[Effects of canagliflozin on amino acid metabolism in atherosclerotic mice].

Objective: To explore the possible anti-atherosclerotic mechanisms of glucose co-transporter-2 inhibitor canagliflozin. Methods: ApoE-/-mice fed on Western diet were randomly assigned into the model group (n=10) and the canagliflozin group (n=10). C57BL/6J mice fed on normal diet were chosen as the control group (n=10). Mice in the canagliflozin group were gavaged with canagliflozin for 14 weeks. The presence and severity of atherosclerosis were evaluated with HE and oil red O stainings in aortic root section slices. PCR assay was performed to determine the mRNA expression levels of nitric oxide synthase. Hepatic transcriptome analysis and hepatic amino acid detection were conducted using RNA-seq and targeted LC-MS, respectively. Results: HE staining and oil red O staining of the aortic root showed that AS models were successfully established in ApoE-/-mice fed on Western diet for 14 weeks. Canagliflozin alleviated the severity of atherosclerosis in pathology. Hepatic transcriptome analysis indicated that canagliflozin impacted on amino acid metabolism, especially arginine synthesis in ApoE-/-mice. Targeted metabolomics analysis of amino acids showed that canagliflozin reduced hepatic levels of L-serine, L-aspartic acid, tyrosine, L-hydroxyproline, and L-citrulline, but raised the hepatic level of L-arginine. Compared to the model group, the canagliflozin group exhibited higher serum arginine and nitric oxide levels as well as elevated nitric oxide mRNA expression in aortic tissues (P<0.05). Conclusion: Canagliflozin regulated the amino acid metabolism, reduced the levels of glucogenic amino acids,and promoted the synthesis of arginine in atherosclerotic mice.

[Report content and prenatal diagnosis of non-invasive prenatal testing for sex chromosome aneuploidy].

Objective: To analyze the report content, the methods and results of prenatal diagnosis of high risk of sex chromosome aneuploidy (SCA) in non-invasive prenatal testing (NIPT). Methods: A total of 227 single pregnancy pregnant women who received genetic counseling and invasive prenatal diagnosis at Drum Tower Hospital Affiliated to the Medical School of Nanjing University from January 2015 to April 2022 due to the high risk of SCA suggested by NIPT were collected. The methods and results of prenatal diagnosis were retrospectively analyzed, and the results of chromosome karyotype analysis and chromosome microarray analysis (CMA) were compared. The relationship between NIPT screening and invasive prenatal diagnosis was analyzed. Results: (1) Prenatal diagnosis methods for 277 SCA high risk pregnant women included 73 cases of karyotyping, 41 cases of CMA and 163 cases of karyotyping combined with CMA, of which one case conducted amniocentesis secondly for further fluorescence in situ hybridization (FISH) testing. Results of invasive prenatal diagnosis were normal in 166 cases (59.9%, 166/277), and the abnormal results including one case of 45,X (0.4%, 1/277), 18 cases of 47,XXX (6.5%, 18/277), 36 cases of 47,XXY (13.0%, 36/277), 20 cases of 47,XYY (7.2%, 20/277), 1 case of 48,XXXX (0.4%, 1/277), 20 cases of mosaic SCA (7.2%, 20/277), 5 cases of sex chromosome structural abnormality or large segment abnormality (1.8%, 5/277), and 10 cases of other abnormalities [3.6%, 10/277; including 9 cases of copy number variation (CNV) and 1 case of balanced translocation]. Positive predictive value (PPV) for SCA screening by NIPT was 34.7% (96/277). (2) Among the 163 cases tested by karyotyping combined with CMA, 11 cases (6.7%, 11/163) showed inconsistent results by both methods, including 5 cases of mosaic SCA, 1 case of additional balanced translocation detected by karyotyping and 5 cases of additional CNV detected by CMA. (3) NIPT screening reports included 149 cases of "sex chromosome aneuploidy"(53.8%, 149/277), 54 cases of "number of sex chromosome increased" (19.5%, 54/277), and 74 cases of "number of sex chromosome or X chromosome decreased" (26.7%, 74/277). The PPV of "number of sex chromosome increased" and "number of sex chromosome or X chromosome decreased" were 72.2% (39/54) and 18.9% (14/74), respectively, and the difference was statistically significant (χ2=34.56, P<0.01). Conclusions: NIPT could be served as an important prenatal screening technique of SCA, especially for trisomy and mosaicism, but the PPV is comparatively low. More information of NIPT such as the specific SCA or maternal SCA might help improving the confidence of genetic counseling and thus guide clinic management. Multi technology platforms including karyotyping, CMA and FISH could be considered in the diagnosis of high risk of SCA by NIPT.

