Monitoring Astrocytic Proteome Dynamics by Cell Type-Specific Protein Labeling

The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetase^L274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics.     

In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry

Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non‐canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non‐canonical amino acid L‐azidohomoalanine (AHA), a surrogate for l ‐methionine, followed by fluorescent labelling of AHA‐containing cellular proteins by azide‐alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)‐targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT‐FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano‐scale secondary ion mass spectrometry (¹⁵NH₃ assimilation) for individual cells within a sediment‐sourced enrichment culture showed concordance between AHA‐positive cells and ¹⁵N enrichment. BONCAT‐FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single‐cell level.     

Multifunctionalization of cetuximab with bioorthogonal chemistries and parallel EGFR profiling of cell-lines using imaging,FACS and immunoprecipitation approaches

The ability to derivatize antibodies is currently limited by the chemical structure of antibodies as polypeptides. Modern methods of bioorthogonal and biocompatible chemical modifications could make antibody functionalization more predictable and easier, without compromising the functions of the antibody. To explore this concept, we modified the well-known anti-epidermal growth factor receptor (EGFR) drug, cetuximab (Erbitux®), with 5-azido-2-nitro-benzoyl (ANB) modifications by optimization of an acylation protocol. We then show that the resulting ANB–cetuximab can be reliably modified with dyes (TAMRA and carboxyrhodamine) or a novel synthesized cyclooctyne modified biotin. The resulting dye- and biotin-modified cetuximabs were then tested across several assay platforms with several cell lines including U87, LN229, F98_EGFR, F98_WT and HEK293 cells. The assay platforms included fluorescence microscopy, FACS and biotin–avidin based immunoprecipitation methods. The modified antibody performs consistently in all of these assay platforms, reliably determining relative abundances of EGFR expression on EGFR expressing cells (LN229 and F98_EGFR) and failing to cross react with weak to negative EGFR expressing cells (U87, F98_WT and HEK293). The ease of achieving diverse and assay relevant functionalizations as well as the consequent rapid construction of highly correlated antigen expression data sets highlights the power of bioorthogonal and biocompatible methods to conjugate macromolecules. These data provide a proof of concept for a multifunctionalization strategy that leverages the biochemical versatility and antigen specificity of antibodies.     

Genetic code expansion to enable site‐specific bioorthogonal labeling of functional G protein‐coupled receptors in live cells

For use in site‐specific bioorthogonal labeling of expressed G protein‐coupled receptors (GPCRs) in live cells, we developed a luciferase‐based reporter assay. The assay was used to compare amber codon suppression efficiency, receptor functionality, and efficiency of different bioorthogonal labeling chemistries. We used the assay system to compare side‐by‐side the efficiency of incorporation of three different noncanonical amino acids [4‐azido‐l‐phenylalanine (azF), cyclopropene‐l‐lysine (CpK), and trans‐cyclooct‐2‐en‐l‐lysine (TCOK)] at three different sites on a GPCR using three different genetic code expansion plasmid systems. As a model GPCR, we engineered an epitope‐tagged C‐C chemokine receptor 5 (CCR5)‐RLuc3 fusion for expression in HEK293T cells. Satisfactory incorporation of azF, CpK, and TCOK into heterologously expressed CCR5 was achieved. We also carried out cell‐based calcium mobilization assays to measure the function of the engineered CCR5, and in the same cells, we performed bioorthogonal labeling of the engineered mutants using heterobivalent compounds containing bioorthogonal tethering groups linked to either a small‐molecule fluorophore or a peptide. Favorable reaction kinetics of tetrazine‐containing compounds with CCR5 harboring TCOK was observed. However, bioorthogonal labeling in live cells of CCR5 harboring CpK with tetrazine‐containing compounds using the inverse electron demand Diels‐Alder ligation was overall slightly more efficient than other reactions tested.     

Insects on isolated colonies of common rock-rose Helianthemum chamaecistus

ABSTRACT.

