A review of studies on the influence of impurities on protein crystallization is given.The possible sources of impurities and its effect on the protein crystallization are presented.The effects of impurities on protein crystallization,including nucleation,macroscopic morphologies,microscopic surface morphologies,growth rates,kinetics,quality,and repartitioning of impurities are reviewed.

Abstract: Objective　To explore the antiviral effect of baricitinib in the SARS-CoV-2 infection and influence on cytokine levels. Methods　Calu-3 cells were infected with SARS-CoV-2 at MOI of 0.1, and the levels of inflammatory cytokines (IL-6, IL-8, TNF-α and IL-1β), interferon β (IFN-β) and interferon-stimulated gene, IFIT2 in the infected cells were analyzed by qRT-PCR methods. At the same time, Calu-3 cells were infected with SARS-CoV-2 (MOI=0.1) after being treated with baricitinib for 2 hours. Cells were collected at 0, 24, 36, and 48 hours, and analyzed for the mRNA of the above genes in the drug-treated and untreated groups. Results　The mRNA levels of IL-6, TNF-a, IL-1β, IFN-β and IFIT2 in Calu-3 infected by SARS-CoV-2 cells were increased significantly. These cytokines were increased by nearly 100-fold post-infection 48 h compared with the control (P<0.000 1), and continued to increase with the infection time (P<0.001 or P<0.000 1). The increase of IL-8 mRNA level was not as significant as IL-6, TNF-α, IL-8, IL-1β, but it also showed a 2-4 folds increase. Baricitinib does not affect the level of viral RNA in Calu-3 cells after SARS-CoV-2 infection (P>0.05). However, baricitinib can significantly inhibit the up-regulation of IL-6 and TNF-α levels induced by SARS-CoV-2 infection (5.25-fold and 3.90-fold down-regulation, respectively, P<0.01), and has little effect on the levels of IL-8 and IL-1β . In addition, the drug could also significantly down-regulate the increase in IFN-β and IFIT2 levels caused by viral infection (10.51-fold and 90.78-fold down-regulation, respectively, P<0.000 1). Conclusions　Baricitinib inhibits the release of inflammatory cytokines to some extent, but it drastically down-regulates the expression of interferons and interferon-stimulated genes (ISGs), and has limited antiviral effect on SARS-CoV-2. Considering that interferon signal pathways play important roles on viral infection, caution should be exercised when using baricitinib to treat COVID-19 patients.

BACKGROUND:Mouse pluripotent stem cel s are induced to differentiate into insulin-secreting cel s that can effectively improve blood glucose levels in diabetic mice. OBJECTIVE:To detect mRNA and protein levels of insulin-like cel clusters from induced pluripotent stem cel s and to investigate the function of insulin-secreting cel s in vitro and in vivo. METHODS:Mouse induced pluripotent stem cel s cultured in vitro were induced to differentiate into insulin-secreting cel s using combined inducers through three stages. The morphology of endodermal cel s, islet-derived progenitor cel s and mature islet cel s in each stage was observed and relative gene expression levels were detected by PCR. Mature insulin-like cel clusters underwent dithizone staining and functions of insulin released in vitro were observed by ELISA assay. Final y, the insulin-secreting cel s were transplanted into the subrenal capsule of diabetic mice, and then blood glucose levels were observed. RESULTS AND CONCLUSION:The mature spherical insulin-like cel clusters were successful y obtained in vitro, which were in iron red by dithizone staining, and expression of insulin mRNA was determined by PCR. The insulin-like cel clusters could secrete insulin in response to various blood glucose levels by ELISA assay. In addition, after the cel s clusters were transplanted into the subrenal capsule of mice with type 1 diabetes, the blood glucose levels were marbedly improved.

AIM: To look for harmfulless anti-leukemia drug with selective high performance, lethal effect of small hairpin RNA (shRNA) on VEGFR2 gene expression of tumor cell line HL60 in vitro.METHODS: The most effective VEGFR2 siRNA was designed and screened. The shRNA oligo was designed and pU6/VEGFR2 entry clone was constructed. HL60 was transfected transiently and vascular endothelial growth factor receptor 2(VEGFR2) expression was tested with MTT assay, RT-PCR and Western blotting. The expression clone was constructed and cotransfected with ViraPowerTM Packaging Mix into 293FTTM cells to produce Lentiviral vectors harboring Lenti6/shVEGFR2. The virion supernatant was added into HL60 cells and VEGFR2 gene inhibitory effect was determined. RESULTS: The inhibitory rates of VEGFR2 siRNA c were high. VEGFR2 expression in HL60 was inhibited by using pU6/VEGFR2 entry clone constructed with shRNA and pENTRTM/U6. For HL60 cells, the inhibitory rate was 84.9%. The expression of VEGFR2 mRNA and protein decreased significantly. 48 hours after transfection of pU6/shVEGFR2 entry clone and transduction of Lenti6/shVEGFR2 expression clone, the cell inhibitory rates were similar. Cell growth inhibitory rate of entry clone descended rapidly after this time point, the expression clone changed slowly, reaching the peak at 96 hours, dropped slightly, having no significance deviation. CONCLUSION: in vitro, VEGFR2 shRNA using lentiviral vector blocks VEGF/VEGFR2 self-secretion in HL60 cells, which inhibits leukemia development.

