Membrane proteins are integral components of cellular membranes, accounting for approximately 30% of the mammalian proteome and serving as targets for 60% of FDA-approved drugs. They are critical to both physiological functions and disease mechanisms. Their functional protein-protein interactions form the basis for many physiological processes, such as signal transduction, material transport, and cell communication. Membrane protein interactions are characterized by membrane environment dependence, spatial asymmetry, weak interaction strength, high dynamics, and a variety of interaction sites. Therefore, in situ analysis is essential for revealing the structural basis and kinetics of these proteins. This paper introduces currently available in situ analytical techniques for studying membrane protein interactions and evaluates the characteristics of each. These techniques are divided into two categories: label-based techniques (e.g., co-immunoprecipitation, proximity ligation assay, bimolecular fluorescence complementation, resonance energy transfer, and proximity labeling) and label-free techniques (e.g., cryo-electron tomography, in situ cross-linking mass spectrometry, Raman spectroscopy, electron paramagnetic resonance, nuclear magnetic resonance, and structure prediction tools). Each technique is critically assessed in terms of its historical development, strengths, and limitations. Based on the authors’ relevant research, the paper further discusses the key issues and trends in the application of these techniques, providing valuable references for the field of membrane protein research. Label-based techniques rely on molecular tags or antibodies to detect proximity or interactions, offering high specificity and adaptability for dynamic studies. For instance, proximity ligation assay combines the specificity of antibodies with the sensitivity of PCR amplification, while proximity labeling enables spatial mapping of interactomes. Conversely, label-free techniques, such as cryo-electron tomography, provide near-native structural insights, and Raman spectroscopy directly probes molecular interactions without perturbing the membrane environment. Despite advancements, these methods face several universal challenges: (1) indirect detection, relying on proximity or tagged proxies rather than direct interaction measurement; (2) limited capacity for continuous dynamic monitoring in live cells; and (3) potential artificial influences introduced by labeling or sample preparation, which may alter native conformations. Emerging trends emphasize the multimodal integration of complementary techniques to overcome individual limitations. For example, combining in situ cross-linking mass spectrometry with proximity labeling enhances both spatial resolution and interaction coverage, enabling high-throughput subcellular interactome mapping. Similarly, coupling fluorescence resonance energy transfer with nuclear magnetic resonance and artificial intelligence (AI) simulations integrates dynamic structural data, atomic-level details, and predictive modeling for holistic insights. Advances in AI, exemplified by AlphaFold’s ability to predict interaction interfaces, further augment experimental data, accelerating structure-function analyses. Future developments in cryo-electron microscopy, super-resolution imaging, and machine learning are poised to refine spatiotemporal resolution and scalability. In conclusion, in situ analysis of membrane protein interactions remains indispensable for deciphering their roles in health and disease. While current technologies have significantly advanced our understanding, persistent gaps highlight the need for innovative, integrative approaches. By synergizing experimental and computational tools, researchers can achieve multiscale, real-time, and perturbation-free analyses, ultimately unraveling the dynamic complexity of membrane protein networks and driving therapeutic discovery.

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.

ObjectiveTo carry out a health technology assessment （HTA） of acupuncture for secondary dysmenorrhea （SD） caused by adenomyosis and endometriosis， in order to provide a reference for relevant medical decision-making. MethodsFrom the perspective of the health system， the assessment covers seven areas， including the technical characteristics， safety， effectiveness， economics， ethical fairness， organizational adaptability， and impact on patients and society. The results are reported accordingly. ResultsThe operational specifications of acupuncture are standardized， and the conditions for its use are clearly defined. Acupuncture has a lower overall incidence of adverse events. The main adverse events are localized pain， subcutaneous bleeding， and dizziness， with most symptoms being mild， all of which have corresponding standard treatments. No reports on occupational or environmental safety were found， and the safety operation specifications are available for reference. Compared with conventional Western medicine， acupuncture demonstrates higher effectiveness. Acupuncture may improve the quality of life scores of patients， though no significant difference was observed. The cost of acupuncture is higher than that of conventional Western medicine， but its overall economic value is greater. The informed consent information is relatively comprehensive. Most patients are aware of the potential benefits and risks of acupuncture and voluntarily opt for it. The treatment process fully respects patient privacy and human rights. The clinical application of acupuncture follows the current acupuncture medical service model， with no special requirements for the level of medical institutions. Patient accessibility and affordability are suitable. Patient satisfaction is high. Most patients indicated they would choose acupuncture again for SD. The main barriers to choosing acupuncture are psychological factors （such as fear of acupuncture）， cost， and transportation issues. Nearly 70% of patients receiving acupuncture treatment benefit from medical insurance reimbursement， with reimbursement rates generally above 50%， indicating strong social security support. ConclusionThe implementation of HTA for acupuncture in the treatment of SD， using the standards for traditional Chinese medicine （TCM）， is feasible. The implementation steps are clear， the data sources for each evaluation domain are adequate， the analysis methods are practical， and the evaluation results are comprehensive. Experts recommend that the findings be used as a reference for relevant medical decision-making.

