Terminal Transferase Research Articles

Extracellular Vesicles Derived from Adipose-Derived Mesenchymal Stem Cells Alleviate Apoptosis and Oxidative Stress of Retinal Pigment Epithelial Cells Through Activation of Nrf2 Signaling Pathway.

Purpose: To examine the potential protective effects of adipose-derived mesenchymal stem cell-derived extracellular vesicles (ASC-EVs) on ARPE-19 cells exposed to hydrogen peroxide (H2O2) stress and to evaluate their ability to delay retinal degeneration in Royal College of Surgeons (RCS) rats. Methods: ARPE-19 cells were pre-treated with ASC-EVs for 24 h, followed by exposure to 200 μM H2O2 for an additional 24 h. RCS rats received an intravitreal injection of phosphate-buffered saline in one eye and ASC-EVs in the other eye. Results: ASC-EV pretreatment significantly protected against H2O2 in the Cell Counting Kit-8 assay and was also effective in the lactate dehydrogenase-release assay. It notably reduced early apoptosis (Annexin V-fluorescein isothiocyanate/propidium iodide assay) and late apoptosis (Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling assay), while significantly decreasing intracellular reactive oxygen species, glutathione levels, and superoxide dismutase activity. NFE2L2, HMOX1, and NQO1 mRNA levels, along with Nrf2, HO-1, and NQO1 protein levels, were significantly elevated with ASC-EV pretreatment. Compared with ARPE-19-derived EVs, 11 miRNAs were upregulated and 34 were downregulated in ASC-EVs. In RCS rats, intravitreal injections of ASC-EVs led to significant preservation of the outer nuclear layer and photoreceptor segments, along with increased nuclear Nrf2 expression and elevated HO-1 and NQO1 levels in the inner retina. Eyes that received intravitreal injections of ASC-EVs demonstrated significantly preserved electroretinography a- and b-wave amplitudes at 1 week post-injection, though this effect faded by 2 weeks. Conclusions: ASC-EVs mitigated apoptosis and oxidative stress in ARPE-19 cells subjected to H2O2 exposure and temporarily slowed retinal degeneration in RCS rats via Nrf2 pathway activation by miRNAs.

Role of BIX01294 in the intracranial inhibition of H3K9 methylation lessens neuronal loss in vascular dementia model.

Dementia develops as a result of multiple factors, including cerebrovascular disease which is called vascular dementia (VD). Histone-3 lysine-9 dimethylation (H3K9me2) broadly increases during VD and inhibits neuroprotective gene expressions. So, we aimed to determine how H3K9me2 inhibitor (BIX01294) affects neuronal damage in VD. An in vivo model of VD was used followed by BIX01294 treatment. Behavioral tests, hematoxylin, and eosin (H&E), Congo red, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were carried out. Hippocampal phosphorylated cyclic-AMP responsive element binding protein (p-CREB), c-fos, brain-derived neurotrophic factor (BDNF), and H3K9me2, were detected by western blot analysis technique. Neurological deficit and anxiety-related behavior significantly reduced in the treatment group compared to the VD group (p < 0.05). BIX01294 improved spatial and passive avoidance memory (p < 0.01 and p < 0.05, respectively) compared to the VD group. Treatment with BIX01294 restored the level of p-CREB/CREB ratio (p < 0.05), cfos (p < 0.01), BDNF (p < 0.01), and suppressed H3K9me2 (p < 0.001) when compared to the VD group. BIX01294 microinjection reduced the apoptosis level in TUNEL staining (p < 0.05), and raised neural cell count in H&E staining (p < 0.01); amyloid beta accumulation significantly decreased in the treatment group (p < 0.05) compared to the VD group. In conclusion, long-term treatment with a low dose of BIX01294 can prevent the progression of neuronal loss in VD model by raising the expression of neurotrophic factors, and reducing the apoptosis level.

The involvement of lidocaine in amyloid-β1-42-dependent mitochondrial dysfunction and apoptosis in hippocampal neurons via nerve growth factor-protein kinase B pathway.

