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.

Tumor drug resistance is an important problem in the failure of chemotherapy and targeted drug therapy, which is a complex process involving chromatin remodeling. SWI/SNF is one of the most studied ATP-dependent chromatin remodeling complexes in tumorigenesis, which plays an important role in the coordination of chromatin structural stability, gene expression, and post-translation modification. However, its mechanism in tumor drug resistance has not been systematically combed. SWI/SNF can be divided into 3 types according to its subunit composition: BAF, PBAF, and ncBAF. These 3 subtypes all contain two mutually exclusive ATPase catalytic subunits (SMARCA2 or SMARCA4), core subunits (SMARCC1 and SMARCD1), and regulatory subunits (ARID1A, PBRM1, and ACTB, etc.), which can control gene expression by regulating chromatin structure. The change of SWI/SNF complex subunits is one of the important factors of tumor drug resistance and progress. SMARCA4 and ARID1A are the most widely studied subunits in tumor drug resistance. Low expression of SMARCA4 can lead to the deletion of the transcription inhibitor of the BCL2L1 gene in mantle cell lymphoma, which will result in transcription up-regulation and significant resistance to the combination therapy of ibrutinib and venetoclax. Low expression of SMARCA4 and high expression of SMARCA2 can activate the FGFR1-pERK1/2 signaling pathway in ovarian high-grade serous carcinoma cells, which induces the overexpression of anti-apoptosis gene BCL2 and results in carboplatin resistance. SMARCA4 deletion can up-regulate epithelial-mesenchymal transition (EMT) by activating YAP1 gene expression in triple-negative breast cancer. It can also reduce the expression of Ca²⁺ channel IP3R3 in ovarian and lung cancer, resulting in the transfer of Ca²⁺ needed to induce apoptosis from endoplasmic reticulum to mitochondria damage. Thus, these two tumors are resistant to cisplatin. It has been found that verteporfin can overcome the drug resistance induced by SMARCA4 deletion. However, this inhibitor has not been applied in clinical practice. Therefore, it is a promising research direction to develop SWI/SNF ATPase targeted drugs with high oral bioavailability to treat patients with tumor resistance induced by low expression or deletion of SMARCA4. ARID1A deletion can activate the expression of ANXA1 protein in HER2+ breast cancer cells or down-regulate the expression of progesterone receptor B protein in endometrial cancer cells. The drug resistance of these two tumor cells to trastuzumab or progesterone is induced by activating AKT pathway. ARID1A deletion in ovarian cancer can increase the expression of MRP2 protein and make it resistant to carboplatin and paclitaxel. ARID1A deletion also can up-regulate the phosphorylation levels of EGFR, ErbB2, and RAF1 oncogene proteins.The ErbB and VEGF pathway are activated and EMT is increased. As a result, lung adenocarcinoma is resistant to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Although great progress has been made in the research on the mechanism of SWI/SNF complex inducing tumor drug resistance, most of the research is still at the protein level. It is necessary to comprehensively and deeply explore the detailed mechanism of drug resistance from gene, transcription, protein, and metabolite levels by using multi-omics techniques, which can provide sufficient theoretical basis for the diagnosis and treatment of poor tumor prognosis caused by mutation or abnormal expression of SWI/SNF subunits in clinical practice.

