Objective To investigate the influence of repeated open field tests， novel object recognition tests， and Barnes maze behavioral tests within a short term on cognitive and anxiety assessment in 3xTg-AD mice.Methods Four groups of 3xTg-AD mice， aged 12 months， were obtained using different pretreatment regimens， and the mice in the experimental groups 1-3 were treated with different drugs， while those in the control group were treated with normal saline. The open field test， the novel object recognition test， and the Barnes maze test were performed on mice successively. A behavioral video analysis system was used to record the locomotor trajectories of the mice and analyze the parameters such as time spent in the central area， exploration time for novel versus familiar objects， and latency to reach the target hole. After one session of complete tests， three sessions were performed repeatedly， and all tests were completed within one month.Results With the increase in the number of repeated tests， there was a significant reduction in the time spent in the central area in the open field test （P<0.05）； in the novel object recognition test， there was an increase in the coefficient of variation for object recognition index and a reduction in exploratory behavior towards both the new environment and the novel objects； in the Barnes maze test， there was no significant difference in the latency to reach the target hole across the four tests， suggesting good reproducibility.Conclusion Repeated tests within a short term can interfere with the evaluation of anxiety status in 3xTg-AD mice in the open field test and their preference for novel objects in the novel object recognition test， but it has no obvious influence on the latency to reach the target hole in the Barnes maze test. Whether behavioral tests can be performed repeatedly in 3xTg-AD mice should be determined based on different tests.

OBJECTIVE To establish a mouse model of diabetes mellitus(DM)combined with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection to investigate the important pathophysiological changes in the development of DM combined with SARS-CoV-2 infection.METHODS Wild-type(WT)mice and transgenic mice expressing the human angiotensin-converting enzyme 2 receptor driven by the cytokeratin-18 gene promoter(K18-hACE2)were randomly divided into the control group,DM group,SARS-CoV-2 spike protein(S)infection group and DM combined with S protein infection group,with 10 to 12 mice in each group.All the mice were induced by 10 weeks of high-fat diet combined with 40 mg·kg-1 streptozotocin(STZ)for 3 days by ip,except those in the control group or S protein infection group.The control group was given the same volume of 0.1 mol·L-1 sodium citrate buffer.Mice in the S protein infection group and DM+S protein infection group were additionally given 50 μL mixture of 15 μg SARS-CoV-2 spike protein and 1 g·L-1 polyinosinic-polycytidylic acid(poly[I:C])via intranasal drops,while the control group was given an equal volume of sterile water.The glucose tolerance level and pancreatic islet β cell function of mice were evaluated via oral glucose tolerance test at the 6th week of high-fat feeding and 1 week after the administration of STZ by ip.From the 6th week of high-fat feeding to 2 weeks after the administration of STZ,the random blood glucose and fasting blood glucose of mice were measured by a blood glucose meter.Blood samples were taken from subman-dibular veins of 3 mice in each group at 24,48 and 120 h after S protein infection,and lung tissues were taken after euthanization.The pathological changes of lungs of DM mice before and after S protein infection were observed by HE staining.Except for the DM group,blood samples were collected before S protein infection and at 6,24,48,72 and 120 h after infection.The levels of plasma interleukin 1β(IL-1β),IL-2,IL-6,IL-10,IL-17,interferon gamma-induced protein 10(IP-10),interferon γ(IFN-γ),tumor necrosis factor α(TNF-α),monocyte chemotactic protein-1(MCP-1)and granulocyte-colony stimulating factor(G-CSF)were detected by Luminex.The plasma levels of heparan sulfate(HS)were measured by enzyme-linked immunosorbent assay.The levels of cytokines and HS were correlated with the degree of pathological damage by Spearman correlation analysis.RESULTS STZ and high-fat diet could induce DM-like expression in mice,and the random blood glucose(P<0.01)and fasting blood glucose(P<0.05)after 1 week in the hACE2-DM group were significantly higher than in the WT-DM group,and the degree of islet function damage in hACE2-DM mice was significantly higher than that of WT-DM mice(P<0.05).Compared with the DM group,the DM+S group showed more severe pulmonary pathological changes after S protein infection,accompanied by a large number of inflammatory infiltrations and thickening of lung interstitial.Compared with the control group,the levels of pro-inflammatory cytokines G-CSF,IL-6 and IP-10 in the plasma of the WT-S group were significantly increased at 6 h after S pro-tein infection(P<0.01),and those of pro-inflammatory cytokine IL-17 and anti-inflammatory cytokine IL-10 were significantly increased at 24 h after S protein infection(P<0.05).Compared with the control group,the plasma levels of pro-inflammatory cytokines IL-1β,IL-6,TNF-α,MCP-1,G-CSF and IP-10 in the hACE2-S group were significantly increased at 6 h after S protein infection(P<0.05,P<0.01).IL-17 was significantly increased at 24 h and 6 h after S protein infection in the WT-DM+S group and hACE2-DM+S group,respectively(P<0.01,P<0.05).In the hACE2-DM+S group,IFN-γ and IL-1β were signifi-cantly increased in delay to 48 h(P<0.05,P<0.01),and MCP-1 was significantly increased in delay to 72h(P<0.05).Compared with the control group,the level of HS in the plasma of the WT-S group increased significantly(P<0.05,P<0.01)at 6 h and 24 h after S protein infection,but began to decrease at 48 h.At the same time,compared with the WT-S group,the HS level in the WT-DM+S group was slightly increased at 6 h after infection and decreased at 24 h.Compared with the control group,the HS level in the hACE2-S group was significantly increased at 24 h(P<0.01),as was the case with the WT-S group 24 h,48 h and 120 h after S protein infection.At 6 h,24 h and 48 h after S protein infection,the plasma HS level of the hACE2-DM+S group was significantly increased(P<0.01,P<0.05),and the duration of the increase was longer than in the hACE2-S group.Moreover,the levels of IL-1β,IL-10,MCP-1,IP-10,G-CSF and HS in plasma were positively correlated with the degree of lung dam-age in the DM+S group.CONCLUSION In this study,the mouse model of diabetes combined with SARS-CoV-2 spike protein infection has mimicked part of the pathophysiological features of clinical patients,mainly manifested as blunted immune response and elevated HS levels with longer duration to infection alone.IL-1β,IL-10,MCP-1,IP-10,G-CSF and HS may keep track of the course of disease in patients with diabetes combined with SARS-CoV-2 infection.

