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.

The rupture of carotid vulnerable plaques is the core pathological basis for major cardiovascular and cerebrovascular events. However， the insufficient alignment between existing animal models and the clinical disease and syndrome characteristics of traditional Chinese and western medicine has limited research progress. In this study， biomedical databases in China and abroad were systematically searched， and the modeling mechanisms and evaluation systems of carotid vulnerable plaque animal models were systematically assessed based on diagnostic criteria of both traditional Chinese and western medicine. Analysis of the clinical correspondence indicated that existing animal models can be categorized into four types： simple high-fat diet， surgical induction combined with high-fat feeding， genetic engineering combined with high-fat feeding， and drug induction combined with high-fat feeding. Among these， the compound strategy of surgical induction combined with high-fat feeding has become the current mainstream approach， showing good concordance with western medicine. The study found that the double balloon injury rabbit model and the ApoE^-/- mouse carotid artery tandem constriction combined with high-fat feeding model demonstrated a high degree of clinical correspondence with both traditional Chinese and western medicine in terms of vulnerable plaque imaging and pathological features. Nevertheless， existing models still face significant technical limitations in faithfully simulating plaque pathology and in translating findings to clinical applications. To address these challenges， integrating complex comorbidity mechanism construction， multimodal dynamic mechanism monitoring， and collaborative evaluation systems of traditional Chinese and western medicine could enable the development of highly concordant carotid vulnerable plaque disease-syndrome combination animal models. Such models would provide a reproducible experimental platform for targeted drug development to regulate plaque stability and for individualized precision treatment， as well as a theoretical basis for innovation in clinical diagnostic and therapeutic strategies.

Donors with a serum sodium concentration of >155 mmol/L are extended criteria donors for liver transplantation (LT). Elevated serum sodium of donors leads to an increased incidence of hepatic dysfunction in the early postoperative period of LT; however, the exact mechanism has not been reported. We constructed a Lewis rat model of 70% hepatic parenchymal area subjected to ischemia-reperfusion (I/R) with hypernatremia and a BRL-3A cell model of hypoxia-reoxygenation (H/R) with high-sodium (HS) culture medium precondition. To determine the degree of injury, biochemical analysis, histological analysis, and oxidative stress and apoptosis detection were performed. We applied specific inhibitors of the epithelial sodium channel (ENaC) and Na⁺/Ca²⁺ exchanger (NCX) in vivo and in vitro to verify their roles in injury. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) levels and the area of hepatic necrosis were significantly elevated in the HS+I/R group. Increased reactive oxygen species (ROS) production, myeloperoxidase (MPO)‍-positive cells, and aggravated cellular apoptosis were detected in the HS+I/R group. The HS+H/R group of BRL-3A cells showed significantly increased cellular apoptosis and ROS production compared to the H/R group. The application of amiloride (Amil), a specific inhibitor of ENaC, reduced ischemia-reperfusion injury (IRI) aggravated by HS both in vivo and in vitro, as evidenced by decreased serum transaminases, inflammatory cytokines, apoptosis, and oxidative stress. SN-6, a specific inhibitor of NCX, had a similar effect to Amil. In summary, hypernatremia aggravates hepatic IRI, which can be attenuated by pharmacological inhibition of ENaC or NCX.
Animals ; Reperfusion Injury/drug therapy* ; Hypernatremia/complications* ; Rats ; Liver/metabolism* ; Rats, Inbred Lew ; Male ; Apoptosis ; Sodium-Calcium Exchanger/antagonists & inhibitors* ; Reactive Oxygen Species/metabolism* ; Oxidative Stress ; Epithelial Sodium Channel Blockers/pharmacology* ; Epithelial Sodium Channels ; Cell Line ; Liver Transplantation

