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.

Hypercholesterolemia is pathologically characterized by abnormal accumulation of low-density lipoprotein cholesterol,which is closely associated with metabolic dysfunction-associated fatty liver disease and increased cardiovascular risks.Hepatocytes maintain cholesterol homeostasis through LDL receptor-mediated uptake and esterification storage mechanisms.However,chronic cholesterol overload induces mitochondrial dysfunction,reactive oxygen species accumulation,and endoplasmic reticulum stress,leading to hepatocyte injury.Moreover,systemic hypercholesterolemia disrupts gut microbiota balance and impairs short-chain fatty acid and ketone metabolism,exacerbating metabolic disturbances and aggravating hepatic injury through enhanced metabolic stress.In this article,we review the advance of studies on hypercholesterolemia in recent years and summary its association with hepatic injury,which can provide theoretical support for further research.

Objective:To investigate the differences in resistance to chemical disinfectants between bacteriophage Phi-X174 and disinfection effect indicator microorganisms, Staphylococcus aureus and Escherichia coli. Methods:Three commonly used disinfectants, including chlorine-containing disinfectants, alcohol-based disinfectants and quaternary ammonium salt disinfectants, were selected to analyze the differences in resistance of phage Phi-X174, Staphylococcus aureus and Escherichia coli by using the quantitative sterilization test of suspension. Results:The 250 mg/L sodium dichloroisocyanurate solution treated for 10 minutes yielded log reduction values of 3.39, 7.25 and 7.37 against phage Phi-X174, Staphylococcus aureus and Escherichia coli respectively. The 40% ethanol for 1 minute yielded log reduction values of 0.03, 2.46 and 7.30 against phage Phi-X174, Staphylococcus aureus and Escherichia coli, respectively. The 100 mg/L benzalkonium chloride for 10 minutes yielded log reduction values of 1.41, 6.84 and 0.93 for phage Phi-X174, Staphylococcus aureus and Escherichia coli, respectively. Conclusion:Phage Phi-X174 has stronger resistance to chlorine-containing disinfectants and alcohol-based disinfectants than Escherichia coli and Staphylococcus aureus. Its resistance to quaternary ammonium salt disinfectants is between that of Staphylococcus aureus and Escherichia coli.

Aim To study the mechanism of adipose mesenchymal stem cell exosomes(ASC-exo)inhibition of fluorescein isothiocyanate(FITC)-induced atopic dermatitis(AD).Methods The mouse age,extrac-tion method,and the concentration of a solution of typeⅠ collagen enzyme and other conditions were compared to study the effects on the morphology and quantity of adipose mesenchymal stem cells(ASCs)after extrac-ted.FITC-induced mouse model in vivo was estab-lished and different doses of ASC-exo were given to measure ear thickness,ear weight and ear scratching times of mice.HE staining was used to observe the pathological changes of ear tissue of mice.The non-toxicity of ASC-exo was detected.IgE,IL-5,IL-13 and other cytokines were detected by ELISA.The gene ex-pressions of TSLP,IL-33,occludin,Claudin-1(CLDN-1)and E-cadherin were detected by RT-qPCR.The protein expression was detected by immunohistochemis-try.Results An efficient method for extracting ASCs was established.Compared with the blank group,mice in the model group showed obvious AD symptoms.Compared with the model group,ASC-exo administra-tion group significantly reduced the number of ear scratches,epidermal thickening,inflammatory cell infil-tration and the secretion of Th2 cytokines IL-5 and IL-13.Meanwhile,ASC-exo administration group signifi-cantly increased the expression of structural proteins CLDN-1 and occludin in epithelial cells and decreased the expression of TSLP and IL-33.Conclusions ASC-exo can significantly improve Th2 skin inflamma-tion in AD mice,and its mechanism may be through in-creasing the expression of tight junction proteins and adhesion link protein in epithelial cells,repairing the skin barrier,and inhibiting the key promoters of allergy TSLP and IL-33.

