Biosilica structures obtained from Nitzschia, Ditylum, Skeletonema, and Coscinodiscus diatom by a filtration-aided acid cleaning method

A filtration-aided acid cleaning method was used to collect biosilica structures from a diatom culture medium, natural seawater, or water bloom. Cell extraction, acid cleaning, and acid removal were all performed on a polytetrafluoroethylene (PTFE) filter cloth, significantly improving the treatment capacity and efficiency of the traditional acid wash method. Five typical diatoms were cultivated in the laboratory for acid cleaning. Different growth speeds were introduced, and different process parameters for acid cleaning were utilized. After the acid cleaning, biosilica structures were collected from the frustules of diatoms using different methods. Girdle bands and valves of Coscinodiscus sp. were separated by floating of the valves. Central spines of Ditylum brightwellii and valves of Skeletonema costatum were separately collected by settling or filtration. Rod-like frustules, such as those of Bacillaris paradoxa, are not suitable for large quantities of acid wash. The silica structures were observed and tested using an AFM-calibrated glass needle to determine their elasticity. Elasticity tests showed that ringent girdle bands are more flexible than complete ones (Coscinodiscus sp.) and that both long-chain clusters of Nitzschia palea and central spines of D. brightwellii have certain elasticities. The required pressure for deforming or breaking the biosilica structures of diatoms was also determined.     

BIOCHEMISTRY OF SILICA BIOMINERALIZATION IN DIATOMS

Diatoms are well known for the intricate patterns of their silica-based cell walls. The complex structures of diatom cell walls are species specific and become precisely reproduced during each cell division cycle, indicating a genetic control of silica biomineralization. Therefore, the formation of the diatom cell wall has been regarded as a paradigm for controlled production of nanostructured silica. However, the mechanisms allowing biosilicification to proceed at ambient temperature at high rates have remained enigmatic. Recently, we have shown that a set of highly cationic peptides (called silaffins) isolated from Cylindrotheca fusiformis shells are able to generate networks of silica nanospheres within seconds when added to a solution of silicic acid. Different silaffin species produce different morphologies of the precipitated silica. Silaffins contain covalently modified Lys-Lys elements. One of these lysine residues bears a novel type of protein modification, a polyamine consisting of 6–11 repeats of the N-methyl-propylamine unit. In addition to the silaffins, additional polyamine-containing substances have been isolated from a number of diatom species that may be involved in the control of biosilica morphology. Scanning electron microscopic analysis of diatom shells isolated in statu nascendi provide insights into the processes of pattern formation in biosilica. A model will be discussed that explains production of nanostructured biosilica in diatoms on the basis of these experimental results.     

Cationic amino acids specific biomimetic silicification in ionic liquid: a quest to understand the formation of 3-D structures in diatoms

The intricate, hierarchical, highly reproducible, and exquisite biosilica structures formed by diatoms have generated great interest to understand biosilicification processes in nature. This curiosity is driven by the quest of researchers to understand nature's complexity, which might enable reproducing these elegant natural diatomaceous structures in our laboratories via biomimetics, which is currently beyond the capabilities of material scientists. To this end, significant understanding of the biomolecules involved in biosilicification has been gained, wherein cationic peptides and proteins are found to play a key role in the formation of these exquisite structures. Although biochemical factors responsible for silica formation in diatoms have been studied for decades, the challenge to mimic biosilica structures similar to those synthesized by diatoms in their natural habitats has not hitherto been successful. This has led to an increasingly interesting debate that physico-chemical environment surrounding diatoms might play an additional critical role towards the control of diatom morphologies. The current study demonstrates this proof of concept by using cationic amino acids as catalyst/template/scaffold towards attaining diatom-like silica morphologies under biomimetic conditions in ionic liquids.     

Microsystem for the single molecule analysis of membrane transport proteins

Micro-chamber arrays enable highly sensitive and quantitative bioassays at the single-molecule level. Accordingly, they are widely used for ultra-sensitive biomedical applications, e.g., digital PCR and digital ELISA. However, the versatility of micro-chambers is generally limited to reactions in aqueous solutions, although various functions of membrane proteins are extremely important. To address this issue, microsystems using arrayed micro-sized chambers sealed with lipid bilayers, referred to here as a “biomembrane microsystems”, have been developed by many research groups for the analysis of membrane proteins. In this review, I would like to introduce recent progress on the single molecule analysis of membrane transport proteins using a biomembrane microsystem, and discuss the future prospects for its use in analytical and pharmacological applications.     

