Objective:To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis. Methods:Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein (Hcp) in wild type (WT), Δ aphA, and corresponding complementary strains. Quantitative reverse transcription PCR and luminescence activity assay of the promoter- lux fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes tssB2, hcp ( tssD2) and vgrG ( tssI2), and the quorum sensing regulator HapR in WT and Δ aphA strains. A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the tssD2b promoter region. Electrophoretic mobility shift assay (EMSA) was used to determine AphA binding to the hapR promoter. Results:The mRNA expression levels of tssB2, hcp( tssD2), vgrG ( tssI2), and hapR as well as the protein expression and secretion levels of Hcp in Δ aphA strain, were significantly higher than those in the WT strain. The promoter activities of the VflT6SS2 core cluster, tssD2a, tssI2a, and hapR were higher in Δ aphA strain than in the WT strain, while the promoter activity of tssD2b showed the opposite trend. The promoter sequence analysis of tssD2a and tssD2b found significant differences in the region from -335 bp to -229 bp, and two potential AphA binding sites on tssD2b. The promoter activity of tssD2b decreased significantly after the point mutation of the two potential AphA binding sites. EMSA results showed that AphA binds directly to the promoter region of hapR. Conclusions:AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of hapR. AphA showed opposite regulation patterns for tssD2a and tssD2b, and AphA could positively regulate the expression of tssD2b by directly binding to the tssD2b promoter region (-335 bp to -229 bp).

Objective:To establish a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) assay for the identification of common Salmonella serotypes and provide etiology evidence for the early precise treatment of salmonellosis. Methods:A total of 500 strains were collected from different regions and sources and five predominant Salmonella serotypes ( Salmonella Typhi , Salmonella Paratyphi A , Salmonella Typhimurium , Salmonella Enteritidis , and Salmonella Indiana) of each strain was identified by agglutination test and whole-genome sequencing. The protein complex of the strains was extracted by using optimized pretreatment method to establish the fingerprint database of peptides for each Salmonella serotype. The new serotyping assays were established by using different modules based on the mass spectra database. Additional 155 strains with specified serotypes and variant sources were used to test and evaluate the accuracy of the new typing assays. Results:Five MALDI-TOF MS databases were established, and two new serotyping assays were established via peptide fingerprint mapping/matching and machine learning of the neuronal convolutional network respectively based on the databases. The results showed that the fingerprint matching approach could quickly identify five common Salmonella serotypes in clinical practice compared with the machine learning method, the accuracy of fingerprint matching assay to identify five Salmonella serotypes reached 100.00% and the serotyping can be conducted within a short time (15-20 minutes) and had a good reproducibility, while the machine learning method could not completely identify these serotypes. Moreover the sensitivity and specificity of fingerprint matching assay were all 100.00% respectively, while they were only 82.23% and 95.81% for machine learning method. Conclusion:The established Salmonella serotyping assay based on MALDI-TOF MS in this study can easily, rapidly and accurately identify different serotypes of Salmonella.

