ObjectiveTo investigate the effects of different fermentation methods and times on the fungal flora and chemical composition of Aurantii Fructus， in order to obtain the optimal fermentation conditions and flora structure， and to ensure the stability and controllability of the fermented varieties. MethodsScanning electron microscopy was used to observe and analyze the colony characteristics on the surface of Aurantii Fructus under different fermentation conditions. Internal transcribed spacer 2（ITS2） high-throughput sequencing， combined with fungal community diversity analysis and fungal community structure analysis， were used to obtain the fungal flora microbial categories of Aurantii Fructus under the conditions of traditional pressure-shelf fermentation and non-pressure-shelf natural fermentation for 7， 14， 21 d（numbered Y1-Y3 for the former， and numbered F1-F3 for the latter）， respectively. At the same time， the chemical components in the fermentation process were detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， combined with principal component analysis（PCA）， partial least squares-discriminant analysis（PLS-DA） and compound retention time， parent ions， characteristic fragment ions and other information， the differential compounds between the different fermentation samples were screened and identified. ResultsThe analysis of fungal community diversity showed that the dominant flora did not change at different fermentation time points in the traditional pressure-shelf fermentation method， while in the non-pressure-shelf natural fermentation method， there was a significant difference with the fermentation process， and at the genus level， the dominant genus of samples Y1， Y2， Y3 and F2 was Aspergillus， while the dominant genera of samples F1 and F3 were both Rhizopus. This indicated that the microbial growth environment provided by the traditional fermentation method was more stable， and the microbial community structure was more stable， which was more conducive to the stable and controllable fermentation process and fermented products. A total of 155 compounds were identified by compositional analysis， including 70 flavonoids， 38 coumarins， 10 alkaloids， 34 organic acids and 3 other compounds. After fermentation， two new components of ribalinine and pranferin were produced. Different fermentation conditions also brought about differences in chemical composition， multivariate statistical analysis obtained 26 differential compounds under two different fermentation methods， mainly including flavonoids， organic acids and coumarins. Comprehensively， the microbial community structure of samples fermented by the traditional pressure-shelf method of Aurantii Fructus for 14 d was stable， the species richness was high and the overall content of differential compounds was high， which was the optimal processing condition. ConclusionCompared with non-pressure-shelf natural fermentation， the traditional method has obvious advantages in terms of the stability of the microbial community structure and the content of chemical compounds， and the optimal condition is 14 days of fermentation. This study is helpful to promote the quality stability and fermentation bioavailability of fermented products of Aurantii Fructus， as well as to provide an experimental basis for the further improvement of the quality control methods of this variety.

ObjectiveTo investigate the effects of different fermentation methods and times on the fungal flora and chemical composition of Aurantii Fructus， in order to obtain the optimal fermentation conditions and flora structure， and to ensure the stability and controllability of the fermented varieties. MethodsScanning electron microscopy was used to observe and analyze the colony characteristics on the surface of Aurantii Fructus under different fermentation conditions. Internal transcribed spacer 2（ITS2） high-throughput sequencing， combined with fungal community diversity analysis and fungal community structure analysis， were used to obtain the fungal flora microbial categories of Aurantii Fructus under the conditions of traditional pressure-shelf fermentation and non-pressure-shelf natural fermentation for 7， 14， 21 d（numbered Y1-Y3 for the former， and numbered F1-F3 for the latter）， respectively. At the same time， the chemical components in the fermentation process were detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， combined with principal component analysis（PCA）， partial least squares-discriminant analysis（PLS-DA） and compound retention time， parent ions， characteristic fragment ions and other information， the differential compounds between the different fermentation samples were screened and identified. ResultsThe analysis of fungal community diversity showed that the dominant flora did not change at different fermentation time points in the traditional pressure-shelf fermentation method， while in the non-pressure-shelf natural fermentation method， there was a significant difference with the fermentation process， and at the genus level， the dominant genus of samples Y1， Y2， Y3 and F2 was Aspergillus， while the dominant genera of samples F1 and F3 were both Rhizopus. This indicated that the microbial growth environment provided by the traditional fermentation method was more stable， and the microbial community structure was more stable， which was more conducive to the stable and controllable fermentation process and fermented products. A total of 155 compounds were identified by compositional analysis， including 70 flavonoids， 38 coumarins， 10 alkaloids， 34 organic acids and 3 other compounds. After fermentation， two new components of ribalinine and pranferin were produced. Different fermentation conditions also brought about differences in chemical composition， multivariate statistical analysis obtained 26 differential compounds under two different fermentation methods， mainly including flavonoids， organic acids and coumarins. Comprehensively， the microbial community structure of samples fermented by the traditional pressure-shelf method of Aurantii Fructus for 14 d was stable， the species richness was high and the overall content of differential compounds was high， which was the optimal processing condition. ConclusionCompared with non-pressure-shelf natural fermentation， the traditional method has obvious advantages in terms of the stability of the microbial community structure and the content of chemical compounds， and the optimal condition is 14 days of fermentation. This study is helpful to promote the quality stability and fermentation bioavailability of fermented products of Aurantii Fructus， as well as to provide an experimental basis for the further improvement of the quality control methods of this variety.

