Flux Assay Research Articles

Histamine stimulates human microglia to alter cellular prion protein expression via the HRH2 histamine receptor

Although the cellular prion protein (PrPC) has been evolutionarily conserved, the role of this protein remains elusive. Recent evidence indicates that PrPC may be involved in neuroinflammation and the immune response in the brain, and its expression may be modified via various mechanisms. Histamine is a proinflammatory mediator and neurotransmitter that stimulates numerous cells via interactions with histamine receptors 1-4 (HRH1-4). Since microglia are the innate immune cells of the central nervous system, we hypothesized that histamine-induced stimulation regulates the expression of PrPC in human-derived microglia. The human microglial clone 3 (HMC3) cell line was treated with histamine, and intracellular calcium levels were measured via a calcium flux assay. Cytokine production was monitored by enzyme-linked immunosorbent assay (ELISA). Western blotting and quantitative reverse transcription-polymerase chain reaction were used to determine protein and gene expression of HRH1-4. Flow cytometry and western blotting were used to measure PrPC expression levels. Fluorescence microscopy was used to examine Iba-1 and PrPC localization. HMC3 cells stimulated by histamine exhibited increased intracellular calcium levels and increased release of IL-6 and IL-8, while also modifying PrPC localization. HMC3 stimulated with histamine for 6 and 24 hours exhibited increased surface PrPC expression. Specifically, we found that stimulation of the HRH2 receptor was responsible for changes in surface PrPC. Histamine-induced increases in surface PrPC were attenuated following inhibition of the HRH2 receptor via the HRH2 antagonist ranitidine. These changes were unique to HRH2 activation, as stimulation of HRH1, HRH3, or HRH4 did not alter surface PrPC. Prolonged stimulation of HMC3 decreased PrPC expression following 48 and 72 hours of histamine stimulation. HMC3 cells can be stimulated by histamine to undergo intracellular calcium influx. Surface expression levels of PrPC on HMC3 cells are altered by histamine exposure, primarily mediated by HRH2. While histamine exposure also increases release of IL-6 and IL-8 in these cells, this cytokine release is not fully dependent on PrPC levels, as IL-6 release is only partially reduced and IL-8 release is unchanged under the conditions of HRH2 blockade that prevent PrPC changes. Overall, this suggests that PrPC may play a role in modulating microglial responses.

The important role of the Wnt/β-catenin signaling pathway in small molecules mediated gingival mesenchymal stem cells transdifferentiate into neuron-like cells

ObjectiveGiven their neural crest origin, gingival mesenchymal stem cells (GMSCs) possess high neurogenic potential, which makes them suitable for cell replacement therapy against neurodegenerative diseases. This study investigated whether GMSCs can be transdifferentiated into neurons in vitro using a protocol involving small molecules VCRFY (VPA, CHIR99021, Repsox, Forskolin, and Y-27632). The regulatory mechanisms of key signaling pathways were also investigated. MethodsNeuronal induction of GMSCs was conducted using a small molecules-based protocol over 7 days, which included the evaluation of cell morphology, proliferation, expressions of neurogenic markers, and intracellular calcium oscillation. The activation of canonical the Wnt signaling pathway was assessed by examining the protein content and subcellular localization of β-catenin. ResultsSmall molecules-treated GMSCs displayed neuronal morphology and increased expression of neurogenic markers, including class III beta-tubulin (TUJ1), neuron-specific enolase (NSE), microtube-associated protein 2 (MAP2), and neurofilament medium (NFM), verified through RT-qPCR, western blotting, and immunocytochemistry. Based on the results of Fluo-4 AM calcium flux assay, small molecules-treated GMSCs exhibited enhanced electrophysiological activity. GMSC proliferation halted after 2 days of treatment. Among the small molecules, CHIR99021 exhibited the highest neuronal induction efficiency. Furthermore, activation of the Wnt/β-catenin signaling pathway augmented neuronal differentiation. ConclusionsSmall molecule-based cellular reprogramming can efficiently generate neurons from GMSCs, with Wnt/β-catenin signaling to play a critical role in neuronal induction.

Heightened mitochondrial respiration in CF cells is normalised by triple CFTR modulator therapy through mechanisms involving calcium

