Objective To investigate the impact of Toxoplasma gondii type I, II and III rhoptry protein 16 (ROP16) on programmed cell death ligand 1 (PD-L1) expression in lung adenocarcinoma cells, and to examine the effects of T. gondii type I ROP16 protein on the relative PD-L1 expression, the relative PD-L1 distribution on the cell membrane surface, and the binding of programmed cell death 1 (PD-1) to PD-L1 in lung adenocarcinoma cells. Methods Lentiviral vectors overexpressing T. gondii type I, II and III ROP16 proteins were generated, and transfected into the human lung adenocarcinoma A549 cell line. A549 cells were used as a blank control group, and A549 cells transfected with an empty lentiviral expression vector were used as a negative control group, while A549 cells transfected with lentiviral vectors overexpressing T. gondii type I, II and III ROP16 proteins served as experimental groups. Stably transfected cells were selected with puromycin and verified using Western blotting, quantitative real-time PCR (RT-qPCR), and immunofluorescence assays. The PD-L1 expression was quantified at translational and transcriptional levels using Western blotting and RT-qPCR assays in A549 cells in the five groups, and the relative PD-L1 distribution was detected on the A549 cell membrane surface using flow cytometry. In addition, the effect of T. gondii type I ROP16 protein on the PD-1/PD-L1 binding was measured in A549 cells using enzyme-linked immunosorbent assay (ELISA). Results The relative ROP16 protein expression was 0, 0, 1.546 ± 0.091, 1.822 ± 0.047 and 2.334 ± 0.089 in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 1 339.00,P < 0.001), and the relative ROP16 mRNA expression was 2.153 ± 0.949, 2.436 ± 1.614, 14.343 ± 0.020, 12.577 ± 0.285 and 15.090 ± 0.420 in the blank control group, negative control group and the T. gondii type I, II and III ROP16 protein overexpression groups, respectively (F = 483.50,P < 0.001). The ROP16 expression was higher in the T. gondii type I, II and III ROP16 protein overexpression groups than in the blank control group at both translational and transcriptional levels (allP values < 0.001). Immunofluorescence assay revealed that T. gondii type I, II and III ROP16 proteins were predominantly localized in A549 cell nuclei. Western blotting showed that the relative PD-L1 protein expression was 0.685 ± 0.109, 0.589 ± 0.114, 1.007 ± 0.117, 0.572 ± 0.151, and 0.426 ± 0.116 in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 9.46,P < 0.05), and RT-qPCR assay quantified that the relative PD-L1 mRNA expression was 1.012 ± 0.190, 1.281 ± 0.465, 1.950 ± 0.175, 0.889 ± 0.251, and 0.230 ± 0.192 in the blank control group, negative control group, and the T. gondii type I, II and III ROP16 protein overexpression groups (F = 14.18,P < 0.05). The PD-L1 expression was higher in the T. gondii type IROP16 protein overexpression group than in the blank control group at both translational and transcriptional levels (both P values < 0.05). Flow cytometry detected that the relative distributions of PD-L1 protein were (10.83 ± 0.60)%, (11.23 ± 0.20)%, and (14.61 ± 0.50)% on the A549 cell membrane surface (F = 28.31, P < 0.05), and the relative distribution of PD-L1 protein was higher in the T. gondii type IROP16 protein overexpression group than in the blank control group and negative control group (both P values < 0.001). ELISA measured significant differences in the absorbance (A) value among the T. gondii type IROP16 protein overexpression group, the blank control group and the negative control group if the concentrations of the recombinant PD-1 protein were 0.04 (F = 10.45, P < 0.05), 0.08 μg/mL (F = 11.68, P < 0.05) and 0.12 μg/mL (F = 52.68, P < 0.05), and the A value was higher in the T. gondii type IROP16 protein overexpression group than in the blank control group and the negative control group (both P values < 0.05), indicating that T. gondii type IROP16 protein promoted the PD-L1/PD-1 binding in A549 cells in a concentration-dose manner. Conclusions T. gondii type IROP16 protein overexpression may up-regulate PD-L1 expression in A549 cells at both transcriptional and translational levels and the relative PD-L1 distribution on the A549 cell membrane surface, and affect the PD-1/PD-L1 binding in a concentration-dependent manner.

