Objective:To identify and characterize one Spiroplasma strain (designated as DGKH1) isolated from the blood of a patient with sepsis. Methods:The traditional bacterial culture, staining, morphological observation, physiological and biochemical identification, 16S rRNA gene sequencing, phylogenetic analysis, genome sequencing, and the genome-related index analysis were performed to accurately determine the taxonomic status of the strain DGKH1. Antibiotic susceptibility testing was performed using a specific kit for culturing and testing Ureaplasma urealyticum/ Metamycoplasma hominis. Results:The strain DGKH1 could weakly grow on Columbia blood agar, chocolate agar, and Haemophilus chocolate 2 agar. However, it did not grow in liquid culture medium containing tetracycline (4 μg/ml), doxycycline (1 μg/ml), minocycline (1 μg/ml), josamycin (2 μg/ml), roxithromycin (1 μg/ml), clarithromycin (1 μg/ml), or telithromycin (1 μg/ml). DGKH1 resembling Metamycoplasma hominis formed "fried egg-like colonies" on Mycoplasma solid culture medium. DGKH1 could not be stained by Gram staining. When observed under transmission electron microscopy (TEM) using phosphate buffer as the matrix, the bacteria were spiral-shaped. Results of 16S rRNA gene sequence alignment showed that DGKH1 was highly similar (99.85%) to Spiroplasma eriocheiris CCTCC M 207170 T. However, the urea decomposition test was positive, which was different from all of the known Spiroplasma species. The phylogenetic analysis based on whole genome showed that DGKH1 was clustered in a small branch along with Spiroplasma eriocheiris CCTCC M 207170 T. However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains were 94.14% and 56.00%, respectively, both below the threshold for prokaryotic species identification. Conclusions:DGKH1 represented a potential new species of genus Spiroplasma, closely related to Spiroplasma eriocheiris. Some microbiological characteristics of DGKH1 were similar to Mycoplasmas. However, the natural host and epidemiological data of DGKH1 need to be further studied.

Objective:To analyze the biological characteristics, phylogenic features and clinical significance of SQ219 and SQ220 isolated from clinical sputum and midstream urine specimens.Methods:The culture and biochemical characteristics of the two strains were observed. VITEK2 System, drug sensitivity testing and MALDI-TOF mass spectrometry were used for bacterial identification. Phylogenetic analysis based on 16S rRNA and core genome was performed. The average nucleotide identity (ANI) based on whole genome sequences was calculated.Results:SQ219 and SQ220 were Gram-stain-negative, aerobic, catalase- and oxidase-positive, and non-motile bacteria. Their optimum growth was observed in NaCl-free medium at 30℃ and pH7. Flexirubin-type pigments were produced by SQ220 on Colombia blood agar, but not by SQ219. Both SQ219 and SQ220 were resistant to aztreonam, amikacin, tobramycin and colistin, which was consistent with the drug resistance phenotype of genus Chryseobacterium. The genome sequences of SQ219 and SQ220 were 5.08 Mb and 4.80 Mb in length, and the G+ C contents were 36.72% and 36.36%, respectively. Both strains carried β-lactam resistance gene ( blaCGA). 16S rRNA phylogenetic analysis showed that SQ219 and SQ220 were closely related to Chryseobacterium gambrini DSM18014 T with the similarities of 98.93% and 98.36%, respectively. Core genome phylogenetic analysis revealed that SQ219 and SQ220 were highly homologous to Chryseobacterium gambrini DSM18014 T. However, the ANI values between the two strains and Chryseobacterium gambrini DSM18014 T were 92.49% and 93.27%, respectively, below the threshold for prokaryotic species identification. Conclusions:Based on the phenotypic and phylogenetic data, SQ219 and SQ220 represent a novel species of the genus Chryseobacterium. This study would help promote the understanding of the evolution of Chrysobacterium and provide reference for the identification of new species of Chrysobacterium.

