Comparison of 18F-FDG PET/CT and 18F-FAPI PET/CT in Systemic Staging of Newly Diagnosed Breast Cancer.

RATIONALE AND OBJECTIVES: The purpose of this study was to compare the performance of 18F-FAPI PET/CT and 18F-FDG PET/CT in systemic staging of newly diagnosed breast cancer. METHODS: Breast cancer patients with initial clinical stage IIB-IIIC who have consequently underwent both 18F-FAPI and 18F-FDG PET/CT from June 2022 to June 2023 were retrospectively analyzed. New clinical stage was assigned to each patient if unsuspected level III axillary and extraaxillary regional lymph node metastases (URNM) and/or distant metastases were disclosed after PET/CT. Sensitivity for both tests was calculated on patient basis and lesion basis using histology or follow-up imaging as reference standard. RESULTS: 38 patients were included. The overall upstaging rate was 47.4% for 18F-FAPI PET/CT (18/38) and 34.2% for 18F-FDG PET/CT (13/38). The rate of distant metastases disclosed by 18F-FAPI PET/CT was 5.5% in stage IIB patients, 30% in stage IIIA patients, 50% in stage IIIB patients, and 75% in stage IIIC patients. On patent-based analysis, the sensitivity of 18F-FAPI PET/CT was significantly different from that of 18F-FDG PET/CT in detecting URNM [100% (13/13) vs 53.8% (7/13), (P = 0.031)], but not for distant metastases [100% (10/10) vs 90% (9/10), (P = 1.000)]. On lesion-based analysis, the sensitivity of 18F-FAPI PET/CT was significantly higher than that of 18F-FDG PET/CT in detecting URNM [97.6% (41/42) vs 52.4% (22/42), (P < 0.001)] and distant metastases [98.1% (51/52) vs 67.3% (35/52), (P < 0.001)]. CONCLUSION: 18F-FAPI PET/CT leads to significant upstaging in newly diagnosed breast cancer, in a rate higher than 18F-FDG PET/CT. The significantly higher lesion-based sensitivity in unsuspected metastases implies a future role of 18F-FAPI PET/CT in evaluation of metastatic disease burden.

Exploring the impact of lipid nanoparticles on protein stability and cellular proteostasis.

Lipid nanoparticles (LNPs) have become pivotal in advancing modern medicine, from mRNA-based vaccines to gene editing with CRISPR-Cas9 systems. Though LNPs based therapeutics offer promising drug delivery with satisfactory clinical safety profiles, concerns are raised regarding their potential nanotoxicity. Here, we explore the impacts of LNPs on protein stability in buffer and cellular protein homeostasis (proteostasis) in HepG2 cells. First, we show that LNPs of different polyethylene glycol (PEG) molar ratios to total lipid ratio boost protein aggregation propensity by reducing protein stability in cell lysate and blood plasma. Second, in HepG2 liver cells, these LNPs induce global proteome aggregation, as imaged by a cellular protein aggregation fluorescent dye (AggStain). Such LNPs induced proteome aggregation is accompanied by decrease in cellular micro-environmental polarity as quantified by a solvatochromic protein aggregation sensor (AggRetina). The observed local polarity fluctuations may be caused by the hydrophobic contents of LNPs that promote cellular proteome aggregation. Finally, we exploit RNA sequencing analysis (RNA-Seq) to reveal activation of unfolded protein response (UPR) pathway and other proteostasis genes upon LNPs treatment. Together, these findings highlight that LNPs may induce subtle proteome stress by compromising protein stability and proteostasis even without obvious damage to cell viability.

Isophorone-based crystallization-induced-emission sensors detect proteome aggregation in live cells and tissues with breast cancer.

BACKGROUND: Protein misfolding and aggregation can lead to various diseases. Recent studies have shed light on the aggregated protein in breast cancer pathology, which suggests that it is crucial to design chemical sensors that visualize protein aggregates in breast cancer, especially in clinical patient-derived samples. However, most reported sensors are constrained in cultured cell lines. RESULTS: In this work, we present the development of two isophorone-based crystallization-induced-emission fluorophores for detecting proteome aggregation in breast cancer cell line and tissues biopsied from diseased patients, designated as A1 and A2. These probes exhibited viscosity sensitivity and recovered their fluorescence strongly at crystalline state. Moreover, A1 and A2 exhibit selective binding capacity and strong fluorescence for various aggregated proteins. Utilizing these probes, we detect protein aggregation in stressed breast cancer cells, xenograft mouse model of human breast cancer and clinical patient-derived samples. Notably, the fluorescence intensity of both probes light up in tumor tissues. SIGNIFICANCE: The synthesized isophorone-based crystallization-induced-emission fluorophores, A1 and A2, enable sensitive detection of protein aggregation in breast cancer cells and tissues. In the future, aggregated proteins are expected to become indicators for early diagnosis and clinical disease monitoring of breast cancer.