AIM: To evaluate whether Wnt pathway-related genes previously implicated in high myopia(HM)could serve as candidate genes for HM in the Chinese Han population, and to identify risk loci associated with HM susceptibility.METHODS: A case-control association analysis was conducted, involving 530 HM patients(HM group)and 1 087 healthy controls. The test efficacy was estimated using Quanto software. Peripheral blood DNA was extracted using the magnetic bead method, and seven candidate single nucleotide polymorphisms(SNPs)were genotyped using the Sequenom MassARRAY system, including HIVEP3 rs17365632, rs35134694, rs11210537, CTNNB1 rs13072632, CAMK2N1 rs10753502, TCF4 rs41396445 and Wnt7B rs73175083. Differences in allele and genotype frequencies between the HM and healthy control groups were compared under different inheritance models. Haplotype analysis was performed using SHEsis plus.RESULTS: All 7 SNPs had a genotyping detection rate exceeding 90%, and were in Hardy-Weinberg equilibrium(P>0.05). The test efficacy of the sample size was above 90.13%, indicating that the samples were representative of the population. In the HM group, the A allele frequency of HIVEP3 rs11210537 was significantly reduced(Pc=0.003, OR=0.889). Conversely, the G allele frequency was significantly elevated(Pc=0.003, OR=1.176). In an additive genetic model(AA vs GG), the AA genotype frequency was significantly lower than the GG genotype frequency(Pc=0.003, OR=0.583). Additionally, the frequency of the CCA haplotype of rs17365632, rs35134694, and rs11210537 in HIVEP3 was decreased in the HM group compared to the control group(Pc=0.008, OR=0.791).CONCLUSION: The SNP locus rs11210537 in the HIVEP3 gene is associated with genetic susceptibility to HM in the Chinese Han population, with the G allele identified as risk genetic markers. The CCA haplotype of rs17365632, rs35134694, and rs11210537 in the HIVEP3 gene represents a protection haplotype for HM.

Purpose: The genomic characteristics of uterine sarcomas have not been fully elucidated. This study aimed to explore the genomic landscape of the uterine sarcomas (USs). Materials and Methods: Comprehensive genomic analysis through RNA-sequencing was conducted. Gene fusion, differentially expressed genes (DEGs), signaling pathway enrichment, immune cell infiltration, and prognosis were analyzed. A deep learning model was constructed to predict the survival of US patients. Results: A total of 71 US samples were examined, including 47 endometrial stromal sarcomas (ESS), 18 uterine leiomyosarcomas (uLMS), three adenosarcomas, two carcinosarcomas, and one uterine tumor resembling an ovarian sex-cord tumor. ESS (including high-grade ESS [HGESS] and low-grade ESS [LGESS]) and uLMS showed distinct gene fusion signatures; a novel gene fusion site, MRPS18A–PDC-AS1 could be a potential diagnostic marker for the pathology differential diagnosis of uLMS and ESS; 797 and 477 uterine sarcoma DEGs (uDEGs) were identified in the ESS vs. uLMS and HGESS vs. LGESS groups, respectively. The uDEGs were enriched in multiple pathways. Fifteen genes including LAMB4 were confirmed with prognostic value in USs; immune infiltration analysis revealed the prognositic value of myeloid dendritic cells, plasmacytoid dendritic cells, natural killer cells, macrophage M1, monocytes and hematopoietic stem cells in USs; the deep learning model named Max-Mean Non-Local multi-instance learning (MMN-MIL) showed satisfactory performance in predicting the survival of US patients, with the area under the receiver operating curve curve reached 0.909 and accuracy achieved 0.804. Conclusion USs harbored distinct gene fusion characteristics and gene expression features between HGESS, LGESS, and uLMS. The MMN-MIL model could effectively predict the survival of US patients.

