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.

Cancer stem cell(CSC)are the"seed"cells in the occurrence,development,metastasis and recurrence of colorectal cancer.Targeted killing of CSC provides a new target for anti-colorectal cancer therapy.Ferroptosis is an iron-dependent cell death mode due to the abnormal accumulation of intracellular i-ron ions,which results in the massive reactive oxygen species(ROS)and lipid peroxides,leading to cell death.Studies have shown that cancer stem cells are more enriched in iron ions than non-CSC,which provides a new perspective for targeting ferropto-sis in cancer stem cells against colorectal cancer.This article re-views the research progress of inducing CSC ferroptosis in the treatment of colorectal cancer,such as targeted regulation of SLC7A11 expression in CSC,chelating iron in CSC lysosomes,targeting CSC phenotypic plasticity,reversing CSC iron homeo-stasis,and targeting CSC lipid droplet metabolism induce CSC ferroptosis,which provides new ideas for anti-tumor therapy.

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.

Aim To investigate the effect of recombi-nant glycoprotein hormone β5/α2(rCGH)on lipolysis in 3T3-L1 adipocytes,and explore the underlying mechanism.Methods 3T3-L1 preadipocytes were cultured and induced to differentiate into mature adipo-cytes,then treated with different concentrations of rCGH for 24 h in vitro.Cell viability of 3T3-L1 adipo-cytes was evaluated by CCK-8 assay,the levels of in-tracellular triglyceride(TG)and glycerol in the culture supernatant were measured by enzymatic method,and the changes of lipid droplets were observed by oil red O staining.The expression levels of HSL and ATGL lipo-lytic proteins in adipocytes were detected by Western blot.To carry out the intervention experiment with dif-ferent concentrations of rCGH with or without the PKA inhibitor,H89,on the mature 3T3-L1 adipocytes,the cultured cells were divided into the control group,H89 pre treatment group,1 μmol·L-1 rCGH group,and(1 μmol·L-1 rCGH+H89)combined intervention group.The contents of intracellular TG and free glycer-ol were measured by enzymatic method,and the ex-pression of CREB and lipolysis-related proteins was de-tected using Western blot.Results Different concen-trations of rCGH(0.25,0.5,1,and 2 μmol·L-1)had no significant effect on the cell viability of adipo-cytes(P＞0.05).Compared with the control group,the treatment with rCGH significantly decreased the size of lipid droplets and intracellular TG content,while significantly elevated glycerol concentration in cell supernatant.rCGH treatment also stimulated the protein expression of p-HSL,ATGL,and p-PKA.In addition,the addition of a PKA inhibitor,H89,atten-uated the effects of rCGH on free glycerol level,intra-cellular TG content,and the expression of p-HSL,p-PLIN1,and p-CREB.Conclusions rCGH enhances the lipolysis of 3T3-L1 adipocytes by up-regulating the activities of HSL,ATGL and PKA,promoting glycerol release,inhibiting TG synthesis and lipid accumula-tion,and its mechanism of action is related to the acti-vation of cAMP/PKA/CREB signaling pathway.

Aim To investigate the intracellular up-take and target protein binding characteristics of two Bruton's tyrosine kinase inhibitors(BTKi)with differ-ent selectivities to provide further insights into the mechanisms of drug off-target-related bleeding risk.Methods Ibrutinib(non-selective BTKi)and za-nubrutinib(selective BTKi)were used as study drugs.After incubation of MEC-1 cells and human platelets with drugs,the cellular thermal shift assay(CETSA)was combined with Western blot to obtain the melting curve and isothermal curve to analyze the binding char-acteristics of the two drugs with the target protein BTK.After incubation of MEC-1 cells and human platelets with drugs,the concentrations of the two drugs were detected by liquid chromatography-tandem mass spectrometry(LC-MS/MS)to analyze the intracellular uptake of the two drugs.Results CETSA analysis confirmed that zanubrutinib was more selective for the target protein BTK compared to ibrutinib.LC-MS/MS analysis showed that both drugs were uptaken intracel-lularly by MEC-1 cells and platelets in a concentration-dependent manner.Conclusions While BTKi targe-ting BTK to B lymphocytes exerts therapeutic effects,off-target effects on platelets due to differences in their intracellular uptake,and target-binding characteristics may be one of the reasons for the differences in bleed-ing risk across selective BTKi.