Objective:To analyze the identification and antibiotics susceptibility of Herbaspirillum in catheter-related bloodstream infection, and improve the awareness and attention of the rare pathogenic microorganisms for clinicians and microbiologists. Methods:The bacterium was isolated from a positive blood culture of a hemodialysis patient with chronic renal failure. The smear of isolate was prepared, stained and observed by microscope. The single colonies were identified by mass spectrometry and VITEK 2 Compact identification and antibiotics sensitivity analysis system, respectively. Then, 16S ribosomal DNA (rDNA) was amplified and sequenced, and bacterial genome was sequenced.Results:The gram-negative bacilli was found in the positive blood culture bottle. After incubated on blood agar for 16 hours, milky white, bulging and non-haemolytic colonies were observed. The identification result was Burkholderia cepacian by VITEK 2_Compact and antimicrobial susceptibility testing showed resistance to aztreonam and polymyxin but sensitive to other drugs in N335 card. The isolate could not be identified by VITEK MS with clinical database. However, it was identified as Herbaspirillum huttiense/Herbaspirillum aquaticum with research database. The 16S rDNA of the strain was consistent with Herbaspirillum huttiense and Herbaspirillum aquaticum (more than 99%). High-throughput bacterial genome sequencing revealed that the isolate in this case shared 100% homology with Herbaspirillum huttiense subsp putei IAM 15032 in Genbank database, which confirmed that the isolate was Herbaspirillum huttiense. Conclusions:There are more and more environmental microorganisms evolved into human pathogenic bacteria. Herbaspirillum species are easy to be misidentified because its biochemical characteristics are similar to other strains.

Objective To investigate the feasibility and efifcacy of endoscopic submucosal dissection (ESD) for gastrointestinal neuroendocrine neoplasms (GI-NENs).Methods 52 patients with conifrmed histological diagnosis of GI-NENs performed ESD from January 2011 to December 2015 were included. The endoscopic morphology of tumor was summarized. Complete resection rate, complications, clinicopathological characteristics, and follow-up results were evaluated.Results There were 16 cases of stomach, 9 cases of colon and rectum 27 cases. Most of the lesions were submucosal uplift. A few of lesions looked like polyps. All the lesions were one-time whole diseased. 44 lesions were NET-G1, 8 lesions were NET-G2. Complete resection rate was 94.23%. 2 cases of rectal lesions infringemented intrinsic muscle layer, and got additional surgery. 1 case of rectal perforation, which was managed by endoscopic treatment and conservative treatment. All cases did not appear haemorrhage. During a mean follow-up period of 22.6 months, local recurrences occurred in 1 case of stomach, and treated with second line ESD. No cases lymph node and distant metastasis were found.Conclusion ESD appears to be a feasible, safe and effective treatment for GI-NENs with strict endoscopic treatment indications.

OBJECTIVETo investigate the effect of CCM3 gene defection on lead induced cell genotoxicity in mouse embryonic fibroblasts.

METHODSC57 female mice were mated with CCM3 gene heterozygous male mice. E13.5 embryos were taken to isolate primary mouse embryonic fibroblasts. After genotyping, wild type and heterozygous cells were treated with different doses of lead acetate. Cell viability, genotoxicity and protein expression were detected by MTS assay, CB micronucleus method and Western blot, respectively.

