A simple and direct pre-embedding technique for ultrastructure of scarce biological specimens

A simple and rapid technique for pre-embedding scarce biological specimens for Transmission electron microscopy (TEM) is reported. It is based on pre-embedding biological samples in bovine serum albumin (BSA) and bis-acrylamide (BA), cross-linked and polymerized with paraformaldehyde, glutaraldehyde, ammonium persulfate and Temed. Pre-embedding in BSA and BA offers several advantages over traditional pre-embedding techniques for TEM including the ability to visualize the sample and a more resistant matrix. This results in more reproducible and consistent analysis. It can be applied to tissues, cells, and subcellular structures handled as pellets or suspensions. In addition, use of the pre-embedding matrix for light microscopy is reported. The ability to pre-embed scarce biological specimens efficiently and reproducibly provides a valuable way to study and characterize cytological tissues such as biopsies or cystic and amniotic fluid cells.     

Single-molecule visualization in cell biology

Recent progress in single-molecule detection techniques has allowed us to visualize the dynamic behaviour and reaction kinetics of individual biological molecules inside living cells. Single-molecule visualization provides a direct way to quantify, with a high spatial and temporal resolution, biological events inside cells at the single-molecule level. In this article, we discuss how single-molecule visualization can be used in cell biology.     

Rejuvenating old fluorophores with new chemistry

The field of organic chemistry began with 19th century scientists identifying and then expanding upon synthetic dye molecules for textiles. In the 20th century, dye chemistry continued with the aim of developing photographic sensitizers and laser dyes. Now, in the 21st century, the rapid evolution of biological imaging techniques provides a new driving force for dye chemistry. Of the extant collection of synthetic fluorescent dyes for biological imaging, two classes reign supreme: rhodamines and cyanines. Here, we provide an overview of recent examples where modern chemistry is used to build these old-but-venerable classes of optically responsive molecules. These new synthetic methods access new fluorophores, which then enable sophisticated imaging experiments leading to new biological insights.     

Visualization of chromatin higher-order structures and dynamics in live cells

Chromatin has highly organized structures in the nucleus, and these higher-order structures are proposed to regulate gene activities and cellular processes. Sequencing-based techniques, such as Hi-C, and fluorescent in situ hybridization (FISH) have revealed a spatial segregation of active and inactive compartments of chromatin, as well as the non-random positioning of chromosomes in the nucleus, respectively. However, regardless of their efficiency in capturing target genomic sites, these techniques are limited to fixed cells. Since chromatin has dynamic structures, live cell imaging techniques are highlighted for their ability to detect conformational changes in chromatin at a specific time point, or to track various arrangements of chromatin through long-term imaging. Given that the imaging approaches to study live cells are dramatically advanced, we recapitulate methods that are widely used to visualize the dynamics of higher-order chromatin structures.     

Peptide conjugates as tools for the study of biological signal transduction

Today, many biological phenomena are being investigated and understood in molecular detail, and organic chemistry is increasingly being directed towards biological phenomena. This review is intended to highlight this interplay of organic chemistry and biology, using biological signal transduction as an example. Lipo-, glyco-, phospho- and nucleoproteins play key roles in the processes whereby chemical signals are passed across cell membranes and further to the cell nucleus. For the study of the biological phenomena associated with these protein conjugates, structurally well-defined peptides containing the characteristic linkage region of the peptide backbone with the lipid, the carbohydrate or the phosphoric acid ester can provide valuable tools. The multi-functionality and pronounced acid- and base-lability of such compounds renders their synthesis a formidable challenge to conventional organic synthesis. However, the recent development of enzymatic protecting groups, provides one of the central techniques which, when coupled with classic chemical synthesis, can provide access to these complex and sensitive biologically relevant peptide conjugates under particularly mild conditions and with high selectivity.     

Biological pathway kinetic rate constants are scale-invariant

MOTIVATION: Scale-free networks have had a profound impact in Biology. Network theory is now used routinely to visualize, navigate through, and help understand gene networks, protein-protein interactions, regulatory networks and metabolic pathways. Here we analyse the numerical rather than topological properties of biological networks and focus on the study of kinetic rate constants within pathways. RESULTS: We have analysed all current entries in the BioModels database and show that the kinetic rate parameters follow Benford's; law closely. The cumulative histogram plot reveals an underlying power-law. This implies that these data are scale-invariant, thus placing biological network topology and their chemistry on an equivalent 'scale-free' power-law foundation.     

