Objective The purpose of this study was to investigate the bacterial communities of biting midges and ticks collected from three sites in the Poyang Lake area,namely,Qunlu Practice Base,Peach Blossom Garden,and Huangtong Animal Husbandry,and whether vectors carry any bacterial pathogens that may cause diseases to humans,to provide scientific basis for prospective pathogen discovery and disease prevention and control. Methods Using a metataxonomics approach in concert with full-length 16S rRNA gene sequencing and operational phylogenetic unit(OPU)analysis,we characterized the species-level microbial community structure of two important vector species,biting midges and ticks,including 33 arthropod samples comprising 3,885 individuals,collected around Poyang Lake. Results A total of 662 OPUs were classified in biting midges,including 195 known species and 373 potentially new species,and 618 OPUs were classified in ticks,including 217 known species and 326 potentially new species.Surprisingly,OPUs with potentially pathogenicity were detected in both arthropod vectors,with 66 known species of biting midges reported to carry potential pathogens,including Asaia lannensis and Rickettsia bellii,compared to 50 in ticks,such as Acinetobacter lwoffii and Staphylococcus sciuri.We found that Proteobacteria was the most dominant group in both midges and ticks.Furthermore,the outcomes demonstrated that the microbiota of midges and ticks tend to be governed by a few highly abundant bacteria.Pantoea sp7 was predominant in biting midges,while Coxiella sp1 was enriched in ticks.Meanwhile,Coxiella spp.,which may be essential for the survival of Haemaphysalis longicornis Neumann,were detected in all tick samples.The identification of dominant species and pathogens of biting midges and ticks in this study serves to broaden our knowledge associated to microbes of arthropod vectors. Conclusion Biting midges and ticks carry large numbers of known and potentially novel bacteria,and carry a wide range of potentially pathogenic bacteria,which may pose a risk of infection to humans and animals.The microbial communities of midges and ticks tend to be dominated by a few highly abundant bacteria.

Objective:To discuss the inhibitory effect of Schisandrin B on the proliferation of pancreatic cancer Pan02 cells,and to clarify the mechanism.Methods:CCK-8 method was used to detect the proliferation rates of the Pan02 cells after treated with different concentrations(0,0.78,1.56,3.12,6.25,12.50,and 25.00 mg·L-1)of Schisandrin B to select the optimal concentration and treatment time of Schisandrin B.The mouse pancreatic cancer Pan02 cells were divided into control group(0 mg·L-1 Schisandrin B),2.5 mg·L-1 Schisandrin B group,5.0 mg·L-1 Schisandrin B group,and 10.0 mg·L-1 Schisandrin B group.The morpholoy of Pan02 cells invarious groups was observed with light microscope;5-ethynyl-2'-deoxyuridine(EdU)staining assay was used to detect the positive expression rates of the Pan02 cells in various groups;flow cytometry was used to detect the percentages of the Pan02 cells at different cell cycles and the apoptotic rates of the cells in various groups;Western blotting method was used to detect the expression levels of cell cycle and apoptosis-related proteins in the cells in various groups.Results:The CCK-8 method results showed that after treated with Schisandrin B for 48 and 72 h,compared with 0 mg·L-1 Schisandrin B,the proliferation rates of the Pan02 cells after treated with different concentrations of Schisandrin B were decreased(P<0.01),especially at 72 h.0.25,5.0,and 10.0 mg·L-1 Schisandrin B were selected to treat the Pan02 cells,and 72 h was the treatment time.In control group,the Pan02 cells had a spindle shape,with good condition,and grew closely adhered to the wall with normal organelles and cytoplasm,in 2.5 and 5.0 mg·L-1 Schisandrin B groups,the cell volume was decreased,the intercellular adhesion was disappeared,and the cell membrane was intact but more permeable;the cytoplasm shrank and vacuolar structures appeared inside the cells,with some fragmented and floating on the surface of the solution;in 10.0 mg·L-1 Schisandrin B group,the