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.

This study aims to explore the impact of host plants on the quality of Taxilli Herba and provide a theoretical basis for the quality control of Taxilli Herba. The components of Taxilli Herba from three different host plants(Morus alba, Salix babylonica, and Cinnamomum cassia) and its 3 hosts(mulberry branch, willow branch, and cinnamon branch) were detected by widely targeted metabolomics based on ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). Principal component analysis(PCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and Venn diagram were employed for analysis. A total of 717 metabolites were detected in Taxilli Herba from the three host plants and the branches of these host plants by UPLC-MS/MS. The results of PCA and OPLS-DA of Taxilli Herba from the three different host plants showed an obvious separation trend due to the different effects of host plants. The Venn diagram showed that there were 32, 8, and 26 characteristic metabolites in samples of Taxilli Herba from M. alba host, S. babylonica host, and C. cassia host, respectively. It was found by comparing the characteristic metabolites of Taxilli Herba and its hosts that each host transmits its characteristic components to Taxilli Herba, so that the Taxilli Herba contains the characteristic components of the host. The Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis showed that the differential metabolites of Taxilli Herba from the three hosts were mainly enriched in flavonoid biosynthesis, arginine and proline metabolism, and glycolysis/gluconeogenesis pathways. Furthermore, the differential metabolites enriching pathways of Taxilli Herba from the three hosts were different depending on the host. In a word, host plants have a significant impact on the metabolites of Taxilli Herba, and it may be an important factor for the quality of Taxilli Herba.
Metabolomics/methods* ; Drugs, Chinese Herbal/chemistry* ; Chromatography, High Pressure Liquid ; Tandem Mass Spectrometry ; Quality Control ; Salix/chemistry* ; Cinnamomum aromaticum/metabolism* ; Principal Component Analysis

OBJECTIVE: To explore the application of targeted mRNA sequencing in fusion gene diagnosis of hematologic diseases. METHODS: Bone marrow or peripheral blood samples of 105 patients with abnormally elevated eosinophil proportions and 291 acute leukemia patients from January 2015 to June 2023 in the First Affiliated Hospital of Soochow University were analyzed and gene structural variants were detected by targeted mRNA sequencing. RESULTS: Among 105 patients with abnormally elevated eosinophil proportions, 6 cases were detected with gene structural variants, among which fusion gene testing results in 5 cases could serve as diagnostic indicators for myeloid neoplasms with eosinophilia. In addition, a IL3∷ETV6 fusion gene was detected in one patient with chronic eosinophilic leukemia, not otherwise specified. Among 119 patients with acute myeloid leukemia (AML), 38 cases were detected structural variants by targeted mRNA sequencing, accounting for 31.9%, which was significantly higher than 20.2% (24/119) detected by multiple quantitative PCR (P < 0.05). We also found one patient with AML had both NUP98∷PRRX2 and KCTD5∷JAK2 fusion genes. A total of 104 patients were detected structural variants by targeted mRNA sequencing in 172 cases with acute B-lymphoblastic leukemia who were tested negative by multiple quantitative PCR, with a detection rate of 60.5% (102/172). CONCLUSION Targeted mRNA sequencing can effectively detect fusion gene and has potential clinical application value in diagnosis and classificatation in hematologic diseases.
Humans ; Hematologic Diseases/diagnosis* ; RNA, Messenger/genetics* ; Oncogene Proteins, Fusion/genetics* ; Sequence Analysis, RNA ; Leukemia, Myeloid, Acute/diagnosis*

