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 mechanism of Forsythiae Fructus-Lonicerae Japonicae Flos（FF） in the treatment of acute lung injury（ALI） by investigating the effects of FF on serum metabolomics of rats with ALI. MethodsThirty male SD rats were acclimated for 1 week， and 6 rats were randomly selected as the blank group. The other 24 rats were injected with lipopolysaccharide（LPS） solution by tracheal drip to establish an ALI model. After successful model establishment， the rats were randomly divided into the model group， the FF low-dose group（3.0 g·kg^-1）， the FF high-dose group（6.0 g·kg^-1）， and the dexamethasone group（5 mg·kg^-1）， with six rats in each group. The FF low- and high-dose groups and the dexamethasone group were received daily oral administration of the corresponding drug solution， and the blank group and the model group were gavaged with an equal amount of saline， treatment was administered continuously for 3 d. The pathological conditions of rat lung tissues were evaluated by hematoxylin-eosin（HE） staining， wet/dry mass ratio（W/D） of the lung tissues， and protein concentration in rat bronchoalveolar lavage fluid（BALF）. Metabolomic analysis of rat serum was performed by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， combined with multivariate statistical analysis， the potential biomarkers of FF in treating ALI were screened by variable importance in the projection（VIP） value>1， P<0.05 from t-test， and log₂fold change（FC）>1 or log₂FC<-1. Kyoto Encyclopedia of Genes and Genomes（KEGG） database combined with MetaboAnalyst were used for pathway analysis of the screened differential metabolites. The protein expression levels of sphingosine-1-phosphate（S1P）， phosphatidylinositol 3-kinase（PI3K）， protein kinase B1（Akt1）， and phosphorylated Akt1（p-Akt1） were examined by Western bolt. The expression levels of interleukin（IL）-6， IL-1β， and tumor necrosis factor（TNF）-α in BALF were detected by enzyme-linked immunosorbent assay（ELISA）. ResultsCompared with the blank group， rats in the model group showed ALI pathological features such as alveolar lumen dilatation， interstitial hemorrhage and massive inflammatory cell infiltration， and the protein concentration in BALF and W/D of the lung tissues were significantly elevated（P<0.01）. Compared with the model group， the low- and high-dose groups of FF as well as the dexamethasone group exhibited reduced pulmonary bronchial hemorrhage in rats， and the protein concentration in BALF and W/D were significantly decreased（P<0.05）， and the lung injury was significantly alleviated. Analysis of rat serum metabolomics revealed that FF downregulated 38 biomarkers. Pathway enrichment analysis showed that FF primarily exerted therapeutic effects through 7 key metabolic pathways， including arginine biosynthesis， sphingomyelin metabolism， alanine， aspartate and glutamate metabolism， taurine and hypotaurine metabolism， α-linolenic acid metabolism， niacin and nicotinamide metabolism， and retinol metabolism. The results of Western bolt and ELISA showed that， compared with the blank group， the model group exhibited significantly elevated expression levels of S1P， PI3K， Akt1 and p-Akt1 proteins in the lung tissues， as well as increased expression levels of IL-6， IL-1β and TNF-α in BALF（P<0.01）. Compared with the model group， the expression levels of the aforementioned indicators were significantly downregulated in the low- and high-dose FF groups as well as the dexamethasone group（P<0.05， P<0.01）. ConclusionFF may play a role in ALI by regulating amino acid metabolism and lipid metabolism， and its mechanism may be related to the inhibition of S1P/PI3K/Akt1 signaling pathway to attenuate the inflammatory response caused by ALI.

This study aims to explore the pharmacodynamic material basis of Jinbei Oral Liquid(JBOL) against idiopathic pulmonary fibrosis(IPF) based on serum pharmacochemistry and network pharmacology. The ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(UHPLC-Q-TOF-MS/MS) technology was employed to analyze and identify the components absorbed into rat blood after oral administration of JBOL. Combined with network pharmacology, the study explored the pharmacodynamic material basis and potential mechanism of JBOL against IPF through protein-protein interaction(PPI) network construction, "component-target-pathway" analysis, Gene Ontology(GO) functional enrichment, and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. First, a total of 114 compounds were rapidly identified in JBOL extract according to the exact relative molecular mass, fragment ions, and other information of the compounds with the use of reference substances and a self-built compound database. Second, on this basis, 70 prototype components in blood were recognized by comparing blank serum with drug-containing serum samples, including 28 flavonoids, 25 organic acids, 4 saponins, 4 alkaloids, and 9 others. Finally, using these components absorbed into blood as candidates, the study obtained 212 potential targets of JBOL against IPF. The anti-IPF mechanism might involve the action of active ingredients such as glycyrrhetinic acid, cryptotanshinone, salvianolic acid B, and forsythoside A on core targets like AKT1, TNF, and ALB and thereby the regulation of multiple signaling pathways including PI3K/AKT, HIF-1, and TNF. In conclusion, JBOL exerts the anti-IPF effect through multiple components, targets, and pathways. The results would provide a reference for further study on pharmacodynamic material basis and pharmacological mechanism of JBOL.