This project is conceived to reveal the role of lidocaine in the process of Alzheimer's disease (AD) and its possible downstream targets. After the employment of AD cell model in mice hippocampal neuronal HT-22 cells in the presence of amyloid-β1-42 (Aβ1-42), Cell Counting Kit-8 method investigated cell viability. Oxidative damage was assayed based on a dichloro-dihydro-fluorescein diacetate fluorescent probe and commercially available kits. The 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide fluorescent probe estimated mitochondrial function. Terminal-deoxynucleotidyl transferase mediated nick end labeling, western blotting, and immunofluorescence appraised the apoptotic level. Western blot also ascertained the alternations of nerve growth factors (NGF)-protein kinase B (Akt) pathway-related proteins. Aβ1-42 concentration dependently triggered the viability loss, oxidative damage, and apoptosis in HT-22 cells. Lidocaine promoted the viability and reduced the mitochondrial impairment and mitochondria-dependent apoptosis in Aβ1-42-treated HT-22 cells in a concentration-dependent manner. Besides, lidocaine activated the NGF-Akt pathway and NGF absence blocked NGF-Akt pathway, aggravated mitochondrial dysfunction as well as mitochondria-dependent apoptosis in lidocaine-administrated HT-22 cells in response to Aβ1-42. Altogether, these observations concluded that lidocaine might stimulate NGF-Akt pathway to confer protection against mitochondrial impairment and apoptosis in Aβ1-42-mediated cellular model of AD.

Anticancer Effects of Weizmannia coagulans MZY531 Postbiotics in CT26 Colorectal Tumor-Bearing Mice by Regulating Apoptosis and Autophagy

Weizmannia coagulans has been shown to have anticancer properties. However, there is limited research on the effects of postbiotic W. coagulans on colorectal cancer cell proliferation. Additionally, the exact mechanisms through which it influences apoptosis- and autophagy-related signaling pathways are yet to be thoroughly elucidated. This study explored the role of W. coagulans MZY531 as a postbiotic in inhibiting tumor growth by modulating apoptosis and autophagy in tumor cells. During the experimental period in the model group, tumors proliferated, tumor markers increased significantly, and immunofluorescence results showed that caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end labeling were significantly decreased. Conversely, supplementation with W. coagulans MZY531 postbiotics significantly reduced the levels of tumor markers carcinoembryonic antigen, colon cancer antigen, and extracellular protein kinase A and promoted cell apoptosis by increasing the caspase-3-positive count and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells in tumor tissue. Mechanistically, W. coagulans MZY531 postbiotics inhibit tumor growth through the modulation of the Bax/Bcl-2/caspase-3 and JAK2/STAT3 apoptosis pathways and PI3K/AKT/mTOR and TGF-β/SMAD4 cell autophagy pathways. W. coagulans MZY531 postbiotics had a more significant effect than that of W. coagulans MZY531 alone. Probiotics are expected to become effective natural functional foods for the treatment of colorectal cancer.

Enzymatic reaction and competitive prereduction enable label-free and separation-free inductively coupled plasma mass spectrometry detection of leukemia cells

The enzyme terminal deoxynucleotidyl transferase (TdT) exhibits significant activity in various leukemia cells, including human acute lymphoblastic leukemia cells (CCRF). It demonstrates substantial potential as a diagnostic biomarker for acute lymphoblastic leukemia (ALL). Herein, a highly sensitive method for the homogeneous quantification of TdT and CCRF cells, without the need for labeling or separation, was established based on inductively coupled plasma mass spectrometry (ICP-MS). The simple sensor relied on the TdT-induced polymerization process, lengthening the DNA strand and producing large amounts of the by-product phosphates of pyrophosphate (PPi). The PPi formed complexes with Cu²⁺, altering its morphology in homogeneous systems. The remaining Cu²⁺ competed with Hg²⁺ in consuming ascorbic acid (AA) through the pre-reduction of Hg²⁺, affecting the ICP-MS signal of Hg²⁺. Under optimal conditions, this approach exhibited excellent specificity and ultrahigh sensitivity, with limits of detection (LODs) as low as 0.05 U/L for TdT and 5 cells/mL for CCRF cells, respectively. The proposed method offers significant advantages, including high sensitivity and precision, operation at room temperature without the involvement of proteases, and the ability to accurately quantify in complex biological matrices. Consequently, the proposed method shows great promise as a valuable tool for TdT-related biological research and leukemia diagnosis.

EGCG enhances antitumor effect of apatinib in nonsmall cell lung cancer by targeting VEGF signaling to inhibit glycolysis.