Aim To design the pyrimidoindole deriva-tive N-butyl-9H-pyrimido[4,5-b]indole-2-carboxamide(BFPI)and synthesize it to investigate whether it in-hibits macrophage pyroptosis and foaming effects through the NLRP3/Caspase-1 pathway.Methods BFPI was synthesized using 2,4,6-triethoxycarbonyl-l,3,5-triazine and 2-aminoindole as starting materials and structurally characterized by 1H NMR,13C NMR,and ESI-MS.The in vitro cultured mouse monocyte macro-phage cell line RAW264.7 was divided into blank,model(PA)and therapeutic(BFPI)groups,and the cells in each group were treated with the corresponding culture medium for 24 h.The proliferative viability was detected by MTT assay,and the formation of intracel-lular lipid droplets was detected by oil red O staining,and NLRP3 was detected by Western-blot and RT-qPCR,caspase-1 and MCP-1 mRNA and protein ex-pression levels by Western blot and RT-qPCR.Results Compared with the blank group,the proliferation vi-ability of cells in the model group significantly de-creased and the formation of lipid droplets significantly increased;compared with the model group,the prolif-eration viability of cells in the treatment group signifi-cantly increased and the formation of lipid droplets sig-nificantly decreased,and the differences were statisti-cally significant(P＜0.01);compared with the blank group,the cellular NLRP3,caspase-1 and MCP-1 mR-NA and protein expression levels of cells in the model group significantly increased;compared with the model group,the expression levels of the above indexes of the cells in the treatment group significantly decreased,and the difference was statistically significant(P＜0.01).Conclusions BFPI contributes to delaying macrophage-derived foam cell formation during athero-genesis by inhibiting macrophage NLRP3,caspase-1,and MCP-1 expression and thereby promoting their pro-liferation and inhibiting lipid phagocytosis.

This study aimed to investigate the therapeutic effect of Leonuri Herba aqueous decoction on primary dysmenorrhea(PD) and explore the underlying mechanism in conjunction with untargeted metabolomics. Forty adult female rats were randomly divi-ded into a normal group, a model control group, ibuprofen(0.12 g·kg~(-1)) group, and high-and low-dose Leonuri Herba aqueous decoction(5 and 2.5 g·kg~(-1)) groups, with eight rats in each group. The PD rat model was prepared using intramuscular injection of estradiol benzoate combined with intraperitoneal injection of pitocin. Drugs were administered by gavage from the 4th day of modeling for 7 d. After the last administration, pitocin was injected intraperitoneally, and the writhing latency and writhing times within 30 min were recorded. The uterine and ovarian coefficients were determined. Estradiol(E_2), progesterone(Prog), oxytocin(OT), cyclooxyge-nase 2(COX-2), prostaglandin E_2(PGE_2), prostaglandin F_(2α)(PGF_(2α)), and Ca~(2+) levels in uterine tissues were measured by ELISA and biochemical kits. Morphological changes in uterine and ovarian tissues were observed by hematoxylin-eosin(HE) staining. The protein expression of oxytocin receptor(OTR), prostaglandin E_2 receptor 3(EP3), and estrogen receptor alpha(ERα) in uterine tissues was detected by immunohistochemistry. The mRNA expression of OTR, PGE_2 receptors 1-4(EP1, EP2, EP3, and EP4), and PGF_(2α) receptor(FP) in uterine tissues was detected by quantitative real-time PCR. Untargeted metabolomics analysis was performed by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(LC-QTOF-MS) technology to screen potential biomarkers and enrich metabolic pathways. The results showed that Leonuri Herba was able to significantly reduce the writhing times in PD rats(P<0.05 or P<0.01), significantly reduce the uterine and ovarian coefficients(P<0.01), and improve their histomorphology. After treatment with Leonuri Herba, PGE_2 content was significantly increased(P<0.05), COX-2, PGF_(2α) and Ca~(2+) content, and PGF_(2α)/PGE_2 was significantly decreased(P<0.05 or P<0.01), and OT content was decreased, while E_2 and Prog content tended to further increase in uterine tissues of PD rats. Correspondingly, OTR and EP3 protein expression was significantly downregulated(P<0.05 or P<0.01) and ERα protein expression was upregulated(P<0.05) in uterine tissues. The mRNA expression of FP and EP4 in uterine tissues was significantly downregulated(P<0.01), and the mRNA expression of EP1, EP3, and OTR showed a decreasing trend. The untargeted metabolomics results showed that 10 differential metabolites were restored in the plasma of PD rats after Leonuri Herba treatment. The results indicate that Leonuri Herba is effective in the prevention and treatment of PD, and the underlying mechanism may be attributed to the regulation of PGs synthesis and corresponding receptor binding.
Humans ; Rats ; Female ; Animals ; Estrogen Receptor alpha ; Oxytocin ; Dysmenorrhea/metabolism* ; Cyclooxygenase 2 ; Dinoprostone ; RNA, Messenger/metabolism* ; Dinoprost

