Objective:To evaluate the synergistic antibacterial effects of lysozyme with ethylenediami-netetraacetic acid disodium salt (EDTA-2Na) on Enterococcus faecalis (E.faecalis) and Porphyromonas endodontalis ( P.endodontalis) .Methods:E.faecalis and P.endodontalis were cultured and adjusted to 108 CFU/mL.Then 0.3, 0.5, 1, 2, 5, 10, 50, 100, 150 and 300 g/L of lysozyme were prepared with deionized water;and the lysozyme solutions were mixed with 0.5, 1.0, 2.0 g/L of EDTA-2Na, re-spectively.The bacteria and lysosome with/without EDTA-2Na interacted for 15 min, then water-soluble tetrazolium (WST) working solution was added and the activity of the bacteria was calculated by mea-suring optical densities at 450 nm and 630 nm with microplate spectrophotometer .Results:Regarding the pure lysozyme from 0.5 g/L to 150 g/L, more E.faecalis and P.endodontalis were inhibited when the concentration of lysozyme was higher , especially for E.faecalis.There was synergistic effect of lysozyme with EDTA-2Na on antibacterial activity , which was related to the concentration of lysozyme .On E.fae-calis, the antibacterial activity of lysozyme with EDTA-2Na was 1.2-3.7 folds than the pure lysozyme when the concentration of lysozyme was 0.5-50 g/L (P<0.05), and on P.endodontalis, the antibac-terial activity of lysozyme with EDTA-2Na was 1.3 -3.5 folds than the pure lysozyme when the concentration of lysozyme was 0.5-10 g/L (P<0.05).When the concentration of lysozyme was higher than 100 g/L, EDTA-2Na did not show synergistic effect on the antibacterial activity (P>0.05). Conclusion: For E.faecalis and P.endodontalis, a low concentration of lysozyme with EDTA-2Na showed significant synergistic antibacterial activity , while a high concentration of lysozyme with EDTA-2 Na did not .

Teaching model plays an important role in clinical education. Problem based learning (PBL) model, with an outstanding feature of stimulating the learning enthusiasm of students, was applied by many American colleges. American College of Acupuncture&Oriental Medicine (ACAOM) combined PBL teaching model with traditional lecture based learning (LBL) model, which had achieved good results. In this article, PBL model and its characters were explored via qualitative case analysis, to provide reference for universities and colleges of traditional Chinese medicine on clinical education.

Objective To investigate clinical features and therapeutic protocols of vulvovaginal-gingival syndrome(VVGS). Methods Clinical data were collected from 11 female patients with VVGS and analyzed. Results Among the 11 patients, the mean and median ages at a definite diagnosis of VVGS were both 46 years, and the average disease duration was 4 years. Painful erosive erythema was found on the vaginal vestibule in 7 patients, on the gingiva in 10, as well as on the bilateral buccal mucosa in 5. Additionally, 8 and 4 patients reported dyspareunia and vaginal hemorrhage, respectively. Seven patients were treated with low-dose oral methylprednisolone combined with hydroxychloroquine or colchicine, and the other 4 were treated with oral hydroxychloroquine alone. Furthermore, all of the 11 patients applied topical tacrolimus 0.1%ointment. Finally, 8 patients were cured or markedly improved. No obvious adverse reactions were observed in any of the patients. Conclusions VVGS mostly occurs in middle-aged women, with the vaginal vestibule and gingiva as predilection sites of painful erythema and dyspareunia as common symptoms. Patients have good responses to methylprednisolone combined with hydroxychloroquine, and hydroxychloroquine alone also shows good efficacy in patients with mild VVGS. Tacrolimus 0.1%ointment can be one of drugs for topical maintenance treatment of VVGS.