Objective:To establish an HPLC method for benzyl benzoate related substances and propose revision suggestions for the quality standard of this excipient.Methods:Phenylsilane bond and silica gel were used as fill-ers,and methanol-1％acetic acid(60:40)was used as the mobile phase at the flow rate of 1.0 mL·min-1.The detection wavelength was 254 nm and the injection amount was 20 μL.Results:Based on the results of the de-struction test and taking into account the separation requirements,the chromatographic conditions for benzyl benzo-ate related substances were confirmed and methodological validation was conducted.The detection limits for benzoic acid and benzaldehyde in related substances were 0.2 μg·mL-1 and 0.01 μg·mL-1.The correction factors for benzoic acid and benzaldehyde were 1.0 and 0.1,respectively.According to the newly established method,six batches of benzyl benzoate samples from four enterprises were inspected for related substances,and benzaldehyde was detected with content from 0.02％to 0.1％.The total amount of impurities was from 0.02％to 0.9％.Conclusion:The newly established method can be added to the quality standards of benzyl benzoate,and different impurity limits can be formulated according to the different administration routes in order to better control drug quality.

Objective:To explore the factors affecting and improvement recommendations about the congealing temperature of benzyl benzoate as pharmaceutical excipient by the method in the Chinese pharmacopoeia.Methods:The influence of seven factors such as measuring device,sample size,melting temperature,condensation tempera-ture,stop stirring time,stirring frequency and reading of congealing temperature on the determination of congealing temperature of benzyl benzoate was investigated.Results:The key factor for determination of benzyl benzoate con-gealing temperature was the stop stirring time,which was more reasonable when the sample temperature drops to constant or starts to rise slightly was reasonable.Conclusion:The improved method has better accuracy and repeat-ability,and is suitable for the measuring the congealing temperature of pharmaceutical excipient benzyl benzoate.

