Purification of cell culture-derived influenza A virus via continuous anion exchange chromatography on monoliths

The continuously increasing demand for potent and safe vaccines and the intensifying economic pressure on health care systems underlines the need for further optimization of vaccine manufacturing. Here, we focus on downstream processing of human influenza vaccines, investigating the purification of serum-free cell culture-derived influenza virus (A/PR/8/34 H1N1) using continuous chromatography. Therefore, quaternary amine anion exchange monoliths (CIM® QA) were characterized for their capacity to capture virus particles from animal cells cultivated in different media and their ability to separate virions from contaminating host cell proteins and DNA. The continuous chromatography was implemented as simulated moving bed chromatography (SMB) in a three zone open loop configuration with a detached high salt zone for regeneration.SMBs exploiting 10% and 50% of the monoliths’ dynamic binding capacity, respectively, allowed the depletion of >98% of the DNA and >52% of the total protein. Based on the hemagglutination assay (HA assay), the virus yield was higher at 10% capacity use (89% vs. 45%). Both SMB separations resulted in a ratio of total protein to hemagglutinin antigen (based on single radial diffusion assay, SRID assay) below the required levels for manufacturing of human vaccines (less than 100 µg of protein per virus strain per dose). The level of contaminating DNA was five-times lower for the 10% loading, but still exceeded the required limit for human vaccines. A subsequent Benzonase® treatment step, however, reduced the DNA contamination below 10 ng per dose. Coupled to continuous cultivations for virus propagation, the establishment of integrated processes for fully continuous production of vaccines seems to be feasible.     

On the photodegradation of azithromycin,erythromycin and tylosin and their transformation products – A kinetic study

In this study, the photo-induced degradation of azithromycin (Azi), erythromycin (Ery) and tylosin (Tyl) was investigated. The three macrolides are regularly found in different kinds of water, and are thus considered a potential environmental risk. In search of efficient ways for elimination, the compounds were systematically irradiated with a polychromatic UVC light source emitting 185, 254 and discretely further up to 580 nm. Due to their specific structural features, the macrolides possess different optical absorption characteristics leading to photodegradation pathways with dissimilar kinetic properties. Hence, the photodegradation products and their kinetics were analyzed using high-performance liquid chromatography (HPLC) coupled to high-resolution time-of-flight mass spectrometry. Among the degradants, i.e. the products formed during UV irradiation, both sugar moieties and lactone macrocycles were observed. Applying a first order kinetic model, the half-life of Azi was determined as 1.0?3.7 min, that of Ery as 1.0?14.2 min depending on the reaction conditions. The two compounds possess much lower absorption cross-sections than Tyl, in particular at 254 nm. Their half-lives appeared at least three times higher than that of Tyl that has t_1/2 of 0.2?1.0 min. Based on quantum efficiency considerations, it was assumed that the degradation of Ery and Azi was mainly due to hydroxyl radical formation, which originated from water irradiation below 200 nm, whereas Tyl experienced predominantly photo induced degradation. The photodegradants of Azi exhibited half-lives of over 200 min, whereas most of the photo-products of Ery and Tyl showed half-lives of less than 10 min. Photodegradation processes were investigated at pH 3, pH 7 and pH 9 and in the presence of hydrogen peroxide. Both the educts and the photo-products were degraded more rapidly under neutral and acid conditions.     

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??Pediatrics fulminant hepatic failure is a result of massive necrosis of liver cells in a short time?? which is a serious acute illness with impaired liver function. In recent years?? blood purification play a very important role in terms of the treatment of the disease. But the disease is prone to serious bleeding complications. Therefore??it is very important to conduct coagulation monitoring during blood purification     

人源瞬时受体电位M2型通道Nudix水解酶9同源结构域的提取和纯化

摸索人源瞬时受体电位M2型通道（TRPM2）羧基端Nudix水解酶9（NUDT9）同源结构域（NUDT9-H）的蛋白提取和纯化方法。 异丙基硫代半乳糖苷诱导表达的Rosetta（DE3）大肠埃希菌菌株经超声破碎和离心后，将收集到的上清液与GST磁珠结合并用还原型谷胱甘肽洗脱以抽提GST-NUDT9-H融合蛋白，浓缩离心后再经分子排阻色谱层析纯化，最后经凝血酶酶切并与GST磁珠结合即得NUDT9-H蛋白。 含有0.5%的十二烷基-β-D-麦芽糖苷（DDM）裂解溶液体系和0.025%的DDM分子排阻层析色谱溶液体系能够提高GST-NUDT9-H融合蛋白的稳定性，再按每2 mg融合蛋白加入1 U凝血酶切割24 h，即获得高纯度NUDT9-H蛋白。 NUDT9-H蛋白稳定性较差，提取和纯化体系中需添加DDM以稳定其构象从而提高目的蛋白的产量和纯度。      

Assessment of extravascular lung water by measuring the number of pulmonary ultrasound B-lines before and after CBP in patients with MODS

