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 establish a low density, high purity and high stability in vitro culture method of primary hippocampal neurons of fetal rats by co-culturing hippocampal and cortical cells, so as to obtain higher purity and better vitality of primary hippocampal neurons disease. Methods The fetal rat hippocampal tissue was isolated from 16-18 days pregnant SD rats, then cut and digested by 0.125% trypsin. The obtained cell suspension was filtered by 200 mesh cell sieve, and then the obtained cell suspensions were then inoculated into the inner layer and outer ring of the culture plate in a surrounding form. They were co-cultured in DMEM/F12 medium containing 10% horse serum. After 4-6 hours of cell adhesion, the culture medium was changed to maintenance medium (Neurobasal+2% B27+0.5 mmol/L glutamine). Then the cell viability was detected with CCK-8 kit and the purity of hippocampal neurons was detected by immunofluorescent staining. Results Hippocampal neurons grew well and formed crisscross neural networks after 5 days. And it could survive for 3 weeks. The purity of hippocampal neurons was up to 98%. Conclusion The method of co-culturing hippocampal and cortical cells can obtain high-purity, high activity, high survival rate, and high stability primary hippocampal neurons from fetal rats, which can provide certain experimental conditions for the study of hippocampal neuron related diseases in the nervous system and is worthy of promotion and application.

Objective To investigate the effect of Ophiopogonin D on lipopolysaccharide(LPS)-induced apoptosis of alveolar epithelial cells by regulating the interleukin-6(IL-6)/Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway.Methods A549 AT Ⅱ cells cultured in vitro were randomly divided into four groups:control group,LPS group,LPS+Ophiopogonin D group,LPS+Ophiopogonin D+colivelin(JAK2/STAT3 signal activator)group,except for the control group,and cells in all other groups were established injury models while being grouped with Ophiopogonin D and colivelin for treatment.Cell counting kit-8(CCK-8)experiment and flow cytometry were applied to detect cell proliferation and apoptosis in each group;Western blotting was applied to detect the expression of IL-6/JAK2/STAT3 signaling pathway proteins of cells in each group.Results The apoptosis rates of A549 cells in control group,LPS group,LPS+Ophiopogonin D group and LPS+Ophiopogonin D+colivelin group were(2.52±0.73)％,(52.43±4.14)％,(1.67±0.52)％and(47.94±3.43)％;IL-6 protein levels were 0.14±0.03,0.49±0.05,0.17±0.04 and 0.45±0.06,and p-JAK2/JAK2 protein levels were 0.17±0.04,0.64±0.08,0.19±0.06 and 0.61±0.07;p-STAT3/STAT3 protein levels were 0.20±0.06,0.69±0.10,0.22±0.07 and 0.65±0.09;the apoptosis rates of AT Ⅱ cells were(3.01±0.69)％,(55.16±3.94)％,(2.35±0.71)％and(50.28±3.78)％;the levels of IL-6 protein were 0.11±0.03,0.87±0.13,0.19±0.04 and 0.84±0.12;the p-JAK2/JAK2 protein levels were 0.13±0.04,0.56±0.08,0.15±0.03 and 0.53±0.07;p-STAT3/STAT3 protein levels were 0.30±0.08,0.79±0.14,0.33±0.09 and 0.75±0.13.The above indexes:control group,LPS+Ophiopogonin D group compared with LPS group,LPS+Ophiopogonin D+colivelin group compared with LPS+Ophiopogonin D group,the differences were statistically significant(all P＜0.05).Conclusion Ophiopogonin D can reduce LPS induced inflammation and oxidative stress levels by inhibiting the activation of IL-6/JAK2/STAT3 signaling pathway,ultimately reducing LPS-induced apoptosis of alveolar epithelial cells.