Nonsmall cell lung cancer (NSCLC), one of the most aggressive malignancies globally, is characterized by poor prognosis and limited life expectancy. Epigallocatechin-3-gallate (EGCG), a natural polyphenol found in green tea, has emerged as a promising anticancer agent due to its potent antitumor properties. However, the role and the underlying mechanisms of EGCG in NSCLC remain poorly understood. Hence, this research aimed to explore the effect of EGCG on the antitumor effect of apatinib in NSCLC throughvascular endothelial growth factor(VEGF)-regulated glycolysis. Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine staining, wound healing, transwell, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and flow cytometry assays were carried out to evaluate the proliferation, migration, invasion, and apoptosis of H1299 cells, respectively. Furthermore, western blot analysis was used to detect the expressions of VEGF, p-vascular endothelial growth factor receptor-2, hypoxia-inducible factor 1α, neuropilin-1, phosphorylated-phosphatidylinositol 3-kinase, and phosphorylated-AKT. The transfection efficiency of H1299 cells with VEGF overexpression plasmid was also assessed by western blot analysis. Glycolysis was analyzed by estimating extracellular acidification rate, lactate concentration, glucose uptake, and the expressions of lactate dehydrogenase A, pyruvate kinase M2, and hexokinase 2. The results demonstrated that VEGF activated glycolysis in NSCLC cells. EGCG alone and apatinib alone or in combination inhibited cell viability, proliferation, invasion, migration, and glycolysis whereas promoted apoptosis in NSCLC cells. EGCG regulated glycolysis levels in NSCLC through VEGF overexpression, and enhanced the antitumor effect of apatinib in NSCLC through VEGF-regulated glycolysis. Taken together, EGCG strengthened the protective effects of apatinib in NSCLC through glycolysis mediated by VEGF.

Between Life and Death: Sea Urchin Embryos Undergo Peculiar DNA Fragmentation after Exposure to Vanadium, Cadmium, Gadolinium, and Selenium.

Exogenous DNA damage represents one of the most harmful outcomes produced by environmental, physical, or chemical agents. Here, a comparative analysis of DNA fragmentation was carried out on Paracentrotus lividus sea urchin embryos exposed to four common pollutants of the marine environment: vanadium, cadmium, gadolinium and selenium. Using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, fragmented DNA was quantified and localized in apoptotic cells mapping whole-mount embryos. This is the first study reporting how different chemicals are able to activate distinctive apoptotic features in sea urchin embryos, categorized as follows: (i) cell-selective apoptosis, showing DNA fragmentation restricted to a subset of extremely damaged cells, acting as an embryo survival mechanism; or (ii) total apoptosis, with fragmented DNA widespread throughout the cells of the entire embryo, leading to its death. Also, this is the first report of the effects of Se exposure on P. lividus sea urchin embryos. These data confirm the TUNEL assay as the most suitable test to study DNA fragmentation in the sea urchin embryo model system. Taken together, this research highlights embryos' ability to find alternative pathways and set physiological limits for development under stress conditions.

MiRNA506 Activates Sphk1 Binding with Sirt1 to Inhibit Brain Injury After Intracerebral Hemorrhage via PI3K/AKT Signaling Pathway.

Intracerebral hemorrhage (ICH) is an acute neurological disorder characterized by high mortality and disability rates. Previous studies have shown that 75% of patients who survive ICH experience varying degrees of neurological deficits. Sphk1 has been implicated in a multitude of phylogenetic processes, including innate immunity and cell proliferation. An in vivo rat model of ICH and an in vitro model of neuronal oxyhemoglobin (OxyHb) were constructed. The expression level of Sphk1 was assessed using western blotting and immunofluorescence, whereas cell death following ICH was evaluated using fluoro-Jade B and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Immunofluorescence facilitated the examination of microglial phenotypic alterations, while enzyme-linked immunosorbent assays were used to determine the concentrations of inflammatory markers. Behavioral assays were employed to assess the overall behavioral modifications of animals. Neuronal Sphk1/Sirt1 protein levels gradually increased following the induction of ICH. Elevated Sphk1 expression resulted in increased levels of anti-inflammatory microglia and reduced levels of pro-inflammatory factors. In contrast, suppression of Sphk1 expression resulted in an increased number of dead cells, thereby exacerbating neurological deficits. In vitro findings indicated that the levels of phosphorylated PI3K and AKT proteins increased in conjunction with Sphk1 expression. This study established that after ICH, Sphk1 interacts with Sirt1 to mitigate neuroinflammation, cell death, oxidative stress, and brain edema via the PI3K/AKT signaling pathway. Augmenting expression of Sphk1 significantly can ameliorate neurological impairments induced by ICH, offering novel targets and perspectives for therapeutic interventions in ICH treatment.

