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.

Electroencephalogram (EEG) is a non-invasive, high temporal-resolution technique for monitoring brain activity. However, affected by the volume conduction effect, EEG has a low spatial resolution and is difficult to locate brain neuronal activity precisely. The surface Laplacian (SL) technique obtains the Laplacian EEG (LEEG) by estimating the second-order spatial derivative of the scalp potential. LEEG can reflect the radial current activity under the scalp, with positive values indicating current flow from the brain to the scalp (“source”) and negative values indicating current flow from the scalp to the brain (“sink”). It attenuates signals from volume conduction, effectively improving the spatial resolution of EEG, and is expected to contribute to breakthroughs in neural engineering. This paper provides a systematic overview of the principles and development of SL technology. Currently, there are two implementation paths for SL technology: current source density algorithms (CSD) and concentric ring electrodes (CRE). CSD performs the Laplace transform of the EEG signals acquired by conventional disc electrodes to indirectly estimate the LEEG. It can be mainly classified into local methods, global methods, and realistic Laplacian methods. The global method is the most commonly used approach in CSD, which can achieve more accurate estimation compared with the local method, and it does not require additional imaging equipment compared with the realistic Laplacian method. CRE employs new concentric ring electrodes instead of the traditional disc electrodes, and measures the LEEG directly by differential acquisition of the multi-ring signals. Depending on the structure, it can be divided into bipolar CRE, quasi-bipolar CRE, tripolar CRE, and multi-pole CRE. The tripolar CRE is widely used due to its optimal detection performance. While ensuring the quality of signal acquisition, the complexity of its preamplifier is relatively acceptable. Here, this paper introduces the study of the SL technique in resting rhythms, visual-related potentials, movement-related potentials, and sensorimotor rhythms. These studies demonstrate that SL technology can improve signal quality and enhance signal characteristics, confirming its potential applications in neuroscientific research, disease diagnosis, visual pathway detection, and brain-computer interfaces. CSD is frequently utilized in applications such as neuroscientific research and disease detection, where high-precision estimation of LEEG is required. And CRE tends to be used in brain-computer interfaces, that have stringent requirements for real-time data processing. Finally, this paper summarizes the strengths and weaknesses of SL technology and envisages its future development. SL technology boasts advantages such as reference independence, high spatial resolution, high temporal resolution, enhanced source connectivity analysis, and noise suppression. However, it also has shortcomings that can be further improved. Theoretically, simulation experiments should be conducted to investigate the theoretical characteristics of SL technology. For CSD methods, the algorithm needs to be optimized to improve the precision of LEEG estimation, reduce dependence on the number of channels, and decrease computational complexity and time consumption. For CRE methods, the electrodes need to be designed with appropriate structures and sizes, and the low-noise, high common-mode rejection ratio preamplifier should be developed. We hope that this paper can promote the in-depth research and wide application of SL technology.

OBJECTIVE To investigate the effects of jaceosidin on osteoarthritis （OA） of rats by regulating adenosine monophosphate-activated protein kinase （AMPK）/NOD-like receptor protein 3 （NLRP3） pathway. METHODS Rats were randomly separated into OA group， jaceosidin group （33.33 mg/kg）， AMPK inhibitor （Compound C， 20 mg/kg） group， jaceosidin （33.33 mg/kg）+Compound C （20 mg/kg） group， and sham operation group， with 12 rats in each group. Except for the sham operation group， the OA model was induced with modified Hulth method in all other groups. After successful modeling， they were given a relevant dose of jaceosidin or normal saline intragastrically， and Compound C or normal saline intraperitoneally， once a day， for consecutive 8 weeks. Twenty-four h after the last medication， the degree of knee joint swelling in rats from each group was measured. The pathological changes of the articular cartilage tissue in the knee joints， and the Mankin score were assessed. The levels of tumor necrosis factor-α （TNF-α）， interleukin-18 （IL-18）， and IL-6， as well as the protein expressions of collagen Ⅱ， aggrecan （ACAN）， and a disintegrin and metalloproteinase with thrombospondin 5 （ADAMTS5）， phosphorylated AMPK （p-AMPK）， AMPK， NLRP3， cleaved-caspase-1， and cleaved-IL-1β were detected in the articular cartilage tissue of rats’ knees. RESULTS Compared with OA group， the cartilage tissue defect of jaceosidin group was relieved， the cartilage matrix staining was deepened， and the number of chondrocytes was increased. Knee swelling， Mankin score， the levels of TNF- α， IL-18 and IL-6， and protein expressions of ADAMTS5， NLRP3， cleaved-caspase-1 and cleaved-IL-1β in knee cartilage were significantly decreased or down-regulated. Protein expressions of collagen Ⅱ， ACAN and phosphorylation level of AMPK were significantly increased or up-regulated （P＜0.05）. Compound C significantly reversed the improvement effects of jaceosidin on the above indexes of OA rats （P＜0.05）. CONCLUSIONS Jaceosidin may inhibit inflammation and extracellular matrix degradation in OA rats by regulating the AMPK/NLRP3 signaling pathway.

