Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Circadian rhythm is a biological endogenous process regulated by the suprachiasmatic nucleus of the hypothalamus, which transmits light signals to peripheral clocks and synchronizes the body with the external environment through balanced expression of circadian rhythm genes. Working the night shift, sleep disorders, and exposure to artificial light can lead to disturbances in circadian rhythm and genetic imbalances. A substantial body of research has demonstrated that circadian rhythm plays a significant role in the treatment of autoimmune diseases and neurodegenerative disorders, with increasing attention being directed toward their impact on oral health. Disturbances in circadian rhythm primarily affect psycho-neuro-immune mechanisms, oxidative stress responses, and oral microflora through pathways such as the hypothalamic-pituitary-adrenal axis (HPA axis), brain and muscle ARNT-like 1 (BMAL1)-brain-derived neurotrophic factor (BDNF) signaling, and BMAL1-nuclear factor kappa-B (NF-κB) interactions. These disruptions may influence the progression of oral diseases. Certain pharmacological agents (e.g., melatonin, vitamin D, nobiletin, and propofol) have been shown to regulate mood disorders, immune function, and sleep-wake cycles by upregulating BMAL1 expression, thus alleviating disturbances in circadian rhythm. In addition, non-pharmacological interventions, such as sleep management strategies, psychotherapy approaches, and light therapy, also modulate these processes through HPA axis regulation. Currently, the specific mechanisms by which circadian rhythm regulates BDNF levels, T cell subsets, and inflammatory signals—thereby influencing both pathogenesis and treatment outcomes for oral diseases—remain unclear. Future research should focus on elucidating these molecular mechanisms as well as identifying therapeutic targets related to circadian rhythm within the oral health context. Further, multidisciplinary collaboration encompassing pharmacy, sleep behavior studies, and psychology will be instrumental in advancing prevention strategies and treatments for oral diseases.

ObjectiveTo investigate the effect of blueberry-derived tectorigenin （TEC） on hepatocellular carcinoma cell lines HepG2 and Huh7 and its mechanism. MethodsTEC was extracted from blueberries and purified， and a bioinformatics analysis was performed to identify potential target genes and signaling pathways. HepG2 and Huh7 cell lines were used and divided into 0， 30， 60， and 90 μg/mL groups according to the concentration of TEC. CCK-8 assay was used to measure cell viability； wound healing assay and Transwell assay were used to assess the migration ability of cells； flow cytometry was used to measure cell apoptosis rate； Western Blot was used to measure the protein expression levels of CCNB1， p53， MDM2， Bax， Bcl-2， and active-Caspase 3. Cell models with low CCNB1 expression （NC group， si-NC group， si-NC+TEC group， si-CCNB1 group， and si-CCNB1+TEC group） and CCNB1 overexpression （OE-NC group， OE-NC+TEC group， OE-CCNB1 group， and OE-CCNB1+TEC group） were established to validate the targets. A one-way analysis of variance or two factors analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. The Wilcoxon signed rank sum test was used to compare the expression levels of genes between cancer tissue and paracancerous tissue. ResultsIn HepG2 and Huh7 cells under the same concentration of TEC， cell viability at 24 hours of TEC intervention was significantly lower than that at 12 and 48 hours （all P<0.05）， and at 24 hours of intervention， the TEC 90 μg/mL group had a significantly lower cell viability than the other groups （all P<0.05）. Therefore， TEC intervention for 24 hours at a concentration of 90 μg/mL was used for subsequent studies. Compared with the TEC 0 μg/mL group， the 30， 60， and 90 μg/mL groups had significant reductions in the number of migrated cells and wound healing rate （all P<0.05）， and compared with the NC group and si-NC group， the si-NC+TEC group and the si-CCNB1 group for HepG2 and Huh7 cells had significant reductions in the number of migrated cells and wound healing rate （all P<0.05）. Compared with the NC group and si-NC group， the si-NC+TEC group and the si-CCNB1 group for HepG2 and Huh7 cells had a significant increase in cell apoptosis rate （all P<0.05）. For HepG2 cells， compared with the 0 μg/mL group， the 30， 60， and 90 μg/mL groups had significant reductions in the protein expression levels of CCNB1 and Bcl-2 （all P<0.05）， and the 60 and 90 μg/mL groups had significant increases in the protein expression levels of p53， Bax， and active-Caspase 3 （all P<0.001） and a significant reduction in the protein expression level of MDM2 （both P<0.05）. For Huh7 cells， compared with the 0 μg/mL group， the 30， 60， and 90 μg/mL groups had a significant reduction in the protein expression level of CCNB1 （all P<0.01）； the 60 and 90 μg/mL groups had significant increases in the protein expression levels of p53 and Bax and a significant reduction in the protein expression level of MDM2 （all P<0.05）； the 90 μg/mL group had a significant reduction in the protein expression level of Bcl-2 and a significant increase in the protein expression level of active-Caspase 3 （both P<0.01）. Compared with the si-NC group， the si-NC+TEC group and the si-CCNB1 group for HepG2 and Huh7 cells had significant reductions in the protein expression levels of CCNB1， MDM2， and Bcl-2 and significant increases in the protein expression levels of p53 and Bax （all P<0.05）. Compared with the OE-NC group， the OE-NC+TEC group for HepG2 and Huh7 cells had significant reductions in the protein expression levels of CCNB1 and MDM2 and a significant increase in the protein expression level of p53 （all P<0.05）， while the OE-CCNB1 group had significant increases in the protein expression levels of CCNB1 and MDM2 and a significant reduction in the protein expression level of p53 （all P<0.05）， and there were no significant differences in the protein expression level of CCNB1， MDM2， and p53 between the OE-CCNB1 group and the OE-CCNB1+TEC group （all P>0.05）. ConclusionTEC can inhibit the proliferation and migration of HepG2 and Huh7 cells and promote their apoptosis in vitro， possibly by downregulating the expression of CCNB1 and activating the p53 signaling pathway.

