Objective To generate a dense granule protein 3 (GRA3) gene-deficient mutant of the Toxoplasma gondii ME49 strain and to test the virulence of the mutant. Methods Gene-deficient parasites were generated with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Guide RNA (gRNA) was designed using the E-CRISPR software, and mutated on the pSAG1::Cas9-U6::sgUPRT plasmid using the Q5 site-directed mutagenesis kit to generate the pSAG1::Cas9-U6::sgGRA3 plasmid. A GRA3 donor plasmid containing GRA3 gene upstream sequences, pyrimethamine resistant gene dihydrofolate reductase-thymidylate synthase (DHFR-TS) and GRA3 gene downstream sequence was generated, and GRA3 donor DNA was amplified using PCR assay. The pSAG1::Cas9-U6::sgGRA3 plasmid and GRA3 donor DNA were electroporated into tachyzoites of the wild-type T. gondii ME49 strain. Then, parasite suspensions were inoculated into human foreskin fibroblast (HFF) cells and screened with pyrimethamine to yield pyrimethamine-resistant parasites for monoclonal screening. The GRA3 gene deficient monoclonal strain (ME49Δgra3) of T. gondii was identified using PCR and Western blotting assays, and the expression of GRA3 protein was determined in the T. gondii ME49Δgra3 strain using Western blotting. Subsequently, 1 000 freshly lysed tachyzoites of T. gondii ME49 and ME49Δgra3 strains were transferred to 12-well plates seeded with HFF cells, and incubated at 37 °C containing 5% CO₂ for 7 days, and the number of plaques was counted by staining with crystal violet solutions. HFF cells infected with tachyzoites of T. gondii ME49 and ME49Δgra3 strains were stained using Giemsa solutions, and the numbers of cells containing 1, 2, 4, and > 4 T. gondii parasitophorous vacuoles were counted. In addition, the survival rates of C57BL/6 mice infected with T. gondii ME49 and ME49Δgra3 strains were compared 35 days post-infection. Results PCR assay revealed successful amplification of both the upstream and downstream homologous arm bands of the DHFR-TS gene in the T. gondii ME49Δgra3 strain, and no corresponding bands were amplified in the ME49 strain. The GRA3 band was amplified in the ME49 strain, and the DHFR-TS band, rather than GRA3 band, was amplified in the ME49Δgra3 strain. Western blotting determined absence of GRA3 protein expression in the ME49Δgra3 strain. Crystal violet staining showed that the T. gondii ME49 strain produced more plaques than the ME49Δgra3 strain [(352.67 ± 26.39) plaques vs. (235.00 ± 26.29) plaques; t = 5.472, P < 0.01], and Giemsa staining revealed that the proportion of T. gondii parasitophorous vacuoles containing at least four T. gondii tachyzoites was higher in HFF cells infected with the ME49 strain than in those infected with the T. gondii ME49Δgra3 strain [(75.67 ± 2.52)% vs. (59.67 ± 2.31)%; t = 8.113, P < 0.01], and the proportion of T. gondii parasitophorous vacuoles containing at least 1 or 2 T. gondii tachyzoites was higher in HFF cells infected with the T. gondii ME49 strain than in those infected with the T. gondii ME49Δgra3 strain [(24.33 ± 2.52)% vs. (40.33 ± 2.31)%; t = −8.113, P < 0.01]. In addition, mice infected with the T. gondii ME49 and ME49Δgra3 strains started to die 8 and 9 days post-infection, and the 35-day mortality rates of mice infected with T. gondii ME49 and ME49Δgra3 strains were 10.00% and 70.00% post-infection (χ² = 6.762, P < 0.01). Conclusions The T. gondii ME49Δgra3 strain has been successfully generated, and GRA3 protein may increase the virulence of the T. gondii ME49 strain.

China is a high-incidence region for gastric cancer globally. The disease is characterized by a high morbidity rate, low early diagnostic rate, and poor long-term outcomes, imposing a significant burden on both patients and society. Therefore, exploring the pathogenesis of gastric cancer, developing novel therapeutic strategies, and identifying new drug targets is of great importance. Telomerase expression is broadly associated with cancer cell targeting, and its up-regulation is one of the key factors driving the initiation and progression of gastric cancer. Additionally, telomerase is intricately involved in the regulation of autophagy and autophagy-associated cell death. While autophagy can induce chemoresistance, excessive autophagy may lead to cell death, which also constitutes one of the mechanisms of chemotherapy. Telomerase not only directly contributes to gastric cancer pathogenesis but also indirectly influences its development and treatment by modulating autophagy and autophagic cell death. Therefore, telomerase holds promise as a novel therapeutic target in gastric cancer.
Humans ; Stomach Neoplasms/genetics* ; Telomerase/genetics* ; Autophagy/physiology*