[Objective] To explore the feasibility of using whole blood as a source of NK cells for allogeneic CAR NK cell therapy and activated NK cell reinfusion therapy, and initially construct a technical system for the separation and purification of NK cells from whole blood. [Methods] All peripheral blood mononuclear cells (PBMCs) were enriched from 400 mL of whole blood by manual separation and machine separation, respectively. The erythrocyte loss rate, PBMCs number, NK cell purity of the two methods were compared. NK cells were sorted from PBMCs by three separation and enrichment methods as immunomagnetic bead negative selection method, platelet lysate culture expansion and PERCOLL density gradient separation method, and the purity and yield of NK cells, the activity of NK cells and the tumor-killing ability of the three separation and enrichment methods were compared. [Results] The proportion of NK cells in the lymphocyte population was higher in the manual separation method than in the machine separation method[(13.16±5.16)% vs (8.56±3.92)%, P<0.05]; the number PBMCs was lower in the manual separation method than in the machine separation method[(4.09±1.80)×10⁸vs (6.49±2.16)×10⁸, P<0.05], and there was no difference in the red blood cell loss between the two methods (P>0.05). The purity of NK cells isolated and enriched from PBMCs by manual separation method using immunomagnetic was (96.77±2.31)%; the yield was (56.27±10.47)%; the inhibition of tumor proliferation was (38.67±14.05)%; and the tumor killing rate was (19.90±8.05)%. The purity of NK cells isolated and enriched from PBMCs by manual separation method using platelet lysis culture expansion method was the highest at day 7, which was (54.84±15.80)%; the cell expansion multiple could reach 16.92±6.28 at day 7; the in vitro tumor killing rate of NK cells was (15.83±5.5)%; the tumor inhibition rate was (44.33±13.5)%; and there was no difference in the toxicity and activity of NK cells between the two methods (P>0.05). The purity of NK cells isolated and enriched by PERCOLL density gradient separation method was (15.83±5.82)%, and the yield was (14±6.25)%, which was significantly lower than the other two methods. [Conclusion] PBMCs isolated from whole blood by manual separation and NK cells enriched by negative selection with immunomagnetic beads have the potential to provide NK cell materials for CAR-NK cell therapy, and NK cells enriched by platelet lysate-conditioned medium have the potential to provide NK cells for large-scale NK cell activation reinfusion therapy.

