase cascade including the cleavage of caspase 3 and the inactivation of poly ADP ribose polymerase 1 (PARP1). The inhibition of PARP by olaparib also increased CML death in combination with TKIs. Our findings suggest a rationale for combining TKIs with 8-Cl-Ado or olaparib for future clinical studies in CML.

Interaction between mitochondrial homeostasis and barrier function in lipopolysaccharide-induced endothelial cell injury.

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.

Proliferating Astrocytes in Primary Culture Do Not Depend upon Mitochondrial Respiratory Complex I Activity or Oxidative Phosphorylation.

Understanding the role of astrocytes in the development of the nervous system and neurodegenerative disorders implies a necessary knowledge of the oxidative metabolism of proliferating astrocytes. The electron flux through mitochondrial respiratory complexes and oxidative phosphorylation may impact the growth and viability of these astrocytes. Here, we aimed at assessing to which extent mitochondrial oxidative metabolism is required for astrocyte survival and proliferation. Primary astrocytes from the neonatal mouse cortex were cultured in a physiologically relevant medium with the addition of piericidin A or oligomycin at concentrations that fully inhibit complex I-linked respiration and ATP synthase, respectively. The presence of these mitochondrial inhibitors for up to 6 days in a culture medium elicited only minor effects on astrocyte growth. Moreover, neither the morphology nor the proportion of glial fibrillary acidic protein-positive astrocytes in culture was affected by piericidin A or oligomycin. Metabolic characterization of the astrocytes showed a relevant glycolytic metabolism under basal conditions, despite functional oxidative phosphorylation and large spare respiratory capacity. Our data suggest that astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.

Relative Importance of Different Elements of Mitochondrial Oxidative Phosphorylation in Maintaining the Barrier Integrity of Retinal Endothelial Cells: Implications for Vascular-Associated Retinal Diseases.

PURPOSE: Mitochondrial dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy and retinopathy of prematurity. Therefore, we aimed to investigate the role of different mitochondrial constituents, specifically those of oxidative phosphorylation (OxPhos), in maintaining the barrier function of RECs. METHODS: Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real time the role of different mitochondrial components in the total impedance (Z) of human RECs (HRECs) and its components: capacitance (C) and the total resistance (R). HRECs were treated with specific mitochondrial inhibitors that target different steps in OxPhos: rotenone for complex I, oligomycin for complex V (ATP synthase), and FCCP for uncoupling OxPhos. Furthermore, data were modeled to investigate the effects of these inhibitors on the three parameters that govern the total resistance of cells: Cell-cell interactions (Rb), cell-matrix interactions (α), and cell membrane permeability (Cm). RESULTS: Rotenone (1 µM) produced the greatest reduction in Z, followed by FCCP (1 µM), whereas no reduction in Z was observed after oligomycin (1 µM) treatment. We then further deconvoluted the effects of these inhibitors on the Rb, α, and Cm parameters. Rotenone (1 µM) completely abolished the resistance contribution of Rb, as the Rb became zero immediately after the treatment. Secondly, FCCP (1 µM) eliminated the resistance contribution of Rb only after 2.5 h and increased Cm without a significant effect on α. Lastly, of all the inhibitors used, oligomycin had the lowest impact on Rb, as evidenced by the fact that this value became similar to that of the control group at the end of the experiment without noticeable effects on Cm or α. CONCLUSION: Our study demonstrates the differential roles of complex I, complex V, and OxPhos coupling in maintaining the barrier functionality of HRECs. We specifically showed that complex I is the most important component in regulating HREC barrier integrity. These observed differences are significant since they could serve as the basis for future pharmacological and gene expression studies aiming to improve the activity of complex I and thereby provide avenues for therapeutic modalities in endothelial-associated retinal diseases.

OXPHOS deficiency induces mitochondrial DNA synthesis through non-canonical AMPK-dependent mRNA compartmentalization.

Eukaryotic cells contain multiple copies of mitochondrial DNA (mtDNA) in discrete organelles or as tubular networks throughout the cytoplasm. The mtDNA copy number is dynamically regulated by mitochondrial biogenesis and mitophagy processes. However, the conditions regulating mtDNA replication, an essential component of biogenesis, are unknown. We observed that short-term (2 h) treatment of rat myoblasts with oligomycin, a specific inhibitor of the mitochondrial F1F0 ATP synthase, resulted in stimulation of mtDNA synthesis from the OH replication origin. This effect was abrogated by Compound C, an antagonist of the AMP-dependent protein kinase (AMPK), a universal intracellular energy sensor, and in AMPK-knockdown cells, indicating that mtDNA replication is regulated by AMPK under oxidative phosphorylation (OXPHOS)- deficient conditions. Using antibody decoration, enzymatically active AMPK, phosphorylated at T172 of the α1 subunit, was found to be located on the mitochondrial surface. Furthermore, oligomycin induced the compartmentalization of several mRNAs encoding OXPHOS components and mtDNA replication factors to mitochondria. Compartmentalization of mRNAs was inhibited by Compound C. We infer that AMPK is locally activated by inhibition of the F1F0 ATP synthase to stimulate association of mtDNA replication factor mRNAs, leading to stimulation of mtDNA synthesis. The findings have implications for the clonal expansion of OXPHOS-deficient mtDNA mutant mitochondria in human patients, with clinical consequences.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

Mitochondrial dysfunction and epithelial to mesenchymal transition in head neck cancer cell lines.