Serial femtosecond crystallography at the SACLA: breakthrough to dynamic structural biology

X-ray crystallography visualizes the world at the atomic level. It has been used as the most powerful technique for observing the three-dimensional structures of biological macromolecules and has pioneered structural biology. To determine a crystal structure with high resolution, it was traditionally required to prepare large crystals (> 200 μm). Later, synchrotron radiation facilities, such as SPring-8, that produce powerful X-rays were built. They enabled users to obtain good quality X-ray diffraction images even with smaller crystals (ca. 200–50 μm). In recent years, one of the most important technological innovations in structural biology has been the development of X-ray free electron lasers (XFELs). The SPring-8 Angstrom Compact free electron LAser (SACLA) in Japan generates the XFEL beam by accelerating electrons to relativistic speeds and directing them through in-vacuum, short-period undulators. Since user operation started in 2012, we have been involved in the development of serial femtosecond crystallography (SFX) measurement systems using XFEL at the SACLA. The SACLA generates X-rays a billion times brighter than SPring-8. The extremely bright XFEL pulses enable data collection with microcrystals (ca. 50–1 μm). Although many molecular analysis techniques exist, SFX is the only technique that can visualize radiation-damage-free structures of biological macromolecules at room temperature in atomic resolution and fast time resolution. Here, we review the achievements of the SACLA-SFX Project in the past 5 years. In particular, we focus on: (1) the measurement system for SFX; (2) experimental phasing by SFX; (3) enzyme chemistry based on damage-free room-temperature structures; and (4) molecular movie taken by time-resolved SFX.     

Exploring biologically relevant chemical space with metal complexes

Altering biological processes with small synthetic molecules is a general approach for the design of drugs and molecular probes. Medicinal chemistry and chemical biology are focused predominately on the design of organic molecules, whereas inorganic compounds find applications mainly for their reactivity (e.g. cisplatin as a DNA-reactive therapeutic) or imaging properties (e.g. gadolinium complexes as MRI diagnostics). In such inorganic pharmaceuticals or probes, coordination chemistry in the biological environment or at the target site lies at the heart of their modes of action. However, past and very recent results suggest that it is also worth exploring a different aspect of metal complexes: their ability to form structures with unique and defined shapes for the design of 'organic-like' small-molecule probes and drugs. In such metal-organic compounds, the metal has the main purpose to organize the organic ligands in three-dimensional space. It is likely that such an approach will complement the molecular diversity of organic chemistry in the quest for the discovery of compounds with superior biological activities.     

The evolution of photosynthesis…again?

‘Replaying the tape’ is an intriguing ‘would it happen again?’ exercise. With respect to broad evolutionary innovations, such as photosynthesis, the answers are central to our search for life elsewhere. Photosynthesis permits a large planetary biomass on Earth. Specifically, oxygenic photosynthesis has allowed an oxygenated atmosphere and the evolution of large metabolically demanding creatures, including ourselves. There are at least six prerequisites for the evolution of biological carbon fixation: a carbon-based life form; the presence of inorganic carbon; the availability of reductants; the presence of light; a light-harvesting mechanism to convert the light energy into chemical energy; and carboxylating enzymes. All were present on the early Earth. To provide the evolutionary pressure, organic carbon must be a scarce resource in contrast to inorganic carbon. The probability of evolving a carboxylase is approached by creating an inventory of carbon-fixation enzymes and comparing them, leading to the conclusion that carbon fixation in general is basic to life and has arisen multiple times. Certainly, the evolutionary pressure to evolve new pathways for carbon fixation would have been present early in evolution. From knowledge about planetary systems and extraterrestrial chemistry, if organic carbon-based life occurs elsewhere, photosynthesis—although perhaps not oxygenic photosynthesis—would also have evolved.     

Neurocan-GFP fusion protein: a new approach to detect hyaluronan on tissue sections and living cells.

