Objective To explore the biosynthesis of Dipsacus asper Wall.ex Henry triterpenoid saponin,and the UGT gene in Dipsacus asper Wall.ex Henry has been analyzed by the identification of whole genome,genome and prokaryotic expression.Methods The laboratory self-tested sequenced protein sequence files of the Dipsacus asper Wall.ex Henry genome were used.To validate the conserved domains of the sequence of the Dipsacus asper Wall.ex Henry UGT gene,BLASTP and hmmsearch were utilized.Prot-Param,SOMPA,MAGA7.0,Tbtools and other tools were used to investigate the protein physicochemical properties,protein structure,and covariance analysis of the Dipsacus asper Wall.ex Henry UGT gene family,and using the joint analysis of transcriptomic data and metabolomics data,two glycosyltransferases that might be related to triterpene saponin biosynthesis were screened,and expression vectors were constructed for prokaryotic expression.Results 44 Dipsacus asper Wall.ex Henry UGT genes were identified from the Dipsacus asper Wall.ex Henry genome.The length of Dipsacus asper Wall.ex Henry UGT proteins ranged from 49 to 1083 amino acids,with an average molecular weight of 24.86 kDa and an isoelectric point of 4.31-8.59.Dipsacus asper Wall.ex Henry UGT gene family was distributed on eight chromosomes.The phylogenetic tree constructed from the sequences of Dipsacus asper Wall.ex Henry,Arabidopsis thaliana and identified UGTs showed that glycosyltransferase gene families in Dipsacus asper Wall.ex Henry were mainly in the UGT73,UGT81,UGT85,and UGT80 families.Cis-acting element analysis showed that light-responsive elements were the most prevalent elements in the promoter regions of UGT gene family members.Two glycosyltransferases potentially related to triterpene saponin biosynthesis were screened using combined transcriptomics and metabolomics analysis,and were successfully expressed in prokaryotic form.Conclusion In this study,two candidate genes related to the biosynthesis of Dipsacus asper Wall.ex Henry triterpenoid saponins were jointly screened for prokaryotic expression using multi-omics,and were subjected to prokaryotic expression for further validation of the function of the genes.

BACKGROUND:Treadmill training is one of the effective ways to promote the recovery of motor function after spinal cord injury.Treadmill training can promote neurogenesis,but the effect of different intensities of treadmill training on the activation of endogenous stem cells is still unclear. OBJECTIVE:To analyze the activation effect of different intensities of treadmill training on endogenous neural stem cells in the spinal cord of mice after spinal cord injury. METHODS:Fifty female C57BL/6J mice were divided into control group,spinal cord injury group,low-,moderate-,and high-intensity exercise groups with 10 mice in each group by random number table method.T10 segment spinal cord injury model was constructed by the clamp method in spinal cord injury group,low-,moderate-,and high-intensity exercise groups.On day 7 after spinal cord injury,mice in the low-,moderate-,and high-intensity exercise groups were respectively trained on the treadmill with corresponding intensity,3 times/d,10 min/times,6 times a week for 28 consecutive days.At 3,7,14,21,and 28 days after treadmill training,the hind limb motor function was evaluated by BMS score.At 28 days after treadmill training,the spinal cord tissue of the injured area was obtained,and the expression of epidermal growth factor receptor,glial fibrillary acidic protein,and 5-Ethynyl-2'-deoxyuridine(EdU),a proliferative marker,was detected.Hematoxylin-eosin staining was used to observe the morphology of spinal cord. RESULTS AND CONCLUSION:(1)The BMS score of mice in the spinal cord injury group was lower than that in the control group(P<0.05).With the extension of treadmill training time,the BMS scores of mice with spinal cord injury gradually increased,and the BMS scores of mice in moderate-intensity exercise group on days 14 and 21 after treadmill training were higher than those in spinal cord injury group and low-and high-intensity exercise groups(P<0.05).The BMS score of mice in moderate-and high-intensity exercise group was higher than that in spinal cord injury group and low-intensity exercise group at 28 days after treadmill training(P<0.05).(2)Compared with the control group,the proportion of epidermal growth factor receptor and EdU positive cells was increased in spinal cord injury group(P<0.05).Compared with spinal cord injury group,the proportion of epidermal growth factor receptor and EdU positive cells was increased in low-,moderate-,and high-intensity exercise groups(P<0.05),and the highest was found in moderate-intensity exercise group.Compared with control group,the proportion of glial fibrillary acidic protein positive cells was increased in spinal cord injury group(P<0.05).Compared with spinal cord injury group,the proportion of glial fibrillary acidic protein positive cells was lower in low-,moderate-,and high-intensity exercise groups(P<0.05),and the moderate-intensity exercise group was the lowest.(3)Hematoxylin-eosin staining showed that a large cavity was formed in the injured area of mice with spinal cord injury,and the cavity in the injured area of mice with spinal cord injury decreased after different intensities of treadmill training,and the decrease was most obvious in the moderate-intensity exercise group.(4)These results indicate that low-,moderate-,and high-intensity treadmill training can promote the recovery of motor function of mice with spinal cord injury by activating endogenous neural stem cells,and the effect of moderate-intensity exercise training is the most obvious.

