Objective To prepare human Fab fragment antibodies against Interleukin-2 and identify their antigenic specificity and combining activities with antigens. Methods The specific Anti-interleukin-2 clones were screened from a natural human Fab fragment antibodies phage display library against the immobilized interleukin-2 antigens. Then the phagemid DNA from the specific clones was digested with Spe I and Nhe I to delete gⅢ (about 660bp). The digested 4.7kb DNA, which was purified after separation of bands from agarose gel using purification kit, was ligated with T4-DNA ligase and the ligated reaction mixture were transformed to the BL21 (DE3) pLysS. Positive clones on the LB agar plates were inoculated to liquid LB culture medium, and when the bacteria were grown to OD600≈0.5 at 37℃ with continuous shaking, IPTG was added to induce the expression of soluble Fab fragment antibodies at 30℃. The expressed products containing Fab fragment antibodies were determined by SDS-PAGE, Western blot and ELISA. Results The soluble products were identified as containing human Fab fragment antibodies against Interleukin-2 by Western blot and formed a Mr 47?103 band under non-reducing condition on SDS-PAGE. The band was then proved as anti-human Fab fragment antibodies by Western blotting. ELISA demonstrated that Fab fragments possessed good antigenic specificity as well as excellent combining activity with interleukin-2 antigens, and the fragments did not react with bovine serum albumin and IL-4 in ELISA. Conclusions The soluble human anti-interleukin-2 Fab fragment antibodies have been highly expressed and successfully identified, and an effective way has been searched out for constructing the engineering antibodies. All of the results may lay a potentially good foundation for engineering human Fab antibodies, and for the clinical application of the antibodies on the immunotherapy of tumor diseases.

Based on analysis of researches in China and abroad on financial burden of hospital acquired infection,the paper named key setbacks in such a study in China and advocated prospective studies in this regard.Furthermore,the authors proposed studies on the financial burden incurred by years of life lost due to such infection,and that on the burden from the hospital perspectives.On the basis of direct and indirect financial burdens,the assessment and dynamic analysis of overall financial burden were proposed,to establish a uniform evaluation method or guidance for such infection and ensure the compatibility of various research outcomes.

Acute pancreatitis (AP) is an acute inflammatory disease of various severity, characterized by upper abdominal pain, elevated pancreatic enzymes, and changes in imaging features of the pancreas. According to the degree of pancreatic injury and the presence and duration of systemic organ failure, AP is classified into mild, moderate, or severe disease. Most AP patients experience mild disease and recover quickly, while up to 20% progress to moderate or severe disease, with an estimated risk of death as high as 30%. Severe acute pancreatitis (SAP) is a clinical emergency with a critical condition and poor prognosis, especially in patients with pancreatic and/or peripancreatic tissue infection and necrosis. AP is essentially an inflammatory process that can lead to protein catabolism and increased metabolic rates, further resulting in negative nitrogen balance. The goal of nutritional support therapy for AP is to correct negative nitrogen balance, reduce inflammation, and improve prognosis. Enteral nutrition therapy is an important component of clinical treatment of SAP. This review aims to summarize the nutritional support treatment in AP based on the existing clinical data and experience.

Objective To explore potential genes associated with the pathogenesis of hypospadias using weighted Gene co-expression network analysis(WGCNA).Methods The WGCNA algorithm was used to process the hypospadias-related dataset GSE35034,and then a gene co-expression weight network was constructed to screen the modules with the highest correlation with hypospadias,and the genes in the modules were enriched and detected by gene ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG).Differential analysis was also performed to screen out differential genes.The differential genes were imported into the String database.Using Cytoscape software,the hub genes in the network were identified.The results screened by the above three methods were combined and analyzed,and the core genes in the intersection set were screened.Using the external dataset GSE121712,the core genes were verified by mRNA expression changes and subject work characterization receiver operating characteristic(ROC)curve diagnosis.Results Fifteen co-expression modules were obtained based on the WGCNA method.Ninety-three common differential genes meeting the conditions were obtained by differential analysis.Ten core genes were finally obtained by Cytoscape software analysis.Finally MEbrown module,differential genes and the 10 core genes yielded a total of 2 intersecting genes:FBXL16 and SYNDIG1.ROC curves verified that the intersecting genes were differentially expressed in patients with hypospadias versus normal subjects.Conclusion In this study,two key genes with significant correlation with hypospadias were obtained by WGCNA,which may be used for the early diagnosis and treatment of hypospadias after further study.

Objective To optimize the method of assay for silver sulfadiazine, and to investigate its stability in high temperature, humidity and salt environment. Methods Agilent ZORBAX SB-C₁₈ column was used in HPLC for the determination of silver sulfadiazine with acetonitrile and 0.1% phosphoric acid as mobile phase.The flowing rate was 1.0 ml/min and column temperature was 30℃. Salt spray test chamber was used to simulate the high temperature, humidity and salt environment. The silver sulfadiazine content was assayed at 0, 2, 4, 6, 8, 10, 12 weeks. Results The calibration curve was linear within the range of 4.00～20.00 μg/ml (r=0.9999). The average recovery was 101.47% (RSD=2.33%). The content of silver sulfadiazine cream began to decrease significantly after 4 weeks. Conclusion This method is convenient, accurate and reliable. It can be used for the content determination of silver sulfadiazine in the cream.The results showed that the silver sulfadiazine cream was unstable in the environment of high temperature, humidity and salt. Therefore, environmental impact should be fully considered in transportation, storage and application. For the long-distance navigation mission, protective measures should be taken for its packaging or replace it with more stable products.

