Objective To investigate the role and mechanism of ubiquitin-specific protease 46 (USP46) in hypoxia/reoxygenation (H/R)-induced pyroptosis of renal tubular epithelial cells. Methods Renal tubular epithelial cells were divided into negative control siRNA group (si-CTL group), USP46 knockdown group (si-USP46 group), negative control siRNA + H/R treatment group (si-CTL+H/R group), and USP46 knockdown + H/R treatment group (si-USP46+H/R group). Flow cytometry was used to detect cell apoptosis in each group. Real-time quantitative polymerase chain reaction (RT-qPCR) was employed to measure the messenger RNA (mRNA) expression of USP46, NOD-like receptor protein 3 (NLRP3), gasdermin D (GSDMD), interleukin (IL)-18, and IL-1β. Western blotting was used to detect the protein expression of USP46, NLRP3, GSDMD, and cleaved cysteinyl aspartate specific proteinase (C-Caspase)-1. The levels of inflammatory factors and lactate dehydrogenase (LDH) in the cell supernatants were detected, and the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the cells were detected. Co-immunoprecipitation was used to verify the interaction between USP46 and NLRP3. Results Compared with the si-CTL group, the si-CTL+H/R group exhibited increased cell apoptosis, elevated protein expression of USP46, NLRP3, GSDMD-N and C-Caspase-1, increased mRNA expression of USP46, NLRP3, GSDMD, IL-18 and IL-1β, higher levels of IL-18, IL-1β, TNF-α and LDH, and increased ROS and MDA levels (all P < 0.05). Compared with the si-CTL+H/R group, the si-USP46+H/R group showed decreased cell apoptosis, reduced protein expression of USP46, NLRP3, GSDMD-N and C-Caspase-1, decreased mRNA expression of USP46, GSDMD and IL-18, lower levels of IL-18, IL-1β, TNF-α and LDH, and decreased ROS and MDA levels (all P < 0.05). Co-immunoprecipitation results indicated that USP46 could bind to NLRP3. Conclusions Downregulation of USP46 alleviates H/R-induced pyroptosis in renal tubular epithelial cells, possibly by inhibiting USP46-dependent NLRP3 deubiquitination and promoting NLRP3 ubiquitination and degradation.

Subunit 5B of constitutively photomorphogenic 9 signalosome(CSN5B)is an inhibitory factor for the biosynthesis of vitamin C in the L-galactose synthesis pathway in plants.To create mutants with richer vitamin C in tomato fruits,a dual-target vector of pKSE402-SlCSN5Bwas constructed and trans-formed into the breeding parent of 1912.Based on the molecular biological assay and DNA sequencing,17 transgenic positive lines were determined,and 6 lines of them were genetically mutated at the target site of SlCSN5B,with an editing efficiency of 35.3％.Among the mutants,2 lines were homozygous mu-tants,csn5b-6 with 180 bp deletion and csn5b-8 with 3 bp deletion.The biological traits of two types of deficiency homozygotes without exogenous T-DNA insertion derived from the T1 generation were observed,and there were no significant differences in phenotypes of the plant and fruit.Through physiological tes-ting of red-ripe fruits from T,generation lines of csn5b-6,the GMPase activity and the vitamin C content significantly increased by 43％and 37.8％,respectively,the content of hydrogen peroxide significantly decreased by 25.9％,and the content of soluble solids did not obviously change,compared with the wild type.There were no significant differences in plant traits and physiological characteristics between T1 gen-eration lines of csn5b-8 and the wild type.Based on gene expression analysis and protein structure predic-tion,the results showed that the gene of SlCSN5B normally transcribed,and the loss of large peptide seg-ments of CSN5B encoded by SlCSN5B caused changes in its structure to affect functioning,which led to an increase of vitamin C content in the line of csn5b-6-1 1.The findings suggest that the vitamin C content of tomato fruit can be improved by CRISPR/Cas9-mediated SlCSN5B gene editing,which provides the valuable resources for high-quality breeding in tomato.