The well-known insulin-like growth factor 1 (IGF1)/IGF-1 receptor (IGF-1R) signaling pathway is overexpressed in many tumors, and is thus an attractive target for cancer treatment. However, results have often been disappointing due to crosstalk with other signals. Here, we report that IGF-1R signaling stimulates the growth of hepatocellular carcinoma (HCC) cells through the translocation of IGF-1R into the ER to enhance sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) activity. In response to ligand binding, IGF-1Rβ is translocated into the ER by β-arrestin2 (β-arr2). Mass spectrometry analysis identified SERCA2 as a target of ER IGF-1Rβ. SERCA2 activity is heavily dependent on the increase in ER IGF-1Rβ levels. ER IGF-1Rβ phosphorylates SERCA2 on Tyr⁹⁹⁰ to enhance its activity. Mutation of SERCA2-Tyr⁹⁹⁰ disrupted the interaction of ER IGF-1Rβ with SERCA2, and therefore ER IGF-1Rβ failed to promote SERCA2 activity. The enhancement of SERCA2 activity triggered Ca²⁺_ER perturbation, leading to an increase in autophagy. Thapsigargin blocked the interaction between SERCA2 and ER IGF-1Rβ and therefore SERCA2 activity, resulting in inhibition of HCC growth. In conclusion, the translocation of IGF-1R into the ER triggers Ca²⁺_ER perturbation by enhancing SERCA2 activity through phosphorylating Tyr⁹⁹⁰ in HCC.

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

Background and purpose: Docetaxel plus prednisone chemotherapy can improve the patients' survival for castrate-resistant prostate cancer. Angiogenesis inhibitors can also inhibit the growth of tumor. The curative effect of combined treatment is still not clear. This study aimed to evaluate the efficacy of docetaxel plus prednisone combined with thalidomide in treating castrate-resistant prostate cancer (CRPC) patients with bone metastasis. Methods:A total number of 78 CRPC patients were selected in Fuzhou General Hospital from Dec. 2008 to Jun. 2015. Seventy-eight patients were divided into two groups: 40 patients in chemotherapy group (docetaxel plus prednisone) and 38 patients in combined treatment group (docetaxel plus prednisone combined with thalidomide). A total number of 78 subjects were evaluated by the effective rate, the remission rate of bone pain, the prostate specific antigen (PSA) progression-free surviv-al, the overall survival and adverse effect. Results: The response rate (65.79%) and the remission rate of bone pain (86.84%) in combined treatment group were both higher than those in chemotherapy group (40.00% and 60.00%, P<0.05). The PSA progression-free survival (4.13 months), progression-free survival (4.25 months) and the overall survival (18.06 months) in combined treatment group were all longer than those in chemotherapy group (3.54, 3.75 and 16.26 months). The PSA pro-gression-free survival was significantly longer in combined treatment group (P<0.05). There was no significant difference in the overall survival between two groups (P>0.05). The rates of adverse effects including peripheral neuritis and lethargy in combined treatment group (26.32% and 55.26%) were higher than those in chemotherapy group (5.00% and 17.50%, P<0.05). Conclusion: Thalidomide combined with docetaxel plus prednisone in CRPC patients with bone metastasis can prolong the PSA progression-free survival and overall survival. The adverse effects are mild. It may become a new choice of treatment for CRPC.

Objective To understand the current status of learning motivation of high vocational nursing students, In order to increase their learning effect and teach quality. Methods 334 high vocational nursing students were investigated with college students learning motivation questionnaire, the results underwent analysis. Results High vocational nursing students had low learning motivation, their learning motivation was diversified. Significant difference existed between top students and low students. Conclusions High vocational nursing students had weak learning motivation, and is not related with grades and source places. It had individual orientation and utility color, but low social property. Learning motivation is an important factors for academic achievement.

OBJECTIVE: To discuss the feasibility of the General Regulations for the Processing of Crude Drugs in the supplement in China Pharmacopeia. METHODS: The problems existing in the General Regulations for the Processing of Crude Drugs in the supplement in China Pharmacopeia were analyzed, aimed at which, some constructive suggestions were presented. RESULTS & CONCLUSION: It's urgent to improve the feasibility of the General Regulations for the Processing of Crude Drugs in the supplement in China Pharmacopeia and standardize the production of Chinese cut crude drugs.