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 To develop GTKO (α-1,3-galactosyltransferase gene-knockout, GTKO)/hCD55 (human CD55) gene-edited xenotransplant donor pigs and verify their function. Methods In this study, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated nuclease 9), PiggyBac transposon technology and somatic cell nuclear transfer technology were used to construct GTKO/hCD55 gene-edited Diannan miniature pigs. The phenotype and function of GTKO/hCD55 pigs were analyzed by Sanger sequencing, real-time fluorescence quantitative PCR, flow cytometry, immunofluorescence, bisulfite sequencing, antigen-antibody binding assays, and complement-dependent cytotoxicity assays. Results After transfection of PX458 and PiggyBac gene editing vectors into wild-type fetal pig fibroblasts, 48 single-cell colonies were obtained through puromycin drug screening. Two single-cell colonies were selected for somatic cell nuclear transfer, resulting in two fetal pigs at 33 days of gestation. The GGTA1(α-1,3-galactosyltransferase) genotypes of fetal pig F01 were -17 bp and wild type (WT), while the GGTA1 genotypes of fetal pig F02 were -26 bp/+2 bp and -3 bp. The hCD55 mRNA expression levels of both fetal pigs were significantly higher than those of WT pigs (P＜0.01). The fetal pig F02 was selected as the donor cell source for recloning, 11 surviving piglets were obtained, all identified as GTKO/hCD55 gene-edited pigs. These pigs showed absence of α-Gal antigen expression, but weak or no expression of hCD55 was observed. Methylation analysis of the hCD55 gene's CpG island showed hypermethylation in kidney tissue lacking hCD55 expression, whereas it was not methylated or partially methylated in kidney tissue expressing hCD55. Moreover, codon optimization of the CpG island of the hCD55 gene to reduce CG content could achieve stable expression of the hCD55 gene. In addition, antigen-antibody binding experiment showed that the amount of human IgM binding to GTKO/hCD55 gene-edited pig fibroblasts was significantly lower than that of WT pigs (P＜0.01). Complement-dependent cytotoxicity experiment showed that the survival rate of fibroblasts in GTKO/hCD55 pigs was significantly higher than that in WT pigs (P＜0.01). Conclusion This study demonstrates the successful generation of GTKO/hCD55 gene-edited xenotransplant donor pigs. Methylation-induced gene silencing of the hCD55 gene can be effectively avoided by reducing the CG content of the CpG island through codon optimization. This study provides a reference for the development of xenotransplant donor pigs and guides subsequent research on xenotransplantation.

The accurate segmentation of breast ultrasound images is an important precondition for the lesion determination. The existing segmentation approaches embrace massive parameters, sluggish inference speed, and huge memory consumption. To tackle this problem, we propose T ²KD Attention U-Net (dual-Teacher Knowledge Distillation Attention U-Net), a lightweight semantic segmentation method combined double-path joint distillation in breast ultrasound images. Primarily, we designed two teacher models to learn the fine-grained features from each class of images according to different feature representation and semantic information of benign and malignant breast lesions. Then we leveraged the joint distillation to train a lightweight student model. Finally, we constructed a novel weight balance loss to focus on the semantic feature of small objection, solving the unbalance problem of tumor and background. Specifically, the extensive experiments conducted on Dataset BUSI and Dataset B demonstrated that the T ²KD Attention U-Net outperformed various knowledge distillation counterparts. Concretely, the accuracy, recall, precision, Dice, and mIoU of proposed method were 95.26%, 86.23%, 85.09%, 83.59%and 77.78% on Dataset BUSI, respectively. And these performance indexes were 97.95%, 92.80%, 88.33%, 88.40% and 82.42% on Dataset B, respectively. Compared with other models, the performance of this model was significantly improved. Meanwhile, compared with the teacher model, the number, size, and complexity of student model were significantly reduced (2.2×10 ⁶ vs. 106.1×10 ⁶, 8.4 MB vs. 414 MB, 16.59 GFLOPs vs. 205.98 GFLOPs, respectively). Indeedy, the proposed model guarantees the performances while greatly decreasing the amount of computation, which provides a new method for the deployment of clinical medical scenarios.
Humans ; Breast Neoplasms/diagnostic imaging* ; Female ; Ultrasonography, Mammary/methods* ; Image Processing, Computer-Assisted/methods* ; Algorithms ; Neural Networks, Computer ; Breast/diagnostic imaging*