AIM To observe the effects of Yiqi Jiedu Tongluo Formula(YQJDTL)on renal microvascular endothelial function and prevention of renal injury in a rat model of type 2 diabetes mellitus(T2DM).METHODS The SD rats were randomly divided into a normal group and a model group.The model group was administered with high-fat diet combined with a single intraperitoneal injection of STZ to establish the T2DM model.The successfully modeled rats were randomly divided into the model group,the canagliflozin group(9 mg/kg),and the low-dose and high-dose YQJDTL groups(4.77,9.45 g/kg).The corresponding doses of the drug were administered by gavage for a total of 12 weeks,during which the rats underwent observation of their general condition and blood glucose changes.After the end of administration,the rats had their levels of renal index,24-hour UP,serum SCr,BUN,TC,TG,HDL-C,LDL-C,ET-1 and NOS measured;their changes in renal microvasculature and the degree of renal fibrosis observed using HE staining,Masson staining,PAS staining,and PASM staining;their ultrastructure of the glomeruli observed using transmission electron microscopy;their renal protein expressions of TGF-β,SMAD2,SMAD3,Col-1,VEGFA and PKC detected by immunohistochemical staining and Western blot;and their renal mRNA expressions of VEGFA,TGF-β,SMAD2 determined by RT-qPCR.RESULTS Compared with the model group,the high-dose YQJDTL group showed decreased levels of renal index,blood glucose,TG,TC,HDL,24 h UP,BUN,SCr and ET-1(P＜0.05,P＜0.01);increased LDL and NOS levels(P＜0.05,P＜0.01);reduced renal inflammatory infiltration and fibrosis degree,inhibited fusion of foot processes and thickening of basement membrane;decreased renal protein expressions of TGF-β,SMAD2,SMAD3,VEGFA,PKC and Col-1(P＜0.05,P＜0.01);and decreased mRNA expressions of VEGFA,TGF-β and SMAD2(P＜0.01).CONCLUSION In the rat models of T2DM,YQJDTL can reduce their levels of blood glucose and lipids by improving the renal indices levels and the renal microvascular endothelial functions to alleviate renal fibrosis and microangiopathy as well,and the mechanism may be associated with the down-regulated expressions of TGF-β/SMAD and VEGF pathway-related proteins.

Objective:To explore the effect of Pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) with chemotherapy on breast cancer patients who got agranulocytosis of 3 to 4 degree, agranulocytosis with fever (FN) and the influential factors of relative dose intensity (RDI) chemotherapy scheme. Meantime, the value of CD34 + and CD45 in peripheral blood on predicting agranulocytosis of 3-4 degree were investigated.Methods:A total of 104 women with breast cancer were treated at Huzhou Maternal and Child Health Hospital and Cancer Hospital of Zhejiang Province from Jan. 2022 to Sep. 2023. All subjects received primary prevention with PEG-rhG-CSF during chemotherapy. The clinical risk factors of agranulocytosis, FN and RDI were analyzed. The levels of CD34 + and CD45 in peripheral blood samples were analyzed by flow cytometry. Then the predictive value of the receiver characteristic curve (ROC) for Grade 3 to 4 agranulocytosis after primary prophylaxis with PEG-rhG-CSF for breast cancer chemotherapy was evaluated.Results:Among 104 breast cancer patients who received primary prevention of PEG-rhG-CSF during chemotherapy, 28 patients had agranulocytosis of 3 to 4 grade, 10 patients got FN, and 12 patients developed RDI<85%. The results of single factor analysis showed that CD34 +, CD45 and chemotherapy scheme were the influential factors of agranulocytosis of 3 to 4 degree, and low RDI of chemotherapy scheme ( OR=0.584, OR=0.999, OR=2.299, OR=0.100, OR=0.999, OR=3.088, P<0.05) . It also showed that CD34 + and chemotherapy scheme were the influential factors of FN ( OR=0.099, OR=2.667, P<0.05) . Multivariate Logistic regression analysis showed that CD34 +, CD45 and intensive chemotherapy were the independent risk factors of agranulocytosis of 3 to 4 degree after primary prevention with PEG-rhG-CSF ( OR=0.602, OR=0.999, OR=20.174, P<0.05) . CD34 + and intensive chemotherapy scheme were the independent influential factors of FN and RDI of chemotherapy scheme after primary prevention with PEG-RHG-CSF ( OR=0.072, OR=33.934, OR=0.086, OR=54.788, P<0.05) . The area under the curve (AUC) of CD34 + were 0.767 (95% CI:0.659-0.876) , AUC of CD45 were 0.743 (95% CI:0.644-0.842) , and the AUC of combined two indexes was 0.825 (95% CI:0.730-0.920) , which was higher than that of single index. So AUC of CD34 + and CD45 can be used for predicting agranulocytosis of grade 3 to 4 in breast cancer patients receiving primary prophylaxis with PEG-rhG-CSF. Conclusions:The levels of CD34 + and CD45 in peripheral blood of breast cancer patients with agranulocytosis of grade 3 to 4 receiving primary prevention with PEG-rhG-CSF during chemotherapy are lower. Combined detection of CD34 + and CD45 in peripheral blood can predict the occurrence of agranulocytosis of grade 3 to 4 in breast cancer patients after primary prevention with PEG-rhG-CSF. Also it can provide a reliable basis for assessing the risk of grade 3 to 4 agranulocytosis.