BackgroundCystic fibrosis (CF) is associated with increased resting energy expenditure. However, the introduction of elexacaftor/tezacaftor/ivacaftor (ETI) has resulted in a paradigm shift in nutritional status for many people with CF, with increase body mass index and reduction in the need for nutritional support. While these changes are likely to reflect improved clinical status and an associated downregulation of energy expenditure, they may also reflect drug-induced alterations in metabolic perturbations within CF cells. We hypothesise that some of these changes relate to normalisation of mitochondrial respiration in CF. MethodsUsing wild-type (WT) and F508del/F508del CFTR human bronchial epithelial cell lines (HBE cell lines) and baby hamster kidney (BHK) cells we examined the impact of ETI on cellular metabolism. We monitored mitochondrial respiration, using Seahorse extracellular flux assays and monitored mitochondrial reactive oxygen species (mROS) and intracellular calcium levels by flow cytometry. ResultsIncreased mitochondrial respiration was found in HBE cell lines and BHK cells expressing CFTR F508del/F508del when assessing basal, maximal, spare respiratory capacities and ATP production, as well as increased mitochondrial ROS generated via forward electron transport. ETI significantly decreased basal, maximal, spare respiratory capacity and ATP production to WT levels or below. Calcium blocker, BAPTA-AM normalised mitochondrial respiration, suggesting a calcium-mediated mechanism. ETI decreased intracellular calcium levels in CF cells to the same extent as BAPTA-AM, highlighting the importance of calcium and chloride in mitochondrial respiration in CF. ConclusionsCF cell lines exhibit increased mitochondrial respiration, which can be downregulated by ETI therapy through mechanisms involving calcium.

Unexpected and Synergistical Effects of All-Trans Retinoic Acid and TGF-β2 on Biological Aspects of 2D and 3D Cultured ARPE19 Cells.

Objectives: To study the effects of all-trans retinoic acid (ATRA) on TGF-β2-induced effects of human retinal pigment epithelium cells under normoxia and hypoxia conditions. Methods: Two-dimensionally (2D) and three-dimensionally (3D) cultured ARPE19 cells were subjected to cellular functional analyses by transepithelial electrical resistance (TEER) and an extracellular flux assay (2D), measurement of levels of reactive oxygen species (ROS), gene expression analyses of COL1, αSMA, Zo-1, HIF1α, and PGC1α (2D), and physical property analyses (3D). Results: Under a normoxia condition, treatment with 100 nM ATRA substantially decreased barrier function regardless of the presence of 5 ng/mL TGF-β2 in 2D ARPE19 monolayer cells. Under a hypoxia condition, treatment with ATRA conversely increased barrier function, but the effect was masked by a marked increase in effects induced by TGF-β2. Although ATRA alone did not affect cellular metabolism and ROS levels in 2D ARPE cells, treatment with ATRA under a hypoxia condition did not affect ROS levels but shifted cellular metabolism from mitochondrial respiration to glycolysis. The changes of cellular metabolism and ROS levels were more pronounced with treatment of both ATRA and TGF-β2 independently of oxygen conditions. Changes in mRNA expressions of some of the above genes suggested the involvement of synergistical regulation of cellular functions by TGF-β2 and hypoxia. In 3D ARPE spheroids, the size was decreased and the stiffness was increased by either treatment with TGF-β2 or ATRA, but these changes were unexpectedly modulated by both ATRA and TGF-β2 treatment regardless of oxygen conditions. Conclusions: The findings reported herein indicate that TGF-β2 and hypoxia synergistically and differentially induce effects in 2D and 3D cultured ARPE19 cells and that their cellular properties are significantly altered by the presence of ATRA.

Fluorescence-Based HTS Assays for Ion Channel Modulation in Drug Discovery Pipelines.

Ion channels represent a druggable family of transmembrane pore-forming proteins with important (patho)physiological functions. While electrophysiological measurement (manual patch clamp) remains the only direct method for detection of ion currents, it is a labor-intensive technique. Although automated patch clamp instruments have become available to date, their high costs limit their use to large pharma companies or commercial screening facilities. Therefore, fluorescence-based assays are particularly important for initial screening of compound libraries. Despite their numerous disadvantages, they are highly amenable to high-throughput screening and in many cases, no sophisticated instrumentation or materials are required. These features predispose them for implementation in early phases of drug discovery pipelines (hit identification), even in an academic environment. This review summarizes the advantages and pitfalls of individual methodological approaches for identification of ion channel modulators employing fluorescent probes (i.e., membrane potential and ion flux assays) with emphasis on practical aspects of their adaptation to high-throughput format.

Differential roles for the oxygen sensing enzymes PHD1 and PHD3 in the regulation of neutrophil metabolism and function.

Neutrophils are essential in the early innate immune response to pathogens. Harnessing their antimicrobial powers, without driving excessive and damaging inflammatory responses, represents an attractive therapeutic possibility. The neutrophil population is increasingly recognised to be more diverse and malleable than was previously appreciated. Hypoxic signalling pathways are known to regulate important neutrophil behaviours and, as such, are potential therapeutic targets for regulating neutrophil antimicrobial and inflammatory responses. We used a combination of in vivo and ex vivo models, utilising neutrophil and myeloid specific PHD1 or PHD3 deficient mouse lines to investigate the roles of oxygen sensing prolyl hydroxylase enzymes in the regulation of neutrophilic inflammation and immunity. Mass spectrometry and Seahorse metabolic flux assays were used to analyse the role of metabolic shifts in driving the downstream phenotypes. We found that PHD1 deficiency drives alterations in neutrophil metabolism and recruitment, in an oxygen dependent fashion. Despite this, PHD1 deficiency did not significantly alter ex vivo neutrophil phenotypes or in vivo outcomes in mouse models of inflammation. Conversely, PHD3 deficiency was found to enhance neutrophil antibacterial properties without excessive inflammatory responses. This was not linked to changes in the abundance of core metabolites but was associated with increased oxygen consumption and increased mitochondrial reactive oxygen species (mROS) production. PHD3 deficiency drives a favourable neutrophil phenotype in infection and, as such, is an important potential therapeutic target.

