Pichia pastoris is one of the most widely used recombinant protein expression systems. In this study, a novel method for rapid screening of P. pastoris strains capable of efficiently expressing recombinant proteins was developed. Firstly, the ability to express recombinant proteins of the modified strain GS115-E in which a functional Sec63-EGFP (Enhanced green fluorescent protein) fusion protein replaced the endogenous endoplasmic reticulum transmembrane protein Sec63 was tested. Next, the plasmids carrying different copy numbers of phytase (phy) gene or xylanase (xyn) gene were transformed into GS115-E to obtain recombinant strains with different expression levels of phytase or xylanase, and the expression levels of EGFP and recombinant proteins in different strains were tested. Finally, a flow cytometer sorter was used to separate a mixture of cells with different phytase expression levels into sub-populations according to green fluorescence intensity. A good linear correlation was found between the fluorescence intensities of EGFP and the expression levels of the recombinant proteins in the recombinant strains (0.8<|R|<1). By using the flow cytometer, high-yielding P. pastoris cells were efficiently screened from a mixture of cells. The expression level of phytase of the selected high-fluorescence strains was 4.09 times higher than that of the low-fluorescence strains after 120 h of methanol induction. By detecting the EGFP fluorescence intensity instead of detecting the expression level and activity of the recombinant proteins in the recombinant strains, the method developed by the present study possesses the greatly improved performance of convenience and versatility in screening high-yielding P. pastoris strains. Combining the method with high-throughput screening instruments and technologies, such as flow cytometer and droplet microfluidics, the speed and throughput of this method will be further increased. This method will provide a simple and rapid approach for screening and obtaining P. pastoris with high abilities to express recombinant proteins.
6-Phytase/genetics* ; Pichia/genetics* ; Plasmids ; Recombinant Proteins/genetics* ; Saccharomycetales

Objective:To study the effect of psoralen on the proliferation, invasion and migration of HTB-47 and CRL-1932 renal cancer cells cultured in vitro, and to further explore the internal mechanism of psoralen inhibiting renal cancer.Methods:The experimental group was HTB-47 and CRL-1932 renal cancer cells treated with dimethyl sulfoxide solution containing 30 μg/ml psoralen, and the control group was renal cancer cells treated with dimethyl sulfoxide. Scratch test, CCK8, Transwell, and Western blot were used to detect the effect of psoralen on renal cancer cells.Results:Compared with the control group, the proliferation, invasion and migration of renal cancer cells treated with psoralen in the experimental group were significantly inhibited. In the renal cancer cells treated with psoralen, the protein expression levels of MKI67, PCNA, MMP2 and MMP9 were significantly decreased (all P<0.05). Conclusions:Psoralen can significantly inhibit the proliferation, invasion and migration of HTB-47 and CRL-1932 renal cancer cells in vitro. The mechanism may be to inhibit the progression of renal cancer by regulating MKI67, PCNA, MMP2 and MMP9.

Objective To investigate the clinical features and prognosis of antimyelin oligodendrocyte glycoprotein-IgG （MOG-IgG） associated disorders （MOGAD）.Methods Eight cases met the diagnostic criteria of MOGAD were reviewed，and the demographics，clinical manifestations，laboratory and imaging results，treatment and prognosis were analyzed. The Expanded Disability Status Scale （EDSS） and the modified Rankin Scale （mRS） were used to dynamically evaluate the neurological disability. Results The ratio of male to female in this 8-case series was 1∶1，the median onset age was 34 years old，and the median disease duration was 42 months. The most common initial symptom is optic neuritis （ON，50%），followed by meningitis and/or encephalitis （25%）. Recurrence occurs in 50% of cases，and the recurrence rate of patients with high serum MOG IgG titer （≥1∶32） is 60%. The white blood cells in the cerebrospinal fluid in cases with meningitis/encephalitis or longitudinally extensive myelitis is often>100×10-6/L，which is higher than other phenotypes. There are 6 cases （75%） of patients with different MRI abnormalities，including hypertensity on T-2/Flair image with enhancement at affected optic nerve and orbital tissue，asymptomatic diencephalon/thalamus lesions on T2 and contrasted image，and MRI findings mimic typical multiple sclerosis may also be present. Cases with myelitis can show short-segment eccentric lesions or long-segment central lesions on MRI. Cases with meningitis/encephalitis showed unilateral cortical high T-2/Flair lesions on cranial MRI with various enhancements. After medication and rehabilitation，the peak EDSS of this case series was 5±1.069，and 2.19±1.689 points at the last visit；mRS was 3.25±1.165 points at the peak and 1.25±1.035 points at the last visit. The difference was statistically significant.Conclusions MOGAD is a rare type of demyelinating disease in central nervous with various manifestations，which is highly treatable. Fully understanding of the clinical characteristics，making a early diagnosis through comprehensive tests，and initiating immunization and rehabilitation treatment in time，will contribute to better prognosis for patients.