BACKGROUND:We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro,but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons.There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age.OBJECTIVE:To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro,and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro.METHODS:Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse (3 months of age) in adherent culture and transfected with lentivirus fused with multimodal imaging genes.High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium.The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons,and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed.An equal number (5×103/mL) of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5％ CO2,37℃ thermostat,respectively.The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation,CCK8 assay,qPCR,and western blot assay.RESULTS AND CONCLUSION:(1) High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse,which expressed Nestin and SOX2 in vitro,and were able to form neurospheres and pass on continuously.(2) The in vitro self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse,and the neurospheres formed by day 4 of cell culture in neonatal mouse were about 150 μm in diameter,whereas the neurospheres formed by adult mouse tactile neurons were only 40 μm in diameter (P<0.0001).(3) CCK8 proliferation assay showed that the proliferative activity of adult mouse cerebrospinal fluid-contacting neurons was significantly weaker than that of neonatal mouse at all time points after culture (P<0.0001).(4) qPCR and western blot assay revealed that the mRNA (P<0.0001) and protein expression levels (P<0.01) of Nestin and SOX2 in cerebrospinal fluid-contacting neurons of adult mouse were significantly decreased compared with those of neonatal mouse.(5) The above results indicated that the self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse in vitro.

Objective To explore the biosynthesis of Dipsacus asper Wall.ex Henry triterpenoid saponin,and the UGT gene in Dipsacus asper Wall.ex Henry has been analyzed by the identification of whole genome,genome and prokaryotic expression.Methods The laboratory self-tested sequenced protein sequence files of the Dipsacus asper Wall.ex Henry genome were used.To validate the conserved domains of the sequence of the Dipsacus asper Wall.ex Henry UGT gene,BLASTP and hmmsearch were utilized.Prot-Param,SOMPA,MAGA7.0,Tbtools and other tools were used to investigate the protein physicochemical properties,protein structure,and covariance analysis of the Dipsacus asper Wall.ex Henry UGT gene family,and using the joint analysis of transcriptomic data and metabolomics data,two glycosyltransferases that might be related to triterpene saponin biosynthesis were screened,and expression vectors were constructed for prokaryotic expression.Results 44 Dipsacus asper Wall.ex Henry UGT genes were identified from the Dipsacus asper Wall.ex Henry genome.The length of Dipsacus asper Wall.ex Henry UGT proteins ranged from 49 to 1083 amino acids,with an average molecular weight of 24.86 kDa and an isoelectric point of 4.31-8.59.Dipsacus asper Wall.ex Henry UGT gene family was distributed on eight chromosomes.The phylogenetic tree constructed from the sequences of Dipsacus asper Wall.ex Henry,Arabidopsis thaliana and identified UGTs showed that glycosyltransferase gene families in Dipsacus asper Wall.ex Henry were mainly in the UGT73,UGT81,UGT85,and UGT80 families.Cis-acting element analysis showed that light-responsive elements were the most prevalent elements in the promoter regions of UGT gene family members.Two glycosyltransferases potentially related to triterpene saponin biosynthesis were screened using combined transcriptomics and metabolomics analysis,and were successfully expressed in prokaryotic form.Conclusion In this study,two candidate genes related to the biosynthesis of Dipsacus asper Wall.ex Henry triterpenoid saponins were jointly screened for prokaryotic expression using multi-omics,and were subjected to prokaryotic expression for further validation of the function of the genes.

