Astragalus membranaceus(A. membranaceus), a traditional tonic, contains flavonoids as one of its main bioactive components and key indicators for quality standard detection. These compounds predominantly exist in glycosylated forms after glycosylation modification within the plant. The catalytic products of flavonoid glycosyltransferases in A. membranaceus have been reported to be mostly monoglycosides, and only AmUGT28 catalyzes luteolin to form diglycosides. In this study, we cloned a glycosyltransferase gene, AmGT90, from A. membranaceus, with an ORF length of 1 335 bp, encoding 444 amino acids, and the protein had a relative molecular mass of 50.5 kDa. Phylogenetic tree analysis indicated that AmGT90 belongs to the UGT74 family. In vitro enzymatic reaction showed that AmGT90 had broad substrate specificity and could catalyze the glycosylation of various flavonoids, including isoflavones, flavones, flavanones, and chalcones. AmGT90 not only catalyzed the formation of monoglycosides but also diglycosides. In addition, the mechanism of AmGT90 catalyzing the formation of diglycosides from luteolin was preliminarily explored. The experimental results showed that AmGT90 may preferentially recognize C4'-OH of luteolin and then recognize C7-OH to form diglycosides. This study reported a glycosyltransferase from A. membranaceus capable of converting flavonoids into monoglycosides and diglycosides. This finding not only enhances our understanding of the biosynthetic pathways of flavonoid glycosides in A. membranaceus but also introduces a new component for glycoside production through synthetic biology.
Glycosyltransferases/chemistry* ; Flavonoids/chemistry* ; Astragalus propinquus/classification* ; Phylogeny ; Glycosylation ; Plant Proteins/chemistry* ; Substrate Specificity ; Cloning, Molecular ; Amino Acid Sequence

OBJECTIVE: To study the results, re-donation situation and characteristics of single-reagent reactive blood donors who were put into the reentry strategy in Wuhan area, explore the rationality and effectiveness of the current reentry strategy, and provide data support for the improvement of the reentry process of blood donors. METHODS: From January 2020 to December 2023, blood donors who conform the reentry criteria and voluntarily applied for returning to Wuhan Blood Center were tested and the results were analyzed. According to the reentry strategy, serological testing and nucleic acid testing were carried out in parallel, serological testing was performed by ELISA with reagents from two different manufacturers, and the primary reactive samples were tested by double-well retest, and HBV/HCV/HIV nucleic acid detection was performed by RT-PCR with an individual donor test mode. Supplementary HBcAb testing was applied for HBV single reagent reactivity by chemiluminescence method. Supplementary TP-WB testing was applied for returning blood donors with repeated TP single reagent reactivity. If returning blood donors with HIV single reagent reactivity were repeated single reagent reactivity, the samples were sent to local CDC for confirmatory test. RESULTS: 7 098 blood donors were qualified for reentry, 716 donors voluntarily applied for reentry, 436 donors successfully reentry, 251 donors entered the next round, 29 donors could not reentry. The reentry rates for the past four years were 66.67%（42/63）, 54.73%（81/148）, 60.71%（136/224） and 62.99%（177/281）, respectively. Up to December 31, 2023, 275 donors donated blood again, and the donation rates for past four years were 76.19%（32/42）, 72.84%（59/81）, 61.76%（84/136） and 56.50%（100/177）, respectively. After donating blood, 31 donors were disqualified again by blood screening and subjected to permanent deferral. The results of returning to the team had statistical differences in reentry items, educational level, age, and marriage（P < 0.05）. CONCLUSION The current reentry strategy adopted by the blood donation and supply institution can effectively retain part of blood donors, reduce the negative emotions of blood donors and increase blood resources.
Humans ; Blood Donors ; China ; Hepatitis B ; Enzyme-Linked Immunosorbent Assay ; Hepatitis C ; Male