To determine whether the change in the number of pulmonary ultrasound B-line can accurately assess the extravascular lung water (EVLW) before and after continuous bedside blood purification (CBP) in patients with multiple organ dysfunction syndrome (MODS).Seventy-six patients with MODS who underwent CBP were examined within 24 hours before and after CBP using pulmonary ultrasound to detect the number of ultrasound B-line or using pulse indicator continuous cardiac output method to examine extravascular lung water, blood oxygenation index, and B-type natriuretic peptide (BNP) content. The correlation of the change in the number of B lines before and after CBP treatment with the negative balance of 24 hours liquid, the change of oxygenation index, and BNP content were analyzed.In the 76 patients, CBP treatment significantly decreased EVLW, the number of B-line, and BNP (P < .05 for all), while it significantly increased the oxygenation index (P < .05). Correlation analysis showed that the decrease in B-line number after CBP treatment was positively correlated with the 24 hours negative lung fluid balance, decrease of EVLW, oxygenation index improvement, and decreased BNP content. The change in the numbers of pulmonary ultrasound B-line can accurately assess the change of EVLW before and after CBP treatment and reflect the efficiency of ventilation in the lungs and the risk of heart failure.Thus, it can replace pulse indicator continuous cardiac output as an indicator for evaluating EVLW in patients with MODS treated with CBP.     

重症急性胰腺炎患者经血液净化治疗后的护理

目的：探讨临床上对经过血液净化治疗的重症急性胰腺炎患者实施细化护理的方法及措施，以求护理质量的持续改进。方法回顾性分析15例患者的临床资料，对其护理方案、相关要点进行疏理、归纳，加以总结。结果护理人员对具体患者的病情状况能够熟悉掌握、有据可循，对护理上的难点能够早期预见、适时应对，制定的护理方案切实可行，应用的护理措施趋于细化，使存在的问题得到了及时发现和有效干预，显现了预期效果，无一例护理并发症发生。结论细化护理能充分体现临床护理的个体化、动态化、多样化、精细化等特点，其护理措施更具针对性、缜密性、时效性和整体性，对促进护理质量的持续改进、护理效果的明显改观具有积极意义。     

豇豆胰蛋白酶抑制剂的大量纯化和急性毒性研究

目的用大肠杆菌大量表达豇豆胰蛋白酶抑制剂蛋白(CpTI)并检测其急性毒性。方法将重组表达载体pET41-CpTI转化大肠杆菌,用IPTG诱导目的蛋白表达,通过疏水层析和阴离子交换层析等色谱技术纯化大肠杆菌表达的CpTI蛋白。取60只ICR小鼠,随机分成6组,每组10只,雌雄各半,经口灌胃法分别一次性给予10.0、4.64、2.15和1.00g/kg BW的重组CpTI蛋白,并设5.00g/kg的BSA对照蛋白组和水空白对照组,连续观察14d。结果所有的小鼠在观察期间体重、脏器比重、进食量等均未出现显著变化,在15d大体解剖也未发现明显的病理变化。小鼠对CpTI蛋白的最大耐受剂量(MTD)应大于10.0g/kg BW。结论在该试验条件下,经口灌胃法给予ICR小鼠CpTI蛋白,未发现其毒性。     

Revolutionary Impact of Nanodrug Delivery on Neuroscience

Brain research is the most expanding interdisciplinary research that is using the state of the art techniques to overcome limitations in order to conduct more accurate and effective experiments. Drug delivery to the target site in the central nervous system (CNS) is one of the most difficult steps in neuroscience researches and therapies. Taking advantage of the nanoscale structure of neural cells (both neurons and glia); nanodrug delivery (second generation of biotechnological products) has a potential revolutionary impact into the basic understanding, visualization and therapeutic applications of neuroscience. Current review article firstly provides an overview of preparation and characterization, purification and separation, loading and delivering of nanodrugs. Different types of nanoparticle bioproducts and a number of methods for their fabrication and delivery systems including (carbon) nanotubes are explained. In the second part, neuroscience and nervous system drugs are deeply investigated. Different mechanisms in which nanoparticles enhance the uptake and clearance of molecules form cerebrospinal fluid (CSF) are discussed. The focus is on nanodrugs that are being used or have potential to improve neural researches, diagnosis and therapy of neurodegenerative disorders.     

New developments in lentiviral vector design,production and purification

Introduction: Lentiviruses are a very potent class of viral vectors for which there is presently a rapidly growing interest for a number of gene therapy. However, their construction, production and purification need to be performed according to state-of-the-art techniques in order to obtain sufficient quantities of high purity material of any usefulness and safety.

Areas covered: The recent advances in the field of recombinant lentivirus vector design, production and purification will be reviewed with an eye toward its utilization for gene therapy. Such a review should be helpful for the potential user of this technology.

Expert opinion: The principal hurdles toward the use of recombinant lentivirus as a gene therapy vector are the low titer at which it is produced as well as the difficulty to purify it at an acceptable level without degrading it. The recent advances in the bioproduction of this vector suggest these issues are about to be resolved, making the retrovirus gene therapy a mature technology.