ObjectiveTo evaluate the antitumor activity of Periplaneta americana extract（PAE） against human-derived lung adenocarcinoma organoids（LUAD-PDOs） and to elucidate its potential mechanism based on transcriptomics. MethodsFresh tumor and adjacent normal tissues from patients with LUAD were collected to construct LUAD-PDOs and normal lung organoid（Nor-PDOs） models using 3D organoid culture technology. The effective intervention concentration of PAE was determined using the cell counting kit-8（CCK-8） assay. Experimental groups included the model group（LUAD-PDOs）， normal group， model administration group（LUAD-PDOs+PAE）， and normal administration group（Nor-PDOs+PAE）. Hematoxylin-eosin（HE） staining was used to observe the pathological structures of PDOs， immunohistochemistry（IHC） was performed to detect the expressions of the proliferation marker Ki-67 and lung adenocarcinoma differentiation markers cytokeratin-7（CK-7） and Napsin A， TUNEL staining was applied to detect cell apoptosis. RNA sequencing（RNA-Seq） was conducted to identify differentially expressed genes（DEGs）， followed by Gene Ontology（GO）， Kyoto Encyclopedia of Genes and Genomes（KEGG）， and Gene Set Enrichment Analysis（GSEA）， alongside protein-protein interaction（PPI） network analysis to screen core mechanisms. Finally， key targets were validated by integrating external database analysis with immunofluorescence（IF）. ResultsNor-PDOs and LUAD-PDOs that highly recapitulated the pathological characteristics of the primary tissues were successfully established. The CCK-8 assay determined that the effective intervention concentration of PAE was 16 g·L^-1. Morphological observation showed that Nor-PDOs exhibited lumen-forming structures， whereas LUAD-PDOs displayed dense， solid structures. CCK-8 and TUNEL assays revealed that， compared with the model group， PAE intervention inhibited the proliferation of LUAD-PDOs and promoted apoptosis in LUAD cells， while showing no significant effect on the viability of Nor-PDOs. Transcriptomic analysis identified 719 DEGs that were significantly reversed after PAE intervention（347 up-regulated and 372 down-regulated）（P<0.05）. GO enrichment analysis indicated that DEGs in the model administration group were significantly enriched in biological processes related to cell cycle regulation compared to the model group. KEGG pathway analysis revealed that PAE affected pathways related to proliferation and metabolism， including pathways in cancer and the p53 signaling pathway. GSEA further confirmed that PAE significantly enhanced the activity of the p53 signaling pathway（P<0.05）. PPI network analysis indicated that breast cancer type 1 susceptibility protein（BRCA1） and checkpoint kinase 1（CHEK1） were the core down-regulated targets in the p53 pathway. IF verified the high expression of BRCA1 and CHEK1 in LUAD-PDOs and their significant downregulation after PAE intervention（P<0.05）. Furthermore， survival analysis based on The Cancer Genome Atlas（TCGA） database indicated that low expression of BRCA1 and CHEK1 was significantly associated with prolonged overall survival in patients with LUAD（P<0.05）. ConclusionPAE effectively inhibits proliferation of LUAD-PDOs and promotes their apoptosis， its anti-tumor mechanism is potentially associated with the activation of the p53 signaling pathway， with BRCA1 and CHEK1 genes likely serving as key downstream targets for the effects of PAE.

Acute lung injury (ALI) has been a kind of acute and severe disease that is mainly characterized by systemic uncontrolled inflammatory response to the production of huge amounts of reactive oxygen species (ROS) in the lung tissue. Given the critical role of ROS in ALI, a Fe₃O₄ loaded bovine serum albumin (BSA) nanocluster (BF) was developed to act as a nanomedicine for the treatment of ALI. Combining with NIR irradiation, it exhibited excellent ROS scavenging capacity. Significantly, it also displayed the excellent antioxidant and anti-inflammatory functions for lipopolysaccharides (LPS) induced macrophages (RAW264.7), and Sprague Dawley rats via lowering intracellular ROS levels, reducing inflammatory factors expression levels, inducing macrophage M2 polarization, inhibiting NF-κB signaling pathway, increasing CD4⁺/CD8⁺ T cell ratios, as well as upregulating HSP70 and CD31 expression levels to reprogram redox homeostasis, reduce systemic inflammation, activate immunoregulation, and accelerate lung tissue repair, finally achieving the synergistic enhancement of ALI immunotherapy. It finally provides an effective therapeutic strategy of BF + NIR for the management of inflammation related diseases.

