Membrane Potential Measurements Research Articles

Shikonin Stimulates Mitochondria-Mediated Apoptosis by Enhancing Intracellular Reactive Oxygen Species Production and DNA Damage in Oral Cancer Cells.

Phytotherapy has rendered a new insight towards the treatment of various cancers, including oral cancer with fewer side effects, over the traditional chemotherapeutic drugs to overcome chemoresistance. Shikonin (Shk) is a natural biologically active alkaloid found in the Lithospermum erythrorhizon plant's root. It has potent cytotoxic activities against various cancers. Our study revealed the release time and anticancer potential of Shk on the SCC9 and H357 oral cancer cell lines. We investigated the antiproliferative, antimigratory, cell cycle arresting and apoptosis promoting activity of Shk in oral cancer cells by performing MTT and morphological assay, colony, and tumor sphere formation assay, AO/EtBr and DAPI staining, Annexin V-FITC/PI staining, assay for reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) measurement, comet assay, qRT-PCR, and western blot analysis. We also checked the interaction of DNA and Shk by docking and CD spectroscopy and EtBr displacement assay. As a result, we found that Shk reduced the viability, proliferation, and tumorigenicity of SCC9 and H357 cells in a time and concentration-dependent manner. We obtained half-maximal inhibitory concentration (IC50) at 0.5 µM for SCC9 and 1.25 µM for H357. It promotes apoptosis via overexpressing proapoptotic Bax and caspase 3 via enhancing ROS that leads to MMP depletion and DNA damage and arrests cells at the G2/M & G2/S phase. The antimigratory activity of Shk was performed by analyzing the expression of markers of epithelial-mesenchymal transition like E-cadherin, ZO-1, N-cadherin, and vimentin. These overall results recommended that Shk shows potent anticancer activity against oral cancer cell lines in both in vitro and ex vivo conditions. So, it could be an excellent agent for the treatment of oral cancer.

Investigation of Apoptotic and Anticancer Effects of 2-substituted Benzothiazoles in Breast Cancer Cell Lines: EGFR Modulation and Mechanistic Insights.

Benzothiazole derivatives, a class of heterocyclic compounds, exhibited diverse biological activities influenced by substituents in the thiazole ring. This study aimed to synthesize these compounds with two functional groups to investigate their potential as anticancer agents, particularly against breast cancer. While previous research demonstrated the efficacy of 2-substituted benzothiazoles against glioma and cervical and pancreatic cancer cells, there is a gap in studies targeting breast cancer. The synthesized compounds were tested in vitro using MCF-7, MDA-MB-231, and MCF-10A cell lines, with Doxorubicin as the positive control. Various assays were conducted, including Annexin V/PI, cell cycle analysis, wound healing, and measurement of mitochondrial membrane potential. Protein expression of EGFR and transcription levels of apoptosis-related genes (Bax and Bcl-xL) and cancer progression-related genes (JAK, STAT3, ERK, AKT, mTOR) were analyzed. Additionally, the balance between antioxidants and oxidants was evaluated by measuring TAS and TOS levels. Our findings revealed that benzothiazole compounds significantly inhibited breast cancer cell growth by reducing cell motility, disrupting mitochondrial membrane potential, and inducing cell cycle arrest in the sub-G1 phase. These compounds increased reactive oxygen species accumulation, leading to cell death. Furthermore, they decreased EGFR protein levels, increased Bax gene transcription, and downregulated the expression of genes such as JAK, STAT3, ERK, AKT, and mTOR. In conclusion, benzothiazole derivatives exhibited potent inhibitory effects on breast cancer in vitro by promoting apoptosis, downregulating EGFR activity, and modulating key signaling pathways, including JAK/STAT, ERK/MAPK, and PI3K/Akt/mTOR. These results highlighted the potential of benzothiazole derivatives as novel therapeutic agents for breast cancer treatment.

Mitochondrial Dysfunction and Ion Imbalance in a Rat Model of Hemodialysis-Induced Myocardial Stunning.