RESULTSMouse embryonic fibroblasts with lead acetate treatment for 24 h, wild-type cells 100.00 µmol/L lead acetate-treated group (69.16±1.36) and the control group (100.00±2.33) compared to cells decreased by 30%, CCM3 heterozygous type cell 100.00 µmol/L lead acetate-treated group (87.16±5.50) and the control group (100.00±2.06) compared to cells decreased by 13%, the difference was statistically significant (F values were 98.59, 82.63, P<0.001). Lead acetate treatment after 48 h, wild-type cells 100.00 µmol/L lead acetate-treated group (51.99±5.62) and the control group (100.00±3.11) compared to cells decreased by 50%, heterozygous type cells 100.00 µmol/L lead acetate treatment group (66.33±4.06) and the control group (100.00±5.72) compared to cells decreased by 35%, the differences were statistically significant (F values were 82.63, 36.86, P < 0.001). The results of CBMN test showed that with increased dose, micronucleus cell rate of two genotypes showed an increasing trend, in the wild-type cells, the micronucleus cell rate (/1 000) for the control group, 29.6±2.2, 6.25 µmol/L dose group 47.3±6.6, 25 µmol/L dose group 55.5±9.1, 100.00 µmol/L dose group 66.8±3.5; heterozygous cells micronucleus cell rate (/1 000) for the control group, 35.3±5.6, 6.25 µmol/L dose of 50.0±8.3, 25.00 µmol/L dose group 57.0±8.5, 100.00 µmol/L dose group 58.8±2.1. Micronucleus cell rates (/1 000) were significant differences, in 100.00 µmol/L dose groups of two genotypes. Western blot results showed that wild-type cells CCM3 expression 100.00 µmol/L lead acetate-treated group (0.70±0.03) was 1.32 times higher than the control group (0.53±0.07), heterozygous cells CCM3 expression 100.00 µmol/L lead acetate-treated group (0.48±0.02) was 1.77 times higher than control group that of 0.27±0.04, there was statistically significant difference (F values were 14.77, 25.74, P < 0.001); wild-type cells γ-H2AX expression 100.00 µmol/L lead acetate-treated group (0.69±0.03) was 1.06 times higher than the control group (0.65±0.07), heterozygous cells γ-H2AX expression 100.00 µmol/L lead acetate-treated group (0.99±0.04) was 1.55 times higher than the control group CCM3 expression levels (0.64±0.06), there was statistically significant difference (wild-type cells: F = 7.08, P = 0.012, heterozygous type cell: F = 13.49, P = 0.002).

CONCLUSIONCCM3 gene may play a role in lead-induced genetic toxicity of mouse embryonic fibroblasts, CCM3 gene-lead interactions effects on mouse embryonic fibroblasts cell toxicity.

Animals ; Apoptosis Regulatory Proteins ; DNA Damage ; Embryo, Mammalian ; Female ; Fibroblasts ; Genotype ; Male ; Membrane Proteins ; Mice ; Mice, Inbred Strains ; Micronuclei, Chromosome-Defective ; Organometallic Compounds ; Proto-Oncogene Proteins

Objective To investigate the different distribution of regulatory T cells (Tregs) and CD8+T cells in the local immune microenvironment of mucosal malignant melanoma and cutaneous malignant melanoma, and analyze the relationship between the two indicators and the prognosis of patients. Methods Immunohistochemistry staining was used to assess the ex?pression of Foxp3+Tregs and CD8+T cells in tumor microenvironment of 58 patients with malignant melanoma. The correlation between two factors, clinicopathological characteristics, and prognosis were analyzed. Results There is no correlation be?tween the expression of Foxp3 and CD8. The number of Foxp3+Tregs was significantly higher in mucosa malignant melanoma than that in cutaneous malignant melanoma (t=2.648, P=0.011). The proportion of Foxp3highTregs was significantly higher in pa?tients with tumor diameter≥3 cm, lymph node metastasis and distant metastasis than that in patients with tumor diameter<3 cm, no lymph node metastasis and no distant metastasis (P<0.05). In addition, in patients with ulceration that proportion was significantly higher in CD8high group than that in patients without ulceration (33.3%vs 5.9%, P<0.05). The median progres?sion-free surial (PFS) was 12 months in Foxp3high group, which was significantly longer than that of Foxp3low group (31 months, P<0.05). The median PFS was significantly higher in CD8high group (25 months) than that of CD8low group (12 months, P<0.05). Subgroup analysis showed that the median PFS was 7 months in Foxp3high CD8low group, which was significantly lower than that of Foxp3highCD8high group (25 months) and Foxp3lowCD8low group (18 months, P=0.003). Univariate analysis showed that median PFS was different in patients with different tumor location, different number of Foxp3+Treg, different number of CD8+T cells, and distant metastases. Conclusion The number of Tregs is closely associated with metastasis in patients with malig?nant melanoma. Compared with cutaneous malignant melanoma, our results indicate that the poor prognosis of mucosal malig?nant melanoma may be associated with the infiltration of more Tregs.

Objective Prostate cancer is one of the most common cancer in males .The article was to investigate the expres-sion and clinicopathological significance of VEGF-C mRNA in prostate cancer , finding the molecular marker for early diagnosis and prognosis assessment of prostate cancer . Methods TaqMan real-time polymerase chain reaction ( PCR) analysis was used to detect the expression of VEGF-C mRNA in 3 prostate cancer cell lines(PC-3, DU145 and LNCap), 32 cases of prostate cancer (Pca) sam-ples and 15 cases of benign prostatic hyperplasia (BPH).Analysis was also made on the correlations of VEGF-C mRNA expression, clinicopathological features and prognosis . Results High levels of VEGF-C mRNA were detected in PC-3 ( 153 .31 ±26 .24 ) and DU145(194.62 ±41.36)compared to LNcap(1.00 ±0.00).The expression of VEGF-C mRNA in prostate cancer tissues was 3.43 folds higher than that in the benign prostatic hyperplasia tissues ([13.67 ±1.95] vs [11.89 ±1.63], P=0.004).The high expres-sion of VEGF-C mRNA in prostate cancer was associated with high Gleason score ( P =0.004 ) and lymph node metastasis ( P =0.015).In patients with high expression and low expression of VEGF-C mRNA, the 3-year survival rate was 12.5%and 40.0%re-spectively(P=0.033). Conclusion The VEGF-C mRNA expression may be related to the biological behavior of prostate cancer .It is suggested that VEGF-C mRNA can be used as a prognostic marker for prostate cancer .