Pan02 cells exhibited notable apoptotic bodies,indicating an apoptotic state.The EdU staining results showed that compared with control group,the rates of EdU positive cells in 2.5,5.0,and 10.0 mg·L-1 Schisandrin B groups were significantly decreased(P<0.01).The flow cytometry results showed that compared with control group,the percentages of the cells at S phase in 2.5,5.0,and 10.0 mg·L-1 Schisandrin B groups were significantly increased(P<0.01),while the percentages of the cells at G2/M phase were significantly decreased(P<0.01),and the percentages of the cells at G0/G1 phase in 5.0 amd 1.0 mg·L-1 Schisandrin groups were decreased(P<0.01);compared with control group,the apoptotic rates of the cells in 2.5,5.0,and 10.0 mg·L-1 Schisandrin B groups were significantly increased(P<0.01).The Western blotting results showed that compared with control group,the expression levels of p27,B-cell lymphoma 2(Bcl-2)associated X protein(Bax),cleaved cysteine aspartic acid protease-3(cleaved Caspase-3),and cleaved poly adenosine diphosphate(ADP)ribose polymerase(cleaved PARP)proteins in the cells in 2.5 mg·L-1 Schisandrin B group were significantly increased(P<0.05 or P<0.01),the expression levels of cyclin A2,cyclin E2,and Bcl-2 proteins in the cells in 5.0 and 10.0 mg·L-1 Schisandrin B groups were significantly decreased(P<0.05 or P<0.01),while the expression levels of p27,Bax,cleaved Caspase-3,and cleaved PARP proteins in the cells in 5.0 and 10.0 mg·L-1 Schisandrin B groups were significantly increased(P<0.01).Conclusion:Schisandrin B has an inhibitory effect on proliferation of the pancreatic cancer Pan02 cells,and its mechanism may be related to the activation of the cysteine aspartic acid protease-3(Caspase-3)pathway to induce the apoptosis and activating p27 protein to induce the arrest of cell cycle at S phase.

Objectives:To discuss the inhibitory effect of Lactobacillus reuteri on the replication of rotavirus(RV)strain SA11 in vivo and in vitro,and to clarify its effect on the expression of related immune factors.Methods:For in vitro experiments,Lactobacillus reuteri was cultured and identified,and the standard curve and growth curve were plotted to screen the optimal time and concentration for Lactobacillus reuteri cultivation.The cells were infected with Lactobacillus reuteri at the concentrations of 5×108,10×108,50×108,100×108,200×108,and 500×108 CFU·mL-1,and the surival rates of Caco-2 cells were detected by trypan blue staining method.Various concentrations of Lactobacillus reuteri were co-incubated with RV in vitro and applied to the Caco-2 cells.The cells were divided into negative control group(NC group),positive control group(PC group),and 107,108,109,and 1010 CFU·mL-1 Lactobacillus reuteri groups.Immunofluorescence focus method was used to detect the viral titers in the Caco-2 cells after treated with Lactobacillus reuteri and real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the copy numbers of RV VP6 gene in the Caco-2 cells after treated with various concentrations of Lactobacillus reuteri.In in vivo experiments,25 litters of SPF suckling mice were divided into control group,RV group(infected with SA11 strain),Ab-NC group(treated with antibiotic to deplete gut microbiota),Ab-RV group(depleting gut microbiota and then infected with SA11 strain),and Ab-Lac-RV group(depleting gut microbiota,treated with Lactobacillus reuteri,and then infected with SA11 strain).The fecal samples were collected on days 2,4,6,8,and 10 gavage,colon tissue sample were collected on day 4 of and RT-qPCR method was used to detect the copy numbers of RV VP6 gene in feces and the mRNA expression levels of interleukin(IL)-1β,IL-8,IL-10,interferon-γ(IFN-γ),and tumor necrosis factor-α(TNF-α)in colon tissue of the suckling mice in vartious groups.Results:The Lactobacillus reuteri grew well,with round,smooth,and milky white convex colonies and neat edges.After Gram staining,the bacteria appeared purple,irregular,and square-shaped rods.16SrDNA sequencing showed 99%sequence homology,indicating successful activation of Lactobacillus reuteri.The number of live Lactobacillus reuteri was linearly related to the