Objective To study the level of umbilical plasma erythropoietin (EPO) in normal and abnormal pregnancy and analyze the influence factors of EPO. Methods EPO concentration in 106 umbilical plasma were measured by enzyme immonoassay. Samples were divided into four groups: control group with healthy full term newborns; hypertension group with neonates born to mothers with hypertension; diabetiec group with neonates born to mothers with diabetes; asphyxia group with neonates with fetal distress or asphyxia. The relationship between the concentration of EPO and gestational age, mode of delivery and pregnant complications were analysed. Results Mothers with hypertension, diabetes, fetal distress or neonatal asphyxia were important factors influencing umbilical blood EPO production ( P

Objective To assess the preventive effects of recombinant human erythropoietin (rHu EPO) on the anemia of premature birth infant. To determine whether prophylactic treatment with rHu EPO and iron would reduce the postnatal hemoglobin (Hb) decline and blood transfusion of premature infants. Methods Fifty one infants of less than 35 weeks of gestation and 2000 g of birth weight from multicenter were randomly assigned to EPO group ( n =31) and control group ( n =20). Infants in EPO group received rHuEPO 250 IU/(kg?t) intravenously or subcutaneously once every second day, started between 2 and 10 days of age, maintained for 4 weeks. Oral iron and vitamin E supplements were given to all infants. Hb, hematocrit (Hct), reticulocyte counts (Ret), serum iron and erythropoietin (EPO) were detected in both groups. Results Postnatal decline of Hb and Hct were less in EPO group than that in the control group at the end of study (129.9?21.0 vs 103.2?14.3, P

Recently, the droplet microfluidic system attracts interests due to its high throughput and low cost to detect and screen. The picoliter micro-droplets from droplet microfluidics are uniform with respect to the size and shape, and could be used as monodispensed micro-reactors for encapsulation and detection of single cell or its metabolites. Therefore, it is indispensable to characterize micro-droplet and its application from droplet microfluidic system. We first constructed the custom-designed droplet microfluidic system for generating micro-droplets, and then used the micro-droplets to encapsulate important amino acids such as glutamic acid, phenylalanine, tryptophan or tyrosine to test the droplets' properties, including the stability, diffusivity and bio-compatibility for investigating its application for amino acid detection and sorting. The custom-designed droplet microfluidic system could generate the uniformed micro-droplets with a controllable size between 20 to 50 microm. The micro-droplets could be stable for more than 20 h without cross-contamination or fusion each other. The throughput of detection and sorting of the system is about 600 micro-droplets per minute. This study provides a high-throughput platform for the analysis and screening of amino acid-producing microorganisms.
Amino Acids ; isolation & purification ; Microfluidic Analytical Techniques ; Microfluidics ; instrumentation

Objective:To observe the expression of the tumor suppressor gene (P53),apoptosis signal receptor (Fas),tumor necrosis factor alpha ( TNF-α) and Cyclin E ( Cyclin E) in serum and cancer tissues with papillary thyroid cancer patients ,and explore their relationship with the clinical pathology characteristics of thyroid papillary carcinoma .Methods:The puncture diagnosis in patients with papillary thyroid carcinoma as the experimental group (n=74),physical examination of healthy people as the normal control group (n=26).The two groups were fasting venous blood samples ,the experimental group in postoperative specimens from cancer tissue , adjacent normal tissue and 7 days after the fasting venous blood was sampled again.Protein content of P53,Fas,TNF-αand Cyclin E was detected by ELISA in serum , cancer adjacent normal tissue and cancer tissue;using real-time fluorescent quantitative assay to observe the gene expression of P53,Fas,TNF-αand Cyclin E in thyroid papillary carcinoma and adjacent normal tissues ; protein expression by immunohistochemical methods in papillary thyroid carcinoma and adjacent normal tissues P 53,Fas,TNF-αand Cyclin E analysis;the clinical expression with papillary thyroid cancer staging , pathological Type and has no relationship to lymph node metastasis.Results:The protein concentration in serum of patients with papillary thyroid carcinoma P 53 , Fas and TNF-αwere significantly lower than that of the normal control group ,Cyclin E protein content was significantly higher than that of normal control group ,the differences were statistically significant (P<0.01);thyroid papillary carcinoma P53,Fas and TNF-αprotein content,protein expression strength and gene expression levels were significantly lower than the normal tissues adjacent to cancer ,protein content ,Cyclin E protein expression and gene expression intensity was significantly higher than that in normal tissues ,the difference was statistically significant (P<0.05).Conclusion:The expression of p53,Fas and TNF-αin papillary thyroid carcinoma lower and expression level of Cyclin E increase ,may play an important role in papillary thyroid cancer invasion and metastasis.Combined detection of the four can be used as markers for early diagnosis of papillary thyroid cancer ,enhance the rate of early diagnosis of thyroid papillary carcinoma.