Background/Objectives: Hemodialysis-induced myocardial stunning (HIMS) is a frequent complication in patients undergoing maintenance hemodialysis, characterized by transient left ventricular dysfunction due to ischemic episodes. Mitochondrial dysfunction and fluctuations in key ions such as potassium (K+) and calcium (Ca2+) are implicated in the pathogenesis of HIMS. This study aims to investigate the role of mitochondrial dysfunction and the protective potential of mitochondrial ATP-sensitive potassium channels (mitoKATP) in mitigating HIMS. Methods: A 5/6 nephrectomy rat model was established to mimic chronic kidney disease and the subsequent HIMS. The effects of mitoKATP channel modulators were evaluated by administering diazoxide (DZX), a mitoKATP opener, and 5-hydroxydecanoate (5-HD), a mitoKATP blocker, before hemodialysis. Mitochondrial function was assessed by measuring membrane potential, ATP synthase activity, and intramitochondrial Ca2+ levels. Myocardial function was evaluated using speckle tracking echocardiography. Results: Rats undergoing hemodialysis exhibited significant reductions in left ventricular strain and synchrony. DZX administration significantly improved mitochondrial function and reduced myocardial strain compared to controls. Conversely, 5-HD worsened mitochondrial swelling and disrupted myocardial function. Higher K+ and Ca2+ concentrations in the dialysate were associated with improved mitochondrial energy metabolism and myocardial strain. Conclusions: Mitochondrial dysfunction and ion imbalances during hemodialysis are key contributors to HIMS. The activation of mitoKATP channels provides mitochondrial protection and may serve as a potential therapeutic strategy to mitigate HIMS.

Active fraction of ground cherry (Physalis angulata L.) calyces attenuates azoxymethane dextran sulfate sodium‑induced colon carcinogenesis in mice.

Physalis angulata L., commonly known as wild tomato or ground cherry, is widely used in tropical and subtropical areas to treat health disorders including inflammation, hepatitis, dermatitis, cancer and diabetes. In Colombia, anti-cancer and anti-inflammatory activity are the most common ethnopharmacological applications of P. angulata calyces. P. angulata dichloromethane fraction (PADF) has significant anti-inflammatory activity. The present study assessed the pharmacological effect of PADF on colorectal cancer (CRC) using cancer and normal human cells and an azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model. MTT and clonogenic assay, cell cycle and apoptosis analysis and mitochondrial membrane potential measurement were employed to evaluate in vitro activity of PADF. PADF selectively induced a cytotoxic effect against CRC cells via apoptosis and G2/M arrest. In the AOM/DSS model, treatment with PADF diminished tumor number and size, affected area and expression of proliferating cell nuclear antigen and promoted colon tissue repair. These effects might be related to the increased expression of p38 pro-apoptotic protein in addition to anti-inflammatory activity of PADF demonstrated by decreased levels of TNF-α, IL-6, and IL-1β. PADF may serve as a potential treatment for CRC. Further investigation is warranted to identify the bioactive components in PADF.

Inhibition of ACSS2 triggers glycolysis inhibition and nuclear translocation to activate SIRT1/ATG5/ATG2B deacetylation axis, promoting autophagy and reducing malignancy and chemoresistance in ovarian cancer