Objective : To investigate the effects of melatonin ( MEL) on the proliferation of hepatic stellate cells (HSCs) induced by platelet - derived growth factor (PDGF⁃BB) and explore its correlation with the regulation of autophagy levels . Methods : The HSC⁃T6 cells were divided into the following groups : control group , model group and MEL (low , medium and high) treatment groups . After 24 hours culture , the cells adhered to the wall and were changed into serum⁃free DMEM medium to synchronize the cells to the G0 stage . After 24 hours culture , all groups were given with PDGF ⁃ BB ( 10 ng/ml) excepted the control group . Besides , melatonin of different concentrations ( 1 nmol/L , 1 μmol/L and 0. 1 mmol/L) were added immediately in three treated groups . After incubated for 48 hours , the effect of MEL on the proliferation of hepatic stellate cells activated by PDGF⁃BB was detected by CCK8 method . The protein expression levels of LC3b and α ⁃SMA in hepatic stellate cells were determined by Western blot . The expression levels of LC3b mRNA and α ⁃SMA mRNA in hepatic stellate cells were determined by qRT⁃PCR . The ultrastructure of HSCs was observed by transmission electron microscopy to understand the autophagy level. Results : Compared with control group , PDGF⁃BB could induce the proliferation of HSCs ( P < 0. 01) . Compared with model group , MEL inhibited the proliferation of HSCs activated by PDGF⁃BB ( P < 0. 01) . Compared with the control group , LC3b and α ⁃SMA protein expressions significantly increased in the model group ( all P < 0. 05) , and LC3b mRNA and α ⁃SMA mRNA expressions significantly increased in the model group (P < 0. 05 , P < 0. 01) . Compared with the model group , MEL could inhibit such effects (LC3b : P < 0. 05 , P < 0. 01 ; α ⁃SMA : P < 0. 01) . Transmission electron microscopy ( TEM) showed that compared with the control group , autopolysosome significantly increased in the model group (P < 0. 05) . Compared with model group , autopolysosome significantly decreased in MEL treatment group (P < 0. 01) . Conclusion The up⁃regulation of autophagy level can promote the proliferation of hepatic stellate cells and the inhibition of hepatic stellate cell proliferation by MEL may be related to the down⁃regulation of autophagy level .

Objective: To explore the mechanism of maternal vitamin D deficiency(VDD)-induced fetal intrauterine growth restriction( IUGR) during pregnancy. Methods: Four weeks female CD-1 mice were divided into two groups: control mice( CTRL) and vitamin D deficiency mice( VDD). VDD mice were fed with diet with low concentration of vitamin D and mated with normal male mice. Several pregnant mice were killed on gestational sixteenth day,and the placentas,serum of maternal and fetal mice were collected. Proteins of placental key enzymes of steroid hormone synthesis and signaling pathways of oxidative stress were measured using Western blot. Serum estrogen,progesterone and 25-( OH)-D were measured via radioimmunoassay. The remaining pregnant mice were killed. Placentas and fetus were weighted,fetus height and placental diameter were calculated. The pregnancy outcomes were observed. Results: Diet with low concentration of vitamin D were used and VDD mice model were established after five weeks. Maternal VDD during pregnancy decreased the weight of placenta and fetus,reduced the height of fetus and the diameter of placentas. The expression of placental aromatase cytochrome p450( CYP19) and the level of estrogen were increased in VDD pregnant mice. Besides,maternal VDD during pregnancy increased the expressions of placental heme oxygenase 1( HO-1),NADPH oxidase 4( NOX-4) and 3-nitrotyrosine( 3-NT).VDD promoted placental nuclear factor E2-related factor 2( Nrf-2) nuclear translocation. Conclusion Maternal vitamin D deficiency during pregnancy causes IUGR partially through evoking placental oxidative stress and promoting estrogen synthesis.