Drugs, Chinese Herbal/pharmacokinetics* ; Animals ; Tandem Mass Spectrometry ; Network Pharmacology ; Rats ; Chromatography, High Pressure Liquid ; Rats, Sprague-Dawley ; Male ; Idiopathic Pulmonary Fibrosis/metabolism* ; Humans ; Administration, Oral ; Protein Interaction Maps/drug effects* ; Signal Transduction/drug effects*

Objective The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains is exacerbating the global burden of tuberculosis (TB), highlighting the urgent need for new treatment strategies for TB. Methods The recombinant adenovirus vaccine expressing cyclic di-adenosine monophosphate (c-di-AMP) phosphodiesterase B (CnpB) (rAd-CnpB), was administered to normal mice via mucosal immunization, either alone or in combination with drug therapy, to treat Mtb respiratory infections in mice.Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of antibodies in serum and bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was performed to assess the transcription levels of cytokines interferon γ(IFN-γ) and interleukin 10(IL-10) in mouse lungs. Flow cytometry was used to determine the proportions of CD4⁺ and CD8⁺ T cell subsets in the lungs and spleens. ELISA was employed to measure the levels of cytokines IFN-γ, IL-2, IL-10, inflammatory factors IL-6, and tumor necrosis factor α (TNF-α) secreted by spleen cells following antigen stimulation. The bacteria loads in the lungs and spleens of Mtb-infected mice were enumerated by plate counting methods. Resluts Intranasal immunization with rAd-CnpB induced high titers of IgG in mouse serum and the production of IgG and IgA in BALF, along with alterations in T lymphocyte subsets in the lungs and spleens. Administration of rAd-CnpB, either alone or in combination with drugs, to Mtb-infected mice significantly increased serum IgG levels as well as IgA and IgG levels in BALF. rAd-CnpB immunization promoted the secretion of CnpB-specific cytokines and inflammatory factors by splenocytes in Mtb-infected mice. However, rAd-CnpB immunotherapy, either alone or combined with drugs, did not significantly affect the bacterial loads in the lungs and spleens of mice with Mtb respiratory infections. Conclusion Mucosal immunization with rAd-CnpB induced significant mucosal, humoral and cellular immune responses in mice, and significantly enhanced CnpB-specific cellular immune responses in Mtb-infected mice.
Animals ; Adenoviridae/immunology* ; Mycobacterium tuberculosis/genetics* ; Mice ; Female ; Phosphoric Diester Hydrolases/genetics* ; Tuberculosis Vaccines/administration & dosage* ; Tuberculosis/prevention & control* ; Mice, Inbred BALB C ; Cytokines ; Lung/microbiology* ; Immunization ; Bronchoalveolar Lavage Fluid/immunology* ; Immunity, Mucosal

OBJECTIVE: Cigarette smoking exacerbates the progression of pulmonary tuberculosis (TB). The role of tertiary lymphoid structures (TLS) in chronic lung diseases has gained attention; however, it remains unclear whether smoking-exacerbated lung damage in TB is associated with TLS. This study aimed to analyze the characteristics of pulmonary TLS in smokers with TB and to explore the possible role of TLS in smoking-related lung injury in TB. METHODS: Lung tissues from 36 male patients (18 smokers and 18 non-smokers) who underwent surgical resection for pulmonary TB were included in this study. Pathological and immunohistological analyses were conducted to evaluate the quantity of TLS, and chest computed tomography (CT) was used to assess the severity of lung lesions. The correlation between the TLS quantity and TB lesion severity scores was analyzed. The immune cells and chemokines involved in TLS formation were also evaluated and compared between smokers and non-smokers. RESULTS: Smoker patients with TB had significantly higher TLS than non-smokers ( P < 0.001). The TLS quantity in both the lung parenchyma and peribronchial regions correlated with TB lesion severity on chest CT (parenchyma: r = 0.5767; peribronchial: r = 0.7373; both P < 0.001). Immunohistochemical analysis showed increased B cells, T cells, and C-X-C motif chemokine ligand 13 (CXCL13) expression in smoker patients with TB ( P < 0.001). CONCLUSION Smoker TB patients exhibited increased pulmonary TLS, which was associated with exacerbated lung lesions on chest CT, suggesting that cigarette smoking may exacerbate lung damage by promoting TLS formation.