Objective: To detect the red blood cell lifespan in patients with polycythemia vera (PV), and explore the influencing factors. Methods: From February 2017 to December 2018, 27 patients with PV at Blood Diseases Hospital, Chinese Academy of Medical Science and 18 normal controls were recruited. Red blood cell lifespan was detected by endogenous carbon monoxide (CO) breath test. The related factors were analyzed. Results: The average red blood cell lifespan of 27 PV patients was 80 (range, 35-120) days (d), which was significantly shorter than that of the normal controls [110.5(69-166) d, P<0.05], namely 35.3 d shorter. The red blood cell lifespan of ten newly diagnosed patients and 17 patients who were treated with hydroxyurea and/or interferon were 98 (35-117) d and 69 (45-120) d, respectively, which were both shorter than that of the normal control (P=0.010, 0.000). Correlation analysis showed that red blood cell lifespan of patients with newly diagnosed PV was associated with JAK2 mutation allele burden (r=0.900, P=0.037), peripheral blood lymphocyte count (r=-0.742, P=0.014) and the level of serum vitamin B₁₂ (r=-0.821, P=0.023). Conclusion The lifespan of red blood cells in patients with PV is about one-third shorter than normal, and is related to JAK2 mutation allele burden, absolute lymphocyte count, and serum vitamin B₁₂ level.

Objective To evaluate the feasibility and clinical efficacy of retroperitoneal laparoscopic adrenalectomy for the treatment of Cushing disease.Methods Clinical data collected from 38 cases retroperitonel laparoscopic adrenalectomy for Cushing disease,from February 2006 to February 2017 were analyzed retrospectively.Among them,there were 6 males and 32 females aged from 13 to 66 years old,with an average age of 38 years old.The disease history ranged from 2 to 96 months,with an average of 28.3 months.Besides,there were 32 cases that had previous history of transsphenoidal pituitary tumor surgery and or radiotherapy.All 38 cases underwent retroperitoneal laparoscopic adrenalectomy therapy.There were 8 cases accepted right adrenalectomy and left subtotal adrenalectomy at the same time,24 cases accepted unilateral adrenalectomy,another 6 cases of pituitary tumors without lesions in medical imaging underwent unilateral adrenalectomy.Results All operations were completed sucessfully without conversion to open surgery and mortality.There was no blood transfusion during the period of operation.The operation time ranged from 30 min to 270 min (mean 88.3 min),the amount of bleeding was among the range of 10 ml to 200 ml (mean 38.33 ml).During 2 to 13 years of the follow up (mean 7 years),the 24 h urine cortisol concentration in the first day after surgery of piatients with subtotal resection was 90.35-220.84 μg/24h (mean 102.83 μg/24h),and 6 cases were in the normal range.After surgery 6-11 months,the clinical symptoms completely disappeared.The hormone replacement therapy was discontinued 1-3 months after surgery.However,There were 3 cases treated with residual adrenalectomy because of the recurrence after 1,3.5,5.0 years respectively.30 cases underwent unilateral adrenalectomy,whose 24 h urine cortisol concentration in the first day after surgery was 99.80-550.84 μg/24h (mean 372.83 μg/24h),among which 24 cases were beyond normal range,and another 6 cases were within the normal range.Reviewing 24 h urine cortisol concentration one month later after operation,it was 382.16-520.34 μg/24h (mean 461.62 μg/24h),with the results of all cases being higher than the normal range,the clinical symptoms were not relieved satisfactorily.During 2 to 7 months after surgery,there were 22 cases further underwent contralateral subtotal adrenalectomy (80％),the remaining 8 cases underwent contralateral adrenalectomy and autologous transplantation of adrenal tissue.Immediately one day after surgery,the 24 h urine cortisol concentration level of patients was ranged from 62.58 to 182.34 μg/24h (mean 92.83 μg/24h),and all 22 cases were within the normal range.The clinical symptoms completely disappeared during next 6 to 9 months after surgery,and hormone replacement therapy was discontinued 3 months after surgery.There were 2 cases received residual adrenalectomy because of the recurrence.Lifelong hormone replacement therapy after surgery occurred in 13 cases and 5 cases developed Nelson syndrome.Conclusions Retroperitoneal laparoscopic of unilateral adrenalectomy and contralateral subtotal adrenalectomy for the treatment of Cushing disease was safe and effective.The contralateral adrenal subtotal resection could be performed at an appropriate time in the case of that the clinical symptoms not obvious.By doing so,it could significantly alleviate the clinical symptoms as well as avoiding lifelong hormone replacement therapy.Once recurrence,residual adrenalectomy can be considered.