Subunit 5B of constitutively photomorphogenic 9 signalosome(CSN5B)is an inhibitory factor for the biosynthesis of vitamin C in the L-galactose synthesis pathway in plants.To create mutants with richer vitamin C in tomato fruits,a dual-target vector of pKSE402-SlCSN5Bwas constructed and trans-formed into the breeding parent of 1912.Based on the molecular biological assay and DNA sequencing,17 transgenic positive lines were determined,and 6 lines of them were genetically mutated at the target site of SlCSN5B,with an editing efficiency of 35.3％.Among the mutants,2 lines were homozygous mu-tants,csn5b-6 with 180 bp deletion and csn5b-8 with 3 bp deletion.The biological traits of two types of deficiency homozygotes without exogenous T-DNA insertion derived from the T1 generation were observed,and there were no significant differences in phenotypes of the plant and fruit.Through physiological tes-ting of red-ripe fruits from T,generation lines of csn5b-6,the GMPase activity and the vitamin C content significantly increased by 43％and 37.8％,respectively,the content of hydrogen peroxide significantly decreased by 25.9％,and the content of soluble solids did not obviously change,compared with the wild type.There were no significant differences in plant traits and physiological characteristics between T1 gen-eration lines of csn5b-8 and the wild type.Based on gene expression analysis and protein structure predic-tion,the results showed that the gene of SlCSN5B normally transcribed,and the loss of large peptide seg-ments of CSN5B encoded by SlCSN5B caused changes in its structure to affect functioning,which led to an increase of vitamin C content in the line of csn5b-6-1 1.The findings suggest that the vitamin C content of tomato fruit can be improved by CRISPR/Cas9-mediated SlCSN5B gene editing,which provides the valuable resources for high-quality breeding in tomato.

Objective:To explore the spatial correlation between gray matter volume (GMV) changes and neurotransmitter receptors/transporters in patients with first-episode schizophrenia (FES) .Methods:Fifty-four FES patients(FES group) and fifty-nine healthy controls (HC group) were selected from June 2014 to May 2020 in the Psychiatry Department of Renmin Hospital of Wuhan University. Structural magnetic resonance imaging (sMRI) was conducted on all subjects. Differences of GMV were compared across 400 cortical regions and 32 subcortical regions. Based on the positron emission tomography(PET) data from Neuromaps, which provides the density of 19 different neurotransmitter receptors and transporters, Spearman correlation analysis was performed to evaluate the spatial correlation between GMV changes and neurotransmitter systems.Results:Compared to the HC group, FES group exhibited significant GMV reductions in widespread cortical (90/400) and subcortical (6/32) regions (all FDR-corrected P<0.05). The effect size of GMV reduction (Cohen’s d) showed significant positive correlations with the density of 5-hydroxytryptamine 1a(5HT1a) ( r=0.400, Pspin=0.002), γ-aminobutyric acid type A receptor(GABA A)( r=0.307, Pspin=0.002), and metabotropic glutamate receptor 5(mGluR5) ( r=0.275, Pspin=0.020) receptors (all FDR-corrected P<0.05). Conclusion:GMV reductions in a wide range of brain regions existed in patients with FES. There are significant correlations between 5HT1a, GABA A and mGluR5 receptors and gray matter reduction in patients with FES. The disorder of these neurotransmitter receptors may be the potential neurobiological mechanism of gray matter structural abnormalities in the early stage of schizophrenia.

