Vampire bat saliva contains a plasminogen activator that presumably assists these hematophagous animals during feeding. Bat-PA (H), the full-length form of Vampire Bat Salivary Plasminogen Activator (DSPAalpha1), is homologous and similar efficacy to tissue-type plasminogen activator (t-PA). The strict fibrin dependence of activity is a characteristic which could be desirable in the fibrinolytic therapy. It is a unique fibrinolytic enzyme that does not promote neurodegeneration. In this study, according to the reported gene sequence (GenBank Accession No. J05082) of Vampire bat (D. rotundus) plasminogen activator. It was the first time to synthesize the full sequence of DSPAalpha1 in vitro and clone it into the expression vector pPIC9K, the recombinant plasmid was linearized and transformed into Pichia pastoris GS115 strain. Secreted expression of recombinant DSPAalpha1 was attained by methanol induction and its molecular mass is 47 kD. To get recombinant GS115 with high amount of protein, hundreds of His+ transformants had been screened to isolate clones resistant to high levels G418 (2-4 mg/mL), the selected clones mini-expressed in Pichia pastoris, and tested their fibrinolytic activities and expressed protein bands by fibrin plate assay and SDS-PAGE. DSPAalpha1 was determined by optical density after SDS-PAGE, the yield is about 30 mg per liter of fermentation culture. DSPAalpha1 derived often from mammalian cells: Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, COS cells, which might be produced at high cost. In Pichia pastoris, it is expected to higher yield and lower cost, thus it might be able to serve as new thrombolytic candidate.
Animals ; Base Sequence ; Chiroptera ; genetics ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Pichia ; genetics ; metabolism ; Plasminogen Activators ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Sequence Analysis, DNA

Objective: To define a new subtype of vascular anomaly, named fibro-adipose vascular anomaly(FAVA)and to discuss the methods of diagnosis and treatment in order to decrease the misdiagnosis rate and improve the recovery rate. Methods: From Jan. 2011 to Dec. 2015, 34 cases with FAVA on the lower extremities received surgical treatment in our center. The clinical data were collected to summarize the imaging and pathological characteristics for diagnosis. Results: The misdiagnosis rate was 76.5%(26/34) in all these 34 patients. The cure rate was 100% after operation. The patients were followed-up for 1-3 years(average, 19 months) with normal function and no recurrence. Conclusions FAVA is usually misdiagnosed as hemangioma or vascular malformation. The disease should be well defined to help the diagnosis and treatment. Surgical excision is one of the optional treatments.

Objective To observe the expression of parathyroid hormone - related peptide (PTHrp) receptor in tibial growth plate and its effects on tibial extension in chronic renal insufficiency rats. Methods Two-week-old male Sprague-Dawley rats were randomly divided into sham group, model group and enalapril group, each with 20 rats. In model group and enalapril group rats had chronic renal insufficiency induced by left ureteral obstruction, and rats were respectively given saline and enalapril by gavage after the operation. In sham group, left ureter was only exposed without ligation, and rats were given saline. The urine was collected 4 weeks after the operation and the total protein content was measured. Then all rats were killed. The concentrations of PTHrp, creatinine and urea nitrogen in intracardiac blood were detected. HE staining and Masson staining were performed on the left kidney to observe pathological changes of glomeruli and renal tubules. The total length of bilateral tibia was measured. The number of columnar cells in the growth plate proliferative zone was measured by safranin O staining and the expression of PTHrp receptor in the growth plate was detected by immunohistochemistry. Results The 24 h urine total protein, creatinine and urea nitrogen in model group were higher than those in sham group (all P＜0.05), while these 3 renal functional parameters in enalapril group were lower than those in model group (all P＜0.05). In model group and enalapril group rats had higher blood concentrations of PTHrp than that in sham group (all P＜0.05), but blood PTHrp in enalapril group was lower than that in model group (P＜0.05). HE staining and Masson staining showed that in the model group rats had severe tubular dilation, inflammatory cell infiltration and the tissue fibrosis, while in enalapril group renal tubules slightly dilated and had a few inflammatory cell infiltration and tissue fibrosis. Compared with those in the sham group, in model group the tibia length, the chondrocyte number of column structure in the growth plate proliferative zone and the PTHrp receptor decreased (all P＜0.05). But in enalapril group those indexes increased than model group (all P＜0.05). Conclusions Chronic renal insufficiency rats had increased PTHrp concentration in the blood but decreased PTHrp receptors expression in tibial growth plate, which lead to their limited tibial extension.

