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.

Achieving periodontal regeneration in class III furcation defects is challenging. Many studies have applied growth factors to periodontal defects, including fibroblast growth factors (FGFs), which demonstrate angiogenic activity and mitogenic ability. This study aimed to evaluate periodontal regeneration following the application of FGF-2 to deproteinized bovine bone mineral (DBBM) in surgically created supra-alveolar class III furcation defects of the mandibular premolars of beagles. The defects were divided into the control, DBBM, and FGF/DBBM groups. For the control group, only root planing was performed. For the DBBM group, only DBBM particles were implanted into the furcation. For the FGF/DBBM group, DBBM was soaked with 0.3% FGF-2 solution, and FGF-2/ DBBM was then positioned into the furcation. After 8 weeks, the dogs were euthanized. The micro-computed tomography analysis revealed that the changes in the bone volume of the furcation area were significantly greater in the FGF/DBBM group than in the DBBM group. In the histomorphometric analysis, the area of the newly formed bone was significantly greater in the FGF/DBBM group than in the DBBM or control group. The cementum extension was significantly longer in the FGF/DBBM or DBBM group than in the control group. The epithelial area was significantly less in the FGF/DBBM group than in the DBBM or control group. The application of FGF combined with DBBM to a class III defect enhanced the regeneration of periodontal tissues and increased the healing rate. This finding indicates that FGF-2 combined with DBBM can be applied to class III defects clinically.

GPR126, also known as ADGRG6, is one of the most deeply studied aGPCRs. Initially, GPR126 was thought to be a receptor associated with muscle development and was primarily expressed in the muscular and skeletal systems. With the deepening of research, it was found that GPR126 is expressed in multiple mammalian tissues and organs, and is involved in many biological processes such as embryonic development, nervous system development, and extracellular matrix interactions. Compared with other aGPCRs proteins, GPR126 has a longer N-terminal domain, which can bind to ligands one-to-one and one-to-many. Its N-terminus contains five domains, a CUB (complement C1r/C1s, Uegf, Bmp1) domain, a PTX (Pentraxin) domain, a SEA (Sperm protein, Enterokinase, and Agrin) domain, a hormone binding (HormR) domain, and a conserved GAIN domain. The GAIN domain has a self-shearing function, which is essential for the maturation, stability, transport and function of aGPCRs. Different SEA domains constitute different GPR126 isomers, which can regulate the activation and closure of downstream signaling pathways through conformational changes. GPR126 has a typical aGPCRs seven-transmembrane helical structure, which can be coupled to Gs and Gi, causing cAMP to up- or down-regulation, mediating transmembrane signaling and participating in the regulation of cell proliferation, differentiation and migration. GPR126 is activated in a tethered-stalk peptide agonism or orthosteric agonism, which is mainly manifested by self-proteolysis or conformational changes in the GAIN domain, which mediates the rapid activation or closure of downstream pathways by tethered agonists. In addition to the tethered short stem peptide activation mode, GPR126 also has another allosteric agonism or tunable agonism mode, which is specifically expressed as the GAIN domain does not have self-shearing function in the physiological state, NTF and CTF always maintain the binding state, and the NTF binds to the ligand to cause conformational changes of the receptor, which somehow transmits signals to the GAIN domain in a spatial structure. The GAIN domain can cause the 7TM domain to produce an activated or inhibited signal for signal transduction, For example, type IV collagen interacts with the CUB and PTX domains of GPR126 to activate GPR126 downstream signal transduction. GPR126 has homology of 51.6%-86.9% among different species, with 10 conserved regions between different species, which can be traced back to the oldest metazoans as well as unicellular animals.In terms of diseases, GPR126 dysfunction involves the pathological process of bone, myelin, embryo and other related diseases, and is also closely related to the occurrence and development of malignant tumors such as breast cancer and colon cancer. However, the biological function of GPR126 in various diseases and its potential as a therapeutic target still needs further research. This paper focuses on the structure, interspecies differences and conservatism, signal transduction and biological functions of GPR126, which provides ideas and references for future research on GPR126.

