Inhibitor and protein microarrays for activity-based recognition of lipolytic enzymes

Protein and small-molecule microarrays are useful tools for high-throughput analysis of DNA-protein, protein-protein, and protein-small molecule interactions. Here we report on novel microarrays for activity screening of lipases and esterases based on phosphonic acid ester inhibitors. These compounds are activity recognition probes (ARPs) and bind to active serine hydrolases in a stoichiometric and irreversible manner. Protein microarrays were generated by spotting six different lipolytic enzymes onto hydrogel-coated glass slides. The activity of immobilized enzymes was determined after treatment with fluorescently labeled ARPs. Alternatively, biotinylated ARPs were bound to streptavidin slides in order to identify their affinity for enzymes in solution. Both systems, the protein- and ARP microarrays proved to be useful and versatile tools for the rapid identification and characterization of novel and known lipolytic enzymes.     

Protein microarrays and biomarkers of infectious disease

Protein microarrays are powerful tools that are widely used in systems biology research. For infectious diseases, proteome microarrays assembled from proteins of pathogens will play an increasingly important role in discovery of diagnostic markers, vaccines, and therapeutics. Distinct formats of protein microarrays have been developed for different applications, including abundance-based and function-based methods. Depending on the application, design issues should be considered, such as the need for multiplexing and label or label free detection methods. New developments, challenges, and future demands in infectious disease research will impact the application of protein microarrays for discovery and validation of biomarkers.     

Fluorescence-based assay formats and signal amplification strategies for DNA microarray analysis

DNA microarrays are powerful tools for the high throughput analysis of nucleic acids due to their parallel detection capabilities. To realize the apparent power of DNA microarray, an efficient assay format is essential and a variety of assay formats have been developed for nucleic acid detection on microarrays. Many of them employ fluorescence-based methods because fluorescence detection is straightforward and easy to implement. Herein we broadly review fluorescence-based assay formats with a focus on PCR-associated target preparation. For the heterogeneous assay of nucleic acid on DNA microarrays, sensitivity is one of the most important factors and special emphasis has been given to recently developed signal amplification strategies aimed at achieving high sensitivity.     

Automated Glycan Assembly of Plant Oligosaccharides and Their Application in Cell-Wall Biology

The plant cell wall provides the richest available resource of fermentable carbohydrates and biobased materials. The main component of plant cell walls is cellulose, which is the most abundant biomolecule on earth. Apart from cellulose, which is constructed from relatively simple β-1,4-glucan chains, plant cell walls also contain structurally more complex heteropolysaccharides (hemicellulose and pectin), as well as lignin and cell-wall proteins. A detailed understanding of the molecular structures, functions, and biosyntheses of cell-wall components is required to further promote their industrial use. Plant cell-wall research is, to a large degree, hampered by a lsack of available well-defined oligosaccharide samples that represent the structural features of cell-wall glycans. One technique to access these oligosaccharides is automated glycan assembly; a technique in which monosaccharide building blocks are, similarly to automated peptide and oligonucleotide chemistry, successively added to a linker-functionalized resin in a fully automated manner. Herein, recent research into the automated glycan assembly of different classes of cell-wall glycans used as molecular tools for cell-wall biology is discussed. More than 60 synthetic oligosaccharides were prepared and printed as microarrays for screening monoclonal antibodies that recognize plant cell-wall polysaccharides. The synthesized oligosaccharides have also been used to investigate glycosyltransferases and glycoside hydrolases, which are involved in synthesis and degradation of plant cell walls, as well as for the analysis of cell-wall-remodeling enzymes.     