BACKGROUND:The development of calcium phosphate bone cement is limited due to its poor mechanical properties and weak osteogenic ability.Silicate bioactive glass is highly favored due to its excellent biological activity and osteogenic ability.Simultaneously,fiber structures can enhance the mechanical strength of materials.OBJECTIVE:To investigate the mechanical properties,biocompatibility,and osteogenic effect of silicate bioactive glass fiber composite calcium phosphate bone cement.METHODS:Different mass percentages(0％,10％,and 20％)of silicate bioactive glass fiber were added to the solid phase of calcium phosphate bone cement,mixed with the liquid phase and cured for 48 hours to obtain silicate bioactive glass fiber composite calcium phosphate bone cement.The mechanical properties,setting time,and ion precipitation of the cement were characterized.The three groups of bone cement extracts were co-cultured with MC3T3-E1 cells.The cell compatibility of the materials was evaluated by CCK-8 assay,live/dead staining,and phalloidin staining.After osteogenic induction,the osteogenic induction ability of the materials was evaluated by alkaline phosphatase staining,alizarin red staining,RUNX2 immunofluorescence staining,and RT-PCR.RESULTS AND CONCLUSION:(1)With the increase of silicate bioactive glass fiber content,the compressive strength and flexural strength of bone cement increased,and the setting time was prolonged.When bone cement was immersed in simulated body fluid,the precipitation of silicon ions,calcium ions,and phosphorus ions could be detected.Moreover,with the increase of silicate bioactive glass fiber content,the mass concentration of silicon ions and phosphorus ions released by bone cement increased,and the mass concentration of calcium ions decreased.(2)Live/dead staining and phalloidin staining results exhibited that silicate bioactive glass fiber composite calcium phosphate bone cement had no toxic effect on MC3T3-E1 cells.CCK-8 assay results showed that silicate bioactive glass fiber composite calcium phosphate bone cement could promote the proliferation of MC3T3-E1 cells.(3)With the increase of silicate bioactive glass fiber content in bone cement,the alkaline phosphatase activity and extracellular calcium deposition of MC3T3-E1 cells increased,the expression of RUNX2 protein increased,and the expression of alkaline phosphatase,osteocalcin,osteopontin,and RUNX2 mRNA expression increased.(4)The results indicate that silicate bioactive glass fibers can enhance the mechanical properties and osteogenic induction ability of calcium phosphate bone cement,among which 20％silicate bioactive glass fibers have a more obvious effect.

Objective To investigate the clinical efficacy and safety of facilitated percutaneous coronary intervention(PCI)with half-dose recombinant staphylokinase(r-SAK)in patients with ST-segment elevation myocardial infarction(STEMI)who are expected to undergo PCI within 120 minutes.Methods From October 2021 to August 2022,a total of 200 STEMI patients in eight centers were included and randomly assigned in a 1﹕1 ratio to either r-SAK group or control group.Patients received loading doses of aspirin and ticagrelor and intravenous heparin and were randomized to receive an intravenous bolus of either 5 mg r-SAK or normal saline prior to PCI.The outcomes were set as ST-segment resolution(STR)at 60-90 minutes after PCI,the proportion and transition of pathological Q waves on the 5th day after PCI,and the proportion of high-sensitivity cardiac troponin T(hs-cTnT)peaking within 12 hours of onset.The safety outcome was major bleeding events defined as Bleeding Academic Research Consortium(BARC)≥type 3 bleeding during hospitalization.Results Compared with the control group,the r-SAK group had a higher proportion of STR≥70%within 60-90 minutes after PCI(58.3%vs.40.3%,P=0.009);a lower proportion of pathological Q waves(59.1%vs.74.1%,P=0.040);a lower rate of Q wave progression(14.8%vs.43.2%,P＜0.001);a higher rate of Q wave disappearance(12.5%vs.3.7%,P=0.027);and a higher proportion of hs-cTnT peaking within 12 hours of symptom onset[31/40(77.5%)vs.17/33(51.5%),P=0.027].Regarding the safety outcome,no significant difference in BARC≥type 3 bleeding was found between the two groups during hospitalization(P＞0.05).Conclusions For STEMI patients who were expected to undergo primary PCI within 120 minutes of symptom onset,the facilitated PCI with half-dose r-SAK significantly increased the proportion of STR≥70%at 60-90 minutes after PCI,reduced the formation of pathological Q waves,and shortened the time to peak hs-cTnT,without increasing the risk of bleeding,which should be an alternative reperfusion strategy worthy of further study.