Hyperglycemic environments directly compromise intestinal epithelial barrier function in an organoid model and hyaluronan (∼35 kDa) protects via a layilin dependent mechanism

BackgroundMetabolic syndrome and diabetes in obese individuals are strong risk factors for development of inflammatory bowel disease (IBD) and colorectal cancer. The pathogenic mechanisms of low-grade metabolic inflammation, including chronic hyperglycemic stress, in disrupting gut homeostasis are poorly understood. In this study, we sought to understand the impact of a hyperglycemic environment on intestinal barrier integrity and the protective effects of small molecular weight (35 kDa) hyaluronan on epithelial barrier function. MethodsIntestinal organoids derived from mouse colon were grown in normal glucose media (5 mM) or high glucose media (25 mM) to study the impact of hyperglycemic stress on the intestinal barrier. Additionally, organoids were pretreated with 35 kDa hyaluronan (HA35) to investigate the effect of hyaluronan on epithelial barrier under high glucose stress. Immunoblotting as well as confocal imaging was used to understand changes in barrier proteins, quantitative as well as spatial distribution, respectively. Alterations in barrier function were measured using trans-epithelial electrical resistance and fluorescein isothiocyanate flux assays. Untargeted proteomics analysis was performed to elucidate mechanisms by which HA35 exerts a protective effect on the barrier. Intestinal organoids derived from receptor knockout mice specific to various HA receptors were utilized to understand the role of HA receptors in barrier protection under high glucose conditions. ResultsWe found that high glucose stress decreased the protein expression as well as spatial distribution of two key barrier proteins, zona occludens-1 (ZO-1) and occludin. HA35 prevented the degradation or loss of ZO-1 and maintained the spatial distribution of both ZO-1 and occludin under hyperglycemic stress. Functionally, we also observed a protective effect of HA35 on the epithelial barrier under high glucose conditions. We found that HA receptor, layilin, was involved in preventing barrier protein loss (ZO-1) as well as maintaining spatial distribution of ZO-1 and occludin. Additionally, proteomics analysis showed that cell death and survival was the primary pathway upregulated in organoids treated with HA35 under high glucose stress. We found that XIAP associated factor 1 (Xaf1) was modulated by HA35 thereby regulating apoptotic cell death in the intestinal organoid system. Finally, we observed that spatial organization of both focal adhesion kinase (FAK) as well as F-actin was mediated by HA35 via layilin. ConclusionOur results highlight the impact of hyperglycemic stress on the intestinal barrier function. This is of clinical relevance, as impaired barrier function has been observed in individuals with metabolic syndrome. Additionally, we demonstrate barrier protective effects of HA35 through its receptor layilin and modulation of cellular apoptosis under high glucose stress.

The polyunsaturated fatty acid docosahexaenoic affects mitochondrial function in prostate cancer cells

BackgroundProstate cancer (PCa) shows a rewired metabolism featuring increased fatty acid uptake and synthesis via de novo lipogenesis, both sharply related to mitochondrial physiology. The docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (PUFA) that exerts its antitumoral properties via different mechanisms, but its specific action on mitochondria in PCa is not clear. Therefore, we investigated whether the DHA modulates mitochondrial function in PCa cell lines.MethodsHere, we evaluated mitochondrial function of non-malignant PNT1A and the castration-resistant (CRPC) prostate 22Rv1 and PC3 cell lines in response to DHA incubation. For this purpose, we used Seahorse extracellular flux assay to assess mitochondria function, [14C]-glucose to evaluate its oxidation as well as its contribution to fatty acid synthesis, 1H-NMR for metabolite profile determination, MitoSOX for superoxide anion production, JC-1 for mitochondrial membrane polarization, mass spectrometry for determination of phosphatidylglycerol levels and composition, staining with MitoTracker dye to assess mitochondrial morphology under super-resolution in addition to Transmission Electron Microscopy, In-Cell ELISA for COX-I and SDH-A protein expression and flow cytometry (Annexin V and 7-AAD) for cell death estimation.ResultsIn all cell lines DHA decreased basal respiratory activity, ATP production, and the spare capacity in mitochondria. Also, the omega-3 induced mitochondrial hyperpolarization, ROS overproduction and changes in membrane phosphatidylglycerol composition. In PNT1A, DHA led to mitochondrial fragmentation and it increased glycolysis while in cancer cells it stimulated glucose oxidation, but decreased de novo lipogenesis specifically in 22Rv1, indicating a metabolic shift. In all cell lines, DHA modulated several metabolites related to energy metabolism and it was incorporated in phosphatidylglycerol, a precursor of cardiolipin, increasing the unsaturation index in the mitochondrial membrane. Accordingly, DHA triggered cell death mainly in PNT1A and 22Rv1.ConclusionIn conclusion, mitochondrial metabolism is significantly affected by the PUFA supplementation to the point that cells are not able to proliferate or survive under DHA-enriched condition. Moreover, combination of DHA supplementation with inhibition of metabolism-related pathways, such as de novo lipogenesis, may be synergistic in castration-resistant prostate cancer.