Objective To detect the red blood cell lifespan in patients with polycythemia vera (PV), and explore the influencing factors. Methods From February 2017 to December 2018, 27 patients with PV at Blood Diseases Hospital, Chinese Academy of Medical Science and 18 normal controls were recruited. Red blood cell lifespan was detected by endogenous carbon monoxide (CO) breath test. The related factors were analyzed. Results The average red blood cell lifespan of 27 PV patients was 80 (range, 35-120) days (d), which was significantly shorter than that of the normal controls [110.5(69-166) d, P<0.05], namely 35.3 d shorter. The red blood cell lifespan of ten newly diagnosed patients and 17 patients who were treated with hydroxyurea and/or interferon were 98 (35-117) d and 69 (45-120) d, respectively, which were both shorter than that of the normal control (P=0.010, 0.000). Correlation analysis showed that red blood cell lifespan of patients with newly diagnosed PV was associated with JAK2 mutation allele burden (r=0.900, P=0.037), peripheral blood lymphocyte count (r=-0.742, P=0.014) and the level of serum vitamin B12 (r=-0.821, P=0.023). Conclusion The lifespan of red blood cells in patients with PV is about one?third shorter than normal, and is related to JAK2 mutation allele burden, absolute lymphocyte count, and serum vitamin B12 level.

Objective: To evaluate the impact of the targeted next-generation sequencing (NGS) assay for difficult congenital anemias. Methods: Blood Disease Hospital Anemia Panel 2014 (BDHAP-2014) including 217 known genes of congenital anemias was developed. NGS and parental verification were performed for patients who were suspected diagnosed with congenital anaemia from August 2014 to July 2017. Results: A total of 46 patients were enrolled in this study, the clinical suspection were 11 cases Fanconi anemia (FA), 8 cases congenital dyserythropoietic anemia (CDA), 6 cases congenital sideroblast anemia (CSA), 12 cases congenital hemolytic anemia (CHA), 1 case dyskeratosis congenital (DC), 4 cases iron-refractory iron deficiency anemia and 4 cases unexplained cytopenia (Uc), respectively. 28 (60.9%) of 46 patients became confirmed cases after targeted NGS, corresponding to 44 mutations of which 33 were new. 26(56.5%) patients with results of the assay matching to clinical suspection, including FA (5/11, 45.5%), CSA (6/6, 100.0%), CDA (3/8, 37.5%) and CHA (12/12, 100.0%). 2 (4.3%) cases not matching to clinical suspection, including dyskeratosis congenital (DC) was made in 1(2.2%) patients with suspected FA and familial hemophagocytic lymphohistiocytosis (FHL) was made in 1(2.2%) patients with suspected unexplained cytopenia (Uc). In 12 CHA patients, the hemolytic type was further clarified by the NGS. The remaining 18 cases were not clearly diagnosed. Conclusion Targeted NGS assay is of major impact on congenital anemias. The assay should be used routinely in congenital anemias.