Booster vaccinations are highly recommended in combating the SARS-CoV-2 Omicron variant and its subvariants. However, the optimal booster vaccination strategies and related immune mechanisms with different prior vaccinations are under-revealed. In this study, we systematically evaluated the immune responses in mice and hamsters with different prime-boost regimens before their protective efficacies against Omicron were detected. We found that boosting with Ad5-nCoV, S_WT-2P or S_Omicron-6P induced significantly higher levels of neutralization activities against Omicron variants than CoronaVac and ZF2001 by eliciting stronger germinal center (GC) responses. Specifically, S_Omicron-6P induced even stronger antibody responses against Omicron variants in CoronaVac and Ad5-nCoV-primed animals than non-Omicron-specific vaccines but with limited differences as compared to Ad5-nCoV and S_WT-2P. In addition, boosting with a specific vaccine has the potential to remodel the existing immune profiles. These findings indicated that adenovirus-vectored vaccines and mRNA vaccines would be more effective than other types of vaccines as booster shots in combating Omicron infections. Moreover, the protective efficacies of the vaccines in booster vaccinations are highly related to GC reactions in secondary lymphatic organs. In summary, these findings provide timely important information on prime-boost regimens and future vaccine design.

Objective To investigate the mechanism of micellar curcumol (MC) regulating the immune microenvi-ronment of ovarian cancer by promoting the polarization of M2-type macrophages to M1-type in ovarian cancer asci-tes.Methods ① After the mice were divided into groups, a mouse ovarian cancer ascites model was constructed by using the mouse ovarian cancer cell line ID8.Then weight changes were observed, tumor tissue and ascites were collected.The expression of CD86 and CD206 on macrophages of the tumor tissue and ascites was detected by flow cytometry.The expression of protein kinase B (PKB/Akt)/mammalian target of rapamycin (mTOR) was detected by Western blot.②A human monocytic leukemia cell line (THP-1) was induced to transform into M2 macrophage (THP-1 M2 macrophage) in vitro, and then treated with 10μg/ml MC.The apoptosis was detected by flow cytom-etry.The mRNA levels of macrophage mannose receptor (CD206), transforming growth factor-β(TGF-β), inter-leukin (IL)-1β and tumor necrosis factor-α (TNF-α) were detected by qRT-PCR.The expression of CD86 and CD206 was detected by flow cytometry, and Akt/mTOR expression and phosphorylation was detected by Western blot.Results ① In vitro study showed that the average body weight of the MC group was lower than that of the control group.Compared with the control group, CD206 expression of macrophages decreased in tumor tissue and ascites in the MC group, while the expression of CD86 increased.The Akt and mTOR phosphorylation level of mac-rophages in the MC group's ascites was lower than that in control group.②In vivo study showed that there was no difference in apoptosis rate among the groups detected by flow cytometry.The mRNA expression level of CD206, TGF-β and the protein expression level of CD206 in MC group were significantly lower than those in the control group, while the mRNA expression of IL-1β, TNF-α and the protein expression level of CD86 were significantly higher than those in the control group.Compared with the control group, the phosphorylation level of Akt and mTOR in the MC group decreased.Conclusion MC promotes M1 polarization of macrophages in ascites to regulate the immune microenvironment of ovarian cancer, which may be related to the Akt/mTOR pathway.

AIM: To analyze the causes of blindness and low vision in patients with visual disability in Yangpu District of Shanghai from 2019 to 2022.METHODS:Cross-sectional study. A total of 1 604 patients who participated in the evaluation of visual disability in Shanghai Yangpu District Kongjiang Hospital, from April 2019 to December 2022 were selected for the study. The grade of visual disability and the main causes of blindness and low vision were determined by trained doctors.RESULTS:A total of 804 patients with visual disabilities were identified, with 87.31% aged 60 and above. The causes of visual disability were high myopic retinopathy(30.47%), age-related macular degeneration(23.26%), glaucoma(17.04%), and diabetic retinopathy(11.07%). Glaucoma(36.96%)is the leading cause of blindness.CONCLUSION: The majority of patients with visual disability are aged 60 years and above. More attention should be paid to the elderly population. Comprehensive prevention, treatment and rehabilitation measures should be applied in different diseases based on classification, so as to early reduce the occurrence of visual disability.

Objective To construct a domain knowledge graph of dementia care,so as to provide the foundation and guarantee for the next intelligent application based on the knowledge graph.Methods A top-down approach was adopted to construct a domain knowledge graph of dementia care.Firstly,the ontology concept is constructed from the top level,namely the schema layer of knowledge graph.Then,instances are filled,and knowledge extraction is carried out from the existing data sources,and the extracted entities and relationships are filled into the pattern layer ontology database to complete the data layer construction of the knowledge graph.Finally,the"entity relationship entity"triplet data was input into the Neo4j graph database for storage.Results In this study,the personalized care plan set of 1 012 dementia cases was used as the corpus to construct a domain knowledge graph of dementia care.The knowledge graph takes people with dementia as the core,and unfolds,one by one,around basic characteristics,care problems,and care plans in a standardized"entity-relationship-entity"triplet format,forming a large knowledge network,which contains a total of 1 522 specific dementia care knowledge entities and 8 kinds of inter-entity relationships.Conclusion The domain knowledge graph of dementia care constructed in this study clearly and intuitively shows the global pedigree and logical path of knowledge,which provides an efficient and intelligent basic guarantee for the browsing,retrieval and application of dementia care knowledge,so as to realize personalized and intelligent management of people with dementia,break through the bottleneck of lack of professionals,improve the health outcomes of people with dementia,promote the implementation of inclusive pension services,and promote healthy aging.

