Brain-derived neurotrophic factor induces neuron-like cellular differentiation of mesenchymal stem cells derived from human umbilical cord blood cells in vitro

Human umbilical cord blood was collected from full-term deliveries scheduled for cesarean section. Mononuclear cells were isolated, amplified and induced as mesenchymal stem cells. Isolated mesenchymal stem cells tested positive for the marker CD29, CD44 and CD105 and negative for typical hematopoietic and endothelial markers. Following treatment with neural induction medium containing brain-derived neurotrophic factor for 7 days, the adherent cells exhibited neuron-like cellular morphology. Immunohistochemical staining and reverse transcription-PCR revealed that the induced mesenchymal stem cells expressed the markers for neuron-specific enolase and neurofilament. The results demonstrated that human umbilical cord blood-derived mesenchymal stem cells can differentiate into neuron-like cells induced by brain-derived neurotrophic factor in vitro.

The potential of ionic liquids in biopharmaceutical engineering

Biopharmaceuticals,such as proteins,peptides,nucleic acids and vaccines,bring about great hopes for the prevention and treatment of various diseases,but the industrialization of these products still faces challenges such as structural instability,inefficient bioactivity and low bioavailability.Ionic liquids(ILs),the marvelous solvent media with inimitable and tunable properties,may provide alternative solutions to overcome the above problems of biopharmaceutical industry.Progress has gradually been made through studies by combination of ILs with biomacromolecules.The applications involved the stabilization,protection,and delivery of biopharmaceuticals.Recent trends are being forwarded to using ILs in vaccines and nucleic acid drugs.However,challenges remain on the toxicity and safety issues.Besides,the cost of adding ILs to the benefits of biopharmaceuticals need to be considered.

Impact of wastewater treatment plant effluent discharge on the antibiotic resistome in downstream aquatic environments:a mini review

Antimicrobial resistance(AMR)has emerged as a significant challenge in human health.Wastewater treatment plants(WWTPs),acting as a link between human activities and the environment,create ideal conditions for the selection and spread of antibiotic resistance genes(ARGs)and antibioticresistant bacteria(ARB).Unfortunately,current treatment processes are ineffective in removing ARGs,resulting in the release of large quantities of ARB and ARGs into the aquatic environment through WWTP effluents.This,in turn,leads to their dispersion and potential transmission to human through water and the food chain.To safeguard human and environmental health,it is crucial to comprehend the mechanisms by which WWTP effluent discharge influences the distribution and diffusion of ARGs in downstream waterbodies.In this study,we examine the latest researches on the antibiotic resistome in various waterbodies that have been exposed to WWTP effluent,highlighting the key influencing mechanisms.Furthermore,recommendations for future research and management strategies to control the dissemination of ARGs from WWTPs to the environment are provided,with the aim to achieve the“One Health”objective.

Positional assembly of multi-enzyme cascade reaction in polyelectrolyte doped microcapsule through electrospray and layer-by-layer assembly

Polyelectrolyte-doped microcapsules(PDM)was fabricated by coaxial electrospray of a mixture of glycerol and water containing 10 mg/mL cationic polyelectrolyte poly(allylamine hydrochloride)(PAH)fed as the core phase solution,and a N,N-dimethylacetylamide solution of 10 wt%polyurethane fed as the shell phase solution.Multienzyme system involving Candida Antarctica lipase B(CALB),glucose oxidase(GOD),and horseradish peroxidase(HRP)for cascade reaction was assembled in the PDM at three different places,namely,surface,shell,and lumen.Placing of enzyme inside aqueous lumen of the PDM was realized by in situ encapsulation through adding the enzyme in the core-phase solution for coaxial electrospray.By ion-pairing of enzyme with cationic surfactant CTAB,an organic soluble enzyme-CTAB complex was prepared,so that in situ embedding of enzyme in the shell of the PDM was realized by adding it into the shell phase solution.Surface attachment of enzymes was achieved by layer-by-layer(LbL)technology,which is based on the ion-exchange interactions between oppositely charged enzymes and PAH that was doped in PDM.The enzyme-decorated microcapsule was then studied as a microbioreactor,in which 1-Oxododecyla-α-glucopyranoside was converted by CALB to glucose,which was oxidised by GOD to gluconolactone in a second step.The hydrogen peroxide produced was then used by HRP to oxidize ABTS to form coloured radical cation ABTS•+for activity analysis.The successful fabrication of the PDM and precise localization of enzymes in the PDM by different strategies were fully characterized.By varying the immobilization strategy,totally six PDM bioreactors with three enzymes precisely positional assembled in different strategies were constructed and their activities for the cascade reaction were investigated and compared.The PDM micro-bioreactor prepared by novel electrospray technologies provide a smart platform for positional assembly of multi-enzyme cascade reaction in a precise and well-controlled manner.

