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.

Objective: This study aimed to determine the predictive performance of non-contrast CT (NCCT) signs for hemorrhagic growth after intracerebral hemorrhage (ICH) when stratified by onset-to-imaging time (OIT). Materials and Methods: 1488 supratentorial ICH within 6 h of onset were consecutively recruited from six centers between January 2018 and August 2022. NCCT signs were classified according to density (hypodensities, swirl sign, black hole sign, blend sign, fluid level, and heterogeneous density) and shape (island sign, satellite sign, and irregular shape) features. Multivariable logistic regression was used to evaluate the association between NCCT signs and three types of hemorrhagic growth: hematoma expansion (HE), intraventricular hemorrhage growth (IVHG), and revised HE (RHE). The performance of the NCCT signs was evaluated using the positive predictive value (PPV) stratified by OIT. Results: Multivariable analysis showed that hypodensities were an independent predictor of HE (adjusted odds ratio [95% confidence interval] of 7.99 [4.87–13.40]), IVHG (3.64 [2.15–6.24]), and RHE (7.90 [4.93–12.90]). Similarly, OIT (for a 1-h increase) was an independent inverse predictor of HE (0.59 [0.52–0.66]), IVHG (0.72 [0.64–0.81]), and RHE (0.61 [0.54– 0.67]). Blend and island signs were independently associated with HE and RHE (10.60 [7.36–15.30] and 10.10 [7.10–14.60], respectively, for the blend sign and 2.75 [1.64–4.67] and 2.62 [1.60–4.30], respectively, for the island sign). Hypodensities demonstrated low PPVs of 0.41 (110/269) or lower for IVHG when stratified by OIT. When OIT was ≤ 2 h, the PPVs of hypodensities, blend sign, and island sign for RHE were 0.80 (215/269), 0.90 (142/157), and 0.83 (103/124), respectively. Conclusion Hypodensities, blend sign, and island sign were the best NCCT predictors of RHE when OIT was ≤ 2 h. NCCT signs may assist in earlier recognition of the risk of hemorrhagic growth and guide early intervention to prevent neurological deterioration resulting from hemorrhagic growth.

OBJECTIVES: Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer, with highmorbidity and mortality rate. Nove drug development for NSCLC is urgently needed.This study aims to investigate the activity of lathyrol derivatives and the mechanism for its inhibitory effect on the growth of NSCLC cells. METHODS: Three lathyrol derivatives were synthesized from lathyrol and their structures were verified by nuclear magnetic resonance. MTT assay was used to detect the effects of the lathyrol derivatives on the proliferation activity of NSCLC cells (A549 and H1299 cells), and the compound with the best activity was selected for subsequent experiments. Colony forming assay, wound-healing assay, and transwell assay were applied to detect in vitro cell proliferation, migration and invasion ability in A549 and H1299 cells, respectively. Quantitative real-time RT-PCR and Western blotting were performed to detect mRNA and protein levels of E-cadherin, N-cadherin, β-catenin, and MMP2 in A549 cells, respectively. RESULTS: Three lathyrol derivatives inhibited the growth of A549 and H1299 cells in a dose-dependent manner, and they showed a weak inhibitory effect on normal cells Beas-2B and 16HBE, indicating that they possessed certain selective toxic effects. Therefore, C-5 benzoylated lathyrol with the best activity was selected as the ideal drug for the subsequent experiments. Compared with the control group, the number and size of cell clusters in the treatment group of A549 and H1299 cells were significantly decreased, the relative mobility were significantly decreased, and the number of invaded cells were significantly decreased (all P<0.05), indicating that the in vitro cell proliferation, migration and invasion ability were decreased. The mRNA levels of integrin α2, integrin β1, MMP2, MMP9, β-catenin, and N-cadherin were decreased, while the expression of E-cadherin was increased (all P<0.05). The protein levels of N-cadherin, β-catenin, MMP2, and integrin αV were decreased, while the expression of E-cadherin was increased (all P<0.05). CONCLUSIONS The lathyrol derivatives synthesized in this study possess good inhibitory activity against NSCLC. Among them, C-5 benzoylated lathyrol significantly inhibits the proliferation, migration, and invasion ability of NSCLC cells in vitro through regulating the process of epithelial-mesenchymal transition.
Cadherins/genetics* ; Carcinoma, Non-Small-Cell Lung/drug therapy* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Epithelial-Mesenchymal Transition ; Humans ; Lung Neoplasms/drug therapy* ; Matrix Metalloproteinase 2/genetics* ; RNA, Messenger ; beta Catenin/genetics*

Objective:To establish a method for the determination of three optical isomers in bortezomib. Methods: An HPLC method was used with a ChiralPAKAY-H normal phase chiral column (250 mm × 4. 6 mm, 5 μm). The mobile phase was n-hexane-ethanol-methanol-trifluoroaceticacid (90 ∶7. 5∶2. 5∶0. 1) and the flow rate was 0. 8 ml·min-1 . The detection wavelength was set at 270 nm. The column temperature was 40℃ and the injection volume was 5 μl. Results: The separation of bortezomib from the three optical isomers was more than 2. 0. The linear range of the three optical isomers was 0. 6-20μg·ml-1(r≥0. 999 7). The average re-covery was 104. 1%, 105. 5% and 92. 0% with RSD of 2. 3%, 2. 4% and 2. 7%, respectively (n=9). The limit of quantification and detection limit was 3 ng and 1 ng, respectively. Conclusion:The method is rapid and accurate, and can be used for the determi-nation of optical isomers in bortezomib.

