Objective To construct a targeted drug delivery system, Ang-BEVs@Dox, based on Angiopep-2 peptide-modified bacterial extracellular vesicles （BEVs） loaded with doxorubicin （Dox）, overcome the challenges of blood-brain barrier （BBB） penetration and systemic toxicity in chemotherapy for glioblastoma （GBM）, enhance drug targeting to brain tumors and reduce its toxic side effects. Methods BEVs derived from Escherichia coli were isolated using ultracentrifugation. The targeting ligand Angiopep-2, specific for the LRP-1 receptor, was conjugated onto the surface of BEVs to construct the targeted carrier （Ang-BEVs）. Dox was loaded into Ang-BEVs using low-frequency sonication to form Ang-BEVs@Dox. The physicochemical properties （morphology and size） of the carriers were characterized by transmission electron microscopy （TEM） and dynamic light scattering （DLS）. The BBB-penetrating capability, in vitro/in vivo anti-tumor efficacy, and biosafety of the system were evaluated using cellular uptake assays, 3D tumor spheroid models, and orthotopic tumor-bearing mouse models. Results ① Carrier characterization and in vitro efficacy: Ang-BEVs@Dox exhibited a particle size of approximately 100 nm and maintained structural stability after Dox loading. It significantly enhanced cellular uptake efficiency in U87MG cells and achieved deep penetration within 3D tumor spheroids. Cytotoxicity assays demonstrated synergistic anti-tumor effects between the BEVs and Dox in the Ang-BEVs@Dox system. ② In vivo targeting and anti-tumor efficacy: In orthotopic tumor-bearing mouse models, Ang-BEVs@Dox effectively penetrated the BBB and significantly inhibited tumor growth, extending the median survival time of tumor-bearing mice to 33.5 days （compared to 23.5 days in the blank control group, P<0.001）. Immunohistochemical analysis revealed significant suppression of the tumor cell proliferation marker Ki-67 and enhancement of the apoptosis marker TUNEL staining signals. ③ Biosafety: Major organs from mice in the Ang-BEVs@Dox treatment group showed no observable pathological damage, indicating good biosafety. Conclusion This study successfully constructed an Angiopep-2 peptide-modified engineered BEVs delivery system （Ang-BEVs@Dox）. Through Angiopep-2-mediated BBB penetration and tumor targeting, it significantly enhanced the accumulation and therapeutic efficacy of BEVs at the GBM site. This method combined efficient delivery, low systemic toxicity, and clinical translation potential, which provided an innovative solution to overcome the therapeutic bottleneck in GBM treatment.

Objective:To analyze the optical coherence tomography (OCT) imaging characteristics in patients with Coats disease and their value in predicting macular fibrosis.Methods:A nested case-control study was performed.A total of 43 patients (43 eyes) diagnosed with Coats disease through color fundus photography, ocular B-scan ultrasonography, fundus fluorescein angiography, and spectral-domain OCT examination were enrolled from January 2008 to October 2021 at the Xijing Hospital.Among them, there were 40 males and 3 females, aged from 2 to 60 years old, with a median age of 13 years.Macular fibrosis was used as an indicator of poor prognosis, and patients were divided into two groups based on whether macular fibrosis occurred at the end of follow-up.The differences in OCT characteristics between two groups were compared and logistic regression analysis was used to identify the risk factors for macular fibrosis.This study adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Xijing Hospital of Fourth Military Medical University (No.KY20202009-C-1).Results:The OCT clinical features of 43 cases of Coats disease included intraretinal hard exudates in 43 eyes (100%), subretinal fluid in 21 eyes (48.8%), macular cysts in 17 eyes (27.9%), subretinal exudates in 9 eyes (20.9%), anterior retinal hyperreflective dots in 7 eyes (16.3%), epiretinal membrane in 21 eyes (48.8%), and intraretinal fluid in 22 eyes (51.2%).In color fundus photos of 41 eyes, 38 eyes (93.0%) had hard exudates distributed in the posterior pole and 27 eyes (65.9%) had the mid-peripheral region.OCT examination showed that hard exudates were distributed in the inner nuclear layer in 35 eyes (81.4%) and the outer nuclear layer in 33 eyes (76.7%).Among 21 eyes with exudative retinal detachment detected by OCT, 9 eyes (42.9%) were detected by fundus photography and 18 eyes (85.7%) were detected by B-scan ultrasonography.The proportions of eyes with subretinal fluid and subretinal exudates were higher in the macular fibrosis group than in the non-macular fibrosis group, and the differences were statistically significant ( χ2=20.755, P<0.001; χ2=6.133, P=0.013).Logistic regression analysis showed that the presence of subretinal fluid was a risk factor for macular fibrosis (odds ratio=48.345, 95% confidence interval: 4.272-547.066, P=0.002). Conclusions:OCT examination can detect subretinal fluid, subretinal exudates, macular cysts, macular exudates, and hyperreflective spots in the retina of patients with Coats disease.Subretinal fluid is a risk factor for macular fibrosis.