Cellular shellization: Surface engineering gives cells an exterior

Unlike eggs and diatoms, most single cells in nature do not have structured shells to provide extensive protection. It is a challenge to artificially confer shell structures on living cells to improve their inherent properties and functions. We discuss four different types of cellular shellizations: man‐made hydrogels, sol‐gels, polyelectrolytes, and mineral shells. We also explore potential applications, such as cell storage, protection, delivery, and therapy. We suggest that shellization could provide another means to regulate and functionalize cells. Specifically, the integration of living cells and non‐living functional shells may be developed as a novel strategy to create “super” or intelligent cells. Unlike biological approaches, this material‐based bio‐interface regulation is inexpensive, effective, and convenient, opening up a novel avenue for cell‐based technologies and practices.     

Enzyme immobilization in a biomimetic silica support

Robust immobilization techniques that preserve the activity of biomolecules have many potential applications. Silicates, primarily in the form of sol-gel composites or functionalized mesoporous silica, have been used to encapsulate a wide variety of biomolecules but the harsh conditions required for chemical synthesis limit their applicability. Silaffin polypeptides from diatoms catalyze the formation of silica in vitro at neutral pH and ambient temperature and pressure. Here we show that butyrylcholinesterase entrapped during the precipitation of silica nanospheres retained all of its activity. Ninety percent of the soluble enzyme was immobilized, and the immobilized enzyme was substantially more stable than the free enzyme. The mechanical properties of silica nanospheres facilitated application in a flow-through reactor. The use of biosilica for enzyme immobilization combines the excellent support properties of a silica matrix with a benign immobilization method that retains enzyme activity.     

Formation of Asymmetrical Structured Silica Controlled by a Phase Separation Process and Implication for Biosilicification

Biogenetic silica displays intricate patterns assembling from nano- to microsize level and interesting non-spherical structures differentiating in specific directions. Several model systems have been proposed to explain the formation of biosilica nanostructures. Of them, phase separation based on the physicochemical properties of organic amines was considered to be responsible for the pattern formation of biosilica. In this paper, using tetraethyl orthosilicate (TEOS, Si(OCH₂CH₃)₄) as silica precursor, phospholipid (PL) and dodecylamine (DA) were introduced to initiate phase separation of organic components and influence silica precipitation. Morphology, structure and composition of the mineralized products were characterized using a range of techniques including field emission scanning electron microscopy (FESEM), transmission electron microscope (TEM), X-ray diffraction (XRD), thermogravimetric and differential thermal analysis (TG-DTA), infrared spectra (IR), and nitrogen physisorption. The results demonstrate that the phase separation process of the organic components leads to the formation of asymmetrically non-spherical silica structures, and the aspect ratios of the asymmetrical structures can be well controlled by varying the concentration of PL and DA. On the basis of the time-dependent experiments, a tentative mechanism is also proposed to illustrate the asymmetrical morphogenesis. Therefore, our results imply that in addition to explaining the hierarchical porous nanopatterning of biosilica, the phase separation process may also be responsible for the growth differentiation of siliceous structures in specific directions. Because organic amine (e.g., long-chair polyamines), phospholipids (e.g., silicalemma) and the phase separation process are associated with the biosilicification of diatoms, our results may provide a new insight into the mechanism of biosilicification.     

Purification of biosilica from living diatoms by a two-step acid cleaning and baking method

Due to their sustainability, intact cell walls, availability of pure cultures, and others, living diatoms show a lot of promise for the application in various fields in particular for micro/nano-devices. In order to purify the biosilica structures of diatoms called frustules, a two-step acid cleaning and baking method was employed. By this path, organic matter and inorganic impurities can be removed very effectively. In addition, the highest quality of frustules was achieved when the samples were cleaned in an excess of boiling 10~15 % HCl and subsequently heated to 600 °C at a heating rate of 3 °C min^?1 for 6 h. In our operation, the native frustule morphology was maintained completely, and dry frustules with more than 90 % SiO₂ in weight can be obtained, and furthermore, the surface area of them reached a good value of 48.47 m² g^?1.     