Knee osteoarthritis (KOA) is a prevalent degenerative joint disease characterized by synovial inflammation, cartilage loss. Often manifesting as joint pain and limited mobility, it severely affects the quality of life of patients. Traditional treatment methods such as pharmacological injections and surgical interventions primarily aim to alleviate symptoms but have limited effects on cartilage repair. Human umbilical cord mesenchymal stem cells (hUC-MSCs), due to their anti-inflammatory and chondrogenic capabilities, is considered a new hope for the treatment of KOA. This article synthesizes the latest research findings from both domestic and international sources to discuss the theoretical basis for the clinical application of hUC-MSCs in treating KOA, clinical study design, and efficacy evaluation. It also addresses the challenges in the clinical application of hUC-MSCs and explores future directions, in the hope of providing feasible theoretical support for the treatment of KOA with hUC-MSCs.

ObjectiveThis study employed the scoping review method to systematically retrieve and analyze the basic information and clinical research evidence of expensive Chinese patent medicines （CPMs）， aiming to provide a basis for future related research and clinical applications. MethodsEight Chinese and English databases were systematically searched for the clinical research evidence on expensive CPMs. ResultsEleven expensive CPMs （Angong Niuhuang Wan， Jufang Zhibao Wan， Suhexiang Wan， Pien Tze Huang， Niuhuang Qingxin Wan， Qinggong Shoutao Wan， Compound Realgar Natural Indigo Tablets， Xihuang Wan， Dingkun Wan， Babao Wan， and Guilingji Capsules） were selected. A total of 365 related studies were included in this review， comprising 331 clinical studies （of which 291 were randomized controlled trials）， 30 systematic reviews and Meta-analyses， 3 expert consensus， and 1 rapid health technology assessment. Among the 11 CPMs， 2（Angong Niuhuang Wan and Jufang Zhibao Wan） had a daily price over 500 yuan. The famous and precious Chinese medicinal materials involved included Moschus （frequency of 7）， Bovisc Alculus （7）， and Borneol （5）. The dosage forms included pills， capsules， oral liquid， tablets， and lozenges. The diseases treated by these CPMs mainly included malignant tumors， cerebrovascular diseases， gynecological diseases， and hepatobiliary system diseases. The sample sizes of the clinical studies were mainly concentrated within the range of 51-100 cases， and the main control form was CPM + basic Western medicine treatment vs. basic Western medicine treatment. The 331 clinical studies reported a total of 44 adverse events occurred， of which 36 were determined to be adverse reactions. ConclusionThe scarcity of raw materials leads to the high prices of expensive CPMs. The difficulty of conducting clinical research and the critical and severe cases treated lead to a lack of clinical research evidence with large sample sizes. The uneven distribution of existing studies， incomplete information on medicine package， and non-standard clinical research designs remain to be addressed in the future.