Objective: To evaluate the clinical outcomes of a series of patients who have undergone reconstruction of craniofacial defects after resection of intracranial tumors or craniofacial trauma with free composite anterolateral thigh flaps. Methods: Retrospective analyses the clinical cases from September 2007 to September 2016. Data included flap survival rate, complication, satisfaction survey was reviewed to evaluate the efficacy and safety of this surgical strategy. Results: Totally 10 free anterolateral thigh flaps including 3 cases of fasciocutaneous flaps, 2 case of adipofascial flaps, 4 cases of myocutaneous flaps, 1 case of chimeric flap, were adopted to reconstruct craniofacial defects. Follow-up ranged from 3 to 17 months (average, 12 months). All flaps were transferred successfully. There were no cranial spinal fluid(CSF) leaks, intracranial infections or donor site complications. All patients were satisfied. Conclusions Because of its abundance of tissue, matched vessels to recipient site, versatility of muscular flaps to fill irregularly intracranial defects, reliable blood supply, feasibility of simultaneous fascia lata harvesting, free composite anterolateral thigh flap is the reconstructive method of choice for craniofacial defects reconstruction after resection of intracranial tumors or craniofacial trauma. The use of ALT flap was reliable in the decrease of CSF leak and infection rate and dependable according to long time follow-up.

Objective:To manufacture one kind of biphasic calcium phosphate (BCP) ceramic by hydroxyapatite (HA) and β-tricalciumphosphate (β-TCP), and to further investigate its compatibility and its efficacy of ectopic bone formation.Methods:BCP was prepared with the ratio of HA and β-TCP at 6/4 using precipitation and H 2O 2 foaming method and then sintered at 1 100 ℃ for 3 hours. The chemical composition of BCP was investigated by X-ray diffraction(XRD). BMSCs were isolated from the bone marrow of Sprague-Dawley (SD) rat and seeded on BCP. The adhesion and morphology of BMSCs on BCP was observed under scanning electron microscope(SEM) and special staining. Cell proliferation was quantified by cell counting kit-8(CCK8) assay. Alkaline phosphatase(ALP)activity of BMSCs was measured by ALP assay kit. For further confirmation, the intramuscularly ectopic implantation models of Beagle were used, general observation, histological, and histomorphometric analyses were conducted at 4, 8, 12 weeks after implantation. Results:BCPs were successfully manufactured. XRD analysis showed the specific diffraction peaks of HA and β-TCP. SEM showed that the surface of the BCP ceramics was widely distributed with macropores and connections, and the pore walls were rough, and the micropores were evenly distributed in the macropores. Phalloidin and DAPI staining showed that the BMSCs extended and adhered to the surface of the material, and the shape gradually changed from irregularity to uniform long spindle. CCK8 method showed that although the cell viability decreased on the first day after coculture, on the third, fourth, fifth and seventh days, the cell viability gradually increased. The assay of alkaline phosphatase activity indicated that BMSCs cultured on the BCP could secrete more alkaline phosphatase on day 1 and 7 compared with the control group. BCP implanted in the muscle could generate osteoid/bone tissue at 8 weeks and 12 weeks, the number of osteoid/bone filled pores were 0.77±0.11, the percentage of osteoid/bone tissue inside the pores were 0.71±0.14.Conclusions:The BCP had a good biocompatibility and favorable efficacy of ectopic osteoinduction.

Objective:To manufacture one kind of biphasic calcium phosphate (BCP) ceramic by hydroxyapatite (HA) and β-tricalciumphosphate (β-TCP), and to further investigate its compatibility and its efficacy of ectopic bone formation.Methods:BCP was prepared with the ratio of HA and β-TCP at 6/4 using precipitation and H 2O 2 foaming method and then sintered at 1 100 ℃ for 3 hours. The chemical composition of BCP was investigated by X-ray diffraction(XRD). BMSCs were isolated from the bone marrow of Sprague-Dawley (SD) rat and seeded on BCP. The adhesion and morphology of BMSCs on BCP was observed under scanning electron microscope(SEM) and special staining. Cell proliferation was quantified by cell counting kit-8(CCK8) assay. Alkaline phosphatase(ALP)activity of BMSCs was measured by ALP assay kit. For further confirmation, the intramuscularly ectopic implantation models of Beagle were used, general observation, histological, and histomorphometric analyses were conducted at 4, 8, 12 weeks after implantation. Results:BCPs were successfully manufactured. XRD analysis showed the specific diffraction peaks of HA and β-TCP. SEM showed that the surface of the BCP ceramics was widely distributed with macropores and connections, and the pore walls were rough, and the micropores were evenly distributed in the macropores. Phalloidin and DAPI staining showed that the BMSCs extended and adhered to the surface of the material, and the shape gradually changed from irregularity to uniform long spindle. CCK8 method showed that although the cell viability decreased on the first day after coculture, on the third, fourth, fifth and seventh days, the cell viability gradually increased. The assay of alkaline phosphatase activity indicated that BMSCs cultured on the BCP could secrete more alkaline phosphatase on day 1 and 7 compared with the control group. BCP implanted in the muscle could generate osteoid/bone tissue at 8 weeks and 12 weeks, the number of osteoid/bone filled pores were 0.77±0.11, the percentage of osteoid/bone tissue inside the pores were 0.71±0.14.Conclusions:The BCP had a good biocompatibility and favorable efficacy of ectopic osteoinduction.