Impaired function of skeletal stem cells derived from growth plates in ovariectomized mice.

INTRODUCTION: Mouse skeletal stem cells (mSSCs, CD45-Ter119-Tie2-CD51+Thy-6C3-CD105-CD200+population) are identified in growth plates (GP) and play important roles in bone regeneration. However, the role of mSSCs in osteoporosis remains unclear. MATERIALS AND METHODS: The GP were stained by HE staining, and the mSSC lineage was analyzed by flow cytometry at postnatal of 14 days and 30 days in wild-type mice. The mice (8 weeks) were either sham operated or ovariectomy (OVX) and then sacrificed at 2, 4 and 8 w. The GP were stained by Movat staining, and mSSC lineage was analyzed. Then, mSSCs were sorted by fluorescence-activated cell sorting (FACS); the clonal ability, chondrogenic differentiation and osteogenic differentiation were evaluated, and the changed genes were analyzed by RNA-seq. RESULTS: The percentage of mSSCs were decreased with the narrow GP. Heights of GP were decreased significantly in 8w-ovx mice compared with 8w-sham mice. We found the percentage of mSSCs were decreased in mice at 2w after ovx, but the cell numbers were not changed. Further, the percentage and cell numbers of mSSCs were not changed at 4w and 8w after ovx. Importantly, the clonal ability, chondrogenic differentiation and osteogenic differentiation of mSSCs were impaired at 8w after ovx. We found 114 genes were down-regulated in mSSCs, including skeletal developmental genes such as Col10a1, Col2a1, Mef2c, Sparc, Matn1, Scube2 and Dlx5. On the contrary, 526 genes were up-regulated, including pro-inflammatory genes such as Csf1, Nfkbla, Nfatc2, Nfkb1 and Nfkb2. CONCLUSION: Function of mSSCs was impaired by up-regulating pro-inflammatory genes in ovx-induced osteoporosis.

Exploring the nursing effect of application Albizia bark on autism in children based on network pharmacology and molecular docking.

OBJECTIVE: Autism is a disorder that manifests itself in early childhood. Early diagnosis of autism may not only help the affected children themselves, but also affect family well-being and social stability. The natural drug Albizia bark has been reported to have some effect in the prevention and treatment of autism in children. Therefore, we used network pharmacology and molecular docking to explore the possible mechanism. MATERIALS AND METHODS: TCMID and BATMAN-TCM was used to retrieve the chemical constituents of Albizia bark, and then obtained the relevant targets about autism by TTD, Gene Cards and OMIM. The resulting ingredients and targets were predicted, then a protein interaction network was constructed, and finally bioinformatics analysis was performed. Finally, molecular docking was used to verify the effective ingredients and targets obtained from the screening. RESULTS: Leucaena saponin B, luteolin, 3', 4', 7-trihydroxyflavone, which may be the key compounds for the treatment of autism. BP mainly involving signal transduction, G protein coupled receptor signal pathway, protein phosphorylation. CC, mainly involving plasma membrane, integral component of plasma membrane, MF, including protein binding, adenosine triphosphate binding, protein kinase activity. Molecular docking showed that AKT1, HRAS, PIK3CA, PIK3R1 and SRC, five potential targets, had good binding ability to Leucaena saponin B. CONCLUSIONS: The natural drug Albizia bark exerts pharmacological effects in a multi-component, multi-target and multi-channel manner, including neural regulation, inflammatory response and immune regulation.