Silica morphogenesis by alternative processing of silaffins in the diatom Thalassiosira pseudonana

For almost 200 years scientists have been fascinated by the ornate cell walls of the diatoms. These structures are made of amorphous silica, exhibiting species-specific, mostly porous patterns in the nano- to micrometer range. Recently, from the diatom Cylindrotheca fusiformis unusual phosphoproteins (termed silaffins) and long chain polyamines have been identified and implicated in biosilica formation. However, analysis of the role of silaffins in morphogenesis of species-specific silica structures has so far been hampered by the difficulty of obtaining structural data from these extremely complex proteins. In the present study, the five major silaffins from the diatom Thalassiosira pseudonana (tpSil1H, -1L, -2H, -2L, and -3) have been isolated, functionally analyzed, and structurally characterized, mak- ing use of the recently available genome data from this organism. Surprisingly, the silaffins of T. pseudonana and C. fusiformis share no sequence homology but are similar regarding amino acid composition and post-translational modifications. Silaffins tpSil1H and -2H are higher molecular mass isoforms of tpSil1L and -2L, respectively, generated in vivo by alternative processing of the same precursor polypeptides. Interestingly, only tpSil1H and -2H but not tpSil1L and -2L induce the formation of porous silica patterns in vitro, suggesting that the alternative processing event is an important step in morphogenesis of T. pseudonana biosilica.     

New tools and new biology: Recent miniaturized systems for molecular and cellular biology

Recent advances in applied physics and chemistry have led to the development of novel microfluidic systems. Microfluidic systems allow minute amounts of reagents to be processed using μm-scale channels and offer several advantages over conventional analytical devices for use in biological sciences: faster, more accurate and more reproducible analytical performance, reduced cell and reagent consumption, portability, and integration of functional components in a single chip. In this review, we introduce how microfluidics has been applied to biological sciences. We first present an overview of the fabrication of microfluidic systems and describe the distinct technologies available for biological research. We then present examples of microsystems used in biological sciences, focusing on applications in molecular and cellular biology.     

Biochemical Composition and Assembly of Biosilica-associated Insoluble Organic Matrices from the Diatom Thalassiosira pseudonana

The nano- and micropatterned biosilica cell walls of diatoms are remarkable examples of biological morphogenesis and possess highly interesting material properties. Only recently has it been demonstrated that biosilica-associated organic structures with specific nanopatterns (termed insoluble organic matrices) are general components of diatom biosilica. The model diatom Thalassiosira pseudonana contains three types of insoluble organic matrices: chitin meshworks, organic microrings, and organic microplates, the latter being described in the present study for the first time. To date, little is known about the molecular composition, intracellular assembly, and biological functions of organic matrices. Here we have performed structural and functional analyses of the organic microrings and organic microplates from T. pseudonana. Proteomics analysis yielded seven proteins of unknown function (termed SiMat proteins) together with five known silica biomineralization proteins (four cingulins and one silaffin). The location of SiMat1-GFP in the insoluble organic microrings and the similarity of tyrosine- and lysine-rich functional domains identifies this protein as a new member of the cingulin protein family. Mass spectrometric analysis indicates that most of the lysine residues of cingulins and the other insoluble organic matrix proteins are post-translationally modified by short polyamine groups, which are known to enhance the silica formation activity of proteins. Studies with recombinant cingulins (rCinY2 and rCinW2) demonstrate that acidic conditions (pH 5.5) trigger the assembly of mixed cingulin aggregates that have silica formation activity. Our results suggest an important role for cingulins in the biogenesis of organic microrings and support the hypothesis that this type of insoluble organic matrix functions in biosilica morphogenesis.     

Ultra‐sensitive immunoassay biosensors using hybrid plasmonic‐biosilica nanostructured materials

We experimentally demonstrate an ultra‐sensitive immunoassay biosensor using diatom biosilica with self‐assembled plasmonic nanoparticles. As the nature‐created photonic crystal structures, diatoms have been adopted to enhance surface plasmon resonances of metal nanoparticles on the surfaces of diatom frustules and to increase the sensitivity of surface‐enhanced Raman scattering (SERS). In this study, a sandwich SERS immunoassay is developed based on the hybrid plasmonic‐biosilica nanostructured materials that are functionalized with goat anti‐mouse IgG. Our experimental results show that diatom frustules improve the detection limit of mouse IgG to 10 pg/mL, which is ?100× better than conventional colloidal SERS sensors on flat glass.

