Objective To investigate the involvement of calmodulin(CaM)in the pathogenesis of Alzheimer disease(AD)and the mechanism by which CaM binds to amyloid-β(Aβ).Methods The hub genes expressed in AD and predicted to be the target proteins for AD prevention and treatment were obtained using bioinformatics methods.The GST-Aβ1-42 recombinant plasmid was constructed through genetic recombination and was then sequenced.The recombinant plasmids were identified using agarose gel electrophoresis,while the extracted and purified GST-Aβ1-42 fusion protein was confirmed using SDS-PAGE gel electrophoresis.GST pull-down assay was used to detect the interaction between GST-Aβ1-42 protein and CaM,expressed in the plasmid.Results The top 20 hub genes in degree ranking were obtained.The DNA sequencing results of the plasmid proved that the recombinant plasmid was successfully constructed.The agarose gel electrophoresis results indicated that the fragment digested by the enzyme was similar to the molecular weight of the Aβ1-42 gene seg-ments,further proving the successful construction of the recombinant plasmid.Binding of GST-Aβ1-42 protein to CaM in a concentration dependent manner was revealed through the GST pull down experiment.Conclusion The GST-Aβ1-42 recombinant plasmid is success-fully constructed and is shown to bind to CaM.

Objective:To learn about the single nucleotide polymorphism (SNP) population structure and regional distribution characteristics of Yersinia pestis in the natural focus of plague in Qinghai-Tibet Plateau. Methods:A total of 319 representative strains of Yersinia pestis isolated from natural focus of plague in Qinghai-Tibet Plateau from 1954 to 2020 were selected, and 2 298 SNP loci included in the global Yersinia pestis phylogenetic tree were compared by whole genome sequencing technology. MEGA 6.0 software was used to construct phylogenetic trees of 319 strains of Yersinia pestis from Qinghai-Tibet Plateau, determine the SNP population structure of Yersinia pestis in the focus, and describe its regional distribution characteristics. Results:The 319 strains of Yersinia pestis isolated from Qinghai-Tibet Plateau natural plague foci were distributed in 5 clades, namely 1.IN, 2.ANT, 3.ANT, 0.PE and 2.MED. The 1.IN clade contained 209 strains (65.52%, 209/319), which was the dominant population of strains in Qinghai Province, accounting for 90.51% (143/158). The 2.ANT clade contained 83 strains (26.02%, 83/319), which was the dominant population in Tibet Autonomous Region, accounting for 67.24% (78/116). The 3.ANT, 0.PE, and 2.MED clades contained 12 (3.76%, 12/319), 9 (2.82%, 9/319) and 6 strains (1.88%, 6/319), respectively, which were scattered in Qinghai Province, Gansu Province, Sichuan Province, Tibet Autonomous Region, and Xinjiang Uygur Autonomous Region under the jurisdiction of Qinghai-Tibet Plateau. Conclusion:The SNP population structure of Yersinia pestis in natural focus of plague in Qinghai-Tibet Plateau is relatively rich, and the strains are distributed in 5 clades: 1.IN, 2.ANT, 3.ANT, 0.PE and 2.MED, showing the distribution characteristics of specific regions.

