Fluorescent Probe for Investigating the Mitochondrial Viscosity and Hydrogen Peroxide Changes in Cerebral Ischemia/Reperfusion Injury.

Cerebral ischemia-reperfusion injury (CIRI), a cause of cerebral dysfunction during cerebral infarction treatment, is closely associated with mitochondrial viscosity and hydrogen peroxide (H2O2). However, the accurate measurement of mitochondrial viscosity and H2O2 levels in CIRI is challenging because of the lack of sufficient selectivity and blood-brain barrier (BBB) penetration of existing monitoring tools related to CIRI, hampering the exploration of the role of mitochondrial viscosity and H2O2 in CIRI. To address this issue, we designed an activatable fluorescent probe, mitochondria-targeting styryl-quinolin-ium (Mito-IQS), with excellent properties including high selectivity, mitochondrial targeting, and BBB penetration, for the visualization of mitochondrial viscosity and H2O2 in the brain. Based on the real-time monitoring capabilities of the probe, bursts of mitochondrial viscosity and H2O2 levels were visualized during CIRI. This probe can be used to monitor the therapeutic effects of butylphthalein treatment. More importantly, in vivo experiments further confirmed that CIRI was closely associated with the mitochondrial viscosity and H2O2 levels. This discovery provides new insights and tools for the study of CIRI and is expected to accelerate the process of CIRI diagnosis, treatment, and drug design.

Exploring deep neural networks via layer-peeled model: Minority collapse in imbalanced training.

In this paper, we introduce the Layer-Peeled Model, a nonconvex, yet analytically tractable, optimization program, in a quest to better understand deep neural networks that are trained for a sufficiently long time. As the name suggests, this model is derived by isolating the topmost layer from the remainder of the neural network, followed by imposing certain constraints separately on the two parts of the network. We demonstrate that the Layer-Peeled Model, albeit simple, inherits many characteristics of well-trained neural networks, thereby offering an effective tool for explaining and predicting common empirical patterns of deep-learning training. First, when working on class-balanced datasets, we prove that any solution to this model forms a simplex equiangular tight frame, which, in part, explains the recently discovered phenomenon of neural collapse [V. Papyan, X. Y. Han, D. L. Donoho, Proc. Natl. Acad. Sci. U.S.A. 117, 24652-24663 (2020)]. More importantly, when moving to the imbalanced case, our analysis of the Layer-Peeled Model reveals a hitherto-unknown phenomenon that we term Minority Collapse, which fundamentally limits the performance of deep-learning models on the minority classes. In addition, we use the Layer-Peeled Model to gain insights into how to mitigate Minority Collapse. Interestingly, this phenomenon is first predicted by the Layer-Peeled Model before being confirmed by our computational experiments.

Pure tone audiogram classification using deep learning techniques.

OBJECTIVE: Pure tone audiometry has played a critical role in audiology as the initial diagnostic tool, offering vital insights for subsequent analyses. This study aims to develop a robust deep learning framework capable of accurately classifying audiograms across various commonly encountered tasks. DESIGN, SETTING, AND PARTICIPANTS: This single-centre retrospective study was conducted in accordance with the STROBE guidelines. A total of 12 518 audiograms were collected from 6259 patients aged between 4 and 96 years, who underwent pure tone audiometry testing between February 2018 and April 2022 at Tongji Hospital, Tongji Medical College, Wuhan, China. Three experienced audiologists independently annotated the audiograms, labelling the hearing loss in degrees, types and configurations of each audiogram. MAIN OUTCOME MEASURES: A deep learning framework was developed and utilised to classify audiograms across three tasks: determining the degrees of hearing loss, identifying the types of hearing loss, and categorising the configurations of audiograms. The classification performance was evaluated using four commonly used metrics: accuracy, precision, recall and F1-score. RESULTS: The deep learning method consistently outperformed alternative methods, including K-Nearest Neighbors, ExtraTrees, Random Forest, XGBoost, LightGBM, CatBoost and FastAI Net, across all three tasks. It achieved the highest accuracy rates, ranging from 96.75% to 99.85%. Precision values fell within the range of 88.93% to 98.41%, while recall values spanned from 89.25% to 98.38%. The F1-score also exhibited strong performance, ranging from 88.99% to 98.39%. CONCLUSIONS: This study demonstrated that a deep learning approach could accurately classify audiograms into their respective categories and could contribute to assisting doctors, particularly those lacking audiology expertise or experience, in better interpreting pure tone audiograms, enhancing diagnostic accuracy in primary care settings, and reducing the misdiagnosis rate of hearing conditions. In scenarios involving large-scale audiological data, the automated classification system could be used as a research tool to efficiently provide a comprehensive overview and statistical analysis. In the era of mobile audiometry, our deep learning framework can also help patients quickly and reliably understand their self-tested audiograms, potentially encouraging timely consultations with audiologists for further evaluation and intervention.

