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 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.

ObjectiveTo observe the effect of transcranial direct current stimulation (tDCS) combined with task-oriented rehabilitation training single pellet reaching and grasping (SPG) on the motor function of forelimb in rats with unilateral contusion of C₅ spinal cord. MethodsA total of 60 adult male Sprague-Dawley rats were randomly divided into sham operation group (sham group), spinal cord injury (SCI) group, tDCS group, SPG group, false group and tDCS+SPG group, with ten rats in each group. Only C₅ lamina was removed in the sham group, and the C₅ spinal cord contusion model was established by IH spinal cord impactor in the other five groups. The rats received tDCS in tDCS group, SPG in SPG group, tDCS without current in false group, tDCS combined with SPG in tDCS+SPG group, and no treatment in the SCI and the sham groups. The rats were evaluated with Rearing and Grooming tests, and motor-evoked potential (MEP). ResultsFour weeks after operation, compared with SCI group, the scores of Rearing and Grooming increased in tDCS group and tDCS+SPG group (P < 0.05), and they were more in the tDCS+SPG group than in tDCS group and SPG group (P < 0.05); the score of Grooming increased in SPG group (P < 0.05); while the amplitude of MEP increased in tDCS group, SPG group and tDCS+SPG group (P < 0.05), and the latency shortened in tDCS group and tDCS+SPG group (P < 0.05); and the amplitude increased more in tDCS+SPG group than in tDCS group and SPG group (P < 0.01). ConclusiontDCS could promote the recovery of motor function in rats with SCI, and the combination therapy of tDCS and task-oriented rehabilitation training is more effective.

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed L-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual L-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of L-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.
Animals ; Dopa Decarboxylase/genetics* ; Dopamine/biosynthesis* ; Drosophila melanogaster/genetics* ; Escherichia coli/metabolism* ; Humans ; Metabolic Engineering

Metabolic engineering has been developed for nearly 30 years since the early 1990s, and it has given a great impetus to microbial strain breeding and improvement. Aromatic chemicals are a variety of important chemicals that can be produced by microbial fermentation and are widely used in the pharmaceutical, food, feed, and material industry. Microbial cells can be engineered to accumulate a variety of useful aromatic chemicals in a targeted manner through rational engineering of the biosynthetic pathways of shikimate and the derived aromatic amino acids. This review summarizes the metabolic engineering strategies and biosynthetic pathways for the production of aromatic chemicals developed in the past 30 years, with the aim to provide a valuable reference and promote the research in this field.
Biosynthetic Pathways ; Fermentation ; Metabolic Engineering ; Shikimic Acid

OBJECTIVE To study the efficacy of sinapine thiocyanate dissoluble microneedle （ST-DMN） for acupoint administration against bronchial asthma （BA）. METHODS The network pharmacology and molecular docking techniques were used to screen the core targets of sinapine thiocyanate （ST） against BA， and the pharmacodynamics of the top 3 core targets was studied. Firstly， ST-DMN was prepared （drug loading of ST was 1 mg/tablet）； secondly， 30 rats were divided into blank control group， model control group， blank microneedle group， Sinapine powder plaster group （positive control group） and ST-DMN group. Except for the blank control group， rats of other groups were sensitized with 10% ovalbumin （containing aluminum hydroxide adjuvant） and nebulized with 1% ovalbumin to induce the BA model. After modeling， blank control group did not receive any intervention； normal saline was applied to the Feishu acupoint and Dazhui acupoint of the rats in the model control group， while the blank microneedle group， Sinapine powder plaster group and ST-DMN group were given blank microneedle， Sinapis alba powder （plaster， 1.5 g） and ST-DMN （3 tablets at 2 acupoints） at same acupoint， once a day， for 28 consecutive days. After administration， the general symptoms were observed and the body mass of the rats was measured.pathological changes of lung tissues in rats was observed； the levels of prostaglandin endoperoxide synthase 2 （PTGS2）， GNYL matrix metalloproteinase-9 （MMP-9） and interleukin-2 （IL-2）in serum， bronchoalveolar lavage fluid （BALF） and lung tissues were determined. RESULTS Results of network pharmacology and molecular docking showed that the key targets of ST against BA were identified as PTGS2， MMP-9， IL-2， epidermal growth factor receptor， heat shock protein90AA1， etc. Pharmacodynamic experiments showed that compared with model control group， relieved cough， restored hair color， sensitive behavior， stable respiration and increased body weight were all found in ST-DMN group； the histopathological changes as the structure of lung tissue， infiltration of alveolar epithelial cells and pulmonary interstitial inflammatory cells were improved to different extent； the levels of PTGS2， MMP-9 and IL-2 in serum， BALF and lung tissue were significantly reduced （P＜0.05 or P＜0.01）. CONCLUSIONS The anti-BA effect of ST-DMN acupoint administration is good， the mechanism of which may be associated with decreasing the levels of PTGS2， MMP-9 and IL-2 in serum， BALF and lung tissue.