K+-Specification with Flavone P0 Probe in a G-Quadruplex DNA.

G-quadruplex (G4) DNA is considered as a prospective therapeutic target due to its potential biological significance. To understand G4 biological roles and function, a G4-specific fluorescent probe is necessary. However, it is difficult for versatile G4 to precisely recognize without perturbing their folding dynamics. Herein, we reported that flavone P0 can be a fluorescent probe for G4 DNA-specific recognition and have developed a highly selective detection of K+ ion by dimeric G4/P0 system. When comparing various nucleic acid structures, including G4, i-motif, ss/ds-DNA, and triplex, an apparent fluorescence enhancement is observed in the presence of G4 DNA for 85-fold, but only 8-fold for non-G4 DNA. Furthermore, based on fluorescent probe of flavone P0 for G4 DNA screening, the noncovalent dimeric G4/P0 system is exploited as a K+ sensor, that selectively responds to K+ with a 513-fold fluorescence enhancement and a detection range for K+ ion concentration from 0 to 500 mM. This K+ sensor also has a remarkably anti-interference ability for other metal cations, especially for a high concentration of Na+. These results have demonstrated that flavone P0 is an efficient tool for monitoring G-quadruplex DNA and endows flavone P0 with bioanalytical and medicinal applications.

Beyond Frequency Bands: Complementary-Ensemble-Empirical-Mode-Decomposition-Enhanced Microstate Sequence Non-Randomness Analysis for Aiding Diagnosis and Cognitive Prediction of Dementia.

Exploring the spatiotemporal dynamic patterns of multi-channel electroencephalography (EEG) is crucial for interpreting dementia and related cognitive decline. Spatiotemporal patterns of EEG can be described through microstate analysis, which provides a discrete approximation of the continuous electric field patterns generated by the brain cortex. Here, we propose a novel microstate spatiotemporal dynamic indicator, termed the microstate sequence non-randomness index (MSNRI). The essence of the method lies in initially generating a sequence of microstate transition patterns through state space compression of EEG data using microstate analysis. Following this, we assess the non-randomness of these microstate patterns using information-based similarity analysis. The results suggest that this MSNRI metric is a potential marker for distinguishing between health control (HC) and frontotemporal dementia (FTD) (HC vs. FTD: 6.958 vs. 5.756, p < 0.01), as well as between HC and populations with Alzheimer's disease (AD) (HC vs. AD: 6.958 vs. 5.462, p < 0.001). Healthy individuals exhibit more complex macroscopic structures and non-random spatiotemporal patterns of microstates, whereas dementia disorders lead to more random spatiotemporal patterns. Additionally, we extend the proposed method by integrating the Complementary Ensemble Empirical Mode Decomposition (CEEMD) method to explore spatiotemporal dynamic patterns of microstates at specific frequency scales. Moreover, we assessed the effectiveness of this innovative method in predicting cognitive scores. The results demonstrate that the incorporation of CEEMD-enhanced microstate dynamic indicators significantly improved the prediction accuracy of Mini-Mental State Examination (MMSE) scores (R2 = 0.940). The CEEMD-enhanced MSNRI method not only aids in the exploration of large-scale neural changes in populations with dementia but also offers a robust tool for characterizing the dynamics of EEG microstate transitions and their impact on cognitive function.

Chemical sensors detect and resolve proteome aggregation in peripheral neuropathy cell model induced by chemotherapeutic agents.