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 × 10-3 to 33.2 ± 10.2 × 10-3 (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.

Subacute and sublethal ingestion of microcystin-LR impairs lung mitochondrial function by an oligomycin-like effect.

Microcystin-LR (MC-LR) is a potent cyanotoxin that can reach several organs. However subacute exposure to sublethal doses of MC-LR has not yet well been studied. Herein, we evaluated the outcomes of subacute and sublethal MC-LR exposure on lungs. Male BALB/c mice were exposed to MC-LR by gavage (30 µg/kg) for 20 consecutive days, whereas CTRL mice received filtered water. Respiratory mechanics was not altered in MC-LR group, but histopathology disclosed increased collagen deposition, immunological cell infiltration, and higher percentage of collapsed alveoli. Mitochondrial function was extensively affected in MC-LR animals. Additionally, a direct in vitro titration of MC-LR revealed impaired mitochondrial function. In conclusion, MC-LR presented an intense deleterious effect on lung mitochondrial function and histology. Furthermore, MC-LR seems to exert an oligomycin-like effect in lung mitochondria. This study opens new perspectives for the understanding of the putative pulmonary initial mechanisms of damage resulting from oral MC-LR intoxication.

Contributing role of mitochondrial energy metabolism on platelet adhesion, activation and thrombus formation under blood flow conditions.

Platelets have an active energy metabolism mediated by mitochondria. However, the role of mitochondria in platelet adhesion, activation, and thrombus formation under blood flow conditions remains to be elucidated. Blood specimens were obtained from healthy adult volunteers. The consumption of glucose molecules by platelets was measured after 24 hours. Platelet adhesion, activation, and thrombus formation on collagen fibrils and immobilized von Willebrand factor (VWF) at a wall shear rate of 1,500 s-1 were detected by fluorescence microscopy with an ultrafast laser confocal unit in the presence or absence of mitochondrial functional inhibitors of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A, and oligomycin. Consumption of glucose molecules within the first 24 h of 4.21 × 10-15 ± 4.46 x 10-15 (n = 6) increased to 13.82 × 10-15 ± 3.46 x 10-15 (n = 4) in the presence of FCCP, 12.11 × 10-15 ± 2.33 x 10-15 (n = 4) in the presence of antimycin A, and 11.87 × 10-15 ± 3.56 x 10-15 (n = 4) in the presence of oligomycin (p < .05). These mitochondrial functional blockers did not influence both surface area coverage by platelets and the 3-dimensional size of platelet thrombi formed on the collagen fibrils. However, a rapid increase in the intracellular calcium ion concentration ([Ca2+]i) upon adhering on immobilized VWF decreased significantly from 405.5 ± 86.2 nM in control to 198.0 ± 79.2 nM in the presence of FCCP (p < .005). A similar decrease in the rapid increase in ([Ca2+]i) was observed in the presence of antimycin A and oligomycin. Mitochondrial function is necessary for platelet activation represented by a rapid increase in [Ca2+]i after platelet adhesion on VWF. However, the influence could not be detected as changes in platelet adhesion or 3-dimensional growth of platelet thrombi on collagen fibrils.

An imbalance of the IL-33/ST2-AXL-efferocytosis axis induces pregnancy loss through metabolic reprogramming of decidual macrophages.