BackgroundMetabolic reprogramming is a hallmark of cancer, characterized by a high dependence on glycolysis and an enhanced utilization of acetate as an alternative carbon source. ACSS2 is a critical regulator of acetate metabolism, playing a significant role in the development and progression of various malignancies. ACSS2 facilitates the conversion of acetate to acetyl-CoA, which participates in multiple metabolic pathways and functions as an epigenetic regulator of protein acetylation, thereby modulating key cellular processes such as autophagy. However, the roles and intrinsic connections of ACSS2, glycolysis, protein acetylation, and autophagy in ovarian cancer (OC) remain to be elucidated. Basic proceduresUtilizing clinical specimens and online databases, we analysed the expression of ACSS2 in OC and its relationship with clinical prognosis. By knocking down ACSS2, we evaluated its effects on the malignant phenotype, acetate metabolism, glycolysis, and autophagy. The metabolic alterations in OC cells were comprehensively analysed using Seahorse assays, transmission electron microscopy, membrane potential measurements, and stable-isotope labeling techniques. CUT&TAG and co-immunoprecipitation techniques were employed to explore the deacetylation of autophagy-related proteins mediated by ACSS2 via SIRT1. Additionally, through molecular docking, transcriptome sequencing, and metabolomics analyses, we validated the pharmacological effects of paeonol on ACSS2 and the glycolytic process in OC cells. Finally, both in vitro and in vivo experiments were performed to investigate the impact of paeonol on autophagy and its anti-OC effects mediated through the ACSS2/SIRT1 deacetylation axis. Main findingsACSS2 is significantly upregulated in OC and is associated with poor prognosis. Knockdown of ACSS2 inhibits OC cells proliferation, migration, invasion, angiogenesis, and platinum resistance, while reducing tumour burden in vivo. Mechanistically, inhibiting ACSS2 reduces acetate metabolism and suppresses glycolysis by targeting HXK2. This glycolytic reduction promotes the translocation of ACSS2 from the cytoplasm to the nucleus, leading to increased expression of the deacetylase SIRT1. SIRT1 mediates the deacetylation of autophagy-related proteins, such as ATG5 and ATG2B, thereby significantly activating autophagy in OC cells and exerting antitumor effects. Paeonol inhibits acetate metabolism and glycolysis in OC cells by targeting ACSS2. Paeonol activates autophagy through the ACSS2/SIRT1/ATG5/ATG2B deacetylation axis, demonstrating inhibition of OC in vitro and in vivo. Principal conclusionsPae can serve as an effective, low-toxicity, multi-targeted drug targeting ACSS2 and glycolysis. It activates autophagy through the ACSS2/SIRT1/ATG5/ATG2B deacetylation signalling cascade, thereby exerting anti-OC effects. Our study provides new insights into the malignant mechanisms of OC and offers a novel strategy for its treatment.

Integrated stress response activation induced by usnic acid alleviates BCL-2 inhibitor ABT-199 resistance in acute myeloid leukemia

IntroductionABT-199 (venetoclax) is a BCL-2 suppressor with pronounced effects on acute myeloid leukemia (AML). However, its usefulness as a monotherapy or in combination with hypomethylating medicines like azacitidine is debatable due to acquired resistance. Usnic acid, a dibenzofuran extracted from lichen Usnea diffracta Vain, exhibits anticancer properties and may counteract multidrug resistance in leukemia cells. ObjectiveThis study investigated whether usnic acid at low-cytotoxicity level could enhance sensitivity of AML cells with acquired resistance to ABT-199 by targeting the integrated stress response pathways. MethodsTo investigate the combined effects on AML cells, we used a cell viability test, flow cytometry to quantify apoptosis, cell cycle analysis, and mitochondrial membrane potential measurement. RNA-seq and immunoblot were used to determine the potential mechanisms of ABT-199 + usnic acid combination. ResultsUsnic acid, at a low cytotoxicity level, successfully restored ABT-199 sensitivity in AML cell lines that had developed ABT-199 resistance and increased ABT-199′s antileukemic activity in a xenograft model. Mechanistically, the combination of usnic acid and ABT-199 cooperated to boost the expression of the integrated stress response (ISR)-associated genes ATF4, CHOP, and NOXA through the heme-regulated inhibitor kinase (HRI), while also promoting the degradation of the anti-apoptotic protein MCL-1. ISRIB, a compound that blocks the ISR, was able to reverse the growth suppression and cell death, the increase in expression of genes related with the ISR, and the inhibition of MCL-1 protein caused by combination therapy. Additionally, the downregulation of MCL-1 was linked to an increase in MCL-1 phosphorylation at serine 159 and subsequent destruction by the proteasome. ConclusionIn summary, usnic acid improves chemosensitivity to ABT-199 by triggering the integrated stress response, leading to decreased levels of MCL-1 protein, suggesting a potential treatment for AML cases resistant to Bcl-2 inhibitors.

Enhancement of gold-curcumin nanoparticle mediated radiation response for improved therapy in cervical cancer: a computational approach and predictive pathway analysis.

Radiotherapy is prevalently applied for highly effective cancer therapy while the low specificity of radiation is deleterious to the nearby healthy cells. High-Z-based nanomaterials offer excellent radio-enhancement properties while natural products provide radioprotection. Modulation of the radiotherapeutic index via applying nanomaterials is feasible for effective treatment however, the scenario changes when simultaneous protection of non-cancerous cells is required. Here, we report the modulatory radiotherapeutic effect of curcumin conjugated gold nanoparticles in a single nanoformulation to pave the long-awaited hope of a single combination-based, cell-selective radio enhancer, and protectant for cancer radiotherapy. We have validated the effective radiation dose along with the combination of the radio-nano-modulator by a reverse experimentation statistical model. The concept was supported by different sets of experiments, like quantification of ROS generation, cell cycle monitoring, mitochondrial membrane potential measurement, etc. along with gene expression study, and predictive modeling of molecular pathways of the killing mechanism. In conclusion, the nanoconjugate showed a promise to become a candidate for the pH-dependent cell-specific radio-modulator.

