Objective To construct a risk prediction model by integrating the molecular subtypes of pan-creatic ductal adenocarcinoma(PDAC)and immune-related genes.Methods With GSE71729 data set(n=145)as the training set,the differentially expressed genes and differential immune-related genes between the squamous and non-squamous subtypes of PDAC were integrated to construct a regulatory network,on the basis of which five immune marker genes regulating the squamous subtype were screened out.An integrated immune score(IIS)model was constructed based on patient survival information and immune marker genes to predict the clinical prog-nosis of PDAC patients,and its predictive performance was tested with 5 validation sets(n=758).Results PDAC patients were assigned into high risk and low risk groups according to the IIS.In both training and validation sets,the overall survival of patients in the high risk group was shorter than that in the low risk group(both P＜0.001).The multivariable Cox regression showed that IIS was an independent prognostic factor for PDAC(HR=2.16,95％CI=1.50-3.10,P＜0.001).Conclusion IIS can be used for risk stratification of PDAC patients and may become a potential prognostic marker for PDAC.

Objective:To establish a rapid method for the detection of varicella-zoster virus (VZV) by recombinase-aid amplification (RAA).Methods:The whole genome sequences of VZV were downloaded from the global shared database for comparison and analysis. Specific primers and probe were designed for the four conserved genes respectively and the optimal combination was selected. The optimal reaction system was selected through the concentration gradient of primers and probes, and a fluorescence RAA detection method was established. The sensitivity of the method was evaluated with VZV positive plasmid standard and clinical samples with gradient dilution, the repeatability of the method was evaluated with the lowest detectable limit concentration of positive plasmid standard, and the specificity of other viral nucleic acid method was evaluated. At the same time, this method and quantitative real-time PCR (qPCR) were used to detect clinical samples and the result were compared.Results:The optimal combination of primer pair F2/R2 and probe P2 targeting open reading frame (ORF) 28 gene was selected. Considering the cost factor, the optimal primer concentration was set at 500 nmol/L and the optimal probe concentration was 280 nmol/L. The minimum detection limit was 10 1 copies/μL, and the minimum clinical positive samples with a Ct value of 36.027 could be detected, and the result of repeated experiments were consistent. The method has no cross-reaction with other viral nucleic acids. The detection rate of clinical positive samples was 93.33%, which was almost identical to that of qPCR. Conclusions:This method is simple to operate with high sensitivity, strong specificity, low requirements for experimental conditions, visual detection result, and can detect VZV nucleic acid in samples within 20 minutes, which is a rapid VZV detection method that can be considered for clinical use for detection.

Objective:To study the effects of modified citrus pectin (MCP) on the viability and gene expressions of synovial fibroblasts (SF) as well as SF treated by galectin-3 (Gal-3).Methods:Rabbit SF was isolated and cultured in vitro. Then SF was treated with different concentrations of MCP (0, 250, 500, and 750 mg/L). In addition, SF was further treated with the same different concentrations of MCP after treatment with 10 μg/ml Gal-3 for 24 h. The viability of SF was detected by CCK-8 on the first, third, and fifth day after treatment. The mRNA expression of transforming growth factor-β1 (TGF-β1), type I collagen (COL1A2), and Gal-3 in SF was detected by real-time quantitative PCR. The synthesis of type I collagen in SF was investigated by immunofluorescence staining. Results:MCP, especially at a concentration of 500 mg/L can inhibit the proliferation of SF significantly (all P < 0.05) on the first, third, and fifth day after treatment. Compared with the control group, MCP at different concentrations induced different gene expression profiles. In particular, MCP at high concentrations can upregulate the expression of TGF-β1, COL1A2 and Gal-3 in SF. However, MCP shows no significant effect on the synthesis of type I collagen in SF. MCP can down-regulate the expression of TGF-β1, COL1A2, and significantly reduce the synthesis of type I collagen in SF after Gal-3 treatment. Particularly, the effect of MCP at a concentration of 500 mg/L on inhibiting the expression of TGF-β1, COL1A2, and Gal-3 in SF is significant. Conclusions:MCP can inhibit the excessive proliferation of SF and regulate gene expression in SF.

Three-dimensional(3D)cell spheroid models combined with mass spectrometry imaging(MSI)enables innovative investigation of in vivo-like biological processes under different physiological and patho-logical conditions.Herein,airflow-assisted desorption electrospray ionization-MSI(AFADESI-MSI)was coupled with 3D HepG2 spheroids to assess the metabolism and hepatotoxicity of amiodarone(AMI).High-coverage imaging of＞1100 endogenous metabolites in hepatocyte spheroids was achieved using AFADESI-MSI.Following AMI treatment at different times,15 metabolites of AMI involved in N-desethylation,hydroxylation,deiodination,and desaturation metabolic reactions were identified,and according to their spatiotemporal dynamics features,the metabolic pathways of AMI were proposed.Subsequently,the temporal and spatial changes in metabolic disturbance within spheroids caused by drug exposure were obtained via metabolomic analysis.The main dysregulated metabolic pathways included arachidonic acid and glycerophospholipid metabolism,providing considerable evidence for the mechanism of AMI hepatotoxicity.In addition,a biomarker group of eight fatty acids was selected that provided improved indication of cell viability and could characterize the hepatotoxicity of AMI.The combination of AFADESI-MSI and HepG2 spheroids can simultaneously obtain spatiotemporal infor-mation for drugs,drug metabolites,and endogenous metabolites after AMI treatment,providing an effective tool for in vitro drug hepatotoxicity evaluation.

