ATP Assay Research Articles

Extracellular vesicles from cartilage progenitors stimulate type II collagen expression and wound healing in meniscal cells.

Knee meniscus tearing is a common orthopaedic injury that can heal poorly if left untreated, increasing the risk of post-traumatic Osteoarthritis. Intraarticular injection of human cartilage-derived progenitor cells (CPCs) has been shown to promote meniscus healing after injury. However, the mechanism by which CPCs stimulated this effect was unclear. The purpose of this study was to determine the paracrine effects that CPC-derived extracellular vesicles (EVs) have on native meniscal cells during healing. EVs from human CPCs and marrow-derived stromal cells were isolated via ultracentrifugation. EVs produced by each cell type were quantified, and their sizes were determined via NanoSight. EV protein expression was characterized via western blot. Meniscal fibrochondrocyte cellular metabolic activity (as an indicator of cell viability and proliferation) following treatment with EVs, was quantified using MTT and ATP assays. A 2D wound healing assay was used to determine the effects of treating inner meniscal fibrochondrocytes with EVs in a dose-dependent manner. Gene expression analysis for chondrogenesis genes was performed via RT-qPCR on inner meniscal fibrochondrocytes following treatment with EVs. Our results showed that CPCs produced a wide size range of EVs expressing CD9, CD81, and HSP70. Treatment of inner meniscal fibrochondrocytes with CPC-EVs improved 2D wound healing, in comparison to EVs isolated from marrow-derived stromal cell controls. CPC-EV treatment increased Type II Collagen mRNA expression in inner meniscal fibrochondrocytes. These findings demonstrate that CPC-EVs stimulate chondrogenic matrix production and wound healing in meniscal cells at the optimal dose of 1.0 × 107 particles/mL, significantly outperforming the effects of marrow stromal cell-derived EVs.

Berberine ameliorates septic cardiomyopathy through protecting mitochondria and upregulating Notch1 signaling in cardiomyocytes

IntroductionSeptic cardiomyopathy (SCM) arises as a consequence of sepsis-associated cardiovascular dysfunction, for which there is currently no specific targeted therapy available. Previous studies have demonstrated the beneficial therapeutic effect of berberine (BBR) on SCM; however, the underlying mechanisms of action remain unclear. The objective of this is to elucidate how BBR alleviates SCM.MethodsSeptic cardiomyopathy rat model was established by performing cecal ligation and puncture (CLP), while a cardiomyocyte injury model was provoked in H9C2 cells using lipopolysaccharide (LPS). Cardiac function was assessed through echocardiography, and myocardial histopathology was examined with hematoxylin-eosin (HE) staining. Cardiomyocyte viability was determined through Cell Counting Kit-8 (CCK8) assay, and measurement of ATP levels was done with an ATP assay kit. Mitochondrial ultrastructure was observed using transmission electron microscopy. Real-time polymerase chain reaction (RT-PCR) and Western blotting were employed to analyze the expression of Notch1 signaling pathway components and downstream molecules in myocardial tissues and cells.ResultIn vivo, BBR markedly improved symptoms and cardiac function in SCM rats, leading to enhanced ATP content, and ameliorated mitochondrial structure. Additionally, BBR increased Notch1 protein expression in myocardial tissue of the rats. In vitro, BBR elevated the survival rates of H9C2 cell, improved mitochondrial morphology, and raised ATP levels. The mRNA expression of Notch1, Hes1, and Hes2, and Notch1 protein expression was upregulated by BBR. While these effects were reversed upon inhibiting the Notch1 signaling pathway.ConclusionBBR improves septic cardiomyopathy by modulating Notch1 signaling to protect myocardial mitochondria.

Assessing effectiveness of cleaning and disinfection of equipment and environmental surfaces in cystic fibrosis clinics using an ATP assay.

Infection control guidelines for cystic fibrosis (CF) stress cleaning of environmental surfaces and patientcare equipment in CF clinics. This multicenter study measured cleanliness of frequently touched surfaces in CF clinics using an ATP bioluminescence assay to assess the effectiveness of cleaning/disinfection and the impact of feedback. Eight surfaces were tested across 19 clinics (10 pediatric, 9 adult) over 5 rounds of testing. Rounds 1 and 2 served as uncleaned baseline, and Round 3 occurring after routine cleaning. Rounds 4 and 5 were performed after feedback provided to staff and measured after cleaning. Pass rates defined as <250 relative light units were the primary outcome. Of the 750 tests performed, 72% of surfaces passed at baseline, and 79%, 83%, and 85% of surfaces passed in Rounds 3, 4, and 5, respectively. The overall pass-rate was significantly higher in adult compared to pediatric clinics (86% vs 71%; P < 0.001). In pediatric clinics, blood pressure equipment and computer keyboards in the pulmonary function lab consistently passed, but the exam room patient/visitor chairs consistently failed in all rounds. In adult clinics blood pressure equipment, keyboards in exam rooms and exam tables passed in all rounds and no surface consistently failed. We demonstrate the feasibility of an ATP bioluminescence assay to measure cleanliness of patient care equipment and surfaces in CF clinics. Pass rates improved after cleaning and feedback for certain surfaces. We found that surfaces are more challenging to keep clean in clinics taking care of younger patients.