Objective To study the correlation between CD8 + T cells' balance and prognosis for patients with diffuse axonal injury(DAI).Methods To collect the 41 patients with DAI as observation group,with 33 cases light craniocerebral injury (LCI) patients and 35 healthy volunteers as control group.We detect the peripheral blood CD8 + CD28 + T cells and CD8 + CD28-T cell percentage,and calculate the ratio of both.The subjects working curve method (ROC) of.the above CD8 + T cells and its ratio were used to predict the prognosis of patients with DAI efficiency evaluation.Results (1) Compared with control group,CD8 + CD28 + T cells in patients with DAI and LCI increased significantly,and DAI was significantly higher than that of LCI group (P ＜ 0.05).Compared with control group,CD8 + CD28-T cells in patients with DAI were decreased,but no statistical difference was found between LCI and the control group,but DAI group was significantly lower than the LCI group (P ＜ 0.05).(2) CD8 + CD28-T cells and CD8 + CD28 +/CD8 + CD28-ratio were correlated with DAI diagnosis (P =0.003,0.000).When the percentage of CD8 + CD28-T cells =2.95％,the sensitivity of the diagnosis of DAI was 87.49％,86.21％;When the ratio of CD8 + CD28 +/CD8 + CD28-=7.39,the sensitivity was 92.63％,90.71％.(3) a total of 7 cases of patients died within 1 week after injury,CD8 + CD28 + T cells and CD8 + CD28 +/CD8 + CD28-ratio were significantly negative correlated with survival time (r =-0.739,-0.834,P =0.021,0.002),and CD8 + CD28-T cells were significantly positive correlated with survival time (r =0.782,P =0.006).Conclu sions Decreasing CD8 + CD28-T cells and higher CD8 + CD28 +/CD8 + CD28-ratio in patients with DAI immunological characteristics,especially the ratio (balance) is closely corrected with the diagnosis and prognosis of DAI.

Objective To study influencing factors in patients with rheumatic diseases.Methods One hundred and twenty patients with rheumatic diseases were selected from November 2013 to February 2014 in our hospital for the study.The influencing factors in patients with rheumatic diseases were obtained by proposed questionnaires,and then the univariate and multivariate Logistic regression analysis were performed to study factors affecting the treatment.Results Patients older than 50 of the observation group accounted for 38％ (18/48),the proportion of farmer,patients with primary school education level or below,and monthly income ＜3 000 RMB was 35％(17/48),60％(29/48),and 46％ (22/48) respectively.Compared with the control group,they were significantly higher than the control group,which were 64％(46/72),58％ (42/72),79％(57/72),65％(47/72) (x2=8.058,P=0.005; x2=6.025,P=0.014; x2=4.986,P=0.226; x2=4.456,P=0.035) respectively.Using the treatment as the dependent variable,age ≥50 years of age,farmer,primary school and lower education level,and monthly income ＜3 000 RMB as the independent variable,we carried out the regression analysis.Age (OR=1.124,95％CI:1.084-7.236),occupation (OR=1.871,95％CI:1.054-7.243),education level (OR=1.982,95％CI:1.157-6.256),monthly income (OR=1.363,95％CI:1.012-8.227) are the influencing factors of rheumatism's treatment.Conclusion The treatment of rheumatic diseases are influenced by many factors,and more societal support should be provided to help patients with rheumatic disease receive professional treatment and better control of the disease.