Humans ; Male ; Tuberculosis, Pulmonary/immunology* ; Middle Aged ; Tertiary Lymphoid Structures/pathology* ; Adult ; Lung/pathology* ; Smoking/adverse effects* ; Smokers ; Aged ; Tomography, X-Ray Computed

Background::Accurately and efficiently extracting microbial genomic sequences from complex metagenomic data is crucial for advancing our understanding in fields such as clinical diagnostics, environmental microbiology, and biodiversity. As sequencing technologies evolve, this task becomes increasingly challenging due to the intricate nature of microbial communities and the vast amount of data generated. Especially in intensive care units (ICUs), infections caused by antibiotic-resistant bacteria are increasingly prevalent among critically ill patients, significantly impacting the effectiveness of treatments and patient prognoses. Therefore, obtaining timely and accurate information about infectious pathogens is of paramount importance for the treatment of patients with severe infections, which enables precisely targeted anti-infection therapies, and a tool that can extract microbial genomic sequences from metagenomic dataset would be of help.Methods::We developed MetaGeneMiner to help with retrieving specific microbial genomic sequences from metagenomes using a k-mer-based approach. It facilitates the rapid and accurate identification and analysis of pathogens. The tool is designed to be user-friendly and efficient on standard personal computers, allowing its use across a wide variety of settings. We validated MetaGeneMiner using eight metagenomic samples from ICU patients, which demonstrated its efficiency and accuracy.Results::The software extensively retrieved coding sequences of pathogens Acinetobacter baumannii and herpes simplex virus type 1 and detected a variety of resistance genes. All documentation and source codes for MetaGeneMiner are freely available at https://gitee.com/sculab/MetaGeneMiner. Conclusions::It is foreseeable that MetaGeneMiner possesses the potential for applications across multiple domains, including clinical diagnostics, environmental microbiology, gut microbiome research, as well as biodiversity and conservation biology. Particularly in ICU settings, MetaGeneMiner introduces a novel, rapid, and precise method for diagnosing and treating infections in critically ill patients. This tool is capable of efficiently identifying infectious pathogens, guiding personalized and precise treatment strategies, and monitoring the development of antibiotic resistance, significantly impacting the diagnosis and treatment of severe infections.

ObjectiveSerum metabolomics of acute lung injury（ALI） in rats was conducted using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） to explore the similarities and differences in the mechanism of Qingqiao（harvested when the fruits of Forsythiae Fructus were initially ripe and still green in color） and Laoqiao（harvested when the fruits of Forsythiae Fructus were ripe） in the treatment of ALI. MethodA total of 24 SD male rats were acclimatized and fed for 1 week， 6 of them were randomly selected for the blank group and 18 for the experimental group. The ALI model was induced in the experimental group by tracheal intubation with lipopolysaccharide（LPS）. After successfully constructing the ALI model， these rats was randomly divided into model group， Qingqiao group and Laoqiao group， with 6 rats in each group. The Qingqiao and Laoqiao groups were administered orally once a day at a dose of 1.5 g·kg^-1， while the blank and model groups received an equivalent volume of saline for 3 consecutive days. The pathological conditions of rat lung tissues were comprehensively assessed by hematoxylin-eosin（HE） staining， wet-to-dry mass ratio（W/D） of lung tissues， and protein concentration in rat bronchoalveolar lavage fluid（BALF）. The levels of interleukin（IL）-6， IL-1β and tumor necrosis factor（TNF）-α in BALF were quantified using enzyme-linked immunosorbent assay（ELISA）. UPLC-Q-TOF-MS was used to identify and analyze the chemical compositions of Qingqiao and Laoqiao， and serum metabolomics of rats in each group was analyzed， combined with multivariate statistical analysis with variable importance in the projection（VIP） value>1， P<0.05 from t-test， and fold change（FC）≥1.5 or FC≤0.5 to screen the differential metabolites Qingqiao and Laoqiao for the treatment of ALI. The Kyoto Encyclopedia of Genes and Genomes（KEGG） database was used in combination with MetaboAnalyst for the metabolic pathway analysis of the screened differential metabolites. ResultCompared with the blank group， rats in the model group exhibited enlarged alveolar lumen， ruptured