Spatheliachromen mitigates methylglyoxal-induced myotube atrophy by activating Nrf2, inhibiting ubiquitin-mediated protein degradation, and restoring mitochondrial function

BackgroundMethylglyoxal (MGO) is a potent precursor of glycative stress that leads to oxidative stress and muscle atrophy in diabetes. Spatheliachromen (FPATM-20), derived from Ficus pumila var. awkeotsang, exhibited potential antioxidant activity. PurposeThis study aimed to evaluate the potential impact and underlying mechanisms of FPATM-20 on MGO-induced myotube atrophy and mitochondrial dysfunction in mouse skeletal C2C12 myotubes. MethodsAtrophic and antioxidant factors were evaluated using immunofluorescence, enzyme-linked immunosorbent assay, and western blotting. Mitochondrial function was assessed using the ATP assay and Seahorse Cell Mito Stress Test. The glycogen content was determined using periodic acid-Schiff staining. Molecular docking was performed to determine the interaction between FPATM-20 and Keap1. ResultsIn myotubes treated with MGO, FPATM-20 activated the Nrf2 pathway, reduced ROS levels, enhanced antioxidant defense, and increased glycogen content. FPATM-20 improved myotube viability and size, upregulated myosin heavy chain (MyHC) expression, modulated ubiquitin-proteasome molecules (nuclear FoxO3a, atrogin-1, MuRF-1, and p62/SQSTM1), and inhibited apoptosis (Bax/Bcl-2 ratio and cleaved caspase 3). Moreover, FPATM-20 restored mitochondrial function, including mitochondrial membrane potential, mitochondrial oxygen consumption rate, and mitochondrial biogenesis pathway (nuclear PGC-1α/TFAM/FNDC5). The inhibition of Nrf2 with ML385 reversed the effects of FPATM-20 on MGO. Furthermore, molecular docking confirmed the binding of FPATM-20 to Keap1, a suppressor of Nrf2, showing the crucial role of Nrf2 in protective effects. ConclusionsFPATM-20 protects myotubes from MGO toxicity by activating the Nrf2 antioxidant defense, reducing protein degradation and apoptosis, and enhancing mitochondrial function. Thus, FPATM-20 may be a novel agent for preventing skeletal muscle atrophy.

Synergistic combination of perphenazine and temozolomide suppresses patient-derived glioblastoma tumorspheres.

Glioblastoma (GBM), a primary malignant brain tumor, has a poor prognosis, even with standard treatments such as radiotherapy and chemotherapy. In this study, we explored the anticancer effects of the synergistic combination of perphenazine (PER), a dopamine receptor D2/3 (DRD2/3) antagonist, and temozolomide (TMZ), a standard treatment for GBM, in patient-derived human GBM tumorspheres (TSs). The biological effects of the combination of PER and TMZ in GBM TSs were assessed by measuring cell viability, ATP, stemness, invasiveness, and apoptosis. Changes in protein and mRNA expression were analyzed using western blotting and RNA sequencing. Co-administration of PER and TMZ was evaluated in vivo using a mouse orthotopic xenograft model. The Severance dataset showed that DRD2 and DRD3 expression was higher in tumor tissues than in the tumor-free cortex of patients with GBM. DRD2/3 knockout by CRISPR/Cas9 in patient-derived human GBM TSs inhibited cell growth and ATP production. The combined treatment with PER and TMZ resulted in superior effects on cell viability and ATP assays compared to those in single treatment groups. Flow cytometry, western blotting, and RNA sequencing confirmed elevated apoptosis in GBM TSs following combination treatment. Additionally, the combination of PER and TMZ downregulated the expression of protein and mRNA associated with stemness and invasiveness. In vivo evaluation showed that combining PER and TMZ extended the survival period of the mouse orthotopic xenograft model. The synergistic combination of PER and TMZ has potential as a novel combination treatment strategy for GBM.

The Marine Antimicrobial Peptide AOD with Intact Disulfide Bonds Has Remarkable Antibacterial and Anti-Biofilm Activity.