Objective To analyze the correlation between hip joint musculoskeletal ultrasound score and ankylosing spondylitis(AS)disease activity,as well as to investigate the value of high frequency ultrasound in the assessment of hip joint involvement in AS.Methods The clinical data of 244 patients with AS who were treated in the rheumatology department of from March 2019 to March 2022 were retrospectively analyzed.Among them,there 174 males and 70 females,aged from 19 to 58 years old with an average of(34.22±9.49)years old;the disease duration of AS patients ranged from 8 months to 26 years,with an average of(13.68±4.04)years.The 244 patients were divided into disease group(83 cases)and control group(161 cases)based in the presence of hip joint involuement.According to the the disease activity,patients in the disease group were further categorezed into active phase(45 cases)and stable phase(38 cases).The ultrasound scores of patients in the active and stable phases of the disease group and the control group were compared.Relevant factors of hip joint involvement in AS patients were analyzed,and analyze the correlation between ultrasound score and Bath ankylosing spondylitis disease activity score index(BASD AI),Bath ankylosing spondylitis functional index(BASFI),visual analogue score of pain(VAS),C-reactive protein(CRP),erythrocyte sedimentation rate(ESR),and the correlation between hip joint capsule score and tendon attach-ment end score and BASDAI,BASFI,VAS,CRP and ESR.Results The hip joint capsule score(3.06±1.12),femoral head score(1.45±0.43),tendon attachment end score(3.28±1.30)and total ultrasound score(6.65±2.31)of the disease group were higher than those of the control group(1.51±0.48)、(0.66±0.27)、(1.61±0.53)、(3.81±1.44)scores(P＜0.05).Multifactor Logstic re-gression analysis showed that the course of disease,hip joint capsule score and total ultrasound score were independent risk factors for hip involvement in AS patients.The hip capsule score(3.65±1.22)and total ultrasound score(8.28±2.33)in the ac-tive phase of the disease group were higher than those in the stable phase(2.48±1.04)、(6.82±1.96)scores(P＜0.05).The hip joint capsule score and total ultrasonic score of AS patients were positively correlated with BASDAI,BASFI,VAS,CRP,and ESR(P＜0.05,P＜0.01).The score of tendon attachment end was positively correlated with CRP(P＜0.05).The score of joint capsule effusion in AS patients was positively correlated with BASDAI,BASFI and VAS(P＜0.05,P＜0.01).The synovial blood flow score was positively correlated with BASDAI,VAS,CRP and ESR(P＜0.05,P＜0.01).The synovial thickening score was positively correlated with BASDAI,BASFI,VAS,CRP and ESR(P＜0.05,P＜0.01).There was no correlation between the score of tendon attachment end and BASDAI,BASFI,VAS,CRP and ESR.Conclusion There is a correlation between hip joint ul-trasonic score of hip joint and clinical indexes in AS patients.Hip joint capsule score and total ultrasonic score were indepen-dent risk factors for hip involvement in AS patients.High frequency ultrasound exhibits clinical value in the diagnosis of hip joint involvement in AS patients.

In order to understand the prevalence and genetic variation of H9N2 subtype avian influ-enza virus in Shandong,a total 492 tracheal and lung tissue samples collected from chicken farms with respiratory symptoms in partial areas in Shandong were detected by H9 subtype AIV real-time RT-PCR,and the positive samples were inoculated with chicken embryos for two generations.Whole genome sequences of the positive strains by applying Illumina Miaseq platform,and genetic evolution and mutation at positions associating with viral pathogenicity and transmissibility were analyzed.The results showed that there were 72 samples were positive for H9 subtype AIV among the 492 samples,with a positive rate of 14.63％.Thirty-four strains of H9 subtype AIV were ob-tained from the positive samples after passing through chicken embryo,meanwhile,the 34 isolates were all H9N2 subtype AIV by whole genome sequencing analysis.By analyzing the evolutionary tree of HA and NA genes,HA and NA genes of the 34 H9N2 AIV strains belonged to Y280-like branch and F/98-like branch,respectively.Meanwhile,based on above branches,there were obvious time node subbranch,which one was"isolates before 2013",another one was"isolates after 2013".The HA cleavage sites of thirty-four H9N2 strains were all 325PSRSSR↓GLF333,which met the se-quence characteristics of the lowly pathogenic avian influenza virus,and the HA receptor binding site 226 amino acid was leucine,which had the characteristics of blinding to a-2,6 mammalian sialic acid receptors.Among the internal amino acid sites that are key to mammalian adaptation,all strains had an I368V mutation in the PB1 gene that enhanced viral transmissibility in mammals and the PB2 genes of some strains were mutated to enhance the mammalian adaptation of I292 V and A588 V.The above results illustrated that the H9N2 subtype AIV gene segments in Shandong have different degrees of recombination and gene variation,so it is necessary to strengthen the monito-ring of virus variation.