Objective To explore the long-term and short-term therapeutic effects and mechanisms of electroacupuncture at Baihui(GV20)point and Dazhui(GV14)point in rats with ischemic stroke.Methods Thirty rats were randomly divided into a sham-operated group,a model group,and an electroacupuncture group,with 10 rats in each group.The model group and the electroacupuncture group were subjected to middle cerebral artery occlusion(MCAO)using the modified Longa method.The electroacupuncture group received electroacupuncture at Baihui point and Dazhui point once daily for one week.Long-term neurological recovery was assessed using the adhesive removal test and the forelimb placement test at 14,21 and 28 days after MCAO.On the second day after MCAO,cerebral cortical histopathological changes were observed using transmission electron microscopy,the expression of Iba-1 in cortical tissue was detected by immunofluorescence,and the expression levels of hypoxia-inducible factor 1α(HIF-1α),vascular endothelial growth factor(VEGF),interleukin 1β(IL-1β),and tumor necrosis factor α(TNF-α)in cortical tissue were measured by Western Blot.Results(1)Compared with the sham-operated group at the same time points,the model group showed prolonged adhesive removal time and increased forelimb stepping times at 14,21 and 28 days after MCAO(P＜0.05).Compared with the model group at the same time points,the electroacupuncture group exhibited significantly shortened adhesive removal time and reduced forelimb stepping times at 14,21 and 28 days after MCAO(P＜0.05).Within 28 days after MCAO,the adhesive removal time was gradually decreased,and forelimb stepping times were gradually reduced.(2)On the second day after MCAO:compared with the sham-operated group,the model group showed degeneration and necrosis of microglia in cortical ischemic penumbra,mitochondrial damage,increased expression of Iba-1 in cortical tissue,and elevated levels of HIF-1α,VEGF,IL-1β and TNF-α,with statistically significant differences(P＜0.05).Compared with the model group,the electroacupuncture group exhibited the improvement of microglia injury in cortical ischemic penumbra,reduced expression of Iba-1 in cortical tissue and decreased levels of HIF-1α,VEGF,IL-1β and TNF-α,with statistically significant differences(P＜0.05).Conclusion Electroacupuncture at Baihui point and Dazhui point can improve symptoms in rats with ischemic stroke in the long term and ameliorate degeneration and necrosis of cortical microglia in the ischemic penumbra in the short term.The mechanism may be related to the downregulation of HIF-1α,VEGF,IL-1β and TNF-α expressions in cerebral cortical tissue and the inhibition of microglial transformation to the M1 phenotype.

BACKGROUND: Lung cancer is one of the most common malignant tumors in the world. The accuracy of intraoperative frozen section (FS) in the diagnosis of lung adenocarcinoma infiltration cannot fully meet the clinical needs. The aim of this study is to explore the possibility of improving the diagnostic efficiency of FS in lung adenocarcinoma by using the original multi-spectral intelligent analyzer. METHODS: Patients with pulmonary nodules who underwent surgery in the Department of Thoracic Surgery, Beijing Friendship Hospital, Capital Medical University from January 2021 to December 2022 were included in the study. The multispectral information of pulmonary nodule tissues and surrounding normal tissues were collected. A neural network model was established and the accuracy of the neural network diagnostic model was verified clinically. RESULTS: A total of 223 samples were collected in this study, 156 samples of primary lung adenocarcinoma were finally included, and a total of 1,560 sets of multispectral data were collected. The area under the curve (AUC) of spectral diagnosis in the test set (10% of the first 116 cases) of the neural network model was 0.955 (95%CI: 0.909-1.000, P<0.05), and the diagnostic accuracy was 95.69%. In the clinical validation group (the last 40 cases), the accuracy of spectral diagnosis and FS diagnosis were both 67.50% (27/40), and the AUC of the combination of the two was 0.949 (95%CI: 0.878-1.000, P<0.05), and the accuracy was 95.00% (38/40). CONCLUSIONS The accuracy of the original multi-spectral intelligent analyzer in the diagnosis of lung invasive adenocarcinoma and non-invasive adenocarcinoma is equivalent to that of FS. The application of the original multi-spectral intelligent analyzer in the diagnosis of FS can improve the diagnostic accuracy and reduce the complexity of intraoperative lung cancer surgery plan.
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Humans ; Lung Neoplasms/surgery* ; Adenocarcinoma of Lung/surgery* ; Adenocarcinoma/surgery* ; Hospitals ; Multiple Pulmonary Nodules