Purpose: The genomic characteristics of uterine sarcomas have not been fully elucidated. This study aimed to explore the genomic landscape of the uterine sarcomas (USs). Materials and Methods: Comprehensive genomic analysis through RNA-sequencing was conducted. Gene fusion, differentially expressed genes (DEGs), signaling pathway enrichment, immune cell infiltration, and prognosis were analyzed. A deep learning model was constructed to predict the survival of US patients. Results: A total of 71 US samples were examined, including 47 endometrial stromal sarcomas (ESS), 18 uterine leiomyosarcomas (uLMS), three adenosarcomas, two carcinosarcomas, and one uterine tumor resembling an ovarian sex-cord tumor. ESS (including high-grade ESS [HGESS] and low-grade ESS [LGESS]) and uLMS showed distinct gene fusion signatures; a novel gene fusion site, MRPS18A–PDC-AS1 could be a potential diagnostic marker for the pathology differential diagnosis of uLMS and ESS; 797 and 477 uterine sarcoma DEGs (uDEGs) were identified in the ESS vs. uLMS and HGESS vs. LGESS groups, respectively. The uDEGs were enriched in multiple pathways. Fifteen genes including LAMB4 were confirmed with prognostic value in USs; immune infiltration analysis revealed the prognositic value of myeloid dendritic cells, plasmacytoid dendritic cells, natural killer cells, macrophage M1, monocytes and hematopoietic stem cells in USs; the deep learning model named Max-Mean Non-Local multi-instance learning (MMN-MIL) showed satisfactory performance in predicting the survival of US patients, with the area under the receiver operating curve curve reached 0.909 and accuracy achieved 0.804. Conclusion USs harbored distinct gene fusion characteristics and gene expression features between HGESS, LGESS, and uLMS. The MMN-MIL model could effectively predict the survival of US patients.

Purpose: The genomic characteristics of uterine sarcomas have not been fully elucidated. This study aimed to explore the genomic landscape of the uterine sarcomas (USs). Materials and Methods: Comprehensive genomic analysis through RNA-sequencing was conducted. Gene fusion, differentially expressed genes (DEGs), signaling pathway enrichment, immune cell infiltration, and prognosis were analyzed. A deep learning model was constructed to predict the survival of US patients. Results: A total of 71 US samples were examined, including 47 endometrial stromal sarcomas (ESS), 18 uterine leiomyosarcomas (uLMS), three adenosarcomas, two carcinosarcomas, and one uterine tumor resembling an ovarian sex-cord tumor. ESS (including high-grade ESS [HGESS] and low-grade ESS [LGESS]) and uLMS showed distinct gene fusion signatures; a novel gene fusion site, MRPS18A–PDC-AS1 could be a potential diagnostic marker for the pathology differential diagnosis of uLMS and ESS; 797 and 477 uterine sarcoma DEGs (uDEGs) were identified in the ESS vs. uLMS and HGESS vs. LGESS groups, respectively. The uDEGs were enriched in multiple pathways. Fifteen genes including LAMB4 were confirmed with prognostic value in USs; immune infiltration analysis revealed the prognositic value of myeloid dendritic cells, plasmacytoid dendritic cells, natural killer cells, macrophage M1, monocytes and hematopoietic stem cells in USs; the deep learning model named Max-Mean Non-Local multi-instance learning (MMN-MIL) showed satisfactory performance in predicting the survival of US patients, with the area under the receiver operating curve curve reached 0.909 and accuracy achieved 0.804. Conclusion USs harbored distinct gene fusion characteristics and gene expression features between HGESS, LGESS, and uLMS. The MMN-MIL model could effectively predict the survival of US patients.