AIM:Xaf1-Saos inducible cell lines,which contain "gene switch" system were used to detect the effect of Xaf1 on tumor necrosis factor receptor(TNFR) signal pathway and to investigate the mechanism of cooperation between Xaf1 and TNF-? in inducing cell apoptosis.METHODS:Xaf1 on TNFR1 expression was measured by RT-PCR and Western blotting.The effect of NF-?B on Xaf1 induced apoptosis was detected by DNA content flow cytometry after co-transfection.DNA binding activity of NF-?B was identified by gel mobility shift assay and transcription activity of NF-?B was analyzed by luciferase assay and RT-PCR.SAPK/JNK activity was checked by SAPK/JNK assay.RESULTS:Xaf1 did not modulate TNFR1 at protein and mRNA levels.Increased NF-?B activity in cells inhibited Xaf1 induced apoptosis.Expression of Xaf1 impaired modestly TNF-? induced NF-?B DNA binding activation and transcription activation,also modestly reduced SAPK/JNK activity.CONCLUSION:Xaf1 inhibits TNFR signal pathway,partly contributing to cooperation with TNF-? to induce apoptosis.

Objective To investigate the incidence of and clinical factors influencing neonatal birth defects from different assisted reproductive technology. Methods Between October 1998 and December 2006,1271 newborns from mothers treated by in vitro fertilization techniques [ including in vitro fertilization (IVF), intracytoplasmic sperm injection (1CSI) and thaw embryo transfer (Thaw-ET) ] matched with 269 newborns from mothers treated by artificial insemination were enrolled in Reproductive Medicine Center in First Hospital Affiliated to Zhengzhou University. Their medical information was analyzed retrospectively to compared neonatal characteristics, the incidence of birth defect and anomalous organs involved between in vitro fertilization group and artificial insemination group. Results In group of in vitro fertilization, those newborns with low birth weight from IVF, ICSI and Thaw-ET were 20. 0% ( 134/671 ), 22. 4% (92/410), 18.9% (36/190)respectively, which were more than 11.5% (31/269) cases in group of artifical semination with statistical significance (P < 0. 05 ). The rates of multiple pregnancy of 23.8% ( 160/671 ), 25.4% (104/410) ,21.1% (40/190) in subgroup of 1VF, ICSI and Thaw-ET were significantly higher than 10. 0% ( 27/269 ) in group of artifical insemination( P < 0. 05 ). The rate of macrosomia in group of in vitro fertilization was significantly lower than that of artificial insemination group (3.9% vs 8. 2%, P <0.05). However, the incidence of birth defect involved in various organs did not show significant difference between two groups ( P>0.05 ). Conclusions The incidence of multiple pregnancy demonstrated obviously increasing trends born with various In Vitro Fertilization techniques, which pave the way to high risk pregnancy. However, the incidence of newborn birth defect was not increased significantly. Thus, to lower occurrence of multiple pregnancy was the key approach to obtain neonates health.

Objective To evaluate the feasibility of assessing osteoarthritis (OA) in hip dysplasia using 3D delayed gadolinium-enhanced MRI of cartilage (dGEMRIC).Methods Thirty-five hips in 20 patients with radiographic evidence of hip dysplasia underwent 3D-dGEMRIC scanning.Clinical symptoms were assessed with the Western Ontario and McMaster Universities Osteoarthritis ( WOMAC ) questionnaire.Radiographic measurement of lateral center-edge angle and T(o)nnis grading were performed on the X-rays.Hips of T(o)nnis grade 1were included in the group of hips with early OA,while the hips with no evidence of OA and without pain symptom were included in the group of hips with normal morphology.The 3D-dGEMRC scans were completed on a 1.5 T MR scanner.The data of 3D-dGEMRIC was reconstructed radically.The dGEMRIC indices were measured on six sites of periphery zones of hip cartilage on reconstructed images.The dGEMRIC indices among different groups were analyzed by non-parametric tests.The differences of dGEMRIC indices among six sites in the group of early OA or the group of normal morphology were analyzed by Wilcoxon test.Results The mean dGEMRIC indices of six sites were lower in group of T(o)nnis grade 1than in group of T(o)nnis grade 0 ( Z =- 2.149,P =0.032 ),and lower in group of T(o)nnis grade 2 than in group of T(o)nnis grade 1( Z =- 1.990,P =0.047 ).The dGEMRIC indices of the anterior site,anterosuperior site,superior-anterior site,and superior site were significantly different between the group of hips with early OA and the group of hips with normal morphology (Z =-2.333--2.041,all of the P values were lower than 0.05).In the group of hips with normal morphology,the dGEMRIC indices of superior-anterior site of hip were lower than superior site(P =0.028).In the group of hips with early OA,the dGEMRIC indices of superior-anterior site were lower than the other sites except for anterior-superior site ( Z =- 3.041- - 2.277,all of the P values were lower than 0.05 ).Conclusions 3 D-dGEMRIC might be a sensitive technique for detection of glycosaminoglycans alteration in early OA and staging of OA in hip dysplasia.Radial reconstruction could provide an accurate assessment of OA,and the results demonstrated that early cartilage alteration could be detected in the anterior to superior sites of hips,and the earliest cartilage alteration may occur in the superior-anterior site of hips.