Characterization of a Novel Pathogenic PLCG2 Variant Leading to APLAIDSyndrome Responsive to a TNF Inhibitor.

Autoinflammation and phospholipase C (PLC) γ2-associated antibody deficiency and immune dysregulation (APLAID) syndrome is an autoinflammatory disease caused by gain-of-function variants in PLCG2. This study investigates the pathogenic mechanism of a novel variant of PLCG2 in a patient with APLAID syndrome. Whole-exome sequencing and Sanger sequencing were used to identify the pathogenic variant in the patient. Single-cell RNA sequencing, immunoblotting, luciferase assay, inositol monophosphate enzyme-linked immunosorbent assay, calcium flux assay, quantitative PCR, and immunoprecipitation were used to define inflammatory signatures and evaluate the effects of the PLCG2 variant on protein functionality and immune signaling. We identified a novel de novo variant, PLCG2 p.D993Y, in a patient with colitis, pansinusitis, skin rash, edema, recurrent respiratory infections, B-cell deficiencies, and hypogammaglobulinemia. The single-cell transcriptome revealed exacerbated inflammatory responses in the patient's peripheral blood mononuclear cells. Expression of the D993Y variant in HEK293T, COS-7, and PLCG2 knock-out THP-1 cell lines showed heightened PLCγ2 phosphorylation; elevated inositol-1,4,5-trisphosphate production and intracellular Ca2+ release; and activation of the MAPK, NF-κB, and NFAT signaling pathways compared with control-transfected cells. In vitro experiments indicated that the D993Y variant altered amino acid properties, disrupting the interaction between the catalytic and autoinhibitory domains of PLCγ2, resulting in PLCγ2 autoactivation. Our findings demonstrated that the PLCG2 D993Y variant is a gain-of-function mutation via impairing its autoinhibition, activating multiple inflammatory signaling pathways, thus leading to APLAID syndrome. This study further broadens the molecular underpinnings and phenotypic spectrum of PLCγ2-related disorders.

Bioequivalence Analysis of Terazosin Hydrochloride Tablets Based on Parallel Artificial Membrane Permeability Analysis.

Parallel artificial membrane permeability analysis (PAMPA) is used to determine the permeability of compounds through concentrated negatively charged phospholipid bilayer barriers. We employed MacroFlux (a scaled-up version of PAMPA) to test the permeation rate of terazosin hydrochloride (TH) tablets and predict in vivo bioequivalence. The dissolution profiles and permeability of one reference formulation, and seven generic TH tablets, were compared. The dissolution profiles of these generic tablets were equivalent to that of the reference drug in four different media. However, the flux and the total permeated amount of some generic TH tablets were below the lower limit of the confidence interval of the original acceptance range in MacroFlux, which implied risk in the bioequivalence test in vivo. We further evaluated potential factors responsible for this discrepancy by µFlux, including active pharmaceutical ingredient (API) permeability and excipient prescriptions. The analysis showed that different properties of API were a main factor leading to biological inequivalence in the MacroFlux assay, while excipient prescriptions did not have an impact on bioequivalence risk. These data indicated that the flux assay may be a helpful as an auxiliary method for predicting bioequivalence of generic drugs and analyze the factors responsible for bioequivalence risk.

Abstract Mo098: LMNA R190W Mutation Associated With Dilated Cardiomyopathy Causes Hypercontractility in iPSC-derived Cardiomyocytes and Engineered Heart Muscles