BACKGROUND:Previous studies on cervical instability failed to explain the dynamic and static interaction relationship and pathological characteristics changes in the development of cervical lesions under the traditional imaging examination.In recent years,the emerging nuclear magnetic resonance imaging(MRI)radiomics can provide a new way for in-depth research on cervical instability. OBJECTIVE:To investigate the application value of MRI radiomics in the study of cervical instability. METHODS:Through recruitment advertisements and the Second Department of Spine of Wangjing Hospital,China Academy of Chinese Medical Sciences,young cervical vertebra unstable subjects and non-unstable subjects aged 18-45 years were included in the cervical vertebra nuclear magnetic image collection.Five specific regions of interest,including the intervertebral disc region,the facet region,the prevertebral muscle region,the deep region of the posterior cervical muscle group,and the superficial region of the posterior cervical muscle group,were manually segmented to extract and screen the image features.Finally,the cervical instability diagnosis classification model was constructed,and the effectiveness of the model was evaluated using the area under the curve. RESULTS AND CONCLUSION:(1)A total of 56 subjects with cervical instability and 55 subjects with non-instability were included,and 1 688 imaging features were extracted for each region of interest.After screening,300 sets of specific image feature combinations were obtained,with 60 sets of regions of interest for each group.(2)Five regions of interest diagnostic classification models for cervical instability were initially established.Among them,the support vector machine model for the articular process region and the support vector machine model for the deep cervical muscle group had certain accuracy for the classification of instability and non-instability,and the average area under the curve of ten-fold cross-validation was 0.719 7 and 0.703 3,respectively.(3)The Logistic model in the intervertebral disc region,the LightGBM model in the prevertebral muscle region,and the Logistic model in the superficial posterior cervical muscle region were generally accurate in the classification of instability and non-instability,and the average area under the curve of ten-fold cross-validation was 0.650 4,0.620 7,and 0.644 2,respectively.(4)This study proved the feasibility of MRI radiomics in the study of cervical instability,further deepened the understanding of the pathogenesis of cervical instability,and also provided an objective basis for the accurate diagnosis of cervical instability.

Objective:To explore and validate the targets of Salvia miltiorrhiza in myopia using network pharmacology and molecular docking technology. Methods:The TCMSP database was used to extract the targets of Salvia miltiorrhiza.GeneCards, DisGeNET, Malacard and OMIM databases were used to extract the myopia-related targets.The target intersection was taken, and the intersecting targets were selected to extract the corresponding active ingredients of traditional Chinese medicine (TCM) and construct the pharmacological regulatory network of TCM using Cytoscape.The protein interaction network map for the key target genes was constructed using the String database, and the relevant proteins were selected to download the three-dimensional structures of the active ingredients from the PubChem database, and molecular docking was performed using AutoDockvina software.Twelve 3-week-old guinea pigs were induced with lens-induced myopia (LIM) in the right eye and randomly divided into normal saline group and sodium danshensu group, with 6 animals in each group.During the maintenance of LIM, periocular injection of 1 ml normal saline or sodium danshensu was performed daily.The contralateral eye was used as a negative control.On days 0, 14, and 28 of the experiment, the axial length of both eyes was measured by A-scan ultrasonography, and the refractive status was assessed with a streak retinoscope.To avoid individual differences, relative spherical equivalent (treated eye-contralateral eye) and relative axial length (treated eye-contralateral eye) were compared.On day 28, the relative expression levels of hypoxia-inducible factor-1α (HIF-1α) and transforming growth factor-β1 (TGF-β1) proteins were determined by Western blot.The feeding and use of laboratory animals followed the 3R principle, and the research program was approved by the Ethics Committee of Experimental Animal Center of Zhengzhou University (No.ZZU-LAC 202320405[02]). Results:Sixteen intersecting key targets were screened for myopia and TCM components derived from Salvia divinorum.A TCM network pharmacology map and protein interaction map were constructed with Salvia divinorum as a drug candidate, and the corresponding proteins of target genes, such as MMP2, TGFB1, and MMP9 were screened to perform molecular docking with the active ingredients, such as lignocellulosic acid, danshensu, tanshinone ⅡA, and so on.After 14 days of induction, the relative spherical equivalent and relative axial length were (-4.67±1.03)D and (0.67±0.26)mm in sodium danshensu group, and (-6.30±1.22)D and (1.08±0.34)mm normal saline group, indicating slower myopia progression and axial elongation in sodium danshensu group, and the differences were statistically significant ( t=2.412, P=0.039; t=2.750, P=0.049). The relative expression levels of HIF-1α protein were 0.20±0.01, 1.29±0.05 and 0.63±0.02, and the relative expression levels of TGF-β1 protein were 0.93±0.05, 0.25±0.01 and 0.74±0.05 in the negative control, normal saline and sodium danshensu groups, respectively.The expression of HIF-1α protein was higher in sodium danshensu group than in negative control group but lower than in the normal saline group, and the expression of TGF-β1 protein was lower in sodium danshensu group than in negative control group but higher than in the normal saline group, showing statistically significant differences (all at P<0.05). Conclusions:Natural compounds extracted from Salvia divinorum extracts may serve as potential drug candidates to combat scleral hypoxia and improve scleral extracellular matrix remodeling.

