Immobilized enzyme-based enzyme electrode biosensors, characterized by high sensitivity and efficiency, strong specificity, and compact size, demonstrate broad application prospects in life science research, disease diagnosis and monitoring, etc. Immobilization of enzyme is a critical step in determining the performance (stability, sensitivity, and reproducibility) of the biosensors. Random immobilization (physical adsorption, covalent cross-linking, etc.) can easily bring about problems, such as decreased enzyme activity and relatively unstable immobilization. Whereas, directional immobilization utilizing amino acid residue mutation, affinity peptide fusion, or nucleotide-specific binding to restrict the orientation of the enzymes provides new possibilities to solve the problems caused by random immobilization. In this paper, the principles, advantages and disadvantages and the application progress of enzyme electrode biosensors of different directional immobilization strategies for enzyme molecular sensing elements by specific amino acids (lysine, histidine, cysteine, unnatural amino acid) with functional groups introduced based on site-specific mutation, affinity peptides (gold binding peptides, carbon binding peptides, carbohydrate binding domains) fused through genetic engineering, and specific binding between nucleotides and target enzymes (proteins) were reviewed, and the application fields, advantages and limitations of various immobilized enzyme interface characterization techniques were discussed, hoping to provide theoretical and technical guidance for the creation of high-performance enzyme sensing elements and the manufacture of enzyme electrode sensors.

ObjectiveChemotherapy is one of the important therapeutic approaches for cancer treatment. However, the emergence of multidrug resistance and side effects significantly limit its application. To address these challenges, chemotherapy is often combined with other drugs or therapies. Among the 13 human fucosyltransferases (FUTs) identified, FUT8 (alpha-(1,6)-fucosyltransferase) is the only enzyme responsible for core fucosylation. Core fucosylation plays an important role in cancer occurrence, metastasis and chemotherapy resistance, making the suppression of FUT8 a potential strategy for reversing multidrug resistance. This study aims to evaluate the feasibility of combining the small molecule FUT8 inhibitor 2FF (2-deoxy-2-fluoro-L-fucose) with the clinical chemotherapeutic drug doxorubicin (DOX) for treating malignant tumors. MethodsThe human hepatocellular carcinoma cell line HepG2 and mouse colon cancer cell line CT26 cells were treated with 2FF, DOX or their combination and core fucosylation levels were assessed using Lectin blot. HepG2 and CT26 cells were exposed to 50 μmol/L 2FF for 72 h, followed by treatment with a gradient concentration of DOX for 24 h. Cell viability and IC₅₀ values were determined via the CCK-8 assay. Transwell invasion assays were conducted to evaluate the combined effect of 2FF and DOX on the invasion ability of HepG2 cells. Flow cytometry was performed to analyze the impact of 2FF, DOX and their combination on membrane PD-L1 expression of HepG2 cells. To assess the in vivo effect, 6- to 8-week-old female BALB/c mice (20-25 g), were subcutaneously injected with 1×10⁶ CT26 cells into the right axilla (four groups, six mice in each group). After the average tumor volume reached 100 mm³, mice were treated with DOX, 2FF, their combination, or saline (mock group) every other day. DOX was administrated intraperitoneally (2 mg/kg), 2FF intravenously (5 mg/kg), and the combination group, received the both treatment. Tumor size was measured every other day using a vernier caliper. ResultsThis study demonstrated that DOX upregulates the core fucosylation levels in HepG2 and CT26 cells,while 2FF effectively inhibits this DOX-induced effect. Furthermone, 2FF enhanced the sensitivity of HepG2 and CT26 cells to DOX. The combination of 2FF and DOX synergistically inhibited the invasion ability of HepG2 cells, and enhanced the anti-tumor efficacy of CT26 subcutaneous tumor model in BALB/c mice. However the combination treatment led to weight loss in mice. In addition, DOX increased the cell surface PD-L1 expression in HepG2 cells, which was effectively suppressed by 2FF. ConclusionThe FUT8 inhibitor 2FF effectively suppresses DOX-induced upregulation of core fucosylation and PD-L1 levels in tumor cells, and 2FF synergistically enhances the anticancer efficacy of DOX.

