A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants.

Despite the well-known crucial role of intradomain disulfide bridges for immunoglobulin folding and stability, the single-chain variable fragment of the anti-viral antibody F8 is functionally expressed when targeted to the reducing environment of the plant cytoplasm. We show here that this antibody fragment is also functionally expressed in the cytoplasm of Escherichia coli. A gel shift assay revealed that the single-chain variable fragment (scFv) accumulating in the plant and bacterial cytoplasm bears free sulfhydryl groups. Guanidinium chloride denaturation/renaturation studies indicated that refolding occurs even in a reducing environment, producing a functional molecule with the same spectral properties of the native scFv(F8). Taken together, these results suggest that folding and functionality of this antibody fragment are not prevented in a reducing environment. This antibody fragment could therefore represent a suitable framework for engineering recombinant antibodies to be targeted to the cytoplasm.     

Can S-LCA methodology support responsible sourcing of raw materials in EU policy context?

Purpose

Access, affordability and sustainability of raw material supply chains are crucial to the sustainable development of the European Union (EU) for both society and economy. The study investigates whether and how the social life cycle assessment (S-LCA) methodology can support responsible sourcing of raw materials in Europe. The potential of social indicators already available in an S-LCA database is tested for the development of new metrics to monitor social risks in raw material industries at EU policy level.

Methods

The Product Social Impact Life Cycle Assessment (PSILCA) database was identified as a data and indicators source to assess social risks in raw material industries in EU-28 and extra-EU countries. Six raw material country sectors in the scope of the European policy on raw materials were identified and aggregated among those available in PSILCA. The selection of indicators for the assessment was based on the RACER (Relevance, Acceptance, Credibility, Ease, Robustness) analysis, leading to the proposal of 9 social impact categories. An S-LCA of the selected raw material industries was, thus, performed for the EU-28 region, followed by a contribution analysis to detect direct and indirect impacts and investigate related supply chains. Finally, the social performance of raw material sectors in EU-28 was compared with that of six extra-EU countries.

Results and discussion

Considering the overall social risks in raw material industries, “Corruption”, “Fair salary”, “Health and safety” and “Freedom of association and collective bargaining” emerged as the most significant categories both in EU and extra-EU. EU-28 shows an above-average performance where the only exception is represented by the mining and quarrying sector. An investigation of the most contributing processes to social impact categories for EU-28 led to the identification of important risks originating in the supply chain and in extra-EU areas. Therefore, the S-LCA methodology confirmed the potential of a life cycle perspective to detect burdens shifting and trade-offs. However, only a limited view on the sectoral social performance could be obtained from the research due to a lack of social data.

Conclusions

The S-LCA methodology and indicators appear appropriate to perform an initial social sustainability screening, thus enabling the identification of hotspots in raw material supply chains and the prioritization of areas of action in EU policies. Further methodological developments in the S-LCA field are necessary to make the approach proposed in the paper fully adequate to support EU policies on raw materials.

     

构建基于CRISPR/Cas9技术敲除ALOX5的质粒

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因(Arachidonate 5-lipoxygenase gene,ALOX5)的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5。     

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.     

High Frequency Ultrasound for In Vivo Pregnancy Diagnosis and Staging of Placental and Fetal Development in Mice

Background

Ultrasound is a valuable non-invasive tool used in obstetrics and gynecology to monitor the growth and well being of the human fetus. The laboratory mouse has recently emerged as an appropriate model for fetal and perinatal studies because morphogenetic processes in mice exhibit adequate homology to those in humans, and genetic manipulations are relatively simple to perform in mice. High-frequency ultrasound (HFUS) has recently become available for small animal preclinical imaging and can be used to study pregnancy and development in the mouse. The objective of the current study was to assess the main applications of HFUS in the evaluation of fetal growth and placental function and to better understand human congenital diseases.

Methodology/Principal Findings

On each gestational day, at least 5 dams were monitored with HFUS; a total of ∼200 embryos were examined. Because it is not possible to measure each variable for the entire duration of the pregnancy, the parameters were divided into three groups as a function of the time at which they were measured. Univariate analysis of the relationship between each measurement and the embryonic day was performed using Spearman’s rank correlation (Rs). Continuous linear regression was adopted for multivariate analysis of significant parameters. All statistical tests were two-sided, and a p value of 0.05 was considered statistically significant.

Conclusions/Significance

The study describes the main applications of HFUS to assess changes in phenotypic parameters in the developing CD1 mouse embryo and fetus during pregnancy and to evaluating physiological fetal and placental growth and the development of principal organs such as the heart, kidney, liver, brain and eyes in the embryonic mouse. A database of normal structural and functional parameters of mouse development will provide a useful tool for the better understanding of morphogenetic and cardiovascular anomalies in transgenic and mutant mouse models.     

A closer look at prion strains

Prions are infectious proteins that are responsible for transmissible spongiform encephalopathies (TSEs) and consist primarily of scrapie prion protein (PrP^Sc), a pathogenic isoform of the host-encoded cellular prion protein (PrP^C). The absence of nucleic acids as essential components of the infectious prions is the most striking feature associated to these diseases. Additionally, different prion strains have been isolated from animal diseases despite the lack of DNA or RNA molecules. Mounting evidence suggests that prion-strain-specific features segregate with different PrP^Sc conformational and aggregation states.

Strains are of practical relevance in prion diseases as they can drastically differ in many aspects, such as incubation period, PrP^Sc biochemical profile (e.g., electrophoretic mobility and glycoform ratio) and distribution of brain lesions. Importantly, such different features are maintained after inoculation of a prion strain into genetically identical hosts and are relatively stable across serial passages.

This review focuses on the characterization of prion strains and on the wide range of important implications that the study of prion strains involves.