Objective    To investigate the optimal administration combination of β-aminopropionitrile (BAPN) and Angiotensin Ⅱ (Ang-Ⅱ) in the establishment of SD rat aortic dissection (AD) model and the related complications. Methods    Forty-two three-week-old male SD rats were randomly divided into 7 groups: a group A (0.25% BAPN), a group B (0.40% BAPN), a group C (0.80% BAPN), a group D [1 g/(kg·d) BAPN], a group E [1 g/(kg·d) BAPN+ 1 μg/(kg·min) saline], a group F [1 g/(kg·d) BAPN+1 μg/(kg·min) Ang-Ⅱ] and a group G (control group). There were 6 rats in each group. The intervention period was 4 weeks (groups E and F were 4 weeks+5 days). Rats were dissected immediately if they died during the experiment. After the intervention, the surviving rats were sacrificed by pentobarbital sodium, and the whole aorta was separated and retained. Hematoxylin-eosin staining was used to observe the changes of aorta from the pathological morphology. Results    There was no statistical difference in the survival rate among the groups after 4 weeks of BAPN intervention (P>0.05). After 5 days of mini-osmotic pumps implantation, the survival rate of rats was higher in the group E than that in the group F (P=0.008), and the incidence of AD in the group E was lower than that in the group F (P=0.001). BAPN could affect the food and water intake of rats. After BAPN intervention for 4 weeks, the body weight of rats in the group G was higher than those in the intervention groups (P<0.05). BAPN combined with Ang-Ⅱ could make the aortic intima thick, elastic fiber breakage, arrangement disorder, and inflammatory cell infiltration in rats, which conformed to the pathological and morphological changes of AD. BAPN could also affect mental state and gastrointestinal tract. Conclusion    The combination of BAPN [1 g/(kg·d)] and Ang-Ⅱ [1 μg/(kg·min)] can stably establish AD model in rats, which will provide a stable carrier for further study of the pathogenesis and therapeutic targets of AD. However, the complications in this process are an unstable factor. How to balance the influence of BAPN on other tissues and organs in the process of AD model establishment remains to be further studied.

The pre-metabolic disease state is the body state of substance metabolism disorder that has not yet reached the physical and chemical indicators of the disease, and abnormal glucose metabolism is often the key link of metabolic disorder. In TCM, the healthy function of the spleen is the cornerstone of the production and distribution of fine substances. This article discussed the pre-metabolic disease state based on the theory of "overflow of Five Qi" in the Nei Jing, taking the loss of spleen preparedness as the starting point, in order to provide new ideas and directions for the prevention and treatment of clinical metabolic diseases.

This study was performed to screen the sequence of specific nanobody targeting SARS-CoV-2 S protein,and to express and purify recombinant anti-SARS-CoV-2 nanobodies,and evaluate their characteristics and application potential.Based on the previously constructed natural bacteriophage nanobody display library,the anti-SARS-CoV-2 S protein nanobody sequences were screened,with biotinylated SARS-CoV-2 S protein receptor-binding domain antigen as target,by biotin-streptavidin liquid phase screening method,phage-ELISA and sequencing.Recombinant anti-SARS-CoV-2 S protein nanobody expression vectors were constructed,the protein was induced by IPTG and then purified,and their characteristics and application potential were analyzed by SDS-PAGE,Westem blot and ELISA methods.Two anti-SARS-CoV-2 S protein nanobody sequences were successfully obtained,and the corresponding prokaryotic expression bacteria were constructed.The recombinant anti-SARS-CoV-2 S protein nanobody was expressed and purified successfully,which can specifically bind to SARS-CoV-2 S protein.In conclusion,the recombinant anti-SARS-CoV-2 S protein nanobodies have screened and expressed successfully,which provides a basis for its application in SARS-CoV-2 detection and treatment,and facilitates related researches.