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.

Aim To investigate the effect and role of neuronal restriction silencing factor(REST/NRSF)in epilepsy disorder.Methods Immunohistochemistry,immunofluorescence,Western blot and qPCR tech-niques were used to detect REST/NRSF expression levels in hippocampal tissues of mice induced by kainic acid and human brain tissue.Viral injections,EEG re-cordings and behavioral methods were used to test the effects on epileptic mice after knockdown and overex-pression of REST/NRSF in the hippocampal CA1 re-gion,respectively.Results The positive rate of REST/NRSF in the lesions of epileptic patients was significantly higher compared with that in the control group.The levels of REST/NRSF protein and mRNA in the CA1 region of the hippocampus of mice in the KA model group were significantly higher.Kv7.2 and Kv7.3 potassium channel mRNA expression levels were significantly down-regulated.Significant up-regu-lation of REST/NRSF expression levels was observed in mouse hippocampus after NMDA injection.Knock-down of REST/NRSF in the CA1 region of hippocam-pus significantly elevated the expression levels of Kv7.2 and Kv7.3 potassium channel mRNAs.The fre-quency of EEG spiking and sharp-wave issuance and epileptic seizure grade were significantly lower.Over-expression of REST/NRSF in the CA1 region of hippo-campus significantly reduced the mRNA expression lev-els of Kv7.2 and Kv7.3 potassium channels.The fre-quency of EEG spiking and sharp-wave issuance was significantly higher and epileptic symptoms were exac-erbated.Conclusion REST/NRSF in mouse hipp-ocampal brain regions is involved in epileptic disease development through transcriptional regulation of Kv7.2 and Kv7.3 potassium channels.

Objective:To investigate the efficacy and prognosis of chemotherapy regimen containing Bruton's tyrosine kinase(BTK)inhibitor in the treatment of relapsed/refractory mantle cell lymphoma(R/R MCL).Methods:The clinical data of 134 patients with R/R MCL were collected and analyzed retrospectively.The clinical characteristics of patients and effect of chemotherapy regimen on efficacy,overall survival(OS)and progression-free survival(PFS)were observed.Results:The median age of the patients was 58(56-61)years old,and male to female ratio was about 2.9∶1.Patients with Ann Arbor stage Ⅲ-Ⅳ accounted for 77.6％,extranodal involvement＞2 for 43.3％,bone marrow involvement for 60.4％,gastrointestinal involvement for 24.6％,and hepatosplenomegaly for 38.1％.The median follow-up time was 30(2-103)months,overall response rate(ORR)was 41.8％,3-year PFS was not reached,and 3-year and 5-year OS rate was 62.7％and 53.8％,respectively.The ORR of BTK inhibitor group was 56.9％,which was higher than 32.5％of non-BTK inhibitor group(P=0.006).The difference was statistically significant in PFS between the two groups(P=0.002),but was not in OS(P＞0.05).The difference was statistically significant in OS between classical and special morphology(P＜0.001),but was not in PFS(P＞0.05).Ki-67 was an influencing factor for OS and PFS.Multivariate analysis showed that Ki-67,B symptoms,MIPI score,and Ann Arbor stage were independent prognostic factors affecting patients'OS.The second-line treatment regimen was an independent prognostic factor affecting patients'PFS.Conclusions:The chemotherapy regimen containing BTK inhibitors can effectively improve the efficacy and prolong the PFS of R/R MCL patients.Ki-67,B symptoms,MIPI score,and Ann Arbor stage are independent prognostic factors for R/R MCL patients.

Objective:To investigate the protective effect of endogenous ω-3 polyunsaturated fatty acid(PUFA)against cisplatin-induced myelosuppression and the mechanism of reducing apoptosis in bone marrow nucleated cells using mfat-1 transgenic mice.Methods:The experimental animals were divided into 4 groups:wild-type mice normal control group,mfat-1 transgenic mice normal control group,wild-type mice model group and mfat-1 transgenic mice model group.The mice in the model group were injected intraperitoneally with 7.5 mg/kg cisplatin on day 0 and day 7 to construct a myelosuppression model,while the mice in the normal control group were injected intraperitoneally with an equal amount of saline,and their status was observed and their body weight was measured daily.Peripheral blood was taken after 14 day for routine blood analysis,and the content and proportion of PUFA in peripheral blood were detected using gas chromatography.Bone marrow nucleated cells in the femur of mice were counted.The histopathological changes in bone marrow were observed by histopathological staining.The apoptosis of nucleated cells and the expression level changes of apoptosis-related genes in the bone marrow of mice were detected by flow cytometry and fluorescence quantitative PCR.Results:Compared with wild-type mice,mfat-1 transgenic mice showed significantly increased levels of ω-3 PUFA in peripheral blood and greater tolerance to cisplatin.Peripheral blood analysis showed that endogenous ω-3 PUFA promoted the recovery of leukocytes,erythrocytes,platelets and haemoglobin in peripheral blood of myelosuppressed mice.The results of HE staining showed that endogenous ω-3 PUFA significantly improved the structural damage of bone marrow tissue induced by cisplatin.Flow cytometry and PCR showed that,compared with wild-type mice model group,the apoptosis rate of bone marrow nucleated cells in mfat-1 transgenic mice was significantly reduced(P＜0.001),and the expression of anti-apoptotic genes Bcl-2 mRNA was significantly increased(P＜0.01),while the expressions of pro-apoptotic genes Bax and Bak mRNA were significantly reduced(P＜0.001,P＜0.05).Conclusion:Endogenous ω-3 PUFA can reduce cisplatin-induced apoptosis in bone marrow nucleated cells,increase the number of peripheral blood cells and exert a protective effect against cisplatin-induced myelosuppression by regulating the expression of apoptosis-related genes.

