Background Subretinal transplantation of retinal pigment epithelium (RPE) cells for the treatment of age-related macular degeneration (AMD) have accelerated the drive to develop xeno-free cultivation system that support the rapid differentiation of human embryonic stem cells (hESCs) into ES-RPE cells.Objective This study was to report a modified xeno-free culture system and method for accelerating derivation of hESCs to differentiate into RPE cells.Methods This study was approved by Ethic Committee of Zhongshan Ophthalmic Center.HESC H1 line was cloned and cuhured in Vitronectin XFTM-coated 6-well dish with xenogenetic-free medium.Cells were cultured in 50 ng/ml noggin,10 ng/ml DKK-1 and 10 ng/ml insulin like growth factor-1 (IGF-1) medium for 2 days,and then the concentration of noggin was decreased to 10 ng/ml and 5 ng/ml basic fibroblast growth factor (bFGF) and cultured for the following 2 days.Sequentially,noggin and bFGF were removed and cultured for 2 days.Finally,1 μmol/L CHIR99021 was added in medium for 6 days.Morphological changes in the progress of ESCs differentiation into RPE were observed by Living Cell Imaging System.The expression of Mitf and RPE65,RPE cellsspecific markers,in the cells were detected by immunofluorescence technique,and the relative expression levels of RPE cells-specific marker mRNA were assayed using real time fluorescent quantitation PCR.Results Polygonalshape monolayer cells which contained pigments were initially observed at day 14 after cultured with the cobblestonelike arrangement.Mitf and RPE65 were strongly expressed in the hES-derived RPE cells 35 days after induced,showing red fluorescence,and the cells presented hexagonal shape at cultured day 60 with numerous pigment granules in cytoplasm.Compared with before differentiation,the expression levels of Mitf mRNA in hES-RPE cells increased by (3.43±2.77) folds and (8.91 ± 2.83) folds,and the expression levels of RPE65 mRNA increased by (14.60 ± 3.94) folds and (87.16 ±9.32) folds at day 7 and day 14 after differentiation,respectively (all at P＜0.05).Conclusions A defined xeno-free culture system is successfully established by adding niacinamide,DKK-l,noggin,IGF-1 and CHIR99021 in xeno-free medium,and this system can accelerate the derivation and differentiation of hESCs into RPE-like cells.

Objective To explore the effects of homogeneity management on the nursing quality in regional medical consortium. Methods A new nursing command system was established in the regional medical consortium. During October 2014 to October 2016, 45 union hospitals affiliated to Zhengzhou Central Hospital had carried out diversified training, unified culture construction and other measures to achieve homogeneity management, appling PDCA cycle management model all through. Ten nurses and ten patients were randomly selected from each department to conduct the regional medical consortium care status survey and patient satisfaction survey respectively. After two years intervention, the difference of nursing quality and patient satisfaction in the affiliated hospitals of the regional medical consortium were analyzed and compared. Results Compared with those who did not have homogeneity management, the qualified rate of first aid, the pass rate of ward management and patient satisfaction were significantly improved in the affiliated hospitals of the regional medical consortium, and the differences were statistically significant (P< 0.01). Conclusions Nursing homogeneity management can effectively improve the quality of nursing care in the affiliated hospitals of the regional medical consortium, ensure the safety of care and improve patient satisfaction.

Objective　To analyze clinical features of nerve injury caused bythe abuse of nitrous oxide.Methods　We retrospectively analyzed the clinical data of eight patients with nitrous oxide poisoning and summarized their clinical features.Results　Eight patients with nitrous oxide poisoning were all young and middle-aged with a history of exposure to nitrous oxide.Peripheral nervous system was impaired in eight patients,central nervous system was impaired in five patients,autonomic nerve was impaired in two patients,and two patient had neuropsychiatric symptoms.Vitamin B12 deficiency and homocysteine elevation were detected in eight patients.High doses of vitamin B12,methylcobalamin or adenosine cobalamin were effective.Conclusion　Long-term consumption of N2O can lead to megaloblastic anemia,nervous system injury and even fatal.

Objective:To explore the performance of the commonly used whole blood C-reactive protein (CRP) detection systems and give related recommendation on the performance requirements of detection systems.Methods:A total of 7 540 venous blood samples from 26 maternal, child and children′s hospitals were collected to conduct this multi-center study on the analytical performance of 5 commonly used whole blood CRP detection systems from March to April in 2019. The blank check, carryover, repeatability, intermediate precision, linearity, sample stability, influence of hematocrit/triglyceride/bilirubin, comparison with SIEMENS specific protein analyzer and trueness were evaluated. The 5 systems included BC-5390CRP autohematology analyzer, AstepPLUS specific protein analyzer, Ottoman-1000 Automated Specific Protein POCT Workstation, i-CHROMA Immunofluorometer equipment Reader and Orion QuikRead go detecting instrument. The 5 systems were labeled as a, b, c, d and e randomly.Results:Within the 5 systems, all values of blank check were less than 1.00 mg/L, the carryovers were lower than 1.00%. The repeatability of different ranges of CRP concentrations including 3.00-10.00, 10.00-30.00 and>30.00 mg/L were less than 10.00%, 6.00% and 5.00%, respectively, and the intermediate precision was less than 10.00%. The linearity correlation coefficients of the 5 systems were all above 0.975, while the slope was within 0.950-1.050. Whole blood samples were stable within 72 hours both at room temperature (18-25 ℃) and refrigerated temperature (2-8 ℃). The CRP results were rarely influenced by high triglyceride or bilirubin, except for the immmunoturbidimetric test based on microparticles coated with anti-human CRP F(ab) 2 fragments. When triglyceride was less than 15.46 mmol/L, the deviation of CRP was less than 10.00%. When bilirubin was less than 345.47 μmol/L, the deviation of CRP was less than 10.00%. CRP was more susceptible to Hct on the systems without Hct correction. The deviation of CRP between different Hct dilution concentration and 40% dilution concentration can reach as high as 67.48%. The correlation coefficients ( r) of 5 systems were all more than 0.975 in the range of 0-300.00 mg/L compared with Siemens specific protein analyzer. All systems passed the trueness verification using the samples with specified values of 12.89 and 30.60 mg/L. Conclusion:The performance of 5 systems can basically meet the clinical needs, but it is suggested that the whole blood CRP detection system without automatic Hct correction should be modified manually.

COVID-19 has swept globally and Pakistan is no exception.To investigate the initial introductions and transmissions of the SARS-CoV-2 in Pakistan,we performed the largest genomic epidemiology study of COVID-19 in Pakistan and generated 150 complete SARS-CoV-2 genome sequences from samples collected from March 16 to June 1,2020.We identified a total of 347 mutated positions,31 of which were over-represented in Pakistan.Meanwhile,we found over 1000 intra-host single-nucleotide variants(iSNVs).Several of them occurred concurrently,indicating possible interactions among them or coevolution.Some of the high-frequency iSNVs in Pakistan were not observed in the global population,suggesting strong purifying selections.The genomic epidemiology revealed five distinctive spreading clusters.The largest cluster consisted of 74 viruses which were derived from different geographic locations of Pakistan and formed a deep hierarchical structure,indicating an extensive and persistent nation-wide transmission of the virus that was probably attributed to a signature mutation(G8371T in ORF 1ab)of this cluster.Further-more,28 putative international introductions were identified,several of which are consistent with the epidemiological investigations.In all,this study has inferred the possible pathways of introduc-tions and transmissions of SARS-CoV-2 in Pakistan,which could aid ongoing and future viral surveillance and COVID-19 control.