Objective To cultivate glioblastoma U87 stem-like cells(SLCs)and to detect the level of stemness bio-markers,mitochondrial respiratory capacity and the capacity of in vivo tumorigenesis.Methods B-27,growth factors EGF and bFGF was added into DMEM/F-12 culture in serum-free stem cell culture medium for U87 SLCs.Suspended culture of U87 SLCs was suspended using the neuro-sphere formation assay,while adherent culture of U87 SLCs was achieved by coating Matrigel matrix on the culture surface.The mRNA and protein level of stemness biomarkers in culture were detected using real-time quantitative PCR and Western blot.The proportion of CD133+cells in culture was detected by flow cytometry.The changes of cell oxygen consumption rate were detected by Seahorse cell metabo-lism analysis.Cell tumorigenesis ability was verified by subcutaneous tumor transplantation in animals.Results U87 SLCs in stem cell culture medium would grow into typical sphere morphology within one week,and the spheres would continue to grow as the culture process prolongs.At the appropriate concentration of adhesive,U87 SLCs adhered to and grow well in stem cell culture medium.The mRNA transcription of stemness biomarkers such as CD133,nes-tin,OLIG2,CD44,CD15,and integrin α6(ITGA6)was significantly increased as found in both culture methods,and the protein levels of CD133 and nestin were also increased under both methods(P＜0.05).U87 SLCs showed higher mitochondrial reserve respiratory capacity(P＜0.05).U87 SLCs could form larger subcutaneous tumors with fewer inoculated cells(P＜0.05),and grew faster in vivo with stronger tumorigenic ability.Conclusions U87 SLCs have typical stemness characteristics and may function as tumor cell model with higher stemness properties.

Objective To study the effect of carboxyamidotriazole-orotate(CTO)on the proliferation and fatty acid anabolism regulation of human pancreatic cancer cells.Methods Human pancreatic cancer cell lines AsPC-1,AsPC-1/GEM(AR),PANC-1 and MiaPaCa-2 were used as the study subjects;cell survival rate was detected by sulfo-nylrhodamine B(SRB);the mRNA level of key genes for fatty acid synthesis was detected by qPCR;the protein level of the AMPK/ACC pathway was detected by Western blot;intracellular lipid metabolites were examined by liquid chromatography-mass spectrometry(LC-MS).Results Comparing to control group,CTO significantly de-creased the cell viability of AsPC-1,AR,PANC-1,and MiaPaCa-2(P＜0.05).CTO down-regulated the mRNA level of key fatty acid synthesis genes(P＜0.05).CTO significantly reduced the protein expression of AMPK,ACC and c-Myc(P＜0.05),while increasing the protein expression of p-AMPK and p-ACC(P＜0.05).CTO decreased lipid metabolite content in AR cells(P＜0.05).Conclusions CTO attenuates cellular fatty acid anabolism by inhibition of oncogene c-Myc expression and AMPK/ACC pathway,down-regulates the expression of fatty acid synthesis-related genes,and then inhibits proliferation of the human pancreatic cancer cell lines AsPC-1,AR,PANC-1 and MiaPaCa-2.

BACKGROUND:Osteoporosis vertebral compression fracture is a common fracture secondary to osteoporosis,and there is currently a lack of effective predictive indicators and methods for osteoporosis vertebral compression fracture. OBJECTIVE:To investigate the predictive effects of paravertebral muscle degeneration,functional cross-sectional area,and percentage of fat infiltration on osteoporotic vertebral compression fractures. METHODS:The 224 patients with osteoporosis diagnosed from January 2018 to June 2022 were included.They were followed up for more than 2 years.They were divided into fracture group and non-fracture group according to the presence and absence of vertebral fracture.The detailed information of demographics,body mass index,bone mineral density and so on were collected.The functional cross-sectional area and percentage of fat infiltration of bilateral Psoas major muscle and extensor dorsi(Erector spinae muscles muscle and multifidus muscle)at the level of lower endplate of L2 vertebral body were measured and calculated. RESULTS AND CONCLUSION:(1)224 patients were ultimately included,of which 126 had fractures as the fracture group and 98 had no fractures as the non-fracture group.There was no statistically significant difference in age,gender,height,body mass,body mass index,and fracture segment between the two groups(P>0.05).(2)The bone mineral density of the fracture group was significantly lower than that of the non-fracture group(P<0.05).Functional cross-sectional areas of Psoas major muscle and extensor dorsi in the fracture group were significantly lower than those in the non-fracture group(P<0.05).The percentage of fat infiltration of the extensor dorsi in the fracture group was significantly higher than that in the non-fracture group(P<0.05).There was no significant difference in percentage of fat infiltration of Psoas major muscle between the two groups(P>0.05).(3)Receiver operating characteristic analysis showed that the vertebral bone mineral density,percentage of fat infiltration of extensor dorsi,functional cross-sectional area of extensor dorsi and percentage of fat infiltration of Psoas major muscle were 0.903 g/cm2,35.426%,418.875 mm2,and 6.375%,respectively.The areas under curve were 0.634,0.755,0.876,and 0.585,respectively.(4)These findings indicate that paravertebral muscle degeneration is strongly associated with the occurrence of osteoporotic vertebral compression fractures.The functional cross-sectional area of extensor dorsi muscle can effectively predict the occurrence of osteoporotic vertebral compression fractures,which is helpful for early prevention and treatment of osteoporotic vertebral compression fractures.