Objective : To study the mechanism of Pirfenidone (PFD) inhibiting the invasion of biliary tract tumors through cancer associated fibroblasts ( CAF) ． Methods : Primary CAF were extracted from the tumor tissues of patients with biliary tract tumor，and the marker proteins of CAF，including vimentin (VIM) ，α-smooth muscle actin ( α-SMA) and fibroblast activating protein ( FAP) were detected by Western blot．Phalloidin experiment showed the function of fibroblast cytoskeleton．ELISA and Western blot were used to verify the difference of TGF-β expression between normal fibroblasts ( NF) and CAF．The functional change of CAF was observed by adding PFD to CAF．The expression of TGF-β in CAF was verified by ELISA，quantitative realtime PCR (qRT-PCR) and West- ern blot．The change of TGF-β in serum was verified by subcutaneous tumor mouse model．The change of collagen contractile function in CAF was observed by collagen contractile test．The changes of MMP2 and MMP9 in CAF me- dium were observed by gelatin enzyme assay．The changes of SMAD signaling pathway protein in CAF were detec- ted by Western blot. Results : The related marker proteins VIM，α-SMA and FAP of CAF were highly expressed， and the filamentous actin (F-actin) of CAF was abundant．ELISA showed that the expression of TGF-β in CAF was enhanced．Western blot experiment confirmed that CAF had stronger collagen function．Western blot，PCR and related phenomenon experiments showed that PFD could inhibit collagen production and TGF-β expression in CAF. SMAD signaling pathway-related protein experiments demonstrated that PFD could affect tumor invasion by inhibi- ting TGF-β/ SMAD signaling pathway Conclusion The function of CAF extracted from cancer patients is dominated by collagen production，while PFD inhibits the collagen production and collagen remodeling related processes of CAF through TGF-β/ SMAD signaling pathway to inhibit tumor invasion.

Objective: To study the mechanism of Pirfenidone (PFD) inhibiting the invasion of biliary tract tumors through cancer associated fibroblasts ( CAF) ． Methods: Primary CAF were extracted from the tumor tissues of patients with biliary tract tumor，and the marker proteins of CAF，including vimentin (VIM) ，α-smooth muscle actin ( α-SMA) and fibroblast activating protein ( FAP) were detected by Western blot．Phalloidin experiment showed the function of fibroblast cytoskeleton．ELISA and Western blot were used to verify the difference of TGF-β expression between normal fibroblasts ( NF) and CAF．The functional change of CAF was observed by adding PFD to CAF．The expression of TGF-β in CAF was verified by ELISA，quantitative real-time PCR (qRT-PCR) and Western blot．The change of TGF-β in serum was verified by subcutaneous tumor mouse model．The change of collagen contractile function in CAF was observed by collagen contractile test．The changes of MMP2 and MMP9 in CAF medium were observed by gelatin enzyme assay．The changes of SMAD signaling pathway protein in CAF were detected by Western blot. Results : The related marker proteins VIM，α-SMA and FAP of CAF were highly expressed， and the filamentous actin (F-actin) of CAF was abundant．ELISA showed that the expression of TGF-β in CAF was enhanced．Western blot experiment confirmed that CAF had stronger collagen function．Western blot，PCR and related phenomenon experiments showed that PFD could inhibit collagen production and TGF-β expression in CAF. SMAD signaling pathway-related protein experiments demonstrated that PFD could affect tumor invasion by inhibiting TGF-β/ SMAD signaling pathway． Conclusion The function of CAF extracted from cancer patients is dominated by collagen production，while PFD inhibits the collagen production and collagen remodeling related processes of CAF through TGF-β/ SMAD signaling pathway to inhibit tumor invasion.

Objective:To explore the microbiological characteristics of donor blood culture and donor liver perfusion culture and summarize the clinical experiences to provide basic rationales for preventing donor-derived infections.Methods:From August 1, 2018 to November 26, 2018 and November 27, 2018 to December 31, 2020 at First Affiliated Hospital, Zhengzhou University, culture results of donor blood and donor liver perfusate were retrospectively reviewed.According to whether or not donor liver was obtained without breaking diaphragm, removing gallbladder intraoperatively and flushing bile through cystic duct, two stages were assigned: before and after improvement measures of liver donor, i.e.August 1, 2018 to November 26, 2018 and November 27, 2018 to December 31, 2020.The culture results of donor blood samples and donor liver perfusion fluid samples in two stages of liver transplantation were statistically analyzed and infection preventing measures during donor liver maintenance and obtaining donor liver examined.Results:A total of 486 cases of blood culture from potential donors and 478 cases of liver perfusion culture were analyzed.The results showed that the incidence of blood culture infection was 4.5% and 4.3% before and after improvement measures( χ2=0.008; P=0.927)while the incidence of perfusion fluid infection was 56.8% and 46.2%( χ2=4.569; P=0.031); Klebsiella pneumoniae was a major pathogen cultured in perfusion solution before improvement measures and Staphylococcus epidermidis after improvement measures. Conclusions:Before organ donation, infection screening and prevention of potential donors and corresponding measures during donor liver acquisition can reduce donor source infection and effectively lower the mortality of recipients.