Benchmark Models for Elucidating Ligand Effects: Thiols Ligated Isostructural Cu6 Nanoclusters.

Atomically precise copper nanoclusters (Cu NCs) have attracted tremendous attention for their huge potential in many applications. However, the uncertainty of the growth mechanism and complexity of the crystallization process hinder the in-depth understanding of their properties. In particular, the ligand effect has been rarely explored at the atomic/molecular level due to the lack of feasible models. Herein, three isostructural Cu6 NCs ligated with diverse mono-thiol ligands (2-mercaptobenzimidazole, 2-mercaptobenzothiazole, and 2-mercaptobenzoxazole, respectively) are successfully synthesized, which provide an ideal platform to unambiguously address the intrinsic role of ligands. The overall atom-by-atom structural evolution process of Cu6 NCs is mapped out with delicate mass spectrometry (MS) for the first time. It is intriguingly found that the ligands, albeit only atomic difference (NH, O, and S), can profoundly affect the building-up processes, chemical properties, atomic structures, as well as catalytic activities of Cu NCs. Furthermore, ion-molecule reactions combined with density functional theory (DFT) calculations demonstrate that the defective sites formed on ligand can significantly contribute to the activation of molecular oxygen. This study provides fundamental insights into the ligand effect, which is vital for the delicate design of high-efficient Cu NCs-based catalysts.

Long-term SARS-CoV-2 neutralizing antibody level prediction using multimodal deep learning: A prospective cohort study on longitudinal data in Wuhan, China.

The ongoing epidemic of SARS-CoV-2 is taking a substantial financial and health toll on people worldwide. Assessing the level and duration of SARS-CoV-2 neutralizing antibody (Nab) would provide key information for government to make sound healthcare policies. Assessed at 3-, 6-, 12-, and 18-month postdischarge, we described the temporal change of IgG levels in 450 individuals with moderate to critical COVID-19 infection. Moreover, a data imputation framework combined with a novel deep learning model was implemented to predict the long-term Nab and IgG levels in these patients. Demographic characteristics, inspection reports, and CT scans during hospitalization were used in this model. Interpretability of the model was further validated with Shapely Additive exPlanation (SHAP) and Gradient-weighted Class Activation Mapping (GradCAM). IgG levels peaked at 3 months and remained stable in 12 months postdischarge, followed by a significant decline in 18 months postdischarge. However, the Nab levels declined from 6 months postdischarge. By training on the cohort of 450 patients, our long-term antibody prediction (LTAP) model could predict long-term IgG levels with relatively high area under the receiver operating characteristic curve (AUC), accuracy, precision, recall, and F1-score, which far exceeds the performance achievable by commonly used models. Several prognostic factors including FDP levels, the percentages of T cells, B cells and natural killer cells, older age, sex, underlying diseases, and so forth, served as important indicators for IgG prediction. Based on these top 15 prognostic factors identified in IgG prediction, a simplified LTAP model for Nab level prediction was established and achieved an AUC of 0.828, which was 8.9% higher than MLP and 6.6% higher than LSTM. The close correlation between IgG and Nab levels making it possible to predict long-term Nab levels based on the factors selected by our LTAP model. Furthermore, our model identified that coagulation disorders and excessive immune response, which indicate disease severity, are closely related to the production of IgG and Nab. This universal model can be used as routine discharge tests to identify virus-infected individuals at risk for recurrent infection and determine the optimal timing of vaccination for general populations.

Artificial oocyte activation with ionomycin compared with A23187 among patients at risk of failed or impaired fertilization.