Apoferritin nanoparticle based dual-antigen influenza conjugate vaccine with potential cross-protective efficacy against heterosubtypic influenza virus

Ferritin nanoparticles with self-assembling properties have been widely explored as vaccine carrier by displaying foreign antigens through genetic fusion strategy.In the present work,an apoferritin(AFt)nanoparticle was tested as influenza vaccine carrier by chemically conjugating a matrix protein 2 ectodomain(M2e)antigen peptide or/and the full-length hemagglutinin(HA)antigen on the outer surface of the AFt,with heterobifunctional sSMCC or SM(PEG)_(24) containing PEG chain as linkers.To each AFt nanoparticle,about 30-32 M2e or 1.8 HA antigen could be coupled.The AFt-(PEG)24-M2e,in which the M2e was coupled through SM(PEG)_(24) containing PEG chain,conferred higher protective efficacy in immunized mice than AFt-M2e did,but was less effective than AFt-(PEG)_(24)-HA.When both M2e and HA were coupled,the synthesized dual-antigen vaccine candidate AFt-(PEG)_(24)-M2e/HA elicited high level of M2e and HA antigen-specific antibodies and conferred 100%protection against lethal infection of homologous PR8 HI N1 virus strain and 70%protection against a heterologous A/FM/1/47(FM1,H1N1)strain,which was more effective than the M2e or HA single antigen vaccine candidates.The potential cross-protective effect of the dual-antigen vaccine was further demonstrated by significant specific hemagglutination inhibition(HAI)titers in serum of the immunized mice against three other heterologous viral strains including A/Singapore/GPl908/2015(IVR-180)H1N1,A/Anhui/1/2005 H5N1,and A/Hong Kong H3N2.

高效体积排阻色谱法定量检测口蹄疫疫苗中146S的疫苗预处理方法

旨在建立一种口蹄疫灭活病毒疫苗的处理方法,去除尺寸排阻色谱法(HPSEC)检测146S抗原过程中的杂质干扰,获得最佳的检测信号并实现146S抗原含量的准确测定。分别考察了超速离心法、PEG沉淀法、核酸酶消化法对两批疫苗样品中的HPSEC检测干扰杂质的去除效果。在优化条件下,超速离心法处理后146S检测结果分别为7.1、7.6 μg/mL,PEG沉淀法为9.7、10.4 μg/mL;酶消化法处理后的检测值最高,分别为10.5、10.4 μg/mL,且杂质去除完全、处理速度快、操作条件温和。通过响应面法确定最优酶消化处理条件如下:200 μL水相中添加终浓度421 U/mL的Benzonase,25.1 ℃下反应1.29 h。在该最优条件下,对4家企业各3种不同血清型共12批疫苗样品进行146S含量检测,结果表明建立的方法对不同疫苗均有良好适用性,且重复性好(RSD<5.3%,n=3)。通过该处理方法,解决了杂质对146S定量检测的干扰,扩大了HPSEC检测技术的应用范围,进一步确保了检测结果的准确可靠。

核糖核酸酶A的错流超滤复性

针对重组蛋白高表达形成的包涵体,基于中空纤维膜的错流超滤复性具有较大的应用潜力。为研究不同条件对基于中空纤维膜的错流超滤复性的影响,以核糖核酸酶A(RNase A)为例,以复性过程的活性收率和质量收率作为考察指标,设计了RNase A复性初始浓度(A)、跨膜压力(B)、循环流速(C)的3因素3水平正交实验。结果表明,错流超滤复性过程中,以上复性条件对RNase A的质量收率基本没有影响,对RNase A的活性收率具有显著影响,其较优组合为A1B1C2,即RNase A复性初始浓度为0.3 mg/mL、跨膜压力34.0 kPa、循环流速935 cm/min。在以上复性条件下,RNase A的活性收率可达92.31%,质量收率为77.56%。

