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.

ObjectiveTo investigate the triglyceride-glucose (TyG) index and the level of serum homocysteine (Hcy) in association with the incidence of stroke in type 2 diabetes mellitus (T2DM) patients. MethodsBased on the chronic disease risk factor surveillance cohort in Pudong New Area, Shanghai, excluding those with stroke in baseline survey, T2DM patients who joined the cohort from January 2016 to October 2020 were selected as the research subjects. During the follow-up period, a total of 318 new-onset ischemic stroke patients were selected as the case group, and a total of 318 individuals matched by gender without stroke were selected as the control group. The Cox proportional hazards regression model was used to adjust for confounding factors and explore the serum TyG index and the Hcy biochemical indicator in association with the risk of stroke. ResultsThe Cox proportional hazards regression results showed that after adjusting for confounding factors, the risk of stroke in T2DM patients with 10 μmol·L⁻¹-¹ and Hcy>15 μmol·L⁻¹ was 1.532 times (95%CI: 1.017‒2.309 times, P=0.041) and 1.738 times (95%CI: 1.119‒2.699 times, P=0.014) higher respectively, compared to T2DM patients with Hcy≤10 μmol·L⁻¹. T2DM patients with TyG>9.074 had 1.396 times higher risk of stroke (95%CI: 1.091‒1.787, P=0.008) compared to those with TyG≤9.074. The study found that urea and α1-antitrypsin (α1-AT) were independent risk factors for the incidence of stroke in T2DM patients. For every unit increase in urea andα1-AT, the risk of stroke in T2DM patients increased by 233.6% (HR=3.336, 95%CI: 1.588‒7.009, P=0.001) and 11.3% (HR=1.113, 95%CI: 1.028‒1.205, P=0.008), respectively. Cumulative risk curves showed that Hcy>10 μmol·L⁻¹ and TyG>9.074 accelerated the time to stroke occurrence in T2DM patients. ConclusionElevated levels of Hcy>10 μmol·L⁻¹ and a higher TyG index are risk factors for stroke in T2DM patients. Effective interventions for Hcy and TyG should be conducted for diabetic patients to reduce the incidence of stroke.

Objective To construct a lentiviral vector for overexpression of bone morphogenetic protein 7(BMP7)in mice,and the effect of BMP7 overexpression on the expression of Jagged1 in mouse aortic endothelial cells and the calcification of the co-cultured vascular smooth muscle cells(VSMCs)were analyzed.Methods According to the target gene information Mouse-BMP7(NM_007557.3)and plasmid information pLVX-zsGreen-C1,gene sequence synthesis was carried out to construct BMP7 overexpression lentivirus.The efficiency of BMP7 overexpression lentivirus infection was detected by qPCR;the expression of Jagged1 protein in aortic endothelial cells from infected mice was detected by Western blot.The endothelial cells with lentivirus overexpressing BMP7 were co-cultured with VSMCs,and the calcification of VSMCs was observed by alizarin red staining.Results BMP7 overexpression lentiviral vector was successfully constructed and transfected into aortic endothelial cells.qPCR test results showed that the expression level of BMP7 mRNA was significantly increased in the BMP7 overexpression group than that in the normal control group(P<0.01),while there was no significant difference in the expression of BMP7 mRNA between the empty vector control group and the normal control group(P>0.05).Western blot results showed that the expression level of Jagged1 protein in endothelial cells of mouse in the BMP7 overexpression group was significantly lower than that in the normal control group(P<0.01),while there was no significant difference in the expression level of Jagged1 protein in endothelial cells between the empty vector control group and the normal control group(P>0.05).The results of alizarin red staining showed that the calcification of VSMCs was significantly increased after co-cultured with endothelial cells infected with BMP7 lentivirus.Conclusion Mouse BMP7 overexpression lentiviral vector was successfully constructed,and overexpression of BMP7 can reduce the expression of Jagged1 in mouse aortic endothelial cells and promote the calcification of co-cultured VSMCs.