Attenuation of a virulent swine acute diarrhea syndrome coronavirus strain via cell culture passage.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly identified enteric alphacoronavirus that causes fatal diarrhea in newborn piglets in China. Here, we propagated a virulent strain SADS-CoV/CN/GDWT/2017 in Vero cells for up to 83 passages. Four strains of SADS-CoV/GDWT-P7, -P18, -P48 and -P83 were isolated and characterized. Sequence alignments showed that these four novel strains exhibited 16 nucleotide mutations and resultant 10 amino acid substitutions in open reading frame 1a/1b, spike, NS3a, envelope, membrane and nucleocapsid proteins. Furthermore, a 58-bp deletion in NS7a/7b was found in P48 and P83 strains, which led to the loss of NS7b and 38 amino acid changes of NS7a. Pig infection studies showed that the P7 strain caused typical watery diarrhea, while the P83 strain induced none-to-mild, delayed and transient diarrhea. This is the first report on cell adaption of a virulent SADS-CoV strain, which gives information on the potential virulence determinants of SADS-CoV.

12 novel atypical porcine pestivirus genomes from neonatal piglets with congenital tremors: A newly emerging branch and high prevalence in China.

Atypical porcine pestivirus (APPV), a newly discovered member of the genus Pestivirus, is considered to be associated with congenital tremors (CT) in piglets. From June 2016 to January 2018, 440 serum and tissue samples from CT-affected piglets in Anhui, Guangdong and Guangxi provinces were collected to detect APPV. The results showed a high level of 63.4% preference for APPV in 27 swine farms and complicated co-infection cases between APPV and other 12 swine viruses. Meanwhile, 12 novel APPV genomes were screened and identified. Results showed that complete genomes, Npro and NS5A genes of these novel 12 APPV sequences revealed 80.5%-99.8%, 78%-100% and 76.9%-99.8% nucleotide identities, respectively. Phylogenetic analyses based on sequences of full-length genomes, Npro and NS5A genes of APPV indicated three well-defined clades including a newly emerging branch in China. This study provides novel epidemiological information of APPV in China.

[Clinical trial of simplified drilling method for dental implant site preparation].

Objective: To investigate the clinical effect of simplified drilling method and conventional drilling method in implants. Methods: A total of 46 patients (62 implants) were enrolled in this study that with dentition defect from May 2015 to May 2016 in the Implant department of Xi'an Jiao Tong University. The experimental group and the control group were randomly assigned according to the random number method, 23 cases in each group. The experimental group used the simplified drilling method (guided drill+ final drill), the control group using the conventional drilling method (step by step drill). The operation time, implant stability, marginal bone resorption rate and implant retention rate were compared between the two drilling methods. Results: The retention of the experiment group was 97% (31/32), the the control group was 100% (30/30). The operative time in the experiment group [(4.9±0.5) min] was significantly lower from the control group [(8.9±2.0) min] (P=0.000). There was no significant difference between the two methods in bone resorption (P=0.197), implant stability (P>0.05) and implant survival rate (P=0.492). Conclusions: The simplified drilling method can significantly reduce the operation time without compromising the clinical outcomes, and the osseointegration is well. The simplified drilling method should be used when sufficient bone mass, careful use in class II bone, forbidden in class I bone.

Detection and genome sequencing of porcine circovirus 3 in neonatal pigs with congenital tremors in South China.

Porcine circovirus 3 (PCV3) is a novel circovirus first discovered in the United States in piglets and sows with porcine dermatitis and nephropathy syndrome, reproductive failure, cardiac and multisystemic inflammation. Here, seven PCV3 strains were identified for the first time from neonatal pigs with clinical signs of congenital tremors (CT) in South China. The tissue tropism of PCV3 in CT-affected piglets was analysed by the real-time quantitative PCR, and the result showed that high loads of viral genomes were detected in the brains and hearts. The complete genomes of seven new PCV3 revealed 96.8%-99.6% nucleotide identities with eleven other PCV3 strains previously reported from the United States and China. Phylogenetic analysis based on the complete genome sequences showed that all PCV3 strains clustered together and were clearly separated from other circovirus species. This study reports on the first identification of PCV3 in CT-affected newborn piglets and provides the epidemiological information of neonatal piglets with CT in Guangdong and Guangxi Provinces of China.