Objective To investigate the predictive value of serum placental growth factor(PLGF)/soluble fms-like tyrosine kinase-1(sFlt-1),combined with the placental three-dimensional energy Doppler index(3D-PDI)in preeclampsia(PE).Methods From January 2021 to December 2022,120 pregnant women with PE risk factors were selected and followed up until 1 week after delivery.Serum PLGF and sFlt-1 levels were measured at routine prenatal check-ups at 14 to 20 weeks gestation.The PLGF/sFlt-1 ratio was calculated,and placental 3D-PDI was detected by ultrasound,including the vascularization index(VI),blood flow index(FI),and vascularization-blood flow index(VFI).Based on whether PE occurred after 20 weeks of pregnancy,cases were divided into PE(55 cases)and control groups(65 cases).The PE group was further divided into mild PE(35 cases)and severe PE groups(20 cases)based on the severity of the di-sease.The differences in PLGF/sFlt-1 and 3D-PDI between the groups were compared in terms of a statistical analysis of the correlation between PLGF,sFlt-1,and 3D-PDI.The receiver operating characteristic curve(ROC)was plotted,and the predictive value of each index on PE alone or in combination was analyzed.Results The systolic blood pressure(SBP),diastolic blood pressure(DBP),24 h protein-uria level,preterm birth rate,NICU admission rate,and preconception BMI in the PE group were higher than those in the control group(P＜0.05).The two groups had no differences in age,gestational age,pregnancy history,and fertility history(P＞0.05).The serum PLGF/sFlt-1 of the PE group was lower than that of the control group,and the serum PLGF/sFlt-1 of the severe group was lower than that of the mild group(P＜0.05).The 3D-PDI index of the PE group was lower than that of the control group,and the 3D-PDI index of the severe group was lower than that of the mild group(P＜0.05).Pearson's correlation analysis indicated that PLGF and VFI were signifi-cantly positively correlated(P＜0.01),and sFlt-1 was significantly negatively correlated with VFI(P＜0.01).ROC curve analysis showed that PLGF/sFlt-1,VI,FI,and VFI all had predictive value for PE and the value of VI,FI,and VFI jointly predicted PE,and was higher than that of various parameters(AUC = 0.951).Serum PLGF/sFlt-1,VI,FI,and VFI combined predicted the highest value(AUC=0.987).Conclusion In patients with PE,serum PLGF,sFlt-1,and placental VFI are significantly correlated.Serum PLGF/sFlt-1,placenta VI,FI,and VFI are reduced in early pregnancy,and the combined application of the four indicators has the highest efficacy in predicting PE,providing a possible reference for the early clinical screening or prediction of PE.

The goal of the study was to investigate the protective effect and mechanism of Artemisia argyi ethanol extract on chemotherapeutic vancomycin (VAN)-induced acute kidney injury (AKI).The acute kidney injury model of male ICR mice was induced by intraperitoneal injection (ip) of VAN.Thirty mice were divided into the blank group, model group, high dose group, middle dose group and low dose group, which were given medicine by gastric perfusion (ig).Serum levels of cystain C (Cys C), blood urea nitrogen (BUN) and serum creatinine (Scr) were measured, which could reflect renal function of mice.Serum oxidative stress and inflammation indices were also determined, including muscular dystrophy association (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), tumor necrosis factor-alpha (TNF-α), Interleukin-1β (IL-1β), and high-sensitive c-reactive protein (hs-CRP).In addition, hematoxylin-eosin staining (HE) was employed for measuring the damage of renal tissues and the content of apoptosis b-cell lymphoma-2 (Bcl-2), Bcl-2 assaciated X protein (Bax) and caspase-3 were measured too.All results showed that Artemisia argyi extract exhibits protective effect on chemotherapeutic VAN-induced AKI, whose mechanism could be related to the oxidative stress, inflammatory reaction and apoptosis.

Objective:To investigate the correlation between TNFAIP3 gene polymorphism and osteoporotic fractures in the elderly and bone metabolism indexes.Methods:A total of 115 patients with senile osteoporotic fractures admitted to Peace Hospital Affiliated to Shanxi Changzhi Medical College from Jan. 2019 to Jun. 2021 were enrolled as the observation group, and 120 patients with senile osteoporotic fractures matched with gender, age and body mass index of the observation group were selected as the control group. The levels of blood calcium, blood phosphorus, alkaline phosphatase, 25-hydroxyvitamin D and other bone metabolism indexes of all subjects were recorded. The genotypes of rs10499194 and rs13207033 in TNFAIP3 gene were analyzed by capillary electrophoresis and fragment analysis (SNaPshot) . The mRNA relative expression level of TNFAIP3 gene in peripheral blood of all subjects was determined by quantitative real-time fluorescence quantitative polymerase chain reaction.Results:There were statistically significant differences in genotype distribution and gene frequency of RS10499194 and RS13207033 between the observation group and the control group ( P<0.05) . For rs10499194, CT genotype carriers had a significantly higher risk of fracture than wild-type CC genotype carriers [OR=3.253 (1.223-8.652) , P=0.014]. The dominant model was also statistically significant ( P=0.003) . For rs13207033, GA carriers had a significantly higher risk of fracture than wild-type GG carriers [OR=3.775 (1.192-11.952) , P= 0.016]. The dominant model was statistically significant ( P=0.009) . The blood calcium level in AA+GA group was significantly higher than that in GG group at rs13207033 site ( P=0.006) . The mRNA expression level of TNFAIP3 gene in the observation group was 1.41±0.09, which was significantly lower than that in the observation group (2.07±0.12, t=6.69, P<0.001) . TNFAIP3 mRNA expression level of rs10499194 site TT+CT group was 1.35±0.11, significantly lower than CC group 1.43±0.13 ( t=2.82, P=0.007) . Conclusion:The polymorphism of TN-FAIP3 gene is related to the occurrence of osteoporotic fractures and bone metabolism, and may affect the gene expression level.