Objective:To explore the genetic and clinical characteristics of Guizhou patients with enlarged vestibular aqueduct（EVA） syndrome through combined SLC26A4 variant analysis and clinical phenotype analysis. Methods:Seventy-two EVA patients underwent comprehensive genetic testing using a multiplex PCR-based deafness gene panel and next-generation sequencing（NGS）. The audiological and temporal bone imaging characteristics were compared across mutation subtypes. Results:A total of 27 pathogenic loci of SLC26A4 were detected in 72 patients, including c.919-2A>G in 79.2%（57/72）. A novel deletion（c.1703_1707+6del） was discovered. Among 65 cases, truncated mutations were 89.2%（58/65）, 52.3%（34/65）, 28（43.1%） and 7（10.8%）. No significant differences were observed in the midpoint diameter of the vestibular aqueduct and the incidence of incomplete partitioning typeⅡ（IP-Ⅱ） of the cochlea among the three groups of patients. Moreover, there was no difference in the midpoint diameter of different vestibular pipes or the combination with IP-Ⅱ. Conclusion:The most common mutation site of SLC26A4 in EVA patients in Guizhou is c.919-2A>G, though genotype-phenotype correlations remain elusive. The detection of 27 mutation sites and the discovery of new mutation sites suggested the precise diagnostic significance of NGS technology in EVA patients in Guizhou.
Humans ; Sulfate Transporters ; Vestibular Aqueduct/abnormalities* ; Mutation ; Membrane Transport Proteins/genetics* ; Hearing Loss, Sensorineural/genetics* ; Male ; Female ; Child ; Adolescent ; Child, Preschool ; Adult ; Young Adult ; Phenotype ; High-Throughput Nucleotide Sequencing

Objective : To investigate the role of caspase recruitment domain-containing protein 9(Card9) inAspergillus fumigatus(A.fumigatus) keratitis and the effect of its deficiency on macrophage resistance to fungal infection. Methods : (1) C57BL/7 mice aged 6-8 weeks were selected and the mice pretreated Card9 siRNA and Blank siRNA, respectively, and the expression of Card9 in each group was detected by Western blot and RT-PCR. The corneal epithelium of the mice was scraped away 72 hours later, andA.fumigatusspore suspension was injected into the corneal stroma. The corneal scores were recorded at 1 d, 3 d, 5 d and 7 d after infection. The expression of Card9, nuclear factor κB(NF-κB), interleukin 1β(IL-1β), interleukin 6(IL-6) and tumor necrosis factor α(TNF-α) in each group was detected by RT-PCR and immunohistochemical(IHC).(2) Human corneal epithelial cells(HCECs) and human monocytic-leukemia cells(THP-1)in vitro, RT-PCR was used to examine the expression of Card9 gene in the two cells, and a stable cell line of THP-1 cells was constructed using shRNA vectors. The expression of Card9 in the cell line was detected by Western Blot and RT-PCR. The cells were induced into macrophages and stimulation byA.fumigatus, and the expression of Card9, NF-κB, IL-1β, IL-6 and TNF-α was detected by RT-PCR. Results : Card9 expression increased inA.fumigatuskeratitis, mainly distributed in cytoplasm of immune cells. The expression of Card9 in the cornea of mice treated with Card9 siRNA was significantly reduced. After inhibiting the expression of Card9 gene, the expressions of Card9, NF-κB, IL-1β, IL-6 and TNF-α significantly decreased and the changes of IL-1β were most significant. Inin vitrostudies, Card9 exhibited negligible expression in human corneal epithelial cells, contrasting with its pronounced expression in THP-1 cells. After the induction of macrophages, Card9, NF-κB, IL-1β, IL-6, and TNF-α were significantly upregulated under the stimulation ofA.fumigatus. After inhibiting the expression of Card9, the stimulated expression of these factors was significantly reduced, with the most notable change observed in IL-1β. Conclusion Card9 is involved in the inflammatory development and healing process ofA.fumigatuskeratitis. Card9 deficiency can cause functional impairment of macrophages and inhibit the expression of inflammatory factors to a certain extent, in which IL-1β has the greatest effect.

Objective To propose a measurement method based on improved YOLOv8 for the anterior segment parameters of ultrasonic biological microscopy(UBM)images to solve the problems of the ophthalmic ultrasound images in low quantity,high annotation cost and weak model generalization ability.Methods Firstly,data enhancement using StyleGAN3 was carried out to improve YOLOv8 model in decreased sensitivity to UBM images and increased generalization ability;secondly,the pseudo-labels of virtual UBM images were generated based on the YOLOv8 model with the original UBM images as the dataset and the method of pseudo-label self-training in semi-supervised learning;finally,the YOLOv8 model was trained with the expan-ded dataset and improved with the global context network(GCNet)module,and the prediction results of the YOLOv8 model were sorted using the key-point ranking algorithm,and the measurements of the anterior segment physiological parameters were computed after screening qualified images based on prior knowledge.Results Compared with the hand-labeled results by the ophthalmologists,the localization error of the StyleGAN3 data-enhanced and self-trained YOLOv8 model was(61.94±40.66)μm,and the mean relative errors for the measurements of anterior chamber angle distance,pupil diameter,ciliary sulcus distance,central corneal thickness,anterior chamber depth and lens thickness were 0.62％,1.35％,0.68％,4.87％,0.93％and 0.75％,respectively.Conclusion The method proposed enhances the accuracy of the measurement method for anterior segment parameters of UBM images,and can meet real-time requirements.[Chinese Medical Equipment Journal,2024,45(2):8-16]