Glycosyltransferases and their Assays

Glycosyltransferases (GTs) are a large family of enzymes that are essential in all domains of life for the biosynthesis of complex carbohydrates and glycoconjugates. GTs catalyse the transfer of a sugar from a glycosyl donor to a variety of acceptor molecules, for example, oligosaccharides, peptides, lipids or small molecules. Such glycosylation reactions are central to many fundamental biological processes, including cellular adhesion, cell signalling and bacterial‐ and plant‐cell‐wall biosynthesis. GTs are therefore of significant interest as molecular targets in chemical biology and drug discovery. In addition, GTs have found wide application as synthetic tools for the preparation of complex carbohydrates and glycoconjugates. In order to exploit the potential of GTs both as molecular targets and synthetic tools, robust and operationally simple bioassays are essential, especially as more and more protein sequences with putative GT activity but unknown biochemical function are being identified. In this minireview, we give a brief introduction to GT biochemistry and biology. We outline the relevance of GTs for medicinal chemistry and chemical biology, and describe selected examples for recently developed GT bioassays, with a particular emphasis on fluorescence‐based formats.     

A lateral flow protein microarray for rapid and sensitive antibody assays

Protein microarrays are useful tools for highly multiplexed determination of presence or levels of clinically relevant biomarkers in human tissues and biofluids. However, such tools have thus far been restricted to laboratory environments. Here, we present a novel 384-plexed easy to use lateral flow protein microarray device capable of sensitive (<30 ng/mL) determination of antigen-specific antibodies in ten minutes of total assay time. Results were developed with gold nanobeads and could be recorded by a cell-phone camera or table top scanner. Excellent accuracy with an area under curve (AUC of 98% was achieved in comparison with an established glass microarray assay for 26 antigen-specific antibodies. We propose that the presented framework could find use in convenient and cost-efficient quality control of antibody production, as well as in providing a platform for multiplexed affinity-based assays in low-resource or mobile settings.     

Engineering the stability and the activity of a glycoside hydrolase

Glycosidases, the enzymes responsible in nature for the catabolism of carbohydrates, are well-studied catalysts widely used in industrial biotransformations and oligosaccharide synthesis, which are also attractive targets for drug development. Glycosidases from hyperthermophilic organisms (thriving at temperatures > 85 °C) are also interesting models to understand the molecular basis of protein stability and to produce robust tools for industrial applications. Here, we review the results obtained in the last two decades by our group on a β-glycosidase from the hyperthermophilic Archaeon Sulfolobus solfataricus. Our findings will be presented in the general context of the stability of proteins from hyperthermophiles and of the chemo-enzymatic synthesis of oligosaccharides.     

Enzyme Therapy: Current Challenges and Future Perspectives

In recent years, enzymes have risen as promising therapeutic tools for different pathologies, from metabolic deficiencies, such as fibrosis conditions, ocular pathologies or joint problems, to cancer or cardiovascular diseases. Treatments based on the catalytic activity of enzymes are able to convert a wide range of target molecules to restore the correct physiological metabolism. These treatments present several advantages compared to established therapeutic approaches thanks to their affinity and specificity properties. However, enzymes present some challenges, such as short in vivo half-life, lack of targeted action and, in particular, patient immune system reaction against the enzyme. For this reason, it is important to monitor serum immune response during treatment. This can be achieved by conventional techniques (ELISA) but also by new promising tools such as microarrays. These assays have gained popularity due to their high-throughput analysis capacity, their simplicity, and their potential to monitor the immune response of patients during enzyme therapies. In this growing field, research is still ongoing to solve current health problems such as COVID-19. Currently, promising therapeutic alternatives using the angiotensin-converting enzyme 2 (ACE2) are being studied to treat COVID-19.     