A study was conducted on rural women of childbearing age aged 20-49 who underwent the National Free Preconception Health Examination Project (NFPHEP)in Yunnan Province from 2013 to 2019. Descriptive analysis was conducted to determine the negative rate of IgG antibodies against the rubella virus and its differences among various socio-demographic characteristics. Among the 1 511 203 study subjects, the negative rate of IgG antibodies against the rubella virus was 24.36%. Only 2.64% of the population had received rubella virus vaccine. The negative rate of IgG antibodies among rural childbearing-age women in the preconception period in Yunnan Province decreased with age and educational level (P_trend<0.001). The negative rate of IgG antibodies in ethnic minority women of childbearing age in the preconception period (25.19%) was higher than that of Han women (23.88%). Among the 22 ethnic groups with over 1 000 participants, the negative rates of IgG antibodies in women of childbearing age from the Blang (32.85%), Bouyei (31.03%), Zhuang (31.01%), and Miao (30.70%) ethnic groups were higher than those of women from other ethnic groups. Among the 16 states (cities) in Yunnan Province, the negative rate of IgG antibodies in pregnant women of childbearing age in Wenshan Zhuang and Miao Autonomous Prefecture (38.06%) and Lincang City (32.63%) was higher than that in other states (cities). The negative rate of serum IgG antibodies in women who reported having received rubella virus vaccine (18.60%) was lower than that in other non-vaccinated populations (24.52%). The proportion of rural women of childbearing age in Yunnan Province who were susceptible to the rubella virus before pregnancy was still high. It is necessary to promote rubella vaccination among people susceptible to rubella, especially pregnant women, to prevent rubella virus infection and reduce the incidence rate and disease burden of rubella people.
Pregnancy ; Female ; Humans ; Rubella virus ; Immunoglobulin G ; Ethnicity ; Minority Groups ; China ; Rubella/prevention & control* ; Antigens, Viral ; Vaccines

A study was conducted on rural women of childbearing age aged 20-49 who underwent the National Free Preconception Health Examination Project (NFPHEP)in Yunnan Province from 2013 to 2019. Descriptive analysis was conducted to determine the negative rate of IgG antibodies against the rubella virus and its differences among various socio-demographic characteristics. Among the 1 511 203 study subjects, the negative rate of IgG antibodies against the rubella virus was 24.36%. Only 2.64% of the population had received rubella virus vaccine. The negative rate of IgG antibodies among rural childbearing-age women in the preconception period in Yunnan Province decreased with age and educational level (P_trend<0.001). The negative rate of IgG antibodies in ethnic minority women of childbearing age in the preconception period (25.19%) was higher than that of Han women (23.88%). Among the 22 ethnic groups with over 1 000 participants, the negative rates of IgG antibodies in women of childbearing age from the Blang (32.85%), Bouyei (31.03%), Zhuang (31.01%), and Miao (30.70%) ethnic groups were higher than those of women from other ethnic groups. Among the 16 states (cities) in Yunnan Province, the negative rate of IgG antibodies in pregnant women of childbearing age in Wenshan Zhuang and Miao Autonomous Prefecture (38.06%) and Lincang City (32.63%) was higher than that in other states (cities). The negative rate of serum IgG antibodies in women who reported having received rubella virus vaccine (18.60%) was lower than that in other non-vaccinated populations (24.52%). The proportion of rural women of childbearing age in Yunnan Province who were susceptible to the rubella virus before pregnancy was still high. It is necessary to promote rubella vaccination among people susceptible to rubella, especially pregnant women, to prevent rubella virus infection and reduce the incidence rate and disease burden of rubella people.
Pregnancy ; Female ; Humans ; Rubella virus ; Immunoglobulin G ; Ethnicity ; Minority Groups ; China ; Rubella/prevention & control* ; Antigens, Viral ; Vaccines