Objective:To investigate the values of serum neuron specific enolase (NSE), circulating tumor cells (CTC) and lactate dehydrogenase (LDH) levels in the diagnosis and treatment of small cell lung cancer (SCLC).Methods:Ninety patients with SCLC who received treatment in the Second Affiliated Hospital of Zhejiang Chinese Medical University, China between December 2017 and December 2019 were retrospectively included in the observation group. Ninety healthy subjects who concurrently received lung examination in the same hospital were included in the healthy control group. An additional 90 patients with benign lung disease were included in the benign lung disease group. Serum NSE, CTC and LDH levels were determined in each group. The values of serum NSE, CTC and LDH levels in the diagnosis of SCLC were analyzed. Serum NSE, CTC and LDH levels were compared between before and after chemotherapy and their values in the treatment of SCLC were analyzed.Results:There were significant differences in serum NSE, CTC and LDH levels between three groups ( F = 359.789, 188.873 and 768.704, all P < 0.001). Serum NSE, CTC and LDH levels in the benign lung disease group were significantly greater than those in the healthy control group and significantly lower than those in the observation group. The receiver operating characteristic curve (ROC curve) analysis showed that the AUC values of serum NSE, CTC and LDH levels in the diagnosis of SCLC were 0.995, 0.953 and 0.987, respectively. The diagnostic accuracy was very high. The value at the maximum tangent point of Youden's index of serum NSE, CTC and LDH levels at the left-upper corner of the ROC curve was taken as the most appropriate cut-off value. The sensitivity and specificity of the most appropriate cut-off value of serum NSE, CTC and LDH levels in the prediction of SCLC were 100.0%/94.4%/91.1% and 94.4%/88.3%/100.0%, respectively. Therefore, serum NSE, CTC and LDH levels were of high values in the predication of SCLC. After chemotherapy, serum NSE, CTC and LDH levels in patients with SCLC were significantly lower than those before chemotherapy [NSE: (12.26 ± 3.26) μg/L vs. (18.36 ± 4.64) μg/L; CTC: (3.54 ± 1.08) counts/5 mL vs. (7.34 ± 1.30) counts/5 mL; LDH: (24.61 ± 9.66) U/L vs. (50.29 ± 16.29) U/L, t = 10.205, 12.864, 21.330, all P < 0.001). Serum NSE, CTC and LDH levels in SCLC patients in whom treatment was effective were significantly lower than those in SCLC patients in which treatment was not effective ( t = 8.111, 7.347, 10.731, all P < 0.001). Spearman correlation results showed that serum NSE, CTC and LDH levels were significantly negatively correlated with curative effects ( r = -0.562, -0.562, -0.758, all P < 0.05). Conclusion:Serum NSE, CTC and LDH levels are highly expressed in SCLC patients, which can be used as markers for early clinical diagnosis and treatment of SCLC.

Objective:To investigate the risk factors associated with anastomotic leakage after robotic surgery in mid-low rectal cancer.Methods:A retrospective case-control study method was conducted. Inclusion criteria: (1) 18 to 80 years old; (2) pathologically confirmed rectal cancer; (3) distance <10 cm from tumor to anal margin; (4) robotic anterior rectal resection. Patients with previous history of colorectal cancer surgery, distant metastases or other malignant tumors, undergoing emergency surgery, with severe abdominal adhesions or those receiving combined organ resection were excluded. Based on the above criteria, 636 patients undergoing robotic radical sphincter-preserving surgery for mid-low rectal cancer in Zhongshan Hospital from January 2015 to December 2018 were included in this study, including 398 males (62.6%) and 238 females (37.4%) with a mean age of (61.9±11.3) years. Sixty-eight cases (10.7%) received neoadjuvant chemoradiotherapy. Amony the 636 included patients, 123(19.3%) underwent natural orifice specimen extraction surgery (NOSES) and 15 (2.3%) underwent preventive stoma. According to the cirteria developed by the International Rectal Cancer Research Group in 2010, the anastomotic leakage was classified as grade A (no requirement of intervention), B (requirement of intervention), and C (requirement of operation). Logistic regression was used to analyze the relationship between anastomotic leakage and clinicopathological factors. Factors in univariate analysis with P<0.05 were included in the multivariate analysis. Results:Anastomotic leakage occurred in 38 cases (6.0%). The grading of anastomotic leakage was grade A in 13 cases (2.0%), grade B in 19 cases (3.0%), and grade C in 6 cases (0.9%). The 3-year disease-free survival rate of patients with anastomotic leakage and without anastomotic leakage was 83.5% and 83.6% respectively ( P=0.862); the 3-year overall survival rate of the two group was 85.1% and 87.5% respectively ( P=0.296). The results of univariate logistic regression analysis showed that male ( P=0.011), longer operation time ( P=0.042), distance ≤5 cm from tumor to anal margin ( P=0.012), more intraoperative blood loss ( P=0.048) were associated with anastomotic leakage (all P<0.05). NOSES was not associated with anastomotic leakage ( P=0.704). Multivariate analysis confirmed that male (OR=3.03, 95%CI: 1.37 to 7.14, P=0.010), operation time ≥180 minutes (OR=2.04, 95%CI: 1.03 to 3.99, P=0.040), distance ≤5 cm from tumor to anal margin (OR=2.56, 95%CI:1.28 to 5.26, P=0.008) were independent risk factors for anastomotic leakage. Conclusion:Male, short distance from tumor to anal margin, and long operation time are independent risk factors for anastomotic leakage in patients undergoing robotic mid-low rectal cancer radical surgeries. These patients need to be cautiously treated during surgery.