During embryo implantation, apoptosis is inevitable. These apoptotic cells (ACs) are removed by efferocytosis, in which macrophages are filled with a metabolite load nearly equal to the phagocyte itself. A timely question pertains to the relationship between efferocytosis-related metabolism and the immune behavior of decidual macrophages (dMΦs) and its effect on pregnancy outcome. Here, we report positive feedback of IL-33/ST2-AXL-efferocytosis leading to pregnancy failure through metabolic reprogramming of dMΦs. We compared the serum levels of IL-33 and sST2, along with IL-33 and ST2, efferocytosis and metabolism of dMΦs, from patients with normal pregnancies and unexplained recurrent pregnancy loss (RPL). We revealed disruption of the IL-33/ST2 axis, increased apoptotic cells and elevated efferocytosis of dMΦs from patients with RPL. The dMΦs that engulfed many apoptotic cells secreted more sST2 and less TGF-ß, which polarized dMΦs toward the M1 phenotype. Moreover, the elevated sST2 biased the efferocytosis-related metabolism of RPL dMΦs toward oxidative phosphorylation and exacerbated the disruption of the IL-33/ST2 signaling pathway. Metabolic disorders also lead to dysfunction of efferocytosis, resulting in more uncleared apoptotic cells and secondary necrosis. We also screened the efferocytotic molecule AXL regulated by IL-33/ST2. This positive feedback axis of IL-33/ST2-AXL-efferocytosis led to pregnancy failure. IL-33 knockout mice demonstrated poor pregnancy outcomes, and exogenous supplementation with mouse IL-33 reduced the embryo losses. These findings highlight a new etiological mechanism whereby dMΦs leverage immunometabolism for homeostasis of the microenvironment at the maternal-fetal interface.

Transcytosis mechanisms of cell-penetrating peptides: Cation-independent CC12 and cationic penetratin.

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.

Unmasking of CgYor1-Dependent Azole Resistance Mediated by Target of Rapamycin (TOR) and Calcineurin Signaling in Candida glabrata.

In this study, 18 predicted membrane-localized ABC transporters of Candida glabrata were deleted individually to create a minilibrary of knockouts (KO). The transporter KOs were analyzed for their susceptibility toward antimycotic drugs. Although CgYOR1 has previously been reported to be upregulated in various azole-resistant clinical isolates of C. glabrata, deletion of this gene did not change the susceptibility to any of the tested azoles. Additionally, Cgyor1Δ showed no change in susceptibility toward oligomycin, which is otherwise a well-known substrate of Yor1 in other yeasts. The role of CgYor1 in azole susceptibility only became evident when the major transporter CgCDR1 gene was deleted. However, under nitrogen-depleted conditions, Cgyor1Δ demonstrated an azole-susceptible phenotype, independent of CgCdr1. Notably, Cgyor1Δ cells also showed increased susceptibility to target of rapamycin (TOR) and calcineurin inhibitors. Moreover, increased phytoceramide levels in Cgyor1Δ and the deletions of regulators downstream of TOR and the calcineurin signaling cascade (Cgypk1Δ, Cgypk2Δ, Cgckb1Δ, and Cgckb2Δ) in the Cgyor1Δ background and their associated fluconazole (FLC) susceptibility phenotypes confirmed their involvement. Collectively, our findings show that TOR and calcineurin signaling govern CgYor1-mediated azole susceptibility in C. glabrata. IMPORTANCE The increasing incidence of Candida glabrata infections in the last 40 years is a serious concern worldwide. These infections are usually associated with intrinsic azole resistance and increasing echinocandin resistance. Efflux pumps, especially ABC transporter upregulation, are one of the prominent mechanisms of azole resistance; however, only a few of them are characterized. In this study, we analyzed the mechanisms of azole resistance due to a multidrug resistance-associated protein (MRP) subfamily ABC transporter, CgYor1. We demonstrate for the first time that CgYor1 does not transport oligomycin but is involved in azole resistance. Under normal growing conditions its function is masked by major transporter CgCdr1; however, under nitrogen-depleted conditions, it displays its azole resistance function independently. Moreover, we propose that the azole susceptibility due to removal of CgYor1 is not due to its transport function but involves modulation of TOR and calcineurin cascades.

Mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells.

Intestinal epithelial cells (IECs) are crucial to maintain intestinal function and the barrier against the outside world. To support their function they rely on energy production, and failure to produce enough energy can lead to IEC malfunction and thus decrease intestinal barrier function. However, IEC metabolic function is not often used as an outcome parameter in intervention studies, perhaps because of the lack of available methods. We therefore developed a method to isolate viable IECs, suitable to faithfully measure their metabolic function by determining extracellular glycolytic and mitochondrial flux. First, various methods were assessed to obtain viable IECs. We then adapted a previously in-house generated image-analysis algorithm to quantify the amount of seeded IECs. Correcting basal respiration data of a group of piglets using this algorithm reduced the variation, showing that this algorithm allows for more accurate analysis of metabolic function. We found that delay in metabolic analysis after IEC isolation decreases their metabolic function and should therefore be prevented. The presence of antibiotics during isolation and metabolic assessment also decreased the metabolic function of IECs. Finally, we found that primary pig IECs did not respond to Oligomycin, a drug that inhibits complex V of the electron transport chain, which may be because of the presence of drug exporters. A method was established to faithfully measure extracellular glycolytic and mitochondrial flux of pig primary IECs. This tool is suitable to gain a better understanding of how interventions affect IEC metabolic function.