Objective:To establish an HPLC method to determine the content of carfilzomib for injection. Methods:The chroma-tographic column was a Waters symmetry C18(250 mm ×4.6 mm, 5 μm)column, the sample solvent was methanol, the mobile phase was 0. 05% trifluoroacetic acid-acetonitrile(57 ∶43) at a flow rate of 1. 0 ml · min-1 , the detection wavelength was 210 nm, the col-umn temperature was 25℃ and the injection volume was 10 ml. Results:The linear range of carfilzomib was 120-600 μg·ml-1 ( r=0. 999 7). The average recovery was 100. 4%( RSD=0. 24%,n=9). Conclusion:The method is rapid and effective, which can be used for the content determination of carfilzomib for injection.

Objective: To establish a determination method for the enantiomer in safinamide mesilate. Methods: A Chiralpak ASH (250 mm ×4. 6 mm, 5 μm) column was used with the mobile phase of n-hexane-ethanol-diethylamine (75 ∶ 25 ∶ 0. 1). The flow rate was 1. 0 ml·min-1 . The wavelength was set at 225 nm. The column temperature was 35℃. Results: The resolution of safinamide mesilate and the enantiomer was above 2. 0. The linear range of them was 1. 007-2 517. 500 μg·ml-1 and 0. 909-2273. 200 μg·ml-1 ,respectively(r = 0. 999 0). The average recovery of the enantionmer was 104. 9% with RSD of 2. 3% (n = 9). Conclusion: The method is accurate and rapid, and suitable for the determination of the enantiomer in safinamide mesilate.

Objective:To evaluate the effects of magnetron-sputtered ZrSiN/ZrO2 gradient coating on the bonding strength of commercially pure titanium to porcelain.Methods:50 cast titanium specimens were prepared according to the ISO 9693 standard,and divided into 2 groups(n =25).The ZrSiN/ZrO2 gradient coating was deposited on specimens by magnetron sputtering technique,and subsequently a low-fusing porcelain was applied for the samples in the expermental group,the samples in the control group were treated by surface sandblasting.The roughness and surface energy of the samples were measured(n =10).The bonding strength of titanium-porcelain specimens was analyzed by three-point bending test (n =10).Scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDS) were employed to examine the properties of the delaminated and cross-sectioned surfaces.The results were analyzed by paired t-test (α =0.05) with SPSS 1 1.0 software.Results:The roughness of the experimantal group was lower(P ＜ 0.05) and the contact angle was larger(P ＜ 0.05) than those in the control group.The titanium-porcelain bond strength of gradient-coated group was greater than that of control group(P ＜0.05).The SEM and EDS examination results of delaminated and cross-sectioned surfaces also indicated that the gradient-coated group showed more porcelain residues.Conclusion:The bonding strength of porcelain-fused-to-titanium can be improved by magnetron sputtered ZrSiN/ZrO2 gradient coating.

Objective:To establish an HPLC method for the determination of optical isomers in carfilzomib .Methods:The sample was separated on a Chiralpak OX-H column.The mobile phase consisted of n-hexane∶isopropanol∶alcohol (89 ∶5 ∶6, v/v/v) with a flow rat of 1.0 ml· min-1 .The detection wavelength was 220 nm.Results:The linear rang of enantiomer , diastereomer Ⅰand di-astereomer Ⅱwas 0.54-2.14 μg· ml -1,0.11-1.80 μg· ml-1 and 0.11-1.81 μg· ml-1(r≥0.998), respectively.The lower limit of quantification was 0.07-0.27 μg· ml-1 .The recoveries of optical isomers were within the range of 99.6%-100.9% with RSD of 1.13%-1.59%(n=9).Conclusion:The method is sensitive, simple, fast, accurate and specific, and suitable for the study of opti-cal isomers in carfilzomib .

Objective:To establish a method for the determination of enantiomer in rivaroxaban by HPLC. Methods:A Chiralpak IA column (250 mm × 4. 6 mm, 5 μm) was used with the mobile phase of 100% methanol. The flow rate was 1. 0 ml·min-1 . The detection wavelength was set at 250 nm and the column temperature was 40℃. Results:The resolution of rivaroxaban and the enanti-omer was above 2. 0. The linear range of them was 0. 5-1 000. 00 μg·ml-1(r=0. 999 9). The detection limit of the enantiomer was 0. 17ng and the average recovery was 102. 9% with RSD of 1. 54%(n=6). Conclusion:The method is accurate and rapid, and suit-able for the determination of the enantiomer in rivaroxaban.

Objective:To establish the determination method for four lurasidone hydrochloride enantiomers by HPLC. Methods:Lurasidone hydrochloride enantiomers were separated on a CHIRALPAK AD-H column (250 mm × 4. 6 mm, 5μm). The mobile phase consisted of hexane-ethanol-diethylamine ( 90∶10∶0. 1) at a flow rate of 1. 0 ml·min-1 and the column temperature was at 40℃. The detection wavelength was 230nm. Results:The resolution of lurasidone hydrochloride enantiomers was above 2. 0. The linear calibra-tion curves were obtained over the range of 5-120 μg· ml-1 for all the enantiomers (r=0. 999 9). The recovery was above 99. 0%with RSD below 0. 5%. The detection limits were 5ng. Conclusion:The method is simple, accurate and rapid, and suitable for the de-termination and quality control.