Objective:To evaluate the efficacy of acute T-cell lymphoblastic leukemia(T-ALL)in children and explore the prognostic risk factors.Methods:The clinical data of 127 newly diagnosed children with T-ALL admitted to five hospitals in Fujian province from April 2011 to December 2020 were retrospectively analyzed,and compared with children with newly diagnosed acute precursor B-cell lymphoblastic leukemia(B-ALL)in the same period.Kaplan-Meier analysis was used to evaluate the overall survival(OS)and event-free survival(EFS),and COX proportional hazard regression model was used to evaluate the prognostic factors.Among 116 children with T-ALL who received standard treatment,78 cases received the Chinese Childhood Leukemia Collaborative Group(CCLG)-ALL 2008 protocol(CCLG-ALL 2008 group),and 38 cases received the China Childhood Cancer Collaborative Group(CCCG)-ALL 2015 protocol(CCCG-ALL 2015 group).The efficacy and serious adverse event(SAE)incidence of the two groups were compared.Results:Proportion of male,age ≥ 10 years old,white blood cell count(WBC)≥ 50 × 109/L,central nervous system leukemia,minimal residual disease(MRD)≥ 1％during induction therapy,and MRD ≥ 0.01％at the end of induction in T-ALL children were significantly higher than those in B-ALL children(P＜0.05).The expected 10-year EFS and OS of T-ALL were 59.7％and 66.0％,respectively,which were significantly lower than those of B-ALL(P＜0.001).COX analysis showed that WBC ≥ 100 x 109/L at initial diagnosis and failure to achieve complete remission(CR)after induction were independent risk factors for poor prognosis.Compared with CCLG-ALL 2008 group,CCCG-ALL 2015 group had lower incidence of infection-related SAE(15.8％vs 34.6％,P=0.042),but higher EFS and OS(73.9％vs 57.2％,PEFS=0.090;86.5％vs 62.3％,PoS=0.023).Conclusions:The prognosis of children with T-ALL is worse than children with B-ALL.WBC ≥ 100 × 109/L at initial diagnosis and non-CR after induction(especially mediastinal mass has not disappeared)are the risk factors for poor prognosis.CCCG-ALL 2015 regimen may reduce infection-related SAE and improve efficacy.

Objective:Serological and molecular biology methods were used to identify the blood type of a patient with forward and reverse ABO typing inconsistency,and to explore the genetic characteristics of this blood type.Methods:The ABO phenotype of the proband was identified by tube method,and the ABO blood group genotype of the proband and her parents was determined by fluorescent PCR.The 7 exons of the ABO gene were directly sequenced and analyzed.Results:According to preliminary serological identification,the ABO phenotype of this patient was Bel subtype.Genotyping tests showed that the ABO genotype of the proband and her father was B/O1,and her mother was O1/O1.Sequencing of exons revealed novel heterozygous variations in exon 1:c.16_17delinsTGTTGCA.Conclusion:The Novel variations in exon 1 led to Bel subtype in the ABO blood group of the proband,and these variations are heritable.

Objective:To investigate the effects of Xiongcan Yishen Formula(XYF)on ferroptosis in mouse TM3 Leydig cells after oxidative stress injury(OSI)induced by H2O2.Methods:An oxidative stress injury model was established in mouse TM3 Leydig cells using H2O2 induction.The modeled TM3 cells were randomly divided into OSI group,XYF group,the ferroptosis inhibitor Ferrostatin-1(F-1)group,and F-1+XYF group,which were respectively intervened with blank serum,20%drug-containing serum,2μmol/L F-1,and2μmol/L F-1+20%drug-containing serum.A control group(normal TM3 cells+blank serum)was also set up.The morphology of cells in each group was observed,and the levels of testosterone,superoxide dismutase(SOD),reactive oxygen spe-cies(ROS),malondialdehyde(MDA),ferritin heavy chain 1(FTH1),solute carrier family 7 member 11(SLC7A11),glutathione(GSH),glutathione peroxidase 4(GPX4),fatty acid CoA ligase 4(FACL4),total iron ions,and ferrous ions were detected.Re-sults:Compared with the model group,the control group showed significantly decreased expression of ROS,MDA,FACL4,total iron,and ferrous ions(P<0.05),and significantly increased levels of testosterone,SOD,GSH,FTH1,SLC7A11,and GPX4(P<0.05).The male silkworm kidney-tonifying formula group significantly promoted testosterone secretion by TM3 cells and upregulated the expression of FTH1,SLC7A11,GPX4,GSH,and SOD in TM3 cells(P<0.05),while significantly downregulating ROS,MDA,FACL4,total iron ions,and ferrous ions(P<0.05).Conclusion:Following H2O2 exposure,oxidative stress can induce ferroptosis in mouse TM3 Leydig cells.XYF can antagonize OSI and ferroptosis in TM3 cells by activating the SLC7A11/GSH/GPX4 axis,which may underlie the mechanism of XYF in the treatment of male late-onset hypogonadism.