RESEARCH QUESTION: Do fertilization rates differ between intracytoplasmic sperm injection (ICSI) cycles treated with artificial oocyte activation (AOA) using 10 µmol/l ionomycin or commercial A23187 in women at risk of failed or impaired fertilization? DESIGN: This single-centre, 7-year retrospective cohort study included 157 couples with a history of total fertilization failure (TFF, 0%) or low fertilization (<30%) after ICSI, or with severe oligo-astheno-teratozoospermia (OAT) in the male partner. Couples and underwent 171 ICSI-AOA cycles using either 10 µmol/l ionomycin or commercial A23187. The embryological and clinical outcomes were compared. RESULTS: Fertilization rates in the ionomycin group were significantly higher than those in the A23187 group for all three subgroups (TFF, 46.9% versus 28.4%, P = 0.002; low fertilization, 67.7% versus 49.2%, P < 0.001; severe OAT, 66.4% versus 31.6%, P < 0.001). AOA with ionomycin significantly increased the day 3 cleavage rate (P = 0.009) when compared with A23187 in the low fertilization group, but not in the TFF or severe OAT group (both P > 0.05). The rates of day 3 good-quality embryos, clinical pregnancy, implantation and live birth, and the cumulative live birth, did not differ between the two groups (all P > 0.05). A total of 64 live births resulted in 72 healthy babies born. CONCLUSIONS: AOA with 10 µmol/l ionomycin may be more effective than commercial A23187 in improving oocyte activation in patients at risk of failed or impaired fertilization, especially in cases of sperm-related defects.

[Protective effect of vitrification and controlled slow freezing on immature testicular tissue].

OBJECTIVE: To explore and compare the current mainstream testicular tissue freezing methods, namely vitrification and controlled slow freezing, and optimize the best testicular tissue freezing methods. METHODS: Testicular tissues of 3-week-old mice and <2-year old prepubertal cynomophage monkeys were collected and cut to 6-26 mm3, and divided into three groups: Fresh group, vitrification group and controlled slow freezing group were resuscitated after 5-7 days of freezing. HE staining, immunofluorescence staining, TUNEL staining and Western blot were used to evaluate the frozen-thawed testicular tissue. RESULTS: 1. In the testes of C57BL6/J mice, the expression level of spermatogonial stem cell marker UCHL1 in the controlled slow freezing group was higher than that in the vitrification group, and the content of apoptotic cells (TUNEL+ cells) was lower than that in the vitrification group. 2. In the testicular tissue of cynomolgus monkeys, the expression levels of spermatogonial stem cell markers UCHL1 and cell proliferation marker PCNA in the CSF group were higher than those in the vitrification group. CONCLUSION: Both vitrification and CSF can successfully preserve the testes of immature mice and cynomolgus monkeys before puberty. However, in the vitrification, there are more frozen damaged areas in the testicular tissue with the frozen volume of 6-26mm3, which may affect the cryopreservation of spermatogonial stem cells in the testicular tissue.

A novel gene mutation in ZP3 loop region identified in patients with empty follicle syndrome.

The zona pellucida (ZP) is an extracellular matrix surrounding mammalian oocytes. It is composed of three to four glycoproteins, ZP1-ZP4. ZP3 is essential for sperm binding and zona matrix formation. Here, we identified a novel heterozygous mutation (NM_001110354.2:c.502_504delGAG) of ZP3, occurring in a pair of sisters with empty follicle syndrome (EFS). A mouse model with the same mutation was established using the CRISPR/Cas9 gene-editing system. As in the above family, F0 -, F1 -, and F2 -generation female mice with the mutation were all infertile. Further analysis using the Chinese hamster ovary cells (CHO-K1) also showed that this mutation weakens the strength of binding between ZP3 and ZP2, which hinders the assembly of ZP and results in unstable ZP formation. Immunohistochemical analysis using ovarian serial sections in both humans and mice demonstrated that the ZP of preantral follicles was thinner than normal control, or even absent. Our study presents a new gene mutation that leads to EFS, providing new evidence and support for the genetic diagnosis of infertile individuals with similar phenotypes. Our results also show that the loop of ZP3 is not only a linker between two amphiphilic helices but may play a critical role in specifying the correct heterodimerization partner.

Modulating the Oxidation State of Titanium via Dual Anions Substitution for Efficient N2 Electroreduction.