Objective:To explore the effectiveness of deep learning image reconstruction (DLIR) algorithm compared to adaptive statistical iterative reconstruction (ASIR-V) algorithm in improving the quality of low-dose brain CT images.Methods:Retrospective inclusion of patients who underwent brain CT examination in the People's Liberation Army General Hospital from November 2021 to August 2022. Four different algorithms were used to reconstruct low-dose CT scans of all patients to obtain 30% intensity ASIR-V (ASIR-V-30%) images, low intensity DLIR (DLIR-L) images, medium intensity DLIR (DLIR-M) images, and high intensity DLIR (DLIR-H) images. The regions of interest were selected from four sets of images, including superficial white matter, superficial gray matter, deep white matter, and deep gray matter, and their CT values and standard deviations were measured for calculating signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR).Subjective evaluation of image quality was conducted by three neuroimaging physicians based on the Likert 5-component scale. The objective and subjective scores of the 4 groups of images were analyzed using ANOVA or Kruskal Wallis. If there are overall differences, pairwise comparisons were conducted within the group.Results:A total of 109 patients were enrolled, including 104 males and 5 females, aged 65-110 years (89.16 ± 9.53) years. The radiation exposure of brain CT low-dose scanning was (0.93 ± 0.01)mSv, significantly lower than that of conventional scanning (2.92 ± 0.01) mSv ( t = 56.15, P < 0.05). The differences in objective image quality analysis of ASIR-V-30%, DLIR-L, DLIR-M, and DLIR-H images of low-dose CT in SNR deep gray matter, SNR deep white matter, SNR superficial gray matter, SNR superficial white matter, CNR deep gray white matter, and CNR superficial gray white matter were statistically significant( F =98.23, 72.95, 68.43, 58.24, 241.13, 289.91, P < 0.05). Among them, DLIR-H images had the lowest noise in deep gray matter, deep white matter, superficial gray matter, and superficial white matter, and had statistically significant differences compared to other image groups ( t = 167.43, 275.46, 182.32, 361.54, P < 0.05). The subjective score of DLIR-H image quality was superior to ASIR-V-30%, DLIR-L, and DLIR-M, with the statistically significant difference ( t = 7.25, 8.32, 9.63, P < 0.05). Conclusions:Compared with ASIR-V, DLIR algorithm can effectively reduce image noise and artifacts in low-dose brain CT, and improve SNR and CNR. The subjective and objective image quality evaluation of DLIR-H is the best.