Terminal deoxynucleotidyl transferase-mediated CRISPR sensing platform for simple and point-of-care detection of cobalt pollution

The excessive use of cobalt in various chemical industries and arbitrary discharge of industrial wastewater have led to increased cobalt pollution in soil and water resources, increasing the risk of human exposure to high concentrations of cobalt and necessitating an urgent need for on-site monitoring platform for cobalt pollution. In this study, the terminal deoxynucleotidyl transferase (TdT)-CRISPR platform has been developed. In this platform, cobalt as a cofactor of TdT, can significantly improve the tailing efficiency of TdT-mediated extension. Therefore, when cobalt is present, the detection probe can be extended with poly(T) tails through the TdT-mediated extension, which can be subsequently served as the DNA activator for Cas12a, leading to the cleavage of fluorescence reporter molecules and triggering turn-on fluorescence signals. Consequently, this dual amplification sensing strategy of TdT-CRISPR platform demonstrated exceptional sensitivity (0.83 nM) and high specificity for cobalt over other ions. Furthermore, the method was successfully employed for the detection of cobalt in tap water and river samples. CRISPR-lateral flow assays (CRISPR-LFAs) were evaluated in this study for the simple and point-of-care detection of cobalt pollution. The assays are capable of detecting cobalt concentrations as low as 50 nM, which is significantly lower than the environmental standards of 16.9 μM, through strip analysis with the naked eye. These results commonly suggest that the TdT-CRISPR platform holds significant promise for monitoring cobalt pollution, providing a robust and sensitive solution for on-site detection and contributing to the mitigation of cobalt contamination risks in environmental matrices.

Construction of a chemiluminescent biosensor based on enzymatic extension and click chemistry for sensitive measurement of MGMT activity in human breast tissues

O6-methylguanine methyltransferase (MGMT) is responsible for dealkylation of naturally occurring O6-methylguanines, and it is closely related with DNA replication, transcription, and cancers. Herein, we develop a chemiluminescent biosensor based on enzymatic extension and click chemistry for sensitive measurement of MGMT activity. When MGMT is present, the MGMT-catalyzed demethylation reaction initiates the cleavage of biotinylated dumbbell probes by PvuII restrictive enzyme, releasing two DNA fragments with 3′-OH end. The resultant DNA fragments can trigger terminal transferase (TdT)- and click chemistry-assisted isothermal amplification to obtain abundant G-rich sequences. The G-rich sequences can be captured by magnetic beads to produce a high chemiluminescence signal. This biosensor can greatly amplify the chemiluminescence signal, facilitating label-free and template-free measurement of MGMT. Especially, the introduction of dumbbell probe and PvuII enzyme can efficiently eliminate the false positive and improve the assay specificity. This biosensor possesses high sensitivity with a detection limit of 1.4 × 10−9 ng/μL, and it may accurately quantify the intracellular MGMT. Importantly, this biosensor can be used to screen the MGMT inhibitors and distinguish the MGMT level in breast tumor tissues and normal tissues, with great potential in drug discovery and cancer diagnosis.

Evaluation of Sperm DNA Fragmentation in Oligoasthenoteratozoospermia Patients Using Two Different Techniques: TUNEL and Sperm Chromatin Dispersion Assays

Background: Oligoasthenoteratozoospermia (OAT) is the most prevalent male infertility condition that is mainly caused by sperm DNA fragmentation (SDF). This study compared the sensitivity and effectiveness of two different approaches for analyzing SDF in patients with OAT: sperm chromatin dispersion (SCD) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Materials and Methods: In this study, which received ethical committee approval, participants were divided in to normal and OAT groups (n=20 for each). both TUNEL and SCD assays were used to analyze the sperm DNA fragmentation. And Malondialdehyde (MDA) levels was measured to determine levels of lipid peroxidation in the seminal plasma. Results: The TUNEL assay showed better ability to predict OAT patients than that of the SCD. For our patient population, the projected cut-off points for the DNA fragmentation index of 29% and 19% were reported using the TUNEL and SCD tests, respectively. Seminal levels of MDA were significantly higher in the OAT group (P=0.002) than that of control group. Conclusion: OAT patients showed higher MDA levels of seminal plasma and DNA fragmentation than the control group. Although sperm DNA fragmentation can be detected with high efficiency and sensitivity using both TUNEL and SCD assays, the TUNEL test was found to be a more accurate predictor for OAT patients.