absorbance(A)value,and the standard curve for regression analysis was Y=0.437 5X+0.000 6,R2=0.999 4.During the 0-2 h cultivation period,the bacteria were at the logarithmic growth phase with slow growth;from 2-14 h,the bacteria grew rapidly and stabilized at 14-16 h,reaching the growth rate peak at 16 h,after which they entered the decline phase.Infection with Lactobacillus reuteri at concentrations of 5×108,10×108,50×108,100×108,and 200×108 CFU·mL-1 resulted in the survival rates of Caco-2 cells were all>90%,so these concentrations were selected for the further experiments.Compared with PC group,the copy numbers of RV VP6 gene in the Caco-2 cells in 5×108,10×108,50×108,100×108,and 200×108 CFU·mL-1 Lactobacillus reuteri groups were significantly decreased(P<0.01).Compared with PC group,the viral titers in the Caco-2 cells in 107,108,109,and 1010 CFU·mL-1 Lactobacillus reuteri groups were significantly decreased(P<0.01).Compared with control group,the numbers of gut microbiota colonies in Ab-NC,Ab-RV,and Ab-Lac-RV groups were significantly decreased,indicating successful depletion of gut microbiota in the suckling mice.On days 2 and 4 after gavage,the RV VP6 gene copy number in the feces in Ab-RV group was significantly lower than that in RV group(P<0.05).On days 4,6,8,and 10 after gavage,the RV VP6 gene copy number in the feces in Ab-Lac-RV group was significantly lower than that in Ab-RV group(P<0.05).Compared with control group,the expression levels of IL-1β,IL-10,IFN-γ,and TNF-α mRNA in colon tissue in Ab-RV and Ab-Lac-RV groups were significantly increased(P<0.05 or P<0.01),while the expression level of IL-8 mRNA was significantly decreased(P<0.05),and the expression level of IL-10 mRNA in colon tissue in Ab-LAC-RV group was significantly increased(P<0.01).Conclusion:Lactobacillus reuteri may inhibit the RV replication by upregulating the expressions of IL-1β,IL-10,IFN-γ,and TNF-α mRNA and downregulating the expression of IL-8 mRNA.

Dental hard tissues lack the ability to self-heal. In dentin and cementum, hydroxyapatite (HA) can exist outside and/or inside collagen fibers. It is difficult to repair or regenerate HA with a highly ordered orientation in the presence of collagen fibers. At present, the biomimetic mineralization of dentin and cementum, mainly carried out by imitating its biological formation process and its physiological structure, can be divided into those originating from the fiber mineralization mechanism and those with HA as the main component. The materials used include natural materials such as demineralized dentin matrix (DDM) and calcined bovine hydroxyapatite (BHA), and synthetic materials such as polymer-induced liquid precursor (PILP) and synthetic HA. In the future, natural materials and synthetic materials should be combined for the restoration and regeneration of dentin and cementum by means of biomimetic mineralization of calcium phosphate released by remineralization solution-HA.

OBJECTIVE: To carry out genetic testing for an abortus suspected with Cornelia de Lange syndrome (CdLS). METHODS: History of gestation and the family was taken. Combined with prenatal ultrasonography and the phenotype of the abortus, a diagnosis was made for the proband. Fetal tissue and peripheral blood samples of its parents were collected for the extraction of genomic DNA. Whole exome sequencing was carried out to detect mutations related to the phenotype. Suspected mutations were verified in the parents through Sanger sequencing. RESULTS: Prenatal ultrasound found that the forearms and hands of the fetus were anomalous, in addition with poorly formed vermis cerebellum, slight micrognathia, and increased echo of bilateral renal parenchyma. Examination of the abortus has noted upper limb and facial malformations. Whole exome sequencing revealed that the fetus carried a heterozygous c.2118delG (p.Lys706fs) frameshift mutation of the NIPBL gene. The same mutation was not found in either parent. CONCLUSION The heterozygous c.2118delG (p.Lys706fs) frameshift mutation of the NIPBL gene probably underlies the CdLS in the fetus. Above finding has provided a basis for the genetic counseling for the family.