The electrocatalytic nitrogen reduction reaction (NRR) is a promising approach for renewable ammonia synthesis but remains significantly challenging due to the low yield and poor selectivity. Herein, a facile N and S dual anions substitution strategy is developed to tune the Ti oxidation states of TiO2 nanohybrid catalyst (NS-TiO2 /C), in which anatase TiO2 nanoplates with dense Ti3+ active sites are uniformly dispersed on porous carbon derived from 2D Ti3 C2 Tx nanosheets. The catalyst NS-TiO2 /C exhibits a superior ambient NRR efficiency with an NH3 yield rate of 19.97 µg h-1 mg-1cat and Faradaic efficiency of 25.49% and is coupled with a remarkable 50 h long-term stability at -0.25 V versus RHE. Both experimental and theoretical results reveal that the N and S dual-substitution effectively regulate the Ti oxidation state and electronical properties of the NS-TiO2 /C via simultaneously forming interstitial and substitutional TiS and TiN bonds in the anatase TiO2 lattice, inducing oxygen vacancies and dense Ti3+ active species as well as better electronic conductivity, which substantially facilitates N2 chemisorption and activation, and reduces the energy barrier of the rate-determining step, thereby essentially boosting NRR efficiency. This work provides a valuable approach to the rational design of advanced materials by modulating oxidation states for efficient electrocatalysis.

Tetrahedral W4cluster confined in graphene-like C2N enables electrocatalytic nitrogen reduction from theoretical perspective.

Exploring the format of active site is essential to further the understanding of an electrocatalyst working under ambient conditions. Herein, we present a DFT study of electrocatalytic nitrogen reduction (eNRR) on W4tetrahedron embedded in graphene-like C2N (denoted as W4@C2N). Our results demonstrate that N-affinity of active sites on W4dominate over single-atom site, rendering *NH2 + (H+ + e-) →*NH3invariably the potential-determining step (PDS) of eNRR via consecutive or distal route (UL = -0.68 V) to ammonia formation. However, *NHNH2 + (H+ + e-) →*NH2NH2has become the PDS (UL = -0.54 V) via enzymatic route towards NH2NH2formation and thereafter desorption, making W4@C2N a potentially promising catalyst for hydrazine production from eNRR. Furthermore, eNRR is competitive with hydrogen evolution reaction (UL = -0.78 V) on W4@C2N, which demonstrated sufficient thermal stability and electric property for electrode application.

Interaction between endometrial microbiota and host gene regulation in recurrent implantation failure.

AIM: To learn about the interaction between endometrial microbiota and host gene regulation in recurrent implantation failure. METHODS: The endometrial microbiota of 111 patients (RIF, 75; CON, 36) was analyzed by using 16 s rRNA sequencing technology. Transcriptome sequencing analysis of the endometrial of 60 patients was performed by using high-throughput sequencing. RESULTS: We found that the structure and composition of endometrium microbiota community of RIF patients were significantly different from those in control group. The abnormality of microbial structure and composition might interfere with the implantation of embryos by affecting the immune adaptation of the endometrium and the formation of endometrial blood vessels. CONCLUSIONS: Our research described the host-microbe interaction in RIF. The structure and composition of endometrium microbiota community of RIF patients were significantly different from those in CON group. The abnormality of microbial structure and composition might interfere with the implantation of embryos by affecting the immune adaptation of the endometrium and the formation of endometrial blood vessels.

Aging endometrium in young women: molecular classification of endometrial aging-based markers in women younger than 35 years with recurrent implantation failure.

BACKGROUND: To explore the differences between a population with premature endometrial aging and a population with normal endometrial status in young women with recurrent implantation failure (< 35 years). METHODS: Systematic analysis of the endometrium transcriptome of 274 RIF women. The NMF algorithm was used for classification based on endometrial-specific aging markers in CellAge, and the endometrial receptivity, gene expression patterns, and clinical data were compared between the classifications. RESULTS: Two hundred forty-five young RIF women could be divided into two clusters, in which the aging gene expression pattern of cluster 2 was closer to the reference cluster. Cluster 1 was characterized by high immune activity, while cluster 2 was characterized by high metabolic activity. Combined with clinical data, cluster 2 was worse than cluster 1 in window of implantation deviation rate and endometrial receptivity. CONCLUSION: Premature aging of the endometrium exists in young women with RIF, and premature aging of the endometrium was associated with poor reproductive outcomes.

A Network-Pharmacology-Combined Integrated Pharmacokinetic Strategy to Investigate the Mechanism of Potential Liver Injury due to Polygonum multiflorum.