Objective:To investigate the expression of miRNAs in thymus of patients with myasthenia gravis(MG)and the re-lated mechanism of action,so as to provide theoretical and experimental basis for clinical diagnosis and treatment.Methods:miRNA microarray technology was used to analyze the differential miRNA expression in MG patients thymus.Fluorescence quantitative PCR and in situ hybridization were used to verify the expression of miR-19a-3p in thymus tissue.The TALL-104 cell line was transfected with miR-19a-3p mimics to observe the effects of miR-19a-3p on cell proliferation,apoptosis and expressions of related molecules(BCL2 and SOCS3).Results:Compared to normal thymus tissue,a total of 282 differentially expressed miRNAs were detected in the thymus of MG patients,among which 103 were up-regulated and 179 were down-regulated.The target genes of differentially expressed miRNAs were mainly related to nuclear molecules,cytoplasmic membrane-like structures and organelle related molecules.Quantita-tive fluorescence PCR and in situ fluorescence hybridization confirmed that the expression of miR-19a-3p in MG patients thymus was significantly lower than that in normal control group.Compared with the control group,miR-19a-3p mimics transfection could signifi-cantly inhibit the apoptosis of TALL-104 cells,increase the expression of BCL2 and decrease the expression of SOCS3(P<0.05).Conclusion:The expression of miRNA in the thymus of MG patients is significantly different from that of non-MG patients,and miR-19a-3p inhibits T cell apoptosis through up-regulation of BCL2 and down-regulation of SOCS3.

Objective To screen the prescription,preparation,quality evaluation of Ganoderma lucidum extract nano-emulsion(GLEN)and study its anti-skin aging effect.Methods The emulsifier and oil phase of the nano-emulsion were preliminarily screened by solubility method.The pseudo-ternary phase diagram method was used to screen the nano-emulsions,and the prepared GLEN were evaluated for quality in terms of the appearance,particle size,structure,drug loading,stability and skin safety.A mouse skin aging model was constructed by injecting D-galactose combined with ultraviolet radiation.GLEN was applied to the skin at the modeling site and investigated for their anti-skin aging efficacy.Results The optimal GLEN prescription was 30%polyoxymethylene castor oil,14.4%glycerol,11.1%medium chain triglycerides,and 44.5%water.GLEN was a dark brown and transparent liquid at room temperature,O/W type nano-emulsion with an average particle size(32.20±2.89)nm,polydispersity coefficient 0.38±0.03,zeta potential value(-4.05±0.27)mV.The content of Ganoderma lucidum polysaccharides was(36.35±0.55)mg·mL-1,and the content of Ganoderma lucidum triterpenes was(5.99±0.46)mg·mL-1,which possessed good stability.Pharmacodynamic experiments showed compared with the model group,the skin scores of mice in the GLEN group significantly improved(P﹤0.05),the water content increased(P﹤0.01),and the contents of SOD,GSH-PX and Hyp increased(P﹤0.05),while the content of MDA was decreased significantly(P﹤0.01).HE,Masson and Sirius crimson staining further indicated that the pathological changes in mice were alleviated to varying degrees in GLEN group.And GLEN had high safety for skin,could be applied to human body.Conclusion In this study,the prepared GLENs were homogeneous in appearance,stable in nature,and exhibited significant anti-aging effects on mouse skin.