Objective To observe the residual of lumbago and leg pain with contained type(CT)and non-contained type(NCT)lumbar disc herniation(LDH)after transforaminal endoscopic treatment,and to explore the role of hypoxia-inducible factor-1α(HIF-1α)and transient receptor potential vanillate 1(TRPV1)pathway.Methods A total of 68 single-segment LDH patients were selected from July 2021 to October 2022,including 44 males and 24 females;aged 26 to 67 years old with an av-erage of(43.63±11.94)years old;course of disease was 4 to 36(18.91±10.34)months;body mass index was(24.45±4.00)kg·m-2;there were 7 cases of L3.4 segments,32 cases of L4,5 segments,and 29 cases of L5S1 segments.All of them were per-formed with percutaneous intervertebral endoscopic extraction of nucleus pulposus and were divided into contained group(CT group)and non-contained group(NCT group)with 34 cases respectively according to the integrity of outer layer of fibrous an-nulus observed during operation.A total of 17 patients who underwent open surgery for scoliosis or vertebral fracture were se-lected as control group,including 12 males and 5 females;aged 21 to 65 years old with an average of(39.41±12.80)years old;body mass index was(24.86±4.11)kg·m-2.The relative mRNA expression quantity of HIF-1α,TRPV1 in nucleus pulposus were measured by quantitative real-time PCR.The contents of neurokinin 1 receptor(NK1R),nerve growth factor(NGF),vascular endothelial growth factor(VEGF)in nucleus pulposus and the serum substance P(SP)and calcitonin gene-related peptide(CGRP)were detected by enzyme linked immunosorbent assay(ELISA).The threshold of lumbar tenderness was de-tected by a pressure pain meter.The degree of lumbago and lumbar function were evaluated by visual analog scale(VAS)and Oswestry disability index(ODI)separately.The residual rate of postoperative lumbago and leg pain was assessed.Results The mRNA relative expression quantity of HIF-1α and TRPV1,and the contents of NK1R,NGF and VEGF in nucleus pulposus,and the levels of serum SP and CGRP before surgery in the NCT group were higher than those in the CT group(P＜0.05),and those in the CT group were higher than the control group(P＜0.05).At day 7 after surgery,the serum SP and CGRP levels,lum-bago and leg pain VAS scores and lumbar ODI index in two LDH groups were lower than before surgery(P＜0.05),and those in the NCT group were higher than the CT group(P＜0.05),and the threshold of lumbar tenderness in the NCT group was lower than the CT group(P＜0.05).The differences of lumbago and leg pain VAS scores,lumbar ODI index and lumbar tenderness threshold between preoperative and postoperative 7 days in the NCT group were lower than those in the CT group(P＜0.05).The residual rate of lumbago and leg pain at 7 days after surgery in the NCT group was higher than that in the CT group(P＜0.05).Conclusion HIF-1α and TRPV1 pathway promoted the excessive production of NGF,VEGF,NK1R in nucleus pulposus and serum neuropeptides SP and CGRP,which may lead to the higher residual rate of lumbago and leg pain with non-contained lumbar disc herniation postoperative.

Objective To compare the application effects of high-frequency electric surgical knife and scalpel in blepharoplasty.Methods A retrospective analysis was performed for 35 patients(70 eyes)who were admitted to the Shanghai Ninth People's Hospital,Shanghai JiaoTong University School of Medicine from January to July 2021,and one side eye was randomly selected as the high-frequency electric surgical knife group(operated by high-frequency electric surgical knife)and the other side eye as the scalpel group(operated by scalpel),with 35 eyes in each group.The effect of blepharoplasty in both eyes after surgery was evaluated.The swelling and ecchymosis of the patients were observed 1 day,1 week,1 month and 3 months after surgery.The Manchester scar scale(MSS)was used to evaluate the scarring of the patients 1 week,1 month and 3 months after surgery.The excised tissues were stained with HE staining and TUNEL staining to assess the degree of necrosis and the extent of thermal damage to the skin and muscle.Results There was no statistically significant difference in swelling,ecchymosis,pain or scarring after surgery between the two groups(P＞0.05).The eyelids of the high-frequency electrosurgical group showed coagulative necrosis of muscle tissue,and the scalpel group had almost no coagulative necrosis.There was statistically significant difference in the proportion of coagulable necrotic muscle after surgery between the two groups(P＜0.05),and the depth of thermal damage in the high-frequency electric surgical knife group was greater than that in the scalpel group(P＜0.05).Conclusion The high-frequency electric surgical knife in blepharoplasty is simple to operate,and safe to use,with timely intraoperative incision and coagulation,which can obtain good surgical field and effect.