Objective To provide pharmaceutical monitoring using the Pharmaceutical Care Network Europe(PCNE)for patients with respiratory diseases,to explore effective pharmaceutical monitoring models in the department of respiratory,and to promote clinical rational drug use.Methods Inpatients diagnosed with chronic obstructive pulmonary disease(COPD)and lung infections in 2022 at the First Affiliated Hospital of Anhui University of Science and Technology were selected and divided into a simple group and an intervention group.According to the PCNE classification system,the types,causes,interventions,acceptance of interventions,and resolution status of drug-related problems(DRPs)were analyzed.Results A total of 120 cases were included,60 cases in the simple group and 60 cases in the intervention group.Regarding the number of DRPs,there were 15 cases in the simple group and 45 cases in the intervention group,and there was a significant difference between the two groups(P<0.05).There were a total of 82 DRPs,which were mainly related to therapeutic efficacy(51.22%)and safety(46.34%),and the reasons for this were that patients'incorrect medication usage method,inappropriate usage and dosage,and unscheduled safety monitoring,etc.The pharmacist interventions were 75(91.46%)at the drug level,38(46.34%)at the physician level,and 43(52.44%)at the patient level;after the pharmacist interventions,the acceptance rate was in the range of 97.56%,and 74.39%of the DRPs were resolved.Conclusion PCNE classification system helps clinical pharmacists to enhance their ability to find and deal with DRPs,reduce the risk of clinical adverse events and promote reasonable and safe drug use.Meanwhile,it is conducive to the standardization of pharmaceutical care records for patients with respiratory diseases and provides reference for pharmaceutical service models for patients in the department of respiratory.

Investigating the structural characteristics of proton tautomers is of great significance for understanding organic reactions and biochemical processes.In this study,a method based on ion mobility mass spectrometry combined with ab initio molecular dynamics calculations was proposed.The structures of proton tautomers were determined by comparing the experimental and theoretical collision-induced dissociation(CID)mass spectrograms of different proton tautomers.Ion mobility mass spectrometry was used to separate proton tautomerism produced during ionization.The CID mass spectra of each isomer could be obtained after mobility separation.The quantum chemical mass spectrometry(QCXMS)program based on ab initio molecular dynamics calculations was used to calculate the fragmentation process and obtain the theoretical CID mass spectra.The results of experiment and calculation showed that this method effectively solved the issue of difficult identification of proton tautomers.This method was also of great significance for the study of ionization mechanism and organic reaction process using mass spectrometry.

The incidence of intrahepatic cholangiocarcinoma (ICC) continues to rise, and there are no effective drugs to treat it. The immune microenvironment plays an important role in the development of ICC and is currently a research hotspot. Icaritin (ICA) is an innovative traditional Chinese medicine for the treatment of advanced hepatocellular carcinoma. It is considered to have potential immunoregulatory and anti-tumor effects, which is potentially consistent with the understanding of "Fuzheng" in the treatment of tumor in traditional Chinese medicine. However, whether ICA can be used to treat ICC has not been reported. Therefore, in this study, sgp19/kRas, an in situ ICC mouse model with intact immune system, was selected to evaluate the efficacy of ICA in the treatment of ICC in vivo for the first time, and the effects of ICA on the tumor immune microenvironment of ICC mice were analyzed by flow cytometry. This experiment was approved by the Experimental Animal Ethics Committee of Capital Medical University (approval number: AEEI-2023-138). In this study, sgp19/kRas ICC mouse model was treated with oral gavage of 100 mg·kg^-1 ICA. We found that ICA significantly inhibited tumor growth and tumor cell proliferation in the sgp19/kRas ICC mouse model after 3 weeks of treatment. Flow cytometry analysis indicated that ICA treatment markedly reduced the proportion of M2 macrophages and increased the number of CD3⁺CD4⁺ T cells. In vitro experiments demonstrated that ICA promoted macrophage polarization towards the M1 phenotype while inhibiting polarization towards the M2 phenotype. Furthermore, transcriptomic analysis suggested that ICA enhanced Toll-like receptor 9 (TLR9) expression, influencing macrophage nitric oxide metabolism synthesis pathways and receptor-related activities, thereby regulating macrophage polarization. In summary, this study demonstrates that ICA treatment significantly delays tumor progression in sgp19/kRas ICC mouse models. Mechanistically, ICA may achieve its anti-ICC effects by upregulating TLR9 receptor expression levels, promoting macrophage polarization towards the M1 phenotype, and altering the tumor immune microenvironment.