Objective:To analyze the efficiency of human resource allocation and its spatial distribution characteristics of district-level Center for Disease Control and Prevention(CDC)in city N of Jiangsu Province in 2020,in order to provide a strong decision-making reference for optimizing and strengthening the CDC talent team.Methods:The efficiency of human resources of district-level CDC of City N in2020 was measured using the Super-Efficiency SBM model,and the spatial association pattern was analyzed using the natural break point classification method and Moran's index,with the visualization presented through LISA maps.Results:The overall level of human resource efficiency in district-level CDC of City N is relatively high.However,spatially,there are significant differences among the regions,showing a trend of high efficiency in the central areas and low efficiency at the ends.Moran's index and LISA maps indicate a negative spatial correlation in efficiency,with a low-high(L-H)cluster centered on Area L and a high-low(H-L)cluster centered on Area J.The high-high(H-H)cluster pattern has not yet formed,exhibiting a characteristic of interspersed high and low efficiency.Conclusions:There are regional differences in the human resource efficiency of the Disease Control Center in City N,and the spatial cluster pattern needs to be optimized.It is recommended to focus on efficiency improvement in Areas P and L,formulate appropriate policies,and promote coordinated regional development.

AIM To control the quality of Bupleurum chinense DC.METHODS The analysis was performed on a 35℃ thermostatic Venusil XBP C18 column(250 mm×4.6 mm,5 μm),with the mobile phase comprising of acetonitrile-water flowing at 1.0 mL/min,and the detection wavelength was set at 210 nm.The HPLC fingerprints were established,after which the contents of saikosaponin A,saikosaponin B2,saikosaponin C,saikosaponin D,saikosaponin E,saikosaponin F and 6″-O-acetylsaikosaponin A were determined,and principal component analysis was made.RESULTS There were thirteen common peaks in the fingerprints for twelve batches of medicinal materials with the similarities of 0.970-0.995.Seven constituents showed good linear relationships within their own ranges(R2≥0.999 8),whose average recoveries were 90.75%-100.91% with the RSDs of 1.6%-4.0% .Various constituents demonstrated similar contents in medicinal materials originated in Inner Mongolia and Shanxi.CONCLUSION This precise,accurate and stable method can be used for the quality evaluation of B.chinense.

Objective:To explore diagnostic value of echocardiography indexes for moderate-severe stenosis in CHD patients and their correlation with cardiac function class.Methods:A total of 90 CHD patients admitted to our hos-pital from Dec 2020 to Dec 2022 were selected as CHD group.According to NYHA classification,CHD group was divided into class Ⅰ～Ⅱ group(n=38)and class Ⅲ～Ⅳ group(n=52).The patients were divided into mild group(n=35)and moderate-severe group(n=55)according to Gensini score.In addition,90 healthy people who sim-ultaneously received physical examination in our hospital were enrolled as control group.LVEF,LVEDd and LVESd were measured in two groups by color ultrasound diagnostic instrument.Cardiac function indexes were com-pared between control group and CHD group,mild group and moderate-severe group,class Ⅰ～Ⅱ group and classⅢ～Ⅳ group.ROC curve was used to analyze the diagnostic value of above cardiac function indexes for moderate-severe stenosis in CHD patients.The correlation among LVEF,LVEDd,LVESd,different stenosis degree and NYHA class in CHD patients was analyzed by Spearman correlation analysis.Results:Compared with the control group,there was significant reduction in LVEF,and significant rise in LVEDd and LVESd in CHD group,P=0.001 all;compared with the mild group,there was significant reduction in LVEF[(45.31±5.08)％vs.(40.34±3.01)％],and significant rise in LVEDd[(53.92±5.09)mm vs.(61.68±4.79)mm]and LVESd[(52.72±4.72)mm vs.(58.06±3.50)mm]in moderate-severe group,P=0.001 all;compared with the class Ⅰ～Ⅱgroup,there was significant reduction in LVEF[(45.07±4.95)％vs.(40.23±3.06)％],and significant rise in LVEDd[(54.50±5.30)mm vs.(61.71±4.91)mm]and LVESd[(52.92±4.63)mm vs.(58.22±3.43)mm]in class Ⅲ～Ⅳ group,P=0.001 all.AUC of combined detection of LVEF,LVEDd and LVESd diagnosing moder-ate-severe stenosis in CHD was 0.909,which was significantly higher than those of single detections(0.733,0.787,0.789)(Z=2.925,2.125,2.043,P＜0.05 or＜0.01).Spearman correlation analysis showed a significant negative correlation between LVEF and NYHA class(r=-0.514),LVEDd and LVESd showed a significant posi-tive correlation with NYHA class(r=0.538,0.546,P=0.001 both),and it showed a significant positive correla-tion between different degrees of coronary stenosis and NYHA class in CHD patients(r=0.875,P=0.001).Con-clusion:The combined detection of LVEF,LVEDd and LVESd possesses high diagnostic value for moderate-se-vere stenosis in CHD patients,and it is significantly correlated with NYHA cardiac function class,which can be used as one of assessing methods for coronary stenosis severity in CHD patients.