ObjectiveIn order to provide a reference for the optimization of preparation process of stir-fried Glycyrrhizae Radix et Rhizoma（sf-GRR）， the quality changes during the processing was studied. MethodsGlycyrrhizae Radix et Rhizoma was processed by stir-frying for 17 min， and samples were collected every 1 min during the processing. The appearance color of the samples was determined by visual analysis technology， the moisture and extract of the process samples were detected by the drying method and the hot extraction method of alcohol-soluble extract in the general rules of the 2020 edition of Chinese Pharmacopoeia（part Ⅳ）， and the contents of liquiritin apioside， liquiritin， isoliquiritin apioside， isoliquiritin， licoricesaponin G₂ and glycyrrhizic acid in the process samples were determined by high performance liquid chromatography（HPLC）. Then principal component analysis（PCA）， partial least squares-discriminant analysis（PLS-DA） and Spearman correlation analysis were used for clustering， discrimination and correlation analysis of the appearance color， moisture， extract and the contents of six internal components. Based on artificial neural network and random forest algorithm， the prediction model of processing degree of sf-GRR was established. On this basis， based on the five principles of quality marker（Q-Maker）， explore the monitoring Q-Maker of sf-GRR. ResultsThe color of Glycyrrhizae Radix et Rhizoma deepened after stir-frying， and the appearance color of the sample changed from light yellow to dark yellow during processing. During the stir-frying process， the moisture content showed a decreasing trend with the extension of processing time， while the extract content showed an increasing trend with the extension of processing time. After stir-frying， the contents of liquiritin apioside， liquiritin and licoricesaponin G₂ showed an overall decreasing trend， while the contents of isoliquiritin apioside and isoliquiritin increased， and the content of glycyrrhizic acid increased slightly. The correlation analysis showed that moisture was positively correlated with brightness（L^*） and red/green value（a^*）， and negatively correlated with yellow/blue value（b^*） and total color difference（E^*ab）. Isoliquiritin apioside and isoliquiritin had negative correlation with L^* and a^*， and positive correlation with b^* and E^*ab. The processing process of sf-GRR could be divided into two stages of the early stage（0-14 min） and the late stage（15-17 min）， and could be divided into three stages of the early stage（0-6 min）， the middle stage（7-14 min） and the late stage（15-17 min） by combining the moisture， extract， the contents of 6 components and color values. Based on artificial neural network analysis and random forest algorithm， isoliquiritin apioside， isoliquiritin， liquiritin and glycyrrhizic acid were selected as monitoring markers for sf-GRR. ConclusionBased on the analysis of the exterior-interior indicators of process samples of sf-GRR， this paper ultimately identifies four processing monitoring markers， which can provide a basis for optimizing the processing technology of sf-GRR.

It is believed that wind pathogen lingering in the lungs is the key factor in the pathogenesis of cough variant asthma. Among these， external wind-induced cough is the main trigger， while internal wind hidden in the lung plays an important role in promoting the disease. Weakness of the zang-fu organs is the fundamental cause of recurrent attacks， especially closely related to deficiencies in the lungs， spleen and kidneys. The primary treatment principles proposed are dispelling wind and relieving cough， as well as tonifying deficiency and supporting the upright qi. Clinical practice should integrate differentiation of the root and branch as well as excess and deficiency， applying staged treatment. During the acute attack phase， the focus is on dispelling wind and stopping cough， supplemented by tonification. During the remission phase， tonifying deficiency and supporting the upright qi is emphasized， with coordinated regulation of the lungs， spleen and kidneys alongside clearing residual pathogens， so that the pathogenic are eliminated and the body restored， leading to natural resolution of cough and reversal of adverse qi flow.

Perihilar cholangiocarcinoma(PHC)is a common malignancy of biliary tract,for which surgery is the most effective treatment.However,its prognosis is not satisfactory even after surgical resection.In recent years,there have been some new advances in the surgical treatment of PHC.In this paper,we reviewed the existing literatures,demonstrated the current situation of preoperative biliary drainage,liver hyperplasia,hepatic resection,liver transplantation and minimally invasive surgery in the treatment of PHC,and prospected the future research direction.

Post-endoscopic retrograde cholangiopancreatography pancreatitis(PEP)is one of the most common complications after endoscopic retrograde cholangiopancreatography(ERCP).Numerous PEP prediction models have been established based on different statistical methods at home and abroad.The PEP prediction model,as a tool for evaluating and screening high-risk populations,can provide a basis for medical staff to find high-risk PEP patients early and take effective preventive measures.In recent years,new PEP prediction models have appeared one after another,but there is still a lack of recognized reliable prediction models in clinic.This article reviews the research status of PEP risk prediction models,aim to provide a direction for establishing a more reliable,accurate,and practical PEP risk prediction model in the later period.