Objective To investigate the correlation between optimal placement depth(OPD)and physical measurement parameters in preterm infants receiving placement of peripherally inserted central catheter(PICC)through the great saphenous vein(GSV),and to establish a predictive formula for OPD during the placement of PICC through the GSV.Methods A retrospective analysis was performed for the preterm infants who received the placement of PICC through the GSV in the Neonatal Intensive Care Unit of the Third Xiangya Hospital of Central South University from December 2022 to February 2024.According to the site of puncture[GSV of the knee joint(KJ)or the ankle joint(AJ)],they were divided into a GSV-KJ placement group(n=38)and a GSV-AJ placement group(n=33).The infants were measured in terms of body weight(BW),body length,the length of the upper and lower parts of the body,head circumference,and abdominal circumference at the time of placement.The Pearson correlation analysis was used to investigate the correlation between the above variables and OPD.A predictive formula was established for OPD in the placement of PICC via the GSV in preterm infants,and the predicted residual between the predicted depth and the ideal OPD was compared between the conventional predictive formula and the new predictive formula.Results The Pearson correlation analysis showed that PICC OPD was significantly positively correlated with BW,body length,the length of the upper and lower parts of the body,head circumference,and abdominal circumference in both the GSV-KJ placement group and GSV-AJ placement group(P<0.05),with the highest degree of correlation between OPD and BW.The univariate linear regression analysis showed a linear relationship between PICC OPD and BW in both groups.The predictive formulas for OPD were as follows:GSV-KJ PICC OPD(cm)=13.1+2.7×BW(kg)and GSV-AJ PICC OPD(cm)=13.4+6.0×BW(kg),and the new predictive formulas had a significantly lower predicted residual than the conventional predictive formula(P<0.05).Conclusions OPD for PICC through the GSV is positively correlated with BW,and the prediction results of the new predictive formula based on BW are closer to the ideal OPD.