Development of Mitoxantrone-Loaded Quercetin Nanoparticles for Breast Cancer Therapy with Potential for Synergism with Bioactive Natural Products

Nanoparticle (NP)-based drug delivery systems have caused a paradigm shift in cancer treatment by enabling drug targeting, sustaining drug release, and reducing systemic toxicity of chemotherapy. Here we developed a novel NP formulation for the anticancer drug mitoxantrone (MTZ) by loading it into an emerging nanomaterial derived from the plant polyphenol quercetin (QCT). QCT was partially oxidized to produce amphiphilic oxQCT which was co-assembled with poly(ethylene glycol) (PEG) and MTZ by nanoprecipitation to form MTZ NPs. The optimal NPs exhibited an average diameter of 128 nm, a polydispersity index of 0.22, and a drug loading efficiency of 76%. While only a small fraction of the loaded drug was released at physiologic pH, a significantly higher fraction was released at acidic pH. The anticancer activity of MTZ NPs was assessed in MCF-7 and MDA-MB-231 breast cancer cell lines, alone and in combination with the bioactive natural products curcumin (CUR) and thymoquinone (TQ). In cell viability assays, MTZ NPs were slightly less potent than free MTZ, most likely due to their sustained release properties, but their cytotoxicity was greatly enhanced in the presence of TQ (in MCF-7 cells) as well as CUR (in MDA-MB-231 cells). The results were corroborated by apoptosis assays such as mitochondrial membrane potential measurement, acridine orange/ethidium bromide staining, in addition to caspase activity assays. The assays revealed that the NPs’ proapoptotic effect was enhanced in the presence of CUR or TQ, depending on the cell line. Our work presents a promising nanocarrier platform for MTZ with the potential to enhance its bioactivity against breast cancer when combined with bioactive natural products.

Optimizing therapeutic efficacy of antifungal peptides via strategic terminal amino acid modification

IntroductionAntifungal peptides (AFPs) have the potential to treat antifungal-resistant infections; however, their structure–function relationship remains unknown, hindering their rapid development. Therefore, it is imperative to investigate and clarify the structure–function relationships of AFPs. ObjectivesThis study aimed to investigate the impact of end-tagging single hydrophobic amino acids and capping the N-terminus with glycine (Gly) on the antifungal activity of peptide W4. MethodsThe antifungal efficacy of the engineered peptides was initially assessed by determining the minimum inhibitory concentration (MIC) /minimal fungicidal concentration (MFC), killing kinetics, and drug resistance induction, in addition to evaluating the biocompatibility and stability. Subsequently, the antifungal mechanism was investigated using fluorescence labeling, electron microscopy, reactive oxygen species (ROS) detection, and measurement of mitochondrial membrane potential and apoptosis. The impact of the engineered peptides on Candida albicans (C. albicans) biofilm and their potential application in the scratch keratomycosis model were investigated. ResultsThe antifungal activity of W4 was significantly enhanced by capping Gly at the N-terminus, resulting in a decrease in average activity from 11.86 μM to 6.25 μM (GW4) and an increase in TI values by 1.9-fold (TIGW4 = 40.99). Mechanistically, GW4 exerted its antifungal effect by disrupting the cellular membrane structure in C. albicans, forming pores and subsequent leakage of intracellular contents. Concurrently, it facilitated intracellular ROS accumulation while decreasing the mitochondrial membrane potential. Additionally, GW4 demonstrated an excellent ability to inhibit and eliminate biofilms of C. albicans. Notably, GW4 demonstrated significant therapeutic potential in a C. albicans-associated keratitis model. ConclusionCapping Gly at the N-terminus increased residue length while significantly enhancing the helical propensity of W4, thereby augmenting its antifungal activity. Our exploratory study demonstrated the potential strategies and avenues for optimizing the structure–function relationships of AFPs and developing highly effective antifungal drugs.