Polygonum multiflorum (PM) has been used as a tonic and anti-aging remedy for centuries in Asian countries. However, its application in the clinic has been hindered by its potential to cause liver injury and the lack of investigations into this mechanism. Here, we established a strategy using a network pharmacological technique combined with integrated pharmacokinetics to provide an applicable approach for addressing this issue. A fast and sensitive HPLC-QQQ-MS method was developed for the simultaneous quantification of five effective compounds (trans-2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucoside, emodin-8-O-ß-d-glucoside, physcion-8-O-ß-d-glucoside, aloe-emodin and emodin). The method was fully validated in terms of specificity, linearity, accuracy, precision, extraction recovery, matrix effects, and stability. The lower limits of quantification were 0.125-0.500 ng/mL. This well-validated method was successfully applied to an integrated pharmacokinetic study of PM extract in rats. The network pharmacological technique was used to evaluate the potential liver injury due to the five absorbed components. Through pathway enrichment analysis, it was found that potential liver injury is primarily associated with PI3K-Akt, MAPK, Rap1, and Ras signaling pathways. In brief, the combined strategy might be valuable in revealing the mechanism of potential liver injury due to PM.

Discovery of Hepatotoxic Equivalent Markers and Mechanism of Polygonum multiflorum Thunb. by Metabolomics Coupled with Molecular Docking.

Polygonum multiflorum Thunb. (PMT), a commonly used Chinese herbal medicine for treating diseases such as poisoning and white hair, has attracted constant attention due to the frequent occurrence of liver injury incidents. To date, its hepatotoxic equivalent markers (HEMs) and potential hepatotoxic mechanisms are still unclear. In order to clarify the HEMs of PMT and further explore the potential mechanisms of hepatotoxicity, firstly, the chemical constituents in PMT extract were globally characterized, and the fingerprints of PMT extracts were established along with the detection of their hepatotoxicity in vivo. Then, the correlations between hepatotoxic features and component contents were modeled by chemometrics to screen HEMs of PMT, which were then further evaluated. Finally, the hepatotoxic mechanisms of PMT were investigated using liver metabolomics and molecular docking. The results show that the chemical combination of 2,3,5,4-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG) and emodin-8-O-glucoside (EG) was discovered as the HEMs of PMT through pre-screening and verifying process. Liver metabolomics revealed that PMT caused liver injury by interfering with purine metabolism, which might be related to mitochondrial function disorder and oxidative injury via the up-regulations of xanthosine and xanthine, and the down-regulation of 5' nucleotidase (NT5E) and adenylate kinase 2 (AK2). This study not only found that the HEMs of PMT were TSG and EG, but also clarified that PMT might affect purine metabolism to induce liver injury, which contributed to our understanding of the underlying mechanisms of PMT hepatotoxicity.

Assessing potential liver injury induced by Polygonum multiflorum using potential biomarkers via targeted sphingolipidomics.

CONTEXT: Polygonum multiflorum Thunb. (Polygonaceae) (PM) can cause potential liver injury which is typical in traditional Chinese medicines (TCMs)-induced hepatotoxicity. The mechanism involved are unclear and there are no sensitive evaluation indicators. OBJECTIVE: To assess PM-induced liver injury, identify sensitive assessment indicators, and screen for new biomarkers using sphingolipidomics. MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats were randomly divided into four groups (control, model with low-, middle- and high-dose groups, n = 6 each). Rats in the three model groups were given different doses of PM (i.g., low/middle/high dose, 2.7/8.1/16.2 g/kg) for four months. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the plasma and liver were quantitatively analyzed. Fixed liver tissue sections were stained with haematoxylin and eosin and examined under a light microscope. The targeted sphingolipidomic analysis of plasma was performed using high-performance liquid chromatography tandem mass spectrometry. RESULTS: The maximal tolerable dose (MTD) of PM administered intragastrically to mice was 51 g/kg. Sphingolipid profiling of normal and PM-induced liver injury SD rats revealed three potential biomarkers: ceramide (Cer) (d18:1/24:1), dihydroceramide (d18:1/18:0)-1-phosphate (dhCer (d18:1/18:0)-1P) and Cer (d18:1/26:1), at 867.3-1349, 383.4-1527, and 540.5-658.7 ng/mL, respectively. A criterion for the ratio of Cer (d18:1/24:1) and Cer (d18:1/26:1) was suggested and verified, with a normal range of 1.343-2.368 (with 95% confidence interval) in plasma. CONCLUSIONS: Three potential biomarkers and one criterion for potential liver injury caused by PM that may be more sensitive than ALT and AST were found.

Single-cell transcriptome and cell-specific network analysis reveal the reparative effect of neurotrophin-4 in preantral follicles grown in vitro.