Purified Astragalus Polysaccharide Combined with Inactivated Vaccine Markedly Prevents Infectious Haematopoietic Necrosis Virus Infection in Rainbow Trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) is experiencing a catastrophic pandemic. In recent years, infectious hematopoietic necrosis virus (IHNV) has spread nationwide, resulting in significant mortality. Currently, there are no available treatments or vaccines for IHNV in China. Here, the Astragalus extract was purified and characterized. Then, we developed an inactivated IHNV vaccine with purified Astragalus polysaccharide (P-APS) as an adjuvant. Safety assays showed that IHNV was successfully inactivated. After a serious IHNV challenge, the cumulative mortality rates were 76.0, 38.0, and 22.1% in control, vaccine, and P-APS + vaccine groups, respectively. P-APS + vaccine was effective at reducing head kidney damage and apoptosis after IHNV challenge by histopathological and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses. The P-APS + vaccine group showed better results in enhancing specific antibodies (IgM) and immune enzyme activities (C3, LZM, GOT, and GPT). RNA-seq revealed that many immune-related pathways were significantly enriched. TLR2, TLR7, C3, IFN-γ, IgM, MHC1, MHC2, MX1, and VIG1 were identified as core genes based on RNA-seq and PPI networks. Mechanistic investigations showed that P-APS + vaccine activates the immune pathway by upregulating the expression of these genes. P-ASP+vaccine induced effective innate and adaptive immune responses that were stronger than single vaccines after vaccination and IHNV challenged. Our findings will provide a promising vaccine candidate against IHNV.

The functional DIAPH3-FOXM1 interaction modulates the aggressive transformation of anaplastic thyroid carcinoma cells and Wnt/β-catenin signalling.

Anaplastic thyroid carcinoma (ATC) is reckoned as an infrequent but extremely advanced neoplasm of the endocrine system. Diaphanous-related formin 3 (DIAPH3) has been extensively implicated in carcinogenic events, but it has not been introduced in ATC. Herein, the role of DAPIH3 and the interrelated functional mechanism are characterised in ATC. The Gene Expression Omnibus (GEO) database was checked for differential DIAPH3 expression in ATC samples and noncancerous samples. Western blotting examined DIAPH3 and forkhead box M1 (FOXM1) expression in ATC cells. In vitro cell counting kit 8 (CCK-8) method, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, Scratch, Matrigel invasion, and terminal-deoxynucleotidyl transferase mediated nick end labelling (TUNEL) assays were used to assess the potential of cells to proliferate, migrate, and invade as well as the cellular apoptotic rate. Co-IP was applied to access DIAPH3-FOXM1 protein interaction. Western blotting also disclosed the expression of proteins associated with apoptosis and Wnt/β-catenin signalling. DIAPH3 was hyper-expressed in papillary cell carcinoma (PTC) tissues and cells. Depleting DIAPH3 strongly eliminated the proliferative, migratory, as well as invasive capabilities of PTC cells while intensifying the apoptotic ability. FOXM1 also harboured elevated expression in PTC cells. FOXM1 was the binding partner with DIAPH3, and the 2 were positively correlated. FOXM1 upregulation again exacerbated the potentials to proliferate, migrate, and invade but it repressed the apoptotic rate of DIAPH3-depleted cells. Furthermore, loss of DIAPH3 downregulated FOXM1 to block Wnt/b-catenin signalling in PTC cells. Combined with these findings, DIAPH3 might favour the aggressive advancement of ATC and motivate the Wnt/β-catenin signalling via binding with FOXM1.