American Oyster Defensin (AOD) is a marine peptide that is derived from North American mussels. It has been demonstrated to exhibit potent antimicrobial activity and high safety in both in vitro and in vivo models. In this study, to facilitate synthesis, mutants of AOD with fewer disulfide bonds were designed and subjected to structural, antimicrobial, and anti-biofilm analysis. The antimicrobial activity of AOD-derived peptides decreased after reduction in the disulfide bond, and among its three derivatives, only AOD-1 inhibited very few bacteria with a MIC value of 64 μg/mL, whereas the others had no inhibitory effect on pathogenic bacteria. The findings demonstrated that full disulfide bonds are indispensable for bactericidal activity, with the α-helix playing a pivotal role in inhibiting bacterial membranes. Furthermore, the results of the ATP, ROS, membrane potential, and membrane fluidity assays demonstrated that intracellular ATP, reactive oxygen species, and membrane fluidity were all increased, while membrane potential was reduced. This indicated that AOD resulted in the impairment of membrane fluidity and induced metabolic disorders, ultimately leading to bacterial death. The inhibitory effect of AOD on the biofilm of S. epidermidis G-81 was determined through the crystal violet and confocal microscopy. The results demonstrated that AOD exhibited a notable inhibitory impact on the biofilm of S. epidermidis G-81. The minimum biofilm inhibitory concentration of AOD on S. epidermidis G-81 was 16 μg/mL, and the minimum biofilm scavenging concentration was 32 μg/mL, which exhibited superior efficacy compared to that of lincomycin. The inhibitory effect on the primary biofilm was 90.3%, and that on the mature biofilm was 82.85%, with a dose-dependent inhibition effect. Concurrently, AOD cleared intra-biofilm organisms and reduced the number of biofilm-holding bacteria by six orders of magnitude. These data indicate that disulfide bonds are essential to the structure and activity of AOD, and AOD may potentially become an effective dual-action antimicrobial and anti-biofilm agent.

Quercetin inhibits cardiomyocyte apoptosis via Sirt3/SOD2/mitochondrial reactive oxygen species during myocardial ischemia–reperfusion injury

BackgroundMyocardial ischemia/reperfusion injury (MI/RI) can lead to impaired cardiac function. Quercetin (Que) has a positive effect and improves MI/RI. Sirtuin-3 (Sirt3) is a deacetylase that ameliorates oxidative stress and is associated with MI/RI. This study aimed to investigate the molecular mechanism by which Que protects cardiac function against MI/RI through the Sirt3 signaling pathway. MethodsWe conducted experiments by constructing hypoxia/reoxygenation (H/R) cardiomyocytes and MI/RI rat models. H9C2 cells were transfected with siRNA-Sirt3. Cardiomyocyte apoptosis was examined by TUNEL and Western blotting. The oxidative stress index was also determined. Mitochondrial reactive oxygen species (ROS) activity assays, ATP assays and mitochondrial membrane potential assays were performed. Evans Blue/TTC staining was used to examine surviving myocardial tissue. ResultsIn the constructed H/R cells and MI/RI animal models, it was found that myocardial cell apoptosis increased (Bcl-2 expression was downregulated; Bax and cleaved caspase-3/8/9 expression were upregulated). In addition, oxidative stress levels increased (MDA levels increased; SOD, CAT, GSH-Px levels decreased), myocardial tissue was damaged (LDH, CK content increased), Sirt3 expression was downregulated, acetylation levels of superoxide dismutase 2 (SOD2) increased (AC-SOD2), and mitochondrial ROS increased. Que treatment alleviated the effects of MI/RI on cardiomyocytes and rats. Sirt3 expression and activity were upregulated, SOD2 acetylation was decreased, and mitochondrial ROS production was reduced by Que treatment. After Sirt3 was knocked down, we found that AC-SOD2 expression was upregulated and mitochondrial ROS were increased in H/R cardiomyocytes, further increased the degree of injury, while Que treatment attenuated the effect of Sirt3 knockdown on H/R cardiomyocytes. ConclusionQue inhibits cardiomyocyte apoptosis, reduces oxidative stress levels, protects mitochondrial function and prevents the impairment of cardiac function during MI/RI via the Sirt3/SOD2/mitochondrial ROS pathway.

TRIM40 interacts with ROCK1 directly and inhibits colorectal cancer cell proliferation through the c-Myc/p21 axis

BackgroundColorectal cancer (CRC) is the most common malignancy of the digestive tract, and to date, morbidity and mortality rates remain high. While existing therapeutic methods have achieved certain effective outcomes, there are still many problems in treating this disease. Therefore, it is still urgent to constantly find new therapeutic targets in CRC that could lead to new therapeutics. MethodsImmunohistochemistry, Real-time PCR and Western Blot were employed to measure mRNA and protein levels of the target protein, respectively. The proliferation ability of CRC cells was evaluated using ATP assay, Soft agar assay, and nude mouse subcutaneous tumorigenesis assay. Protein Degradation Assay was conducted to determine protein degradation rate, while Ubiquitination assay was used to assess the ubiquitination modification level of target proteins. Immunoprecipitation assay was used to study protein interactions, and pull-down assay was employed to investigate direct interactions between proteins. ResultsTRIM40 was significantly down-regulated in CRC tissues, with its expression levels positively correlating with disease prognosis. Using both in vitro and in vivo approaches, it was demonstrated that TRIM40 could significantly inhibit the proliferation of CRC cells. Molecular mechanism studies showed that TRIM40 directly binds to and ubiquitinates ROCK1 protein, accelerating its degradation and subsequently reducing the stability of c-Myc protein. This cascade of events results in the release of transcriptional inhibition of p21 by c-Myc, leading to increased p21 expression and G0/G1 phase arrest in CRC cells. ConclusionThis research suggests that TRIM40 could be a valuable therapeutic target for the treatment of CRC.