Objective:To analyze the molecular evolution characteristics of HA and NA genes of influenza B/Yamagata (BY) and influenza B/Victoria (BV) lineage viruses in Guizhou Province, aiming to provide reference for scientific prevention and control of influenza. Methods:The prevalence of various types of influenza viruses in Guizhou Province from 2017 to 2021 was analyzed. The nucleic acid of influenza B viruses was extracted, and then the HA and NA genes were amplified by RT-PCR. Fourteen strains were sequenced and the sequences of 83 strains were obtained from GISAID. Homologies between the 97 influenza B viruses as well as the phylogenetic characteristics and amino acid site variations were analyzed. Results:Influenza A, BY and BV lineage viruses co-circulated in Guizhou Province and BV lineage was the predominant type. The homologies of HA and NA genes were 98.7%-99.4% and 98.4%-99.6% between BY lineage viruses and the reference vaccine strain B/PHUKET/3073/2013. BV lineage viruses shared 98.3%-99.3% and 98.9%-99.6% homologies with the reference vaccine strain B/Colorado/06/2017. The BY lineage strains in Guizhou Province mainly belonged to Y3 genetic group with HA gene in two branches of Y3-H1-2 and NA gene in three branches of Y3-N1-3. Three reassortant strains were found in Y3 clade. The isolated BV lineage strains mainly belonged to V1A-2 genetic group with HA gene in four branches of V1A-2 H1-4 and NA gene in five branches of V1A-2 N1-5. Twenty reassortant strains were found in V1A-2 clade and no inter-lineage reassortants were found. Analysis of variations at key amino acid sites showed that there was no mutation at epitopes in Y3 genetic group. However, there were point mutations at four main epitopes and a shift mutation in 190 helix in V1A-2 genetic group. There was no mutation in drug resistance sites. Conclusions:Various types of influenza viruses circulated in Guizhou Province. The homology between influenza B viruses and vaccine strains was decreasing. Different branches of HA and NA genes had been evolved and various forms of mutations were detected in the sequences. Intra-lineage reassortant strains and new varieties emerged. Surveillance of influenza B viruses should be strengthened.

Objective:To understand the genetic variation and the prevalence of H9N2 subtype avian influenza virus in Guizhou province, and to provide the scientific evidence for the prevention and control of avian influenza virus.Methods:The results of AIV detection in live poultry market(LPM) environment in Guizhou province from October 2018 to March 2019 were statistically analyzed, RNAs were extracted and sequenced from the HA genes of 13 samples of H9N2 positive screened by real-time PCR. Then the homology, the genetic evolution and the mutations of important amino acid were analyzed by bioinformation softwares. Results:The positive rate of AIV was 52.2% and the positive rate of H9N2 was 83.7% in LPM environment. The homology between nucleotides of the HA gene of 21 strains ranged from 91.6% to 100.0%, and the homology between amino acids of the HA gene ranged from 91.0% to 100.0%. All strains belonged to Y280 sublineage and G57 genotype. Key sites analysis showed that they had a common motif PSRSSRGLF and LSRSSRGLF at the cleavage site, which indicated that they were lentogenic and low pathogenic strains. Mutations H191N, E198T/A and Q234L at the receptor binding sites in the HA was found in 21 strains, while indicated the viruses had the potential to bind human-like receptor. The analysis results of glycosylation motifs showed that all 21 strains had 7 glycosylation sites, but had a site deletion at amino acid site 218 and an addition at 313.There was no significant mutation in the key site compared with the human infected strains. Conclusions:The detection rate of AIV in LPM environment in Guizhou province was high, and the pollution was very serious, and H9N2 subtype is the main subtype, All H9N2 subtype AIVs belonged to Y280 sublineage and G57 genotype, and they were low pathogenic avian influenza viruses in Guizhou province, but the genetic gap were widening and mutations of key amino acid site might enhance susceptibility and pathogenicity to human beings. Hence, It is necessary to strengthen the surveillance of molecular characteristic variation of H9N2 subtype AIV.