Cell Cycle Proteins/genetics* ; DNA Mutational Analysis ; De Lange Syndrome/pathology* ; Female ; Fetus ; Humans ; Male ; Mutation ; Phenotype ; Pregnancy ; Whole Exome Sequencing

Objective:Experimental autoimmune thyroiditis (EAT) rat model was establish to observe the effects of iodine excess on thyroid function, antibody and thyrotropin receptor (TSHR) gene expression in EAT rats, and to explore the role of TSHR gene in autoimmune thyroiditis.Methods:According to body weight (80 - 180 g), 48 rats (4-week-old female Lewis) were randomly divided into control group, thyroglobulin (TG) group, TG + high iodine Ⅰ(TG + HⅠ) group, and TG + high iodine Ⅱ (TG + HⅡ) group, 12 rats per group. The iodine concentration in drinking water given to each group was 50 μg/L, 50 μg/L, 20 mg/L and 200 mg/L, respectively. At the same time, rats in TG, TG + HⅠ and TG + HⅡ groups were immunized once every two weeks for three times using pTg and CFA as immunoreagent. Paraffin embedded sections of thyroid tissues were used to observe the pathological changes of rats. The serum levels of thyroglobulin antibody (TgAb), thyroperoxidase autoantibody (TPOAb), free triiodothyronine (FT 3) and free thyroxine (FT 4) in rats were determined by radioimmunoassay. Serum TSHR content in rats was determined by enzyme linked immunosorbent assay (ELISA). The expression of TSHR mRNA in whole blood and thyroid tissue of rats was determined by RT-PCR. The expression of TSHR protein in thyroid tissue of rats was determined by immunohistochemistry (IHC). Results:Hematoxylin-eosin (HE) showed that the thyroid follicles in control group were complete in structure and regular in shape, and no lymphocyte infiltration was observed. A small number of lymphocytes were observed in TG group and scattered in distribution. Follicular structure destruction, fusion and interfollicular infiltration were observed in TG + HⅠ group and TG + HⅡ group. There were significant differences in serum TgAb, TPOAb, FT 3 and FT 4 levels among all groups ( H = 30.28, 21.99, 12.87, 26.69, P < 0.05). Compared to the control group [6.89 (6.32, 7.27), 11.02 (7.60, 12.53), 5.05 (2.71, 7.99), 7.51 (6.50, 9.24) pmol/L], the levels of TgAb [34.99 (25.39, 41.35), 37.70 (29.06, 43.99), 46.41 (38.52, 55.26)], TPOAb [22.87 (13.65, 31.82), 22.22 (14.82, 28.33), 14.61 (12.95, 19.34)], FT 3 [57.74 (24.56, 64.27), 43.64 (5.69, 80.03), 38.56 (17.73, 47.59) pmol/L], and FT 4 [62.16 (41.22, 91.57), 60.61 (35.52, 103.31), 47.96 (31.84, 112.71) pmol/L] were significantly higher in TG group, TG + HⅠ group, and TG + HⅡ group ( P < 0.05). Compared with the control group [(249.37 ± 38.12) μU/L], TG group [(225.33 ± 41.28) μU/L], and TG + HⅠ group [(218.15 ± 65.51) μU/L], TSHR expression level in TG + HⅡ group [(154.26 ± 25.95) μU/L] were significantly decreased ( P < 0.05). The mRNA expression levels of TSHR gene in the whole blood (0.89 ± 0.19, 0.89 ± 0.30, 0.85 ± 0.24) and thyroid tissue(0.63 ± 0.25, 0.46 ± 0.16, 0.51 ± 0.25) of TG group, TG + HⅠ group and TG + HⅡ group were significantly lower than that of control group (1.00 ± 0.05, 1.13 ± 0.21, P < 0.05). IHC showed that the positive intensity of TSHR protein in control group was significantly higher than that in TG group, TG + HⅠ group and TG + HⅡ group. Conclusions:Long-term exposure to high iodine will eventually lead to the damage of iodine-uptake function in thyroid gland and thyroid diseases. Abnormal expression of TSHR gene may lead to antigenicity of thyrotropin binding site in extracellular receptor region and autoimmune thyroid disease.