• 1 A study was made of the insect species present on twenty-eight colonies of H.chamaecistus Mill, in 1978 ranging in size (cover) from 0.4 m² to about 6500 m² and separated from nearest large neighbours by distances up to 6.5 km. Twenty-three insect species were collected of which nine or ten were more or less monophagous.
• 2 There was a clear relationship between species richness and size of host colony. Sites with less than 30 m² of rock-rose, about 67% of those in the study area, were likely to have less than half of the monophagous species. Distance to neighbouring colonies did not contribute significantly to species richness.
• 3 The consistency of results from year to year was examined at five sites re-sampled in 1979. The 1978 data were also used to compare‘expected’and observed faunas at five new sites sampled in 1979 based upon the species-area relationship and the relative frequency of species at known sites.
• 4 Insect dispersal was examined using a suction trap placed near the largest rock-rose site. Colonization of new sites was studied by experimental outplanting of pot-grown plants.
• 5 These direct observations, and the monitoring of fourteen sites for 5 years, suggested that colonization and interchange of faunas were rare events. The experimental introduction of one to five species at four sites showed in several cases that the sites were suitable for the species, and that introduction could significantly enhance a site's complement.

     

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides^{1, 2}. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy³. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid⁴ and N-acetylgalactosamine^{5, 6}, in developing zebrafish and in other living organisms.Download video file.^{(41M, mov)}     

五种典型藻类对C，N，P吸收动力学分析及种间竞争模拟

目的:获得五种典型藻类(甲藻属微小亚历山大藻(Alexandrium minutum)和锥状斯氏藻(Scrippsiella trochoidea),赤潮硅藻中肋骨条藻(Skeletonema costatum)以及绿藻属杜氏藻(Dunaliella salina)和青岛大扁藻(Platymonas helgolandica tsingtaoensis))对C,N,P营养的吸收动力学参数,并利用经典藻类种间竞争模型,构建一个藻类混合共存的生态平衡体系,揭示藻类种间竞争规律,为赤潮爆发机制和预测的研究提供一个新思路。方法:监测批次培养过程中藻体的生长规律以及培养基中C,N,P营养的消耗,计算出藻类营养吸收动力学参数,将参数代入Huisman-Weissing竞争模型,模拟藻类种间竞争。结果:(1)在单独培养条件下,杜氏藻具有最高的比生长率(0.834 d~(-1))和最大细胞浓度(3.4×10~6 cells/mL),锥状斯氏藻和微小亚历山大藻的比生长率μ和最大细胞浓度与其它三种藻相比均明显偏低,p0.01;(2)随着环境总碳浓度从5 mM提高到20 mM,五种藻的比生长率和最大细胞浓度均显著上升,其中杜氏藻和青岛大扁藻对C浓度改变的响应更加敏感;(3)杜氏藻和中肋骨条藻理论最大比生长率(μmax)明显高于其它三种藻类,锥状斯氏藻和微小亚历山大藻对C,N,P营养盐的需求量相比于其它三种藻明显偏高,p0.01;(4)藻类共生平衡系统中,N营养添加有利于杜氏藻和中肋骨条藻发挥更好的种间竞争优势,P营养添加有利于微小亚历山大藻和锥状斯氏藻发挥种间竞争优势;结论:不同环境条件下,五种藻类最大比生长速率μmax和营养吸收半饱和常数Ks直接影响它们的种间竞争能力,基于藻类动力学参数的种间竞争模型为赤潮爆发机制和预测的研究提供一个新思路。     

Energy absorption during impact on the proximal femur is affected by body mass index and flooring surface

Impact mechanics theory suggests that peak loads should decrease with increase in system energy absorption. In light of the reduced hip fracture risk for persons with high body mass index (BMI) and for falls on soft surfaces, the purpose of this study was to characterize the effects of participant BMI, gender, and flooring surface on system energy absorption during lateral falls on the hip with human volunteers. Twenty university-aged participants completed the study with five men and five women in both low BMI (<22.5 kg/m²) and high BMI (>27.5 kg/m²) groups. Participants underwent lateral pelvis release experiments from a height of 5 cm onto two common floors and four safety floors mounted on a force plate. A motion-capture system measured pelvic deflection. The energy absorbed during the initial compressive phase of impact was calculated as the area under the force–deflection curve. System energy absorption was (on average) 3-fold greater for high compared to low BMI participants, but no effects of gender were observed. Even after normalizing for body mass, high BMI participants absorbed 1.8-fold more energy per unit mass. Additionally, three of four safety floors demonstrated significantly increased energy absorption compared to a baseline resilient-rolled-sheeting system (% increases ranging from 20.7 to 28.3). Peak system deflection was larger for high BMI persons and for impacts on several safety floors. This study indicates that energy absorption may be a common mechanism underlying the reduced risk of hip fracture for persons with high BMI and for those who fall on soft surfaces.     