Objective To analyze the incidence and disease burden of substance use disorder(SUD)in China from 1990 to 2019,to evaluate the impact of different ages,periods and birth cohorts on the disease burden of SUD,and to predict disease burden of SUD from 2020 to 2034,so as to provide strategies for the prevention of SUD.Methods Based on the Global Burden of Disease Study 2019(GBD 2019)database,the disease burden was described by incidence,years of life lost(YLLs),years lived with disability(YLDs)and disability-adjusted life years(DALYs).The Joinpoint regression model was used to analyze the trend of standardized incidence and standardized DALYs rate of SUD.Based on the age-period-cohort model,the age,period and cohort effects of SUD were discussed.The grey prediction model GM(1,1)was used to fit the trend of the incidence and standardized incidence of SUD and the trend of disease burden,and to predict the incidence and disease burden of SUD in 2020-2034.Results From 1990 to 2019,the standardized incidence of SUD of amphetamines[average annual percentage change(AAPC)=-0.9％]and cocaine(AAPC=-0.5％)in China showed a downward trend(P＜0.001),and the standardized incidence of SUD of cannabis(AAPC=0.9％)showed an increasing trend year by year(P＜0.001).The trend of standardized incidence of opioid abuse disorders was not obvious(P＞0.05).The DALYs rate caused by the 4 SUD showed a decreasing trend year by year(AAPCamphetamines=-2.2％,AAPCcocaine=-1.5％,AAPCcannabis=-1.0％,AAPCopioids=-1.0％,P＜0.001).The results of age-period-cohort effect showed that the peak incidence of amphetamine,cocaine,cannabis and opioid use disorders was in the 25-30 age group.The DALYs rate caused by cannabis SUD increased with age,while the DALYs rates of amphetamines,cocaine and opioids SUD reached the peak in the 25-29,30-34 and 35-39 age groups,respectively.The results of period effect showed that the risk of SUD in propylamines,cocaine and cannabis decreased first and then increased,while the risk of SUD in opioids increased and then decreased and increased again.The results of birth cohort effect showed that the risk of SUD of amphetamines,cocaine and opioids showed a decreasing trend as a whole except for a small fluctuation in individual birth cohorts.The risk of DALYs rate caused by SUD of amphetamines,cocaine and opioids showed a decreasing trend as a whole,while the risk of DALYs rate caused by SUD of cannabis showed an increasing trend year by year.The prediction results showed that the incidence of SUD of amphetamines,cocaine and opioids showed a downward trend from 2020 to 2034,and the incidence of SUD of cannabis showed a fluctuating upward trend.The DALYs attributed to SUD of amphetamines,cocaine,cannabis and opioids showed a decreasing trend year by year.Conclusion The disease burden of SUD in China is decreasing year by year in the future.The incidence and disease burden are affected by age effect,period effect and cohort effect to varying degrees.Early prevention and effective intervention are the key measures to control SUD.

Vascular calcification is a highly regulated process of ectopic calcification in cardiovascular system while no effective intervention can be clinically performed up to date.As vascular calcification undergoes a common regulatory mechanism within bone formation,bone morphogenetic protein 7(BMP-7)main-tains contractile phenotype of vascular smooth muscle cells and further inhibits vascular calcification via promoting the process of osteoblast differentiation,reducing ectopic calcification pressure by increasing bone formation and reducing bone resorption.This work systematically reviews the role of BMP-7 in vascular calcifi-cation and the possible mechanism,and their current clinical application as well.The current proceedings may help develope early diagnostic strategy and therapeutic treatment with BMP-7 as a new molecular marker and potential drug target.The expec-tation could achieve early prevention and intervention of vascular calcification and improve poor prognosis on patients.

Aim To explore the mechanism of modified Wuzi Yanzong pill to reduce testicular toxicity of Tripterygium wilfordii by means of network pharmacolo-gy.Methods The effective chemical composition and target of modified Wuzi Yanzong Pill to reduce testicu-lar toxicity of Tripterygium wilfordii were analyzed by using multiple databases to understand the mechanism of action.Results The first five compounds of modi-fied Wuzi Yanzong pill are quercetin,sucrose,kaempferol,galactose and ellagic acid,respectively.The first five targets were TP53,SRC,EP300,MAPK3,STAT3;KEGG pathway enrichment analysis mainly concentrated in cancer pathway,lipid and atherosclero-sis,hepatitis B,apoptosis,diabetes complications AGE-RAGE signaling pathway,PI3K-Akt signaling pathway and other signaling pathways.Conclusions The ac-tive components of modified Wuzi Yanzong pill can re-duce the testicular toxicity of Tripterygium wilfordii mainly by interfering with TP53,SRC,EP300,MAPK3,STAT3 and other targets,regulating apoptosis,PI3K-Akt signaling pathway,diabetes complications AGE-RAGE signaling pathway,lipid metabolism and other pathways.