As a consequence of somatosensory nervous system injury or disease, neuropathic pain is commonly associated with chemotherapies, known as chemotherapy-induced peripheral neuropathy (CIPN). However, the mechanisms underlying CIPN-induced proteome aggregation in neuronal cells remain elusive due to limited detection tools. Herein, we present series sensors for fluorescence imaging (AggStain) and proteomics analysis (AggLink) to visualize and capture aggregated proteome in CIPN neuronal cell model. The environment-sensitive AggStain imaging sensor selectively binds and detects protein aggregation with 12.3 fold fluorescence enhancement. Further, the covalent AggLink proteomic sensor captures cellular aggregated proteins and profiles their composition via LC-MS/MS analysis. This integrative sensor platform reveals the presence of proteome aggregation in CIPN cell model and highlights its potential for broader applications in assessing proteome stability under various cellular stress conditions.

Unraveling Intracellular Protein Corona Components of Nanoplastics via Photocatalytic Protein Proximity Labeling.

Bioaccumulation of nanoplastic particles has drawn increasing attention regarding environmental sustainability and biosafety. How nanoplastic particles interact with the cellular milieu still remains elusive. Herein, we exemplify a general approach to profile the composition of a "protein corona" interacting with nanoparticles via the photocatalytic protein proximity labeling method. To enable photocatalytic proximity labeling of the proteome interacting with particles, iodine-substituted BODIPY (I-BODIPY) is selected as the photosensitizer and covalently conjugated onto amino-polystyrene nanoparticles as a model system. Next, selective proximity labeling of interacting proteins is demonstrated using I-BODIPY-labeled nanoplastic particles in both Escherichia coli lysate and live alpha mouse liver 12 cells. Mechanistic studies reveal that the covalent modifications of proteins by an aminoalkyne substrate are conducted via a reactive oxygen species photosensitization pathway. Further proteomic analysis uncovers that mitochondria-related proteins are intensively involved in the protein corona, indicating substantial interactions between nanoplastic particles and mitochondria. In addition, proteostasis network components are also identified, accompanied by consequent cellular proteome aggregation confirmed by fluorescence imaging. Together, this work exemplifies a general strategy to interrogate the composition of the protein corona of nanomaterials by endowing them with photooxidation properties to enable photocatalytic protein proximity labeling function.

Dual-environment-sensitive probe to detect protein aggregation in stressed laryngeal carcinoma cells and tissues.

The interplay between protein folding and biological activity is crucial, with the integrity of the proteome being paramount to ensuring effective biological function execution. In this study, we report a dual-environment-sensitive probe A1, capable of selectively binding to protein aggregates and dynamically monitoring their formation and degradation. Through in vitro, cellular, and tissue assays, A1 demonstrated specificity in distinguishing aggregated from folded protein states, selectively partitioning into aggregated proteins. Thermal shift assays revealed A1 could monitor the process of protein aggregation upon binding to misfolded proteins and preceding to insoluble aggregate formation. In cellular models, A1 detected stress-induced proteome aggregation in TU212 cells (laryngeal carcinoma cells), revealing a less polar microenvironment within the aggregated proteome. Similarly, tissue samples showed more severe proteome aggregation in cancerous tissues compared to paracancerous tissues. Overall, A1 represents a versatile tool for probing protein aggregation with significant implications for both fundamental research and clinical diagnostics.

Acetamide-Caprolactam Deep Eutectic Solvent-Based Electrolyte for Stable Zn-Metal Batteries.

Aqueous Zn-ion batteries (AZIBs) are promising for grid-scale energy storage. However, conventional AZIBs face challenges including hydrogen evolution reaction (HER), leading to high local pH, and by-product formation on the anode. Hereby the hydrogen bonds in the aqueous electrolyte are reconstructed by using a deep eutectic co-solvent (DES) made of acetamide (H-bond donor) and caprolactam (H-bond acceptor), which effectively suppresses the reactivity of water and broadens the electrochemical voltage stability window. The coordination between Zn2+ and acetamide-caprolactam in DES-based electrolytes produces a unique solvation structure that promotes the preferential growth of Zn crystals along the (002) plane. This will inhibit the formation of Zn dendrites and ensure the uniform deposition of Zn-ions on the anode surface. In addition, it is found that this DES-based electrolyte can form a protective membrane on the anode surface, reducing the risks of Zn corrosion. Compared to conventional electrolytes, the DES-based electrolyte shows a long-term stable plating/stripping performance with a significantly improved Coulombic efficiency from 78.18% to 98.37%. It is further demonstrated that a Zn||VS2 full-cell with the DES-based electrolyte exhibits enhanced stability after 500 cycles with 85.4% capacity retention at 0.5 A g-1 .