Isotopic labeling of the heme cofactor in cytochrome p450 and other heme proteins

A recombinant bacterial expression system that generates ¹³C-labeled heme or ¹⁵N-labeled heme in functional cytochrome P450 enzymes and other heme-containing systems is reported here using a mutant strain of Escherichia coli (HU227) in which the HemA gene is inactive. By synthesizing several isotopomers of aminolevulinic acid with ¹³C or ¹⁵N at different locations, isotopes have been incorporated with high abundance into the heme cofactor of five different cytochrome P450 isoforms, along with one peroxidase. Confirmed both ¹³C- and ¹⁵N-incorporation; spectral and catalytic assays show the labeled enzymes produced in this system are functional.     

The effect of co-cultivation and selection parameters on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese soybean varieties

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l⁻¹) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l⁻¹ hygromycin for 10 days, 5 mg l⁻¹ hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l⁻¹ hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.     

99mTc-bioorthogonal click chemistry reagent for in vivo pretargeted imaging

Metal-free click chemistry has become an important tool for pretargeted approaches in the molecular imaging field. The application of bioorthogonal click chemistry between a pretargeted trans-cyclooctene (TCO) derivatized monoclonal antibody (mAb) and a ^99mTc-modified 1,2,4,5-tetrazine for tumor imaging was examined in vitro and in vivo. The HYNIC tetrazine compound was synthesized and structurally characterized, confirming its identity. Radiolabeling studies demonstrated that the HYNIC tetrazine was labeled with ^99mTc at an efficiency of >95% and was radiochemically stable. ^99mTc–HYNIC tetrazine reacted with the TCO–CC49 mAb in vitro demonstrating its selective reactivity. In vivo biodistribution studies revealed non-specific liver and GI uptake due to the hydrophobic property of the compound, however pretargeted SPECT imaging studies demonstrated tumor visualization confirming the success of the cycloaddition reaction in vivo. These results demonstrated the potential of ^99mTc–HYNIC–tetrazine for tumor imaging with pretargeted mAbs.     

Identification of lysine acetyltransferase p300 substrates using 4-pentynoyl-coenzyme A and bioorthogonal proteomics

Proteomic studies have identified a plethora of lysine acetylated proteins in eukaryotes and bacteria. Determining the individual lysine acetyltransferases responsible for each protein acetylation mark is crucial for elucidating the underlying regulatory mechanisms, but has been challenging due to limited biochemical methods. Here, we describe the application of a bioorthogonal chemical proteomics method to profile and identify substrates of individual lysine acetyltransferases. Addition of 4-pentynoyl-coenzyme A, an alkynyl chemical reporter for protein acetylation, to cell extracts, together with purified lysine acetyltransferase p300, enabled the fluorescent profiling and identification of protein substrates via Cu(I)-catalyzed alkyne-azide cycloaddition. We identified several known protein substrates of the acetyltransferase p300 as well as the lysine residues that were modified. Interestingly, several new candidate p300 substrates and their sites of acetylation were also discovered using this approach. Our results demonstrate that bioorthogonal chemical proteomics allows the rapid substrate identification of individual protein acetyltransferases in vitro.     

Streptomyces leeuwenhoekii sp. nov., the producer of chaxalactins and chaxamycins,forms a distinct branch in Streptomyces gene trees

A polyphasic study was carried out to establish the taxonomic status of an Atacama Desert isolate, Streptomyces strain C34^T, which synthesises novel antibiotics, the chaxalactins and chaxamycins. The organism was shown to have chemotaxonomic, cultural and morphological properties consistent with its classification in the genus Streptomyces. Analysis of 16S rRNA gene sequences showed that strain C34^T formed a distinct phyletic line in the Streptomyces gene tree that was very loosely associated with the type strains of several Streptomyces species. Multilocus sequence analysis based on five house-keeping gene alleles underpinned the separation of strain C34^T from all of its nearest phylogenetic neighbours, apart from Streptomyces chiangmaiensis TA-1^T and Streptomyces hyderabadensis OU-40^T which are not currently in the MLSA database. Strain C34^T was distinguished readily from the S. chiangmaiensis and S. hyderabadensis strains by using a combination of cultural and phenotypic data. Consequently, strain C34^T is considered to represent a new species of the genus Streptomyces for which the name Streptomyces leeuwenhoekii sp. nov. is proposed. The type strain is C34^T (= DSM 42122^T = NRRL B-24963^T). Analysis of the whole-genome sequence of S. leeuwenhoekii, with 6,780 predicted open reading frames and a total genome size of around 7.86 Mb, revealed a high potential for natural product biosynthesis.     