微藻碳水化合物生产生物燃料的研究进展

化石能源的不可再生性和使用过程中造成的环境污染使开发新能源变得非常迫切。微藻因为生长速度快,固定二氧化碳能力强,碳水化合物积累量高,近年来成为生物燃料生产的研究热点。微藻碳水化合物制备生物燃料极具潜力,但规模化培养为制约其发展的瓶颈。本文总结了微藻碳水化合物的组成及代谢,介绍了微藻碳水化合物的酶水解、化学和超声等预处理方法,比较分析了酶糖化和化学糖化特点,简述了传统的厌氧消化、活性污泥发酵技术用于微藻碳水化合物制备生物燃料的循环利用优势,对微藻碳水化物制备生物液、气态燃料的研究进展、经济性和产业化前景进行了综述。提出了微藻碳水化合物制备生物燃料需要以更经济、有效的糖化和发酵技术为未来研发方向。     

Development of a Sensitive Microarray Platform for the Ranking of Galectin Inhibitors: Identification of a Selective Galectin‐3 Inhibitor

Glycan microarrays are useful tools for lectin glycan profiling. The use of a glycan microarray based on evanescent‐field fluorescence detection was herein further extended to the screening of lectin inhibitors in competitive experiments. The efficacy of this approach was tested with 2/3′‐mono‐ and 2,3′‐diaromatic type II lactosamine derivatives and galectins as targets and was validated by comparison with fluorescence anisotropy proposed as an orthogonal protein interaction measurement technique. We showed that subtle differences in the architecture of the inhibitor could be sensed that pointed out the preference of galectin‐3 for 2′‐arylamido derivatives over ureas, thioureas, and amines and that of galectin‐7 for derivatives bearing an α substituent at the anomeric position of glucosamine. We eventually identified a diaromatic oxazoline as a highly specific inhibitor of galectin‐3 versus galectin‐1 and galectin‐7.     

Activity-based protein profiling: new developments and directions in functional proteomics

Proteomic screening has become increasingly insightful with the availability of novel analytical tools and technologies. Detailed analysis and integration of the profound datasets attained from comprehensive profiling studies are enabling researchers to dig deeper into the foundations of genomic and proteomic networks, towards a clearer understanding of the intricate cellular circuitries they manifest. The major difficulty often lies in correlating the patho/physiological state presented with the underlying biological mechanisms; therefore, identification of causal variants as therapeutic targets is extremely important. Herein, we will describe methods that address this challenge through activity-based protein profiling, which applies chemical probes to the comparison and monitoring of protein dynamics across complex proteomes. Over recent years such activity-based probes have been creatively augmented with applications in gel-based separations, microarrays and in vivo imaging. These developments offer a newfound ability to characterise and differentiate cells, tissues and proteomes through activity-dependent signatures; this has expanded the scope and impact of activity-based probes in biomedical research.     

DNA芯片制作的常用方法

DNA芯片是分子生物学研究领域的重要工具。目前最常用的制作方法是将探针固定于载体上。介绍载玻片的氨基硅烷化修饰和探针的五种修饰方法 ,以及两者之间的结合方式。这些方法简单、实用、经济 ,适合国内实验室开展 DNA芯片研究工作     

Chemical Tools for Studying the Biological Function of Glycolipids

Glycolipids play an important role in many biological processes and to this end, synthetic chemists have developed a variety of new techniques and “chemical tools” that allow for the study of glycolipids in vitro and in vivo. The types of probes prepared include fluorescent, radio‐labelled, biotinylated and photoreactive ones, as well as others based on liposomes, microarrays and other supramolecular constructs—each of which offers its own advantages, as is discussed. A number of more specialised probes, such as metabolically engineered glycolipids and photopolymerisable glycolipids, have also been prepared in order to investigate various processes including substrate specificities and binding interactions. The purpose of this review is to present the key approaches that can be used for the development of glycolipid probes, organised according to application, and also to discuss the limitations of such strategies, which include the nontrivial task of ensuring that the probe does not adversely influence the biological activity of the parent compound. On the whole, it is exciting to see what can be achieved through the development of chemical probes as tools to study biological processes, and it is envisioned that the reader will be inspired by the large number of superb studies highlighted here and will be encouraged to undertake further work in this research area.     