Objective:To investigate the molecular expression and pathological features of endothelial cell (EC) in a murine model of choroidal neovascularization (CNV) based on single-cell RNA sequencing (scRNA-seq).Methods:Six C57BL/6 mice aged 6-8 weeks were randomly divided into two groups, with 3 mice in each group.Bilateral eyeballs were enucleated.The choroidal tissues from the two groups were isolated by shearing the complex and scraping the choroid, respectively.Single-cell suspension was prepared by continuous digestion with trypsin/type Ⅰ collagenase at 37 ℃, and the cell viability and EC ratio were detected by flow cytometry to determine the preparation method of single-cell suspension.Another 6 mice were randomly assigned into the control group and the CNV group, with 3 mice in each group.The CNV model was induced by laser photocoagulation and single-cell suspensions were prepared 7 days after modeling.Gene expression library construction was performed using the Chromi-um (10x Genomics) instrument.High throughput sequencing was performed using the Illumina Novaseq6000 to obtain the expression matrix.The EC subpopulations were classified according to previous researches and the Cellmarker database.Pseudo-time analysis was performed in EC, revealing the gene expression matrix of different states.CNV-EC were further selected with preliminary analysis of the expression characteristics.Another 6 mice were selected to establish the CNV model and eyeball frozen sections were prepared 7 days after modeling.Expression and distribution as well as the area percentage of EC marker Pecam1, mitochondrial outer membrane proteins Tomm20 and mt-Co1, and capillary markers Kdr and Plvap were observed by immunofluorescence staining, and the vascular diameter was calculated.The use and care of animals followed the ARVO statement.This study protocol was approved by the Experimental Animal Welfare and Ethics Committee of Air Force Military Medical University (No.20200181).Results:The cell viability of the single-cell suspension prepared from choroidal-scleral fragments and choroidal scrapings was 99.4% and 99.1%, respectively, both of which met the sequencing requirements.The percentage of EC detected by flow cytometry was approximately 1.58%.The scRNA-seq result revealed that both the normal control and CNV groups contained 13 choroidal cell clusters.Compared with the normal control group, the proportions of rod/cone photoreceptor cells, EC and hematopoietic cells all increased, while the retinal pigment epithelium (RPE) and Schwan cells reduced in the CNV group.Among all clusters, EC constituted 18.4%.The pseudo-time analysis demonstrated that EC could be further divided into 4 states.The percentage of state 2 EC was 29.1% in the CNV group, which was significantly higher than 9.5% in the normal control group.Differentially expressed gene analysis showed that the expression of mitochondrion-related genes, including mt-Nd4 and mt-Atp6, were upregulated in state 2 EC, while capillary-related genes, including Kdr and Esm1, were downregulated.Immunofluorescent staining revealed that the area of Tomm20 and mt-Co1 in Pecam1-positive EC in the CNV area was (19.50±4.68)% and (4.64±2.82)%, respectively, which were both higher than (3.00±2.09)% and (0.18±0.34)% in normal area ( t=7.88, 3.84; both at P<0.01). The area of Kdr and Plvap in Pecam1-positive EC in the CNV area was (1.50±0.29)% and (0.79±0.97)%, respectively, which were both lower than (31.30±5.44)% and (10.43±2.28)% in the normal area ( t=13.40, 9.48; both at P<0.01). The vascular diameter in the CNV area was (5.52±1.85)μm, which was larger than (4.21±1.84)μm in the normal area ( t=9.57, P<0.001). Conclusions:When CNV occurs, the proportion of EC in choroid increases, and CNV-EC shows pathologic features of mitochondrial metabolic activation and loss of capillary properties, suggesting the mitochondrial activation of EC may play a role in the formation of CNV.