Objective:To investigate the risk factors associated with anastomotic leakage after robotic surgery in mid-low rectal cancer.Methods:A retrospective case-control study method was conducted. Inclusion criteria: (1) 18 to 80 years old; (2) pathologically confirmed rectal cancer; (3) distance <10 cm from tumor to anal margin; (4) robotic anterior rectal resection. Patients with previous history of colorectal cancer surgery, distant metastases or other malignant tumors, undergoing emergency surgery, with severe abdominal adhesions or those receiving combined organ resection were excluded. Based on the above criteria, 636 patients undergoing robotic radical sphincter-preserving surgery for mid-low rectal cancer in Zhongshan Hospital from January 2015 to December 2018 were included in this study, including 398 males (62.6%) and 238 females (37.4%) with a mean age of (61.9±11.3) years. Sixty-eight cases (10.7%) received neoadjuvant chemoradiotherapy. Amony the 636 included patients, 123(19.3%) underwent natural orifice specimen extraction surgery (NOSES) and 15 (2.3%) underwent preventive stoma. According to the cirteria developed by the International Rectal Cancer Research Group in 2010, the anastomotic leakage was classified as grade A (no requirement of intervention), B (requirement of intervention), and C (requirement of operation). Logistic regression was used to analyze the relationship between anastomotic leakage and clinicopathological factors. Factors in univariate analysis with P<0.05 were included in the multivariate analysis. Results:Anastomotic leakage occurred in 38 cases (6.0%). The grading of anastomotic leakage was grade A in 13 cases (2.0%), grade B in 19 cases (3.0%), and grade C in 6 cases (0.9%). The 3-year disease-free survival rate of patients with anastomotic leakage and without anastomotic leakage was 83.5% and 83.6% respectively ( P=0.862); the 3-year overall survival rate of the two group was 85.1% and 87.5% respectively ( P=0.296). The results of univariate logistic regression analysis showed that male ( P=0.011), longer operation time ( P=0.042), distance ≤5 cm from tumor to anal margin ( P=0.012), more intraoperative blood loss ( P=0.048) were associated with anastomotic leakage (all P<0.05). NOSES was not associated with anastomotic leakage ( P=0.704). Multivariate analysis confirmed that male (OR=3.03, 95%CI: 1.37 to 7.14, P=0.010), operation time ≥180 minutes (OR=2.04, 95%CI: 1.03 to 3.99, P=0.040), distance ≤5 cm from tumor to anal margin (OR=2.56, 95%CI:1.28 to 5.26, P=0.008) were independent risk factors for anastomotic leakage. Conclusion:Male, short distance from tumor to anal margin, and long operation time are independent risk factors for anastomotic leakage in patients undergoing robotic mid-low rectal cancer radical surgeries. These patients need to be cautiously treated during surgery.