Objective:To analyze clinical efficacy, failure mode and prognostic factors of nasopharyngeal carcinoma (NPC) patients undergoing intensity modulated radiation therapy (IMRT).Methods:Clinical data of 951 locally advanced NPC patients who were newly-treated with IMRT in Hunan Cancer Hospital from January 2018 to January 2019 were retrospectively analyzed. The patients' general data, overall survival (OS), local recurrence-free survival (LRFS), regional recurrence-free survival (RRFS), local recurrence-free survival (LRRFS), distant metastasis-free survival (DMFS), and progression-free survival (PFS) were analyzed. Comparison among different groups was performed by one-way ANOVA. Survival rate was calculated by Kaplan-Meier method. Survival difference was compared by log-rank test. Univariate and multivariate analyses were performed by Cox regression model.Results:The median follow-up time was 62.0 months (IQR, 58.0-65.0 months). The 5-year OS, LRFS, RRFS, LRRFS, DMFS, and PFS were 85.4%, 94.0%, 97.7%, 92.6%, 85.7% and 76.9%, respectively. According to the 8th edition staging of American Joint Committee on Cancer (AJCC), there were 10 cases (1.1%) of stage I, 76 cases (8.0%) of stage II, 445 cases (46.8%) of stage III, and 420 cases (44.2%) of stage IVA, respectively. Among them, the OS rates of stage I, II, III and IVA patients were 100%, 97.2%, 88.8% and 79.2%, respectively ( P<0.001); LRRFS rates were 100%, 90.4%, 94.7% and 90.4%, respectively( P=0.104); DMFS rates were 90.0%, 95.9%, 88.0% and 81.1%, respectively ( P<0.001); PFS rates were 90.0%, 89.1%, 80.9% and 70.1% respectively ( P<0.001). There were 183 cases of treatment failure, including 52 cases (5.5%) of local failure, 19 cases (2.0%) of regional failure, 130 cases (13.7%) of distant metastasis, 16 cases of local combined with regional failure (1.7%), 16 cases (1.7%) of local failure combined with distant metastasis, 13 cases (1.4%) of regional failure combined with distant metastasis, and 9 cases (0.9%) of local regional failure combined with distant metastasis, respectively. Multivariate regression analysis suggested that EB virus DNA copy number before treatment, T stage and N stage were the independent prognostic factors affecting OS, DMFS and PFS. Conclusions:Compared with two-dimensional radiotherapy, IMRT has improved the overall therapeutic effect for NPC, especially the local control rate. Distant metastasis is still the main failure mode. Clinical staging, prognostic risk stratification and prognostic biomarkers can be combined to deliver stratified and precise treatment, which may further improve clinical efficacy and reduce treatment-related side effects.

BACKGROUND:It has been found that vascular endothelial growth factor 165 and bone morphogenetic proteins interact with each other during hypoxia-reoxygenation and are involved in the repair process of osteoblast injury by regulating the activation of intracellular signaling pathways. OBJECTIVE:To further investigate the relationship between vascular endothelial growth factor 165/bone morphogenetic protein and hypoxic-reoxygenated osteoblast injury. METHODS:Osteoblasts were selected and the hypoxic-reoxygenated injury model was established.Vascular endothelial growth factor 165 and bone morphogenetic protein expressions at mRNA and protein levels were detected by real-time PCR and western blot before and after modeling.After modeling,osteoblasts were given different concentrations of vascular endothelial growth factor 165 and bone morphogenetic protein 2(10,20,40 ng/mL).Cell proliferation was detected by cell counting kit-8 method and apoptosis was detected by DAPI at 12,24,36,48,and 72 hours after treatment. RESULTS AND CONCLUSION:Compared with before modeling,the mRNA and protein expressions of vascular endothelial growth factor 165 and bone morphogenetic protein 2 in osteoblasts after modeling were significantly decreased(P＜0.05).The proliferation rate of osteoblasts was significantly increased with the increase of vascular endothelial growth factor 165 concentration(P＜0.05),while the apoptosis rate of osteoblasts decreased significantly with the increase of vascular endothelial growth factor 165 concentration(P＜0.05).The proliferation rate of osteoblast was significantly increased with the increase of bone morphogenetic protein 2 concentration(P＜0.05),while the apoptosis rate of osteoblast decreased significantly with the increase of bone morphogenetic protein 2 concentration(P＜0.05).To conclude,vascular endothelial growth factor 165 and bone morphogenetic protein are lowly expressed in hypoxic-reoxygenated osteoblast injury,and treatment with vascular endothelial growth factor 165 and bone morphogenetic protein can reduce the injury of hypoxic-reoxygenated osteoblast in a concentration-dependent manner,suggesting that vascular endothelial growth factor 165 and bone morphogenetic protein have a significant protective effect against the injury of hypoxic-reoxygenated osteoblasts.