Objective To construct an integrated pharmaceutical care model for chronic respiratory system disease patients,and to provide policy recommendations.Methods A field survey,a semi-structured interview,and a questionnaire survey were comprehensively used to investigate the effect of the reform and the status quo of pharmaceutical care for chronic respiratory diseases after the medicine consistency reform in the medical alliance of Chongqing.Subsequently,the rainbow model was used to assess the challenges in the macro,meso,micro,and supporting element's dimensions,and the integrated pharmaceutical care model was established as a consequence.Results The primary issues involved in the medical alliance were as followed:insufficient joint reformation for public health services,medical insurance,and medical production and circulation(JRPMM);deficient supporting policies;a dearth of information support;an inadequately list of medications;insufficient collaboration among chronic disease specialties;and unstandardized pharmaceutical care.Consequently,the following recommendations were:in the macro dimension,to strengthen the JRPMM to enhance the service capacity of primary hospitals and to develop suitable human resource coordination mechanisms.In the meso dimension,to intensify the reform of pharmaceutical care in the medical alliance and enhance specialty collaboration horizontally.In the micro dimension,to establish a pharmaceutical management committee in the medical alliance,to formulate pharmaceutical technical specifications for the hierarchical medical system,and to combine"talent and technology"training innovatively.In the supporting elements dimension,to bolster information support in the area and to develop suitable incentives and assessment institutions.Conclusion This paper reported an integrated pharmaceutical care model for chronic respiratory diseases,which could serve as a reference for standardizing the hierarchical medical system for chronic diseases.

Aim To investigate the effects of 2-dode-cyl-6-methoxycyclohexa-2 , 5-diene-l, 4-dione ( DM-DD) on resisting hepatic fibrosis induced by carbon tetrachloride ( CC14 ) in rats and the underlying mechanisms , with a specific focus on the TGF-pi/Smads signaling pathway. Methods The hepatic fibrosis model was replicated using 50% CC14. Various parameters, including levels of aspartate transferase ( AST) , ala-nine transferase ( ALT ) , albumin/globulin ( A/G ) , total protein (TP) , total bilirubin (T-BIL) , hyaluron-ic acid ( HA ) , laminin ( LN ) , collagen type Ж ( Col Ж) , and collagen type IV(ColIV) in the blood, were measured. Liver tissue lesions and fiber formation were observed using HE and Masson staining. The expression levels of a smooth muscle actin (a-SMA) , collagen type I ( Col I ) , transformed growth factor (TGF-pi), Smad2, and Smad7 proteins were assessed using immunohistochemistry. a-SMA, Coll, TGF-pi, and Smad7 mRNA levels in liver tissue were measured by RT-PCR. Additionally, the expression levels of TGF-pi, Smad4, and Smad7 proteins in liver tissue were determined by Western blot. Results In comparison to the normal control group, the model group exhibited significantly elevated levels of AST, ALT, TP, T-BIL, HA, LN, Col Ш and Col IV in serum. But A/G level notably decreased. Successful modeling was confirmed by the presence of extensive fiber formations observed through HE and Massonstaining in liver tissue. The DMDD administration group demonstrated a notable decrease levels of AST, ALT, TP, T-BIL, HA, LN, Col III, and CollV, but A/G was significantly elevated when compared to the model group. Furthermore, a-SMA, Coll, TGF-f31, Smad2 and Smad4 mRNA and protein levels in the DMDD administration group were significantly reduced, while Smad7 significantly declined. HE and Masson staining results reflected a marked reduction in fibrous hyper-plasia. Conclusion DMDD exhibits a protective effect against CCl4-induced hepatic fibrosis, and its mechanism appears to be associated with the TGF-fJl/ Smads signaling pathway.