Objective To investigate the effect of Liu-Shen-Wan on transplanted tumors in mice with colon cancer based on the polarization of M2 macrophages in the tumor microenvironment. Methods We established a subcutaneous transplantation tumor model of mice with CT26 colon cancer. Mice were randomly divided into vehicle, oxaliplatin, and oxaliplatin combined with Liu-Shen-Wan groups. Treatment was administered for three weeks, and tumor volume was measured. All mice were weighed during the administration. After the end of the treatment, the mice were dissected and tumors were photographed and weighed. Spleen index was calculated. The expression levels of IFN-γ and IL-12P40 in serum and related blood biochemical indices were measured. The expression levels of M2 macrophage polarization indices, namely, IL-10 and TGF-β, in serum and tumor tissues were detected. The infiltration degree of M2 macrophages in each group was observed by immunohistochemical experiments. Results The tumor volume and mouse weight in the oxaliplatin combined with Liu-Shen-Wan group significantly decreased compared with those in the vehicle group. The spleen index increased, and the expression levels of IFN-γ and IL-12P40 in serum also significantly increased. The mice had no obvious side effects after the drug treatment. In addition, the expression levels of IL-10 and TGF-β in the serum and tissues of mice in the oxaliplatin combined with Liu-Shen-Wan group significantly decreased. The expression levels of CD68 and CD206 in tumor tissues also decreased. Conclusion The anti-tumor effect of Liu-Shen-Wan on the transplanted tumors of mice with colon cancer is related to the inhibition of M2 macrophage polarization in the tumor microenvironment.

BACKGROUND: Preoperative diagnosis and differential diagnosis of small solid pulmonary nodules are very difficult. Computed tomography (CT), as a common method for lung cancer screening, is widely used in clinical practice. The aim of this study was to analyze the clinical data of patients with malignant pulmonary nodules and intrapulmonary lymph nodes in the clinical diagnosis and treatment of <1 cm solid pulmonary nodules, so as to provide reference for the differentiation of the two. METHODS: Patients with solid pulmonary nodules who underwent surgery from June 2017 to June 2020 were analyzed retrospectively. The clinical data of 145 nodules (lung adenocarcinoma 60, lung carcinoid 2, malignant mesothelioma 1, sarcomatoid carcinoma 1, lymph node 81) were collected and finally divided into two groups: lung adenocarcinoma and intrapulmonary lymph nodes, and their clinical data were statistically analyzed. According to the results of univariate analysis (χ² test, t test), the variables with statistical differences were selected and included in Logistic regression multivariate analysis. The predictive variables were determined and the receiver operating characteristic (ROC) curve was drawn to get the area under the curve (AUC) value of the area under the curve. RESULTS: Logistic regression analysis showed that the longest diameter, Max CT value, lobulation sign and spiculation sign were important indicators for distinguishing lung adenocarcinoma from intrapulmonary lymph nodes, and the risk ratios were 106.645 (95%CI: 3.828-2,971.220, P<0.01), 0.980 (95%CI: 0.969-0.991, P<0.01), 3.550 (95%CI: 1.299-9.701, P=0.01), 3.618 (95%CI: 1.288-10.163, P=0.02). According to the results of Logistic regression analysis, the prediction model is determined, the ROC curve is drawn, and the AUC value under the curve is calculated to be 0.877 (95%CI: 0.821-0.933, P<0.01). CONCLUSIONS For <1 cm solid pulmonary nodules, among many factors, the longest diameter, Max CT value, lobulation sign and spiculation sign are more important in distinguishing malignant pulmonary nodules from intrapulmonary lymph nodes.