Objective:To analyze the histopathological characteristics of peri-implant soft tissue in re-constructed jaws and the changes after keratinized mucosa augmentation(KM A)with free gingival graft(FGG).Methods:Twenty patients were enrolled in this study.Five patients of them,who were perio-dontal and systemic healthy and referred for crown lengthening before restoration with healthy keratinized gingiva collected were enrolled as healthy controls.15 patients of them were with fibula or iliac bone flaps jaw reconstruction(10 with fibula flap and 5 with iliac flap),who were referred to FGG and implant exposures before restoration.Soft tissue was collected before FGG in reconstructed jaws,and in 5 patients(3 with fibula flap and 2 with iliac flap)8 weeks after FGG if a second surgery was conducted.Histologi-cal analysis with hematoxylin-eosin stain and immunological analysis to interlukin-1(IL-1),interlukin-6(IL-6)and tumor necrosis factor-α(TNF-α)were performed.Results:Thickness from the bottom of stratum basale to the top of stratum granulosum and thickness of keratinized layer in reconstructed jaws were significantly lower compared with that of natural healthy keratinized gingiva[0.27(0.20,0.30)mmvs.0.36(0.35,0.47)mm,P＜0.05;16.49(14.90,23.37)μm vs.26.37(24.12,31.53)μm,P＜0.05].In the reconstructed area,thickness from the bottom of stratum basale to the top of stra-tum granulosum increased after KMA with FGG[0.19(0.16,0.25)mm vs.0.38(0.25,0.39)mm,P=0.059]and the thickness of keratinized layer significantly increased after KMA with FGG[16.42(14.16,22.35)μm vs.28.57(27.16,29.14)μm,P＜0.05],which was similar to that in the con-trol group.Furthermore,the number of positive cells of IL-1,IL-6 and TNF-α significantly increased after KMA[0.67(0.17,8.93)vs.11.00(9.16,18.00);13.00(8.50,14.14)vs.21.89(15.00,28.12);0.22(0.04,0.63)vs.2.83(1.68,5.00),respectively,P＜0.05]as well as the average optical density value[0.15(0.14,0.17)vs.0.18(0.17,0.21);0.28(0.26,0.33)vs.0.36(0.33,0.37);0.23(0.22,0.29)vs.0.30(0.28,0.42),respectively,P＜0.05],which was similar to that in the healthy keratinized gingiva.Conclusion:The lack of rete pegs and inflammato-ry factors were common in soft tissue with jaw reconstruction.FGG can improve the quality of the epithe-lium and may improve the stability of the mucosa around implants.

Objective:A mouse model of pancreatitis induced by coxsackievirus B3 (CVB3) was established. The pathological change of pancreas and the infiltration of Th17/Treg cells were observed.Methods:The BALB/c mice were inoculated intraperitoneally with CVB3 to induce acute viral pancreatitis model. Then the pathological changes of pancreas were observed by HE staining; the viral RNA load and relative expression of cytokines (IFN-γ, IL-6 and IL-17) mRNA were detected by q-PCR; the proportion of infiltrated CD45 + CD3 + T cells, CD4 + and CD8 + T cells, Th17 and Treg cells in the pancreas was determined by flow cytometry. Results:Three days after CVB3 infection, the viral RNA load in pancreas was the highest (0.96±0.18) and gradually decreased with prolongation of infection. Compared with the 3 dpi group, the viral RNA load in pancreas was decreased (0.96±0.18 vs. 0.62±0.14) at 7 dpi, but there was no statistically significant difference. In addition, the infiltration of immune cell in pancreas increased significantly after 7dpi and the pathological score >2. The percent of infiltrated Th17 cells (1.05±0.21 vs. 22.13±5.79) and Treg cells (3.11±0.78 vs. 8.25±1.30) among CD4 + T cells significantly increased after infection (P<0.05), and the Th17/Treg also increased (P<0.01). Compared with the control group, the relative mRNA expression of IFN-γ (1.05±0.23 vs. 672.6±47.67), IL-6 (1.00±0.38 vs. 68.28±4.57), and IL-17 (1.01±0.11 vs. 54.15±7.94) in pancreas increased at 7 days after CVB3 infection ( P<0.01). Conclusions:The infiltration of Th17/Treg cells and the expression of related cytokines related cytokines IL-6 and IL-17 mRNA were upregulated in pancreas, which promoted the process of CVB3-induced pancreatitis.