[Applications and challenges of wearable electroencephalogram signals in depression recognition and personalized music intervention].

Rapid and accurate identification and effective non-drug intervention are the worldwide challenges in the field of depression. Electroencephalogram (EEG) signals contain rich quantitative markers of depression, but whole-brain EEG signals acquisition process is too complicated to be applied on a large-scale population. Based on the wearable frontal lobe EEG monitoring device developed by the authors' laboratory, this study discussed the application of wearable EEG signal in depression recognition and intervention. The technical principle of wearable EEG signals monitoring device and the commonly used wearable EEG devices were introduced. Key technologies for wearable EEG signals-based depression recognition and the existing technical limitations were reviewed and discussed. Finally, a closed-loop brain-computer music interface system for personalized depression intervention was proposed, and the technical challenges were further discussed. This review paper may contribute to the transformation of relevant theories and technologies from basic research to application, and further advance the process of depression screening and personalized intervention.

Plastic nanoparticles cause proteome stress and aggregation by compromising cellular protein homeostasis ex vivo and in vivo.

Decomposition of plastic materials into minuscule particles and their long-term uptake pose increasing concerns on environmental sustainability and biosafety. Besides common cell viability and cytotoxicity evaluations, how plastic nanoparticles interfere with different stress response pathways and affect cellular fitness has been less explored. Here, we provided the first piece of evidence to demonstrate plastic nanoparticles potentially can deteriorate proteome stability, compromise cellular protein homeostasis, and consequently cause global proteome misfolding and aggregation. Polystyrene (PS) nanoparticles of different sizes and surface charges were exploited as model plastic materials. In cell lysate and human blood plasma, naked PS nanoparticles with hydrophobic surface deteriorated proteome thermodynamic stability and exaggerated its aggregation propensity. While no cell viability ablation was observed in cells treated with PS nanoparticles up to 200 µg·mL-1, global proteome aggregation and stress was detected by a selective proteome aggregation sensor. Further proteomics analysis revealed how protein homeostasis network was remodeled by positively charged PS nanoparticles via differential expression of key proteins to counteract proteome stress. In mice model, size-dependent liver accumulation of positively charged PS nanoparticles induced hepatocellular proteome aggregation and compromised protein homeostasis network capacity that were invisible to standard alanine transaminase and aspartate transaminase (ALT/AST) liver function as-say and histology. Meanwhile, long-term liver accumulation of plastic nanoparticles deteriorated liver metabolism and saturated liver detoxification capacity of overdosed acetaminophen. This work highlighted the impact of nanoplastics on cellular proteome integrity and cellular fitness that are invisible to current biochemical assays and clinical tests.

Solvatochromic sensors detect proteome aggregation in stressed liver tissues with hepatic cancer and cirrhosis.

Protein misfolding and aggregation involve complex cellular processes with clinical implications in various diseases. However, the detection of aggregated proteomes without defined 3-D structures in a complex biological milieu is challenging. This study utilizes chromone scaffold-based environment-sensitive fluorophores P1 and P2 to detect misfolded and aggregated proteome in stressed liver cells and the liver tissues diseased patients. The reported crystallization induced emission probes (P1 and P2) exhibit both polarity and viscosity sensitivity, with emission intensity and wavelength linearly correlated to viscosity and polarity. Meanwhile, P1 and P2 selectively and generally fluoresce upon binding to various aggregated proteins. In hepatic cells, P2 outperforms P1 in detecting stress-induced global proteome aggregation. In mouse liver tissue upon drug-induced injury, the fluorescence intensity of P2 correlated with the severity of liver injury, serving as an earlier indicator for liver stress prior to ALT/AST increase. The quantification of emission wavelength reveals lower micro-environmental polarity in liver-injury tissue. In patient-derived tissues with hepatic cancer and cirrhosis, P1 and P2 also report on the presence of aggregated proteome. Together, the reported solvatochromic proteome aggregation sensors can detect hepatic proteome aggregation and analyze its local polarity in cultured cell lines, animal model tissues, and human clinical samples.

Tailoring the Amphiphilicity of Fluorescent Protein Chromophores to Detect Intracellular Proteome Aggregation in Diverse Biological Samples.