Objective:To investigate urinary microalbumin to creatinine ratio (ACR) and α1-microglobulin to creatinine ratio (MCR) of people aged 40 years old and above in Shanxi province, and analyze the influencing factors of abnormal ACR and MCR, and to provide evidence for the prevention and control of chronic kidney diseases.Methods:It was a cross-sectional study. The data came from a screening study of chronic kidney diseases conducted by Shanxi Provincial People's Hospital from April to November 2019, involving aged 40 years old and above from 10 counties (Ningwu county, Yu county, Yangqu county, Lin county, Shouyang county, Zezhou county, Huozhou city, Hejin city, Linyi county and Ruicheng county) in Shanxi province. The related data were collected through questionnaire surveys, physical examinations, and blood and urine sample collection. Urinary α1-microglobulin, creatinine, and microalbuminuria were measured. Urinary ACR and MCR were calculated using urinary creatinine correction. ACR abnormality was defined as ≥30 mg/g, and MCR abnormality was defined as >23 mg/g. Covariate analysis was used to control confounding factors, and adjusted urinary ACR and MCR of 10 counties were calculated. Spearman correlation analysis and chi-square test were performed to analyze the factors associated with abnormal urinary ACR and MCR. Logistic regression analysis model was used to identify the influencing factors of abnormal urinary ACR and MCR.Results:A total of 12 285 residents were enrolled in the study, including 5 206 males (42.4%) and 7 079 females (57.6%). The median age was 58.0 (51.0, 66.0) years old. The median urinary ACR was 7.5 (4.5, 15.7) mg/g, and the median urinary MCR was 10.2 (6.4, 16.2) mg/g. A total of 1 572 individuals (12.80%) had urinary ACR abnormality and 1 450 individuals (11.80%) had urinary MCR abnormality. Yangqu county, Yuxian county, and Ningwu county had higher urinary ACR with (35.58±3.04) mg/g, (34.08±4.50) mg/g and (32.09±3.19) mg/g, respectively. The urinary MCR was generally similar among the 10 counties and Yangqu county had higher urinary MCR with (13.86±0.41) mg/g. In addition to Yu county, female individuals had higher urinary ACR compared to males in other counties, whereas female individuals had lower urinary MCR compared to males in 10 counties. Multivariate logistic regression analysis results showed that elevated triglyceride, fasting blood glucose, glycated hemoglobin, systolic blood pressure, diastolic blood pressure, age, body mass index and gender were independent influencing factors of abnormal urinary ACR and MCR (all P<0.05). Elevated blood homocysteine and low educational level were independent influencing factors of urinary MCR abnormality (both P<0.05). Conclusions:There are differences of gender and region in urinary ACR and MCR among individuals aged 40 years old and above in the 10 counties of Shanxi province. Triglyceride, fasting blood glucose, glycated hemoglobin, systolic blood pressure, diastolic blood pressure, age, gender, and body mass index are independent related factors of abnormal urinary ACR and MCR. Blood homocysteine and education level are independent related factors of abnormal urinary MCR.

Objective:To manufacture one kind of biphasic calcium phosphate (BCP) ceramic by hydroxyapatite (HA) and β-tricalciumphosphate (β-TCP), and to further investigate its compatibility and its efficacy of ectopic bone formation.Methods:BCP was prepared with the ratio of HA and β-TCP at 6/4 using precipitation and H 2O 2 foaming method and then sintered at 1 100 ℃ for 3 hours. The chemical composition of BCP was investigated by X-ray diffraction(XRD). BMSCs were isolated from the bone marrow of Sprague-Dawley (SD) rat and seeded on BCP. The adhesion and morphology of BMSCs on BCP was observed under scanning electron microscope(SEM) and special staining. Cell proliferation was quantified by cell counting kit-8(CCK8) assay. Alkaline phosphatase(ALP)activity of BMSCs was measured by ALP assay kit. For further confirmation, the intramuscularly ectopic implantation models of Beagle were used, general observation, histological, and histomorphometric analyses were conducted at 4, 8, 12 weeks after implantation. Results:BCPs were successfully manufactured. XRD analysis showed the specific diffraction peaks of HA and β-TCP. SEM showed that the surface of the BCP ceramics was widely distributed with macropores and connections, and the pore walls were rough, and the micropores were evenly distributed in the macropores. Phalloidin and DAPI staining showed that the BMSCs extended and adhered to the surface of the material, and the shape gradually changed from irregularity to uniform long spindle. CCK8 method showed that although the cell viability decreased on the first day after coculture, on the third, fourth, fifth and seventh days, the cell viability gradually increased. The assay of alkaline phosphatase activity indicated that BMSCs cultured on the BCP could secrete more alkaline phosphatase on day 1 and 7 compared with the control group. BCP implanted in the muscle could generate osteoid/bone tissue at 8 weeks and 12 weeks, the number of osteoid/bone filled pores were 0.77±0.11, the percentage of osteoid/bone tissue inside the pores were 0.71±0.14.Conclusions:The BCP had a good biocompatibility and favorable efficacy of ectopic osteoinduction.