Structural insights into the human P2X1 receptor and ligand interactions.

The P2X1 receptor is a trimeric ligand-gated ion channel that plays an important role in urogenital and immune functions, offering the potential for new drug treatments. However, progress in this area has been hindered by limited structural information and a lack of well-characterised tool compounds. In this study, we employ cryogenic electron microscopy (cryo-EM) to elucidate the structures of the P2X1 receptor in an ATP-bound desensitised state and an NF449-bound closed state. NF449, a potent P2X1 receptor antagonist, engages the receptor distinctively, while ATP, the endogenous ligand, binds in a manner consistent with other P2X receptors. To explore the molecular basis of receptor inhibition, activation, and ligand interactions, key residues involved in ligand and metal ion binding were mutated. Radioligand binding assays with [3H]-α,β-methylene ATP and intracellular calcium ion influx assays were used to evaluate the effects of these mutations. These experiments validate key ligand-receptor interactions and identify conserved and non-conserved residues critical for ligand binding or receptor modulation. This research expands our understanding of the P2X1 receptor structure at a molecular level and opens new avenues for in silico drug design targeting the P2X1 receptor.

MTS, WST-8, and ATP viability assays in 2D and 3D cultures: Comparison of methodologically different assays in primary human chondrocytes.

Tissue engineering enables the production of three-dimensional microtissues which mimic naturally occurring conditions in special tissues. These 3D culture systems are particularly suitable for application in regenerative medicine or experimental pharmacology and toxicology. Therefore, it is important to analyse the cells in their 3D microenvironment with regard to viability and differentiation. Tetrazolium assays (WST-8 and MTS) are still the methods of choice for estimating the number of living, metabolically active cells, with WST-8 being cell-impermeable compared to MTS. In contrast to these methods, the ATP assay is an endpoint method based on the luciferase-induced reaction of ATP with luciferin after cell lysis. We compared three methodologically different proliferation/toxicity assays (MTS, WST-8, ATP) in monolayer (2D) and 3D culture systems to improve the technically challenging determination of the number of viable cells. Chondrocytes were isolated from human articular cartilage. Three different test systems (MTS, WST-8, ATP) were applied to monolayer cells (2D, varying cell numbers) and spheroids (3D, different sizes) in 96-well plates. The intracellular ATP concentration was determined by luciferase-induced reaction of ATP with luciferin using a luminometer. Formazan formation was measured spectrophotometrically after different incubation periods. Evaluation was performed by phase contrast microscopy (toxicity), correlation of cell count and ATP concentration or absorption signal (Gompertz function) and propidium iodide (PI) staining to proof the cell lysis of all cells in spheroids. In 2D culture, all three assays showed a good correlation between the number of seeded cells and the ATP concentration or absorption data, whereas the MTS-assay showed the lowest specificity. In 3D culture, the spheroid sizes were directly related to the number of cells seeded. The absorption data of the WST-8 and MTS assay correlated only for certain spheroid size ranges, whereas the MTS-assay showed again the lowest specificity. Only the measured intracellular ATP content showed a linear correlation with all spheroid sizes ranging from 100-1000 μm. The WST-8 assay revealed the second-best sensitivity which allows the measurement of spheroids larger than 240 μm. Phase contrast observation of monolayer cells showed toxic effects of MTS after 6 h incubation and no signs of toxicity of WST-8. Staining with propidium iodide showed complete lysis of all cells in a spheroid in the ATP assay. Among tetrazolium-based assays, WST-8 is preferable to MTS because of its non-toxicity and better sensitivity. When determining the number of viable cells in the 2D system, caution is advised when using the ATP assay because of its two-phase slope of the correlation graph concerning cell number and intracellular ATP. In 3D systems of human chondrocytes, the ATP-assay is superior to the other two test systems, as the correlation graph between cell number and intracellular ATP is biphasic. Since differentiation processes or other metabolic events can influence the results of proliferation and toxicity assays (determination of viable cells), this should be taken into account when using these test systems.

Jiedu Xiaozheng Yin extract targets cancer stem cells by Wnt signaling pathway in colorectal cancer