Objective To analyze the prevalence and characteristics of skin diseases in elderly patients with type 2 diabetes mellitus (T2DM).Methods Clinical out patient records for both elderly and non-elderly individuals with T2DM were collected from the dermatology department of Beijing Hospital.Statistical comparisons were performed between the elderly group and the non-elderly group on the prevalence of common skin complications and disease characteristics.Analysis was performed in the elderly group to identify any association of common skin disorders with gender,age,duration of T2DM,or level of glycosylated hemoglobin (HbA1c).Results Among 679 elderly T2DM cases,pruritus was the most common complication (26.1％),followed by eczema (21.t ％) and tinea pedis/onychomycosis (18.3％).Pruritus was more prevalent in the elderly group (177 cases,26.1％) compared to the non-elderly group (34 cases,9.8％) (x2 =6.006,P＜0.01).On the other hand,tinea pedis/onychomycosis was less prevalent in the senior group (124 cases,18.3 ％) than in the non-senior group (112 cases,32.2 ％) (x2 =37.437,P＜0.01).In the senior group,pruritus was more prevalent among male patients (105 out of 358 cases,29.3％) than among female patients(72 out of 321 cases,22.4％) (x2 =4.181,P =0.041).On average,patients with eczema were older[(72.3 ± 2.2)years] and had a longer history of T2DM [(8.3 ± 0.3)years] compared with patients without eczema[age:(65.2±1.9)years,history of T2DM:(5.2±0.1)years] (t=1.250,P=0.023 for age;t=1.680,P=0.033 for disease duration).Patients with tinea pedis/onychomycosis had higher HbA1c levels (6.7 ± 0.4) ％ than patients without tinea pedis/onychomycosis (6.1 ± 0.3) ％ (t =0.790,P＜0.01).Conclusions Pruritus is the most common skin complication in elderly patients with type 2 diabetes and is more common in elderly patients and male patients.Risk factors for developing eczema in elderly patients with type 2 diabetes may include older age and long disease duration.High levels of glycosylated hemoglobin in elderly patients with type 2 diabetes indicates increased risk for foot fungi infections.

Objective To study the discrepancy influence of the sulbactam content on susceptibility testing results of cefoperazone/sulbactam combination disks.Methods Agar dilution method was used to determine MICs of cefoperazone,cefoperazone/sulbactam(2:1 and 1:1),and disk diffusion was used to detect the zone diameters of cefoperazone,cefoperazone/sulbactam(75/30 and 75/75μg/disk)disks against 534 clinical gram-negative isolates.The discrepancy within the results of MICs,zore diameters,the method of agar dilution and disk diffusion was analyzed.Results By standard agar dilution method,MIC_(50) of cefoperazone,cefoperazone/sulbactam(2:1 and 1:1)were 32,16,16μg/ml,and MIC_(90) of those were ≥256.128,64 μg/ml respectively.No statistic discrepancy was found for MICs between the ratios of 2:1 and 1:1 combination by Wilcoxon ranks test(Z=-0.248,P=0.804).Susceptibility rate,resistance rate,and intermediate rate with 75/30μg disk were 55.3%,24.5%and 20.2%respectively,which were similar to those determined by agar dilution method.Susceptibility rate,resistance rate,and intermediate rate(I%)with 75/75μg disk were 72.5%,12.4% and 15.1% respectively,compared with the susceptibility rate from 75/30μg disk was 17.2% higher.Statistic discrepancy were tested by paired t-test (t=21.613,P＜0.01)with two groups of whole strains' zone diameters from 75/30μg and 75/75μg disks,and resulting in the difference of susceptibility or resistance rates for ESBL-producing strains,Acinetobacter bauamnnii and Enterobacteriaceae without ESBL tested isolates.On the contrary,for Pseudomonas aeruginosa,Stenotrophomonas maltophilia and ESBL negative isolates,the zone diameters discrepancy was not statistically significant between the results from 75/30μg and 75/75μg disks.Conclusions There is no statistic discrepancy between the susceptibility results from cefoperazone/sulbactam(2:1 or 1:1 ratios)in dilution method and in diffusion method with 75/30μg disk.When the 75/75μg disk is used to be tested for ESBL-producing strains,Acinetobacter bauamnnii and other Enterobacteriaceae,the results should be shown with sulbactam content.