Hyaluronan is an unsulfated glycosaminoglycan (GAG) that is ubiquitously expressed in the extracellular matrix (ECM) of all vertebrates, where hyaluronan rich matrices constitute a particular permissive environment for the development of complex biological structures and also for tumor progression. Because of its conserved structure and ubiquitous expression, antibodies for its histochemical detection cannot be produced. We have engineered a fusion protein, neurocan-GFP, and expressed it as a secreted molecule in mammalian cells. Neurocan-GFP fusion protein specifically binds to hyaluronan and directly visualizes hyaluronan on tissue sections, revealing a very detailed picture of hyaluronan distribution. The fluorescent fusion protein can be used in combination with antibodies and nuclear markers for double or triple staining. In addition, it is suitable to visualize hyaluronan on living cells by time-lapse video microscopy. The successful production and application of the neurocan-GFP fusion protein opens up new perspectives for using GFP fusion proteins as detection tools in histological and cytological studies complementing conventional antibody and biotin/avidin techniques.     

Antibody microarrays for label-free cell-based applications

The recent advances in microtechnologies have shown the interest of developing microarrays dedicated to cell analysis. In this way, miniaturized cell analyzing platforms use several detection techniques requiring specific solid supports for microarray read-out (colorimetric, fluorescent, electrochemical, acoustic, optical…). Real-time and label-free techniques, such as Surface Plasmon Resonance imaging (SPRi), arouse increasing interest for applications in miniaturized formats. Thus, we focused our study on chemical methods for antibody-based microarray fabrication dedicated to the SPRi analysis of cells or cellular activity. Three different approaches were designed and developed for specific applications. In the first case, a polypyrrole-based chemistry was used to array antibody-microarray for specific capture of whole living cells. In the second case, the polypyrrole-based chemistry was complexified in a three molecular level assembly using DNA and antibody conjugates to allow the specific release of cells after their capture. Finally, in the third case, a thiol-based chemistry was developed for long incubation times of biological samples of high complexity. This last approach was focused on the simultaneous study of both cell type characterization and secretory activity (detection of proteins secreted by cells). This paper describes three original methods allowing a rapid and efficient analysis of cellular sample on-chip using immunoaffinity-based assays.     

Visualizing Escherichia coli sub-cellular structure using sparse deconvolution Spatial Light Interference Tomography

Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. However, tomographic imaging of live cells is challenging mainly because they are transparent, i.e., weakly scattering structures. Therefore, this type of imaging has been implemented largely using fluorescence techniques. While confocal fluorescence imaging is a common approach to achieve sectioning, it requires fluorescence probes that are often harmful to the living specimen. On the other hand, by using the intrinsic contrast of the structures it is possible to study living cells in a non-invasive manner. One method that provides high-resolution quantitative information about nanoscale structures is a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM). In addition to rendering quantitative phase information, when combined with a high numerical aperture objective, SLIM also provides excellent depth sectioning capabilities. However, like in all linear optical systems, SLIM's resolution is limited by diffraction. Here we present a novel 3D field deconvolution algorithm that exploits the sparsity of phase images and renders images with resolution beyond the diffraction limit. We employ this label-free method, called deconvolution Spatial Light Interference Tomography (dSLIT), to visualize coiled sub-cellular structures in E. coli cells which are most likely the cytoskeletal MreB protein and the division site regulating MinCDE proteins. Previously these structures have only been observed using specialized strains and plasmids and fluorescence techniques. Our results indicate that dSLIT can be employed to study such structures in a practical and non-invasive manner.     