Background and Aims:Accurate early pathogen diagnosis is a breakthrough for improving the prognosis of infectious pancreatic necrosis(IPN)patients.However,there is currently a lack of efficient methods for early identification of IPN in clinical settings.This study was performed to assess the application value of next-generation sequencing technology based on metagenomic capture(MetaCAP)in the pathogen diagnosis of IPN. Methods:A prospective study was conducted on 29 patients suspected of having acute necrotizing pancreatitis at Xiangya Hospital of Central South University between January and July 2024.Blood samples were tested using MetaCAP and conventional pathogen culture.The results of peritoneal fluid pathogen culture were used as the gold standard to compare the diagnostic efficacy of the two methods. Results:Due to three cases lacking peritoneal fluid culture results,a total of 26 cases were included in the final analysis.The overall mortality rate was 23.1％(6/26).During hospitalization,9 cases(34.6％)were diagnosed with IPN.The sensitivity and negative predictive value of MetaCAP for diagnosing IPN were significantly higher than those of conventional pathogen culture(77.8％vs.11.1％,P=0.031;86.7％vs.65.2％,P=0.032),while the differences in specificity(76.5％vs.88.2％,P=0.689)and positive predictive value(63.6％vs.33.3％,P=0.347)between the two methods were not statistically significant.The average detection time for MetaCAP was 33(20-49)h,while microbial culture took 125(45-142)h,with a significant difference(P＜0.001).The average cost for blood MetaCAP testing was 2 500 yuan per case,but it accounted for only 1.19％of the average hospitalization cost. Conclusion:MetaCAP has significant value in the early pathogen diagnosis of IPN,with a shorter detection time,good testing efficacy,and health-economic value,demonstrating a promising clinical application prospect.

In order to understand the prevalence and genetic variation of H9N2 subtype avian influ-enza virus in Shandong,a total 492 tracheal and lung tissue samples collected from chicken farms with respiratory symptoms in partial areas in Shandong were detected by H9 subtype AIV real-time RT-PCR,and the positive samples were inoculated with chicken embryos for two generations.Whole genome sequences of the positive strains by applying Illumina Miaseq platform,and genetic evolution and mutation at positions associating with viral pathogenicity and transmissibility were analyzed.The results showed that there were 72 samples were positive for H9 subtype AIV among the 492 samples,with a positive rate of 14.63％.Thirty-four strains of H9 subtype AIV were ob-tained from the positive samples after passing through chicken embryo,meanwhile,the 34 isolates were all H9N2 subtype AIV by whole genome sequencing analysis.By analyzing the evolutionary tree of HA and NA genes,HA and NA genes of the 34 H9N2 AIV strains belonged to Y280-like branch and F/98-like branch,respectively.Meanwhile,based on above branches,there were obvious time node subbranch,which one was"isolates before 2013",another one was"isolates after 2013".The HA cleavage sites of thirty-four H9N2 strains were all 325PSRSSR↓GLF333,which met the se-quence characteristics of the lowly pathogenic avian influenza virus,and the HA receptor binding site 226 amino acid was leucine,which had the characteristics of blinding to a-2,6 mammalian sialic acid receptors.Among the internal amino acid sites that are key to mammalian adaptation,all strains had an I368V mutation in the PB1 gene that enhanced viral transmissibility in mammals and the PB2 genes of some strains were mutated to enhance the mammalian adaptation of I292 V and A588 V.The above results illustrated that the H9N2 subtype AIV gene segments in Shandong have different degrees of recombination and gene variation,so it is necessary to strengthen the monito-ring of virus variation.