Objective:To explore the value of combined detection of urinary kidney injury markers in the diagnosis of early-stage diabetic kidney disease (DKD), and to provide evidence for early-stage DKD screening.Methods:It was a retrospective study. The clinical data of patients with type 2 diabetes mellitus (T2DM) from the Second Affiliated Hospital of Hainan Medicine University from January 2022 to August 2023 were collected. According to urinary microalbumin/creatinine ratio (UACR), the patients were divided into three groups: isolated diabetes group (UACR < 30 mg/g), early-stage DKD group (30 mg/g ≤ UACR < 300 mg/g) and clinical DKD group (UACR ≥ 300 mg/g), and the differences of clinical data among three groups were compared. Glomerular injury markers urinary microalbumin, transferrin, immunoglobulin (Ig) and α2 macroglobulin, and renal tubule injury markers α1 microglobulin (α1-MG), β2 microglobulin (β2-MG), retinol-binding protein (RBP), N-acetyl-β- D-glucosidase (NAG), neutrophil gelatinase-associated lipid carrier protein (NGAL) were measured. Spearman correlation method was used to analyze the correlation between urinary kidney injury markers and clinical indicators. Multivariate logistic regression analysis method was used to analyze the risk factors of DKD occurrence (UACR > 300 mg/g). Receiver-operating characteristic curve was used to analyze the value of individual and combined detection of urinary renal injury markers in the diagnosis of early-stage DKD (30 mg/g ≤ UACR < 300 mg/g). Results:A total of 116 T2DM patients were enrolled in this study, aged (61.99±12.56) years old (30 to 91 years old), with 79 males (68.1%). There were 44 (37.9%) isolated diabetes patients, 27 (23.3%) early-stage DKD patients, and 45 (38.8%) clinical DKD patients. Serum creatinine (Scr, H=34.183, P<0.001) and blood urea nitrogen (BUN, H=34.082, P<0.001) in clinical DKD group were higher than those in isolated diabetes group and early-stage DKD group. Spearman correlation analysis showed that glomerular injury markers urinary microalbumin, transferrin, Ig and α2 macroglobulin were positively correlated with Scr, BUN and UACR, and negatively correlated with estimated glomerular filtration rate and serum albumin (all P<0.05). Renal tubule injury markers urinary α1-MG, β2-MG, NAG, RBP, and NGAL were positively correlated with Scr, BUN and UACR, and negatively correlated with estimated glomerular filtration rate and serum albumin (all P<0.05). Multivariate logistic regression analysis indicated that systolic blood pressure ≥ 140 mmHg ( OR=1.033, 95% CI 1.008-1.060, P=0.010), high urinary microalbumin ( OR=1.018, 95% CI 1.007-1.030, P=0.001), high urinary RBP ( OR=1.309, 95% CI 1.086-1.577, P=0.005), high urinary NGAL ( OR=1.004, 95% CI 1.000-1.008, P=0.037), low serum albumin ( OR=0.833, 95% CI 0.749-0.926, P=0.001) and low urinary Ig ( OR=0.994, 95% CI 0.990-0.999, P=0.018) were independent influencing factors of DKD occurrence. Receiver-operating characteristic curve revealed that the area under the curve ( AUC) was the largest for diagnosing early-stage DKD when urinary microalbumin was detected alone ( AUC=0.976, 95% CI 0.955-0.997, P<0.001), with sensitivity and specificity of 95.6% and 90.1%, respectively. The combined detection of urinary microalbumin + Ig + transferrin + α2 macroglobulin + α1-MG + β2-MG + NAG + RBP + NGAL had an AUC of 0.986 (95% CI 0.971-1.000, P<0.001), with sensitivity and specificity of 93.3% and 98.5%, respectively, which was better than each single index. Further optimized detection combination was urinary microalbumin combined with β2-MG and NGAL, which had the best diagnostic efficacy ( AUC=0.978, 95% CI 0.958-0.999, P<0.001), with sensitivity and specificity of 95.6% and 93.0%, respectively. Conclusions:Compared with the single detection of each index, the combined detection of urinary glomerular injury and renal tubule injury markers has higher value in diagnosing early-stage DKD. The combined detection of urinary microalbumin combined with β2-MG and NGAL has the highest value in diagnosing early-stage DKD.