Objective:To analyze the epidemiological characteristics and genotypes of human rhinovirus (HRV) in patients with upper respiratory tract infection in Qingdao in the winter of 2020.Methods:Throat swab samples were collected from 101 patients with upper respiratory tract infection in Qingdao from November 2020 to January 2021. Quantitative PCR was used to detect 15 common respiratory viruses in the samples. HRV-positive samples were further analyzed with RT-PCR to amplify and sequence HRV VP4/VP2 gene. A phylogenetic tree was constructed based on the sequencing results and homology analysis was conducted.Results:Six common respiratory viruses were detected in the 101 patients. Thirty-four cases (34/101, 33.66%) were single pathogen infection and two cases were multiple infection (2/101, 1.98%). The positive rate of HRV was the highest (21.78%, 22/101). Twenty HRV VP4/VP2 sequences were successfully amplified. Phylogenetic analysis showed that there were 16 strains of HRV-A subtype and four strains of HRV-C subtype and 14 serotypes were involved.Conclusions:HRV was one of the leading viral pathogens causing upper respiratory tract infection in Qingdao in the winter of 2020 and the predominant subtype was HRV-A.

Background::Sepsis is a serious disease caused by infection. Aminophylline has anti-asthma and anti-inflammatory effects. We aimed to explore the safety and effect of aminophylline in sepsis.Methods::We conducted a clinical randomized controlled trial involving 100 patients diagnosed with sepsis within 48 h after intensive care unit (ICU) admission in two sites. All patients were randomized in a 1:1 ratio to receive standard therapy with or without aminophylline. The primary clinical outcome was all-cause mortality at 28 days.Results::From September 27, 2018 to February 12, 2020, we screened 277 septic patients and eventually enrolled 100 patients, with 50 assigned to the aminophylline group and 50 to the usual-care group. At 28 days, 7 of 50 patients (14.0%) in the aminophylline group had died, compared with 16 of 50 (32.0%) in the usual-care group ( P = 0.032). Cox regression showed that the aminophylline group had a lower hazard of death (hazard ratio = 0.312, 95% confidence interval: 0.129-0.753). Compared with the usual-care group, patients in the aminophylline group had a longer survival time ( P = 0.039 by the log-rank test). The effects of aminophylline on vasopressor dose, oxygenation index, and sequential organ failure assessment score were time-dependent with treatment. There were no significant differences in total hospitalization days, ICU hospitalization days, and rates of serious adverse events (all P > 0.05). No adverse events were observed in the trial. Conclusions::Aminophylline as an adjunct therapy could significantly reduce the risk of death and prolong the survival time of patients with sepsis.Trial registration::ChiCTR.org.cn, ChiCTR1800019173.

Objective:To establish a real-time fluorescent quantitative PCR for the detection of torque teno virus types 7 (TTV7), 8 (TTV8) and 10 (TTV10) and analyze its performance in clinical sample detection.Methods:Specific primers were designed based on the gene sequences of TTV7, TTV8 and TTV10 in GenBank. Recombinant plasmids of pMD19-T-TTV7, pMD19-T-TTV8 and pMD19-T-TTV10 were constructed and used as positive standard control to establish a real-time fluorescent quantitative PCR based on FAM-Eclipse probe method. The specificity and sensitivity of the established method were evaluated. Moreover, it was validated in terms of clinical sample detection.Results:The standard curve equations of the real-time fluorescent quantitative PCR for detecting TTV7, TTV8 and TTV10 were y=-0.340 2 x+ 114.780 0 ( R2=0.998 8), y=-0.351 1 x+ 114.940 0 ( R2=0.995 3) and y=-0.348 9 x+ 115.020 0 ( R2=0.991 7), respectively, and there was no cross-reaction with other viruses. The detection sensitivity of the established method for TTV7, TTV8 and TTV10 were 108 copies/μl, 84 copies/μl and 98 copies/μl, and the positive detection rates in clinical pediatric serum samples were 10.9%, 2.1% and 4.3%, respectively. Conclusions:The established real-time fluorescent quantitative PCR for detection of TTV7, TTV8 and TTV10 was featured by strong specificity and high sensitivity, which could be used for rapid TTV detection in clinical serum samples.

Objective:To construct the dual-luciferase reporter vector for identification of internal ribosome entry site (IRES).Methods:The hairpin structure was inserted between Renilla luciferase (R-Luc) and Firefly luciferase (F-Luc) genes based on psiCHECK-2 to form plasmid psiCHECK-IRES. IRES of Encephalomyocarditis virus (EMCV) was inserted between the hairpin structure and F-Luc genes of psiCHECK-IRES to form vector psiCHECK-IRES-EMCV. After psiCHECK-IRES-EMCV or psiCHECK-IRES was transfected into BHK-21 cells respectively, expressions of F-Luc and R-Luc were detected by RT-qPCR. Then Luciferase activity of transfected cells was detected with the dual-luciferase reporter assay system at 24 h post-transfection.Results:The hairpin structure was successfully inserted into psiCHECK-2 to form psiCHECK-IRES by sequencing. RT-qPCR result showed that there were the approximate expressing levels of mRNA between F-Luc and R-Luc. The result indicated that no aberrant monocistronic transcripts, which caused false positive F-Luc readings, were produced. Then IRES of EMCV was introduced into psiCHECK-IRES to form psiCHECK-IRES-EMCV. The F-Luc/R-Luc ratio in psiCHECK-IRES-EMCV-transfected cells was 53.35 times that of psiCHECK-IRES-transfected cells. The result confirmed that IRES of EMCV initiated effectively the translation of F-Luc.Conclusions:Dual-luciferase reporter vector psiCHECK-IRES was successfully constructed, which could be used to validate viruses and eukaryotic genes, the translation thereof was IRES-dependent.