Analysis of the mechanism of berberine against stomach carcinoma based on network pharmacology and experimental validation.

Although the therapeutic effects of berberine have received some attention in recent years, its potential mechanisms underlying its action against stomach carcinoma (SC) remain unclear. In this study, we aimed to elucidate the mechanisms underlying the effects of berberine against SC using a network pharmacology and experimental verification approach. Several publicly available databases were used to collect the targets of berberine and SC. Protein-protein interaction (PPI) network, enrichment analyses and molecular docking were performed based on the potential targets of berberine against SC. The potential clinical significance and prognostic value of the targets were predicted by using nomogram and receiver operating characteristic (ROC) analyses. Then the viability and apoptosis of SC cells treated with berberine were determined. Moreover, reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) measurements and western blot assay were carried out to validate the predicted mechanisms. Seventy-six potential targets of berberine against SC were identified. The construction of PPI network enabled the identification of hub targets, such as AKT1, TP53, IL6, JUN and MAPK1. Enrichment analyses showed that berberine was involved in apoptosis, mitophagy, ROS metabolic process, AMPK and MAPK signaling pathway. The expression levels of hub targets also contributed to the clinical prognosis of patients with SC. Molecular docking revealed the possible patterns of direct interaction between berberine and target proteins, including AMPK, TP53 and MAPK1. Experimental results showed that berberine reduced SC cell viability, promoted apoptosis and ROS generation, and contributed to reductions in MMP and ATP levels. Western blot assay demonstrated that berberine increased AMPK and TP53 expression, while decreased phosphorylated-MAPK3/1 expression. We elucidated the potential action mechanisms of berberine against SC using a network pharmacology approach. Some predicted mechanisms underlying the anti-SC effects were verified based on experimental approaches. Our findings provide a meaningful foundation for berberine as a cellular apoptosis-inducing and energy metabolism-regulating agent against SC. However, in vivo experiments and clinical studies need to be further carried out. Moreover, it is necessary to study the potential negative effects of berberine thoroughly.

Evaluation of Hydrazide-hydrazone and 4-thiazolidinone Derivatives of Etodolac as Potential Anticancer Agents in Leukemia Cells

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs), which are commonly used for their anti-inflammatory and analgesic properties, have also been found to prevent cancer. (±)(R,S) Etodolac is an NSAID that belongs to the class of cyclooxygenase-2 inhibitors. Various derivatives of etodolac are synthesized to boost its anti-proliferative action and lessen its potential negative effects. In our earlier studies, some novel derivatives of etodolac exhibited stronger cytotoxic effects on prostate cell lines and had similar effects on leukemia cells in pre-screening experiments. Objective: Using the K562 leukemia cell line as a model, we sought to investigate the anti-cancer properties of a hydrazide-hydrozone derivative (SGK-205) and a 4-thiazolidinone derivative of etodolac (SGK-216). Materials and Methods: In the current investigation, SGK-205 and SGK-216 compounds were administered to K562 cells for 24 and 48 hours at concentrations of 10, 25, 50, 75 and 100 μM. Cell viability was assessed using the MTT test, and apoptosis by Annexin V-PI staining and mitochondrial membrane potential assays, together with mRNA expressions of apoptotic proteins. The levels of the proteins, HER2 and COX2, were also examined to evaluate COX2 inhibitory capacity. Results: In K562 cells, there was a definite dose-dependent response to SGK-205 and SGK-216 compounds. Results from MTT viability tests, together with mitochondrial membrane potential measurements and Annexin V-PI staining, revealed that SGK-216 and SGK-205 significantly outperformed etodolac in terms of their apoptotic and anti-proliferative activities. The concentration range of 10-20 M for both chemicals was sufficient to start biological responses. Apoptosis was also investigated through the expressions of pro- and anti-apoptotic proteins. Additionally, gene expression research demonstrated SGK-205 to be a beneficial substitute to etodolac in lowering COX-2 and human epidermal growth factor receptor-2 (HER2) expression. Conclusion: Our data indicated both derivatives to have higher anti-proliferative and apoptotic effects compared to etodolac. An overall assessment highlighting apoptotic induction potential, acceptable toxicity levels, a consistent dose-response relationship, and COX2 inhibitory actions, in particular, indicated SGK-205 as a viable novel therapeutic.