Objective:To manufacture one kind of biphasic calcium phosphate (BCP) ceramic by hydroxyapatite (HA) and β-tricalciumphosphate (β-TCP), and to further investigate its compatibility and its efficacy of ectopic bone formation.Methods:BCP was prepared with the ratio of HA and β-TCP at 6/4 using precipitation and H 2O 2 foaming method and then sintered at 1 100 ℃ for 3 hours. The chemical composition of BCP was investigated by X-ray diffraction(XRD). BMSCs were isolated from the bone marrow of Sprague-Dawley (SD) rat and seeded on BCP. The adhesion and morphology of BMSCs on BCP was observed under scanning electron microscope(SEM) and special staining. Cell proliferation was quantified by cell counting kit-8(CCK8) assay. Alkaline phosphatase(ALP)activity of BMSCs was measured by ALP assay kit. For further confirmation, the intramuscularly ectopic implantation models of Beagle were used, general observation, histological, and histomorphometric analyses were conducted at 4, 8, 12 weeks after implantation. Results:BCPs were successfully manufactured. XRD analysis showed the specific diffraction peaks of HA and β-TCP. SEM showed that the surface of the BCP ceramics was widely distributed with macropores and connections, and the pore walls were rough, and the micropores were evenly distributed in the macropores. Phalloidin and DAPI staining showed that the BMSCs extended and adhered to the surface of the material, and the shape gradually changed from irregularity to uniform long spindle. CCK8 method showed that although the cell viability decreased on the first day after coculture, on the third, fourth, fifth and seventh days, the cell viability gradually increased. The assay of alkaline phosphatase activity indicated that BMSCs cultured on the BCP could secrete more alkaline phosphatase on day 1 and 7 compared with the control group. BCP implanted in the muscle could generate osteoid/bone tissue at 8 weeks and 12 weeks, the number of osteoid/bone filled pores were 0.77±0.11, the percentage of osteoid/bone tissue inside the pores were 0.71±0.14.Conclusions:The BCP had a good biocompatibility and favorable efficacy of ectopic osteoinduction.

Objective:The differentially expressed genes were screened from microarray data in the patients with non-syndromic craniosynostosis, and a protein interaction network was established to screen and predict hub genes related to the disease.Methods:The data set of GSE50796 were downloaded from the GEO database, which included seven samples of the closed cranial suture tissues from the non-syndromic craniosynostosis patients, and seven samples of the unclosed cranial suture tissues from the non-syndromic craniosynostosis patients. Analyze the differentially expressed genes were collected and analyzed with GEO2R, a GEO database online tool. P<0.05 and |logFC|> 2 were set as filter criteria. The ggplot2 of R package was applied for GO enrichment analysis, and the KEGG pathway analysis was completed with Enrichr. Gene set enrichment analysis (GSEA) was performed via GSEA 3.0 to analyze the correlation between gene sets and phenotypes. Secondly, the STRING database was used to analyze the interaction relationships between differentially expressed proteins in different tissues, and then Cytoscape and related plug-ins were used to establish the differentially expressed protein interaction network and screen the hub genes. Meanwhile, the key modules, important biological processes, and multiple co-expression relationships were analyzed. Results:A total of 255 differentially expressed genes based on the above screening conditions were obtained. The regulation of neural development screened by GO enrichment analysis, the PI3K-Akt signaling pathway screened by KEGG enrichment analysis, the important biological pathways (DNA replication, cell cycle, cytokine and receptor interaction) screened by GSEA enrichment analysis, and the positive regulation of osteoblast differentiation screened by ClueGO analysis, might be closely related to the etiology of non-syndromic craniosynostosis. The up-regulated hub genes such as CLEC12A, MS4A3 and DNTT in the group with closed sutures were screened by protein-protein interaction network and literature analysis, which might play a vital role in the pathogenic processes of non-syndromic craniosynostosis.Conclusions:With the multi-dimensional enrichment analysis of the differentially expressed genes and the establishment of protein interaction networks, we have deepened our understanding of differentially expressed genes, important biological processes and signaling pathways involved in the pathogenesis of non-syndromic craniosynostosis. The selected hub genes may become early diagnostic markers and potential molecular therapeutic targets.