ObjectiveTo investigate the effect of folic acid-modified crebanine polyethylene glycol-polylactic acid hydroxyacetic acid copolymer（PEG-PLGA） nanoparticles（FA-Cre@PEG-PLGA NPs， hereinafter referred to as NPs） combined with ultrasonic irradiation on subcutaneous tumor of liver cancer in Kunming（KM） mice. MethodsEighty-four healthy male KM mice were utilized to establish a subcutaneous tumor model of mouse hepatocellular carcinoma with H22 cells， then mice were randomly divided into model group， placebo group， hydroxycamptothecin group（8 mg∙kg^-1）， low， medium and high dose crebanine raw material groups（2， 2.5， 3 mg∙kg^-1， hereinafter referred to as the low， medium and high dose crebanine groups， respectively）， low， medium and high dose NPs groups（2， 2.5， 3 mg∙kg^-1）， and low， medium and high dose NPs combined with ultrasonic irradiation groups（2， 2.5， 3 mg∙kg^-1， hereinafter referred to as the low， medium and high dose combination groups， respectively）. The corresponding doses of drugs were administered via tail vein injection， the model group received no treatment， while the placebo group was injected with an equivalent amount of normal saline. Dosing was conducted for a total of 10 times on alternate days. The body mass of the mice was monitored， and parameters such as body mass change rate， thymus index， spleen index， tumor volume， tumor weight， relative tumor growth rate（T/C）， and tumor inhibition rate（TGI） were calculated. Pathological changes in liver and kidney tissues as well as the tumor were observed by hematoxylin-eosin（HE） staining. Additionally， the levels of aspartate aminotransferase（AST）， alanine aminotransferase（ALT）， blood urea nitrogen（BUN） and creatinine（CREA） in serum of mice were detected by biochemical method. Furthermore， the effect of ultrasound on the distribution of NPs in subcutaneous tumors of mouse hepatocellular carcinoma was observed by in vivo imaging technique. ResultsAmong different treatment methods， the combination of NPs and ultrasound irradiation had the best therapeutic effect. Compared with the model group， the body mass growth rates of mice in the medium and high combination groups decreased， while the thymus index and spleen index increased， but there was no statistically significant difference in serum AST， ALT， BUN and CREA levels， indicating that NPs combined with ultrasound irradiation had little effect on the normal physiological state of the body， oth groups had TGI>40% and T/C<60%， indicating a clear anti-tumor effect. Pathological analysis showed that compared with the NPs groups， the combination groups exhibited varying degrees of necrosis in tumor cells， accompanied by less damage to the liver and kidneys. In vivo imaging of small animals showed that compared with the high dose NPs group， the high dose combination group had stronger tumor targeting ability（P<0.01）. ConclusionNPs combined with ultrasonic irradiation can not only effectively targeted the drug to the tumor site， inhibit the subcutaneous tumor growth of mouse liver cancer， but also decrease damage to liver and kidney tissues.

