Trypsiligase-Catalyzed Labeling of Proteins on Living Cells

Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the N terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins’ native N termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.     

英国食品GDAs标签的发展经验及对中国的启发

对英国食品每日摄入量指南（GDAs）标签开展案例研究，结果发现，GDAs标签从包装背面标签转为FOP标签，实施已有20年以上。该标签采用特定营养素体系度量法模型，显示每份食品能量、糖、脂肪、饱和脂肪、盐的含量及其每日参考摄入量占比，既没有颜色编码又没有形象的图标显示，且实施效果不如多数FOP标签。对此，中国可考虑将营养成分表信息以食品份量单位直观显示在包装正面，方便消费者关注与理解。     

Photocaged Hoechst Enables Subnuclear Visualization and Cell Selective Staining of DNA in vivo

Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type- and subcellular-specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking in vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms.     

Human DDX17 Unwinds Rift Valley Fever Virus Non-Coding RNAs

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the Bunyaviridae family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5′ non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17_135–555) and RVFV non-coding RNAs, IGR and 5’ NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17_135–555, IGR, and 5’ NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5’ NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17_135–555 is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17_135–555 can unwind both RNAs. Overall, our study provides direct evidence of DDX17_135–555 interacting with and unwinding RVFV non-coding regions.     

When organic products are tasty: Taste inferences from an Organic = Healthy Association

Previous research has consistently demonstrated that organic food is typically seen as healthier. The aim of the present study is to investigate how these health inferences influence taste perceptions of organic food. In Study 1, we show that a neutral food product with an organic label is perceived as more healthy than the same product without such a label. This higher level of perceived healthiness is paired with an improved perceived taste. In Study 2 and 3, we obtain evidence in Dutch and US samples that an organic label increases perceived taste and attractiveness of healthy (but not unhealthy) food. Whereas previous studies have shown general health halo effects of organic labels, this perspective cannot explain the specific pattern of our results, which speaks towards an ‘organic = healthy = tasty’ intuition.     

Selective Analysis of Sulfur-Containing Species in a Heavy Crude Oil by Deuterium Labeling Reactions and Ultrahigh Resolution Mass Spectrometry

A heavy crude oil has been treated with deuterated alkylating reagents (CD₃I and C₂D₅I) and directly analyzed without any prior fractionation and chromatographic separation by high-field Orbitrap Fourier Transform Mass Spectrometry (FTMS) and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) using electrospray ionization (ESI). The reaction of a polycyclic aromatic sulfur heterocycles (PASHs) dibenzothiophene (DBT), in the presence of silver tetrafluoroborate (AgBF₄) with ethyl iodide (C₂H₅I) in anhydrous dichloroethane (DCE) was optimized as a sample reaction to study heavy crude oil mixtures, and the reaction yield was monitored and determined by proton nuclear magnetic resonance spectroscopy (¹H-NMR). The obtained conditions were then applied to a mixture of standard aromatic CH-, N-, O- and S-containing compounds and then a heavy crude oil, and only sulfur-containing compounds were selectively alkylated. The deuterium labeled alkylating reagents, iodomethane-d₃ (CD₃I) and iodoethane-d₅ (C₂D₅I), were employed to the alkylation of heavy crude oil to selectively differentiate the tagged sulfur species from the original crude oil.     

Compilation of Modern Technologies To Map Genome-Wide Cytosine Modifications in DNA

Over the past few decades, various DNA modification detection methods have been developed; many of the high-resolution methods are based on bisulfite treatment, which leads to DNA degradation, to a degree. Thus, novel bisulfite-free approaches have been developed in recent years and shown to be useful for epigenome analysis in otherwise difficult-to-handle, but important, DNA samples, such as hmC-seal and hmC-CATCH. Herein, an overview of advances in the development of epigenome sequencing methods for these important DNA modifications is provided.     

新加坡食品较健康选择标志系统经验启示

文章介绍了新加坡较健康选择标志系统基本情况,从规格与使用要求、申请程序、适用范围等进行阐述,指出新加坡作为第一个实施食品FOP标签系统的亚洲国家,其经验做法对中国乃至多数亚洲国家有重要借鉴意义,中国FOP标签系统应具有充分的依据,严格规范申请和使用行为并适度扩大应用范围。     

Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. Gd^III−Gd^III distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-Gd^III were indistinguishable from Gd^III−Gd^III distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.