Aim To determine the antiviral and anti-inflammatory effects of the recommended prescription for COVID-2019-lung-spleen qi deficiency(4-1)against in vitro infection of SARS-CoV-2 and common coronaviruses.Methods The main chemical substances of 4-1 were analyzed by LC-MS.The toxicity and antiviral effects of of 4-1 were detected by MTT and by CPE assay, respectively.The viral loads in cell supernatant and the expression of inflammatory factors induced by viral infection were determined by qRT-PCR.Results The recommended prescription 4-1 contained 94 chemical compounds, including flavonoids, steroids, sesquiterpenoids, and so on.The range of selection indexes for SARS-CoV-2 and common coronaviruses was 8.44±0.4952.26±2.3.This prescription could inhibit the proliferation of SARS-CoV-2, the expression of ACE2 and S mRNA, and down regulate IL-1α and CCL-5/RANTES at 10, 5, and 2.5 g•L-1 doses.Further, at doses of 20, 10 and 5 g•L-1, it could inhibit the proliferation of three common coronaviruses and suppress the overexpression of IL-6, CXCL-8/IL-8, CXCL-10/IP-10, TNF-α, IFN-α, CCL-2/MCP-1, MIG and CCL-5/RANTES induced by OC43/229E infection.The inhibitory effects were dose-dependent.Conclusions The prescription 4-1 has antiviral and anti-inflammation effects against multiple coronaviruses.This study provides the research basis for the treatment of common respiratory viral infections and emerging infectious diseases such as COVID-19 by using traditional Chinese medicine.

Inflammatory bowel disease (IBD) is an, intractable inflammatory autoimmune disease characterized by T-cell infiltration to the colon. Mesenchymal stem cells (MSCs), owing to their immunosuppressive capabilities, have the potential to rescue IBD. But the therapeutic effectiveness of MSCs is sometime thwarted by their variable immunomodulatory ability in vivo. In the present study, we produced engineered MSCs that secrete interleukin10 (IL-10) and evaluated their therapeutic potential in IBD mouse model. The MSCs maintained the phenotype and cell proliferation rate after overexpression of IL-10 by lentivirus (LV) infection. Immune cells and MSCs in vitro co-culture systems exhibited that relative to unmodified MSCs, immune cells co-cultured with IL-10-overexpressing MSCs had significantly lower numbers of T helper 1 cells (Th1) and T helper 17 cells (Th17) (P<0.05), the content of TNF-α in the supernatant of macrophage cells co-cultured with MSCs overexpressing IL-10 was significantly decreased (P<0.0001). Tail vein injection of the IL-10 overexpressing MSCs achieved a better therapeutic effect in the dextran sodium sulfate (DSS) induced colitis mouse model than that of the unmodified MSCs, as indicated by colon length, disease activity index (DAI) and colonic cytokines expression. The experimental results were statistically different (P>0.05). Overall, LV induced MSCs overexpressing IL-10 might be a promising alternative therapeutic option for the treatment of IBD.

Antitubercular Agents/therapeutic use* ; China/epidemiology* ; Drug Resistance, Multiple, Bacterial ; Extensively Drug-Resistant Tuberculosis ; Humans ; Kanamycin Resistance ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; Ofloxacin/pharmacology* ; Tuberculosis, Multidrug-Resistant/epidemiology*