Conserved Organization of γ-Aminobutyric AcidAReceptor Genes: Cloning and Analysis of the Chicken β4-Subunit Gene

A series of genomic clones containing DNA that encodes the chicken gamma-aminobutyric acidA (GABAA) receptor beta 4 subunit have been isolated. These have been restriction mapped and partially sequenced to determine the structural organization and the size of the beta 4-subunit gene. This gene, which comprises nine exons, spans more than 65 kb. The organization of the chicken GABAA receptor beta 4-subunit gene has been compared to that of the murine GABAA receptor delta-subunit gene and to those of the genes that encode other members of the ligand-gated ion-channel superfamily, namely muscle and neuronal nicotinic acetylcholine receptors (AChRs). Although the positions of the intron/exon boundaries of GABAA receptor subunit genes are seen to be highly conserved, there are significant differences between the genes that encode GABAA receptor and AChR subunits. These results are discussed in relation to the proposal that this superfamily of ligand-gated ion-channel receptor genes arose by duplication of an ancestral receptor gene.     

A confirmatory analysis of malachite green residues in rainbow trout with liquid chromatography-electrospray tandem mass spectrometry

A quantitative liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed for the determination of malachite green (MG) and its metabolite leucomalachite green (LMG) in fish. Residues were extracted with an acetonitrile-acetate buffer and purified using the automated solid-phase extraction (ASPEC). Residues were analyzed with a reversed-phase LC-MS/MS using a positive-ion electrospray ionisation (ESI). Isotope-labelled leucomalachite green (LMG-D5) was used as an internal standard for the quantification of LMG residues. The related dye, brilliant green (BG) was used as an instrumental standard. Identification and quantification of analytes were based on the ion transitions monitored by multiple reaction monitoring (MRM). The decision limit (CCalpha) for MG and LMG was 0.13 and 0.16 microgkg(-1). The respective detection capabilities (CCbeta) were 0.22 and 0.27 microgkg(-1). The absolute recovery (repeatability SD(r)) was in the range of 58-65% (7.8-11.2%) for MG and 59-68% (9.7-16.9%) for LMG. LMG was quantified also based on the internal standard, giving a recovery (repeatability SD(r)) of 103-110% (4.8-9.3%). The method was further evaluated by analyzing a total of 34 fish residue monitoring samples, of which eight samples were found to be non-compliant containing low residues of LMG.     

Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.     

Functional priorities in LCA and design for environment

Aim, Scope and Background  The interest in environmental questions has increased enormously during the last decade. Environmental protection has become an issue of strategic importance within the manufacturing industry and many companies are now working in the field of Design for Environment (DFE). The main purpose of DFE is to create products and services for achieving a sustainable society. Designers are widely believed to have a key role in adapting products to a sustainable society and one of the major instruments in the context of Design for Environment is Life Cycle Assessment (LCA). However, product development creates particular challenges for incorporating environmental issues that combine functional and environmental assessment. A natural and important part of product design is to define and analyse the functions of the product. Consequently, the functional unit in LCA is a core issue in DFE. Most recent research in DFE has focused on how to reduce the environmental impact of products throughout their life-cycle by addressing environmental aspects, while little attention has been given to the functionality of the product. Additionally, early product development phases, so called re-think phases, are considered to have the influence on major changes in products in general. These phases have thus the highest potential for changing products and product systems towards a sustainable development. Main Features  This paper discusses an extended functional representation in design for environment methods to evaluate sustainable design solutions, especially in early (re-think) phases of product design. Based on engineering-design science and several case studies, a concept has been developed describing how functional preferences can be visualised in design for environment and product development. In addition, the functional unit in LCA is discussed. The concept is called Functional Profile (FP) and is additionally exemplified in a case study on radio equipment. Discussion  The new functional characterisation concept helps identify functional priorities in design for environment. The Functional Profile is a structured, systematic and creative concept for identifying the necessary functions of a new product. The FP is envisioned to complement existing design for environment methods, not to replace them. Instead of being a product-development tool or method, the concept is an approach that increases understanding of inter-reactions between functional characteristics of products and their environmental characteristics, which furthermore facilitates trade-off decisions. One of the objectives behind the concept is to highlight the importance of balancing functional requirements and environmental impacts, presenting both the advantages and disadvantages of the product. Outlook  A second paper will be produced to complement the functional-environmental characterisation concept in early product development phase, presenting the environmental characterisation part and illustrating correlations between the functional and environmental sides.     

Extracellular transglutaminase 2 has a role in cell adhesion, whereas intracellular transglutaminase 2 is involved in regulation of endothelial cell proliferation and apoptosis

Objective: Transglutaminase 2 (TG2) is a multifunctional protein with an important role in vascular biology, where it is involved in cell–matrix interaction, cell attachment and cell population expansion. In efforts to elucidate the role of TG2 in endothelial cell biology, in this study, we measured several endothelial cell characteristics in cells where TG2 was specifically knocked down by RNAi. Materials and methods: The effect of small interfering RNA (siRNA)‐TG2 on human umbilical vein endothelial cells was studied. Adhesion and cell viability were assessed by chemical reduction of MTT, and cell proliferation was analysed by flow cytometry. Apoptosis was evaluated by annexin V/PI dual staining and protein expression level was assayed by western blotting. Results: We found that siRNA‐TG2 reduced endothelial cell number, lead to cell adhesion deficiency, cell cycle arrest in G₁ phase and induction of apoptosis. Our results show that exogenously added TG2 could reverse loss of adhesion but did not overcome the defect in cell proliferation, nor could it inhibit siRNA‐TG2‐induced apoptosis. Conclusion: We conclude that TG2 loss in endothelial cells causes reduction in cell number as a result of cell cycle arrest, flaws in adhesion and induction of apoptosis. Our results imply that reduction in cell number and increased apoptosis in response to TG2 silencing is independent of the cell adhesion process. Altogether, our findings underline the significance of TG2 in endothelial cell cycle progression and cell survival, in vitro.