Deconvolution of potential drug targets of the central nervous system (CNS) is particularly challenging because of the complicated structure and function of the brain. Here, a spatiotemporally resolved metabolomics and isotope tracing strategy was proposed and demonstrated to be powerful for deconvoluting and localizing potential targets of CNS drugs by using ambient mass spectrometry imaging. This strategy can map various substances including exogenous drugs, isotopically labeled metabolites, and various types of endogenous metabolites in the brain tissue sections to illustrate their microregional distribution pattern in the brain and locate drug action-related metabolic nodes and pathways. The strategy revealed that the sedative-hypnotic drug candidate YZG-331 was prominently distributed in the pineal gland and entered the thalamus and hypothalamus in relatively small amounts, and can increase glutamate decarboxylase activity to elevate γ-aminobutyric acid (GABA) levels in the hypothalamus, agonize organic cation transporter 3 to release extracellular histamine into peripheral circulation. These findings emphasize the promising capability of spatiotemporally resolved metabolomics and isotope tracing to help elucidate the multiple targets and the mechanisms of action of CNS drugs.

Objective:To describe the characteristics of molecular transmission network of newly diagnosed HIV-1 infected patients, analyze their risk factors related to network access and provide a scientific basis for precise prevention of HIV infection.Methods:For 340 blood samples collected from confirmed HIV-1 infection cases aged ≥50 years in Pengzhou city of Sichuan province from April 2019 to August 2021, nested PCR amplification was used to amplify, clean up and splice clips the pol gene region. The phylogenetic tree was constructed by multi-sequence comparison to distinguish subtypes, and the pairwise genetic distance was calculated. When the genetic distance threshold was 0.90%, the number of clusters was the largest (41), and the molecular transmission network was constructed.The χ2 test and logistic regression analysis were performed.The software SPSS 19.0 was used for statistical analysis. Results:A total of 340 samples were successfully amplified (97.06%, 330/340) in 330 samples. 6 HIV-1 subtypes identified, including:CRF01_AE(56.67%,187/330), CRF07_BC(27.88%,92/330), B(11.21%,37/330), CRF08_BC(3.33%,11/330), CRF55_01B(0.61%,2/330) and C(0.30%,1/330).The network entry rate was 58.79% (194/330).The results of logistic regression analysis of the risk factors of HIV-1 molecular transmission network in the research subjects showed that compared with illiteracy, junior high school ( OR=0.35, 95% CI:0.13-0.97) and high school/technical secondary school ( OR=0.14, 95% CI: 0.02-0.97) had lower possibility of network entry. Compared with farmers, unknown occupations ( OR=0.40,95% CI: 0.17-0.95) are less likely to enter the network .Compared with CRF01_AE, CRF07_BC ( OR=0.20, 95% CI: 0.11-0.35) and CRF08_BC subtype ( OR=0.09, 95% CI: 0.02-0.45) were less likely to enter the network. Conclusions:The sources of AIDS transmission among middle-aged and elderly people of rural areas are diversified in Pengzhou city of Sichuan province. AIDS intervention should focus on middle-aged and elderly farmers with low educational level, and strengthen detection and traceability investigation.

OBJECTIVE: To investigate the effect of orthodontic traction on the microstructure of dental enamel. METHODS: Forty-eight isolated premolars were randomly divided into 6 groups (=8), including Group A (blank control group), in which the teeth were bonded with the orthodontic brackets without any loading force; Groups B1, B2, and B3 where the teeth were bonded with the orthodontic brackets using clinical adhesives and loaded with 50 g force for 6 months, 200 g force for 6 months, and 200 g force for 1 month, respectively; and Groups C1 and C2, where the teeth were bonded with straight wire brackets using light curing bonding and chemical curing bonding techniques, respectively. All the teeth were embedded with non-decalcified epoxy resin. Scanning electron microscope (SEM), atomic force microscope (AFM), and energy spectrometer (EDS) were used to analyze interface morphology and elemental composition of the teeth sliced with a hard tissue microtome. RESULTS: Compared with those in Group A, the teeth in the other 5 groups showed increased adhesive residue index with microcracks and void structures on the enamel surface under SEM; AFM revealed microcracks on the enamel surface with angles to the grinding direction. A larger loading force on the bracket resulted in more microcracks on the enamel interface. The interface roughness differed significantly between Groups A and C2, and the peak-to-valley distance differed significantly between Groups A, C, and C2. CONCLUSIONS Orthodontic traction can cause changes in the microstructure of normal dental enamel.
Dental Enamel ; Materials Testing ; Orthodontic Brackets ; Resin Cements ; Surface Properties ; Traction