[An analysis of influencing factors for brucellosis in major occupational groups in Bayannur, China].

Objective: To investigate the current status of brucella infection in major occupational groups in Bayannur, China, and to analyze the risk factors for brucellosis. Methods: From January to March, 2015, a questionnaire survey and the serological testing were performed to investigate the current status of brucella infection in 649 workers in 13 slaughter and breeding plants in 7 counties in Bayannur, China. The items in the questionnaire survey included general information, related occupational information, living habits, medical history, and awareness of related knowledge. Results: A total of 112 workers (17.26%) had brucella infection. Brucella infection was associated with sex, age, years of exposure to hazardous substances, job, educational background, usage of protective equipment, the presence or absence of a wound at the exposure site, and whether to smoke during the work clearance (P<0.05) . The multivariate analysis showed that the risk factors for brucellosis were meat processing work (OR=1.812) , >5 years of exposure to hazardous substances (OR=1.363) , improper use or nonuse of protective equipment (OR=1.957) , smoke during work (OR=2.027) , and the presence of a woundat the exposure site (OR= 1.231) ; the protective factors for brucellosis were high school and college education and above (OR=0.521) and high rate of awareness of brucella-related knowledge (OR=0.648) . Conclusion: The brucella infection rate is high in the major occupational groups in Bayannur, China. The main influencing factors for brucellosis are job, years of exposure to hazardous substances, usage of protective equipment, whether to smoke during work, and the presence or absence of a wound at the exposure site.

[Comparison and analysis of minimal residual disease in multiple myeloma patients detected by 4-color and 8-color fluorescence antibody panels].

Objective: To explore and compare 4-color and 8-color fluorescence antibody panels for detecting minimal residual disease of multiple myeloma patients after therapy. Methods: One 8-color antibody panel was built including CD38 and CD138 for the identification of plasma cells (PCs) , membrane antigen CD45, CD19, CD56 and CD117, cytoplasmic Kappa (cκ) and Lambda (cλ) light chain antigen. Six tubes of 4-color panels were built, among them, membrane antigen CD45/CD19, CD56/CD117, CD19/CD56 and light chains were analyzed combined by CD38/CD138 for PCs gate in the tubes M1-3 and tube C-(κ/λ), respectively; CD19 or CD45 and cκ/cλ light chains were detected in the tube MC1-(C)D38 for CD38/SSC identified PCs gate and tube MC2-(CD138) for CD138/SSC identified PCs gate separately. Twenty normal volunteer bone marrows and seventy-three specimens from multiple myeloma patients after complete remission were measured and analyzed. Results: MRD positive samples were discriminated in 82.19% of the specimen evaluated through either abnormal plasma cells (aPCs) or clonal plasma cells (cPCs) by 8-color antibody panel. Among of them, consistency was 89.04%. The median percentage of cPCs was 0.105%. The lowest sensitivity of experiment was 0.004%. Percentage of PCs identified by CD38/ SSC gate was higher than that by CD38/CD138 (P<0.001) and CD138/SSC gate (P=0.001) apparently. The lowest MRD positive rate was found in tube C (65.75%) , which lower than 8-color panel obviously (P=0.024) . The percentages of aPCs measured by tube M2-(56/117) were significantly lower than other tubes (P=0.014) . MRD positive rate determined by cPCs was higher than that by aPCs both in the tube MC1-(C)D38 and tube MC2-(CD138), whose concordance rate were 68.49% and 79.45%, respectively. Compared with 8-color panel, tube MC2-(CD138) the best choice among six tubes of 4-color panels, which has the best sensitivity, accuracy and negative predicted value, higher positive predicted value and specificity. Tube M1-(45/19) was the second choice. Conclusions: CD38/CD138 are the best markers for gating PCs. Membrane antigen and cκ/cλ detected simultaneously is a better method for MM MRD detection and 8-color antibody panel is an ideal approach. Two tubes of 4-color antibody combination, M1-(45/19) and MC2-(CD138) are recommended in the 4-color panels.