Objective:To construct and prepare recombinant virus strains chimeric with human metapneumovirus (HMPV) antigenic epitopes.Methods:Recombinant influenza virus vectors which chimeric with different HMPV antigenic epitopes were rescued by reverse genetics using eight-plasmid system. The recombinant influenza virus strain used the internal genes of A/PR/8/34 (PB1, PB2, PA, NP, NS, M, HA, and NA) as a backbone, with concomitant genetic modifications to insert the B-cell epitopes of HMPV into the HA gene, and the CTL+ Th cell epitopes of HMPV into the NA gene. Preparation of recombinant influenza virus strains using reverse genetics in a " 7+ 1" model. The recombinant virus strains were evaluated by measuring hemagglutinin (HA) titers, half tissue culture infectious dose (TCID 50) and growth curves. Sequencing analysis was conducted to verify whether the rescued viruses carried the chimeric HMPV epitopes. Results:The epitopes of HMPV were inserted into the influenza virus genome and two recombinant influenza virus strains were rescued successfully, named as FLU/HMPV/B and FLU/HMPV/CTL+ Th. HA titers of the recombinant strains were both 2 7, their TCID 50 were 10 5.2/ml and 10 5.0/ml, respectively. After cultured for three passages in chick embryo, these two recombinant strains could proliferate steadily. Whole genome sequencing verified that the FLU/HMPV/B carried the B-cell epitopes of HMPV, the FLU/HMPV/CTL+ Th carried the CTL and Th cell epitopes of HMPV. Growth curve tests also verified that the recombinant strains could proliferate steadily in eggs. Conclusions:Two recombinant influenza virus vector strains carrying the B cell, CTL and Th epitopes of HMPV were rescued successfully. The result of the recombinant virus strains in terms of growth characteristics as well as genetic stability indicate that they meet the requirements for proceeding to the next step of animal experiments. The immunogenicity and immunoprotective effect will be further evaluated by mouse experiments. Ultimately new ideas for the realization of " one vaccine for two uses" or " one vaccine formultiple uses", as well as a new strategy for the development of HMPV vaccine will be proposed.

Objective:To analyze changes in melanocyte density and epidermal thickness in vulvar lichen sclerosus (VLS) lesions.Methods:Vulvar skin tissues were collected from 15 adult female patients with VLS in Department of Dermatology, Beijing Hospital from June to December in 2018. According to pathological manifestations, 15 skin lesions were divided into early-stage VLS group ( n = 7) and late-stage VLS group ( n = 8) , and subjected to immunofluorescent staining. Then, density of melanocytes, and thickness of the whole epidermis and cell layers (from the bottom of the stratum corneum to the bottom of the basal layer) were calculated by using an image analysis software. Normal vulvar skin tissues were obtained from 15 healthy adult females after vulvar plastic surgery in Plastic Surgery Hospital, Chinese Academy of Medical Sciences, and served as control group. Results:The density of melanocytes in the epidermis was significantly lower in the early-stage VLS group (0.170 ± 0.071) and late-stage VLS group (0.110 ± 0.035) than in the control group (0.275 ± 0.036; F = 36.426, P < 0.001) . There was no significant difference in the thickness of the whole epidermis among the early-stage VLS group (203.682 ± 137.997 μm) , late-stage VLS group (150.020 ± 70.914 μm) and control group (194.030 ± 82.996 μm; F = 0.738, P = 0.487) . The thickness of cell layers did not differ between the early-stage VLS group (154.603 ± 121.984 μm) and control group (176.974 ± 80.296 μm; P = 0.899) , but was significantly thinner in the late-stage VLS group (83.455 ± 37.129 μm) than in the control group ( P = 0.003) . Conclusions:The density of epidermal melanocytes decreased in the early-stage and late-stage VLS skin lesions. Compared with the normal skin tissues, the early-stage VLS lesions showed no significant changes in the thickness of the whole epidermis and cell layers, but the late-stage VLS lesions showed decreased thickness of cell layers.