Diabetic cardiomyopathy is a distinct form of cardiomyopathy that can lead to heart failure, arrhythmias, cardiogenic shock, and sudden death. It has become a major cause of mortality in diabetic patients. The pathogenesis of diabetic cardiomyopathy is complex, involving increased oxidative stress, activation of inflammatory responses, disturbances in glucose and lipid metabolism, accumulation of advanced glycation end products (AGEs), abnormal autophagy and apoptosis, insulin resistance, and impaired intracellular Ca²⁺ homeostasis. Recent studies have shown that adenosine monophosphate-activated protein kinase (AMPK) plays a crucial protective role by lowering blood glucose levels, promoting lipolysis, inhibiting lipid synthesis, and exerting antioxidant, anti-inflammatory, anti-apoptotic, and anti-ferroptotic effects. It also enhances autophagy, thereby alleviating myocardial injury under hyperglycemic conditions. Consequently, AMPK is considered a key protective factor in diabetic cardiomyopathy. As part of diabetes prevention and treatment strategies, both pharmacological and exercise interventions have been shown to mitigate diabetic cardiomyopathy by modulating the AMPK signaling pathway. However, the precise regulatory mechanisms, optimal intervention strategies, and clinical translation require further investigation. This review summarizes the role of AMPK in the prevention and treatment of diabetic cardiomyopathy through drug and/or exercise interventions, aiming to provide a reference for the development and application of AMPK-targeted therapies. First, several classical AMPK activators (e.g., AICAR, A-769662, O-304, and metformin) have been shown to enhance autophagy and glucose uptake while inhibiting oxidative stress and inflammatory responses by increasing the phosphorylation of AMPK and its downstream target, mammalian target of rapamycin (mTOR), and/or by upregulating the gene expression of glucose transporters GLUT1 and GLUT4. Second, many antidiabetic agents (e.g., teneligliptin, liraglutide, exenatide, semaglutide, canagliflozin, dapagliflozin, and empagliflozin) can promote autophagy, reverse excessive apoptosis and autophagy, and alleviate oxidative stress and inflammation by enhancing AMPK phosphorylation and its downstream targets, such as mTOR, or by increasing the expression of silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor‑α (PPAR‑α). Third, certain anti-anginal (e.g., trimetazidine, nicorandil), anti-asthmatic (e.g., farrerol), antibacterial (e.g., sodium houttuyfonate), and antibiotic (e.g., minocycline) agents have been shown to promote autophagy/mitophagy, mitochondrial biogenesis, and inhibit oxidative stress and lipid accumulation via AMPK phosphorylation and its downstream targets such as protein kinase B (PKB/AKT) and/or PPAR‑α. Fourth, natural compounds (e.g., dihydromyricetin, quercetin, resveratrol, berberine, platycodin D, asiaticoside, cinnamaldehyde, and icariin) can upregulate AMPK phosphorylation and downstream targets such as AKT, mTOR, and/or the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), thereby exerting anti-inflammatory, anti-apoptotic, anti-pyroptotic, antioxidant, and pro-autophagic effects. Fifth, moderate exercise (e.g., continuous or intermittent aerobic exercise, aerobic combined with resistance training, or high-intensity interval training) can activate AMPK and its downstream targets (e.g., acetyl-CoA carboxylase (ACC), GLUT4, PPARγ coactivator-1α (PGC-1α), PPAR-α, and forkhead box protein O3 (FOXO3)) to promote fatty acid oxidation and glucose uptake, and to inhibit oxidative stress and excessive mitochondrial fission. Finally, the combination of liraglutide and aerobic interval training has been shown to activate the AMPK/FOXO1 pathway, thereby reducing excessive myocardial fatty acid uptake and oxidation. This combination therapy offers superior improvement in cardiac dysfunction, myocardial hypertrophy, and fibrosis in diabetic conditions compared to liraglutide or exercise alone.

Aim To investigate the effect of long non- coding RNA p21 (LncRNA p21) regulating Hippo- Yes-associated protein (Hippo-YAP) signaling pathway on the formation of abdominal aortic aneurysm (AAA) in mice. Methods C57BL/6 ApoE