Confocal-multilabeling, ultrasensitive TUNEL analysis of DNA breaks in individual cells

OBJECTIVE: To visualize and localize fragmented DNA strands within apoptotic cells by means of fluorescence using TdT-mediated dUTP-biotin nick end labeling (TUNEL) techniques, laser scanning confocal microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: For this experiment, lymphoid reverted cells were used as a model. Characteristic DNA breaks inside apoptotic cells were detected using TUNEL techniques by a reaction involving tetramethyl rhodamin isothyocyanate (TRITC). The DNA from cell nuclei was counterstained using chromomycin A3 (CA3). The tandem TRITC-CA3 in CLSM was applied to investigate the ability to detect DNA breaks in individual cells using TUNEL techniques and its amplified variants (TUNEL-CARD). FAMIS was applied on dynamic sequences of images of TUNEL preparations and on four-dimensional (4-D) sequences of images of TUNEL-CARD preparations. RESULTS: Distribution and amplitude of fluorescent structures were characterized on dynamic sequences of images. Characterization was improved when FAMIS was applied on 4-D sequences of images, taking into account differences in photobleaching and/or spectrum of TRITC and CA3. CONCLUSION: It is possible to discriminate targets from CA3. FAMIS and TUNEL methods can be used to visualize and localize multiple DNA breaks in lymphoid reverted cells in improved methods of experimentation.     

质谱和核磁共振成为研究生物大分子的重要方法 ———简介2002年诺贝尔化学奖得主的贡献

2002年诺贝尔化学奖授予了在质谱和核磁共振领域有杰出贡献的三位科学家。Jonh B. Fenn和Koichi Tanaka发展了质谱鉴定生物大分子质量的方法,Kurt Wuethrich建立了核磁共振测定蛋白质分子溶液三维结构的方法。他们的研究成果是质谱和核磁共振方法发展的里程碑,极大地推动了这两种研究方法的进一步发展。如今,质谱和核磁共振已发展成为认识生物大分子的强劲的研究手段。     

Basidiomycetes: A new source of secondary metabolites.

The area of natural products research is the most rapidly growing field of organic chemistry, due to the great technical developments in the isolation and identification techniques. Today, near to one million natural products -isolated from the most diverse living things- are known. Microorganisms are among the least-studied of these. Nevertheless, they offer large possibilities for the discovery of new structures and biological activities. Among the microorganisms, the Basidiomycetes present a production capacity and a range of biologically active metabolites, which have scarcely been investigated. The wide spectrum of natural products with biological activity produced by Basidiomycetes includes antimicrobial agents, antifungal, antiviral and cytotoxic activities, enzymes, plant growth regulators and flavors. These metabolites are generally grouped by their chemical origin, and the relationship between chemical structure and the different biological activities reported. The main objective of this review is to bring an updated revision of the numerous and interesting biosynthetic pathways from basidiomycetes.     

Carotenoid research: History and new perspectives for chemistry in biological systems

The history of carotenoid research as this progressed from chemistry to biochemistry and biology is outlined. Proposed roles of carotenoids in eye health, as antioxidants, and in protection against cancer and other degenerative diseases, as well as stimulatory effects on the immune system and metabolism are covered. Proposed biological actions must be consistent with the chemistry of carotenoids in the largely aqueous biological systems, which may differ from the known chemistry of carotenoids in organic solvents. In particular, carotenoids tend to form aggregates. The effects of this aggregation and of other molecular interactions in vivo are likely to be crucial to biological activity. These perspectives of the chemistry of carotenoids and carotenoid free radicals are examined and the need for carotenoid samples used in experimental work to be pure and free from breakdown products and pro-oxidant peroxides is emphasised.This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.     

Escher: A Web Application for Building,Sharing, and Embedding Data-Rich Visualizations of Biological Pathways

Escher is a web application for visualizing data on biological pathways. Three key features make Escher a uniquely effective tool for pathway visualization. First, users can rapidly design new pathway maps. Escher provides pathway suggestions based on user data and genome-scale models, so users can draw pathways in a semi-automated way. Second, users can visualize data related to genes or proteins on the associated reactions and pathways, using rules that define which enzymes catalyze each reaction. Thus, users can identify trends in common genomic data types (e.g. RNA-Seq, proteomics, ChIP)—in conjunction with metabolite- and reaction-oriented data types (e.g. metabolomics, fluxomics). Third, Escher harnesses the strengths of web technologies (SVG, D3, developer tools) so that visualizations can be rapidly adapted, extended, shared, and embedded. This paper provides examples of each of these features and explains how the development approach used for Escher can be used to guide the development of future visualization tools.     

Lifeact: a versatile marker to visualize F-actin

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.