Objectives:Analyze the clinical characteristics of patients with primary antiphospholipid syndrome (PAPS) progressing to systemic lupus erythematosus (SLE).Explore the risk factors for the progression from PAPS to SLE.Methods:The clinical data of 262 patients with PAPS enrolled in Peking Union Medical College Hospital from February 2005 to September 2021 were evaluated. Assessments included demographic data, clinical manifestations, laboratory tests (serum levels of complement, anti-nuclear antibodies, anti-double-stranded DNA antibodies), treatment, and outcomes. Kaplan-Meier analysis was used to calculate the prevalence of SLE in patients with PAPS. Univariate Cox regression analysis was employed to identify the risk factors for PAPS progressing to SLE.Results:Among 262 patients with PAPS, 249 had PAPS (PAPS group) and 13 progressed to SLE (5.0%) (PAPS-SLE group). Univariate Cox regression analysis indicated that cardiac valve disease ( HR=6.360), positive anti-double-stranded DNA antibodies ( HR=7.203), low level of complement C3 ( HR=25.715), and low level of complement C4 ( HR=10.466) were risk factors for the progression of PAPS to SLE, whereas arterial thrombotic events ( HR=0.109) were protective factors ( P<0.05 for all). Kaplan-Meier analysis showed that the prevalence of SLE in patients suffering from PAPS with a disease course>10 years was 9%-15%. Hydroxychloroquine treatment had no effect on the occurrence of SLE in patients with PAPS ( HR=0.753, 95% CI 0.231-2.450, P=0.638). Patients with≥2 risk factors had a significantly higher prevalence of SLE compared with those with no or one risk factor (13-year cumulative prevalence of SLE 48.7% vs. 0 vs. 6.2%, P<0.001 for both). Conclusions:PAPS may progress to SLE in some patients. Early onset, cardiac-valve disease, positive anti-dsDNA antibody, and low levels of complement are risk factors for the progression of PAPS to SLE (especially in patients with≥2 risk factors). Whether application of hydroxychloroquine can delay this transition has yet to be demonstrated.

Chronic diseases occur repeatedly and progress slowly, and patients with chronic diseases are often accompanied by pressure such as changes in life rhythm, decreased activity ability and complications during treatment.Positive psychosocial factors play an important role in the development of chronic diseases, and the personal mastery, as an important protective psychological resource for individual self-management and stress coping, is of great significance for the improvement of patients ′ prognosis and quality of life.This article mainly reviews the influencing factors and intervention measures of personal control in patients with chronic diseases, aiming at providing reference for clinical nursing staff to develop and implement positive psychological intervention strategies for patients with chronic diseases.

Thyroid-associated ophthalmopathy(TAO)is a rare organ-specific autoimmune disease with an unclear pathogenesis. At present, the treatment still relies mainly on glucocorticoids and traditional immunosuppressants. However, some patients respond poorly to these drugs and experience treatment-related adverse reactions, highlighting the urgent need for novel drugs for TAO treatment. In recent years, with the deepening of research on the pathogenesis of TAO, a multitude of biologics targeting specific targets have emerged. Among them, teprotumumab, which targets the insulin-like growth factor-I receptor(IGF-IR), has been approved by the Food and Drug Administration for the treatment of TAO, and several other biologics are currently in clinical trials. This review provides the latest reference for the clinical prevention, treatment, and research of TAO by summarizing the current clinical research status of biologics targeting IGF-IR, neonatal Fc receptor(FcRn), thyroid-stimulating hormone receptor(TSHR), B cells, cytokines, and other biological agents in TAO and analyzing their impact on clinical treatment and future research trends.

Gastric cancer is one of the major diseases threatening human health, with a high incidence and a low early diagnostic rate. There are many bottlenecks encountered during its treatment. Consequently, improving the early diagnostic rate and exploring new therapeutic targets are currently urgent challenges that need to be addressed. Telomerase is undetectable in normal tissues, but it exhibits high specificity and sensitivity in most cancers and has a definite correlation with prognosis. It may serve as a serum tumor marker and prognostic indicator. Human telomerase reverse transcriptase (hTERT) gene polymorphism can regulate the susceptibility of people to gastric cancer, and affect the occurrence, development, proliferation and apoptosis of gastric cancer through its target gene. Substances such as resistin, visfatin, G-quadruplex and methylenedioxyaniline can affect the occurrence and development of gastric cancer by regulating telomerase expression. The mechanism by which hTERT regulates tumor invasion and metastasis is currently unclear, so elucidating its mechanism is of great significance.This paper will review the research progress of this mechanism in recent years.