The formation of amorphous misfolded and aggregated proteins is a hallmark of proteome stress in diseased cells. Given its lack of defined targeting sites, the rational design of intracellular proteome aggregation sensors has been challenging. Herein, we modulate the amphiphilicity of fluorescent protein chromophores to enable selective detection of aggregated proteins in different biological samples, including recombinant proteins, stressed live cells, intoxicated mouse liver tissue, and human hepatocellular carcinoma tissue. By tuning the number of hydroxyl groups, we optimize the selectivity of fluorescent protein chromophores toward aggregated proteins in these biological samples. In recombinant protein applications, the most hydrophobic P0 (cLogP = 5.28) offers the highest fold change (FC = 31.6), sensitivity (LLOD = 0.1 µM), and brightness (Φ = 0.20) upon binding to aggregated proteins. In contrast, P4 of balanced amphiphilicity (cLogP = 2.32) is required for selective detection of proteome stresses in live cells. In mouse and human liver histology tissues, hydrophobic P1 exhibits the best performance in staining the aggregated proteome. Overall, the amphiphilicity of fluorescent chromophores governs the sensor's performance by matching the diverse nature of different biological samples. Together with common extracellular amyloid sensors (e.g., Thioflavin T), these sensors developed herein for intracellular amorphous aggregation complement the toolbox to study protein aggregation.

Fluorene-based tau fibrillation sensor and inhibitor with fluorogenic and photo-crosslinking properties.

Tau protein aggregation into neurofibrillary tangles often causes tauopathies. Herein, we report fluorene based sensors with fluorogenicity upon binding to tau proteins. Intriguingly, these sensors possess triplet state properties to inhibit tau fibrillation upon photo-induced crosslinking.

The Unique Genetic Mutation Characteristics Based on Large Panel Next-Generation Sequencing (NGS) Detection in Multiple Primary Lung Cancers (MPLC) Patients.

BACKGROUND: With the wide application of multislice spiral computed tomography (CT), the frequency of detection of multiple lung cancer is increasing. This study aimed to analyze gene mutations characteristics in multiple primary lung cancers (MPLC) using large panel next-generation sequencing (NGS) assays. METHODS: Patients with MPLC surgically removed from the Affiliated Hospital of Guangdong Medical University from Jan 2020 to Dec 2021 enrolled the study. NGS sequencing of large panels of 425 tumor-associated genes was performed. RESULTS: The 425 panel sequencing of 114 nodules in 36 patients showed that epidermal growth factor receptor (EGFR) accounted for the largest proportion (55.3%), followed by Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2) (9.6%), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), and Kirsten rat sarcoma viral oncogene (KRAS) (8.8%). Fusion target variation was rare (only 2, 1.8%). ERBB2 Y772_A775dup accounted for 73%, KRAS G12C for about 18%, and BRAF V600E for only 10%. AT-rich interaction domain 1A (ARID1A) mutations were significantly higher in invasive adenocarcinoma (IA) which contained solid/micro-papillary malignant components (p = 0.008). The tumor mutation burden (TMB) distribution was low, with a median TMB of 1.1 MUTS/Mb. There were no differences in the TMB distribution of different driver genes. In addition, 97.2% of MPLC patients (35/36) had driver gene mutations, and 47% had co-mutations, mainly in IA (45%) and invasive adenocarcinoma (MIA) (37%) nodule, with EGFR (39.4%), KRAS (9.1%), ERBB2 (6.1%), tumor protein 53 (TP53) (6.1%) predominately. CONCLUSIONS: MPLC has a unique genetic mutation characteristic that differs from advanced patients and usually presents with low TMB. Comprehensive NGS helps to diagnose MPLC and guides the MPLC clinical treatment. ARID1A is significantly enriched in IA nodules containing micro-papillary/solid components, suggesting that these MPLC patients may have a poor prognosis.

Integrated Imaging and Proteomic Sensors Resolve Proteome Aggregation in Liver Caused by Non-steroidal Anti-inflammatory Drug Overdose.