Objective:To investigate the specimen quality of intersphincteric resection with transabdominal transanal mixed approach for rectal cancer in the Chinese Transanal Total Mesorectal Excision Registry Collaborative (CTRC) database.Methods:The retrospective case-control study was conducted. Based on the concept of real-world research, the clinicopathological data of 281 pati-ents with rectal cancer in the CTRC database who underwent intersphincteric resection with trans-abdominal transanal mixed approach in 19 medical centers, including the Beijing Friendship Hospital of Capital Medical University et al, from November 15,2017 to December 31,2023 were collected. There were 196 males and 85 females, aged 61(range, 27-87)years. Observation indicators: (1) preoperative examinations; (2) neoadjuvant therapy; (3) postoperative examinations; (4) analysis of influencing factors for positive circumferential margin in surgical specimen of intersphincteric resec-tion for rectal cancer. Measurement data with normal distribution were represented as Mean±SD. Measurement data with skewed distribution were represented as M(range). Count data were described as absolute numbers or percentages. The chi-square test was used for univariate analysis. Logistic regression model was used for multivariate analysis. Results:(1) Preoperative examinations. Of the 281 patients, 234 cases underwent preoperative pelvic magnetic resonance imaging (MRI) examina-tion. There were 2 cases in clinical stage T0, 3 cases in clinical stage T1, 58 cases in clinical stage T2, 137 cases in clinical stage T3, 24 cases in clinical stage T4, 3 cases in clinical stage Tx, 7 cases missing clinical T staging data. There were 87 cases in clinical stage N0, 68 cases in clinical stage N1, 60 cases in clinical stage N2, 9 cases in clinical stage Nx, 10 cases missing clinical N staging data. There were 30 cases with mesorectal fascia invasion, 53 cases with extramural venous invasion. The distance from lower margin of tumor to anal margin was 41.9(range, 1.0-80.0)mm. (2) Neoadjuvant therapy. Of the 281 patients, 125 cases underwent neoadjuvant therapy, including 39 cases receiving chemo-therapy alone, 6 cases receiving short-course simultaneous chemoradiotherapy, 5 cases receiving short-course simultaneous chemoradiotherapy and delayed surgery, 48 cases receiving long-course simultaneous chemoradiotherapy, 2 cases receiving other treatments, and 25 cases missing neoadju-vant therapy data. (3) Postoperative examinations. Of the 281 patients, 249 cases achieved R 0 resection, 9 cases achieved R 1 resection, and there were 23 cases missing surgical margin data. The maximum tumor diameter, the number of lymph nodes harvested and positive rate of vessel carcinoma embolus were 30.0(range, 0.5-200.0)mm, 13(range, 0-70) and 27.55%(73/265) in 281 patients. There were 252 patients with circumferential margin records, showing positive in 15 cases, with a positive rate as 5.95%(15/252). The minimum distance from deep part of tumor to circumferential margin was 7.0(range, 0-150.0)mm in 252 patients. There were 85 cases with distal margin records, showing positive in 1 case, and the distance from lower margin of tumor to distal margin was 10.0(range, 0-202.0)mm. There were 273 patients with specimen integrity records, which showed intact specimen in 208 cases, fair specimen in 58 cases, poor specimen in 4 cases, unevaluated specimen in 3 cases. There were 7 cases with rectal perforation. Of the 281 patients, cases in pathological stage T0, Tis, T1, T2, T3, T4 were 14, 5, 22, 107, 113, 12, respectively, and there were 8 cases missing pathological T staging data. Of the 281 patients, cases in pathological stage N0, N1a, N1b, N1c, N2a, N2b were 176, 27, 27, 11,20, 12, respectively, and there were 8 cases missing pathological N staging data. Of the 281 patients, there were 4 cases with distant metastasis, 262 cases without distant metastasis, 5 cases not evaluated, and 10 cases missing tumor metastasis data. Of the 125 patients undergoing neoadjuvant therapy, there were 85 cases with tumor regression grade records, including 16 cases as grade 1, 27 cases as grade 2, 19 cases as grade 3, 15 cases as grade 4, 8 cases as grade 5. (4) Analysis of influencing factors for positive circumferential margin in surgical specimen of intersphincteric resection for rectal cancer. Results of univariate analysis showed that preoperative T staging on preoperative pelvic MRI, mesorectal fascia invasion, extramural venous invasion, pathological T staging, and pathological N staging were related factors for positive circumferential margin in surgical specimen of intersphincteric resection for rectal cancer ( P<0.05). Conclusions:Intersph-incteric resection with transabdominal transanal mixed approach has good specimen quality and low positive rate of surgical margin. T staging on preoperative pelvic MRI may be related to positive circumferential margin after intersphincteric resection for rectal cancer.

Objective:To construct a novel chimeric antigen receptor T (CAR-T) cell targeting CD138 and to investigate its cytotoxicity against myeloma cells.Methods:The hybridoma strain that can stably secrete the CD138 monoclonal antibody (mAb) was prepared and obtained through monoclonal antibody screening technology. The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies, which were then collected and purified to obtain pure CD138 mAb. Further examinations were performed to assess the biological characteristics of CD138 mAb. The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR, which was subsequently expressed on the surface of T cells by lentiviral infection. Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells. In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects.Results:① We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain, 5G2, which could persistently and stably secrete the anti-CD138 antibody. ② The purified CD138 (5G2) mAb can especially recognize CD138 + cells with a binding affinity constant (K D) of 6.011×10 -9 mol/L and showed no significant binding activity with CD138 - cells. ③The variable region sequence of the CD138 (5G2) antibody was obtained using molecular cloning technology, and CD138 (5G2) CAR was successfully constructed and expressed on T cells through lentivirus infection and, concurrently, demonstrated effective binding to recombinant human CD138 protein.④ The proliferation of T cells transduced with the CD138 (5G2) CAR was highly efficient. The phenotype analysis revealed that CD138 (5G2) CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells, with a reduced proportion of terminally differentiated effector memory subsets. ⑤CD138 (5G2) CAR-T cells demonstrated specific cytotoxicity against CD138 + myeloma cell line H929, whereas CD138 - cell line K562 remained unaffected. The percentage of residual H929 cells was (12.92±8.02) % after co-culturing with CD138 (5G2) CAR-T cells, while (54.25±15.79) % was left in the Vector-T group (E∶T=1∶2; P<0.001). ⑥Results of degranulation assays demonstrated a significant activation of CD138 (5G2) CAR-T cells after co-culture with the H929 cell line, whereas no significant activation was observed in Vector-T cells [ (25.78±3.35) % vs (6.13±1.30) %, P<0.001]. ⑦After co-culturing with CD138 + cells, CD138 (5G2) CAR-T cells exhibited a significant increase in cytokine secretion compared to the Vector-T group [interleukin-2: (1 697.52±599.05) pg/ml vs (5.07±1.17) pg/ml, P<0.001; interferon-γ: (3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml, P<0.001; and tumor necrosis factor-α: (1 837.43±640.49) pg/ml vs (8.75±1.65) pg/ml, P<0.001]. However, no significant difference was observed in cytokine secretion levels between the two groups after co-culturing with CD138 - cells. Conclusion:This study successfully prepared a novel monoclonal antibody against CD138, and CAR-T cells constructed with the antigen recognition domain derived from this 5G2 mAb demonstrated effective antitumor activity against myeloma cells. This can be used as a new option for the detection of the CD138 antigen and proposes a novel strategy for multiple myeloma immunotherapy.