BACKGROUND:We have successfully isolated and cultured neonatal mouse cerebrospinal fluid-contacting neurons in vitro,but there is no study that reports an effective method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons.There is no study on whether the self-renewal ability of cerebrospinal fluid-contacting neurons changes with age.OBJECTIVE:To establish a method for isolating and culturing high-purity adult mouse cerebrospinal fluid-contacting neurons in vitro,and to characterize the self-renewal ability of adult mouse cerebrospinal fluid-contacting neurons and neonatal mouse cerebrospinal fluid-contacting neurons in vitro.METHODS:Primary cells containing cerebrospinal fluid-contacting neurons were isolated from the cervical medulla of adult mouse (3 months of age) in adherent culture and transfected with lentivirus fused with multimodal imaging genes.High-purity adult mouse cerebrospinal fluid-contacting neurons were obtained by puromycin screening in suspension culture in complete medium.The expression of neural stem cell markers Nestin and SOX2 was detected by immunofluorescence in adult mouse cerebrospinal fluid-contacting neurons,and the ability of adult mouse cerebrospinal fluid-contacting neurons to form spheres and pass on in vitro was observed.An equal number (5×103/mL) of passage 3 adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons were divided into two groups under the same conditions and inoculated into ultra-low adhesion plates containing complete medium in suspension culture at 5％ CO2,37℃ thermostat,respectively.The self-renewal capacity of adult mouse and neonatal mouse cerebrospinal fluid-contacting neurons was characterized by in vitro spheroid formation,CCK8 assay,qPCR,and western blot assay.RESULTS AND CONCLUSION:(1) High-purity cerebrospinal fluid-contacting neurons were successfully isolated from adult mouse,which expressed Nestin and SOX2 in vitro,and were able to form neurospheres and pass on continuously.(2) The in vitro self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse,and the neurospheres formed by day 4 of cell culture in neonatal mouse were about 150 μm in diameter,whereas the neurospheres formed by adult mouse tactile neurons were only 40 μm in diameter (P<0.0001).(3) CCK8 proliferation assay showed that the proliferative activity of adult mouse cerebrospinal fluid-contacting neurons was significantly weaker than that of neonatal mouse at all time points after culture (P<0.0001).(4) qPCR and western blot assay revealed that the mRNA (P<0.0001) and protein expression levels (P<0.01) of Nestin and SOX2 in cerebrospinal fluid-contacting neurons of adult mouse were significantly decreased compared with those of neonatal mouse.(5) The above results indicated that the self-renewal ability of cerebrospinal fluid-contacting neurons in adult mouse was significantly weaker than that of neonatal mouse in vitro.

Objective:To analyze and evaluate the equity of children's health in countries along the"the Belt and Road",promote further attention to children's health in countries along the route,and promote cooperation and exchanges on children's health between China and countries along the"the Belt and Road".Methods:Using concentration index and concentration curve to measure overall equity,and using the Thiel index for intraregional and interregional euqity measurement.Results:The under-five mortality concentration index is 0.349 7,the concentration curve is below the absolute fair line.The Thiel index shows that inequality in low-income countries,lower-middle-income countries,upper-middle-income countries and high-income countries is the leading cause of child health inequities in the"the Belt and Road"countries.Conclusion:There is inequity in the health of children in countries along"the Belt and Road Initiative",countries along the"the Belt and Road"should take comprehensive measures to reduce the under-five mortality rate,at the same time strengthen international cooperation to further promote equity in children's health in"Belt and Road"countries.