OBJECTIVES: To explore the efficacy of DSA-guided intrathecal drug delivery system combined with Zi Wu Liu Zhu Acupoint Therapy for management of cancer pain and provide reference for its standardized clinical application. Methods and. RESULTS: Recommendations were formulated based on literature review and expert group discussion, and consensus was reached following expert consultation. The consensus recommendations are comprehensive, covering the entire treatment procedures from preoperative assessment and preparation, surgical operation process, postoperative management and traditional Chinese medicine treatment to individualized treatment planning. The study results showed that the treatment plans combining traditional Chinese with Western medicine effectively alleviated cancer pain, reduced the use of opioid drugs, and significantly improved the quality of life and enhanced immune function of the patients. Postoperative follow-up suggested good treatment tolerance among the patients without serious complications. CONCLUSIONS The formulated consensus is comprehensive and can provide reference for clinicians to use DSA-guided intrathecal drug delivery system combined with Zi Wu Liu Zhu Acupoint Therapy. The combined treatment has a high clinical value with a good safety profile for management of cancer pain.
Humans ; Medicine, Chinese Traditional ; Cancer Pain/therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Drug Delivery Systems ; Pain Management/methods* ; China

The identification and optimization of mutations in nanobodies are crucial for enhancing their therapeutic potential in disease prevention and control. However, this process is often complex and time-consuming, which limit its widespread application in practice. In this study, we developed a workflow, named Evolutionary-Nanobody (EvoNB), to predict key mutation sites of nanobodies by combining protein language models (PLMs) and molecular dynamic (MD) simulations. By fine-tuning the ESM2 model on a large-scale nanobody dataset, the ability of EvoNB to capture specific sequence features of nanobodies was significantly enhanced. The fine-tuned EvoNB model demonstrated higher predictive accuracy in the conserved framework and highly variable complementarity-determining regions of nanobodies. Additionally, we selected four widely representative nanobody-antigen complexes to verify the predicted effects of mutations. MD simulations analyzed the energy changes caused by these mutations to predict their impact on binding affinity to the targets. The results showed that multiple mutations screened by EvoNB significantly enhanced the binding affinity between nanobody and its target, further validating the potential of this workflow for designing and optimizing nanobody mutations. Additionally, sequence-based predictions are generally less dependent on structural absence, allowing them to be more easily integrated with tools for structural predictions, such as AlphaFold 3. Through mutation prediction and systematic analysis of key sites, we can quickly predict the most promising variants for experimental validation without relying on traditional evolutionary or selection processes. The EvoNB workflow provides an effective tool for the rapid optimization of nanobodies and facilitates the application of PLMs in the biomedical field.

Objective To explore the efficacy of DSA-guided intrathecal drug delivery system combined with Zi Wu Liu Zhu Acupoint Therapy for management of cancer pain and provide reference for its standardized clinical application.Methods and Results Recommendations were formulated based on literature review and expert group discussion,and consensus was reached following expert consultation.The consensus recommendations are comprehensive,covering the entire treatment procedures from preoperative assessment and preparation,surgical operation process,postoperative management and traditional Chinese medicine treatment to individualized treatment planning.The study results showed that the treatment plans combining traditional Chinese with Western medicine effectively alleviated cancer pain,reduced the use of opioid drugs,and significantly improved the quality of life and enhanced immune function of the patients.Postoperative follow-up suggested good treatment tolerance among the patients without serious complications.Conclusion The formulated consensus is comprehensive and can provide reference for clinicians to use DSA-guided intrathecal drug delivery system combined with Zi Wu Liu Zhu Acupoint Therapy.The combined treatment has a high clinical value with a good safety profile for management of cancer pain.