Familial dilated cardiomyopathy (DCM) caused by mutations in sarcomeric proteins have been extensively studied in vitro , however less is known about DCM caused by alterations in non-sarcomeric genes like Lamin A (LMNA). LMNA is a ubiquitously expressed nuclear intermediate filament protein that regulates nuclear structure, chromatin access and gene expression. LMNA mutations account for 6% of DCM, and affected patients develop drug-therapy resistant severe heart failure and lethal arrythmias that are not amenable to currently available therapies. Most identified LMNA-cardiomyopathy mutations have been associated with severely depressed cardiac systolic function in patients and cardiomyocyte hypocontractility in vitro . However, the underlying mechanisms are incompletely understood. This project examines the mechanisms by which a patient-derived pathogenic mutation, LMNA R190W, leads to cardiac dysfunction at the cellular and tissue levels. We hypothesize that LMNA R190W mutation causes dysregulated expression of calcium handling genes, eventually leading to altered contractility. First, we investigated early disease pathogenesis using iPSC-derived cardiomyocytes. Intriguingly, single cardiomyocyte traction force microscopy studies showed cardiomyocyte hypercontractility, and calcium flux assays revealed prolonged calcium transients. Second, in human engineered heart muscle tissues, LMNA CMs were also consistently hypercontractile. Through gene expression studies, we identified upregulation of a cardiac-specific sarcomeric calcium regulator, calsequestrin 2 (CASQ2), which modulates intracellular calcium availability and localization, and thereby indirectly impacts calcium-dependent contraction and electrophysiology. siRNA-mediated knockdown of CASQ2 in iPSC-CMs decreased cellular calcium content, suggesting Casq2 is necessary for increased calcium and contractility in LMNA R190W cells. These findings suggest that in the initial stages of LMNA R190W-associated cardiomyopathy, cardiomyocyte contractility is not diminished, but actually augmented, and this may be related to aberrant calcium handling. We posit that chronic inappropriate calcium overload may eventually lead to cell death, and predispose to cardiomyopathy. Understanding the initial processes driving the development of LMNA cardiomyopathy will inform more accurate mechanism-based subclassification of mutations, and the rational development of precision therapies.

Abstract We037: GPC3-mediated metabolic rewiring of diabetic mesenchymal stromal enhances their cardioprotective functions via pyruvate kinase M2 activation

Background: Mesenchymal stromal cells (MSC) represent promising stem cell therapy for treating cardiovascular diseases. Diabetes affects the functional capability of MSC and impedes cell-based therapy. However, the impact of diabetes on MSC myocardial reparative activity, metabolic fingerprint, and the mechanism of dysfunction is still poorly understood. Therefore, our study aimed to understand the molecular basis of diabetes-induced MSC functions and their metabolic mechanism, and potential metabolic reprogramming of diabetic MSC to reverse these dysfunctions may represent a strategy to enhance cell-based therapeutics for myocardial repair in diabetic patients. Hypothesis: Our central hypothesis is that increased Glypican-3 (GPC3) mediates diabetic MSC dysfunction and diminishes energy metabolism; metabolic reprogramming of diabetic MSC with GPC3 knockdown restored diabetic MSC reparative and therapeutic activities in post-MI cardiac repair. Methods: MSCs were isolated from the bone marrow of mice from db/+ non-diabetic (WT-MSC) and diabetic mice (db/db-MSC) to compare the MSC metabolic profiles and consequent reparative function. We investigated the effect of GPC3 knockdown on db/db-MSC energy metabolism, immunosuppression, angiogenic potential in vitro, and therapeutic potential for ischemic cardiac tissue repair. Results: Intra-myocardial transplantation of db/db-MSC into the ischemic myocardium of mice does not confer cardiac benefit post-MI. The seahorse metabolic flux assays and 13 C glucose tracing studies identified defective energy metabolism in db/db-MSC. Furthermore, we found that GPC3, a heparan sulfate proteoglycan, is highly upregulated in db/db-MSC. GPC3 knockdown-db/db-MSC restored their energy metabolism, immunomodulation, and angiogenesis. Intramyocardial injection of GPC3 metabolically reprogrammed-db/db-MSC confers cardiac benefit post-MI. Conclusion: Our findings indicate that metabolic rewiring using GPC3 may be used to restore the metabolic state and functions of db/db-MSC for cell therapy applications.

Abstract We006: Metabolic Reprogramming to Increase Mitochodnrial Mass, Fusion and Energetics Represents an Important Rate Limiting Step in Direct Cardiac Reprogramming