alveoli， interstitial hemorrhage， bronchial exudation of a large number of neutrophils and erythrocytes， and a significant increase in the protein concentration in the BALF and the W/D value of the lung tissues（P<0.01）. In contrast， compared with the model group， rats in the Qingqiao group and the Laoqiao group showed reduced bronchial hemorrhage in the lungs， and the protein concentration in the BALF and the W/D value of the lung tissues were significantly decreased（P<0.01）， the lung injury was significantly alleviated， but more obvious in the Qingqiao group. Compared with the blank group， the expression levels of IL-6， IL-1β and TNF-α in the BALF of the model group were significantly higher（P<0.01）. Additionally， compared with the model group， the expression levels of IL-6， IL-1β and TNF-α in the Qingqiao and Laoqiao groups were significantly lower（P<0.01）. The chemical composition analysis of Qingqiao and Laoqiao revealed that 63 components were detected in Qingqiao and 55 components were detected in Laoqiao， with 47 common components， 16 components unique to Qingqiao and 8 components unique to Laoqiao. Characterizing the differences in serum metabolomics in rats， 19 and 12 metabolites were called back by Qingqiao and Laoqiao， respectively. The metabolic pathway enrichment analysis showed that Qingqiao exerted its therapeutic effects by affecting 6 key metabolic pathways， including linoleic acid metabolism， phenylalanine metabolism， phenylalanine， tyrosine and tryptophan biosynthesis， glycerophospholipid metabolism， α-linolenic acid metabolism， and arachidonic acid metabolism， and Laoqiao exerted therapeutic effects by affecting 6 key metabolic pathways， including linoleic acid metabolism， arachidonic acid metabolism， sphingolipid metabolism， phenylalanine metabolism， ascorbate and aldarate metabolism， and glycerophospholipid metabolism. ConclusionQingqiao and Laoqiao have therapeutic effects on ALI， and Qingqiao is more effective. Both of them can play a therapeutic role in ALI by regulating amino acid metabolism and lipid metabolism， but the metabolic pathways affected by them are different.

Objective:To evaluate the efficacy of 6% hydroxyethyl starch (HES) 130/0.4 electrolyte solution for fluid therapy in the patients undergoing meningioma resection.Methods:Ninety-two American Society of Anesthesiologists Physical Status classification Ⅰ or Ⅱ patients of either sex, aged 18-64 yr, with body mass index of 18-30 kg/m 2, with expected operation duration>3 h, undergoing elective meningioma resection, were divided into 2 groups ( n=46 each) using a random number table method: lactated Ringer′s solution (LR) group and HES group. LR was infused throughout operation in group LR, and 6% HES was intravenously infused in group HES, with the maximum dose not exceeding 50 ml/kg, and the excess part was supplemented with LR. Goal-directed fluid therapy was used to maintain stroke volume variation<13% and mean arterial pressure 70-90 mmHg. Arterial blood gas analysis was performed immediately before anesthesia induction (T 0), when 1 000 and 2 000 ml of fluid were infused (T 1, 2), and at the end of surgery (T 3) to record electrolyte and acid-base balance indexes. Thromboelastogram was simultaneously monitored. The occurrence of electrolyte disorder, acid-base imbalance and abnormal coagulation function and consumption of norepinephrine were recorded. Patients were followed up at 3 and 7 days after surgery, and the Chinese quality of recovery-15 scores were recorded. The hospitalization time and occurrence of brain edema, pulmonary edema, nausea and vomiting were recorded. Results:In group L and group H, 4 cases and 6 cases were excluded due to prolonged operation time, and 42 cases and 40 cases were finally included, respectively. Compared with LR group, the plasma Na + concentration was significantly increased at T 3, the plasma Cl - concentration and pH value were increased at T 1-3, the plasma Ca 2+ concentration was decreased at T 2, 3, reaction time was increased at T 3, coagulation time was increased and maximum amplitude was decreasedat T 2, 3, and coagulation Angle was decreased at T 1-3( P<0.05). No electrolyte disorder and abnormal coagulation function was found in the two groups. There was no statistically significant difference in the consumption of norepinephrine, postoperative Chinese quality of recovery-15 score, length of hospital stay and incidence of alkalosis, pulmonary edema, brain edema, and nausea and vomiting between the two groups ( P>0.05). Conclusions:The efficacy of liquid therapy is comparable between HES and LR in the patients undergoing meningioma resection.