Given the extreme heterogeneity and the loss of defined protein structures, misfolded and aggregated proteins are technically challenging to visualize and analyze. Herein, we assembled an integrated sensor system to resolve aggregated proteome in live cells and animal liver tissues that are overdosed by non-steroidal anti-inflammatory drugs (NSAIDs). A fluorogenic protein aggregation sensor (AggStain) first discovered the presence of aggregated proteome upon overdosing liver cells with NSAIDs. A solvatochromic protein aggregation sensor (AggRetina) further quantified the compactness (polarity) inside these cellular aggregates. Importantly, we exploited a proteomic sensor (AggLink) to selectively capture aggregated proteins upon NSAID overdose and profile their composition, revealing global collapse of cellular protein homeostasis. Finally, we detected subtle proteome aggregation in mouse liver tissue without obvious acute injury at a low NSAID dosage. Overall, we demonstrated an integrated sensor toolset for proteome aggregation studies and unveiled for the first time that NSAID overdose can cause proteome aggregation in liver cells and tissues.

Blue light exposure collapses the inner blood-retinal barrier by accelerating endothelial CLDN5 degradation through the disturbance of GNAZ and the activation of ADAM17.

Blue light is part of the natural light spectrum that emits high energy. Currently, people are frequently exposed to blue light from 3C devices, resulting in a growing incidence of retinopathy. The retinal vasculature is complex, and retinal vessels not only serve the metabolic needs of the retinal sublayers, but also maintain electrolyte homeostasis by forming the inner blood-retinal barrier (iBRB). The iBRB, which is primarily composed of endothelial cells, has well-developed tight junctions. However, with exposure to blue light, the risks of targeting retinal endothelial cells are currently unknown. We found that endothelial claudin-5 (CLDN5) was rapidly degraded under blue light, coinciding with the activation of a disintegrin and metalloprotease 17 (ADAM17), even at non-cytotoxic lighting. An apparently broken tight junction and a permeable paracellular cleft were observed. Mice exposed to blue light displayed iBRB leakage, conferring attenuation of the electroretinogram b-wave and oscillatory potentials. Both pharmacological and genetic inhibition of ADAM17 remarkably alleviated CLDN5 degradation induced by blue light. Under untreated condition, ADAM17 is sequestered by GNAZ (a circadian-responsive, retina-enriched inhibitory G protein), whereas ADAM17 escapes from GNAZ by blue light illuminance. GNAZ knockdown led to ADAM17 hyperactivation, CLDN5 downregulation, and paracellular permeability in vitro, and retinal damage mimicked blue light exposure in vivo. These data demonstrate that blue light exposure might impair the iBRB by accelerating CLDN5 degradation through the disturbance of the GNAZ-ADAM17 axis.

Developing an Affinity-Based Chemical Proteomics Method to In Situ Capture Amorphous Aggregated Proteome and Profile Its Heterogeneity in Stressed Cells.

Stress induced amorphous proteome aggregation is a hallmark for diseased cells, with the proteomic composition intimately associated with disease pathogenicity. Due to its particularly dynamic, reversible, and dissociable nature, as well as lack of specific recognition anchor, it is difficult to capture aggregated proteins in situ. In this work, we develop a chemical proteomics method (AggLink) to capture amorphous aggregated proteins in live stressed cells and identify the proteomic contents using LC-MS/MS. Our method relies on an affinity-based chemical probe (AggLink 1.0) that is optimized to selectively bind to and covalently label amorphous aggregated proteins in live stressed cells. Especially, chaotrope-compatible ligation enables effective enrichment of labeled aggregated proteins under urea denaturation and dissociation conditions. Compared to conventional fractionation-based method to profile aggregated proteome, our method showed improved enrichment selectivity, detection sensitivity, and identification accuracy. In HeLa cells, the AggLink method reveals the constituent heterogeneity of aggregated proteome induced by inhibition of pro-folding (HSP90) or pro-degradation (proteasome) pathway, which uncovers a synergistic strategy to reduce cancer cell viability. In addition, the unique fluorogenicity of our probe upon labeling aggregated proteome detects its cellular location and morphology. Together, the AggLink method may help to expand our knowledge of the previously nontargetable amorphous aggregated proteome.