BACKGROUND: In-vitro-grow (IVG) of preantral follicles is essential for female fertility preservation, while practical approach for improvement is far from being explored. Studies have indicated that neurotrophin-4 (NT-4) is preferentially expressed in human preantral follicles and may be crucial to preantral follicle growth. METHODS: We observed the location and expression of Tropomyosin-related kinase B (TRKB) in human and mouse ovaries with immunofluorescence and Western blot, and the relation between oocyte maturation and NT-4 level in follicular fluid (FF). Mice model was applied to investigate the effect of NT-4 on preantral follicle IVG. Single-cell RNA sequencing of oocyte combined with cell-specific network analysis was conducted to uncover the underlying mechanism of effect. RESULTS: We reported the dynamic location of TRKB in human and mouse ovaries, and a positive relationship between human oocyte maturation and NT-4 level in FF. Improving effect of NT-4 was observed on mice preantral follicle IVG, including follicle development and oocyte maturation. Transcriptome analysis showed that the reparative effect of NT-4 on oocyte maturation might be mediated by regulation of PI3K-Akt signaling and subsequent organization of F-actin. Suppression of advanced stimulated complement system in granulosa cells might contribute to the improvement. Cell-specific network analysis revealed NT-4 may recover the inflammation damage induced by abnormal lipid metabolism in IVG. CONCLUSIONS: Our data suggest that NT-4 is involved in ovarian physiology and may improve the efficiency of preantral follicle IVG for fertility preservation.

Efficient and economical approach for flexible photothermal icephobic copper mesh with robust superhydrophobicity and active deicing property.

Facing various problems caused by icing in daily life, preparing photothermal deicing materials with wide applicability in high efficiency and low cost is not only a current research hotspot but also a great challenge. Herein, an economical spray-coating method is applied to prepare high-efficiency flexible photothermal icephobic copper mesh using micro silicon carbide (SiC) particles as photothermal conversion material and nano silica (SiO2) particles as a surface superhydrophobic modifier. Owing to the excellent hierarchical micro-nanostructures, the SiC/SiO2 coated copper mesh exhibits a water contact angle (CA) of 162 ± 2° and a sliding angle (SA) of 3 ± 2°. Interestingly, the coated copper mesh exhibits exceptional mechanical durability against water droplet and water flow impact, repeated bending-twisting and tape-peeling. Benefitting from the robust superhydrophobicity, the SiC/SiO2 coating on the copper mesh can significantly delay the freezing time of the droplets and reduce the ice adhesion strength. Furthermore, the coated copper mesh well retains the good photothermal conversion and thermal conductivity properties of the micro SiC particles. Under NIR irradiation, the surface temperature of the coated copper mesh placed on the ice layer can increase by 35.3 °C in 220 s, so that it can rapidly melt the accumulated frost and ice layer on the inner wall of the refrigerator. The presented flexible photothermal icephobic copper mesh exhibits enormous potential when applied to remove ice from apparatus that is accessible, such as road, overhead transmission lines and power networks owing to its flexibility, economy, and high energy efficiency.

α-Hederin inhibits the growth of lung cancer A549 cells in vitro and in vivo by decreasing SIRT6 dependent glycolysis.

CONTEXT: α-Hederin, a potent bioactive compound of Pulsatilla chinensis (Bunge) Regel (Ranunculaceae), has many pharmacological uses, but its effect on cancer cell metabolism is still unclear. OBJECTIVE: To elucidate the role of α-hederin in the glucose metabolism of lung cancer cells. MATERIALS AND METHODS: Cell Counting Kit 8 and colony formation assays were employed to assess the antiproliferative effects of α-hederin. Glucose uptake, ATP generation, and lactate production were measured. Glycolysis-related proteins were detected using western blotting, and a sirtuin 6 (SIRT6) inhibitor was used to verify A549 cell proliferation. Sixty male BALB/c nude mice were divided into normal control, 5-FU (25 mg/kg), and α-hederin (5 and 10 mg/kg) groups to assess the antitumor effect for 32 days. Glycolysis-related protein expression was evaluated using immunohistochemical analysis. RESULTS: α-Hederin inhibited A549 (IC50 = 13.75 µM), NCI-H460 (IC50 = 17.57 µM), and NCI-H292 (IC50 = 18.04 µM) proliferation; inhibited glucose uptake and ATP generation; and reduced lactate production. Furthermore, α-hederin (10 and 15 µM) markedly inhibited hexokinase 2, glucose transporter 1, pyruvate kinase M2, lactate dehydrogenase A, monocarboxylate transporter, c-Myc, hypoxia-inducible factor-1α, and activated SIRT6 protein expression. Using a SIRT6 inhibitor, we demonstrated that α-hederin inhibits glycolysis by activating SIRT6. A tumour xenograft mouse model of lung cancer confirmed that α-hederin (5 and 10 mg/kg) inhibits lung cancer growth by inhibiting glycolysis in vivo. DISCUSSION AND CONCLUSIONS: α-Hederin inhibits A549 cell growth by inhibiting SIRT6-dependent glycolysis. α-Hederin might serve as a potential agent to suppress cancer.