鸡白痢沙门氏菌兔抗血清多抗IgG的纯化与稳定

建立了离子交换层析介质一步纯化鸡白痢沙门氏菌兔抗血清获得多抗免疫球蛋白G (IgG)的方法,利用IgG与杂蛋白等电点的差异,指导阴阳离子交换层析介质筛选和操作条件优化;针对多抗IgG易失活的难题,采用差示扫描荧光法为纯化后的IgG筛选稳定剂。结果表明,通过毛细管等电聚焦测得兔抗血清多抗IgG的等电点为6.04~7.08,阳离子交换介质CM Sepharose FastFlow层析纯化的IgG最高电泳纯度为63.5%,高效液相尺寸排阻色谱测得IgG回收率为15.5%;阴离子交换层析纯化效果更佳,pH 5.5条件下Q Sepharose XL(Q-XL)介质层析获得的IgG最高纯度达99.3%,回收率达67.5%;200 g/L山梨醇对IgG具有最佳稳定作用,IgG的2个热变性温度分别提高了5.52和8.84℃,70℃时山梨醇的稳定作用更明显;以200 g/L山梨醇为稳定剂、用阴离子交换层析介质Q Sepharose XL一步纯化所得IgG具有高纯度、高收率及高稳定性,且制备工艺简单。

高分子多孔微球产品的制备及其在类病毒颗粒分离纯化中的应用

高分子多孔微球在医药领域的分离纯化中应用广泛,是重要的化工产品。高分子多孔微球的形成是一个复杂的过程,利用聚合物与有机溶剂致孔剂的相分离,可得到小于100 nm孔径的多孔微球;而水和疏水性聚合物之间相分离程度远大于有机溶剂与聚合物间的相分离程度,因此可获得大于100 nm孔径的多孔微球。针对不同应用领域选择不同的方法,设计并制备出合理的微球结构,对多孔微球的实际应用十分关键。本综述介绍了纳孔、介孔、大孔微球产品的制备方法悬浮聚合和种子聚合法,重点介绍本课题组制备超大孔微球的方法——反胶团溶胀法和W/O/W复乳法,上述两种方法解决了常规方法难以制备大孔径微球产品的问题,拓展了多孔微球在类病毒颗粒等大尺寸蛋白分离纯化领域的应用。在此基础上,以HB-VLPs为模型对比了不同孔径微球对类病毒颗粒吸附量、回收率、结构、以及纯化效果的差异,揭示超大孔微球产品在类病毒颗粒分离纯化中的优势。

Occurrence and distribution of antibiotic resistance genes in the sediments of the East China Sea bays

The coastal area of the East China Sea has experienced rapid urbanization and industrialization in China since 1980 s, resulting in severe pollution of its environments.Antibiotic resistance genes(ARGs) are regarded as a kind of emerging pollutant with potential high risk. The sediment samples were collected from Hangzhou Bay(HB),Xiangshan Bay(XB), and Taizhou Bay(TB) to investigate the spatial occurrence and distribution of 27 ARGs and class I integron–integrase gene(intI1) in the coastal area of the East China Sea. The PCR results showed the frequent presence of 11 ARGs and intI1 in the sediments of the three bays. The qPCR results further showed that sulfonamide resistance was the most prevalent ARG type and antibiotic target replacement and protection were the most important resistance mechanisms in the sediments. Regarding the subtype of ARGs, sulI, tetW, and dfrA13 were the most abundant ARGs, in which sulI was higher in TB(based on both the absolute and relative abundances) and dfrA13 was higher in HB(based on the relative abundances). The network analysis revealed that intI1 had significant correlations with tetC, sulI, sulII, and blaPSE-1. Oil was the key connected factor, which had positive connections with sulI, sulII, and blaPSE-1. In addition, the joint effect of heavy metals and nutrients & organic pollutants might be crucial for the fate of ARGs in the coastal sediments.