Objective:To investigate the relationship between serum cholinesterase (SChE) level and the prognosis of patients with septic shock (SS).Methods:A total of 594 patients with SS admitted to the First Affiliated Hospital of Zhengzhou University from June 2013 to June 2017 were enrolled. General data such as gender, age, acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) score were recorded as well as routine blood test, procalcitonin (PCT), hepatic function, renal function, coagulation function and blood gas analysis parameters within 48 hours of SS diagnosis. The patients were followed by telephone from September to October in 2019, and the outcome was recorded. The primary outcome was all-cause death 28 days after discharge. The secondary outcomes were all-cause death in intensive care unit (ICU) and 2 years after discharge, and the length of ICU stay. The patients were divided into two groups according to prognosis of 28 days: the survival group and the death group. The clinical data of the two groups were compared. Multivariate Cox regression analysis was used to screen prognostic risk factors of 28 days in patients with SS. The receiver operating characteristic (ROC) curve was used to explore predictive value of liver function parameter SChE for 28-day prognosis of patients with SS. The patients were divided into two groups according to the levels of SChE: the low SChE group (SChE ≤ 4 000 U/L) and the normal SChE group (SChE > 4 000 U/L). Kaplan-Meier survival curves were used to compare the cumulative survival rates without endpoint event of patients with different SChE levels.Results:A total of 385 patients with SS were enrolled according to the inclusion and exclusion criteria, and a total of 356 patients were followed up successfully, with a follow-up rate of 92.5% (356/385). There were 142 survival patients and 214 death patients at 28 days, with a 28-day mortality rate of 60.1% (214/356). There were 116 survival patients and 240 death patients at 2 years, with a 2-year mortality rate of 67.4% (240/356). Compared with the 28-day survival group, the patients in the death group were older and had higher APACHEⅡ score, partial hepatic and renal function parameters, higher level of blood lactate (Lac) and lower levels of white blood cell count (WBC), platelet count (PLT) and SChE with statistically significant differences. Multivariate Cox regression analysis showed that the age [relative risk ( RR) = 1.444, 95% confidence interval (95% CI) was 1.090-1.914, P = 0.010], APACHEⅡ score ( RR = 2.249, 95% CI was 1.688-2.997, P = 0.000), SChE ( RR = 1.469, 95% CI was 1.057-2.043, P = 0.022), and Lac ( RR = 2.190, 95% CI was 1.636-2.931, P = 0.000) were independent risk factors for 28-day mortality of patients with SS. The ROC curve analysis showed that SChE had a weak prognostic value for 28-day prognosis of patients with SS [the area under ROC curve (AUC) was 0.574]. However, the combined predictive value of SChE, APACHEⅡ score and Lac was greater than APACHEⅡ score or Lac alone for prediction (AUC: 0.807 vs. 0.785, 0.697), with a sensitivity of 79.9% and a specificity of 68.5%. Compared with the normal SChE group ( n = 88), the 28-day mortality of patients in the low SChE group ( n = 268) was significantly increased [63.1% (169/268) vs. 51.1% (45/88), P < 0.05], but ICU mortality [59.7% (160/268) vs. 48.9% (43/88)], 2-year mortality [69.8% (187/268) vs. 60.2% (53/88)] or the length of ICU stay [days: 4 (2, 7) vs. 5 (2, 9)] between the two groups showed no statistical significance (all P > 0.05). Kaplan-Meier survival curve analysis showed that the cumulative survival rate without endpoint event of patients in the low SChE group was significantly lower than that in the normal SChE group (Log-Rank test: χ 2 = 5.852, P = 0.016). Conclusions:Increased risk of 28-day mortality in patients with SS whose SChE is below normal. The level of SChE is an independent risk factor for 28-day death in SS patients, and it is one of the indicators to evaluate the short-term prognosis of patients with SS.