Objective:To design the fourth-generation chimeric antigen receptor-T (CAR-T) cells that secrete interleukin-7 (IL7) and chemokine C legend 19 (CCL19) on the basis of the second-generation CAR, and to analyze and compare the differences in proliferation, chemotaxis, tumor cell clearance and persistence in the microenvironment of multiple myeloma (MM) between them.Methods:The fourth-generation CAR vector plasmid was constructed by using 2A self-cleaving peptide technology. The third-generation lentiviral packaging system was used to prepare high-titer lentivirus. Flow cytometry was used to monitor the transduction efficiency of lentivirus and the subtype changes of CAR-T cells. The enzyme-linked immunosorbent assay (ELISA) was used to quantify the IL7 and CCL19 secreted by CAR-T cells.The calculation of absolute number of CAR-T cells during culture was used to analysis cell proliferation activity. Transwell migration assay was used to verify the chemotactic ability of CAR-T cells. The specific killing activity of CAR-T cells was detected by using the luciferase bioluminescence method. The NOD-Prkdcem26Cd52Il2rgem26Cd22/Nju (NOD) mouse xenograft model was used to verify the anti-myeloma activity and safety of CAR-T cells in vivo.Results:Flow cytometry results showed that the stable CAR expression rates of the second-generation anti-CS1 CAR-T and fourth-generation anti-CS1-IL7-CCL19 CAR-T cells were (91.50±0.29)% and (46.7±0.12)%, respectively. CAR-T cells were successfully constructed. Subtype analysis demonstrated that the ratio of stem memory T cell (TSCM) in anti-CS1-IL7-CCL19 CAR-T cells was (67.58±0.59)%, which was significantly higher than (50.74 ± 1.01)% of anti-CS1 CAR-T ( P=0.000 1), with more strong immune memory function and better durability. Anti-CS1-IL7-CCL19 CAR-T cells can continuously secrete IL7 and CCL19 compared to MOCK-T and anti-CS1 CAR-T ( P<0.000 1). The number of anti-CS1-IL7-CCL19 CAR-T cells reached (22.77±0.79)×10 6 on the 9th day after lentivirus transduction, which was significantly higher than (9.40±0.79)×10 6 of anti-CS1 CAR-T cells ( P=0.000 1), with stronger proliferation ability. The number of chemotaxis cells of anti-CS1-IL7-CCL19 CAR-T cells to reactive T cells was (109.0±4.04), which was significantly higher than (9.33±1.20) of MOCK-T ( P<0.000 1) and (7.33±0.88) of anti-CS1 CAR-T ( P<0.000 1), with stronger chemotactic ability. The specific killing activity showed that both anti-CS1-IL7-CCL19 CAR-T and anti-CS1 CAR-T cells had specific killing efficacies when compared with the MOCK-T cells ( P<0.000 1). Animal experiment indicated that anti-CS1-IL7-CCL19 CAR-T cells significantly reduced the tumor burden ( P<0.000 1) and extended the overall survival time ( P=0.006 1) of tumor-bearing mice. Conclusions:The anti-CS1-IL7-CCL19 CAR-T cells designed in this study show stronger proliferative activity, chemotactic ability, and durability without affecting the anti-myeloma activity in vivo and in vivo, which provides strategies for overcoming the defects of low survival rate, poor durability and inhibition by tumor microenvironment of traditional CAR-T cells, and offers preliminary experimental basis for the clinical application of the fourth-generation CAR-T cells.