Objective:To investigate the analgesic effect and safety of using an epidural analgesia pump versus an intravenous analgesia pump for uterine artery embolization in the treatment of uterine fibroids. Methods:Fifty patients with uterine fibroids undergoing uterine artery embolization admitted to The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University from January 2019 to December 2021 were included in this study. They were divided into an observation group and a control group ( n = 25/group). Patients in the observation group used an epidural analgesia pump for pain relief, while patients in the control group used an intravenous analgesia pump for pain relief. At 1, 6, 12, and 24 hours after surgery, pain severity was compared between the two groups using the Visual Analogue Scale. Comfort level was compared between the two groups using the Bruggemann Comfort scale. Before and after surgery, respiratory rate, heart rate, blood pressure, and adverse reactions were compared between the two groups. Results:At 1 hour after surgery, the Visual Analogue Scale score in the observation group was significantly lower than that in the control group [3.00 (2.00, 5.50) vs. 7.00 (6.00, 8.00), Z = -3.84, P < 0.05]. At 6, 12, and 24 hours after surgery, there was a significant difference in the Visual Analogue Scale score between the two groups (all P > 0.05). Within 24 hours after surgery, the use of opioid analgesics in the observation group was less than that in the control group [16.00% (4/25) vs. 88.00% (22/25), χ2 = 25.96, P < 0.001]. At 1 hour after surgery, the Bruggemann Comfort Scale score in the observation group was significantly higher than that in the control group [0.00 (0.00, 0.50) vs. 0.00 (0.00, 0.00), Z = 2.08, P < 0.05]. At 6, 12, and 24 hours after surgery, there was no significant difference in the Bruggemann Comfort Scale score between the two groups (all P > 0.05). After surgery, heart rate was significantly decreased in each group compared with before surgery (both P < 0.05). There were no significant differences in respiratory rate and mean arterial pressure between the two groups before and after surgery (both P > 0.05). There were no significant differences in the incidences of postoperative nausea, vomiting, and fever between the two groups (all P > 0.05). Conclusion:The epidural analgesia pump used for uterine artery embolization in the treatment of uterine fibroids has a better analgesic effect and provides more comfort and is safer than the intravenous analgesia pump. The former is worthy of clinical promotion.

BACKGROUND: Low-density computed tomography (LDCT) improved early lung cancer diagnosis but introduces an excess of false-positive pulmonary nodules data. Hence, accurate diagnosis of early-stage lung cancer remains challenging. The purpose of the study was to assess the feasibility of using circulating tumour cells (CTCs) to differentiate malignant from benign pulmonary nodules. METHODS: 122 patients with suspected malignant pulmonary nodules detected on chest CT in preparation for surgery were prospectively recruited. Peripheral blood samples were collected before surgery, and CTCs were identified upon isolation by size of epithelial tumour cells and morphological analysis. Laser capture microdissection, MALBAC amplification, and whole-exome sequencing were performed on 8 samples. The diagnostic efficacy of CTCs counting, and the genomic variation profile of benign and malignant CTCs samples were analysed. RESULTS: Using 2.5 cells/5 mL as the cut-off value, the area under the receiver operating characteristic curve was of 0.651 (95% confidence interval: 0.538-0.764), with a sensitivity and specificity of 0.526 and 0.800, respectively, and positive and negative predictive values of 91.1% and 30.3%, respectively. Distinct sequence variations differences in DNA damage repair-related and driver genes were observed in benign and malignant samples. TP53 mutations were identified in CTCs of four malignant cases; in particular, g.7578115T>C, g.7578645C>T, and g.7579472G>C were exclusively detected in all four malignant samples. CONCLUSIONS CTCs play an ancillary role in the diagnosis of pulmonary nodules. TP53 mutations in CTCs might be used to identify benign and malignant pulmonary nodules.
Humans ; Lung Neoplasms ; Exome Sequencing ; Multiple Pulmonary Nodules ; Carcinoma ; DNA Repair

Drug optimization, which improves drug potency/specificity by structure‒activity relationship (SAR) and drug-like properties, is rigorously performed to select drug candidates for clinical trials. However, the current drug optimization may overlook the structure‒tissue exposure/selectivity-relationship (STR) in disease-targeted tissues vs. normal tissues, which may mislead the drug candidate selection and impact the balance of clinical efficacy/toxicity. In this study, we investigated the STR in correlation with observed clinical efficacy/toxicity using seven selective estrogen receptor modulators (SERMs) that have similar structures, same molecular target, and similar/different pharmacokinetics. The results showed that drug's plasma exposure was not correlated with drug's exposures in the target tissues (tumor, fat pad, bone, uterus), while tissue exposure/selectivity of SERMs was correlated with clinical efficacy/safety. Slight structure modifications of four SERMs did not change drug's plasma exposure but altered drug's tissue exposure/selectivity. Seven SERMs with high protein binding showed higher accumulation in tumors compared to surrounding normal tissues, which is likely due to tumor EPR effect of protein-bound drugs. These suggest that STR alters drug's tissue exposure/selectivity in disease-targeted tissues vs. normal tissues impacting clinical efficacy/toxicity. Drug optimization needs to balance the SAR and STR in selecting drug candidate for clinical trial to improve success of clinical drug development.