Ethnopharmacological relevanceThe clinical application of the traditional Chinese medicinal formula Jiedu Xiaozheng Yin (JXY) for gastrointestinal tumors, particularly colorectal cancer (CRC), is well-established, yet the precise biological mechanism underlying its efficacy in CRC treatment remains elusive. Aims of the studyThis study endeavors to unravel the intricate mechanism through which JXY modulates colorectal cancer stem cells, thus elucidating the pathways by which it exerts its potent anti-tumor effects. Materials and methodsIn this study, the regulatory impact of JXY on the signaling pathway and function of CRC cells was analyzed through Network pharmacology. The ethyl acetate extract of JXY was detected the major compounds using HPLC and then treated the HCT-116 cells for RNA-Sequencing (RNA-Seq). Protein expression and stemness of HCT-15 and HCT-116 cells following JXY extract treatment were assessed using Western blot analysis and matrigel spheroid assays. Additionally, the β-catenin transcriptional activity was evaluated using a TOPflash reporter assay with or without Lithium chloride (LiCl) stimulation. Patient-derived organoids of CRC (CRC PDOs) were cultured using a stemness maintenance medium, and their viability was measured using ATP assays after treatment of JXY extract. Furthermore, the anti-tumor efficacy of JXY extract was assessed using a xenograft mice model derived from HCT-15 cells. ResultsNetwork pharmacology emphasized the influence of JXY on cancer stem cells and the Wnt signaling pathway. HPLC analysis confirmed that the JXY extract contained the three most prevalent pharmaceutical compounds among the four herbs documented in the Chinese Pharmacopoeia (rosmarinic acid, quercetin, and kaempferol). RNA-Seq results further elucidated the effect of JXY extract, particularly targeting cancer stem cells and the Wnt signaling pathway. Furthermore, JXY extract inhibited spheroid formation in CRC cells and downregulated CRC CSC markers (CD133, DCLK1, and C-MYC). Additionally, JXY extract suppressed the β-catenin expression and transcriptional activity as well as the Wnt pathway target proteins, including C-MYC and Cyclin D1. Consistent with findings from cell lines, JXY extract suppressed the growth of CRC PDOs exhibiting stemness characteristics. And JXY extract demonstrated a significant inhibitory effect on tumor growth, C-MYC, and β-catenin protein levels in xenograft tumors. ConclusionsThese results highlight the novel function of JXY extract in targeting CRC CSCs by regulating Wnt signaling pathway, underscoring its potential as a therapeutic agent for treating CRC.

Establishment of patient-derived organoids and a characterization based drug discovery platform for treatment of gastric cancer

BackgroundGastric cancer (GC) encompasses many different histological and molecular subtypes. It is a major driver of cancer mortality because of poor survival and limited treatment options. Personalised medicine in the form of patient-derived organoids (PDOs) represents a promising approach for improving therapeutic outcomes. The goal of this study was to overcome the limitations of current models by ameliorating organoid cultivation.MethodsOrganoids derived from cancer tissue were evaluated by haematoxylin and eosin staining, immunohistochemistry, mRNA, and whole-exome sequencing. Three representative chemotherapy drugs, 5-fluorouracil, docetaxel, and oxaliplatin, were compared for their efficacy against different subtypes of gastric organoids by ATP assay and apoptosis staining. In addition, drug sensitivity screening results from two publicly available databases, the Genomics of Drug Sensitivity in Cancer and Cancer Cell Line Encyclopaedia, were pooled and applied to organoid lines. Once key targeting genes were confirmed, chemotherapy was used in combination with poly (ADP ribose) polymerase (PARP)-targeted therapy.ResultsWe successfully constructed GC PDOs surgically resected from GC patient tissue. PDOs closely reflected the histopathological and genomic features of the corresponding primary tumours. Whole-exosome sequencing and mRNA analysis revealed that changes to the original tumour genome were maintained during long-term culture. The drugs caused divergent responses in intestinal, poorly differentiated intestinal, and diffuse gastric cancer organoids, which were confirmed in organoid lines. Poorly differentiated intestinal GC patients benefited from a combination of 5-fluorouracil and veliparib.ConclusionThe present study demonstrates that combining chemotherapy with PARP targeting may improve the treatment of chemotherapy-resistant tumours.

Integrin β3 enhances glycolysis and increases lactate production in endometriosis

BackgroundEndometriosis (EMs) is a chronic disease characterized by endometrial-like tissue present outside of the uterus. Macrophages have been confirmed to participate in the development of EMs. Integrin β3 (ITGB3), a β-subunit of the integrin family, is crucial in tumor progression. In this study, we investigated the pivotal role of ITGB3 in endometrial stromal cells (ESCs) and its influence on the development of EMs, particularly focusing on the regulatory impact of macrophages. MethodsIn this study, we used western blot, Real-time qPCR, Immunohistochemistry to detected the high expression of ITGB3 in ESCs. ITGB3-overexpression ESCs (ITGB3-OE) was constructed and detected by RNA-seq with normal ESCs. ATP and lactate expression assay, transwell migration assay, wound healing, cell adhesion assay and other molecular biology techniques were used to explore the potential mechanisms. In vivo, we constructed the EMs mouse model and injected with cilengitite to inhibit ITGB3. ResultsHere, we found ITGB3 highly expressed in ectopic lesions in EMs. The increasing ITGB3 resulted in activating the glycolysis, which produced more ATP and lactate in ITGB3-OE. After culturing with lactate, the migration, proliferation and invasion ability of ESCs were enhanced, while the result in 2-DG was reversed. In vivo, the results showed that after antagonizing ITGB3, the number of ectopic lesions was decrease. ConclusionsOur findings indicate that ITGB3 up-regulated by macrophages are able to regulate the glycolysis to promote the development of EMs and lactate enhances the ability of proliferation, migration, invasion and adhesion of EMs iv vivo and in vitro.