Objective:To observe the differences in scleral lamina curvature (SLC) of patients with pseudoexfoliation glaucoma (PXG), primary open-angle glaucoma (POAG), and primary chronic angle-closure glaucoma (CPACG) were compared and analyzed and their significance was analyzed.Methods:A retrospective clinical study. From June 2017 to December 2020, 30 PXG (PXG group), POAG (POAG group) and CPACG patients (CPACG group) diagnosed at Eye Center of Cangnan County people's Hospital of Zhejiang Province (Cangnan Hospital Affiliated to Wenzhou Medical University) were included in the study. The age difference between the three groups of patients was statistically significant ( t=17.925, P=0.001); gender composition ratio ( χ2=2.158, P=0.276), intraocular pressure ( t=4.993, P=0.078), and axial length ( t=1.956, P=0.532), central corneal thickness ( t=1.407, P=0.724), average visual field defect ( t=2.725, P=0.496), optic disc retinal nerve fiber layer thickness ( t=2.185, P=0.492) in comparison, the differences were not statistically significant ( P>0.05). The frequency-domain optical coherence tomography deep-enhanced imaging (OCT EDI) technology was used to measure the average and 0°, 30°, 60°, 90°, 120°, 150° SLC of the affected eyes, and calculate the SLC index (SLCI) and SL curve depth (SLCD). Quantitative data comparison between groups used independent sample t test. Count data comparison used χ2 test. Univariate and multivariate logistic regression analysis were used for correlation analysis. Results:The results of OCT EDI examination showed that the SLC of eyes with PXG and CPACG was significantly steep, while the SLC of eyes with POAG was relatively flat. Except for the angle of 150°, the other 6 angles of SLCI and SLCD in the PXG group and CPACG group were higher than those in the POAG group, and the differences were statistically significant ( P<0.05). However, there was no statistically significant difference between PXG group and CPACG group for 7 angles of SLCI and SLCD ( P>0.05). Logistic regression analysis showed that the average SLCI [odds ratio (OR)=1.498, 95% confidence interval ( CI) 1.137-2.018, P=0.001], age ( OR=1.074, 95% CI 1.019-1.143, P=0.016) was significantly correlated with PXG; mean SLCI ( OR=1.625, 95% CI 1.192-1.997, P=0.001), intraocular pressure ( OR=1.383, 95% CI 1.106-1.993, P=0.012) was significantly correlated with CPACG. POAG group ( β=0.143, 95% CI 0.032- 0.208, P=0.016), CPACG group ( β=0.132, 95% CI 0.079-0.315, P=0.043) intraocular pressure was correlated with mean SLCI; all factors of PXG group were correlated with SLCI without correlation ( P>0.05). Conclusion:Compared with POAG, the SLC of eyes with PXG and CPACG is steeper and related to disease occurrence.

Objective:To evaluate the early analgesic effect of new non-steroidal anti-inflammatory drugs 0.1% bromofenac sodium hydrate eye drops in pterygium surgery and the efficacy and safety on relieving the irritative symptoms.Methods:One hundred patients (112 eyes) with pterygium who were treated in Cangnan People′s Hospital (Cangnan Hospital Affiliated of Wenzhou Medical University) from January 2016 to September 2017 were selected and divided into observation group and control group by the hospital sequence, with 50 patients in each group. Before operation and 7 d after operation, the patients in two group were given topical tobramycin eye drops (4 times a day), and the patients in observation group were given 0.1% bromofenac sodium hydrate eye drops twice a day on the base of tobramycin eye drops. The patients were investigated early after operation and the pain scores of the two groups were compared 6, 8, 24, 48 and 72 h after operation.The corneal epithelial healing was compared too.Results:The pain scores in observation group 6, 8, 24, 48 and 72 h after operation were significantly lower than that in control group [(0.96 ± 0.13) scores vs. (2.21 ± 0.07) scores, (0.90 ± 0.05)scores vs. (1.71 ± 0.30) scores, (0.71 ± 0.07) scores vs.(1.54 ± 0.09) scores, (0.51 ± 0.04) scores vs. (1.31 ± 0.10) scores, (0.21 ± 0.02) scores vs. (0.80 ± 0.1) scores], and the differences were statistically significant ( P<0.05). The corneal wound healing rate of the two groups 2 or 3 d after operaion had no significant differences and the corneal wound healing rate 7 d after operaion was 100.0% (57/57, 55/55) in two groups. Conclusions:In pterygium surgery patients, using 0.1% bromfenac sodium hydrate drops before and after operation to relieve pain is valid, and can effectively relieve early postoperative pain.