Objective To explore the mechanism by which diazepam alleviates lipopolysaccharide(LPS)-induced pyroptosis and inflammation to delay the progression of pulmonary fibrosis.Methods MRC-5 cells challenged with LPS were treated with diazepam and transfected with a let-7a-5p mimic alone or co-transfected with pc-DNA-MYD88.The changes in cellular expressions of inflammatory factors were analyzed with ELISA,and the expressions of fibrosis-and pyroptosis-related proteins were detected using Western blotting.In the animal experiment,C57BL/6 mice were randomized for treatment with LPS,LPS+diazepam,LPS+diazepam+let-7a-5p mimic,LPS+diazepam+ST2825(a MYD88 inhibitor),or LPS+diazepam+let-7a-5p mimic+pc-DNA-MYD88,and pulmonary fibrosis and pulmonary expression of α-SMA were examined using Masson staining and immunofluorescence staining,respectively.Results LPS exposure of MRC-5 cells significantly downregulated let-7a-5p expression,up-regulated MYD88 expression,increased the levels of IL-4,IL-6,TGF-β and TNF-α,and enhanced the expressions of fibrosis-related proteins(Col-I,Col-III,and α-SMA)and pyroptosis-related proteins(NLRP3,caspase-1,ASC,and GSDMD-N).Diazepam treatment of LPS-stimulated cells effectively inhibited the expressions of inflammation-related factors and the fibrosis-and pyroptosis-related proteins.In C57BL/6 mice,diazepam treatment obviously alleviated LPS-induced pulmonary fibrosis and reduced and pulmonary expression of α-SMA,and these effects were further enhanced by treatment with let-7a-5p mimic or ST2825,but the effect of let-7a-5p mimic was significantly attenuated by MYD88 over-expression.Conclusion Diazepam can negatively regulate MYD88 by upregulating the expression of let-7a-5p to inhibit LPS-induced pyroptosis and inflammatory response,thereby alleviating lung fibrosis in mice.

Myopia is a significant global health issue,and its exact causes are still not fully understood,leaving a lack of effective and propaqable treatment options.The prevalence of myopia among children and adolescents in China remains high,posing challenges for prevention and control efforts.According to the"theory of imbalance of essence and tendon",an imbalance in the essential elements and tendons can impede the passage of eye essence and blood,resulting in delayed expansion and contraction of the eye meridians and tendons,leading to blurred vision.Modern research indicates that during the development of myopia,there are notable changes in the microstructure,activation range,and signaling of various brain regions,providing a biological basis for the"brain-eye"mechanism.Moreover,abnormal activity in the brain nucleus contributes to alterations in choroid blood flow and the impairment of eye muscle regulation,thereby accelerating the progression of myopia,this phenomenon represents the manifestation of the brain-eye imbalance.Consequently,strategies for myopia prevention and control should prioritize nourishing kidney essence,replenishing brain marrow,promoting liver and blood health,and softening the meridians.These measures aim to optimize the functioning of the brain and eyes,maintain the flexibility of eye tendons,enhance eye regulation,sustain the strength of eye tendons,and delay the advancement of eye axis growth and myopia.By enriching the scientific understanding of the appropriate application of traditional Chinese medicine techniques to prevent and control myopia through the brain,this research provides valuable insights for future explorations in this field.