Objective:By establishing a rat model of experimental autoimmune thyroiditis(EAT), to investigate the effects of different iodine intake on the hippocampal morphology, monoamine neurotransmitters and ethology of the offspring of EAT rats.Methods:A total of 60 female and 20 male Lewis rats with a body weight of 50 - 60 g were selected. Female rats were divided into 4 groups (15 rats in each group) with random number table method according to their body weight: control group (NI group), thyroglobulin group (Tg group), Tg + high iodine Ⅰ group (Tg + HⅠ group), and Tg + high iodine Ⅱ group (Tg + HⅡ group), and the latter three groups were model groups. The contents of iodine in drinking water of the 4 groups were 100 μg/L, 100 μg/L, 20 mg/L and 200 mg/L, respectively. Rats in the model groups were immunized with porcine thyroglobulin (PTg) subcutaneously at multiple sites, and the NI group was injected with normal saline, once every 2 weeks, 3 times in total. The rats in each group were mated in cages according to the ratio of 3 : 1 between female and male. After experiment of the offspring, the urine samples of mother rats were collected within the previous week, urinary iodine concentration was determined by As 3+-Ce 4+ catalytic spectrophotometry; then the mother rats were killed, HE staining was used to observe the changes of thyroid histomorphology and the infiltration of inflammatory cells; serum thyroglobulin antibody (TgAb) and thyroid peroxidase antibody (TPOAb) of mother rats were determined by radioimmunoassay. Brain tissues were collected from 7 days old offspring, hippocampal morphology of 7 days old offspring was observed by toluidine blue staining; the contents of norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) in brain tissues of 7 days old offspring were measured by enzyme-linked immunosorbent assay (ELISA); 30 and 60 days old offspring were used for water maze-location navigation test and open field test. Results:The levels of urinary iodine increased significantly of mother rats in Tg + HⅠ and Tg + HⅡ groups than that in NI group (median, μg/L: 35 380.18, 236 847.16 vs 221.43, P < 0.05). HE staining showed that the thyroid tissue of mother rats in Tg, Tg + HⅠ and Tg + HⅡ groups had different degrees of destruction and inflammatory cells infiltration, and the degree of destruction and infiltration increased with the increase of iodine intake. Compared with NI group, the contents of TgAb and TPOAb in serum of mother rats in Tg, Tg + HⅠ and Tg + HⅡ groups were significantly increased(2.118 4 ± 0.675 1, 2.103 0 ± 0.714 1, 2.783 6 ± 1.084 3 vs 0.790 1 ± 0.101 0, P < 0.05; 1.015 8 ± 0.252 8, 1.019 5 ± 0.202 0, 0.936 6 ± 0.183 4 vs 0.692 2 ± 0.111 9, P < 0.05), and the content of TgAb in Tg + HⅡ group was significantly higher than that in Tg and Tg + HⅠ groups ( P < 0.05). Compared with NI group, the number of hippocampal neurons decreased and relative damage occurred in Tg, Tg + HⅠ and Tg + HⅡ groups of the offspring. Compared with NI group, the NE contents in brain tissues of the offspring in Tg, Tg + HⅠ and Tg + HⅡ groups decreased (pg/ml: 1 232.01 ± 253.45, 1 197.64 ± 222.46, 1 074.40 ± 366.38 vs 1 733.67 ± 158.12, P < 0.05); there were no significant differences in DA and 5-HT contents in brain tissues of offspring in each group ( P > 0.05). In the water maze-location navigation test, the latency of the Tg + HⅡ group on the 4th day of the 30 days old offspring reaching the platform was significantly longer than that of the NI and Tg groups ( P < 0.05). In the open field test, there was no significant difference in 30 and 60 days old offspring in the latency of moving the original quadrant ( P > 0.05). Conclusions:With the increase of iodine intake, the degrees of thyroid tissue destruction and inflammatory cells infiltration in EAT rats increase, and the levels of TgAb in serum increase significantly. Iodine has certain effects on the hippocampal morphology and the level of monoamine neurotransmitters in the brains of the offspring of EAT rats. The effects of different iodine-induced EAT rats on their offspring's learning, memory and spatial exploration are mainly shown in childhood.