China produces about 15 000 tons of expired drugs every year. Standardized destruction of expired drugs consumes a large number of secondary costs， causing huge economic losses. Improper handling will also lead to serious environmental pollution， endanger national health and even endanger public safety. This paper analyzes the hazards， sources and disposal methods， and research status of expired drugs， introduces the Shelf-Life Extension Program of United States， and put forward feasible measures for the recovery， integration and reuse of expired drugs. It is suggested to construct a scientific and reasonable recovery and disposal system of expired drugs in combination with the actual situation in China， explore the strategy of extending the expiration of drugs， and greatly reduce the waste of drug resources.

Porcine epidemic diarrhea virus (PEDV) inhibits the host typeⅠinterferon and cellular antiviral response, but its inhibition mechanism is unclear, and the roles of PEDV nonstructural proteins in regulating typeⅠinterferon responses have been seldom studied. To study the effect of nsp1 on typeⅠinterferon response, nsp1 gene was cloned into a eukaryotic expression vector pCAGGS. The expression of nsp1 in transfected cells was determined by Western blot and indirect immunofluorescence assay. The effects of nsp1 on the induction of typeⅠinterferon were evaluated by dual luciferase reporter gene assay, ELISA and VSV bioassay. Western blot and indirect immunofluorescence assay showed that nsp1 was highly expressed in transfected cells and PEDV-infected cells. Dual luciferase reporter gene assay results indicated that nsp1 strongly inhibited the IFN-β promoter activity, and the inhibitory effect was nsp1 dose-dependent. ELISA results showed that nsp1 significantly inhibited the expression of IFN-β in protein level. And VSV replication-inhibition bioassay revealed that nsp1 significantly inhibited typeⅠIFN antiviral activities induced by poly(I:C). Our results implied that nsp1 was a highly conserved protein of PEDV and exhibited antagonistic function on interferon promoter activity. The results have laid a foundation for further understanding the immune evasion mechanism of PEDV and for developing new effective vaccine against PEDV.

ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） of Qingxin Lianziyin（QXLZY） benchmark samples， in order to clarify the key quality attributes and provide a reference for the quality evaluation of QXLZY. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of QXLZY benchmark samples was developed by using a YMC Hydrosphere C₁₈ column（4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-10 min， 5%-20%A； 10-20 min， 20%A； 20-25 min， 20%-24%A； 25-40 min， 24%-30%A； 40-55 min， 30%-50%A； 55-65 min， 50%-100%A； 65-75 min， 100%A； 75-75.1 min， 100%-5%A； 75.1-90 min， 5%A）， and the detection wavelength was 360 nm. Ultra-high performance liquid chromatography-linear ion trap/orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） with electrospray ionization（ESI） was used to identify the components of QXLZY benchmark samples by accurate relative molecular weight and multilevel MS fragment ion information， the detection conditions were positive and negative ion modes and data dependency scanning mode. TLC identification methods for Ophiopogonis Radix， Lycii Cortex， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma in QXLZY were established. ResultA total of 15 characteristic peaks were identified from Glycyrrhizae Radix et Rhizoma， Plantaginis Semen and Scutellariae Radix， and the relative standard deviations of the retention times of 15 characteristic peaks in 15 batches of QXLZY benchmark samples were≤3% with peak 8（baicalin） as the reference peak. A total of 100 compounds， including flavonoids， organic acids， saponins， amino acids and others， were identified in the benchmark samples by UHPLC-LTQ-Orbitrap MS. The established TLC had good separation and was suitable for the identification of Ophiopogonis Radix， Lycii Cortex， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma in QXLZY. ConclusionThe material basis of QXLZY benchmark samples is basically determined by MS designation and source attribution. The established specific chromatogram and TLC of QXLZY are simple， stable and reproducible， which can provide a reference for the development and quality control of QXLZY.

ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） identification method of Kaixinsan（KXS） samples， in order to clarify the key quality attributes and provide reference for the quality evaluation of KXS. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of KXS was developed with YMC Hydrosphere C₁₈ column（4.6 mm×250 mm， 5 μm）， the mobile phase was acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-15 min， 2%-20%A； 15-25 min， 20%-25%A； 25-30 min， 25%-30%A； 30-45 min， 30%-31%A； 45-50 min， 31%-44%A； 50-65 min， 44%-45%A； 65-73 min， 45%-75%A； 73-95 min， 75%-100%A； 95-105 min， 100%A； 105-105.1 min， 100%-2%A； 105.1-120 min， 2%A）， the detection wavelength was 320 nm. Ultra high performance liquid chromatography-linear ion trap-electrostatic field orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） was used to identify the chemical components of KXS with electrospray ionization（ESI）， negative ion mode and scanning range of m/z 50-2 000. TLC identification methods for Poria and Ginseng Radix et Rhizoma in KXS were established. ResultThere were 11 common peaks in the specific chromatogram of KXS， attributed to Polygalae Radix， Poria and Acori Tatarinowii Rhizoma. Taking peak 9（α-asarone） as the reference peak， the relative standard deviations of the retention times of 15 batches of KXS samples were<0.2%. A total of 34 compounds were identified by UHPLC-LTQ-Orbitrap MS， including terpenoids， phenylpropanoids， oligosaccharides and ketones. The established TLC had good separation and was rapid， reliable， simple， feasible， suitable for the identification of Poria and Ginseng Radix et Rhizoma in KXS. ConclusionThe specific chromatogram and TLC of KXS are stable and reproducible. The material basis of KXS is basically clarified by MS， which can provide a reference for the development and quality control of KXS.