ObjectiveTo investigate the effect of Liuwei Dihuangwan drug-containing serum （LDP） on epithelial-mesenchymal transition （EMT） and the expression of cancer stem cell （CSC） properties in 4T1 cells from triple-negative breast cancer by intervening myeloid-derived suppressor cells （MDSCs）. MethodsSPF-grade female SD rats were randomly divided into three groups， which were given 0.39， 1.94， 3.89 g·kg^-1·d^-1 suspension of Liuwei Dihuangwan for 7 days， respectively， to prepare low-， medium-， and high-dose LDPs. 4T1 cells were inoculated subcutaneously into the mammary glands of SPF-grade female Balb/c mice to construct a transplantation tumor model. Bone marrow cells were extracted from the tibia and femur and induced into MDSCs in vitro. The cell counting kit-8 （CCK-8） assay was used to detect the viability of 4T1 cells and MDSCs. The number of MDSCs and the expressions of CSC surface markers CD44 and CD24 in 4T1 cells were detected by flow cytometry （FC）. The migration， invasion， and proliferation of 4T1 cells were detected by cell scratch assay， Transwell invasion assay， and plate colony-forming assay， respectively. Western blot （WB） was used to detect the protein expression of transforming growth factor-β （TGF-β）， nuclear factor-κB （NF-κB）， C-X-C motif chemokine ligand 2 （CXCL2）， E-cadherin， and N-cadherin. The expression of EMT-related proteins E-cadherin and N-cadherin were detected by immunofluorescence （IF）. ResultsCompared with the normal group， LDP showed no significant inhibitory effect on the cell viability of 4T1 cells， but it significantly reduced the viability and number of MDSCs and reduced the number of MDSCs， as well as the expression of TGF-β （P<0.05， P<0.01）. The migration， invasion， and proliferation of 4T1 cells were increased after co-culture with MDSCs （P<0.05， P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin and the proportion of CSC （CD44⁺CD24^-） were elevated （P<0.05， P<0.01）， while the expression of E-cadherin was decreased （P<0.05）. After the intervention of MDSCs with LDP， followed by co-culture with 4T1 cells， the migration， invasion， and proliferation of 4T1 cells were obviously reduced （P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin were decreased （P<0.05， P<0.01）， and the expression of E-cadherin was increased （P<0.05， P<0.01）. There was no statistical difference in the proportion of CSC （CD44⁺CD24^-） in 4T1 cells. However， the proportion of CSC （CD44⁺CD24^-） was decreased in the co-culture system of 4T1 cells and MDSCs with LDP intervention （P<0.05， P<0.01）. ConclusionLDP can reduce the viability and number of MDSCs and the expression of TGF-β， NF-κB， and CXCL2， reverse EMT， and reduce the characteristic expression of CSC to inhibit the migration， invasion， and proliferation of 4T1 cells.

ObjectiveTo investigate the effect of Liuwei Dihuangwan drug-containing serum （LDP） on epithelial-mesenchymal transition （EMT） and the expression of cancer stem cell （CSC） properties in 4T1 cells from triple-negative breast cancer by intervening myeloid-derived suppressor cells （MDSCs）. MethodsSPF-grade female SD rats were randomly divided into three groups， which were given 0.39， 1.94， 3.89 g·kg^-1·d^-1 suspension of Liuwei Dihuangwan for 7 days， respectively， to prepare low-， medium-， and high-dose LDPs. 4T1 cells were inoculated subcutaneously into the mammary glands of SPF-grade female Balb/c mice to construct a transplantation tumor model. Bone marrow cells were extracted from the tibia and femur and induced into MDSCs in vitro. The cell counting kit-8 （CCK-8） assay was used to detect the viability of 4T1 cells and MDSCs. The number of MDSCs and the expressions of CSC surface markers CD44 and CD24 in 4T1 cells were detected by flow cytometry （FC）. The migration， invasion， and proliferation of 4T1 cells were detected by cell scratch assay， Transwell invasion assay， and plate colony-forming assay， respectively. Western blot （WB） was used to detect the protein expression of transforming growth factor-β （TGF-β）， nuclear factor-κB （NF-κB）， C-X-C motif chemokine ligand 2 （CXCL2）， E-cadherin， and N-cadherin. The expression of EMT-related proteins E-cadherin and N-cadherin were detected by immunofluorescence （IF）. ResultsCompared with the normal group， LDP showed no significant inhibitory effect on the cell viability of 4T1 cells， but it significantly reduced the viability and number of MDSCs and reduced the number of MDSCs， as well as the expression of TGF-β （P<0.05， P<0.01）. The migration， invasion， and proliferation of 4T1 cells were increased after co-culture with MDSCs （P<0.05， P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin and the proportion of CSC （CD44⁺CD24^-） were elevated （P<0.05， P<0.01）， while the expression of E-cadherin was decreased （P<0.05）. After the intervention of MDSCs with LDP， followed by co-culture with 4T1 cells， the migration， invasion， and proliferation of 4T1 cells were obviously reduced （P<0.01）. The expressions of NF-κB， CXCL2， and N-cadherin were decreased （P<0.05， P<0.01）， and the expression of E-cadherin was increased （P<0.05， P<0.01）. There was no statistical difference in the proportion of CSC （CD44⁺CD24^-） in 4T1 cells. However， the proportion of CSC （CD44⁺CD24^-） was decreased in the co-culture system of 4T1 cells and MDSCs with LDP intervention （P<0.05， P<0.01）. ConclusionLDP can reduce the viability and number of MDSCs and the expression of TGF-β， NF-κB， and CXCL2， reverse EMT， and reduce the characteristic expression of CSC to inhibit the migration， invasion， and proliferation of 4T1 cells.