Objective:To explore the correlation and interaction between epidermal growth factor (EGF) rs2237051 and peroxidase proliferators activate receptor-α (PPAR-α) rs4253623 polymorphisms and the susceptibility of generalized aggressive periodontitis (GAgP).Methods:Two hundred and nineteen Chinese patients with GAgP were enrolled from the patients of the Department of Periodontology, Peking University School and Hospital of Stomatology from January 2001 to December 2015. The control group comprised 138 periodontally healthy volunteers recruited from the staff and students of the Peking University School and Hospital of Stomatology. The EGF rs2237051 and PPAR-α rs4253623 polymorphisms were genotyped using time-of-flight mass spectrometry. Logistic regression models were conducted to analyze the correlation between the EGF rs2237051 and PPAR-α rs4253623 variants with GAgP. The likelihood ratio test was used to analyze whether there was an interaction between the two polymorphisms in the susceptibility of GAgP. The interaction model adopted was the multiplication model.Results:The mean ages of GAgP group (male:87; female:132) and control group (male: 53; female: 85) were (27.3±4.5) years and (27.1±4.2) years respectively and there was no significant difference in age and gender distribution between the two groups. For EGF rs2237051, the frequency of AA genotype in the GAgP group [49.5% (107/216)] was significantly higher than that in the control group [37.7% (52/138)], and the frequency of AG/GG genotype in the GAgP group [50.5% (109/216)] was significantly lower than that in the control group [62.3% (86/138)]( P<0.05). Compared with AA genotype, individuals with AG/GG genotype had a 39% lower risk of GAgP after adjustment of age and gender ( OR: 0.61, 95 %CI: 0.40-0.95, P<0.05). For PPAR-α rs4253623, the frequency of AA genotype in the GAgP group [76.2% (160/210)] was significantly higher than that in the control group [65.9%(81/123)], and the frequency of AG/GG genotype in the GAgP group [23.8% (50/210)] was significantly lower than that in the control group [34.1%(42/123)] ( P<0.05). Compared with AA genotype, individuals with AG/GG genotype had a 40% lower risk of GAgP after adjustment of age and gender ( OR: 0.60, 95 %CI: 0.36-0.98, P<0.05). EGF rs2237051 and PPAR-α rs4253623 showed a significant interaction in the susceptibility to GAgP. Compared with AA genotype, the risk of GAgP in individuals with both AG/GG genotypes of EGF rs2237051 and PPAR-α rs4253623 was reduced by 66% ( OR: 0.34, 95 %CI: 0.17-0.66, P<0.01). Conclusions:EGF rs2237051 and PPAR-α rs4253623 are correlated with GAgP susceptibility, and there is a significant interaction between them in the susceptibility of GAgP. The G allele of the two loci has a protective effect on the disease of GAgP.

Objective To evaluate the application of combinatorial probe anchor synthesis (cPAS)?based high?throughput low coverage whole genome sequencing in chromosomal aberration detection in spontaneous miscarriage. Methods From September 2015 to May 2017, spontaneous miscarriage samples were collected from Inner Mongolia Maternal and Child Health Care Hospital. Those samples were further analyzed with two independent methods, fluorescence in situ hybridization (FISH) and low coverage whole genome sequencing on the BGISEQ?500 high?throughput platform. The performance of low coverage whole genome sequencing was assessed by comparing to FISH results. Results In 595 spontaneous miscarried specimens, low coverage whole genome sequencing revealed 144 cases (24.2%, 144/595) chromosomal abnormalities, of which a subset of 137 cases (23.0%, 137/595) were detected as aneuploidies, 2 cases (0.3%, 2/595) as mosaicisms and 5 cases (0.8%, 5/595) as copy number variation (≥5 Mb). Conclusion cPAS?based high?throughput low coverage whole genome sequencing is a reliable method in detecting chromosomal aberrations inspontaneous abortion tissues, including chromosome aneuploidies, mosaicisms and copy number variation (≥5 Mb).

Objective: To evaluate the application of combinatorial probe anchor synthesis (cPAS)-based high-throughput low coverage whole genome sequencing in chromosomal aberration detection in spontaneous miscarriage. Methods: From September 2015 to May 2017, spontaneous miscarriage samples were collected from Inner Mongolia Maternal and Child Health Care Hospital. Those samples were further analyzed with two independent methods, fluorescence in situ hybridization (FISH) and low coverage whole genome sequencing on the BGISEQ-500 high-throughput platform. The performance of low coverage whole genome sequencing was assessed by comparing to FISH results. Results: In 595 spontaneous miscarried specimens, low coverage whole genome sequencing revealed 144 cases (24.2%, 144/595) chromosomal abnormalities, of which a subset of 137 cases (23.0%, 137/595) were detected as aneuploidies, 2 cases (0.3%, 2/595) as mosaicisms and 5 cases (0.8%, 5/595) as copy number variation (≥5 Mb). Conclusion cPAS-based high-throughput low coverage whole genome sequencing is a reliable method in detecting chromosomal aberrations inspontaneous abortion tissues, including chromosome aneuploidies, mosaicisms and copy number variation (≥5 Mb).