Abstract P357: Renal Male and Female Cortical Mitochondria Display Bioenergetic Differences in Response to Varying Substrates

Introduction: In general, renal disease susceptibility is higher in males than in age-matched females until menopause. The damage to renal proximal tubules (PT) is key to the onset of kidney disease development due to their high capacity for reabsorption and intense mitochondrial metabolism. Our previous data identified higher oxygen consumption in the renal cortex of healthy male Sprague Dawley rats and lower H 2 O 2 production compared to females. We hypothesize that sex differences in renal mitochondrial substrate reliance trigger differential bioenergetic responses in PTs, which contributes to kidney disease development later in life. Methods: 11-week-old male and female Sprague Dawley rats were acquired from Charles River Labs. The kidneys were flushed, and the renal cortex was used for ex vivo experiments. Mitochondria were isolated with differential centrifugation, and rovided with different substrate combinations: 10mM pyruvate and 2mM malate (PM), 5mM of glutamate, malate, and succinate (GMS), or 5mM succinate only (S). Spectrofluorimetry was employed to measure membrane potential (TMRM) and H 2 O 2 production (Amplex Red), XF24e Seahorse was used for oxygen consumption rate (OCR). OriginPro was used for statistical analysis (one-way ANOVA with post hoc test) and data displayed as mean ± SEM. Results: Seahorse revealed that male renal cortical mitochondria exhibit higher basal and maximum OCR compared to females when provided with the GMS or PM substrate combinations (39.4 ± 1.18 vs 26.5± 0.88, respectively for basal GMS, and 52.0 ± 2.33 vs 32.6 ± 1.24 maximum respiration GMS, p<0.001). However, similar OCR was observed when mitochondria were provided succinate only (31.2 ± 1.67 vs 28.7 ± 1.30, respectively for basal and 50.5 ± 2.49 vs 51.1 ± 1.76 maximum respiration, p>0.05). Mitochondrial membrane potential was higher in females compared to males when succinate only was provided (p<0.001), whereas similar mitochondrial membrane potential was observed between males and females when GMS or PM were used. Regardless of the substrate combination used, H 2 O 2 production was higher in female renal mitochondria compared to males (p<0.001). Conclusions: We report that male and female renal cortical mitochondria display unique bioenergetic properties in response to varying substrates. These basal bioenergetic differences may contribute to the difference in susceptibility to kidney diseases later in life.

Towards optical measurements of membrane potential values in Bacillus subtilis using fluorescence lifetime.

Membrane potential (MP) changes can provide a simple readout of bacterial functional and metabolic state or stress levels. While several optical methods exist for measuring fast changes in MP in excitable cells, there is a dearth of such methods for absolute and precise measurements of steady-state membrane potentials (MPs) in bacterial cells. Conventional electrode-based methods for the measurement of MP are not suitable for calibrating optical methods in small bacterial cells. While optical measurement based on Nernstian indicators have been successfully used, they do not provide absolute or precise quantification of MP or its changes. We present a novel, calibrated MP recording approach to address this gap. In this study, we used a fluorescence lifetime-based approach to obtain a single-cell resolved distribution of the membrane potential and its changes upon extracellular chemical perturbation in a population of bacterial cells for the first time. Our method is based on (i) a unique VoltageFluor (VF) optical transducer, whose fluorescence lifetime varies as a function of MP via photoinduced electron transfer (PeT) and (ii) a quantitative phasor-FLIM analysis for high-throughput readout. This method allows MP changes to be easily visualized, recorded and quantified. By artificially modulating potassium concentration gradients across the membrane using an ionophore, we have obtained a Bacillus subtilis-specific MP versus VF lifetime calibration and estimated the MP for unperturbed B. subtilis cells to be -65 mV and that for chemically depolarized cells as -14 mV. We observed a population level MP heterogeneity of ~6-10 mV indicating a considerable degree of diversity of physiological and metabolic states among individual cells. Our work paves the way for deeper insights into bacterial electrophysiology and bioelectricity research.