Objective:To investigate the application value of metagenomic next-genera-tion sequencing (mNGS) in pathogenic diagnosis of suspected infected severe acute pancreatitis (SAP).Methods:The prospective study was conducted. The clinical data of 25 patients with suspected infected SAP who were admitted to the Xiangya Hospital of Central South University from May to September 2023 were collected. Upper limb venous blood samples of all the patients were collected for both of mNGS and routine pathogen microbial culture. Observation indicators: (1) grouping situations of the enrolled patients; (2) comparison of the diagnostic efficiency of mNGS and routine pathogen microbial culture; (3) results of peripheral blood pathogen microbial testing and peri-pancreatic effusion microbial culture; (4) testing time and cost. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the independent sample t test. Measurement data with skewed distribution were represented as M( Q1, Q3). Count data were expressed as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test. Results:(1) Grouping situations of the enrolled patients. A total of 25 patients were selected for eligibility. There were 18 males and 7 females, aged 48(40,59)years. The duration of hospital stay of 25 patients was 30(20,50)days. The etiologies of 25 patients included 14 cases of hyperlipidemic pancreatitis, 8 cases of biliary pancreatitis, 1 case of alcohol-induced acute pancreatitis, and 2 cases of pancreatitis caused by other causes. Of the 25 patients, there were 17 cases with infected pancreatic necrosis (IPN) including 7 cases of death, and 8 cases with sterile pancreatic necrosis including no death. (2) Comparison of the diagnostic efficiency of mNGS and routine pathogen microbial culture. The positive rates of mNGS and routine pathogen microbial culture in diagnosis of suspected infected SAP were 72.0%(18/25) and 32.0%(8/25), respectively, showing a significant difference between them ( χ2=8.01, P<0.05). The sensitivity and negative predic-tive value of mNGS and routine pathogen microbial culture in diagnosis of IPN were 94.1%(16/17), 35.3%(6/17) and 85.7%(6/7), 35.3%(6/17), showing significant differences between them ( χ2=12.88, 5.04, P<0.05). The specificity and positive predictive value of mNGS and routine pathogen microbial culture in diagnosis of IPN were 75.0%(6/8), 75.0%(6/8) and 88.9%(16/18), 75.0%(6/8), showing no significant difference between them ( χ2=0, 0.82, P>0.05). (3) Results of peripheral blood pathogen microbial testing and peripancreatic effusion microbial culture. Of the 17 patients with IPN, 36 strains of pathogenic bacteria were detected by mNGS, and 6 strains were detected by routine pathogen microbial culture. There were 16 of 17 patients with IPN showing positive mNGS pathogenic testing, of which 13 cases were consistent with the pathogenic testing results of peri-pancreatic effusion microbial culture, showing a consistency rate of 76.5%(13/17). There were 6 pati-ents with IPN showing positive routine pathogen microbial culture, with a consistency rate of 35.3%(6/17) to peripancreatic effusion microbial culture. (4) Testing time and cost. Testing time of mNGS and routine pathogen microbial culture were (43±17)hours and (111±36)hours, showing a signifi-cant difference between them ( t=9.31, P<0.05). Testing cost of mNGS was (2 267±0)yuan/case, accoun-ting for 1.7% of the hospitalization expenses of (133 759±120 744)yuan/case. Testing cost of routine pathogen microbial culture was (240±0)yuan/case, accounting of 0.2% of the hospitalization expenses. Conclusion:mNGS has important value for early pathogenic diagnosis of suspected infected SAP, and has a high timeliness.

Objective:To analyze the clinical features of postpartum hepatitis flares in pregnant women with hepatitis B virus (HBV) infection.Methods:A retrospective study was conducted. Patients who met the enrollment criteria were included. Liver function and HBV virology tests were collected from pregnant women with chronic HBV infection at delivery, 6, 24, 36, and 48 weeks after delivery through the hospital information and test system. Additionally, antiviral therapy types and drug withdrawal times were collected. Statistical analysis was performed on all the resulting data.Results:A total of 533 pregnant women who met the inclusion criteria were included, with all patients aged (29.5±3.7) years old. A total of 408 cases received antiviral drugs during pregnancy to interrupt mother-to-child transmission. There was no significant difference in the levels of alanine aminotransferase (ALT, z ?=?-1.981, P ?=?0.048), aspartate aminotransferase (AST, z ?=?-3.956, P ?