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.

The accuracy of quantitative hormone testing has been concerned by clinical and laboratory professionals. Most of the small molecule hormones in human body have the characteristics of low concentration and similar structure, and need to be detected by high sensitivity and high specificity detection technology to achieve accurate quantification. In the past 20 years, liquid chromatography-tandem mass spectrometry (LC-MS) has gradually moved from scientific research to clinical application because of its unique sensitivity and specificity, which is considered to be a powerful supplement to traditional immunological methods, and has continuously achieved important fruits in clinical disease diagnosis and treatment. In order to further explore the application value of mass spectrometry technology in the quantitative detection of hormones, the Chinese Journal of Laboratory Medicine invited experts from the fields of clinical endocrinology and laboratory medicine to summarize their experience and opinions on the performance characteristics, challenges and future development direction of mass spectrometry technology in clinical hormone testing. Experts agreed that the advantages of mass spectrometry technology in hormone testing have been very clear, could effectively solve some clinical challenges in the diagnosis and treatment of endocrine diseases, but still faces a series of challenges such as insufficient talent reserve, low sample throughput, high operating costs and lack of standardized management.

Identifying the compound formulae-related xenobiotics in bio-samples is full of challenges.Conventional strategies always exhibit the insufficiencies in overall coverage,analytical efficiency,and degree of automation,and the results highly rely on the personal knowledge and experience.The goal of this work was to establish a software-aided approach,by integrating ultra-high performance liquid chromatography/ion-mobility quadrupole time-of-flight mass spectrometry(UHPLC/IM-QTOF-MS)and in-house high-definition MS2 library,to enhance the identification of prototypes and metabolites of the compound formulae in vivo,taking Sishen formula(SSF)as a template.Seven different MS2 acquisition methods were compared,which demonstrated the potency of a hybrid scan approach(namely high-definition data-independent/data-dependent acquisition(HDDIDDA))in the identification precision,MS1 coverage,and MS2 spectra quality.The HDDIDDA data for 55 reference compounds,four component drugs,and SSF,together with the rat bio-samples(e.g.,plasma,urine,feces,liver,and kidney),were acquired.Based on the UNIFI? platform(Waters),the efficient data processing workflows were estab-lished by combining mass defect filtering(MDF)-induced classification,diagnostic product ions(DPIs),and neutral loss filtering(NLF)-dominated structural confirmation.The high-definition MS2 spectral li-braries,dubbed in vitro-SSF and in vivo-SSF,were elaborated,enabling the efficient and automatic identification of SSF-associated xenobiotics in diverse rat bio-samples.Consequently,118 prototypes and 206 metabolites of SSF were identified,with the identification rate reaching 80.51％and 79.61％,respectively.The metabolic pathways mainly involved the oxidation,reduction,hydrolysis,sulfation,methylation,demethylation,acetylation,glucuronidation,and the combined reactions.Conclusively,the proposed strategy can drive the identification of compound formulae-related xenobiotics in vivo in an intelligent manner.

Objective To investigate the distribution and antimicrobial resistance of Enterobacter cloacae complex(ECC)isolated from a tertiary hospital in Shanghai.Methods Clinical ECC isolates were collected from 2018 to 2020.Antimicrobial susceptibility test was performed with broth microdilution and agar dilution methods.PCR was applied to detect five carbapenemase genes(blaKPC,blaNDM,blaIMP,blaVIM and blaOXA-48).Results A total of 222 ECC isolates were collected from 2018-2020,including 36 strains(16.2％)from general surgery department.The strains were mainly isolated from sputum(41.0％)and urine(20.3％).MIC results showed that the isolates were highly resistant to trimethoprim-sulfamethoxazole,the second-and third-generation cephalosporins,aztreonam and quinolones(31.1％-71.2％resistant),but low resistance rates to tigecycline,amikacin,mecillinam,and ceftazidime-avibactam(0.5％-9.0％resistant).About 9.5％and 10.4％of the strains were resistant to meropenem and imipenem,respectively.A total of 34 ECC strains were carbapenem-nonsusceptible strains,of which 23 strains were resistant to carbapenems.Among the 34 carbapenem-nonsusceptible strains,25(73.5％)strains were susceptible to mecillinam,including 15 strains producing metallo-β-lactamase.PCR assay identified carbapenemase genes in 21 of the 34 carbapenem-nonsusceptible strains,including blaNDM(14 strains),blaIMP(5 strains)and blaKPC(2 strains).Compared with the strains isolated in 2018,the ECC strains isolated in 2019 and 2020 showed significantly higher resistance rates to imipenem and ceftazidime-avibactam(P＜0.05),primarily associated with the production of NDM.Among the ECC strains resistant to third-generation cephalosporins,20.4％were resistant to imipenem and meropenem,while 4.9％and 8.7％were resistant to amikacin and mecillinam,respectively,and 9.7％were nonsusceptible to tigecycline.Conclusions The ECC isolates in 2019 and 2020 showed increasing resistance rates to carbapenems and ceftazidime-avibactam due to the production of metallo-β-lactamases.Some of the metallo-β-lactamase-producing ECC isolates were susceptible to mecillinam.