Preparation of PVA-based charged mosaic membrane using screen-printed method and evaluation of ion transport properties

In this study, a polyvinyl alcohol (PVA)-based charged mosaic membrane (CMM) with a stripe-shaped charged structure from PVA-modified block copolymers was fabricated on a non-fabric support using a screen-printed method. The membrane structure of the stained CMM indicated that the striped negatively (N)- and positively (P)-charged layers with 20 μ m thickness were formed on the support. The domain sizes of the N and P layers were 173 and 203 μ m, respectively. Membrane potential measurements revealed that the charge density in the N layers of the fabricated CMM (CM-1) was almost the same as that in the P layers. A diffusion dialysis test indicated that the CM-1 had an electrolyte permselectivity of 986 between 0.1 M KCl and 0.1 M sucrose; a value which is approximately 20 times higher than that of Desalton® which is the only commercially available CMM. A water permeation test showed that CM-1 exhibited negative osmosis, which is a unique behavior of CMMs with high electrolyte permselectivity. Absolute flux in the negative osmosis was maximum when the electrolyte concentration was 0.1 M. A piezo dialysis test at an initial concentration of 500 ppm NaCl and applied pressure of 0.7 MPa, indicated that the rejection of CM-1 yielded negative value, confirming that the membrane can desalt salty water via piezo dialysis.

Ion-selectivity of galvanic corrosion products formed in multi-material gaps under atmospheric corrosion conditions with anti-freezing salts

Several multi-materials, which are joints of high-strength steels and lightweight materials such as aluminum alloy and carbon fiber reinforced thermoplastic, were subjected to the cyclic corrosion test for simulating atmospheric corrosion of automotive bodies and outdoor exposure tests. The ion-selectivity of the corrosion products formed in the multi-material gaps was examined by a membrane potential measurement. The corrosion products of all examined joints showed anion-selectivity rather than cation-selectivity. Corrosion products formed in joint gaps were less effective in preventing the progression of galvanic corrosion, suggesting that sufficient atmospheric corrosion protection is required for the use of multi-materials.

In vitro and in silico investigations of the pro-apoptotic activity of Opuntia ficus-indica cladode extracts against K562 cells

Abstract Objectives Cladodes of Opuntia ficus-indica (OFI) are thought to be an excellent source of bioactive compounds, making them an aspirant for the manufacture of health-promoting compounds. This study aims at exploring the pro-apoptotic effects of spiny and thornless OFI cladode extracts on the human immortalized myelogenous leukemia cell line (K562). Methods The ethanol extraction method was used for preparing cladode extract. Cytotoxicity was evaluated by MTT assays. Membrane permeability/damage was detected by annexin V-binding assays, and mitochondrial damage/alteration was detected by mitochondrial membrane potential measurements. The protein expression quantities of Bax and Bcl-2 were assessed by Western blotting. The pro-apoptotic potentials of the main spiny and thornless OFI extract constituents were investigated structurally and mechanistically using protein–ligand docking and interaction profiling. Results Spiny OFI extract displayed a stronger cytotoxic effect than thornless OFI extract on K562 cells. In silico findings agreed with the pro-apoptotic action observed in vitro. Conclusions Finally, our findings imply that OFI extracts cause apoptosis in K562 cells in order to have anti-cancer effects.

The activation of AMPK/PGC-1α/GLUT4 signaling pathway through early exercise improves mitochondrial function and mitigates ischemic brain damage.

Mitochondria play a crucial role in maintaining cellular energy supply and serve as a source of energy for repairing nerve damage following a stroke. Given that exercise has the potential to enhance energy metabolism, investigating the impact of exercise on mitochondrial function provides a plausible mechanism for stroke treatment. In our study, we established the middle cerebral artery occlusion (MCAO) model in Sprague-Dawley rats and implemented early exercise intervention. Neurological severity scores, beam-walking test score, and weight were used to evaluate neurological function. The volume of cerebral infarction was measured by MRI. Nerve cell apoptosis was detected by TUNEL staining. Mitochondrial morphology and structure were detected by mitochondrial electron microscopy. Mitochondrial function was assessed using membrane potential and ATP measurements. Western blotting was used to detect the protein expression of AMPK/PGC-1α/GLUT4. Through the above experiments, we found that early exercise improved neurological function in rats after MCAO, reduced cerebral infarction volume and neuronal apoptosis, promoted the recovery of mitochondrial morphology and function. We further examined the protein expression of AMPK/PGC-1α/GLUT4 signaling pathway and confirmed that early exercise was able to increase its expression. Therefore, we suggest that early exercise initiated the AMPK/PGC-1α/GLUT4 signaling pathway, restoring mitochondrial function and augmenting energy supply. This, in turn, effectively improved both nerve and body function in rats following ischemic stroke.