With the rapid development and wide application of traditional Chinese medicine injection (TCMI), a number of adverse events of some TCMIs have incessantly been reported and have drawn broad attention in recent years. Establishing effective and practical analytical methods for safety evaluation and quality control of TCMI can help to improve the safety of TCMIs in clinical applications. In this study, a sensitive and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and validated for the quantitative determination of potentially harmful substance 5,5'-oxydimethylenebis (2-furfural, OMBF) in TCMI samples. Chromatographic separation was performed on a C18 reversed-phase column (150 mm × 2.1 mm, 5 µm) by gradient elution, using methanol-water containing 0.1% formic acid as mobile phase at the flow rate of 0.3 mL/min. MS/MS detection was performed on a triple quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction-monitoring mode. The method was sensitive with a limit of quantification of 0.3 ng/mL and linear over the range of 0.3-30 ng/mL (=0.9998). Intra- and inter-day precision for analyte was <9.52% RSD with recoveries in the range 88.0-109.67% at three concentration levels. The validated method was successfully applied to quantitatively determine the compound OMBF in TCMIs and glucose injections. Our study indicates that this method is simple, sensitive, practicable and reliable, and could be applied for safety evaluation and quality control of TCMIs and glucose injections.

OBJECTIVE:To compare the differences between artificially cultivated and wild Xinjiang Artemisia rupestris,and screen the different components. METHODS:HPLC-MS was adopted to establish the fingerprints of artificially cultivated and wild Xinjiang A. rupestris from different origin and harvest time. Principal component analysis was conducted by Marker ViewTM soft-ware and SIMCA-P 11.5 software,the characteristics of principal components were analyzed,difference variable was screened, and different components of artificially cultivated and wild varieties were obtained. RESULTS:Fingerprints of 22 batches of A. rup-estris(12 batches of wild varieties,10 batches of artificially cultivated varieties)were established. According to the principal com-ponent analysis,artificially cultivated and wild varieties were well grouped,with obvious differences;the principal components of artificially cultivated varieties with different harvest time showed certain difference,mainly before and after flowering,concentrat-ing in to-be flowering and full flowering periods. Wild varieties from different origins had obvious regional difference,showing cer-tain differences in composition and content. 268 variables were found in matrix of positive ion mode and 155 in negative ion mode. 28 groups of variables were extracted by difference variable,and 19 variables were determined. CONCLUSIONS:Artificially culti-vated and wild varieties have obvious difference in principal component,mainly in flowering period and picking places. It can pro-vide theoretical basis for the standardized cultivation and origin protection of Xinjiang A. rupestris.

Objective To evaluate the value of the neutrophil-lymphocyte ratio (NLR) in elderly type 2 diabetic patients (T2DM) with coronary heart disease (CHD).Methods We performed a retrospective observational study on 228 patients undergoing coronary angiography in Guizhou Provincial People's Hospital from April 2014 to July 2015.Patients were divided into three groups:the simple T2DM group (n=77),simple CHD group (n=72),and T2DM complicated with CHD group (n=79).Meanwhile,70 healthy elderly subjects served as the control group.The white blood cell count,high-sensitivity C-reactive protein (hs-CRP) and other clinical and laboratory parameters were collected,and NLR was calculated.Risk factors for CHD in T2DM patients were determined by logistic regression analysis.Multiple stepwise regression analysis was adopted to identify factors influencing NLR.Results The white blood cell count,neutrophil count,NLR and hs-CRP level in the simple T2DM,CHD,and T2DM+CHD groups were higher than in the control group [(7.48 1.81) 109/L,(7.72± 1.89) 109/L,(7.98±2.12) 109/L vs.(6.22± 1.61) 109/L;(4.49±1.38) 109/L,(4.88±1.56) 109/L,(5.35±1.40) 109/L vs.(3.52±0.84) 109/L;(2.84± 0.77),(3.07±0.79),(3.34±0.83) vs.(1.58±0.42);(2.92±0.65) mg/L,(3.20±0.86) mg/ L,(4.98±1.10) mg/L vs.(1.105±0.23) mg/L;respectively,P＜0.05 or P＜0.01].The lymphocyte count in the simple T2DM,CHD,and T2DM+CHD groups were lower than in the control group [(1.57±0.41) × 109/L,(1.58±0.40) × 109/L,(1.61±0.48) × 109/L vs.(2.22± 0.51) × 109/L,P＜0.05].NLR and hs-CRP levels in the T2DM+CHD group were higher than in the former two groups (all P＜0.05).Pearson correlation analysis showed that NLR was positively correlated with the Gensini score and hs-CRP level (r=0.7455 and 0.7792,both P＜0.01).Logistic regression analysis showed that NLR,hs-CRP levels and glycosylated hemoglobin A1c (HbA1c) were the risk factors for CHD in T2DM patients (OR=4.331,3.997 and 2.928,all P＜0.05).Multiple stepwise regression analysis showed that NLR was positively correlated with fasting plasma glucose,HbA1 c levels and systolic blood pressure (β' =0.3133,0.4720 and 0.3069,all P＜0.05).Conclusions NLR may be a valuable predictive factor for CHD in elderly T2DM patients.