新型电荷型纳米盘的可控制备及其与细胞色素P450的结合性能

分别采用氮气吹干法和旋转蒸发法制备由磷脂和膜支架蛋白组成的电荷型纳米盘,用凝胶过滤色谱对其尺寸分级,分析了其性能,考察了其与肝微粒体细胞色素P450的结合能力。结果表明,纳米盘外观澄清透明,微观呈圆盘状,平均直径10nm,在p H 7.4下Zeta电位为-19.86 m V;肝微粒体破碎液与纳米盘能很好结合,CO差示光谱在450 nm出现明显吸收峰,细胞色素P450含量为0.10 nmol/mg,比活比未经纳米盘处理时提高13.0倍,较传统方法提升1.5倍,且操作时间由数日缩短至数小时。电荷型纳米盘在结合膜蛋白细胞色素P450的同时,活性保持良好,在膜蛋白研究领域极具应用潜力。

新型耐碱蛋白A介质的制备与性能

以蛋白A结构中耐碱性更好的C区为基础,构建一种新型耐碱蛋白A,并偶联至琼脂糖基质,获得新型耐碱蛋白A亲和层析介质,分别用激光共聚焦显微镜和石英晶体微天平研究了人免疫球蛋白(h Ig G)与蛋白A介质的结合过程。结果表明,该介质具有较商品配基更高的结合力和稳定的再生性能,hIgG动态载量达62.0 mg/mL,40次再生操作后载量为初始载量的84%。该介质在抗体纯化领域具有较好的应用前景。

RIGID GIGAPOROUS CHROMATOGRAPHIC MEDIA AND THEIR POTENTIAL IMPACT ON DOWNSTREAM PROCESSING

More and more biomolecules are being produced by the biotechnology industry for applications ranging from medicine and food to engineering materials. Liquid chromatography plays a center-stage role in a typical downstream process producing biomolecules such as recombinant proteins. Rigid gigaporous media are porous particles possessing large transecting through-pores with a pore-to-particle diameter ratio of dpore/dparticle〉 0.01. They allow convective flow in the large through-pores, while the smaller diffusion-pores （typically several hundred angstroms in size） supply the needed surface areas. Because of the transecting gigapores, a portion of the mobile phase flows through the pores in addition to fluid flow in the interstitial spaces between the particles in a packed-bed column. This considerably lowers the operating column pressure drop. This lower pressure drop makes axial-direction scale-up of chromatographic columns possible to avoid pancake columns that invariably degrade separation resolution. The large gigapores also make the binding sites on the diffusion pore surfaces more accessible, thus increasing the loading capacity of large protein molecules that can be hindered sterically if only diffusion pores are present. This work discusses the development of rigid gigaporous media and their potential impact on the design of multi-stage downstream process from the angle of multi-scale analysis.

CHROMATOGRAPHIC REFOLDING OF PROTEINS:MOLECULAR ACTION AND COLUMN CONTROL

Protein expression in E coil often results in the formation of a kind of protein aggregate called inclusion body Conversion of the inactive protein aggregate into biologically active protein is a key step in production of recombinant products Convenlional dilution refolding technique suffers from disadvantages of low recovery and low concentration Various chromatographic refolding techniques have been developed over the last few years These include size-exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and different affinity chromatography. A successful strategy is the use of gradient elution in column control which provides a gentle and gradual change of the solution environment for the macromolecule to rsfold at nano-scale, The gradient refolding at column scale could minimize misfolding and aggregation which are induced by sudden change of the solution in conventional refolding operation.

Identifying human-induced influence on microbial community:A comparative study in the effluent-receiving areas in Hangzhou Bay

Microbial community structure is afiected by both natural processes and human activities.In coastal area,anthropegenetic activity can usually lead to the discharge of the effluent from wastewater treatment plant(WWTP)to sea,and thus the water quality chronically turns worse and marine ecosystem becomes unhealthy.Microorganisms play key roles in pollutants degradation and ecological restoration;however,there are few studies about how the WWTP effluent disposal influences coastal microbial communities.In this study,sediment samples were collected from two WWTP effluentreceiving areas(abbreviated as JX and SY)in Hangzhou Bay.First,based on the high-throughput sequencing of 16S rRNA gene,microbial community structure was analyzed.Secondly,several statistical analyses were conducted to reveal the microbial community characteristics in response to the effluent disposal.Using PCoA,the significant difference of in microbial community structure was determined between JX and SY;using RDA,water COD and temperature,and sediment available phosphate and ammonia nitrogen were identified as the key environmental factors for the community difference;using LDA effect size analysis,the most distinctive microbes were found and their correlations with environmental factors were investigated;and according to detrended beta-nearesttaxon-index,the sediment microbial communities were found to follow"niche theory".An interesting and important finding was that in SY that received more and toxic COD,many distinctive microbes were related to the groups that were capable of degrading toxic organic pollutants.This study provides a clear illustration of eco-environmental deterioration under the long-term human pressure from the view of microbial ecology.