The human oral microbiome harbors one of the most diverse microbial communities in the human body, playing critical roles in oral and systemic health. Recent technological innovations are propelling the characterization and manipulation of oral microbiota. High-throughput sequencing enables comprehensive taxonomic and functional profiling of oral microbiomes. New long-read platforms improve genome assembly from complex samples. Single-cell genomics provides insights into uncultured taxa. Advanced imaging modalities including fluorescence, mass spectrometry, and Raman spectroscopy have enabled the visualization of the spatial organization and interactions of oral microbes with increasing resolution. Fluorescence techniques link phylogenetic identity with localization. Mass spectrometry imaging reveals metabolic niches and activities while Raman spectroscopy generates rapid biomolecular fingerprints for classification. Culturomics facilitates the isolation and cultivation of novel fastidious oral taxa using high-throughput approaches. Ongoing integration of these technologies holds the promise of transforming our understanding of oral microbiome assembly, gene expression, metabolites, microenvironments, virulence mechanisms, and microbe-host interfaces in the context of health and disease. However, significant knowledge gaps persist regarding community origins, developmental trajectories, homeostasis versus dysbiosis triggers, functional biomarkers, and strategies to deliberately reshape the oral microbiome for therapeutic benefit. The convergence of sequencing, imaging, cultureomics, synthetic systems, and biomimetic models will provide unprecedented insights into the oral microbiome and offer opportunities to predict, prevent, diagnose, and treat associated oral diseases.
Humans ; Phylogeny ; Biomimetics ; Dysbiosis ; Homeostasis ; Mass Spectrometry

【Objective】 To compare the desalination effects of five desalination methods and their effects on the components for human coagulation factor Ⅷ(FⅧ), and provide reference for selection of protein desalination methods. 【Methods】 Sephadex G-25 Medium gel, Fractogel EMD BioSEC gel, ultrafiltration, room temperature dialysis and 4℃ dialysis were used to desalt human FⅧ. The desalination effect was evaluated by the removal rate of Na +, citrate ion and glycine. FⅧ protein recovery, FⅧ activity (FⅧ∶C), VWF antigen (VWF∶Ag), VWF activity(VWF∶Ac), VWF polymers and SDS-PAGE analysis before and after desalination were compared to evaluate the effect of desalination on FⅧ components. 【Results】 In terms of desalination effect, the removal rate of Na⁺ was the lowest in ultrafiltration desalination, while that of Fractogel EMD BioSEC gel was the highest [(97.90±0.06) % vs (99.82±0.07) %]. Except that there was no statistical significance between Sephadex G-25 Medium gel desalination and Fractogel EMD BioSEC gel desalination (P=0.90), the removal rates of the other four methods were statistically significant. The removal rate of glycine was the lowest in ultrafiltration desalination, wihle that of Fractogel EMD BioSEC gel desalination was the highest [(95.78±0.42) % vs (99.81±0.08) %]. Significant difference in glycine removal was noticed in ultrafiltration desalination, but not among the other four desalination methods. There was no significant difference in the removal rate of citrate ions among the five methods (P=0.85). For the effect of FⅧ components, FⅧ∶C, VWF∶Ag, VWF∶Ac and protein recovery rates of ultrafiltration desalination were the highest, with (18.34±1.99) IU/mL, (11.81±0.33) IU/mL, (12.26±0.58) IU/mL and (97.13±1.37) %, respectively. There was no significant change in VWF∶Ac/VWF∶Ag before and after desalination by the five methods. SDS-PAGE and VWF polymer analysis showed that different desalination methods had no significant impact on protein composition. 【Conclusion】 Although different desalination methods had no significant effect on the composition of FⅧ protein, the desalination effect was different. Moreover, different desalination methods had significant effects on protein recovery, FⅧ∶C, VWF∶Ag and VWF∶Ac. The selection of desalination methods should be more considered during protein processing,