Unleashing the Neurotherapeutic Potential: The Crucial Role of miR-206-3p in Facilitating Hsp90aa1-Mediated Central Nervous System Injuries During Heat Stroke.

This study aims to explore the molecular mechanisms of miR-206-3p in regulating Hsp90aa1 and its involvement in the central nervous system (CNS) injury in heat stroke. Weighted gene co-expression network analysis (WGCNA) was performed on the GSE64778 dataset of heat stroke to identify module genes most closely associated with disease characteristics. Through the selection of key genes and predicting upstream miRNAs using RNAInter and miRWalk databases, the regulatory relationship between miR-206-3p and Hsp90aa1 was determined. Through in vitro experiments, various methods, including bioinformatics analysis, dual-luciferase reporter gene assay, RIP experiment, and RNA pull-down experiment, were utilized to validate this regulatory relationship. Furthermore, functional experiments, including CCK-8 assay to test neuron cell viability and flow cytometry to assess neuron apoptosis levels, confirmed the role of miR-206-3p. Transmission electron microscopy, real-time quantitative PCR, DCFH-DA staining, and ATP assay were employed to verify neuronal mitochondrial damage. Heat stroke rat models were constructed, and mNSS scoring and cresyl violet staining were utilized to assess neural functional impairment. Biochemical experiments were conducted to evaluate inflammation, brain water content, and histopathological changes in brain tissue using H&E staining. TUNEL staining was applied to detect neuronal apoptosis in brain tissue. RT-qPCR and Western blot were performed to measure gene and protein expression levels, further validating the regulatory relationship in vivo. Bioinformatics analysis indicated that miR-206-3p regulation of Hsp90aa1 may be involved in CNS injury in heat stroke. In vivo, animal experiments demonstrated that miR-206-3p and Hsp90aa1 co-localized in neurons of the rat hippocampal CA3 region, and with prolonged heat stress, the expression of miR-206-3p gradually increased while the expression of Hsp90aa1 gradually decreased. Further in vitro cellular mechanism validation and functional experiments confirmed that miR-206-3p could inhibit neuronal cell viability and promote apoptosis and mitochondrial damage by targeting Hsp90aa1. In vivo, experiments confirmed that miR-206-3p promotes CNS injury in heat stroke. This study revealed the regulatory relationship between miR-206-3p and Hsp90aa1, suggesting that miR-206-3p could regulate the expression of Hsp90aa1, inhibit neuronal cell viability, and promote apoptosis, thereby contributing to CNS injury in heat stroke.

P-266 Profiling redox effects induced by micro-nanoplastics in the female reproductive system: insights from human granulosa cells

Abstract Study question Are nano-microplastics (NMPs) taken up into human granulosa cells causing redox alterations? Summary answer NMPs enter into human granulosa cells and influence viability, energy production and antioxidant response. What is known already In recent decades, anthropogenic activities have increased the production of global plastic with millions of tons produced every year. Under the action of different physical, chemical or biological agents, plastic waste breaks down into (NMP) particles that propagate in the environment posing threats for human health. NMPs translocate from digestive tract to circulatory systems reaching organs and cells, including female gonads. Nevertheless, there is insufficient knowledge of their effects on reproductive functions in mammals to allow for an accurate risk assessment to be conducted and any risks managed in animal and human being contexts. Study design, size, duration Human ovarian granulosa cells (KGN cell line) were exposed to NMPs of different sizes (40 nm, 70 nm, 100 nm, and 200 nm) at concentrations ranging from 5 to 1000 μg/ml for 24 h and then processed for cellular and biochemical analyses Participants/materials, setting, methods NMPs uptake by granulosa cells was tested by the employment of fluorescent NMPs and observation under confocal laser scanning microscopy. Cell viability was assed by Cell Counting Kit8. The effects of NMPs on mitochondrial bioenergetics was evaluated by MitoStress kit (Seahorse Xfe96, Agilent). ATP production was evaluated by Cell Titer-Glo ATP assay kit (Promega). Key enzymes of antioxidant response were analysed at transcript and protein level by using real-time Taqman PCR and Western blotting, respectively. Main results and the role of chance We demonstrated that NMPs are taken up by granulosa cells at all tested concentrions along with significant decrease of cell vitality with all sizes and concentrations. Seahorse analysis revealed an altered bioenergetic profiles. ATP levels increase at 70, 100 and 200 nm (one way ANOVA p = 0.001) at all tested concentrations (one way ANOVA p &amp;lt; 0.001). The gene and protein expression of catalase (CAT) superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2) increased at 5 and 100 µg/ml with all sizes except 100 nm, whereas the level of sirtuins (SIRT1 and SIRT3) transcripts and protein decreased. Reduced levels of phosphorylated NRF2, the transcription factor that activates antioxidant responsive elements (ARE)-mediated gene expression were observed. Limitations, reasons for caution The use of human cell lines requires careful consideration when transferring to human testing. Wider implications of the findings Our results contribute to understanding the effects of NMPs on mammalian fertility in order to find possible protective approaches. Considering that the level of susceptibility of mammalian female germ cells to NMPs is still unknown, present results contribute to evidence-based strategies allowing living and working in a health promoting environment. Trial registration number Effects of combined exposure to nano/microplastics and plastic additives on mammalian female fertility (CUP E53D2301100006)