Objective To investigate the effects of total flavones from Oxytropis falcata Bunge on hepatic fibrosis(HF)induced by carbon tetrachloride and liver transforming growth factor(TGF-β)/Smad signaling pathway.Methods Forty-eight male rats were randomly divided into normal group(intraperitoneal injection of peanut oil,intragastric administration of 0.9％NaCl),model group(intraperitoneal injection of 40％CC14 peanut oil solution induced HF model,intragastric administration of 0.9％NaCl),positive control group(modeling,intragastric administration of 0.2 mg·kg-1 of colchicine),experimental-L,-M,-H groups(modeling,intragastric administration of 100,200 and 400 mg·kg-1 of total flavonoid extract of Oxytropis falcata Bunge),8 individuals in each group,for 4 consecutive weeks.The histopathological changes were observed by hematoxylin-eosin and Masson staining.Serum liver function and liver fibrosis were measured;erum inflammatory factors were detected;fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to determine gene expression in liver.Results The pathological injury of liver tissue in the model group was serious,and a large number of inflammatory factors and collagen fibers were accumulated,while the rest of the treatment groups had different degrees of remission.In normal group,model group,positive control group,experimental-L,-M,-H groups,glutamic-pyruvic transaminase levels were(49.28±12.44),(5 885.42±948.37),(4 454.60±489.27),(4 650.47±843.53),(3 761.75±887.30)and(3 544.90±1 066.75)μg·L-1;glutamic-oxaloacetic transaminase levels were(186.90±46.89),(5 936.23±793.81),(3 971.37±780.28),(4 360.30±863.35),(3 943.10±439.47)and(3 971.38±631.08)μg·L-1;hyaluronic acid levels were(45.08±17.16),(104.32±36.06),(66.83±20.09),(70.30±21.07),(60.00±9.68)and(59.02±10.73)μg·L-1;laminin levels were(23.13±3.89),(60.85±13.66),(35.67±9.92),(39.98±9.39),(36.55±12.21)and(34.68±24.83)μg·L-1;type Ⅲ procollagen level were(24.98±5.34),(82.58±30.14),(40.70±16.14),(51.08±23.21),(43.60±12.48)and(44.20±11.66)p±g·L-1;interleukin(IL)-1β levels were(37.63±1.24),(46.10±3.23),(39.22±2.36),(41.33±0.93),(40.25±2.04)and(39.18±2.23)pg·mL-1;tumor necrosis factor-α levels were(314.58±20.56),(383.71±16.97),(349.00±7.93),(348.88±25.11),(325.75±27.84)and(335.07±21.33)pg·mL-1;TGF-β1 mRNA expression of relative quantity respectively were 1.00±0.00,60.99±15.70,9.61±1.59,7.37±1.09,6.41±0.64,6.87±1.09;Smad7 mRNA relative expression were 1.00±0.00,0.34±0.05,0.21±0.03,0.35±0.02,0.38±0.02,0.42±0.03.The above indexes in the model group were compared with the normal group,and the above indexes in the experimental-M,-H groups were compared with the model group,and the differences were statistically significant(P＜0.05,P＜0.01,P＜0.001).Conclusion Total flavonoids of Oxytropis falcata Bunge have protective effects on CC14-induced liver fibrosis in rats,and the mechanism may be related to the regulation of TGF-β/Smad pathway.

Objective:To investigate the clinical efficacy of three-connections and four-screwings technique in the treatment of high double column acetabular fractures through a single ilioinguinal approach.Methods:A retrospective study was conducted to analyze the data of 42 patients who had been treated for high double column acetabular fractures from June 2017 to June 2020 at Trauma Ward 2, Department of Orthopedics and Traumatology, The First Hospital of Traditional Chinese Medicine of Changde. There were 19 males and 23 females with an age of (42.7±25.6) years. 29 injuries were due to a traffic accident, 12 ones to fall from a height, and one to fall. The time from injury to operation was (4.5±2.1) days. All the patients were treated by the three-connections and four-screwings technique through a single ilioinguinal approach. Briefly, the anterior column was connected and secured to the main bone using 3 routes, and the posterior column was attached and fixated to the anterior column reset using 2 or 3 of the 4 screwings. The operation time, intraoperative blood loss, fracture reduction quality, fracture healing time, hip function at the last follow-up and complications during the follow-up were recorded.Results:For this cohort, the operation time was (150.0±30.5) min, and intraoperative blood loss (300.0±50.0) mL. According to the Matta scale for postoperative acetabular fracture reduction, 34 cases were excellent, 6 cases good, and 2 cases acceptable, with an excellent and good rate of 95.2% (40/42). After operation one patient had fat liquefaction and wound exudation which responded to drainage and dressing change. The 42 patients were followed up for (15.0±3.4) months. All fractures healed after (11.0±2.0) months. By the modified Merle d'Aubigné & Postel scoring system, the hip function was evaluated at the last follow-up as excellent in 33 cases, as good in 6 cases, and as fair in 3 cases, yielding an excellent and good rate of 92.9% (39/42).Conclusions:In the treatment of high double column acetabular fractures, the three-connections and four-screwings technique through a single ilioinguinal approach can lead to fine reduction and rigid fixation by lag screw compression and neutralization plate protection. Consequently, early functional exercises can be performed to secure good therapeutic outcomes for the patients.