Hypercholesterolemia is pathologically characterized by abnormal accumulation of low-density lipoprotein cholesterol,which is closely associated with metabolic dysfunction-associated fatty liver disease and increased cardiovascular risks.Hepatocytes maintain cholesterol homeostasis through LDL receptor-mediated uptake and esterification storage mechanisms.However,chronic cholesterol overload induces mitochondrial dysfunction,reactive oxygen species accumulation,and endoplasmic reticulum stress,leading to hepatocyte injury.Moreover,systemic hypercholesterolemia disrupts gut microbiota balance and impairs short-chain fatty acid and ketone metabolism,exacerbating metabolic disturbances and aggravating hepatic injury through enhanced metabolic stress.In this article,we review the advance of studies on hypercholesterolemia in recent years and summary its association with hepatic injury,which can provide theoretical support for further research.

Objective:To observe and analyze the correlation between ectopic foveal inner layer (EIFL) and the EIFL-based idiopathic epiretinal membrane (ERM) staging system and the anatomic and functional prognosis of ERM eyes post pars plana vitrectomy (PPV).Methods:A retrospective study. From January 1, 2020 to October 30, 2023, 345 eyes of 330 patients diagnosed with idiopathic ERM in Department of Ophthalmology of Peking University People's Hospital and treated with standard transciliary flat three-channel 25G PPV combined with ERM and internal limiting membrane exfoliation were included in the study. Among them, 96 were males (111 eyes) and 234 were females (234 eyes). The mean age was (66.8±7.7) years. All study eyes received standard three-port 25G PPV combined with ERM and internal limiting membrane peeling. All study eyes underwent best corrected visual acuity (BCVA) and optical coherence tomography (OCT) examinations. BCVA was performed using a standard logarithmic visual acuity chart and converted to logarithm of the minimum angle of resolution visual acuity for statistical analysis. EIFL thickness and central foveal thickness (CFT) on OCT were measured. ERM eyes were grouped into stage Ⅰ, Ⅱ, Ⅲ and Ⅳ according to ERM staging scheme based on EIFL; disorganization of the retinal inner layers (DRIL) of study eyes were assessed and grouped into no, mild and severe groups. The correlation between ERM staging as well as EIFL thickness and the anatomical and functional prognosis 6 months post-PPV were analyzed.Results:Among 345 study eyes, 12, 87, 174 and 72 eyes were stage Ⅰ-Ⅳ ERM respectively, 63 with no DRIL, 216 with mild DRIL and 66 with severe DRIL. Among the 153 eyes with macular edema, the edema subsided in 66 eyes (43.1%, 66/153) 6 months after the operation. Eighty-seven eyes (56.9%, 87/153) did not regress. The edema subsided 6 months after the operation was not significantly correlated with the ERM stage before the operation ( χ2=3.331, R=?0.145, P=0.304) or the degree of DRIL ( χ2=0.655, R=?0.108, P=0.445). The results of the correlation analysis showed that logMAR BCVA 6 months after the surgery was positively correlated with the degree of DRIL before the surgery ( Tau-b=0.236), ERM stage ( Tau-b=0.194), CFT ( r=0.383), and EIFL thickness ( r=0.317) ( P<0.05). There was no significant correlation with the thickness of the outer nuclear layer before the operation ( r=0.004, P>0.05). Preoperative ERM stage ( Tau-b=0.303, P<0.001) and DRIL severity ( Tau-b= 0.238, P=0.001) were positively correlated with CFT at 6 months after surgery. Conclusion:The ERM stage and EIFL thickness before the operation are positively correlated with logMAR BCVA and CFT 6 months after the operation.

Demethyleneberberine(DMB),one of the active me-tabolite of berberine,displays versatile pharmacological proper-ties.Numerous studies indicate diverse biological activities,such as actions against infection,inflammation,fibrosis,cancer cells,blood lipids,etc.It presents considerable potential in treating neurodegenerative diseases.This article provides a con-cise review of the pharmacological actions and mechanisms made in demethyleneberberine research to facilitate a deeper under-standing of its pharmacology and provide a scientific foundation for its prudent development.