ObjectiveTo investigate the mechanism of Acanthopanacis Senticosi Radix et Rhizoma seu Caulis extract （ASH） in treating Parkinson's disease （PD） in mice by Data-Independent Acquisition （DIA） proteomics. MethodsThe α-Synuclein （α-Syn） transgenic PD mice were selected as suitable models for PD， and they were randomly assigned into PD， ASH （61.25 mg·kg^-1）， and Madopar （97.5 mg·kg^-1） groups. Male C57BL/6 mice of the same age were selected as the control group， with eight mice in each group. Mice were administrated with corresponding drugs by gavage once a day for 20 days. The pole climbing time and the number of autonomic activities were recorded to evaluate the exercise ability of mice. Hematoxylin-eosin staining was employed to observe neuronal changes in the substantia nigra of PD mice. Immunohistochemistry （IHC） was employed to measure the tyrosine hydroxylase （TH） activity in the substantia nigra and assess the areal density of α-Syn in the striatum. DIA proteomics was used to compare protein expression in the substantia nigra between groups. IHC was utilized to validate key differentially expressed proteins， including Lactotransferrin， Notch2， Ndrg2， and TMEM 166. The cell counting kit-8 （CCK-8） method was used to investigate the effect of ASH on the viability of PD cells with overexpression of α-Syn. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were employed to determine the protein and mRNA levels of Lactotransferrin， Notch2， Ndrg2， and TMEM 166 in PD cells. ResultsCompared with the control group， the model group showed prolonged pole climbing time， diminished coordination ability， reduced autonomic activities （P<0.01）， and reduced swelling neurons. Compared with the model group， ASH and Madopar reduced the climbing time， increased autonomic activities （P<0.01）， and ameliorated neuronal damage. Compared with the control group， the model group showed a decrease in TH activity in the substantia nigra and an increase in α-Syn accumulation in the striatum （P<0.01）. Compared with the model group， the ASH group showed an increase in TH activity and a reduction in α-Syn accumulation （P<0.05）. DIA proteomics revealed a total of 464 differentially expressed proteins in the model group compared with the control group， with 323 proteins being up-regulated and 141 down-regulated. A total of 262 differentially expressed proteins were screened in the ASH group compared with the model group， including 85 proteins being up-regulated and 177 down-regulated. Kyoto encylopedia of genes and genomes （KEGG） pathway analysis indicated that ASH primarily regulated the Notch signaling pathway. The model group showed up-regulation in protein levels of Notch2， Ndrg2， and TMEM 166 and down-regulation in the protein level of Lactotransferrin compared with the control group （P<0.01）. Compared with the model group， ASH down-regulated the protein levels of Notch2， Ndrg2， and TMEM 166 （P<0.05） while up-regulating the protein level of Lactotransferrin （P<0.01）. The IHC results corroborated the proteomics findings. The cell experiment results showed that compared with the control group， the modeling up-regulated the mRNA and protein levels of Notch2， Ndrg2， and TMEM 166 （P<0.01）， while down-regulating the mRNA and protein levels of Lactotransferrin （P<0.01）. Compared with the model group， ASH reduced the mRNA and protein levels of Notch2， Ndrg2， and TMEM 166 （P<0.01）， while increasing the mRNA and protein levels of Lactotransferrin （P<0.05， P<0.01）. ConclusionASH may Synergistically inhibit the Notch signaling pathway and mitigate neuronal damage by down-regulating the expression of Notch2 and Ndrg2. Additionally， by up-regulating the expression of Lactotransferrin and down-regulating the expression of TMEM166， ASH can address brain iron accumulation， intervene in ferroptosis， inhibit mitophagy， and mitigate reactive oxygen species damage， thereby protecting nerve cells and contributing to the treatment of PD.

Background: and Purpose Symptomatic intracranial hemorrhage (sICH) following endovascular thrombectomy (EVT) is a severe complication associated with adverse functional outcomes and increased mortality rates. Currently, a reliable predictive model for sICH risk after EVT is lacking. Methods: This study used data from patients aged ≥20 years who underwent EVT for anterior circulation stroke from the nationwide Taiwan Registry of Endovascular Thrombectomy for Acute Ischemic Stroke (TREAT-AIS). A predictive model including factors associated with an increased risk of sICH after EVT was developed to differentiate between patients with and without sICH. This model was compared existing predictive models using nationwide registry data to evaluate its relative performance. Results: Of the 2,507 identified patients, 158 developed sICH after EVT. Factors such as diastolic blood pressure, Alberta Stroke Program Early CT Score, platelet count, glucose level, collateral score, and successful reperfusion were associated with the risk of sICH after EVT. The TREAT-AIS score demonstrated acceptable predictive accuracy (area under the curve [AUC]=0.694), with higher scores being associated with an increased risk of sICH (odds ratio=2.01 per score increase, 95% confidence interval=1.64–2.45, P<0.001). The discriminatory capacity of the score was similar in patients with symptom onset beyond 6 hours (AUC=0.705). Compared to existing models, the TREAT-AIS score consistently exhibited superior predictive accuracy, although this difference was marginal. Conclusions The TREAT-AIS score outperformed existing models, and demonstrated an acceptable discriminatory capacity for distinguishing patients according to sICH risk levels. However, the differences between models were only marginal. Further research incorporating periprocedural and postprocedural factors is required to improve the predictive accuracy.