Objective To investigate the application and the effects of individualized hollow resin plugs in the prevention of external auditory canal stenosis after canaloplasty of external auditory meatus for microtia patients . Methods The clinical data of 65 patients (68 ears) with microtia and aural atresia hospitalized in our department from January ,2006 to December ,2015 were summarized .Otoplasty of external ears and canaloplasty of external auditory meatus were operated on all patients under general anesthesia .Thigh flap transplantation was lined in the bony canal .One month later ,individualized hollow resin plugs were made and worn for 6 to 12 months ,and fol-lowed up for 1～2 years .Results The shapes of the reconstructed auricles were satisfactory .Sixty-four ears with wide external auditory canal openings were recorded in 68 ears .Preoperative hearing was 70 .45 ± 5 .5 dB HL ,and postoperative hearing was 55 .55 ± 5 .2 dB HL .Their hearing was not affected .External auditory canal restenosis occurred in 4 ears .Conclusion The individualized hollow resin plugs do not affect the hearing in use ,and can effec-tively prevent the restenosis of the external auditory canal after microtia reconstruction .

Objective To investigate the effect of intense low frequency noise on the different hearing functions and hair cell damage in bama pigs.Methods Thirteen bama pigs were randomly divided into a normal control group (3 pigs) and an experimental group (10 pigs).The pigs in experimental group were randomly divided into 50 Hz subgroup (5 pigs) and 70 Hz subgroup (5 pigs).The 50 Hz subgroup was exposed to intense low frequency noise at 167～170 dB SPL,50 Hz for 30 min,and the 70 Hz subgroup was exposed to intense low frequency noise at 164 dB SPL,70 Hz for 30 min.Auditory brainstem response(ABR) and distortion product otoacoustic emission (DPOAE) were performed before and after noise exposure.The cochlear were collected and the inner ear morphology changes were studied.Results Before the experiment,the ABR threshold of the pig was 20～50 dB SPL,and the average of 50 Hz group was 33.5±9.4 dB SPL,the average of the 70 Hz group was 34.0±4.6 dB SPL.The level of the above 3 000 Hz DPOAE could be elicited.But they were not elicited after the exposure to the noise.The inner ear structures were damaged.The DAPI showed hair cell missing.Conclusion ABR and DPOAE were elevated after noise exposure.The auditory system of bama pigs had irreversible damages.Hair cells damaged were given priority to with necrosis,and the higher the level of intense low frequency noise,the more serious the damage to the hair cell.

Objective]To study the feasibility of Cartilage engineering using fibrin gel and chondrocyte cell sheets.[Methods]rabbit auricular chondrocytes were isolated and cultured to form cell sheets in flasks. The cell sheets were harvested using cell scrapers,and cut into fragments. The two precursor solutions of Fibrin gel were used to suspend the cell sheet fragments and isolated chondrocytes,and then added into the wells of a 48-well plate to form Gelatinous chondroid disc constructs. After in vitro culture, the constructs were implanted into nude mice. After 8 weeks,the constructs were harvested,and the specimens were evaluated using grossly observing, histological and immunohistochemical observation. [Results]Mature cartilage discs were obtained. The histomorphology of the explanted discs appeared non-uniform cartilaginous tissue comprise of regenerated cartilage islands with different size and irregular shape. Immunohistochemistry staining demonstrated that type II collagen highly expressed in the ECM of the cartilage islands. In 1 of the 8 discs,partial ossification was observed.[Conclusion]Fibrin gel is a favourable carrier. Artificial cartilage with stereochemical structure was constructed via combining the fibrin gel and chondrocyte cell sheets.