Objective:To explore the differences of gene expression profiles of precursors of exhausted T cells (Tpex) and terminal exhausted T cells (Tex) in the peripheral blood of patients with active pulmonary tuberculosis (ATB).Methods:Twenty-five cases of ATB, 13 cases of latent tuberculosis infection (LTBI) and 10 health controls were enrolled from January 2021 to October 2022 in the Fifth People′s Hospital of Wuxi. The proportions of Tpex and Tex in the peripheral blood mononuclear cells (PBMCs) of the three groups were detected by flowcytometry. PBMCs of ATB were separated into Tpex and Tex by fluorescence-activated cell sorting. RNA-sequencing was performed and up-regulated and down-regulated genes were screended. Differently expressed genes were analyzed by gene set enrichment analysis of gene ontology (GO) to find regulatory pathways affecting cell metabolism and function. Wilcoxon matched-pairs signed rank test, Kruskal-Wallis test and Dunn multiple comparsion test were used for statistical analysis.Results:The proportion of Tpex in ATB group was 2.86%(1.74%), which was lower than 7.93%(6.16%) of Tex, and the difference was statistically significant ( Z=-3.91, P<0.001). The proportions of Tpex and Tex in LTBI group were 9.47%(6.26%) and 7.43%(5.48%), respectively, and the difference was not statistically significant ( Z=-0.93, P=0.345). The proportions of Tpex and Tex in healthy control group were 8.42%(2.69%) and 6.49%(5.14%), respectively, with no statistical significance ( Z=-1.36, P=0.170). There was statistical difference of the proportion of Tpex among the three groups ( H=21.93, P<0.001), and the proportion of Tpex in ATB group was lower than those in LTBI and heathy control groups, and the differences were both statistically significant ( Z=4.16, P<0.001 and Z=3.34, P=0.003, respectively), while the proportions of Tex in these three groups were not statistically different ( H=2.17, P=0.338). Compared with Tex, the gene expressions of memory markers, such as B-cell lymphoma 2 of Tpex were up-regulated, and the gene expressions of exhausted markers, such as lymphocyte activation gene 3 were down-regulated. In terms of cellular metabolism, the gene expressions of mitochondrial protein complex, mitochondrial matrix and oxidative phosphorylation of Tpex were up-regulated, and the gene expressions of glycolysis were down-regulated. The gene expressions of pyruvate metabolism in Tex were up-regulated, and the gene expressions of CD4 + T lymphocyte activation and differentiation and glycolytic process in Tpex were down-regulated. Conclusions:Tpex in ATB express more characteristics of memory cells and less features of exhausted markers compared with Tex, and the function of mitochondria of Tpex preserves well.

Objective:To clarify the potential correlation between biological changes of meninges in periodontitis mice and cognitive impairment by analyzing the biological changes of meninges in periodontitis mice using single-cell RNA sequencing.Methods:Thirty C57BL/6 mice were divided into two groups by using random number table method (15 mice in each group). Mice in the control group were locally administered 2% carboxyl methyl cellulose (CMC) without Porphyromonas gingivalis (Pg) on both buccal sides. A mixture of Pg W83 and 2% CMC was applied on both buccal sides in the experimental group mice three times a week, lasting for 16 weeks in total. The absorption of alveolar bone, locomotor activity and cognitive function, the activation of microglia and astrocytes in the cortex were observed and assessed. The mRNA expression levels of Occludin in meninges and brain were detected in two groups. Single-cell RNA sequencing data of meninges were processed by uniform manifold approximation and projection (UMAP). Differential genes expressions of endothelial cells were processed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. In addition, real-time fluorescence quantitative PCR (RT-qPCR) was