Leonurine inhibits ferroptosis in renal tubular epithelial cells by activating p62/Nrf2/HO-1 signaling pathway / 中国中药杂志

To investigate the protective effect and the potential mechanism of leonurine(Leo) against erastin-induced ferroptosis in human renal tubular epithelial cells(HK-2 cells), an in vitro erastin-induced ferroptosis model was constructed to detect the cell viability as well as the expressions of ferroptosis-related indexes and signaling pathway-related proteins. HK-2 cells were cultured in vitro, and the effects of Leo on the viability of HK-2 cells at 10, 20, 40, 60, 80 and 100 μmol·L~(-1) were examined by CCK-8 assay to determine the safe dose range of Leo administration. A ferroptosis cell model was induced by erastin, a common ferroptosis inducer, and the appropriate concentrations were screened. CCK-8 assay was used to detect the effects of Leo(20, 40, 80 μmol·L~(-1)) and positive drug ferrostatin-1(Fer-1, 1, 2 μmol·L~(-1)) on the viability of ferroptosis model cells, and the changes of cell morphology were observed by phase contrast microscopy. Then, the optimal concentration of Leo was obtained by Western blot for nuclear factor erythroid 2-related factor 2(Nrf2) activation, and transmission electron microscope was further used to detect the characteristic microscopic morphological changes during ferroptosis. Flow cytometry was performed to detect reactive oxygen species(ROS), and the level of glutathione(GSH) was measured using a GSH assay kit. The expressions of glutathione peroxidase 4(GPX4), p62, and heme oxygenase 1(HO-1) in each group were quantified by Western blot. RESULTS:: showed that Leo had no side effects on the viability of normal HK-2 cells in the concentration range of 10-100 μmol·L~(-1). The viability of HK-2 cells decreased as the concentration of erastin increased, and 5 μmol·L~(-1) erastin significantly induced ferroptosis in the cells. Compared with the model group, Leo dose-dependently increased cell via-bility and improved cell morphology, and 80 μmol·L~(-1) Leo promoted the translocation of Nrf2 from the cytoplasm to the nucleus. Further studies revealed that Leo remarkably alleviated the characteristic microstructural damage of ferroptosis cells caused by erastin, inhibited the release of intracellular ROS, elevated GSH and GPX4, promoted the nuclear translocation of Nrf2, and significantly upregulated the expression of p62 and HO-1 proteins. In conclusion, Leo exerted a protective effect on erastin-induced ferroptosis in HK-2 cells, which might be associated with its anti-oxidative stress by activating p62/Nrf2/HO-1 signaling pathway.

Clinical characteristics of Danon disease / 中华心血管病杂志

Objective: To review the clinical data of 7 patients with Danon disease and analyze their clinical characteristics. Methods: The medical records of 7 patients with Danon disease, who were hospitalized in Peking Union Medical College Hospital of Chinese Academy of Medical Sciences from April 2008 to July 2021, were reviewed and summarized, of which 6 cases were diagnosed as Danon disease by lysosomal-associated membrane protein-2 (LAMP-2) gene mutation detection and 1 case was diagnosed by clinicopathological features. Clinical manifestations, biochemical indexes, electrocardiogram, echocardiography, skeletal muscle and myocardial biopsy and gene detection results were analyzed, and patients received clinical follow-up after discharge. Results: Six patients were male and average age was (15.4±3.5) years and the average follow-up time was (27.7±17.0) months. The main clinical manifestations were myocardial hypertrophy (6/7), decreased myodynamia (2/7) and poor academic performance (3/7). Electrocardiogram features included pre-excitation syndrome (6/7) and left ventricular hypertrophy (7/7). Echocardiography examination evidenced myocardial hypertrophy (6/7), and left ventricular dilatation and systolic dysfunction during the disease course (1/7). The results of skeletal muscle biopsy in 6 patients were consistent with autophagy vacuolar myopathy. Subendocardial myocardial biopsy was performed in 3 patients, and a large amount of glycogen deposition with autophagosome formation was found in cardiomyocytes. LAMP-2 gene was detected in 6 patients, and missense mutations were found in all these patients. During the follow-up period, implantable cardioverter defibrillator implantation was performed in 1 patient because of high atrioventricular block 4 years after diagnosis, and there was no death or hospitalization for cardiovascular events in the other patients. Conclusion: The main clinical manifestations of Danon disease are cardiomyopathy, myopathy and mental retardation. Pre-excitation syndrome is a common electrocardiographic manifestation. Autophagy vacuoles can be seen in skeletal muscle and myocardial pathological biopsies. LAMP-2 gene mutation analysis is helpful in the diagnose of this disease.