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 μm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 μm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.
Emulsions ; Flow Cytometry ; High-Throughput Screening Assays ; Microfluidics ; methods

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 μL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.
Microfluidics ; Mutagenesis ; Pichia ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins

【Objective】 To explore the application value of different language assessment tools in the assessment of language development of 12-month-old high-risk infants, and to screen out simple and valid language assessment tools. 【Methods】 A total of 217 11- to 13-month-old high-risk infants who were followed up at the outpatient service for high-risk infants at the child health clinic of Guiyang Maternal and Child Health Hospital from March 2022 to May 2023 were selected as the study subjects. Their language was evaluated by Early Language Milestone Scale (ELMS), Putonghua Communicative Development Inventory (PCDI) and Ages and Stages Questionnaire-Third Edition (ASQ-3). With Gesell as the gold standard for the assessment of language, the area under receiver operating characteristic curve (AUC), sensitivity, specificity, accuracy, positive predictive value, negative predictive value, Youden index and Kappa value of the three tools were calculated. Spearman correlation analysis was used to analyze the correlation between the different language assessment scales. The Technique for Order Preference by Similarity to Ideal Solution (TOPSIS) was used to evaluate the three tools. 【Results】 1)Among 217 high-risk infants, 78 preterm infants was the most (35.94%). The rate of delayed language development detected by Gesell, ELMS, PCDI-comprehension, PCDI-expression, ASQ-3 were 5.5%, 7.8%, 36.4%, 30.0% and 11.5%, respectively. 2)ASQ-3 had the strongest correlation with Gesell language region (rs=0.607, P<0.01) and it had the most AUC (AUC=0.812, P<0.05). The consistency between ASQ-3 and Gesell was moderate (Kappa=0.56, P<0.01). ASQ-3 had the highest sensitivity(91.7%), accuracy(93.1%), negative predictive value(99.5%) and Youden index(0.85), and ELMS had the highest specificity(94.6%). 3) Comprehensive evaluation of three tools by the TOPSIS indicated that ASQ-3 was the best, followed by ELMS and PCDI-comprehension was the worse. 【Conclusion】 Among the three assessment tools, ASQ-3 has the highest value in assessing the language development of 11- to 13-month-old high risk infants, and it may be necessary to expand the age range and establish a national norm of ELMS and PCDI in the future.

Objective:To summarize the patient profiles and therapeutic efficacies of ABO-incompatible living-related kidney transplantations at 19 domestic transplant centers and provide rationales for clinical application of ABOi-KT.Methods:Clinical cases of ABO-incompatible/compatible kidney transplantation (ABOi-KT/ABOc-KT) from December 2006 to December 2009 were collected. Then, statistical analyses were conducted from the aspects of tissue matching, perioperative managements, complications and survival rates of renal allograft or recipients.Results:Clinical data of 342 ABOi-KT and 779 ABOc-KT indicated that (1) no inter-group differences existed in age, body mass index (BMI), donor-recipient relationship or waiting time of pre-operative dialysis; (2) ABO blood type: blood type O recipients had the longest waiting list and transplantations from blood type A to blood type O accounted for the largest proportion; (3) HLA matching: no statistical significance existed in mismatch rate or positive rate of PRA I/II between two types of surgery; (4) CD20 should be properly used on the basis of different phrases; (5) hemorrhage was a common complication during an early postoperative period and microthrombosis appeared later; (6) no difference existed in postoperative incidence of complications or survival rate of renal allograft and recipients at 1/3/5/10 years between ABOi-KT and ABOc-KT. The acute rejection rate and serum creatinine levels of ABOi-KT recipients were comparable to those of ABOc-KT recipients within 1 year.Conclusions:ABOi-KT is both safe and effective so that it may be applied at all transplant centers as needed.