Objective: To evaluate the immunogenicity of split influenza H1N1 vaccine formulated with an oil-in-water nano-emulsion adjuvant in aged mice and young mice. Methods: A nano-emulsion adjuvant formulated split influenza H1N1 vaccine was used to immunize aged and young mice through intramuscular injection. Each mouse was immunized with 0.012 μg of hemagglutinin (HA) twice with an interval of 28 d. Hemagglutination inhibition (HI) titers in serum were measured 27 d after first immunization. Serum HI, IgG1 and IgG2a titers were detected 14 d after the last immunization. No adjuvant-formulated vaccine and normal saline (NS) were used to set up control groups. Virus challenge test was carried out using 10 times the median lethal dose (LD₅₀) of A/Puerto Rico/8/34 (H1N1) strain two weeks after the last immunization and the protective effects were assessed through measuring the dynamic changes in body weight and survival rate. Results: Higher levels of serum HI, IgG1 and IgG2a antibodies and higher HI antibody conversion rates were induced in the adjuvant groups, especially in the aged mice group, than in the control groups. Nano-emulsion adjuvant improved the immunogenicity of HA and mouse immunity to A/Puerto Rico/8/34 (H1N1). Conclusions Nano-emulsion adjuvant could enhance the immunogenicity of influenza antigens, especially in aged mice.

Objective To assess the diet quality of pregnant women during the second trimester using the dietary inflammatory index ( DII) and to explore the correlation between the DII in second trimester of preg-nancy and preterm delivery.Methods A total of 253 women with singleton pregnancy in 16-20 gestational weeks who had received routine prenatal care between August 2014 and April 2015 at the First Affiliated Hospi-tal of Anhui Medical University were enrolled with cluster random sampling.The included women were asked to recall daily dietary intake in the 3 days prior to the survey.All dietary data were analyzed for energy and nutri-ents intake with a nutritional analysis software, followed by calculation of DII according to previous reports. Based on quartiles of the DII scores, the participants were divided into three groups, namely the anti-inflam-matory group (DII<-5.10), intermediate group (DII -5.10--2.55), and the pro-inflammatory group (DII>-2.55).The participants were followed up until delivery and the pregnancy outcomes were recorded. The relationship between the DII in second trimester of pregnancy and preterm delivery were analyzed. Results DII scores of the 253 pregnant women during the second trimester of pregnancy ranged from -7.913 to 3.872.The risks of preterm birth in the anti-inflammatory diet group, the intermediate group, and the pro-inflammatory diet group were 0, 1.6%, and 6.3%, respectively, with statistically significant differences among the groups (P=0.034).The higher DII scores (pro-inflammatory) were associated with higher inci-dence of preterm birth ( P<0.05 ) .Conclusion DII may be used to assess diet quality of pregnant women during the second trimester and to predict the risk of preterm birth.

Objective To compare the effect of Solitaire AB and Enterprise stent-assisted endovascular embolization of intracranial wide-necked aneurysms. Methods The clinical data 218 patients with intracranial aneurysm treated with Solitaire AB stent or Enterprise stent in Guangdong General Hospital from January 2012 to December 2014 were analyzed retrospectively. Seventy patients were treated with Solitaire AB stents and 148 were treated with Enterprise stents. They were followed up after procedure (2 months to 2 years). The Raymond grade and the modified Rankin scale (mRS)scores were used to compare the efficacy of the 2 kinds of stents. Results There were no significant differences in patients before procedure in the Hunt-Hess grade (Z = 1. 550,P = 0. 121),Fisher grade (Z =1. 537,P =0. 124),aneurysm size (Z =0. 192,P =0. 848),and position (χ2 = 0. 337,P = 0. 736)between the 2 stent groups,while there were no significant differences in patients after procedure in the instant Raymond grade (Z = 0. 682,P = 0. 495), follow-up Raymond grade (Z = 0. 206,P =0. 837),and mRS score (P =0. 662)between the 2 stent groups. Further multivariate Logistic regression analysis showed that the stent type (Solitaire AB stent or Enterprise stent),Raymond grade follow-up grade (OR,0. 029 ,95% CI 0. 126 - 0. 199,P = 0. 661 ),and postoperative mRS score (OR,2. 000,95% CI 0. 204 - 19. 603,P = 0. 552)had no influence on the
prognosis. Conclusions In the assisted embolization of intracranial wide-necked aneurysms using the Solitaire AB and Enterprise stents,the effects of both stents are good in the assisted embolization of intracranial wide-necked aneurysms.