Objective:To investigate the impact of obstructive sleep apnea(OSA)on cardiac structure and function in elderly patients with type 2 diabetes mellitus(T2DM).Methods:This was a case-control study.Elderly patients with T2DM aged 65 years and above at the Department of Geriatrics of Tianjin Medical University General Hospital were consecutively enrolled in this study, and eventually 61 patients with complete general information were included.All patients were examined with polysomnography(PSG). They were divided into the T2DM group and the T2DM+ OSA group based on whether there was concurrent OSA.Differences in cardiac structure and function were compared between the two groups and between patients with mild OSA and those with moderate-severe OSA.The correlation of OSA with cardiac structure and function in T2DM patients was analyzed by using Pearson correlation and multiple linear regression analysis.Results:The left ventricular ejection fraction(LVEF)was lower in the T2DM+ OSA group than in the T2DM group(52.38±4.70 % vs.56.34±5.92%, t=2.892, P=0.005). The anteroposterior diameter of the right ventricle, inter-ventricular septum thickness and left ventricular posterior wall thickness increased in the T2DM+ OSA group compared with the T2DM group(21.50±1.49 mm vs.20.55±1.05 mm, 10.21±0.88 mm vs.9.52±1.04 mm, 10.42±0.83 mm vs.9.83±0.83 mm, t=-2.670, -2.770 and -2.716, P=0.010, 0.007 and 0.009). LVEF was lower in patients with moderate-severe OSA than in those with mild OSA group(50.58±3.55% vs.55.83±4.83%, t=3.813, P=0.001). The anteroposterior diameter of the right ventricle, inter-ventricular septum thickness and left ventricular posterior wall thickness were greater in patients with moderate-severe OSA than in those with mild OSA(21.86±1.39 mm vs.20.79±1.46 mm, 10.48±0.82 mm vs.9.69±0.75 mm, 10.68±0.80 mm vs.9.92±0.64 mm, t=-2.231, -2.871 and -2.943, P=0.032, 0.007 and 0.006). Pearson correlation analysis showed that the Apnea Hypopnea Index(AHI)was correlated with LVEF, the anteroposterior diameter of the right ventricle, inter-ventricular septum thickness and left ventricular posterior wall thickness( r=-0.425, 0.340, 0.458 and 0.473, P=0.001, 0.007, <0.001 and <0.001). After adjusting for age, body mass index, systolic blood pressure and triglycerides, multiple linear regression analysis showed that AHI was also correlated with LVEF, the anteroposterior diameter of the right ventricle, inter-ventricular septum thickness and left ventricular posterior wall thickness( β=-0.385, 0.520, 0.604 and 0.388, P=0.036, 0.011, 0.001 and 0.039). Conclusions:OSA aggravates cardiac remodeling and systolic insufficiency in T2DM patients and may be related to the severity of intermittent hypoxia.

OBJECTIVETo analyze the impact of platelet count on the prognosis of stage II-III colorectal cancer receiving adjuvant chemotherapy.

METHODSClinical and follow-up data of 286 patients with stage II-III colorectal cancer receiving adjuvant FOLFOX chemotherapy from March 2003 to October 2011 were analyzed retrospectively. Associations of baseline blood platelet count before chemotherapy and nadir blood platelet count during chemotherapy with relapse and death after adjuvant chemotherapy were analyzed by ROC curve and the optimal cutoff was selected. The association of the blood platelet count and the prognosis was analyzed by Kaplan-Meier and Cox regression model.