Objective To investigate the effect of artemisinin(ART)on the malignant biological behavior of colorectal cancer(CRC)cells stimulated by glucose and its mechanism.Methods The concentration gradients of 0,5,10,20,40 and 60 μmol/L of ART were used to treat the human colorectal cancer cell line SW480,and then the cell viability was detected by CCK-8.Cell apoptosis was detected by flow cytometry.Transwell was used to detect the cell migration and invasion.Western blot was used to detect the apoptosis,epithelial-mesenchymal transition(EMT)and Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)related proteins expression.Results Compared with the 0 μmol/L of ART,the viability of SW480 cells decreased under 5,10,20,40,60 μmol/L of ART treatment(P<0.05),and IC50 was 36.91 μmol/L.Therefore,the cells treated with 10,20 and 40 μmol/L of ART were as the low-dose,medium-dose and high-dose ART groups,the cells treated with 0 μmol/L of ART were as the control group,and the cells treated with 40 μmol/L of ART and 10 μmol/L of Coumermycin A1 were as the Coumermycin A1 group.Compared with the control group,the cell scratch wound healing rate,invasion ability,and expression levels of Bcl-2,N-cadherin,Vimentin,p-JAK2,and p-STAT3 in the low-dose ART group,the medium-dose ART group,and the high-dose ART group decreased obviously(P<0.05),while the apoptosis rate,and expression levels of Bax,Caspase-3 and E-cadherin increased(P<0.05).Compared with the high-dose ART group,the cell scratch wound healing rate,invasion ability,and expression levels of Bcl-2,N-cadherin,Vimentin,p-JAK2,and p-STAT3 in the Coumermycin A1 group increased obviously(P<0.05),while the apoptosis rate,and expression levels of Bax,Caspase-3 and E-cadherin decreased(P<0.05).Conclusion ART may inhibit the viability,migration,invasion and EMT of glucose-stimulated CRC cells and promote apoptosis by inhibiting the JAK2/STAT3 signaling pathway.

Objective To evaluate the clinical efficacy of invisible orthodontic appliances without brackets for the distal movement of maxillary molars to improve the ability of orthodontists to predict treatment outcomes.Methods Web of Science,Cochrane Library,Embase,PubMed,Wanfang Database,CNKI Database,and VIP Database were searched for studies investigating the efficacy of invisible orthodontic appliances for distal movement of maxillary molars in adult patients and published from database inception to August 1,2023.A total of three researchers screened the studies and evaluated their quality and conducted a meta-analysis of those that met quality standards.Results This study included 13 pre-and postcontrol trials with a total sample size of 281 patients.The meta-analysis revealed no sig-nificant differences in the sagittal or vertical parameters of the jawbone after treatment when compared with those before treatment(P＞0.05).The displacement of the first molar was MD=-2.34,95％CI(-2.83,-1.85);the displacement was MD=-0.95,95％CI(-1.34,-0.56);and the inclination was MD=-2.51,95％CI(-3.56,-1.46).There was a statistically significant difference in the change in sagittal,vertical,and axial tilt of the first molar before and after treatment.After treatment,the average adduction distance of the incisors was MD=-0.82,95％CI(-1.54,-0.09),and the decrease in lip inclination was MD=-1.61,95％CI(-2.86,-0.36);these values were significantly different from those before treat-ment(P＜0.05).Conclusion Invisible orthodontic appliances can effectively move the upper molars in a distal direc-tion and control the vertical position of the molars.When the molars move further away,there is some degree of com-pression and distal tilt movement,which is beneficial for patients with high angles.The sagittal movement of incisors is beneficial for improving the patient's profile.