The identification and optimization of mutations in nanobodies are crucial for enhancing their thera-peutic potential in disease prevention and control.However,this process is often complex and time-consuming,which limit its widespread application in practice.In this study,we developed a work-flow,named Evolutionary-Nanobody(EvoNB),to predict key mutation sites of nanobodies by combining protein language models(PLMs)and molecular dynamic(MD)simulations.By fine-tuning the ESM2 model on a large-scale nanobody dataset,the ability of EvoNB to capture specific sequence features of nanobodies was significantly enhanced.The fine-tuned EvoNB model demonstrated higher predictive accuracy in the conserved framework and highly variable complementarity-determining regions of nanobodies.Additionally,we selected four widely representative nanobody-antigen complexes to verify the predicted effects of mutations.MD simulations analyzed the energy changes caused by these mu-tations to predict their impact on binding affinity to the targets.The results showed that multiple mu-tations screened by EvoNB significantly enhanced the binding affinity between nanobody and its target,further validating the potential of this workflow for designing and optimizing nanobody mutations.Additionally,sequence-based predictions are generally less dependent on structural absence,allowing them to be more easily integrated with tools for structural predictions,such as AlphaFold 3.Through mutation prediction and systematic analysis of key sites,we can quickly predict the most promising variants for experimental validation without relying on traditional evolutionary or selection processes.The EvoNB workflow provides an effective tool for the rapid optimization of nanobodies and facilitates the application of PLMs in the biomedical field.

Objective:To investigate the effect of astragaloside IV (AS-IV) regulating the signal axis of stromal cell-derived factor-1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) on the mobilization of bone marrow endothelial progenitor cells (EPCs) to peripheral blood in diabetes skin ulcer (DSU) rats.Methods:Twenty four SPF grade male Sprague Dawley (SD) rats were selected to make the model of type 2 diabetes rats by intraperitoneal injection of 30 mg/kg 1% (plastid ratio) streptozotocin, and then round full-thickness skin with a diameter of 2 cm was cut on both sides of the waist and back to make the skin ulcer model of diabetes rats. After that, they were randomly divided into AS-IV group (50 mg/kg AS-IV), blocker group (50 mg/kg AS-IV+ 5 mg/kg AMD3100) and model group. At the same time, a blank group ( n=8) was set up, The drug was administered via intraperitoneal injection, and the model group and blank group were treated with 0.9% NaCl of equal volume. On the 10th day, peripheral blood, femoral bone marrow, and wound neovascularization tissues of rats were collected. The number of EPCs in peripheral blood of each group of rats was measured by flow cytometry, and the protein expression of SDF-1α and CXCR4 in peripheral blood, femoral bone marrow, and wound neovascularization tissues of rats was detected by enzyme-linked immunosorbent assay (ELISA); At the same time, the wound healing rates of each group were tested. Results:On the 10th and 21st day after modeling, the wound healing rate of each group of rats was compared. The blank group healed the fastest, while the model group healed the slowest. The AS-IV group had better healing than the model group and the blocker group, and the difference was statistically significant (all P<0.05). On the 10th day after modeling, the positive rates of peripheral blood EPCs in the white group, AS-IV group, and blocker group were significantly higher than those in the model group (all P<0.05), while the positive rates of peripheral blood EPCs in the blocker group were significantly lower than those in the AS-IV group (all P<0.05). On the 10th day after modeling, the protein expression of SDF-1α and CXCR4 in the wound, serum, and bone marrow of the model group was the lowest, while the protein expression in the blank group was the highest (all P<0.05). The protein expression of SDF-1α and CXCR4 in the wound, serum, bone marrow of the AS-IV group was significantly higher than that of the blocker group and model group, and the difference was statistically significant (all P<0.05). Conclusions:Astragaloside IV can promote the mobilization and migration of endothelial progenitor cells from bone marrow to peripheral blood in diabetes ulcer rats by regulating SDF-1α/CXCR4 signal axis, and can participate in angiogenesis of diabetes ulcer wounds as seed cells to promote the healing of diabetes skin ulcers.

Limbal niche cells (LNCs) in the limbal niche, a type of mesenchymal stem cells closely associated with limbal epithelial stem/progenitor cells (LESCs), heterogeneously express both mesenchymal and putative embryonic stem cell markers and play a critical role in regulating the quiescence, self-renewal, and differentiation of LESCs.Previous studies have shown that LNCs can be isolated by collagenase, dispase, dispase-collagenase and explant culture.Transwell and 3D Matrigel coculture are widely used in ex vivo studies of LNCs and LESCs, and the interactive mechanism may include SDF-1/CXCR4, Notch, BMP, Wnt, Sonic Hedgehog, and KIT/AKT signaling pathways, and various cytokines such as nerve growth factor, keratinocyte growth factor, and insulin-like growth factor.LNCs have become a hot topic in corneal epithelial tissue engineering, ocular surface reconstruction, and corneal regeneration.This review provides an overview of the research background, isolation and culture methods, interaction mechanism of LNCs with LESCs, and its application prospects.