used to verify the expressions of transcription activating factor 3 (Atf3) and apolpoprotein L domain-containing 1 (Apold 1). Results:Methylene blue staining found the distances of buccal and palatal cement-enamel junction-alveolar bone crest in experimental mice [(185.60±17.60), (206.90±13.37) μm] increased significantly compared with the control group [(135.33±9.57), (163.05±14.98) μm] ( t=5.02, P=0.002; t=4.37, P=0.005). Open field experiment showed the total distance and average speed of mice in the experimental group [(971.88±164.57) cm, (3.25±0.55) cm/s] were not statistically significant compared with the control group [(914.24±278.81) cm, (3.05±0.93) cm/s] ( t=0.65, P=0.525; t=0.65, P=0.520). The recognition index of the experimental group [(48.02±16.92) %] was lower than the control group [(66.27±17.90) %] ( t=2.40, P=0.027) by novel object recognition tests. Compared with the control group [(63.56±11.88) %], the alternation of experimental group [(50.99±14.17) %] was significantly decreased in Y maze tests ( t=2.33, P=0.030). Immunohistochemistry results showed microglia and astrocytes were activated in the cortex of experimental mice. Compared with the control group (1.02±0.25, 1.04±0.31), the relative mRNA expressions of Occludin decreased significantly in the meninges and brain of periodontitis mice, respectively (0.61±0.10, 0.64±0.20) ( t=3.47, P=0.010; t=2.66, P=0.024). By single-cell RNA sequencing, meninges cells were divided into 11 types, such as endothelial cells, fibroblasts, immune cells and so on. Endothelial cells were the main cell types in meninges [the control group: 26.47% (1 589/6 004), the experimental group: 26.26% (807/3 073)]. Compared with the control group [5.56% (334/6 004)], the percentage of granulocytes increased in the periodontitis mice [11.65% (358/3 073)]. Using clustering analysis to further focus on endothelial cells, GO enrichment analysis revealed differential genes were mainly related to angiogenesis, cell adhesion, apoptosis and so on. KEGG enrichment analysis revealed that differential genes were related to signaling pathways of interleukin-17, relaxin and so on. The relative mRNA expressions of Atf3 and Apold1 in meninges of periodontitis mice (0.42±0.24, 0.54±0.27) were significantly lower than the control group (1.03±0.26, 1.02±0.23) ( t=3.88, P=0.005; t=3.02, P=0.017). Conclusions:The mice chronically infected with Pg W83 occurred memory impairment, neuroinflammation and changes of barrier function. In the meninges of periodontitis mice, there were infiltration of immune cells and down-regulation expressions of Atf3 and Apold1 by single-cell RNA sequencing. Meningeal immunity and changes of barrier function may play an important role in the cognitive impairment caused by periodontitis.

Enamel formation is a complex physiological process that depends on the coordinated regulation of multiple mechanisms. This process is quite sensitive to various local and systemic interference factors. Therefore, during the long period from the embryonic stage to adolescence or even adulthood, various interference factors may lead to enamel developmental defects. Among them, early life is the most sensitive stage to environmental factors exposure, while it is also the critical period of enamel development of deciduous and permanent teeth. Environmental factors exposure during this period often leads to varying degrees of enamel development defects. In this review, we generalize the research progress of environmental factors affecting enamel developmental defects, summarize the potential mechanisms of environmental factors leading to enamel developmental defects, and conclude the clinical management strategies based on tertiary prevention. This work hopes to provide a theoretical basis for preventing abnormal teeth development from the critical time window of early life, propose eugenics health consultation and promote children ′s oral health management.