Objective To investigate the effect of dorsal repulsive axon guidance protein(Draxin)on the migration of trunk neural crest cells during the early development of embryonic mouse spinal cord.Methods Immunohistochemistry and in situ hybridization were used to detect the expression characteristics of Draxin in early embryonic spinal cord(8 mice each group);In situ hybridization was used to detect the change of migration characteristics of trunk neural crest cells in early embryonic spinal cord of different types of mouse(5 mice each group);in vitro culture method was used to check the effect of Draxin on the migration characteristics of embryonic mouse trunk neural crest cells(16 mice each group).Resultsβ-galactosidase gene Z(LacZ)gene was introduced when Draxin gene was knocked out to produce Draxin gene knockout mice.β-galactosidase staining was used to detect LacZ gene expression in Draxin knockout embryonic mice,and the result showed that Draxin expression was observed in the spinal cord of early embryonic mice since 9.5 days(E9.5).Draxin expression was obvious in the embryonic mice spinal cord in E10.5 period.In situ hybridization was used to detect the expression of Draxin gene in the spinal cord of wild type embryonic mice,and the result further verified the obvious expression of Draxin in the early embryonic mice spinal cord in El0.5 period.Sox10 in situ hybridization was used to detect neural crest cell migration in the spinal cord of embryonic mice in E10.5 period.The result showed that segmental migration of neural crest cells in the early embryonic spinal cord of some Draxin knockout mice was delayed compared with the wild type mice.The effect of Draxin on the migration of wild type early embryonic mice trunk neural crest cells in vitro was tested.The result showed that Draxin reduced the migration distance of neural crest cells in vitro.Conclusion In the early developmental stage of embryonic spinal cord(E9.5-E10.5),neural crest cells migrated exuberant.At the same time,Draxin plays an important inhibitory function in the formation of the specific migration pathways of trunk neural crest cells by promoting neural crest cells migrating away from Draxin expressing regions.

RNA-based gene therapy has been widely used for various diseases, and extensive studies have proved that suitable delivery routes greatly help the development of RNA therapeutics. Identifying a safe and effective delivery system is key to realizing RNA therapeutics' clinical translation. Inhalation is a non-invasive pulmonary delivery modality that can enhance the retention of therapeutic agents in the lungs with negligible toxicity, thereby improving patient compliance. Inhaled RNA therapeutics are increasingly becoming an area of focus for researchers; however, only several clinical trials have explored inhaled delivery of RNA for pulmonary diseases. This review presents an overview of recent advances in inhaled delivery systems for RNA therapeutics, including viral and nonviral systems, highlighting state of the art regarding inhalation in the messenger RNA (mRNA) field. We also summarize the applications of mRNA inhalants in infectious and other lung diseases. Simultaneously, the research progresses on small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and different types of RNA are also discussed to provide new strategies for developing RNA inhalation therapy. Finally, we clarify the challenges inhaled RNA-based therapeutics face before their widespread adoption and provide insights to help advance this exciting field to the bedside.

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) cause a severe neurodevelopmental disorder, yet the impact of truncating mutations remains unclear. Here, we introduce the Cdkl5^492stop mouse model, mimicking C-terminal truncating mutations in patients. 492stop/Y mice exhibit altered dendritic spine morphology and spontaneous seizure-like behaviors, alongside other behavioral deficits. After creating cell lines with various Cdkl5 truncating mutations, we found that these mutations are regulated by the nonsense-mediated RNA decay pathway. Most truncating mutations result in CDKL5 protein loss, leading to multiple disease phenotypes, and offering new insights into the pathogenesis of CDKL5 disorder.
Animals ; Disease Models, Animal ; Mice ; Protein Serine-Threonine Kinases/deficiency* ; Mutation/genetics* ; Epileptic Syndromes/genetics* ; Humans ; Dendritic Spines/pathology* ; Spasms, Infantile/genetics* ; Male ; Seizures/genetics* ; Mice, Inbred C57BL