Objective To discuss the significance of near infrared spectroscopy (NIRS) in evaluation of intrapartum hypoxic-ischemic cerebral injury, and to provide a method to evaluate neonatal brain damage objectively and quantitatively. Methods A total of 63 neonates with fetal distress were divided into hypoxic-ischemic encephalopathy(HIE) group and non-HIE group. Thirtyfive newborns with no fetal distress were chosen as controls. Using NIRS, the brain regional oxygen saturation(rSO2) in these neonates were measured. Evaluation of brain rSO2 in the diagnosis of HIE was analyzed with receiver operating characteristic (ROC) curve. Results At the time of fetal head visible on vulval gapping and 5 min after birth, the HIE group showed decreased brain rSO2[(36. 6±5.0)% and (52. 0±4. 2)%], comparing with control group[(45. 9±4. 6)% and (59. 6±4. 4)%]and non-HIE group[(44.1±3.1) % and (57. 6±3. 5) %](P<0. 01) . The brain rSO2 was positively correlated with the pH and oxygen saturation of umbilical artery blood in all groups (P<0. 01). When the cut-off value of brain rSO2 was <39. 5% at fetal head visible on vulval gapping, the sensitivity and specificity of assessing HIE were 67% and 93%, respectively, while 70% and 86% when the cut-off value was <53. 5% at 5 min after birth. Conclusions The brain rSO2 obtained by NIRS could be used to evaluate brain oxygenation, and may be useful in predicting HIE in neonates with fetal distress.

Objective To investigate the expression and resistant gene of integron in multidrug-resistant Acinetobacter baumannii (MDR-Ab).Methods 51 strains of MDR-Ab isolated from a hospital in August-October 2012 were collected, antimicrobial susceptibility testing was performed.Class I(Int I),II (Int II)and III (Int III)of integrase genes and inte-gron variable region gene cassettes were detected by polymerase chain reaction (PCR),and the homology of integron varia-ble region was analyzed by detection results of restriction fragment length polymorphism (RFLP)and DNA sequencing. Results Positive rate of integrase gene in MDR-Ab was 78.43%(40/51).All genes belonged to Int I,while IntⅡand IntⅢ were not found.Variable region cassettes were detected in 97.50% (n=39)of Int I,there were 5 types of integron gene cassettes:aacA4 in 14 strains,aacA4+catB8 in 22 strains,arr-3 +aacA4 in 1 strain,dfrA15 in 1 strain and arr-3 in 1 strain.Conclusion MDR-Ab isolated from this hospital may be related with Int I expression.Int I carried gene cassettes as follows:aacA4,aacA4+catB8,arr-3+aacA4,dfrA15 and arr-3.

According to the characteristics of sludge samples from full-scale wastewater treatment bioreac-tors, the essential total DNA extraction method for most environmental samples, lysozyme-SDS-phenol/ chloroform method, was modified to improve sample pretreatment, intensify cell lysis and enhance the effi-ciency of impurity removal. Obtain a general total DNA extraction method for industrial sludge samples. Such a method was applied for total DNA extraction of sludge samples from several running full-scale an- aerobic or aerobic bioreactors in Shijiazhuang, China. The results indicated that the modified method was suitable for all the sludge samples in this study, showing the satisfying generality. The extracted total DNA of all sludge samples were pure, with about 1.8 of A260/ A280 ratio. The method was also efficient; with average total DNA yield of over 0.7 mg/g and maximum yield of 0.85 mg/g. Moreover, all the extracted to- tal DNA samples could serve as templates directly to amplify 16S rDNA by PCR. The PCR products could be separated well by denaturing gradient gel electrophoresis (DGGE) and the DGGE band patterns were clear enough to be used for further analysis. All these facts indicated that the total DNA extraction method provided in this study could meet the requirements of sludge samples research, from full-scale wastewater treatment bioreactors, using molecular biology technologies.