Objective: To investigate the transcriptomic changes in primary human oral keratinocytes (pHOKs) after coculture with Prevotella melaninogenica (P.m) and to verify the changes in human oral keratinocyte (HOK) cell lines. Methods: pHOK was isolated and cocultured with P.m for 0, 4 and 24 h. Total RNA was extracted, a gene library was constructed, transcriptional sequencing was performed, differentially expressed genes (DEGs) were analyzed, gene ontology (GO) pathway analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed, and the validation of DEGs was performed by qRT-PCR and Western Blot in the HOK and P.m coculture cell model. Results : 1 788 DEGs were detected between the 4 h group and control group, including upregulated DEGs such as lymphocyte cytosolic protein 1(LCP1), keratin 7 (KRT7) and Cilia and flagella associated protein 251(CFAP251) and downregulated DEGs such as FERM, ARH/RhoGEF and Pleckstrin domain protein 1 (FARP1), WW domain containing transcription regulator 1(WWTR1) and Discoidin, CUB and LCCL domain-containing protein 2 (DCBLD2). 1 832 DEGs were detected between the 24 h group and control group, including upregulated DEGs such as LCP1, complement C1s(C1S), kynureninase (KYNU) and downregulated DEGs such as phosphoserine aminotransferase 1 (PSAT1), FARP1 and FKBP prolyl isomerase 10 (FKBP10). There were 1 090 common differentially expressed genes (cDEGs) in the 4 h and 24 h groups, including LCP1, KYNU and long intergenic nonprotein coding RNA 958 (LINC00958). The GO pathways were mainly enriched in response to lipopolysaccharide and the molecules of bacterial origin and apical part of the cell. KEGG pathway analysis revealed enrichment in the interleukin-17 (IL-17) signaling pathway, tumor necrosis factor (TNF) signaling pathway, Toll-like receptor (TLR) pathway, etc. We verified the expression of a cDEG, Myosin1B (MYO1B), and qRT-PCR and Western Blot analysis showed that MYO1B expression was significantly upregulated between the control group and the P.m cocultured group (P<0.001), and its expression followed a time-dependent and concentration-dependent manner. Conclusion P.m played an important role in the transcriptome of oral keratinocytes.

Objective:To investigate the clinical value of computer tomography (CT)-based three-section formula in identification of Siewert types of adenocarcinoma of esophagogastric junction (AEG).Methods:The retrospective and descriptive study was conducted. The clinicopatho-logical data of 62 AEG patients who were admitted to two medical centers, including 33 patients from Peking University Cancer Hospital and 29 patients from the First Affiliated Hospital of Amy Medical University, between January 2019 and January 2021 were collected. There were 53 males and 9 females, aged (66±9)years. All patients underwent CT examination to obtain the coronal and axial images and determine the upper and lower edges of the tumor and the esophagogastric junction, which were imported into the formula for Siewert classification. Observation indicators: (1) results of CT examination and pathological examination; (2) consistence of judgements for CT examination between doctors; (3) consistence of judgements between CT examination and patholo-gical examination. Results of pathological examination came from intraoperative surgical observa-tion and postoperative histopathological examination. Measurement data with normal distribution were represented as Mean± SD, and count data were described as absolute numbers or percentages. The consistency coefficient Kappa ( κ) was used to evaluate the consistency of diagnosis between resear-chers. The κ≥0.75 was regarded as excellent consistency, 0.40< κ<0.75 as good consistency, κ ≤0.40 as poor consistency. Wilcoxon rank sum test was used to evaluate the statistical difference between results of the CT-based three-section formula method and the pathological examination. Taking the results of histopathological examination as standard, the sensitivity, specifi-city, accuracy and 95% confidence interval of the CT-based three-section formula method were calculated. Results:(1) Results of CT examination and pathological examination: all the 62 patients underwent CT examination successfully to obtain the coronal and axial images and determine the upper and lower edges of the tumor and the esophagogastric junction, which were used for Siewert classifica-tion. There were 3 cases with AEG of Siewert type Ⅰ, 47 cases with AEG of Siewert type Ⅱ and 12 cases with AEG of Siewert type Ⅲ according to doctor's judgement from the Peking University