Background: Residential smoking cessation therapy programs offer intensive treatment for heavy smokers who struggle to quit independently, particularly those with high nicotine dependence and health conditions that necessitate urgent cessation. While previous studies have established the effectiveness of such programs and identified various factors influencing smoking cessation success, it remains unclear how changes in smokers’ thoughts and attitudes following residential therapy correlate with their ability to quit smoking. We investigated the relationship between smoking cessation-related characteristics, smoking-related psychological status, and participants’ smoking cessation success after a residential smoking cessation therapy program. Methods: From January 2017 to December 2018, 291 participants completed the program. All participants completed questionnaires on smoking cessation-related characteristics and smoking-related psychological status before the program and on the 5th day. Six months later, smoking cessation success was assessed using a urine cotinine test. Results: After 6 months, 222 participants successfully quit smoking, while 69 failed. The success and failure groups exhibited statistically significant differences in age, marital status, total smoking duration, stress, and emotion regulation strategies. Participants who used ineffective emotion regulation strategies more frequently had a lower rate of smoking cessation success (odds ratio [OR], 0.969; 95% confidence interval [CI], 0.948–0.991). Moreover, an increase in the perception of the negative effects of smoking cessation (OR, 0.982; 95% CI, 0.967–0.997) and smoking temptation (OR, 0.960; 95% CI, 0.929–0.993) was associated with higher cessation success. Conclusion Emotion regulation strategies, perceptions of the effects of smoking cessation, and smoking temptation were associated with successful smoking cessation.

Background: Skipping breakfast is associated with an increased risk of chronic inflammatory diseases. This study aimed to examine the association between breakfast-eating habits and inflammation, using high-sensitivity C-reactive protein (hs-CRP) as a marker. Methods: A total of 4,000 Korean adult males with no history of myocardial infarction, angina, stroke, diabetes, rheumatoid arthritis, cancer, or current smoking were included. Data from the 2016–2018 Korea National Health and Nutrition Examination Survey were used for analysis. The frequency of breakfast consumption was assessed through a questionnaire item in the dietary survey section asking participants about their weekly breakfast consumption routines over the past year. Participants were categorized into two groups, namely “0–2 breakfasts per week” and “3–7 breakfasts per week”; hs-CRP concentrations were measured through blood tests. Results: Comparing between the “infrequent breakfast consumption (0–2 breakfasts per week)” and “frequent breakfast consumption (3–7 breakfasts per week)” groups, the mean hs-CRP was found to be significantly higher in the “infrequent breakfast consumption” group, even after adjusting for age, body mass index, physical activity, alcohol consumption, systolic blood pressure, blood pressure medication, fasting blood glucose, and triglycerides (mean hs-CRP: frequent breakfast consumption, 1.36±0.09 mg/L; infrequent breakfast consumption, 1.17±0.05 mg/L; P-value=0.036). Conclusion Less frequent breakfast consumption was associated with elevated hs-CRP levels. Further large-scale studies incorporating adjusted measures of daily eating patterns as well as food quality and quantity are required for a deeper understanding of the role of breakfast in the primary prevention of chronic inflammatory diseases.

Background: We investigated whether nutritional intake is associated with physical activity (PA) and handgrip strength (HGS) in individuals with airflow limitation. Methods: This study analyzed data from the 2014 and 2016 Korean National Health and Nutrition Examination Survey. We assessed total protein intake (g/day), caloric intake (kcal/day), and other nutritional intakes, using a 24-hour dietary recall questionnaire. HGS was measured three times for each hand using a digital grip strength dynamometer, and PA was assessed as health-enhancing PA. Airflow limitation was defined as a forced expiratory volume/forced vital capacity ratio of 0.7 in individuals over 40 years of age. Participants were categorized into groups based on their PA levels and HGS measurements: active aerobic PA vs. non-active aerobic PA, and normal HGS vs. low HGS. Results: Among the 622 individuals with airflow limitation, those involved in active aerobic PA and those with higher HGS had notably higher total food, calorie, water, protein, and lipid intake. The correlations between protein and caloric intake with HGS were strong (correlation coefficients=0.344 and 0.346, respectively). The forest plots show that higher intakes of food, water, calories, protein, and lipids are positively associated with active aerobic PA, while higher intakes of these nutrients are inversely associated with low HGS. However, in the multivariate logistic regression analysis, no significant associations were observed between nutritional intake and active aerobic PA or HGS. Conclusion Nutritional intake was found to not be an independent factor associated with PA and HGS. However, the observed correlations suggest potential indirect effects that warrant further investigation.