Objective:To investigate the diagnostic value of nucleic acid matrix-assisted laser desorption ionization-time of flight mass spectrometry（MALDI-TOF MS）in sputum smear-negative patients with nontuberculous Mycobacterial（NTM）pulmonary disease.Methods:Clinical data of 123 patients suspected of NTM pulmonary disease admitted in Public Health Clinical Center Affiliated to Shandong University between July 2022 and November 2023 were retrospectively analyzed. Bronchoalveolar lavage fluid（BALF）specimens were collected for MALDI-TOF MS assay and MGIT 960 culture. The diagnostic efficacy of MALDI-TOF MS for NTM pulmonary disease in patients with negative sputum smears for acid-fast bacilli was evaluated with receiver operating characteristic（ROC）curve. Statistical analysis was performed using SPSS 26.0 software and MedCalc statistical software.Results:Diagnosis of NTM pulmonary disease was finally confirmed in 66 out of the 123 suspected patients. It took 8 to 24 h for MALDI-TOF MS to identify NTM species and resistance. By MALDI-TOF MS，72 NTM strains were identified，with the Mycobacterium avium complex being the most prevalent（34 strains，47.22%），followed by the Mycobacterium abscessus complex（13 strains，18.06%）；resistance to macrolides was detected in 6 cases，while no resistance to aminoglycosides was found. It took 9 to 45 days for BALF MGIT 960 culture to identify NTM，and took 7 to 15 days for NTM typing and drug sensitivity testing. By BALF MGIT 960 culture，28 NTM strains were identified；and 1 case was found to be resistant to macrolides. Using confirmed diagnosis as the gold standard，MALDI-TOF MS demonstrated higher sensitivity，negative predictive value，and agreement rate compared to MGIT 960 culture（84.85% vs. 42.42%，81.13% vs. 56.32%，80.49% vs. 62.60%， χ2=25.667，8.998，9.664， P<0.05 or <0.01）. The area under ROC curve（AUC）for MALDI-TOF MS was significantly higher than that of MGIT 960 culture（0.801 vs. 0.642， Z=3.300， P=0.001）. Conclusion:Compared to MGIT 960 culture，MALDI-TOF MS exhibits superior diagnostic efficiency in detecting NTM pulmonary disease in patients with acid-fast bacilli smear-negative sputum，with advantage of rapidly identifying NTM species and resistance.

Objective: To investigate the use of e-cigarette and analyze its influencing factors among high school students in Wuhan City, so as to provide insights into developing control measures for adolescents. Methods: From September to November 2019, high school students in Wuhan City were selected by the multi-stage stratified cluster random sampling method, and basic information, e-cigarette use and second-hand smoke exposure were collected through questionnaire surveys. Proportions of e-cigarette current use and attempt to use were analyzed, and factors affecting the current use of e-cigarettes were identified using a multivariable logistic regression model. Results: Totally 5 700 questionnaires were allocated, and 5 602 valid questionnaires were recovered, with an effective rate of 98.28%. The respondents included 2 925 males (52.21%) and 2 677 females (47.79%); 4 033 high school students (71.99%) and 1 569 vocational high school students (28.01%). The proportion of attempt to use e-cigarettes was 9.23%, and the proportion of current e-cigarette use was 2.03%. Multivariable logistic regression analysis identified male (OR=2.112, 95%CI: 1.285-3.471), vocational high school (OR=1.967, 95%CI: 1.214-3.186), private high school (OR=9.684, 95%CI: 5.648-16.605), family second-hand smoke exposure (OR=3.064, 95%CI: 1.741-5.392), second-hand smoke exposure in public places (OR=4.402, 95%CI: 1.687-11.484) and having close friends who were smokers (OR=6.432, 95%CI: 3.219-12.852) as factors affecting current e-cigarette use among high school students. Conclusions The proportion of current e-cigarette use among high school students in Wuhan City was 2.03%. Male, vocational high school, private high school, second-hand smoke exposure and having close friends who were smokers may be promoting factors for e-cigarette use.