Direct reprogramming of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) shows promise for treating myocardial infarction (MI). However, low conversion efficiency and relative immaturity of iCMs have so far precluded its clinical translation. Native CM maturation and cell cycle exit is associated with a switch from glycolytic metabolism to mitochondrial fatty acid oxidation (FAO), accompanied by a shift in the dynamic balance of mitochondrial fission (dividing) and fusion (joining) toward fusion. Because CFs lack the energy requirements of CMs, we hypothesized that transdifferentiation of CFs to iCMs requires a similar switch. Combining confocal microscopy with metabolic flux assays, we directly compared native CF and CM mitochondrial morphology and energetics. CMs showed significantly higher mitochondrial mass, fusion and energetics, all metrics relevant for increased reliance on mitochondrial FAO. Applying these techniques to study reprogramming, we found that at later reprogramming time points key metrics move significantly toward the phenotypes of native CMs. We then hypothesized that facilitating metabolic reprogramming by inhibiting fission and/or promoting fusion could improve iCM conversion. A loss of function screen identified certain fission genes, particularly the little studied mitochondrial fission regulator 1-like protein (Mtfr1l), as potent barriers to iCM conversion and maturation. In contrast, the core fusion genes were necessary for successful generation of iCMs, particularly the key fusion effector optical atrophy 1 (Opa1). Mtfr1l has been shown to promote cleavage of Opa1, and indeed concurrent knockdown of Opa1 ablated improvements to reprogramming efficiency. In conclusion, we showed both that iCMs are associated with increased mitochondrial mass, fusion and energetics approaching those of native CMs and that inducing CM-like mitochondria by knocking down Mtfr1l dramatically improved reprogramming, potentially through increased fusion activity of Opa1. By incorporating metabolic reprogramming into traditional direct cardiac reprogramming strategies, we may be able to move the field closer to clinical application to improve outcomes after MI.

Baicalin induces cell death of non-small cell lung cancer cells via MCOLN3-mediated lysosomal dysfunction and autophagy blockage

BackgroundNon-small cell lung cancer (NSCLC) accounts for 85 % of lung cancer, becoming the most mortality of all cancers globally. Blockage of autophagy in NSCLC represents a promising therapeutic strategy that inhibits angiogenesis and overcomes drug resistance. Natural ingredients in anti-tumor adjuvants are increasingly reported to promote cell death with less side effects and the potential to increase chemotherapeutic drugs sensitivity. Baicalin, a Scutellaria baicalensis-extracted flavonoid glycoside, is reported to induce death of NSCLC cells, however, its effects on autophagy in NSCLC cells remain unclear. PurposeThis study aimed to investigate the effect of baicalin on autophagic flux in NSCLC cells, unraveling the underlying mechanism including potential target and its role in cell death of NSCLC cells. MethodsIn vitro anti-cancer effects of baicalin were verified by evaluating proliferation, clone formation, cell cycle, and cell migration in three NSCLC cell lines (A549, H1299, and PC-9). In vivo anti-tumor efficacies of baicalin were evaluated in subcutaneous xenograft tumor model in nude mice. Autophagy characterization in NSCLC cells included autophagic marker detection by western blot and immunofluorescence staining, subcellular structure observation by TEM, lysosomal function by RNA-seq and fluorescence staining (LysoTracker®, LysoSensor®, and acridine orange). Based on RNA-seq and molecular biological verification using apoptotic, autophagic, and lysosomal inhibitors, potential target molecule of baicalin was verified via Ca2+ flux assay, MCOLN3 knockdown by shRNA, and virtual molecular docking. ResultsBaicalin inhibited NSCLC cell proliferation and migration, and suppressed tumor growth in vivo. Baicalin blocked the autophagic flux via activating the membranal cation channel MCOLN3 of lysosome, which disrupted its Ca2+ balance and induced lysosome dysfunction, leading to failure of autolysosome degradation. The cytoplasmic Ca2+ imbalance further resulted in depolarization of mitochondrial membrane potentials and ROS accumulation in NSCLC cells, mediating autophagy-related apoptosis. ConclusionThis study demonstrated that baicalin inhibited autolysosome degradation by activating MCOLN3, leading to dysfunction in lysosomal pH elevation, thereby inhibiting autophagy in NSCLC, leading to apoptotic death of NSCLC cells. These findings enriched the existing theories of cancer therapy based on autophagy inhibition and underlying mechanisms of flavonoids as antitumor agents, paving the way for their clinical application in future.

Molecular and clinical characterization of a founder mutation causing G6PC3 deficiency.

G6PC3 deficiency is a monogenic immunometabolic disorder that causes syndromic congenital neutropenia. Patients display heterogeneous extra-hematological manifestations, contributing to delayed diagnosis. Here, we investigated the origin and functional consequence of the G6PC3 c.210delC variant found in patients of Mexican origin. Based on the shared haplotypes amongst carriers of the c.210delC mutation, we estimated that this variant originated from a founder effect in a common ancestor. Furthermore, by ancestry analysis, we concluded that it originated in the indigenous Mexican population. At the protein level, we showed that this frameshift mutation leads to an aberrant protein expression in overexpression and patient-derived cells. G6PC3 pathology is driven by the intracellular accumulation of the metabolite 1,5-anhydroglucitol-6-phosphate (1,5-AG6P) that inhibits glycolysis. We characterized how the variant c.210delC impacts glycolysis by performing extracellular flux assays on patient-derived cells. When treated with 1,5-anhydroglucitol (1,5-AG), the precursor to 1,5-AG6P, patient-derived cells exhibited markedly reduced engagement of glycolysis. Finally, we compared the clinical presentation of patients with the mutation c.210delC and all other G6PC3 deficient patients reported in the literature to date, and we found that c.210delC carriers display all prominent clinical features observed in prior G6PC3 deficient patients. In conclusion, G6PC3 c.210delC is a loss-of-function mutation that arose from a founder effect in the indigenous Mexican population. These findings may facilitate the diagnosis of additional patients in this geographical area. Moreover, the in vitro 1,5-AG-dependent functional assay used in our study could be employed to assess the pathogenicity of additional G6PC3 variants.