Defined presentation of carbohydrates on a duplex DNA scaffold

A new method for the spatially defined alignment of carbohydrates on a duplex DNA scaffold is presented. The use of an N-hydroxysuccinimide (NHS)-ester phosphoramidite along with carbohydrates containing an alkylamine linker allows for on-column labeling during solid-phase oligonucleotide synthesis. This modification method during solid-phase synthesis only requires the use of minimal amounts of complex carbohydrates. The covalently attached carbohydrates are presented in the major groove of the B-form duplex DNA as potential substrates for murine type II C-type lectin receptors mMGL1 and mMGL2. CD spectroscopy and thermal melting revealed only minimal disturbance of the overall helical structure. Surface plasmon resonance and cellular uptake studies with bone-marrow-derived dendritic cells were used to assess the capability of these carbohydrate-modified duplexes to bind to mMGL receptors.     

The Role of Galectin-1 in Cancer Progression,and Synthetic Multivalent Systems for the Study of Galectin-1

This review discusses the role of galectin-1 in the tumor microenvironment. First, the structure and function of galectin-1 are discussed. Galectin-1, a member of the galectin family of lectins, is a functionally dimeric galactoside-binding protein. Although galectin-1 has both intracellular and extracellular functions, the defining carbohydrate-binding role occurs extracellularly. In this review, the extracellular roles of galectin-1 in cancer processes are discussed. In particular, the importance of multivalent interactions in galectin-1 mediated cellular processes is reviewed. Multivalent interactions involving galectin-1 in cellular adhesion, mobility and invasion, tumor-induced angiogenesis, and apoptosis are presented. Although the mechanisms of action of galectin-1 in these processes are still not well understood, the overexpression of galectin-1 in cancer progression indicates that the role of galectin-1 is significant. To conclude this review, synthetic frameworks that have been used to modulate galectin-1 processes are reviewed. Small molecule oligomers of carbohydrates, carbohydrate-functionalized pseudopolyrotaxanes, cyclodextrins, calixarenes, and glycodendrimers are presented. These synthetic multivalent systems serve as important tools for studying galectin-1 mediated cancer cellular functions.     

细菌胞外物质中糖类对Q235钢的腐蚀研究

细菌及其胞外物质是影响金属腐蚀的重要原因之一。细菌胞外物质的构成较为复杂,各组分对腐蚀的影响也各有不同。主要研究了细菌胞外物质中糖类对Q235钢腐蚀的影响,采用静态失重、电化学等方法研究胞外物质中含量较高的3种常见糖类(葡萄糖、鼠李糖和海藻糖)及其混合物在1 mol/L Na2SO4溶液中对Q235钢的缓蚀作用。结果表明,当糖类的质量浓度在10 g/L时,糖类及其混合物在溶液中对Q235钢都有一定的缓蚀作用,缓蚀率均能达到30%左右,但其效果与铁细菌的氧化作用相比甚微。     

Bioinformatics Tools for Marker Discovery in Plant Breeding

In the last decade, intensive research has been invested in marker-assisted selection (MAS). In parallel, there have been great advances in high-throughput genomic technology, such as microarrays and next-generation sequencing. These technologies overcome barriers for crops that do not have genomic information available and shorten the time from genomic darkness to discovery of an overwhelming number of markers. Consequently, MAS has become an available approach for any crop. Herein, the bioinformatics tools for high-throughput marker discovery using microarrays and high-throughput sequencing are reviewed and options for using them in MAS are suggested.     

Effect of Carbohydrate on the Rheological Parameters of Paste Extrusion

Carbohydrates are often key components in the formulation of extrusion pastes, yet the reasons for selection are presently empirical. The influence on paste extrusion of various types of carbohydrates, such as starch, dextrin, lactose, and glucose, have been studied and related to their water retention capacities. The bulk yield stress and the surface shear stress both decrease as the moisture content increases; however, the way in which the water that is present interacts with the carbohydrate has an important influence. The behavior of some carbohydrates can be substantially accounted for by a consideration of packing effects; however, dextrin behaves differently. The carbohydrates function not only as a binder but also as a means of retaining the liquid phase.