BLCA-4 and UBC combined detection for early diagnosis of bladder cancer.

The objective of the present study was to report the clinical significance of bladder cancer specific nuclear matrix protein 4 (BLCA-4) and urinary bladder cancer (UBC) on early diagnosis of bladder cancers. Enzyme-linked immunosorbent assay (ELISA) was used to detect BLCA-4 and UBC of 56 bladder cancer patients and 26 patients with urinary tract benign diseases, serving as controls. Urine exfoliated cell test was performed, and then the significance of BLCA-4 and UBC on the diagnosis of bladder cancers was analyzed. The sensitivity of BLCA-4 and UBC of the bladder cancer patients was significantly higher than that of the urine exfoliated cell test (P less than 0.05). The difference of BLCA-4 and UBC was not significant (P >0.05). The difference of BLCA-4 and UBC in the tumors with different gradings and stagings was not significant (P >0.05). Combined detection of BLCA-4 and UBC could improve the diagnosis sensitivity and specificity of bladder cancers with the advantages of high maneuverability, repeatability and objective results.

Feasibility of comfortable and secure intubation achieved with the Disposcope endoscope or Macintosh laryngoscope for patients in whom glottis viewing is difficult.

We aimed to study the feasibility of a comfortable and secure intubation achieved with the Disposcope endoscope or Macintosh laryngoscope when glottis viewing is difficult. Forty adults scheduled for elective surgery under general anesthesia, in whom glottis viewing was difficult with the Macintosh laryngoscope (classified as Cormack-Lehane Grade III or IV), were randomized into 2 groups (N = 20 each): Disposcope endoscope (Group D) and Macintosh laryngoscope (Group M). We recorded the successful glottis viewing rate; intubation time; successful intubation rate; incidence of systolic blood pressure (SBP) ≥140 mmHg and heart rate (HR) ≥90 bpm from the beginning of intubation to 5 min after intubation; and postoperative sore throat and hoarseness. Successful glottis viewing rate and successful first intubation rate were higher in Group D than in Group M (P < 0.05); the number of intubations taking >3 min and with SBP ≥140 mmHg and HR ≥90 bpm were less in Group D (P < 0.05). The visual analogue scale of postoperative sore throat 1 day after extubation was higher in Group M than in Group D (P < 0.05). No significant differences were found in other indices (P > 0.05). Better stability of hemodynamics, less intubation time, higher successful first intubation rate, and fewer incidences of postoperative sore throat were achieved in Group D than in Group M. Thus, comfortable and secure intubation can be achieved using the Disposcope endoscope in patients in whom glottis viewing is difficult.

Aurora-A is a determinant of tamoxifen sensitivity through phosphorylation of ERα in breast cancer.

Despite the clinical success of tamoxifen, its resistance remains a major challenge in breast cancer. Here we show that Aurora-A determines tamoxifen sensitivity by regulation of oestrogen receptor (ER)α. Ectopic expression of Aurora-A decreases and depletion of Aurora-A enhances tamoxifen sensitivity in ERα-positive breast cancer. Elevated Aurora-A was significantly associated with the recurrence of ERα-positive tumours. Notably, Aurora-A inhibitor MLN8237, which is currently in clinical trial, synergizes with tamoxifen and overcomes tamoxifen resistance. Furthermore, Aurora-A interacts with and phosphorylates ERα on serine-167 and -305, leading to increase in ERα DNA-binding and transcriptional activity. Elevated levels of Aurora-A are significantly associated with disease-free survival in ERα-positive but not ERα-negative breast cancers. These data suggest that Aurora-A has a pivotal role in tamoxifen resistance and ERα is a bona fide substrate of Aurora-A. Thus, Aurora-A represents a prognostic marker in ERα-positive tumour and a critical therapeutic target in tamoxifen-resistant breast cancer, and Aurora-A inhibitor could be used as either an independent or concurrent agent in tamoxifen-resistant tumour.