Myeloid cannabinoid CB1 receptor deletion confers atheroprotection in male mice by reducing macrophage proliferation in a sex-dependent manner.

Although the cannabinoid CB1 receptor has been implicated in atherosclerosis, its cell-specific effects in this disease are not well understood. To address this, we generated a transgenic mouse model to study the role of myeloid CB1 signalling in atherosclerosis. Here, we report that male mice with myeloid-specific Cnr1 deficiency on atherogenic background developed smaller lesions and necrotic cores than controls, while only minor genotype differences were observed in females. Male Cnr1-deficient mice showed reduced arterial monocyte recruitment and macrophage proliferation with less inflammatory phenotype. The sex-specific differences in proliferation were dependent on oestrogen receptor (ER)α-oestradiol signalling. Kinase activity profiling identified a CB1-dependent regulation of p53 and cyclin-dependent kinases. Transcriptomic profiling further revealed chromatin modifications, mRNA processing, and mitochondrial respiration among the key processes affected by CB1 signalling, which was supported by metabolic flux assays. Chronic administration of the peripherally restricted CB1 antagonist JD5037 inhibited plaque progression and macrophage proliferation, but only in male mice. Finally, CNR1 expression was detectable in human carotid endarterectomy plaques and inversely correlated with proliferation, oxidative metabolism, and inflammatory markers, suggesting a possible implication of CB1-dependent regulation in human pathophysiology. Impaired macrophage CB1 signalling is atheroprotective by limiting their arterial recruitment, proliferation, and inflammatory reprogramming in male mice. The importance of macrophage CB1 signalling appears to be sex-dependent.

Fatty acid oxidation-induced HIF-1α activation facilitates hepatic urate synthesis through upregulating NT5C2 and XDH

Abstract Dyslipidemia affects approximately half of all people with gout, and prior Mendelian randomization analysis suggested a causal role for elevated triglycerides in hyperuricemia (HU), but the underlying mechanisms remain elusive. We hypothesize that dyslipidemia promotes hepatic urate biosynthesis in HU and gout and fatty acid (FA) oxidation (FAO) drives this process. Here we developed a targeted metabolomics to quantify major metabolites in purine metabolic pathway in the sera of a human cohort with HU, gout, and normaluricemic controls. We found that the levels of major purine metabolites and multiple FAs were significantly elevated in HU and gout, compared to normouricemic controls, whereas hypoxathine showed opposite trend. Furthermore, the levels of multiple serum FAs were positively correlated with urate, xanthine, and inosine but negatively with hypoxanthine, which was also observed in a murine model of high-fat diet-induced HU. Using a stable isotope labeled metabolic flux assay, we discovered that exogenous hypoxanthine plays a key role in urate synthesis. Moreover, FAO-induced hypoxia-inducible factor 1 alpha (HIF-1α) activation upregulated 5’-nucleotidase II (NT5C2) and xanthine dehydrogenase (XDH) levels to facilitate hypoxanthine uptake from blood to liver and activation of urate biosynthesis. Our findings was further supported by data in human hepatocytes and 50 paired serum and liver tissues from liver transplant donors.Together, this study uncovers a mechanism by which FAO promotes hepatic urate synthesis by activating HIF-1α-NT5C2/XDH pathways, directly linking lipid metabolism to HU.

Skeletal Muscle Derived Cells from Old Human Donors Show Increased Mitochondrial Lipid Peroxidation and Reduced Mitochondrial Function