Objective·To investigate the predictive value of the Clinical Frailty Scale(CFS)in the long term outcomes in acute myocardial infarction(AMI)patients who completed in-hospital cardiac rehabilitation(CR).Methods·A total of 501 AMI patients treated in the Cardiology Center of Shanghai Sixth People's Hospital,Shanghai Jiao Tong University of Medicine from May 2020 to May 2022 were prospectively enrolled,with their baseline clinical data collected.The patients completed graded in-hospital CR and were assessed by CFS based on their completion of CR before discharge.Patients were then categorized into three groups(norm group,vulnerable group and frail group)according to their CFS level.The difference in 1-year major cardiovascular event(all-cause death and re-hospitalization for heart failure)rates among the three groups was investigated.Logistic regression analysis was performed to explore the effective risk factors relevant to the outcomes,and receiver operator characteristic(ROC)curves were generated to analyze the prognostic value.Finally,an optimal prediction model was developed.Results·The CFS level in AMI patients who completed CR was positively correlated with age and peak pro-B-type natriuretic peptide(peak proBNP),and inversely correlated with gender difference(P<0.05).Accompanied with the elevated CFS level,the incidence of both outcomes increased,and there were significant differences in all-cause death(2.6%,5.6%and 15.2%,P=0.002),and while no significant differences in re-hospitalization for heart failure among the three groups(19.6%,22.2%and 24.2%).All-cause death of the frail group was significantly higher than that of the norm group(P=0.004),while there was no significant difference between the vulnerable group and the norm group.CFS could sensitively predict the 1-year all-cause death in AMI patients(β=1.89,OR=6.61,P=0.001),and the risk model combined with CFS had the best predictive effect(AUC=0.845,P=0.000).Conclusion·Assessment by CFS in AMI patients who completed in-hospital CR contributes to identifying AMI patients with high risk of all-cause death in 1 year.

Objective:Cerebral ischemia reperfusion injury(CIRI)can cause damage to distant organs,such as the gastrointestinal tract,through the gut-brain axis.Melatonin is known to play a neuroprotective role by activating specific receptor pathways.However,the changes of melatonin receptors in the gastrointestinal tract after brain injury and their relationship with intestinal injury are still unclear.Methods:Twenty-four male Sprague-Dawley rats were randomly di-vided into the Sham group and CIRI group.The CIRI model was prepared by Zea-Longa method.The jejunum,ileum,and colon tissues of the rats were collected 2 h after ischemia and 24 h after reperfusion.The damage of intestinal and brain tissues was observed by using 2,3,5-triphenyl tetrazolium chloride(TTC)and HE staining.The positive expres-sion of zonula occludens-1(ZO-1)and Claudin5 was observed by immunofluorescence staining.The melatonin receptor 1(MT1)and melatonin receptor 2(MT2)expression was detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR),immunofluorescence staining and Western Blot.The correlation between the melatonin receptors and intestinal tight junction proteins was analyzed by general linear regression.Results:TTC staining showed that the infarct size of rats in the CIRI group was significantly higher than that in the Sham group.HE staining showed that intestinal villi was broken and shortened,and goblet cells were reduced in the CIRI group.The results of immuno-fluorescence staining revealed that the positive expression of ZO-1 and Claudin5 in the intestinal tissues of rats in the CIRI group was significantly lower than that in the Sham group(P＜0.05).Compared with the Sham group,the RT-qPCR revealed a significantly lower expression of MT1 and MT2 mRNA in the CIRI group(P＜0.05),and the decrease in colon tissue was the most obvious.The results of immunofluorescence staining and WB also showed that the expression of MTl and MT2 in the CIRI group was significantly lower than that in the Sham group(P＜0.05).A gen-eral linear regression analysis revealed the difference in mean fluorescence intensities of MT1+and MT2+between the Sham group and the CIRI group were positively correlated with the difference of ZO-1+and Claudin5+between the two groups.Conclusion:After CIRI,the expression of both MT1 and MT2 receptors in various intestinal segments was de-creased,and the decrease in colon tissue was the most significant.It is suggested that the poor recovery of stroke may be related to the decrease of melatonin receptor.