MiRNA-153 attenuates progression of non-small cell lung cancer through targeting positive regulatory/SET domain 2

Background: To explore whether micro ribonucleic acid (miR)-153 regulates positive regulatory/SET domain 2 (PRDM2) in a targeted manner and affects the proliferation and apoptosis of non-small cell lung cancer (NSCLC) A549 cells. Methodology: The expressions of miR-153 and PRDM2 in NSCLC tissues and A549 cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). TargetScan was utilized to predict miR-153 target genes. Methyl thiazolyl tetrazolium (MTT) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were carried out to study the cell proliferation and apoptosis. Western blotting was performed to examine the changes in the proteins in the Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway following the miR-153 overexpression. Results: The expression of miR-153 was decreased and that of PRDM2 was increased in NSCLC tissues and cells. Target genes regulated by miR-153 participated in self-vascular development, miRNA metabolic process and the Wnt signaling pathway. The overexpression of miR-153 led to an obvious reduction in the proliferation ability of A549 cells, a notable increase in apoptotic cells, and significant decreases in phosphorylated (p)-JAK2 and p-STAT3 proteins. Dual-luciferase reporter gene assay revealed that miR-153 could directly modulate the expression level of PRDM2. Conclusion: MiR-153 directly targets PRDM2 and affects A549 cell proliferation and apoptosis through the JAK/STAT pathway. Keywords: Non-small cell lung cancer; miR-153; positive regulatory/SET domain 2; Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway.

COL6A1 Inhibits the Malignant Development of Bladder Cancer by Regulating FBN1.

Bladder cancer (BLCA) is a prevalent malignancy worldwide with a high recurrence rate. Collagen Type VI Alpha 1 (COL6A1) plays a key role in several cancer types. In this study, we aimed to explore the role of COL6A1 in BLCA. COL6A1 expression in BLCA was determined using The Cancer Genome Atlas database and real-time quantitative polymerase chain reaction (RT-qPCR). Counting Kit-8, wound-healing, and transwell assays were used to assess the effect of COL6A1 on T24 and 5637 cells. Apoptosis in BLCA cell lines was explored using western blotting and flow cytometry. Co-immunoprecipitation was performed to determine interactions between proteins. The role of COL6A1 in tumor growth in nude mice was evaluated by hematoxylin-eosin, immunohistochemical, and terminal deoxynucleotidyl transferase dUTP Nick-End Labeling. In BLCA, COL6A1 expression was downregulated. Moreover, the COL6A1 overexpression suppressed the viability, migration, and invasion, while promoting apoptosis of BLCA cell lines, with increased Caspase-3, Bax, and p53, and decreased Bcl-2. Conversely, silencing of COL6A1 promoted proliferation, migration, and invasion, while inhibiting apoptosis in BLCA cell lines. In vivo, COL6A1 inhibits tumor growth and progression. Fibrillin-1 (FBN1) was positively correlated with COL6A1 expression. COL6A1 could bind to FBN1 in BLCA cell lines. The expression of FBN1 in BLCA cell lines decreased after COL6A1 silencing, whereas COL6A1 overexpression upregulated FBN1 expression. COL6A1 was downregulated and exerted an inhibitory effect on the development of BLCA, and its expression was positively correlated with the expression of FBN1.

Terminal deoxynucleotidyl transferase (TdT) based template-free signal amplification for the detection of exosomes in MUC1-positive cells

The Mucin1 (MUC1) protein, involved in cytoprotective and signaling pathways, is abnormally elevated in various cancers, making it a key cancer indicator. Exosomes, which reflect the status of their originating cells, offer potential for cancer diagnosis. Thus, developing a method to detect MUC1-positive exosomes is crucial for the early diagnosis of certain cancers. In this study, we developed a highly sensitive, specific, and simple UV–visible signal amplification method to detect MUC1-positive exosomes using terminal deoxynucleotidyl transferase (TdT). Initially, exosomes were captured on magnetic beads using a CD63 aptamer(apt). The Primer-AuNPs-MUC1 apt complex which we synthesized by low pH loading method was then attached MUC1 proteins on the surface of the exosomes to create a sandwich structure. TdT catalyzed the extension of Biotin-dATP at the 3′ end of the primer, introducing multiple biotin sites into the sandwich structure. These sites subsequently bound multiple streptavidin-horseradish peroxidase (streptavidin-HRP), which catalyzed the oxidative color change of the substrate, which can be detected by colorimetric method. This method can detect A549 exosomes in the range of 1.4E+6 to 4.2E+8 particles/mL and shows high specificity for cell lines with different MUC1 expression. Additionally, it successfully distinguished cholangiocarcinoma (CCA) patients (n=11) from healthy individuals (n=7) in clinical serum assays, demonstrating good performance in real sample detection.