Establishment of XRD fourier fingerprint identification method of realgar decoction pieces and its anti-tumor activity in tumor-in-situ transplanted mice

Ethnopharmacological relevanceRealgar, a traditional mineral Chinese medicine, has been used in China for more than 2000 years. It has been recorded in many ancient and modern works that it has anti-cancer and anti-tumor effects. Of course, colon cancer is also within the scope of its treatment. Realgar needs to be processed into realgar decoction pieces by water grinding before being used for medicine. To ensure the consistency of efficacy and quality of realgar decoction pieces, modern methods need to be used for further quality control. Aim of the studyThe research of traditional mineral Chinese medicine is relatively difficult, and the related research is less. The purpose of this study is to control the quality of realgar decoction pieces by modern analytical technology and analyze its components. On this basis, its anti-colon cancer activity was discussed. Materials and methodsSeveral batches of realgar decoction pieces were analyzed by XRD, and the components of realgar decoction pieces were obtained. The quality control fingerprints of realgar decoction pieces were established by processing XRD spectra and similarity evaluation. Then, the effects of realgar decoction pieces on apoptosis of CT26 and HTC-116 cells were observed in vitro by Hoechst 33258 staining, flow cytometry, measurement of mitochondrial membrane potential and Western blot; In vivo, the mouse model of tumor-in-situ transplantation of colon cancer was established, and the related indexes were observed. ResultThe explorations showed that the XRD Fourier fingerprints of realgar decoction pieces samples that had the same phase revealed 10 common peaks, respectively. The similarity evaluation of the established XRD Fourier fingerprint was greater than 0.900. We also demonstrated that realgar decoction pieces can promote apoptosis and inhibit tumor growth in colon cancer cells, its activating effect on p53 protein, and its safety when used within reasonable limits. ConclusionThe quality control of realgar decoction pieces by XRD is scientific and has the inhibitory effect on colon cancer, which has the development potential.

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

BackgroundDespite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.MethodsTranscriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.ResultsTranscriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.ConclusionsOverall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

Leptin Induces Evidence of Placental Mitochondrial Dysfunction in a Mouse Model of Preeclampsia

Preeclampsia (PE) affects 5-10% of pregnancies worldwide that increase maternal and fetal morbidity and mortality. PE is often characterized by hypertension, endothelial dysfunction, and restriction of intrauterine fetal growth. Our lab established that mid-gestation leptin infusion in pregnant mice induces a model of PE, notably reducing fetal growth in late gestation. Clinical studies indicate that PE patients demonstrate elevated levels of leptin compared to healthy pregnancies as well as present with evidence of placental mitochondrial dysfunction. Therefore, we hypothesize that mid-late gestation leptin infusion induces placental mitochondrial dysfunction in pregnant mouse placenta. We infused timed-pregnant C57/Bl6 mice with saline (sham) or leptin (0.9 mg/kg/day) via s.c. osmotic minipump from gestation day (GD)11-18 (n=4-8). We collected placentas on GD18 for analysis. We isolate mitochondria from mouse placenta via differential centrifugation, after douncing pulverized tissue. From the isolated mitochondria, we assessed morphology via electron microscopy (EM) and performed amplex red assay for reactive oxygen species (ROS) measurements and tetramethyl rhodamine methyl ester (TMRM) dye was used to measure membrane potential. Pairwise comparisons were by student’s t-test. Our data indicates that leptin decreased membrane potential in placenta mitochondria compared to the sham placentas (1466±16.42 sham vs 1353±32.48 leptin A.U./mg of protein, **p<0.01). In comparison, there were no differences in H2O2 production between the sham and leptin-infused placental mitochondrial isolations (7381±125.2 leptin vs 7311±180.3 sham A.U./mg of protein). EM analysis of one sham and one leptin placenta indicated an increase in mitophagy in the leptin-infused placenta slices as well as decreased mitochondrial circularity, suggesting that leptin increases mitochondrial death. In summary, these data indicate that leptin induces mitochondrial dysfunction which in turn may contribute to placental dysfunction and reduced uteroplacental fetal growth in PE. 1R01HL169576, 4R00HL146948. 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.