Objective:To investigate the predictive value of serum proprotein convertase subtilisin/kexin 9(PCSK9)level for major adverse cardiovascular events(MACE)in patients with acute myocardial infarction(AMI)after per-cutaneous coronary intervention(PCI).Methods:A total of 585 AMI patients who underwent PCI in our hospital from May 2016 to Aug 2020 were enrolled.General data and preoperative serum PCSK9 level were collected.Accord-ing to presence of MACE within one year after PCI,patients were divided into MACE group(n=152)and no MACE group(n=433).Correlation between blood lipids and serum PCSK9 level,and influencing factors for MACE within one year after PCI in AMI patients were analyzed.And the predictive value of serum PCSK9 level for MACE within one year after PCI in AMI patients was analyzed.Results:After one year follow-up,the incidence rate of MACE was 25.98％(152/585).Compared with no MACE group,there were significant rise in preoperative Gensini score,proportions of age≥60 years,multiple infarcted sites,number of diseased vessels ≥2,hypertension,diabe-tes,hyperlipidemia,LVEF＜50％and postoperative slow-flow/no-reflow,serum PCSK9 level[51.95(46.82,56.58)ng/ml vs.72.24(62.37,73.88)ng/ml]in MACE group,P＜0.01 all.Spearman correlation analysis indicated that in AMI patients,serum PCSK9 level was significant positively correlated with levels of total cholesterol(TC)and low den-sity lipoprotein cholesterol(LDL-C)(r=0.728,0.784,P=0.014,0.008).Multivariate Logistic regression analysis indi-cated that age≥60 years,preoperative Gensini score,multiple infarcted sites,number of diseased vessels≥2,hypertension,diabetes,hyperlipidemia,LVEF＜50％,slow-flow/no-reflow after PCI,serum PCSK9 level were independent risk fac-tors for MACE within one year after PCI in AMI patients(OR=2.757～6.888,P＜0.01 all).ROC curve analysis indica-ted that cut-off value,sensitivity,specificity and AUC(95％CI)of serum PCSK9 level was 59.11 ng/ml,88.72％,77.37％and 0.871(0.841～0.897)respectively for predicting MACE within one year after PCI in AMI patients,sugges-ting serum PCSK9 level possessed good predictive value for it.Conclusion:High serum PCSK9 level can increase the risk of MACE after PCI in AMI patients,which has good predictive value for it.

Elemental imaging analysis based on laser induced-breakdown spectroscopy(LIBS)can provide significant reference value for oil and gas exploration activities.Improving the scanning speed and spatial resolution of LIBS elemental imaging analysis instruments contributes to enhancing the efficiency of mineral surface elemental analysis,which is crucial for achieving in-situ,real-time,and rapid LIBS analysis.In this study,a high-speed scanning LIBS elemental imaging analysis instrument was developed based on a scanning mirror device,achieving a scanning speed of 100 Hz and a spatial resolution of 50 μm.The stability of spectral data collected by this instrument was validated using aluminum alloy standard samples with uniform elemental distribution.The experimental results showed that the relative standard deviations(RSD)of the spectral data collected at different locations were 2.76％,2.79％,2.35％and 2.55％,respectively,demonstrating that the instrument's performance met analysis requirements.Analysis of spectral acquisition channels led to the selection of the 337-595 nm spectral range.Imaging analysis of major elements on the surface of meteorite mineral samples with complex matrices was conducted using this instrument,coupled with a multi-element imaging algorithm enabling visualization analysis of four major elements on the same image.The results revealed a higher level of detail and complexity in element distribution.The study demonstrated that this instrument,combined with multi-element imaging analysis algorithms,could provide crucial technical support for rapid imaging of element distribution in minerals at a microscopic scale during geological research.