Cancer Hospital, and there were 3 cases with AEG of Siewert type Ⅰ, 49 cases with AEG of Siewert type Ⅱ and 10 cases with AEG of Siewert type Ⅲ according to doctor's judgement from the First Affiliated Hospital of Amy Medical University, respectively. After arbitration, there were 2 cases with AEG of Siewert type Ⅰ, 48 cases with AEG of Siewert type Ⅱ and 12 cases with AEG of Siewert type Ⅲ determined by the CT based three-section formula. There were 7 cases of stage T1, 10 cases of stage T2, 24 cases of stage T3, 14 of stage T4a and 7 cases of stage T4b of pathological T staging. There were 2 cases with AEG of Siewert type Ⅰ, 48 cases with AEG of Siewert type Ⅱ and 12 cases with AEG of Siewert type Ⅲ determined by pathological examination. (2) Consistence of judgements for CT examination between doctors: the consistency of Siewert classification determined by CT-based three-section formula between 2 doctors was good ( κ=0.74, P<0.001). (3) Consistence of judgements between pathological examination and CT examination: with Siewert classification determined by pathological examination as reference, the accuracy of Siewert classification determined by CT based three-section formula was 90.3%( κ=0.73, P<0.001). The sensitivity and specificity of CT-based three-section formula were 66.7%(95% confidence interval as 20.8%-93.9%) and 100.0%(95% confidence interval as 93.9%-100.0%) for AEG of Siewert type Ⅰ, 97.7%(95% confidence interval as 88.2%-99.6%) and 72.2%(95% confidence interval as 49.1%-87.5%) for AEG of Siewert type Ⅱ, 73.3%(95% confidence interval as 48.0%-89.1%) and 97.9%(95% confidence interval as 88.9%-99.9%) for AEG of Siewert type Ⅲ, respectively. Conclusion:The CT-based three-section formula can be used for identification of Siewert types of AEG, with a high accuracy.

Objective:To investigate the efficacy of CT imaging features in evaluating occult peritoneal metastasis (OPM) of diffuse infiltrating gastric cancer (Borrmann Type Ⅳ).Methods:Totally 101 patients with locally advanced Borrmann type Ⅳ gastric cancer were retrospectively collected who were admitted to Peking University Cancer Hospital from March 2014 to March 2021. The patients were divided into OPM group (53 cases) and the non-OPM group (48 cases) according to the results of preoperative CT and laparoscopic exploration/peritoneal cytology examination. The pathological examination results were recorded, including the degree of histological differentiation and Lauren classification. The evaluation indicators included the tumor center position, the number of tumor-occupied portions, involved orientation, mucosal broadband sign, stratified enhancement, serosa invasion, increased density of peripheral fat tissue, and enlarged lymph nodes. The maximum thickness of the primary tumor, average CT value of the primary tumor (arterial phase, venous phase, and delayed phase), difference between venous phase and arterial phase, difference between delayed phase and venous phase, and pattern of the enhanced curve were recorded. The Mann-Whitney U or Chi-square test was used to compare the differences of pathological and CT features between two groups. The multivariate logistic regression was used to screen independent predictors and establish a nomogram. The receiver operating characteristic curve was used to evaluate the performance of the nomogram in predicting OPM, and the Hosmer-Lemeshow test was used to test the model′s goodness of fit. Results:There was statistical significance in the seven indicators between the OPM and non-OPM groups, including tumor-occupied portions of stomach, mucosal broadband sign, stratified enhancement, serosa invasion, increased density of peripheral fat tissue, the enhanced curve pattern and the degree of histological differentiation ( P<0.05). Among them, the degree of histological differentiation (OR=0.19, P=0.033), stratified enhancement (OR=7.02, P=0.005) and serosa invasion (OR=14.27, P<0.001) were independent predictors of OPM. The nomogram was established based on the three significant features. The area under the curve for predicting OPM was 0.826 (95%CI 0.745-0.908), the sensitivity was 0.566 and the specificity was 0.938. The Hosmer-Lemeshow test showed a good agreement between the OPM risk predicted by the nomogram and the actual risk ( P=0.525). Conclusions:The CT features of Borrmann type Ⅳgastric cancer complicated with OPM have specific characteristics. The diagnosis model based on the degree of histological differentiation, stratified enhancement, and serosa invasion had high efficacy in evaluating OPM.