Drug pairs of Huangqi and Danggui alleviates pyroptosis by promoting autophagy activity via AMPK/mTOR signaling pathway in middle-cerebral artery occlusion/reperfusion in rats

Ethnopharmacological relevanceCerebral ischemia-reperfusion (I/R) injury is a common complication of ischemic stroke, with autophagy and pyroptosis playing key roles. Huangqi and Danggui (HQDG) are a commonly used drug pair of Chinese traditional medicine for clinical treatment of ischemic stroke. Aim of the studyThe study aims to investigate the interaction between autophagy and pyroptosis regulated by HQDG through the AMPK/mTOR signaling pathway during cerebral I/R injury. Materials and methodsModel of middle-cerebral artery occlusion/reperfusion (MCAO/R) in SD rats was established using the Longa suture method. The components of traditional Chinese medicine were detected by liquid chromatography coupled to quadrupole orbitrap high resolution mass spectrometry (LC/MS). Neurological deficits were evaluated by neurological function score. Changes of cerebral blood flow were detected by a laser speckle blood flow imaging instrument. The volume of cerebral infarction was observed by 2,3,5-Chlorotriphenyltetrazolium (TTC) staining. The permeability of the blood-brain barrier was measured by Evans blue test. Neurovascular unit and autophagosomes in brain tissue were assessed by transmission electron microscopy. Neuronal pyroptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL)/Caspase-1 staining. The expression of autophagy related proteins, pyroptosis related proteins, and AMPK/mTOR pathway related proteins were detected by Western blot. ResultsAfter cerebral I/R injury, autophagy and pyroptosis, were characterized by increased number of autophagosomes and pyroptosis cells, upregulated expression of Beclin 1, LC3-II/LC3-I, NLRP3, cleaved Caspase-1, IL-1beta, IL-18 proteins, and downregulated expression of P62 proteins. HQDG significantly improved neurological function, reduced the volume of cerebral infarction, increased cerebral blood flow, improved blood-brain barrier permeability and the function of neurovascular units. Autophagy was further activated and pyroptosis was significantly inhibited by HQDG, which promoted increased number of autophagosomes, enhanced expression of Beclin 1, LC3-II/LC3-I proteins, reduced expression of P62, NLRP3, cleaved Caspase-1, IL-1beta, and IL-18 proteins, and downregulated the number of pyroptosis cells. On the other hand, after administering 3-Methyladenine (3-MA) to inhibit autophagy, the above effects of HQDG were significantly inhibited. Besides, HQDG promoted AMPK phosphorylation, and weakened mTOR phosphorylation. However, after the administration of AMPK inhibitor Compound C, HQDG caused increase in Beclin 1 and LC3-II/LC3-I, reduced P62 and NLRP3, and cleaved Caspase-1 protein expression, whereas cerebral blood flow decreased. ConclusionHQDG alleviated pyroptosis by promoting autophagy via AMPK/mTOR signaling pathway after middle-cerebral artery occlusion/reperfusion in rats, showing its potential for treatment of cerebral I/R injury in humans.

Propofol Ameliorates Sepsis-Induced Myocardial Dysfunction via Anti-Apoptotic, Anti-Oxidative Properties, and mTOR Signaling.

Sepsis often leads to cardiomyopathy, contributing to increased mortality rates. 2,6-Diisopropylphenol (propofol), an anesthetic, has demonstrated efficacy in protecting cardiomyocytes from cell death caused by hypoxia and reoxygenation. This study examined the effects of propofol on sepsis-associated myocardial dysfunction and explored the underlying mechanism of action. Mice and rat cardiomyocytes (H9C2 cell line) were used to establish a sepsis-induced myocardial dysfunction model. Lipopolysaccharides (LPS)-treated mice and H9C2 cells were treated with propofol, with rapamycin used for mechanistic studies in H9C2 cells. Cardiac function was evaluated by echocardiographic measurements. Heart tissues were stained with hematoxylin and eosin, and heart weight/body weight ratio along with the levels of cardiac biomarkers were measured using Enzyme Linked Immunosorbent Assay (ELISA). Activation of the mammalian target of rapamycin (mTOR) pathway was assessed by western blotting. Apoptosis in heart tissues and H9C2 cells was evaluated using Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay, and cell viability was quantified using Cell Counting Kit (CCK)-8 assay. Oxidative stress in H9C2 cells was assessed by measuring reactive oxygen species (ROS) levels through immunofluorescence staining and malondialdehyde (MDA) and superoxide dismutase (SOD) levels using ELISA. Propofol reversed LPS-induced myocardial changes and cardiac dysfunction (p < 0.05). In mouse tissues and H9C2 cells, propofol reversed LPS-induced mTOR pathway inhibition and apoptosis (p < 0.001). Moreover, propofol alleviated oxidative stress in LPS-treated cells. The activation of the mTOR pathway by propofol, along with its inhibitory effects on oxidative stress and apoptosis in cardiomyocytes, was negated by rapamycin (p < 0.001). Propofol ameliorates sepsis-induced myocardial dysfunction triggered by LPS through the mTOR pathway, thereby promoting antioxidative stress and reducing cell apoptosis.