OBJECTIVE: To diagnose a fetus with Papillorenal syndrome by prenatal ultrasonography and genetic testing, and to correlate its genotype with phenotype. METHODS: Ultrasound finding of the fetus was reviewed. Muscle sample of the abortus was taken, and genetic variant related to the clinical phenotype was screened by whole exome sequencing (WES). Suspected pathogenic variant was verified by Sanger sequencing. RESULTS: Prenatal ultrasound revealed severe dysplasia of the fetal kidneys and oligohydramnios. WES revealed that the fetus has carried a c.736G>T (p.Glu246Ter) nonsense variant of the PAX2 gene, which was unreported previously. The result of Sanger sequencing was consistent with that of WES. Both parents of the fetus were of the wild-type, suggesting a de novo origin of the fetal variant. CONCLUSION The novel heterozygous c.736G>T (p.Glu246Ter) variant of the PAX2 gene probably underlay the Papillorenal syndrome in the fetus. Above finding has provided a basis for genetic counseling and clinical decision-making.

OBJECTIVE: To explore the genetic basis for fetus with short limbs detected by prenatal ultrasonography. METHODS: Results of clinical imaging of the fetus was collected. Amniotic fluid sample was collected through amniocentesis for the extraction of fetal DNA. Whole exome sequencing was carried out to detect variants related to the clinical phenotypes. Candidate variant was verified by Sanger sequencing. RESULTS: Prenatal ultrasound showed that the fetus had short limbs but no other abnormality. Whole exome sequencing has identified that the fetus carried two heterozygous pathogenic variants c.484G>T and c.1436dupA of the SLC26A2 gene, for which its mother and father were heterozygous carriers, respectively. CONCLUSION The fetus was diagnosed with atelosteogenesis type 2 by combined prenatal ultrasonography and whole exome sequencing, which may be attributed to the compound heterozygous variants of the SLC26A2 gene. Above findings provided evidence for the diagnosis of the fetus and genetic counseling.

Objective:To explore the application effect of five-in-one anti-cancer support model in patients with breast neoplasms.Methods:A total of 70 patients who were admitted to the Fourth Ward of Department of Breast Surgery in Harbin Medical University Cancer Hospital from May to September 2019 were selected as research objects by using the convenient sampling method. According to the random number table method, they were randomly divided into the study group and the control group, with 35 cases in each group.The control group was given routine nursing, while the study group was given five-in-one anti-cancer support model nursing. The depression, anxiety, social isolation, social support and quality of life scores of patients of the two groups were evaluated before and after the intervention.Results:After intervention, the depression scores of patients in the study group were lower than those in the control group. The total score of quality of life, score of social and family status and score of functional status in the study group were lower than those in the control group. The differences were statistically significant ( P<0.05) . Conclusions:The five-in-one anti-cancer support model is adopted to design body, mind, spirit, and social relaxation courses that meet the characteristics of breast cancer patients to meet their psychological and social needs, which has certain clinical significance.