Objective:To explore the influence of family function and personal characteristics on death anxiety in patients with advanced cancer, providing reference for finding methods and approaches to alleviate death anxiety in advanced cancer patients.Methods:From March to June 2023, convenience sampling was used to select 182 advanced cancer patients admitted to the Cancer Center of the Fifth Affiliated Hospital of Sun Yat-sen University. The Chinese Version of Death and Dying Distress Scale and Family APGAR Index were used to investigate patients' death anxiety and family function. The Numerical Rating Scale and Kamofsky Performance Status were used to assess patients' pain and performance status. Single factor analysis and multiple linear regression were used to analyze the influencing factors of death anxiety in advanced cancer patients.Results:A total of 182 questionnaires were distributed, and 165 valid questionnaires were collected, with a valid response rate of 90.7%. The death anxiety score of advanced cancer patients was (22.52±15.27), and 10.3% (17/165) of patients had moderate or above death anxiety. The patients' total family function score was (8.62±1.97), and 86.7%(143/165) patients self-reported good family function. The death anxiety score was negatively correlated with the family function score ( P<0.05). Multiple linear regression analysis showed that Kamofsky Performance Status score, pre-illness employment, family function, place of residence, and pain score were the influencing factors of death anxiety in advanced cancer patients, and the differences were statistically significant ( R2=0.196, P<0.01) . Conclusions:The advanced cancer patients have low levels of death anxiety in our study. Advanced cancer patients with moderate family dysfunction, living in rural areas, working before illness, and high pain scores have high levels of death anxiety, while patients with good performance status have low levels of death anxiety. It is recommended that clinical workers strengthen the assessment of death anxiety and family function in patients with advanced cancer, take timely and effective measures based on influencing factors, and help alleviate death anxiety in patients with advanced cancer.

Objective:To evaluate the safety and necessity of vasopressor infusion through midline catheter.Methods:A convenient sampling method was used for a controlled study. A total of 88 adult patients who used vasopressors admitted to respiratory intensive care unit (RICU) of Fenyang Hospital in Shanxi Province from June 2022 to June 2023 were enrolled as the research subjects. A total of 44 patients who were infused with vasopressors through peripherally inserted central venous catheter (PICC) from June to December 2022 were enrolled as the PICC group, and 44 patients who were infused with vasopressors through midline catheter from January to June 2023 were enrolled as the midline catheter group. Both groups of patients used the modified Sedinger technique under the guidance of B-ultrasound for puncture and catheter placement. The middle 1/3 site between the cubital fossa and the axilla was selected. The catheters were 5 Fr double lumen. After catheter placement, the patients were followed until catheter removal, death, or 30 days (whichever came first). Based on the Infusion therapy standards of practice revised by American Infusion Nurses Society (INS), and combined with the results of previous preliminary tests, the safety evaluation was conducted on incomplete catheter obstruction, catheter-related bloodstream infection (CRBSI), phlebitis, thrombus within the catheter during extubation, redness of the puncture site (but no infection), and exudation of the puncture site in the two groups of patients.Results:There were no statistical differences in gender, age, catheter indwelling time, and primary disease between the two groups, indicating that the baseline data of the two groups were balanced and comparable. No CRBSI or phlebitis occurred in both groups during the observation period after catheterization. One patient in both groups had exudation at the puncture site [both were 2.27% (1/44)]. Compared with the PICC group, the incidence of incomplete catheter obstruction, thrombus within the catheter during extubation, redness of the puncture site (but no infection) in the midline catheter group were lowered [incomplete catheter obstruction: 4.55% (2/44) vs. 6.82% (3/44), thrombus within the catheter during extubation: 0% (0/44) vs. 2.27% (1/44), redness of the puncture site (but no infection): 0% (0/44) vs. 4.55% (2/44)], the overall incidence was significantly decreased [6.82% (3/44) vs. 15.91% (7/44), P < 0.01]. Conclusion:Administering vasopressor through a midline catheter can reduce the incidence of catheter-related complications, decrease the rate of central venous catheterization, and reduce the financial burden on patients.