Mutation of mpv17 results in loss of iridophores due to mitochondrial dysfunction in tilapia

Abstract Mpv17 (mitochondrial inner membrane protein MPV17) deficiency causes severe mitochondrial DNA depletion syndrome in mammals and loss of pigmentation of iridophores and a significant decrease of melanophores in zebrafish. The reasons for this are still unclear. In this study, we established an mpv17 homozygous mutant line in Nile tilapia. The developing mutants are transparent due to the loss of iridophores and aggregation of pigment granules in the melanophores and disappearance of the vertical pigment bars on the side of the fish. Transcriptome analysis using the skin of fish at 30 dpf (days post fertilization) revealed that the genes related to purine (especially pnp4a) and melanin synthesis were significantly downregulated. However, administration of guanine diets failed to rescue the phenotype of the mutants. In addition, no obvious apoptosis signals were observed in the iris of the mutants by TUNEL staining. Significant downregulation of genes related to iridophore differentiation was detected by qPCR. Insufficient ATP, as revealed by ATP assay, α-MSH treatment, and adcy5 mutational analysis, might account for the defects of melanophores in mpv17 mutants. Several tissues displayed less mtDNA and decreased ATP levels. Taken together, these results indicated that mutation of mpv17 led to mitochondrial dTMP deficiency, followed by impaired mtDNA content and mitochondrial function, which in turn, led to loss of iridophores and a transparent body color in tilapia.

M6A demethylase FTO-stabilized exosomal circBRCA1 alleviates oxidative stress-induced granulosa cell damage via the miR-642a-5p/FOXO1 axis

BackgroundPremature ovarian insufficiency (POI) is an important cause of female infertility and seriously impacts the physical and psychological health of patients. Human umbilical cord mesenchymal stem cell-derived exosomes (HucMSCs-Exs, H-Exs) have exhibited protective effects on ovarian function with unclear mechanisms.MethodsA comprehensive analysis of the Gene Expression Omnibus (GEO) database were used to identify POI-associated circRNAs and miRNAs. The relationship between HucMSC-derived exosomal circBRCA1/miR-642a-5p/FOXO1 axis and POI was examined by RT-qPCR, Western blotting, reactive oxygen species (ROS) staining, senescence-associated β-gal (SA-β-gal) staining, JC-1 staining, TEM, oxygen consumption rate (OCR) measurements and ATP assay in vivo and in vitro. RT-qPCR detected the expression of circBRCA1 in GCs and serum of patients with normal ovarian reserve function (n = 50) and patients with POI (n = 50); then, the correlation of circBRCA1 with ovarian reserve function indexes was analyzed.ResultsHerein, we found that circBRCA1 was decreased in the serum and ovarian granulosa cells (GCs) of patients with POI and was associated with decreased ovarian reserve. H-Exs improved the disorder of the estrous cycles and reproductive hormone levels, reduced the number of atretic follicles, and alleviated the apoptosis and senescence of GCs in rats with POI. Moreover, H-Exs mitigated mitochondrial damage and reversed the reduced circBRCA1 expression induced by oxidative stress in GCs. Mechanistically, FTO served as an eraser to increase the stability and expression of circBRCA1 by mediating the m6A demethylation of circBRCA1, and exosomal circBRCA1 sponged miR-642a-5p to block its interaction with FOXO1. CircBRCA1 insufficiency aggravated mitochondrial dysfunction, mimicking FTO or FOXO1 depletion effects, which was counteracted by miR-642a-5p inhibition.ConclusionH-Exs secreted circBRCA1 regulated by m6A modification, directly sponged miR-642a-5p to upregulate FOXO1, resisted oxidative stress injuries in GCs and protected ovarian function in rats with POI. Exosomal circBRCA1 supplementation may be a general prospect for the prevention and treatment of POI.Graphical

The activation of Piezo1 channel promotes invasion and migration via the release of extracellular ATP in cervical cancer