This work was aimed at predicting and verifying B-cell epitopes of SARS-CoV-2 E protein through bioinformatics methods,and clarifying the dominant B cell epitopes with mouse polyclonal antibody serum prepared through SARS-CoV-2 re-combinant E protein immunization and human positive serum vaccinated with inactivated SARS-CoV-2 vaccine.The structural and B-cell linear epitopes of SARS-CoV-2 E protein were predicted with SOPMA,Expasy,SWISS-MODEL,IEDB database,and Bepid-2.0 software.Candidate epitopes were expressed as GST-tagged recombinant protein fragments in an E.coli sys-tem,and their immunoreactivity with mouse and human poly-clonal positive serum against SARS-CoV-2 E protein was de-tected by western blotting and indirect ELISA,respectively.The epitope prediction results showed that E protein contained linear B cell epitopes Ser6-Val14 and Tyr57-Pro71,and the conformational epitopes of Glu8-Val12,Leu39-Tyr59,and Ser60-Leu65.The GST tagged recombinant E protein fragments of E1 and E3,containing Ser6-Val14 and Tyr57-Pro71 epitopes,respectively,as well as E2 without an epitope sequence as a control,were expressed in an E.coli expression system and successfully purified with an Ni-NTA column.Western blotting and indirect ELISA analysis indicated that all mouse and human SARS-CoV-2 positive sera positively reacted with only E1 and E3 proteins,but negatively reacted with E2 protein,thus indicating that the corresponding epitope prediction with Ser6-Val14 and Tyr57-Pro71 was correct.This study successfully predicted and preliminarily identified two linear B cell epitopes of SARS-CoV-2 E protein,thus providing a reference for the preparation of new coronavirus vaccines and the analysis of immune response characteristics.

Objective:To evaluate the diagnostic value of hematological parameters for PCa with prostate-specific antigen(PSA)of 4-10 μg/L and construct a risk-stratification model with these parameters.Methods:We retrospectively analyzed the da-ta on the males undergoing the initial prostatic biopsy in the Affiliated Hospital of Xuzhou Medical University with PSA of 4-10 μg/L from March 2010 to April 2021.According to the results of biopsy,we classified the patients into a PCa and a non-PCa group,and compared the hematological parameters between the two groups.We performed univariate and multivariate logistic regression analyses,identified the independent risk factors for PCa,constructed a risk-stratification model for the prediction of PCa and evaluated its effi-ciency.Results:A total of 415 cases were included in this study,107(25.8％)in the PCa and 308(74.2％)in the non-PCa group.Compared with the non-PCa males,the PCa patients showed a significantly older age,higher ratios of neutrophil to lymphocyte and platelet to lymphocyte,systemic immune-inflammation index(SII),red blood cell distribution width and cystatin C(CysC)level(all P＜0.05),but lower red blood cell count and hemoglobin and free/total PSA(f/tPSA)levels(all P＜0.05).Multivariate logis-tic regression analysis indicated that age,f/tPSA,SII and CysC were independent risk factors for the prediction of PCa(all P＜0.05).Five prediction models were constructed based on the above risk factors,and the area under the ROC curve(AUC)of the four-parameter(age+f/tPSA+SII+CysC)model was 0.745(95％CI:0.694-0.796),significantly higher than those of the other mod-els(P＜0.05).A risk-stratification model(low-,intermediate-,and high-risk)was also constructed based on the total nomogram scores,which showed a comparable performance to that of the Prostate Imaging Reporting and Data System(PI-RADS)for the predic-tion of PCa(AUC:0.727[95％CI:0.650-0.804]vs 0.734[95％CI:0.658-0.811]).However,the prediction rate by the risk-stratification model was evidently higher in the low-risk males than in those with low PI-RADS scores(1-2)(39.4％vs 22.2％).Conclusion:SII and CysC are independent risk factors for the prediction of PCa in patients with gray-zone PSA levels.The risk-stratification model based on age,SII,CysC and f/tPSA is comparable to PI-RADS in the diagnostic efficiency of PCa,with an even higher prediction rate in low-risk patients than in those with low PI-RADS scores,and contributive to precision screening and reduction of excessive biopsies in the diagnosis of PCa with gray-zone PSA.

Objective:To explore the mechanism of hypertension inducing erectile dysfunction(ED)using transcriptomics and network pharmacology.Methods:We randomly divided 12 male rats with spontaneous hypertension(SHT)into an L-arginine(LA)group(n=6)and an SHT model control(MC)group(n=6),took another 6 Wistar Kyoto male rats as normal controls(NC),and treated the animals in the LA group by intraperitoneal injection of LA at 400 mg/kg and those in the latter two groups with physio-logical saline,once a day,all for 7 days.Then we observed the blood pressure and penile erection of the rats,and determined the ex-pressions of the cGMP/PKG signaling pathway-related proteins and mRNAs in different groups using ELISA,Western blot and RT-qPCR.Results:Transcriptomics combined with network pharmacology showed that the cGMP/PKG signaling pathway played a key role in hypertension-induced ED.In vivo animal experiments revealed a significantly lower frequency of penile erections in the MC than in the NC group(1.33±0.52 vs 2.67±0.51,P＜0.05).The protein expressions of eNOS,PKG and sGC were markedly de-creased in the model controls compared with those the normal controls(P＜0.05),but remarkably upregulated in the LA group com-pared with those in the MC group(P＜0.05).Conclusion:Hypertension decreases the expressions of eNOS,NO,sGC,cGMP and PKG proteins and the level of testosterone by inhibiting the cGMP/PKG signaling pathway,which consequently suppresses the relaxa-tion of the penile vascular smooth muscle and reduces erectile function.