Objective:To investigate the impact of daily step count on glycemic outcomes in community residents with impaired glucose tolerance (IGT).Methods:This was a prospective cohort study, in October 2018, 204 residents who met the criteria of IGT were recruited in the Shijingshan District in Beijing. The subjects were tested for fasting blood glucose, oral glucose tolerance test 2-hour blood glucose (2hBG), glycated hemoglobin A 1c (HbA 1c), lipid profile, liver and kidney function, as well as measurements of height, weight and waist circumference. A dedicated mobile application was used to deliver prediabetes health knowledge monthly. Online guidance was provided to answer questions and daily step count was collected using the application. Three years later, a follow-up was conducted to assess the participants′ glycemic outcomes and other indexes, and a total of 142 participants completed the follow-up review. According to daily step count, the subjects were categorized into high step count group (42 cases,>7 000 steps daily), moderate step count group (54 cases, 5 000-7 000 steps daily), and low step count group (46 cases,<5 000 steps daily). Subjects were categorized into diabetes group (30 cases), prediabetes group (77 cases) and normal glucose tolerance group (35 cases) with glycemic outcomes. Independent sample t test was used to compare the differences in blood glucose, blood lipids, and step counts between the two groups. Kruskal-Wallis H test or one-way ANOVA was used to compare the differences in blood glucose, blood lipids, and step counts between multiple groups. The χ2 test was used to compare the differences in glycemic outcomes between multiple groups. Multivariate logistic regression analysis was used to assess the impact of daily step counts and body mass index on glycemic outcomes. Linear regression analysis was used to evaluate the relationship between daily step counts and 2 h BG. Results:A total of 142 participants completed the 3-year follow-up, including 43 males and 99 females, with a mean age of (60.15±5.67) years. At baseline, males had significantly higher body mass index, waist circumference, and fasting blood glucose when compared to those in females [(26.97±2.43) vs (24.89±2.93) kg/m 2, (92.68±7.75) vs (83.83±8.60) cm, (5.83±0.61) vs (5.62±0.52) mmol/L], the total cholesterol and HDL-C were also significantly lower in males than those in females [(5.10±1.16) vs (5.55±0.95) mmol/L, (1.35±0.34) vs (1.56±0.35) mmol/L] (all P<0.05). After 3-year follow-up, 21.1% (30/142) of IGT participants progressed to diabetes, with an annual conversion rate of approximately 7%. The normal glucose tolerance group showed significantly higher daily step counts when compared with the prediabetes and diabetes groups [(7 886±2 867) vs (5 981±2 655) vs (4 117±2 674) steps] ( H=31.778, P<0.001). Individuals with higher daily step counts exhibited lower body mass index, 2 h BG, and HbA 1c level when compared with those in the ones with moderate and low step counts [(24.26±3.09) vs (25.44±3.38) vs (26.26±3.59) kg/m 2, (7.50±1.71) vs (9.15±3.30) vs (11.19±3.84) mmol/L, 5.97%±0.46% vs 6.14%±0.99% vs 6.40%±0.96%] (all P<0.05). Higher step count was positively correlated with the reversal of prediabetes to normal blood glucose levels (moderate step count, OR=0.297, 95% CI: 0.109-0.804; low step count, OR=0.055, 95% CI: 0.010-0.287), lower daily step count correlated positively with prediabetes progressing to diabetes ( OR=4.857, 95% CI: 1.140-20.689) (all P<0.05). For every additional 1 000 steps per day, the 2 h BG decreased by 0.5 mmol/L. Conclusion:As daily step count increases, the glucose metabolism improves in IGT community residents. Higher daily step count is associated with reversal of IGT to normal glucose tolerance, while lower daily step count may be associated with the progression of IGT to diabetes.

Objective:To understand the epidemiological characteristics of group A rotavirus (RVA) infection in hospitalized children under 5 years of age with diarrhea in 2019, and to provide reference for the surveillance of RVA.Methods:Stool samples and clinical information of hospitalized children under 5 years of age with diarrhea were collected from sentinel hospitals in 20 provinces in 2019. RVA nucleic acid detection and genotyping were performed according to the rotavirus detection method in the National Viral Diarrhea Surveillance Program.Results:A total of 5 395 viral diarrhea samples were collected, 5 038 were tested, and 1 247 diarrhea samples showed RVA positive results (1 247/5 038, 24.75%). The positive rate of RVA in Fujian province was the lowest (30/319, 9.40%), and the positive rate of RVA was the highest in Henan province (182/338, 53.85%). The positive rate of RVA in male and female children was 25.24%(762/3 019)and 24.02%(485/ 2 019), respectively. There was no significant gender distribution of RVA infection ( χ2 = 0.96, P=0.326). Children aged 12 to 17 months were mainly susceptible to RVA (342/1 033, 33.11%), and the positive rate of RVA in children aged 48 to 59 months was lower (35/227, 15.42%). RVA infection showed significant age distribution characteristics ( χ2 = 86.78, P<0.001). RVA infection had significant difference between urban and rural areas ( χ2 = 20.92, P<0.001) and seasonal characteristics ( χ2 =411.42, P<0.001). RVA genotyping showed that G9P[8] type (994/1 122, 88.59%) was the dominant epidemic strain. Conclusions:In 2019, the main genotype of RVA infection in hospitalized children under 5 years of age with diarrhea was G9P[8], and RVA infection had significant age, region and season characteristics.