Ultra‐sensitive immunoassay biosensor using diatom biosilica with self‐assembled plasmonic nanoparticles.     

Live diatom silica immobilization of multimeric and redox-active enzymes

Living organisms are adept in forming inorganic materials (biominerals) with unique structures and properties that exceed the capabilities of engineered materials. Biomimetic materials syntheses are being developed that aim at replicating the advantageous properties of biominerals in vitro and endow them with additional functionalities. Recently, proof-of-concept was provided for an alternative approach that allows for the production of biomineral-based functional materials in vivo. In this approach, the cellular machinery for the biosynthesis of nano-/micropatterned SiO₂ (silica) structures in diatoms was genetically engineered to incorporate a monomeric, cofactor-independent (“simple”) enzyme, HabB, into diatom silica. In the present work, it is demonstrated that this approach is also applicable for enzymes with “complex” activity requirements, including oligomerization, metal ions, organic redox cofactors, and posttranslational modifications. Functional expression of the enzymes β-glucuronidase, glucose oxidase, galactose oxidase, and horseradish peroxidase in the diatom Thalassiosira pseudonana was accomplished, and 66 to 78% of the expressed enzymes were stably incorporated into the biosilica. The in vivo incorporated enzymes represent approximately 0.1% (wt/wt) of the diatom biosilica and are stabilized against denaturation and proteolytic degradation. Furthermore, it is demonstrated that the gene construct for in vivo immobilization of glucose oxidase can be utilized as the first negative selection marker for diatom genetic engineering.     

Pentalysine Clusters Mediate Silica Targeting of Silaffins in Thalassiosira pseudonana

The biological formation of inorganic materials (biomineralization) often occurs in specialized intracellular vesicles. Prominent examples are diatoms, a group of single-celled eukaryotic microalgae that produce their SiO₂ (silica)-based cell walls within intracellular silica deposition vesicles (SDVs). SDVs contain protein-based organic matrices that control silica formation, resulting in species specifically nanopatterned biosilica, an organic-inorganic composite material. So far no information is available regarding the molecular mechanisms of SDV biogenesis. Here we have investigated by fluorescence microscopy and subcellular membrane fractionation the intracellular transport of silaffin Sil3. Silaffins are a group of phosphoproteins constituting the main components of the organic matrix of diatom biosilica. We demonstrate that the N-terminal signal peptide of Sil3 mediates import into a specific subregion of the endoplasmic reticulum. Additional segments from the mature part of Sil3 are required to reach post-endoplasmic reticulum compartments. Further transport of Sil3 and incorporation into the biosilica (silica targeting) require protein segments that contain a high density of modified lysine residues and phosphoserines. Silica targeting of Sil3 is not dependent on a particular peptide sequence, yet a lysine-rich 12–14-amino acid peptide motif (pentalysine cluster), which is conserved in all silaffins, strongly promotes silica targeting. The results of the present work provide the first insight into the molecular mechanisms for biogenesis of mineral-forming vesicles from an eukaryotic organism.     

Grazing-induced changes in cell wall silicification in a marine diatom

In aquatic environments, diatoms (Bacillariophyceae) constitute a central group of microalgae which contribute to about 40% of the oceanic primary production. Diatoms have an absolute requirement for silicon to build-up their silicified cell wall in the form of two shells (the frustule). To date, changes in diatom cell wall silicification have been only studied in response to changes in the growth environment, with consistent increase in diatom silica content when specific growth rates decrease under nutrient or light limitations. Here, we report the first evidence for grazing-induced changes in cell wall silicification in a marine diatom. Cells grown in preconditioned media that had contained both diatoms and herbivores are significantly more silicified than diatoms grown in media that have contained diatoms alone or starved herbivores. These observations suggest that grazing-induced increase in cell wall silicification can be viewed as an adaptive reaction in habitats with variable grazing pressure, and demonstrate that silicification in diatoms is not only a constitutive mechanical protection for the cell, but also a phenotypically plastic trait modulated by grazing. In turn, our results corroborate the idea that plant-herbivore interactions, beyond grazing sensu stricto, contribute to drive ecosystem structure and biogeochemical cycles in the ocean.     