Neuroprotective Role of AQP4 Knockdown in Astrocytes After Oxygen-Glucose Deprivation.

Aquaporin-4 (AQP4), predominantly expressed in astrocytes, has been implicated in the development of brain edema following ischemic events. However, its role in post-stroke neuroinflammation is not fully understood. Using a middle cerebral artery occlusion (MCAO) mouse model, we assessed AQP4's role in post-stroke inflammation. Brain tissue slices from male C57BL/6 mice were subjected to immunohistochemistry and western blot post-MCAO. Additionally, primary astrocytes were isolated for quantitative real-time PCR and immunofluorescence assays to evaluate the expression of inflammatory markers glial fibrillary acidic protein (GFAP) and AQP4. AQP4 modulation was achieved using viral knockdown and overexpression methods. Neuronal damage was assessed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) tests in co-culture studies. MCAO mice exhibited a significant upregulation in GFAP. This reactive astrogliosis corresponded with an elevation in inflammatory markers. AQP4 expression responded to this inflammatory trend, peaking at 6 h after OGD and returning to baseline levels at 24 and 48 h. Co-culture experiments revealed that AQP4(+) astrocytes exacerbated injury in OGD-treated neurons, as evidenced by increased TUNEL positivity and apoptotic events. Conversely, AQP4(-) astrocytes appeared to have a protective effect. Knockdown of AQP4 resulted in reduced post-OGD inflammatory response, whereas AQP4 overexpression intensified the injury to neurons post-OGD. In vivo experiments also confirmed that AQP4 inhibitor TGN-020 reduced and overexpression of AQP4 increased behavioral abnormalities and brain infarcts. Our findings underscore AQP4's pivotal role in modulating post-stroke neuroinflammation. Targeting AQP4 may present a novel therapeutic avenue for mitigating ischemia-induced neuronal damage.

Post-injury treatment with 7,8-dihydroxyflavone attenuates white matter pathology in aged mice following focal traumatic brain injury

Traumatic brain injury (TBI) is a major cause of morbidity and mortality, not least in the elderly. The incidence of aged TBI patients has increased dramatically during the last decades. High age is a highly negative prognostic factor in TBI, and pharmacological treatment options are lacking. We used the controlled cortical impact (CCI) TBI model in 23-month-old male and female mice and analyzed the effect of post-injury treatment with 7,8 dihydroxyflavone (7,8-DHF), a brain-derived neurotrophic factor (BDNF)-mimetic compound, on white matter pathology. Following CCI or sham injury, mice received subcutaneous 7,8-DHF injections (5 ​mg/kg) 30 ​min post-injury and were sacrificed on 2, 7 or 14 days post-injury (dpi) for histological and immunofluorescence analyses. Histological assessment with Luxol Fast Blue (LFB)/Cresyl Violet stain showed that administration of 7,8-DHF resulted in preserved white matter tissue at 2 and 7 dpi with no difference in cortical tissue loss at all investigated time points. Treatment with 7,8-DHF led to reduced axonal swellings at 2 and 7 dpi, as visualized by SMI-31 (Neurofilament Heavy Chain) immunofluorescence, and reduced number of TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labelling)/CC1-positive mature oligodendrocytes at 2 dpi in the perilesional white matter. Post-injury proliferation of Platelet-derived Growth Factor Receptor (PDGFRα)-positive oligodendodrocyte progenitor cells was not altered by 7,8-DHF. Our results suggest that 7,8-DHF can attenuate white matter pathology by mitigating axonal injury and oligodendrocyte death in the aged mouse brain following TBI. These data argue that further exploration of 7,8-DHF towards clinical use is warranted.