Objective　To study the efficacy and safety of Naomaili granule in patients with different subtypes of acute cerebral infarction. Methods　A total of 122 hospitalized patients with acute cerebral infarction from June to December 2019 were randomly divided into control group （n=56） and observation group （n=66）. The control group was treated according to the guidelines for the diagnosis and treatment of acute ischemic stroke，while the observation group was treated with Naomaili granule besides the guidelines. The two groups were classified according to the TOAST criteria. Before and after treatment，NIHSS score and BI score were compared，and adverse reaction was evaluated. Results　Compared with the control group，the NIHSS score of observation group was decreased while BI score was increased after 21 days of treatment （P<0.05）. After the treatment，the NIHSS score of the subtype of large-artery therosclerosis（LAA）、small-artery occlusion（SAO）、cardioembolism（OD）、otherdetermined cause（OD）and undetermined cause（UND）was decreased while BI scores was increased（P<0.05）. However，LAA subtype improved more obviously（P<0.05），while the change of CE subtype had no statistical significance（P>0.05）. Conclusion　Naomaili granule has a good effect on acute cerebral infarction and it can improve the nervous function and activities of daily living for the subtypes of LAA，SAO，OD and UND according to the TOAST criteria.

Objective To investigate the metabolic characteristics,body composition and dietary intake in elderly sarcopenia patients with type 2 diabetes(T2DM).Methods A total of 652 T2DM patients(327 males and 325 females)aged over 60 years were selected from endocrinology department of nine different hospitals in Beijing.Body composition was measured by bioimpedance analysis,and the appendicular skeletal mass index(ASMI)was calculated.Sarcopenia was defined as body height-adjusted skeletal muscle mass (ASMI)below the lower quintile of the young reference group.The diagnostic cutoff points for sarcopenia were 7.18 kg/m2 for men and 5.73 kg/m2 for women.All patients were divided into the sarcopenia group versus the control group(below vs.not below 7.18 kg/m2 for men and 5.73 kg/m2 for women).The anthropometric parameters,body composition,biochemical laboratory results and dietary intake were compared between the two groups.The blood glucose target levels were less than 7 mmol/L of fasting plasma glucose(FPG)or less than 7％ of haemoglobin A1c(HbA1c).The obesity ratio was calculated according to body fat percentage more than 25％ in men and more than 30％ in women as obesity.Results There were 116 (17.8％)patients in the sarcopenia group (men/women,82/34),and 536 (82.2 ％) patients in the control group (men/women,245/291).Age was higher in the sarcopenia group than in the control group(t =4.385,P =0.000),and body mass index and waist hip ratio(WHR)were lower in the sarcopenia group than in the control group(t =11.724 and 4.173,P=0.000 and 0.000).FPG[(7.5±2.4) mmol/L vs.(8.5±2.5)mmol/L,t =-3.450,P=0.001]and HbA1c[(7.0±1.6) ％ vs.(8.2± 1.7) ％,t =-5.745,P =0.000] were higher in male sarcopenia group than in male control group.The control rate of FPG (127.0％ or 51.8％ vs.27.0％ or 32.9％,x2=8.817,P=0.003)and HbA1c(131.0％ or 53.5％ vs.23.0％ or 28.0％,x2 =15.934,P=0.000)were lower in the sarcopenia group than in the control group.The red blood cell counts,hemoglobin and serum albumin levels,estimated glomerular filtr ationrate(eGFR)were lower in male sarcopenia group than in the male control group(P＜0.05).eGFR was lower in female sarcopenia group than in female control group(t =4.090,P =0.000).Both in men and women,ASMI,grip power,fatless circumference on upper arm,bone mineral content and basal metabolic rate were lower in the sarcopenia group than in the control group(P＜0.05).The total daily intake of energy,carbohydrate,protein and fat were lower in male sarcopenia group than in male control group(P＜ 0.05).Conclusions Compared with the control group,sarcopenia patients are older with worse glycemic control and lower levels of BMI,WHR,renal function,muscle mass and muscle strength.Sarcopenia patients are more prone to osteoporosis.Furthermore,they have poorer nutritional status with an imbalance of dietary intake.Appropriate increase of protein especially high quality protein intake should be recommended to sarcopenia patients with T2DM.