BackgroundThe mechanosensitive ion channel Piezo1 has emerged as a potential prognostic and therapeutic target in different types of cancers. The aim of this study was to determine the expression levels and underlying mechanisms of Piezo1 in the invasion and migration processes in cervical cancer. MethodsInitially, we employed qRT-PCR, western blot, and immunohistochemical staining techniques to assess the disparity in Piezo1 expression in cervical cancer tissues and cells. Subsequently, we conducted wound healing, transwell assays and phalloidin staining to observe the effects of stable Piezo1 silencing and Piezo1 selective agonist Yoda1 on the invasion and migration capabilities. The release of extracellular ATP was assessed using the enhanced ATP assay kit. Furthermore, we conducted rescue experiments to investigate whether the activation of Piezo1 facilitates cervical cancer invasion and migration through extracellular ATP. Finally, we constructed xenograft tumor models to determine weather the Piezo1 selective agonist Yoda1 influenced the tumor growth in vivo. ResultsIn our study, we found that Piezo1 expression was elevated in both cervical cancer tissues and cells, with the highest levels observed in patients with lymph node metastasis. Knocking down Piezo1 resulted in a significant reduction in the invasion and migration capabilities of cervical cancer cells, whereas the use of the Piezo1 selective agonist Yoda1 enhanced these capabilities. Moreover, the activation of Piezo1 channels was found to regulate the release of extracellular ATP. Mechanistically, the activation of Piezo1 might facilitate cervical cancer invasion, migration, and pseudopodium formation through the release of extracellular ATP. And Piezo1 was an important molecule for the tumor growth of cervical cancer in vivo. ConclusionOur findings revealed that Piezo1 facilitated the invasion and migration of cervical cancer by releasing extracellular ATP, which might hold potential as a valuable target for prognostic and therapeutic interventions in cervical cancer.

IP3R2 regulates apoptosis by Ca2+ transfer through mitochondria-ER contacts in hypoxic photoreceptor injury

Mitochondria-associated ER membranes (MAMs) are contact sites that enable bidirectional communication between the ER (endoplasmic reticulum) and mitochondria, including the transfer of Ca2+ signals. MAMs are essential for mitochondrial function and cellular energy metabolism. However, unrestrained Ca2+ transfer to the mitochondria can lead to mitochondria-dependent apoptosis. IP3R2 (Inositol 1,4,5-trisphosphate receptor 2) is an important intracellular Ca2+ channel. This study investigated the contribution of IP3R2-MAMs to hypoxia-induced apoptosis in photoreceptor cells. A photoreceptor hypoxia model was established by subretinal injection of hyaluronic acid (1%) in C57BL/6 mice and 1% O2 treatment in 661W cells. Transmission electron microscopy (TEM), ER-mitochondria colocalization, and the MAM reporter were utilized to evaluate MAM alterations. Cell apoptosis and mitochondrial homeostasis were evaluated using immunofluorescence (IF), flow cytometry, western blotting (WB), and ATP assays. SiRNA transfection was employed to silence IP3R2 in 661W cells. Upon hypoxia induction, MAMs were significantly increased in photoreceptors both in vivo and in vitro. This was accompanied by the activation of mitochondrial apoptosis and disruption of mitochondrial homeostasis. Elevated MAM-enriched IP3R2 protein levels induced by hypoxic injury led to mitochondrial calcium overload and subsequent photoreceptor apoptosis. Notably, IP3R2 knockdown not only improved mitochondrial morphology but also restored mitochondrial function in photoreceptors by limiting MAM formation and thereby attenuating mitochondrial calcium overload under hypoxia. Our results suggest that IP3R2-MAM-mediated mitochondrial calcium overload plays a critical role in mitochondrial dyshomeostasis, ultimately contributing to photoreceptor cell death. Targeting MAM constitutive proteins might provide an option for a therapeutic approach to mitigate photoreceptor death in retinal detachment.

Triple Blockade of Oncogenic RAS Signaling Using KRAS and MEK Inhibitors in Combination with Irradiation in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of human malignancies and carries an exceptionally poor prognosis. It is mostly driven by multiple oncogenic alterations, with the highest mutation frequency being observed in the KRAS gene, which is a key oncogenic driver of tumorogenesis and malignant progression in PDAC. However, KRAS remained undruggable for decades until the emergence of G12C mutation specific KRAS inhibitors. Despite this development, this therapeutic approach to target KRAS directly is not routinely used for PDAC patients, with the reasons being the rare presence of G12C mutation in PDAC with only 1-2% of occurring cases, modest therapeutic efficacy, activation of compensatory pathways leading to cell resistance, and absence of effective KRASG12D or pan-KRAS inhibitors. Additionally, indirect approaches to targeting KRAS through upstream and downstream regulators or effectors were also found to be either ineffective or known to cause major toxicities. For this reason, new and more effective treatment strategies that combine different therapeutic modalities aiming at achieving synergism and minimizing intrinsic or adaptive resistance mechanisms are required. In the current work presented here, pancreatic cancer cell lines with oncogenic KRAS G12C, G12D, or wild-type KRAS were treated with specific KRAS or SOS1/2 inhibitors, and therapeutic synergisms with concomitant MEK inhibition and irradiation were systematically evaluated by means of cell viability, 2D-clonogenic, 3D-anchorage independent soft agar, and bioluminescent ATP assays. Underlying pathophysiological mechanisms were examined by using Western blot analyses, apoptosis assay, and RAS activation assay.