Objective To investigate the association between the functional polymorphisms of Foxp3 gene and unexplained recurrent spontaneous abortion (URSA).Methods PCR-restriction fragment length polymorphism (rs3761548,rs2294021 ) and PCR with sequence-specific primers (rs2232365,rs5902434) were used to detect four polymorphisms of Foxp3 in 146 URSA cases and 112 normal controls.Results ( 1 ) The frequencies of rs3761548A/C were 10.3％,22.3％ in genotype C/C,38.4％,40.2％ in genotype A/C and 51.4％,37.5％ in genotype A/A between URSA patients and normal controls; the frequencies of rs2232365A/G were 5.5％,15.2％ in genotype A/A,47.9％,50.0％ in genotype A/G,46.6％,34.8％ in genotype G/G between URSA patients and normal controls; they all reached statistical difference ( P＜0.05 ).The carriers of rs3761548A allele and rs2232365G allele increased the risk of URSA (OR=1.73,1.61 ; all P ＜ 0.05 ).(2) There was no difference in the genotypic distribution of rs5902434del/ArTTpolymorphism between cases and controls ( P =0.10),but the frequency of del allele in URSA was statistically increased than that of controls (71.2％,62.5％ ; OR =1.49,P =0.04 ).(3) There was no different distribution in 3 genotypes (C/C,T/C,T/T) and 2 alleles (T and C) of rs2294021T/C between URSA patients and normal controls (P =0.18 and 0.08 ).(4) Estimated haplotype frequency distribution of rs5902434del/ATT,rs3761548A/C and rs22323565A/G showed haplotype del-A-G conferring the susceptibility to URSA ( OR =2.51,P ＜ 0.01 ) but haplotype del-C-G and ATT-A-A could provide protection on URSA ( OR =0.18,0.22 ; all P ＜ 0.01 ).Conclusion Functional polymorphisms of Foxp3 gene could probably confer the susceptibility to URSA,by altering Foxp3 function and (or) its expression.

Objective: To investigate the expression of TSLP in human lung cancer tissue and the correla-tion between TSLP expression and number of regulatory T cells (Tregs). Methods: The expression of TSLP mRNA and protein was detected in different pathological lesions of the lung by Q-RT-PCR and immunohisto-chemistry. Immunohistochemistry was used to detect Foxp3+ Tregs. The correlation of TSLP with the number of Tregs was analyzed. Results: TSLP gene was expressed in tumor tissues (n=37), latero-tumor tissues (n=29) and non-tumor lung tissues (n=24), without statistical difference (P=0.148). TSLP protein was expressed in the cytoplasm and was observeed in 69.57% of tumor tissues, 13.33% of benign lesions and 30.00% of non-tumor lung tissues, with a significant difference (P<0.05). The expression of TSLP protein was correlated with tumor size (P=0.000) and lymph node metastasis (P=0.018). The number of Tregs in TSLP positive group was more than that in TSLP negative group (P<0.05). Conclusion: The expression of TSLP in lung tu-mor tissues is increased and is correlated with the number of Tregs, indicating that TSLP could induce Treg to play an important role in tumor immunotolerance.

Objective To investigate the effect of expectant management on the maternal and/or infant out-comes of early onset severe preeclamp sia in different gestafional weeks. Methods A retrospective study was carried out on 158 patients with early onset severe preeclampsia. They were divided into three groups according to their onset gestation ago:group A( <28weeks,n=28) ,group B(≥28、<32weeks,n=51) and group C(≥32、< 34weeks,n =79). Results The rates of complications declined along with the postponement of the onset gestation age, but there was no statistical significant difference among these three groups. The neonatal asphyxia rate and perinatal infant mor-tality of these three groups declined along with the postponement of gestation age, and there were statistical significant differences among these three groups ( P<0.05 ). Expectant treatment time of group B was significantly longer than that of the other two groups ( P<0.05 ), and cesarean section was a main method of pregnancy termination for the groups B and C. Conclusion The smaller the gestational ages in the early onset severe preeelampsia,the higher the maternal and/ or infant complication rates, neonatal morbidity and mortality.