OBJECTIVE To investigate the improvement effects of glycyrrhizin （GL） on Helicobacter pylori （HP）-associated gastritis in rats and its mechanism. METHODS HP-associated gastritis rat model was induced by inoculating with 1×109 cfu/mL HP. The model rats were randomly divided into model group， positive control group （HP standard quadruple group）， GL low-dose， medium-dose and high-dose groups （5， 20， 50 mg/kg）， with 12 rats in each group. Another 12 healthy rats were selected as normal control group. Except the normal control group and model group were given constant volume of normal saline intragastrically， the other groups were given corresponding drugs intragastrically， once a day， for 30 consecutive days. After administration， rats received 13C urea breath test， and delta-over-baseline （DOB） was recorded； the pathological and cellular morphological changes of gastric mucosa in rats were observed， and pathological scoring was performed； the levels of interleukin-8 （IL-8）， IL-1β， tumor necrosis factor-α （TNF-α）， reactive oxygen species （ROS） and malondialdehyde （MDA） were detected in gastric mucosa of rats； mRNA expressions of high mobility group box-1 protein （HMGB1） and nuclear factor-κ-B （NF-κB）， relative expressions of nitric oxide synthases （iNOS） and HMGB1， the phosphorylation level of NF- κBp65 were also detected in rats. RESULTS Compared with normal control group， the DOB value， histopathological score of gastric mucosa， the levels of IL-8， IL-1β， TNF-α， ROS and MDA， relative expressions of HMGB1 and NF- κB mRNA， relative expressions of iNOS and HMGB1 protein and the phosphorylation level of NF-κB p65 were all increased significantly in model group （P＜0.05）； the epithelial cells of gastric mucosa in rats were incomplete in structure and decreased in the number， with an increase in cell fragments and vacuoles， and significant cell pyknosis. Compared with model group， the changes of the above indexes in GL groups and positive control group were significantly reversed （P＜0.05）； the changes in the above indicators in the GL high-dose group were more significant than GL low-dose and medium-dose groups （P＜0.05）； the pathological changes of gastric mucosal cells in rats had all improved. CONCLUSIONS GL may inhibit inflammation and oxidative stress by inhibiting the activation of HMGB1/NF-κB pathway， thus relieving HP-induced gastric mucosal injury.

Abstract: Stem cells, which are a type of primitive cells with multipotent differentiation potential and self-renewal ability, have the potential to regenerate various tissues and organs. Stem cell drug development is a frontier research field in life sciences. Extensive clinical trials involving stem cells have been conducted for different complicated diseases. Some stem cells have been approved as drugs for some indications, indicating their broad industrial prospects. This review introduces the progress of stem cell drugs around the world, especially in China, and discusses the main problems in the industrialization of stem cell drugs, such as their effectiveness, quality control and safety, so as to provide some reference and insight for the development and rapid industrialization of stem cell drugs.

Objective:To explore the health economic evaluation methods of disease screening,providing insights for scholars conducting related research.Methods:By integrating relevant content from modem epidemiology,health statistics,and health economics,it investigates the differences in conducting health economic evaluations of disease screening using authenticity indicators(sensitivity,specificity)and predictive value indicators(positive predictive value,negative predictive value),and combines empirical studies with cases from the literature.Results:Authenticity indicators(sensitivity,specificity)are intrinsic measures of diagnostic tests,unaffected by the disease prevalence in the sample population,making them more suitable for conducting health economic evaluations of disease screening.Moreover,authenticity indicators are easy to obtain,and the model calculations are straightforward and convenient.Case study results demonstrate that compared to risk score screening,the Incremental Cost-Effectiveness Ratio(ICER)of fasting plasma glucose(FPG)testing for type 2 diabetes is 626,indicating that for every additional diabetic patient identified through risk score screening,an extra 626 yuan is spent.Conclusion:It is recommended to use authenticity indicators for conducting health economic evaluations of disease screening,standardize the path methods for model selection,and continuously improve the evidence quality of health economic evaluations of disease screening in China.

ObjectiveCellular temperature imaging can assist scientists in studying and comprehending the temperature distribution within cells, revealing critical information about cellular metabolism and biochemical processes. Currently, cell temperature imaging techniques based on fluorescent temperature probes suffer from limitations such as low temperature resolution and a limited measurement range. This paper aims to develop a single-cell temperature imaging and real-time monitoring technique by leveraging the temperature-dependent properties of single-molecule quantum coherence processes. MethodsUsing femtosecond pulse lasers, we prepare delayed and phase-adjustable pairs of femtosecond pulses. These modulated pulse pairs excite fluorescent single molecules labeled within cells through a microscopic system, followed by the collection and recording of the arrival time of each fluorescent photon. By defining the quantum coherence visibility (V) of single molecules in relation to the surrounding environmental temperature, a correspondence between V and environmental temperature is established. By modulating and demodulating the arrival times of fluorescent photons, we obtain the local temperature of single molecules. Combined with scanning imaging, we finally achieve temperature imaging and real-time detection of cells. ResultsThis method achieves high precision (temperature resolution<0.1°C) and a wide temperature range (10-50°C) for temperature imaging and measurement, and it enables the observation of temperature changes related to individual cell metabolism. ConclusionThis research contributes to a deeper understanding of cellular metabolism, protein function, and disease mechanisms, providing a valuable tool for biomedical research.