Skeletal muscle undergoes atrophy with age progression resulting in decreased mitochondrial function. Similarly, age-related increases in mitochondrial oxidant generation can promote mitochondrial oxidative damage, but the relationship between increased oxidant stress and mitochondrial function is not well understood. Here, we profiled changes in mitochondrial function and mitochondrial lipid peroxidation in intact primary skeletal muscle progenitor cells (myoblasts) derived from the Rectus abdominis muscle of healthy young (19.67 ± 1.70 years) and old (67.0 ± 1.41 years) humans. Using metabolic flux assays, we found that cells derived from young humans showed higher basal and maximal oxygen consumption rates compared to cells derived from old humans (basal: 22.67 ± 6.53 vs. 10.81 ± 5.69; maximal: 41.02 ± 15.20 vs. 17.33 ± 4.84 pmol/min/protein, p < 0.001). Similarly, cells derived from young humans also showed higher ATP production and spare respiratory capacity (ATP: 18.79 ± 5.17 vs. 9.15 ± 4.91; SRC: 18.35 ± 9.62 vs. 6.52 ± 3.42 pmol/min/protein, p < 0.001). In contrast, using live-cell fluorescence microscopy, MitoTracker red and the mitochondrial-target lipid peroxide probe MitoPeDPP, we found higher mitochondrial lipid peroxidation levels in cells derived from old than young humans ( p = 0.0007). We then evaluated whether peroxiredoxin 6 (Prdx6), an enzyme that repairs lipid peroxidation through its phospholipid glutathione peroxidase and phospholipase A2 activities, impacts mitochondrial function using CRISPR/Cas9-mediated knockouts of Prdx6 in C2C12 myoblasts and extracellular flux assays. Spare respiratory capacity was lower in cells with full Prdx6-KO than in cells transfected with non-target gRNAs (NT: 295.10 ± 18.59 vs. Prdx6-KO: 242.20 ± 17.93; p = 0.05). These results show that mitochondrial function decreases with age along with increases in mitochondrial lipid peroxidation in primary muscle cells derived from old humans. Similarly, these results show that the Prdx6 supports mitochondrial function. Overall, our data contributes to our understanding of the mechanistic drivers of age-related skeletal muscle mitochondrial dysfunction. NIGMS-R35GM146951 NIA-R01AG059715. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ANGPTL2 knockdown induces autophagy to relieve alveolar macrophage pyroptosis by reducing LILRB2-mediated inhibition of TREM2.

Acute lung injury (ALI) is featured with a robust inflammatory response. Angiopoietin-like protein 2 (ANGPTL2), a pro-inflammatory protein, is complicated with various disorders. However, the role of ANGPTL2 in ALI remains to be further explored. The mice and MH-S cells were administrated with lipopolysaccharide (LPS) to evoke the lung injury invivo and invitro. The role and mechanism of ANGPTL was investigated by haematoxylin-eosin, measurement of wet/dry ratio, cell count, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling, reverse transcription quantitative polymerase chain reaction, immunofluorescence, enzyme-linked immunosorbent assay, detection of autophagic flux and western blot assays. The level of ANGPTL2 was upregulated in lung injury. Knockout of ANGPTL2 alleviated LPS-induced pathological symptoms, reduced pulmonary wet/dry weight ratio, the numbers of total cells and neutrophils in BALF, apoptosis rate and the release of pro-inflammatory mediators, and modulated polarization of alveolar macrophages in mice. Knockdown of ANGPTL2 downregulated the level of pyroptosis indicators, and elevated the level of autophagy in LPS-induced MH-S cells. Besides, downregulation of ANGPTL2 reversed the LPS-induced the expression of leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) and triggering receptor expressed on myeloid cells 2 (TREM2), which was reversed by the overexpression of LILRB2. Importantly, knockdown of TREM2 reversed the levels of autophagy- and pyroptosis-involved proteins, and the contents of pro-inflammatory factors in LPS-induced MH-S cells transfected with si ANGPTL2, which was further inverted with the treatment of rapamycin. Therefore, ANGPTL2 silencing enhanced autophagy to alleviate alveolar macrophage pyroptosis via reducing LILRB2-mediated inhibition of TREM2.

Bioequivalence prediction with small-scale biphasic dissolution and simultaneous dissolution-permeation apparatus—An aripiprazole case study

Both biphasic dissolution and simultaneous dissolution-permeation (D-P) systems have great potential to improve the in vitro-in vivo correlation compared to simple dissolution assays, but the assay conditions, and the evaluation methods still need to be refined in order to effectively use these apparatuses in drug development.Therefore, this comprehensive study aimed to compare the predictive accuracy of small-volume (16–20 mL) D-P system and small-volume (40–80 mL) biphasic dissolution apparatus in bioequivalence prediction of five aripiprazole (ARP) containing marketed drug products.Assay conditions, specifically dose dependence were studied to overcome the limitations of both small-scale systems. In case of biphasic dissolution the in vivo maximum plasma concentration (Cmax) prediction greatly improved with the dose reduction of ARP, while in case of the D-P setup the use of whole tablet gave just as accurate prediction as the scaled dose. With the dose reduction strategy both equipment was able to reach 100 % accuracy in bioequivalence prediction for Cmax ratio. In case of the in vivo area under the curve (AUC) prediction the predictive accuracy for the AUC ratio was not dependent on the dose, and both apparatus had a 100 % accuracy predicting bioequivalence based on AUC results.This paper presents for the first time that not only selected parameters of flux assays (like permeability, initial flux, AUC value) were used as an input parameter of a mechanistic model (gastrointestinal unified theory) to predict absorption rate but the whole in vitro flux profile was used. All fraction absorbed values estimated by Predictor Software fell within the ±15 % acceptance range during the comparison with the in vivo data.