Erzhi pills ameliorate cognitive dysfunction and alter proteomic hippocampus profiles induced by d-galactose and Aß1-40 injection in ovariectomized Alzheimer's disease model rats.

CONTEXT: Erzhi pills are a classic Chinese medicine prescription, but their effects on Alzheimer's disease (AD) are not clear. OBJECTIVE: The protective effects of Erzhi pills in AD rats and their potential mechanisms were investigated. MATERIALS AND METHODS: An AD rat model was established by ovariectomy combined with d-galactose and Aß1-40 injection. Rats were randomly divided into five groups: sham-operated, model, oestradiol valerate (0.80 mg/kg), Erzhi pills high-dose (1.50 g/kg), and Erzhi pills low-dose (0.75 g/kg). Learning and memory abilities were evaluated with the Morris water maze test, oestrogen levels with an ELISA kit, and hippocampal neuron morphology and Nissl bodies in the cytoplasm with H&E and Nissl staining. The expression of ERß, Aß1-40, and p-tau404 was determined by immunohistochemistry. Nano LC-LTQ-Orbitrap Proteomics determined potential targets and related signalling pathways. Western blotting was used to detect the expression of the related proteins. RESULTS: Erzhi pills (1.5, 0.75 g/kg) markedly reduced escape latencies on the MWM, increased numbers of platform crossings, numbers of neurons, Nissl bodies, oestrogen levels (100.18, 43.04 pg/mL), and ERß-positive cells (57.42, 39.83); Aß1-40 (18.85, 36.83)- and p-tau404 (14.42, 29.71)-positive cells were significantly decreased. Proteomics identified more than 100 differentially expressed proteins involved in 48 signalling pathways, five of which are involved in the PI3K/Akt signalling pathway. Western blotting showed decreased expression of GSK3ß and Bad, while Akt, PI3K, 14-3-3, Bcl-xl, and Bcl-2 were upregulated. DISCUSSION AND CONCLUSION: Erzhi pills may serve as a potential agent for AD therapeutics by improving learning and memory.

The role of galectin-3 in spermatozoa-zona pellucida binding and its association with fertilization in vitro.

Human spermatozoa can fertilize an oocyte only after post-testicular maturation and capacitation. These processes involve dynamic modification and reorganization of the sperm plasma membrane, which allow them to bind to the zona pellucida (ZP) of the oocyte. Defective sperm-ZP binding is one of the major causes of male subfertility. Galectin-3 is a secretory lectin in human seminal plasma well known for its action on cell adhesion. The aim of this study was to determine the role of galectin-3 in spermatozoa-ZP interaction and its association with fertilization rate in clinical assisted reproduction. Our studies revealed that the acrosomal region of ejaculated and capacitated spermatozoa possess strong galectin-3 immunoreactivity, which is much stronger than that of epididymal spermatozoa. Expression of galectin-3 can also be detected on seminal plasma-derived extracellular vesicles (EVs) and can be transferred to the sperm surface. Blocking of sperm surface galectin-3 function by antibody or carbohydrate substrate reduced the ZP-binding capacity of spermatozoa. Purified galectin-3 is capable of binding to ZP, indicating that galectin-3 may serve as a cross-linking bridge between ZP glycans and sperm surface glycoproteins. Galectin-3 levels in seminal plasma-derived EVs were positively associated with fertilization rates. These results suggest that galectin-3 in EVs is transferred to the sperm surface during post-testicular maturation and plays a crucial role in spermatozoa-ZP binding after capacitation. Reduced galectin-3 expression in seminal plasma-derived EVs may be a cause behind a low fertilization rate. Further studies with more clinical samples are required to confirm the relationship between galectin-3 levels and IVF outcomes.