RESULTSROC curve showed the baseline blood platelet count was associated with recurrence (AUC=0.588, P=0.034). The optimal cutoff affecting recurrence was 276×10(9)/L. Kaplan-Meier showed those with baseline platelet count >276×10(9)/L receiving adjuvant chemotherapy had worse disease free survival (DFS) than those with baseline platelet count ≤276×10(9)/L, whose 5-year disease free survival(DFS) was 66% and 80% respectively (P=0.013). Cox regression analysis revealed baseline platelet count >276×10(9)/L was an independent unfavorable factor for DFS of adjuvant chemotherapy in colorectal cancer (HR=1.865, 95% CI: 1.108-3.141, P=0.019).

CONCLUSIONColorectal cancer patients receiving adjuvant chemotherapy with baseline platelet count >276×10(9)/L have worse prognosis.

Antineoplastic Combined Chemotherapy Protocols ; Chemotherapy, Adjuvant ; Colonic Neoplasms ; Colorectal Neoplasms ; Disease-Free Survival ; Fluorouracil ; Humans ; Leucovorin ; Neoplasm Staging ; Organoplatinum Compounds ; Platelet Count ; Prognosis ; Recurrence ; Retrospective Studies

Objective To examine the association between uric acid (UA)levels of patients with acute ischemic stroke at ad-mission and discharged outcome.Methods The acute ischemic stroke patients of Xinganmeng People’s Hospital in Inner Mongolia,from June 1,2009 to May 31,2012 were continuity included in the present study,the included analysis sample size were 3 440 cases.Poor discharged outcome was defined as the occurrence of disability or death.With reference to the Modi-fied Rankin's Scale (MRs)Stroke Scale,Scores were recorded in the questionnaires,score of 3 or more (MRs≥3)was de-fined as disability.The patients were all grouped by P20,P60,P90 of UA,binary logistic regression were used in studying of risk factors,calculated the odds ratios (Odds ratio,OR)and 95% confidence interval (95% Confident interval,95%CI).All tests were two-sided test and a significance level of 0.05.Results A total of 359 people occurred poor outcomes in the stud-y,accounting for 10.44%.Univariate logistic regression analysis of poor outcome occurred showed that relative to the lowest group(P20,UA≤222.6 mmol/L),the second and third group (UA:222.7 ~ 310.9 mmol/L and 311.0~419.7 mmol/L) OR (95% CI)were:0.70(0.53~0.91)(P <0.05)and 0.66(0.49~0.88)(P <0.05).After adjusted age,body tempera-ture,high blood pressure,hyperglycemia,history of stroke,high triglycerides,high LDL-C and smoking,relative to the low-est level group,the second and third group occurred poor outcoming OR (95% CI)were:0.70(0.53~0.93)(P <0.05)and 0.66(0.48~0.90)(P <0.05).Conclusion Higher levels of uric acid levels in patients with acute ischemic stroke may inde-pendently related with occurred poor discharged outcome.

Objective To construct the eukaryotic expression plasmid for the expression of human Connexin26 in COS-7 cells.Methods Total RNA was isolated from human peripheral blood lymphocytes and used as template for the PCR cloning of the human Connexin26 gene.The human Cx26 cDNA containing the 678 bp whole coding region of the human Connexin26 gene was amplified by PCR using specific primers and cloned into the pCI-neo vector to construct the recombinant eukaryotic expression plasmid,pCI-Cx26.The recombinant plasmid was identified by restriction endonuclease digestion,and transfected into COS-7 cells by liposome.The expression of Cx26 mRNA and the protein were analyzed by RT-PCR and SDS-PAGE,respectively.Results Restriction endonuclease digestion analysis verified successful construction of the recombinant plasmid,pCI-Cx26.The expression of Cx26 mRNA and protein in the transfected COS-7 cells were detected by RT-PCR and SDS-PAGE,respectively.Conclusion The eukaryotic expression plasmid for human Cx26 has been constructed successfully with the capability of expression in COS-7 cells.