Objective To explore the clinical value of fibrinogen-albumin-ratio (FAR) in adult extracorporeal membrane oxygenation (ECMO) hemorrhage. Methods The clinical data of adult patients receiving ECMO in the West China Hospital from 2018 to 2020 were analyzed retrospectively. Patients were divided into a bleeding group and a non-bleeding group based on whether they experienced bleeding after ECMO. Logistic regression analysis was used to study the relationship between FAR and bleeding, as well as risk factors for death. Receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to analyze the predictive ability of FAR. According to the optimal cut-off value of FAR for predicting hemorrhage, patients were divided into a high-risk group and a low-risk group, and the occurrence of bleeding was compared between the two groups. Results A total of 125 patients were enrolled in this study, including 85 males and 40 females, aged 46.00 (31.50, 55.50) years. Among them, 58 patients received veno-arterial extracorporeal membrane oxygenation (VA-ECMO) and 67 patients received veno-venous extracorporeal membrane oxygenation (VV-ECMO). There were 49 patients having bleeding, and the lactate level was higher (P=0.026), the platelet count before ECMO initiation and 24 h after ECMO initiation was lower (P=0.031, 0.020), the fibrinogen level 24 h after ECMO initiation was lower (P=0.049), and the proportion of myocarditis patients was higher (P=0.017) in the bleeding group than those of the non-bleeding group. In the subgroup analysis of ECMO mode, the higher D-Dimer, lactate level and lower FAR before and 24 h after ECMO initiation were associated with bleeding in the VA-ECMO group (P=0.017, 0.011, 0.033, 0.005). The 24 h FAR was independently correlated with bleeding (P=0.048), and AUC was 0.714. The cut-off value was 55.73. According to this optimal cut-off value, 25 patients were divided into the high-risk group (≤55.73) and 33 into the low-risk group (>55.73). There was a higher incidence of bleeding in the high-risk group compared to the low-risk group (unadjusted P=0.002; P=0.013 for multivariable adjustment). In the VV-ECMO group, the relationship between FAR and bleeding events was not significant (P>0.05). Conclusion Low 24 h FAR is an independent risk factor for bleeding in VA-ECMO patients, and the diagnostic cut-off value is 55.73.

For the treatment of atrial fibrillation,Professor WU Wei innovatively put forward the theory of heart-blood-vessels trinity and the theory of stasis-toxin causing palpitation.It is believed that atrial fibrillation is caused by stasis and toxin,and affects the heart,blood and vessels.The core pathogenesis of atrial fibrillation is due to qi stagnation,blood stasis and toxin.The treatment for atrial fibrillation should be closely based on the pathogenesis,the therapeutic principles of treating from the perspective of stasis and together by removing toxin gradually is advocated.And the therapy of regulating qi,activating blood and removing stasis is also the way to remove toxin.The medication is based on the modified Taoren Honghua Decoction,which is mainly composed of Persicae Semen,Carthami Flos,Chuanxiong Rhizoma,Corydalis Rhizoma,Rehmanniae Radix,Paeoniae Radix Rubra,Salviae Miltiorrhizae Radix et Rhizoma,Jujubae Fructus,Puerariae Lobatae Radix,Nardostachyos Radix et Rhizoma,Ostreae Concha,Poria,and Polygonati Odorati Rhizoma.According to the characteristics of Lingnan climate and atrial fibrillation mostly being easy to affect the emotions,the pungent drugs in the prescription are usually removed,and the specific herbal pair of Puerariae Lobatae Radix-Nardostachyos Radix et Rhizoma is added to remove toxin according to the differentiation of disease.Moreover,for the treatment of atrial fibrillation,Professor WU Wei also adopts traditional Chinese medicine(TCM)external treatment such as foot bath,acupuncture and moxibustion,and physical-breathing exercise as well as health-care methods for comprehensive regulation,relieving the toxin and restoring the original qi.During the treatment atrial fibrillation,Professor WU Wei follows the principle of precise intervention and comprehensive regulation with Chinese medicine,so as to achieve the purpose of eliminating symptoms,restoring sinus rhythm and improving physical constitution.The thoughts of Professor WU Wei for the syndrome differentiation and treatment of atrial fibrillation will provide reference for the treatment of atrial fibrillation with TCM.