Objective:To standardize the bacterial endotoxin testing criteria for zoledronic acid injection and estab-lish a detection method using recombinant factor C(rFC).Methods:The gel-clot method(BET)was utilized to test 13 batches of zoledronic acid injection from national supervision and random inspection.Interference tests were conducted on zoledronic acid injections from three manufacturers at different concentrations(500,100,50,25 μg·mL-1)using rFC test kits from two manufacturers.Results:Detection was performed for the specification of 100 mL∶5 mg and other specifications according to＜0.50 EU per 1 mL and＜5.0 EU per 1 mg,respectively,and all results met the criteria.The recovery rate for 25 μg·mL-1 using rFC kits from both manufacturers ranged between 50％and 200％.Validation of rFC.Methods:Eight batches of zoledronic acid injection were validated at 25 μg·mL-1,five batches of zoledronic acid injection(100 mL∶5 mg)were validated at 0.5 EU·mL-1,and all recovery rates were between 50％and 200％.Conclusion:The bacterial endotoxin testing method for zoledronic acid injection can be established as follows:for large volume injection products with 100 mL or more,each 1 mL should contain less than 0.50 EU of endotoxin(following the Chinese Pharmacopoeia 2020,general chapter 1143).For other specifications,each 1 mg of zoledronic acid should contain less than 10.0 EU of endotoxin.The rFC test kit method for bacterial endotoxins involved diluting the sample with water for bacterial endotoxin testing to contain 25 μg of zoledronic acid per 1 mL,or performing 1∶1 dilution for large volume injections(100 mL∶5.0 mg specifi-cation),with recovery rate between 50％and 200％as per the kit instructions.

Objective To explore the clinical efficacy and safety of early surgical treatment for spinal thoracolumbar fracture without nerve injury.Methods The clinical data of 80 patients with spinal thoracolumbar fracture without nerve injury who were admitted to the department of spinal surgery in our hospital were retrospectively analyzed.According to the different operation timing,those who underwent surgery within 72 hours after fracture were included in the early operation group(n=41),and those who underwent surgery 72 hours to 2 weeks after fracture were included in the elective operation group(n=39).All operations were performed through the Wiltse approach for short-segment pedicle screw fixation on the injured vertebra.The operation time,intraoperative blood loss,hospital stay and incidence of complication of the two groups were compared.The visual analogue scale(VAS)scores,Oswestry disability index(ODI),compression rate of the anterior edge height of the injured vertebra,and the Cobb angle in the sagittal position of the injured vertebra before surgery,1 week after surgery and 1 year after surgery were compared between the two groups.The improvement rates of the anterior edge height compression and the Cobb angle in the sagittal position of the injured vertebra 1 week and 1 year after surgery were compared between the two groups.Results There was no significant difference in the operation time,intraoperative blood loss or total incidence of complications between the two groups(P>0.05).The hospital stay in the early operation group was shorter than that in the elective operation group,and the difference was statistically significant(P<0.05).The VAS scores and ODI 1 week and 1 year after surgery of the two groups were better than those before surgery,and the differences were statistically significant(P<0.05).There was no significant difference in the VAS scores or ODI at each time point before and after surgery between the two groups(P>0.05).The compression rate of the anterior edge height and Cobb angle in the sagittal position of the injured vertebra 1 week and 1 year after surgery in the two groups were lower/smaller than those before surgery,with statistically significant differences(P<0.05).There was no statistically significant difference in the compression rate of the anterior edge height or Cobb angle before surgery in the sagittal position of the injured vertebrae between the two groups(P>0.05).The compression rate of the anterior edge height and Cobb angle in the sagittal position of the injured vertebra 1 week and 1 year after surgery in the early operation group were lower/smaller than those in the elective operation group,and the differences were statistically significant(P<0.05).The improvement rates of the anterior edge height compression and the Cobb angle in the sagittal position of the injured vertebra 1 week and 1 year after surgery in the early operation group were better than those in the elective operation group,and the differences were statistically significant(P<0.05).Conclusion Early surgical treatment for spinal thoracolumbar fracture without nerve damage is safe,it can significantly shorten hospitalization time,obtain good fracture reduction quality and definite therapeutic effects.However,a comprehensive preoperative evaluation of the patients' condition is necessary to ensure surgical safety.