ObjectiveTemporal heterogeneity in lung cancer presents as fluctuations in the biological characteristics, genomic mutations, proliferation rates, and chemotherapeutic responses of tumor cells over time, posing a significant barrier to effective treatment. The complexity of this temporal variance, coupled with the spatial diversity of lung cancer, presents formidable challenges for research. This article will pave the way for new avenues in lung cancer research, aiding in a deeper understanding of the temporal heterogeneity of lung cancer, thereby enhancing the cure rate for lung cancer. MethodsRaman spectroscopy emerges as a powerful tool for real-time surveillance of biomolecular composition changes in lung cancer at the cellular scale, thus shedding light on the disease’s temporal heterogeneity. In our investigation, we harnessed Raman spectroscopic microscopy alongside multivariate statistical analysis to scrutinize the biomolecular alterations in human lung epithelial cells across various timeframes after benzo(a)pyrene exposure. ResultsOur findings indicated a temporal reduction in nucleic acids, lipids, proteins, and carotenoids, coinciding with a rise in glucose concentration. These patterns suggest that benzo(a)pyrene induces structural damage to the genetic material, accelerates lipid peroxidation, disrupts protein metabolism, curtails carotenoid production, and alters glucose metabolic pathways. Employing Raman spectroscopy enabled us to monitor the biomolecular dynamics within lung cancer cells in a real-time, non-invasive, and non-destructive manner, facilitating the elucidation of pivotal molecular features. ConclusionThis research enhances the comprehension of lung cancer progression and supports the development of personalized therapeutic approaches, which may improve the clinical outcomes for patients.

ObjectiveBody fluid stains left at crime scenes are frequently trace amounts, while the identification of body fluids through real time fluorogenic quantitative technique often necessitates the repeated detection within the limited sample, as multiple miRNA markers are the basis for the identification. Based on the goal of both the throughput and efficiency improvement of miRNA analysis in trace samples, a duplex real time fluorogenic quantitative PCR assay system was designed to accurately quantify two miRNAs simultaneously, and the system should be further verified by actual sample for the body fluid identification. MethodsThe duplex real time fluorogenic quantitative PCR system of miR-451a to miR-21-5p was established with specially designed primers and probes, and the concentrations of the primers and probes were both optimized. The specificity, sensitivity and reproducibility of the system were validated, while its capability for body fluid identification was assessed using the miR-451a to miR-21-5p ratio. ResultsThe optimized assay system exhibited excellent specificity and repeatability, with coefficients of variation consistently below 8% for both intra- and inter-batch variability. The amplification efficiency of miR-451a and miR-21-5p reached 71.77% and 74.81%, respectively, with high and relatively consistent results. By utilizing this duplex real time fluorogenic quantitative PCR assay system, a total of 58 body fluid samples were analyzed, exhibiting a discrimination rate of 100% between blood and non-blood samples, as well as between peripheral blood and menstrual blood samples. Moreover, the results, obtained from single real time fluorogenic quantitative PCR assay system and duplex real time fluorogenic quantitative PCR assay system, showed no statistically significant difference with randomly selected blood samples (n=20). Compared to previous single real time fluorogenic quantitative PCR assay system, the sensitivity of duplex real time fluorogenic quantitative PCR assay system exhibited remarkable improvement. A minimum input of only 0.1 ng total RNA was sufficient for accurate detection of peripheral blood and menstrual blood samples, while saliva, semen, and vaginal secretion required only 1 ng total RNA for precise identification purposes. Additionally, the duplex real time fluorogenic quantitative PCR assay system successfully differentiated between different types of body fluids in simulated samples under natural outdoor conditions. ConclusionThe duplex real time fluorogenic quantitative PCR assay system effectively reduced both the time and material costs by half compared to the single system, especially suitable for the examination of body fluid stains left at crime scenes, solving the contradiction between the trace amount and the multiple sample volumes demand of repeated real time fluorogenic quantitative PCR. The duplex real time fluorogenic quantitative PCR assay successfully distinguished blood and other body fluid, as well as peripheral blood and menstrual blood samples, which maintains an equivalent capability for body fluid identification with half sample, time and reagent consumption. This system provides an efficient tool for identifying suspicious body fluids, as well as a foundation for more multiplexed real time fluorogenic quantitative PCR assay system research.