Objective To investigate the risk factors for the failure of ibuprofen treatment in preterm infants with hemodynamically significant patent ductus arteriosus(hsPDA).Methods A retrospective collection of clinical data was conducted on preterm infants with a gestational age of＜34 weeks who were diagnosed with hsPDA and treated at the Department of Neonatology,Maternal and Child Health Hospital of Hubei Province,Tongji Medical College,Huazhong University of Science and Technology,from January 2018 to June 2023.The subjects were divided into two groups based on the treatment approach:the ibuprofen group(95 cases)and the ibuprofen plus surgery group(44 cases).The risk factors for the failure of ibuprofen treatment in preterm infants with hsPDA were identified by binary logistic regression analysis.Results The binary logistic regression analysis revealed that an increased diameter of the ductus arteriosus,a resistance index(RI)value of the middle cerebral artery ≥0.80,and prolonged total invasive mechanical ventilation time were risk factors for the failure of ibuprofen treatment in preterm infants with hsPDA(P＜0.05).Receiver operating characteristic curve analysis showed that a ductus arteriosus diameter＞2.85 mm,a middle cerebral artery RI value ≥0.80,and a total invasive mechanical ventilation time＞16 days had significant predictive value for the failure of ibuprofen treatment in preterm infants with hsPDA(P＜0.05).The combined predictive value of these three factors was the highest,with an area under the curve of 0.843,a sensitivity of 86.5％,and a specificity of 75.0％(P＜0.05).Conclusions A ductus arteriosus diameter＞2.85 mm,a middle cerebral artery RI value ≥0.80,and a total invasive mechanical ventilation time＞16 days are risk factors for the failure of ibuprofen treatment in preterm infants with hsPDA,and they are of significant predictive value for the necessity of surgical treatment following the failure of ibuprofen treatment.[Chinese Journal of Contemporary Pediatrics,2024,26(4):343-349]

Aim To investigate the role of metabolites of eicosapentaenoic acid (EPA) in promoting the transdifferentiation of pancreatic α cells to β cells. Methods Male C57BL/6J mice were injected intraperitoneally with 60 mg/kg streptozocin (STZ) for five consecutive days to establish a type 1 diabetes (T1DM) mouse model. After two weeks, they were randomly divided into model groups and 97% EPA diet intervention group, 75% fish oil (50% EPA +25% DHA) diet intervention group, and random blood glucose was detected every week; after the model expired, the regeneration of pancreatic β cells in mouse pancreas was observed by immunofluorescence staining. The islets of mice (obtained by crossing GCG

Platycodonis Radix is the dry root of Platycodon grandiflorum of Campanulaceae, which has a variety of pharmacological effects and is a commonly used bulk Chinese medicine. In this study, the chloroplast genome sequences of six P. grandiflorum from different producing areas has been sequenced with Illumina HiSeq X Ten platform. The specific DNA barcodes were screened, and the germplasm resources and genetic diversity were analyzed according to the specific barcodes. The total length of the chloroplast genome of 6 P. grandiflorum samples was 172 260-172 275 bp, and all chloroplast genomes showed a typical circular tetrad structure and encoded 141 genes. The comparative genomics analysis and results of amplification efficiency demonstrated that trnG-UCC and ndhG_ndhF were the potential specific DNA barcodes for identification the germplasm resources of P. grandiflorum. A total of 305 P. grandiflorum samples were collected from 15 production areas in 9 provinces, for which the fragments of trnG-UCC and ndhG_ndhF were amplificated and the sequences were analyzed. The results showed that trnG-UCC and ndhG_ndhF have 5 and 11 mutation sites, respectively, and 5 and 7 haplotypes were identified, respectively. The combined analysis of the two sequences formed 13 haplotypes (named Hap1-Hap13), and Hap4 is the main genotype, followed by Hap1. The unique haplotypes possessed by the three producing areas can be used as DNA molecular tags in this area to distinguish from the germplasm resources of P. grandiflorum from other areas. The haplotype diversity, nucleotide diversity and genetic distance were 0.94, 4.79×10^-3 and 0.000 0-0.020 3, respectively, suggesting that the genetic diversity was abundant and intraspecific kinship was relatively close. This study laid a foundation for the identification of P. grandiflorum, the protection and utilization of germplasm resources, and molecular breeding.