Ten compounds were isolated and purified from ethanol extracts of dried roots bark of Polygala tenuifolia Willd. by various chromatography techniques such as silica gel and Sephadex LH-20. Their structures were identified by analysis of physicochemical properties and spectral data, and determined as β-sitosterol (1), tenuifolin (2), 6-methoxy coumarin (3), 7-phenyl-1-hydroxy-2,3,6-trimethoxyxanthone (4), 1,8-dihydroxy-3,4,7-trimethoxyanthone (5), mangiferin (6), quercetin-3-O-β-D-glucoside (7), rutin (8), syringaldehyde (9), salicylicacid (10). Among them, compounds 3, 4 and 5 were isolated from the genus of Ploygala for the first time and compound 4 was a new xanthone. The acetylcholinesterase inhibitory activities of compounds 3, 4 and 5 were evaluated by Ellman colorimetric method, compounds 3 and 5 exhibited moderate inhibitory activity, compound 4 exhibited weak inhibitory activity.

Objective To explore the context and hotspot changes of forensic mixed stain research through bibliometric approach.Methods The literature of forensic mixed stain included in the core col-lection of Web of Science database from 2011 to 2022 were collected as the study object,and the an-nual publication number,countrie(region),institution,journal,keywords,etc.were bibliometrically and visually analyzed using the R-based Bibliometrix 1.1.6 package and VOSviewer 1.6.18 software.Re-sults A total of 732 articles on forensic mixed stain were included from 2011 to 2022,with the an-nual number of articles published and the annual citation frequency showing a steady increase year by year.Among the 59 countries(regions)with the most published articles,the United States ranked first with 246 articles,followed by China with 153 articles.The literature came from 104 journals,and the total number of articles published in the top 10 journals was 633.FORENSIC SCI INT GENET ranked first with 307 articles.Visual analysis using VOSviewer software showed that keywords could be divided into four research clusters,namely the genetic marker development group(blue),the mixed stain typing analysis theory group(red),the sequencing analysis group(yellow),and the case sample research group(green).It can be divided into four development stages in terms of different time peri-ods:early development(2011-2013),middle development(2014-2016),rapid development(2017-2020)and latest development(2021-2022).Conclusion The number of publications by domestic and foreign scholars in the study of mixed stain in forensic science is showing a relatively stable trend.Machine learning,next generation sequencing and other research have been the hottest topics that have attracted the most attention in recent years,which is expected to further develop the theory of mixed stain typing and sequencing analysis in forensic mixed stain research.

Acupuncture, a therapeutic treatment defined as the insertion of needles into the body at specific points (ie, acupoints), has growing in popularity world-wide to treat various diseases effectively, especially acute and chronic pain. In parallel, interest in the physiological mechanisms underlying acupuncture analgesia, particularly the neural mechanisms have been increasing. Over the past decades, our understanding of how the central nervous system and peripheral nervous system process signals induced by acupuncture has developed rapidly by using electrophysiological methods. However, with the development of neuroscience, electrophysiology is being challenged by calcium imaging in view field, neuron population and visualization in vivo. Owing to the outstanding spatial resolution, the novel imaging approaches provide opportunities to enrich our knowledge about the neurophysiological mechanisms of acupuncture analgesia at subcellular, cellular, and circuit levels in combination with new labeling, genetic and circuit tracing techniques. Therefore, this review will introduce the principle and the method of calcium imaging applied to acupuncture research. We will also review the current findings in pain research using calcium imaging from in vitro to in vivo experiments and discuss the potential methodological considerations in studying acupuncture analgesia.
Calcium ; Acupuncture Therapy ; Acupuncture ; Acupuncture Analgesia/methods* ; Acupuncture Points ; Technology