Fabrication,Densification, and Replica Molding of 3D Carbon Nanotube Microstructures

The introduction of new materials and processes to microfabrication has, in large part, enabled many important advances in microsystems, lab-on-a-chip devices, and their applications. In particular, capabilities for cost-effective fabrication of polymer microstructures were transformed by the advent of soft lithography and other micromolding techniques ^{1, 2}, and this led a revolution in applications of microfabrication to biomedical engineering and biology. Nevertheless, it remains challenging to fabricate microstructures with well-defined nanoscale surface textures, and to fabricate arbitrary 3D shapes at the micro-scale. Robustness of master molds and maintenance of shape integrity is especially important to achieve high fidelity replication of complex structures and preserving their nanoscale surface texture. The combination of hierarchical textures, and heterogeneous shapes, is a profound challenge to existing microfabrication methods that largely rely upon top-down etching using fixed mask templates. On the other hand, the bottom-up synthesis of nanostructures such as nanotubes and nanowires can offer new capabilities to microfabrication, in particular by taking advantage of the collective self-organization of nanostructures, and local control of their growth behavior with respect to microfabricated patterns. Our goal is to introduce vertically aligned carbon nanotubes (CNTs), which we refer to as CNT "forests", as a new microfabrication material. We present details of a suite of related methods recently developed by our group: fabrication of CNT forest microstructures by thermal CVD from lithographically patterned catalyst thin films; self-directed elastocapillary densification of CNT microstructures; and replica molding of polymer microstructures using CNT composite master molds. In particular, our work shows that self-directed capillary densification ("capillary forming"), which is performed by condensation of a solvent onto the substrate with CNT microstructures, significantly increases the packing density of CNTs. This process enables directed transformation of vertical CNT microstructures into straight, inclined, and twisted shapes, which have robust mechanical properties exceeding those of typical microfabrication polymers. This in turn enables formation of nanocomposite CNT master molds by capillary-driven infiltration of polymers. The replica structures exhibit the anisotropic nanoscale texture of the aligned CNTs, and can have walls with sub-micron thickness and aspect ratios exceeding 50:1. Integration of CNT microstructures in fabrication offers further opportunity to exploit the electrical and thermal properties of CNTs, and diverse capabilities for chemical and biochemical functionalization ³.     

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.     

Functional proteomics in histone research and epigenetics

Post-translational modifications of histones comprise an important part of epigenetic gene regulation. Mass spectrometry and immunochemical techniques are currently the methods of choice for identification and quantitation of known and novel histone modifications. While peptide-centric mass spectrometry is a well-established tool for identification and quantification of histone modifications, recent technological advances have allowed discrete modification patterns to be assessed on intact histones. Chromatin immunoprecipitation assays (ChIP and ChIP-on-chip) are currently gaining tremendous popularity and are used to explore gene-specific patterns of histone modifications on a genomic scale. In this review, we introduce the basic concepts and recent developments of mass spectrometry, as well as immunochemical techniques and their applications in the analysis of histone modifications.     

Capacitive microsystems for biological sensing

The growing interest in personalized medicine leads to the need for fast, cheap and portable devices that reveal the genetic profile easily and accurately. To this direction, several ideas to avoid the classical methods of diagnosis and treatment through miniaturized and label-free systems have emerged. Capacitive biosensors address these requirements and thus have the perspective to be used in advanced diagnostic devices that promise early detection of potential fatal conditions. The operation principles, as well as the design and fabrication of several capacitive microsystems for the detection of biomolecular interactions are presented in this review. These systems are micro-membranes based on surface stress changes, interdigitated micro-electrodes and electrode-solution interfaces. Their applications extend to DNA hybridization, protein-ligand binding, antigen-antibody binding, etc. Finally, the limitations and prospects of capacitive microsystems in biological applications are discussed.     

抗体信息数据库

抗体是介导体液免疫的重要效应分子。近年来,随着治疗性抗体药物不断上市及其临床应用范围不断拓宽,治疗性抗体已成为生物制药的产业支柱。另一方面,随着抗体相关的基础研究日益深入和抗体序列、结构、功能表位等相关数据大量涌现,作为抗体数据管理、搜索与利用的重要工具,抗体资源库也层出不穷并得到长足发展,在相关的研究、开发、生产与销售中发挥日益重要的作用。本文对包括Kabat、IMGT、abYsis等在内抗体信息数据库进行介绍。