Ninety percent of clinical drug development fails despite implementation of many successful strategies, which raised the question whether certain aspects in target validation and drug optimization are overlooked? Current drug optimization overly emphasizes potency/specificity using structure‒activity-relationship (SAR) but overlooks tissue exposure/selectivity in disease/normal tissues using structure‒tissue exposure/selectivity-relationship (STR), which may mislead the drug candidate selection and impact the balance of clinical dose/efficacy/toxicity. We propose structure‒tissue exposure/selectivity-activity relationship (STAR) to improve drug optimization, which classifies drug candidates based on drug's potency/selectivity, tissue exposure/selectivity, and required dose for balancing clinical efficacy/toxicity. Class I drugs have high specificity/potency and high tissue exposure/selectivity, which needs low dose to achieve superior clinical efficacy/safety with high success rate. Class II drugs have high specificity/potency and low tissue exposure/selectivity, which requires high dose to achieve clinical efficacy with high toxicity and needs to be cautiously evaluated. Class III drugs have relatively low (adequate) specificity/potency but high tissue exposure/selectivity, which requires low dose to achieve clinical efficacy with manageable toxicity but are often overlooked. Class IV drugs have low specificity/potency and low tissue exposure/selectivity, which achieves inadequate efficacy/safety, and should be terminated early. STAR may improve drug optimization and clinical studies for the success of clinical drug development.

Objective To assess the accuracy of CT features of lung nodules (≤3 cm) in predicting the accuracy of the pathological subtype and degree of infiltration of adenocarcinoma. Methods We retrospectively analyzed the clinical data of 333 patients with non-cavitary pulmonary nodules diagnosed as adenocarcinoma by surgery and pathology in the China-Japan Friendship Hospital from 2011 to 2018, including 108 males and 225 females, aged 16-82 (59.57±10.16) years. The basic clinical data and CT characteristics of the patients were recorded. Results When the average CT value was ≥507 Hu, the maximum diameter of the lung window was ≥14.5 mm, and the solid component ratio was ≥5.0%, it indicated more likely the invasive adenocarcinoma (IAC). The higher the average CT value of the nodule, the larger the maximum diameter of the lung window, and the more solid components, the higher the degree of infiltration. CT morphological features (including burrs, lobes, vascular signs, bronchial signs, pleural stretch or depression signs) were more common in IAC. Among them, burrs were more common in acinar adenocarcinoma and papillary adenocarcinoma. In invasive adenocarcinoma, the higher the risk of recurrence of the pathological subtype, the greater the average CT value. When the average CT value of IAC was >−106 Hu, and the proportion of solid components was ≥70.5%, the histological subtypes were more inclined to micropapillary/solid predominant adenocarcinoma. Conclusion The evaluation of CT features of lung nodules can improve the predictive value of histopathological types of lung adeno-carcinoma, thereby optimizing clinical treatment decisions and obtaining more ideal therapeutic effects.

Primary liver cancer is one of the common malignant tumors and its mortality ranks third in the world. Because there are no obvious symptoms in the early stage of liver cancer, most patients are diagnosed as advanced stage, without the opportunity of surgical resection. The authors report a case of hepatocellular carcinoma with portal vein tumor thrombus, which reduced significantly after hepatic artery infusion chemotherapy combined with bevacizumab and atezolizumab, showing the safety and efficacy.