生物正交化学在活体标记及药物传递中的研究进展

生物正交化学反应是一类可以在生理条件下发生的化学反应,具有简单、高效、高特异性的特点,在生物医学的研究中被广泛应用.基于生物体天然生命过程的代谢工程,可对生物分子进行无损、高效的生物代谢修饰,是一种理想的生物修饰技术.通过生物代谢途径可有效地将各种化学报告基团引入靶标物的生物分子中,有利于携带配对基团的标记物与其发生生物正交反应,从而在活体系统中实现生物分子的标记示踪和药物递送.这种基于代谢工程与生物正交化学的标记策略因为具有两者之间的优势,在生物医学工程中的标记、成像示踪、诊断等领域展现出巨大的研究价值与应用潜力.本文介绍了生物正交和代谢工程的原理与生物医学研究进展,阐述了生物正交化学在分子成像和药物传递等方面的研究与应用.     

Genes,enzymes and membrane proteins of the nitrate respiration system ofEscherichia coli

Summary A method was devised to isolate mutants carrying deletions through several genetic loci (chlD ⁺ andchlA ⁺) which are involved in the membrane-bound nitrate respiratory complex ofEscherichia coli. Specific transducing phages were used to reintroduce these genes. Comparisons of membrane fractions from these transduced strains showed five membrane proteins that are necessary for the formation of an active nitrate respiration system. Two particular bacterial genes (chlD ⁺ andchlA ⁺) were shown to control these five membrane proteins.Three of the proteins specified bychlA ⁺, appear to be constitutively controlled and always present in the membrane ofE. coli irrespective of growth conditions, while the other two proteins, specified bychlD ⁺, appear to be induced byanaerobic growth in the presence of nitrate.     

Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for 13C metabolic flux analysis

In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and ¹³C-metabolic flux analysis (¹³C-MFA). Here, cells were grown in parallel cultures with [1-¹³C]glucose and [U-¹³C]glucose as tracers and ¹³C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of ¹³C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for ¹³C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased ¹³C-flux measurements in C. acetobutylicum.     

Metal ion-induced assembly of dipeptide-attached perylenediimide for fluorometric “turn on” detection of biologically important small molecule

A dipeptide-appended perylenediimide (PDI-CFF) fluorescent molecule was designed, synthesized, and characterized. Though the molecule does not dissolve in any individual solvent, it dissolves well in an organic/water mixed solvent system such as tetrahydrofuran/water. This new fluorescent molecule was self-assembled in a tetrahydrofuran/water mixture to form both nanofibrous network structures and a nano ring structure. It has shown nanofibril morphology by the interactions with ferric ions (PDI-CFF/Fe³⁺ system) with diminishing fluorescent property. Interestingly, L-ascorbic acid (LAA) interacts with the PDI-CFF/Fe³⁺ system, showing turn-on fluorescence. Another interesting feature is that the minimum detection limits for Fe³⁺ ions and LAA are at the submicromolar levels of 6.2 × 10⁻⁸ and 3 × 10⁻⁸ M, respectively. Moreover, the fluorescent (10 μM) signals can be monitored by the naked eye under handheld UV lamp irradiation at 365 nm, and this is very convenient for the real application. In this study, the molecule offers the opportunity for processing these sequential fluorescence responses in order to fabricate a implication logic gate that includes NOT, AND, and OR simple logic gates using chemical stimuli (ferric ions and LAA) as inputs and fluorescence emission at 536 nm as output. The detailed mechanism of interactions of Fe³⁺ with PDI-CFF and LAA with the PDI-CFF/Fe³⁺ system is vividly studied by using Fourier transform infrared (FT-IR) analysis and fluorescence. Moreover, this new molecule was reusable for several times without significant loss of its activity. The construction of logic gates using biologically important molecules/ions holds future promise for the design and development of new bio-logic gates.     

Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe²⁺/ascorbate system. Activation of the phosphatase requires both Fe²⁺ ion and ascorbate and the level of activation is dependent on the concentrations of Fe²⁺ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe²⁺ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3mM, respectively. Several common divalent metal ions, including Co²⁺, Ni²⁺, Cu²⁺, Mg²⁺, and Ca²⁺ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe²⁺ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe²⁺/ascorbate system is a specific process. Moreover, H₂O₂, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe²⁺/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe²⁺/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.