Structural Flexibility and Disassembly Kinetics of Single Ferritin Molecules Using Optical Nanotweezers.

Ferritin, a spherical protein shell assembled from 24 subunits, functions as an efficient iron storage and release system through its channels. Understanding how various chemicals affect the structural behavior of ferritin is crucial for unravelling the origins of iron-related diseases in living organisms including humans. In particular, the influence of chemicals on ferritin's dynamics and iron release is barely explored at the single-protein level. Here, by employing optical nanotweezers using double-nanohole (DNH) structures, we examined the effect of ascorbic acid (reducing reagent) and pH on individual ferritin's conformational dynamics. The dynamics of ferritin increased as the concentration of ascorbic acid approached saturation. At pH 2.0, ferritin exhibited significant structural fluctuations and eventually underwent a stepwise disassembly into fragments. This work demonstrated the disassembly pathway and kinetics of a single ferritin molecule in solution. We identified four critical fragments during its disassembly pathway, which are 22-mer, 12-mer, tetramer, and dimer subunits. Moreover, we present single-molecule evidence of the cooperative disassembly of ferritin. Interrogating ferritin's structural change in response to different chemicals holds importance for understanding their roles in iron metabolism, hence facilitating further development of medical treatments for its associated diseases.

Optical Monitoring of In Situ Iron Loading into Single, Native Ferritin Proteins.

Ferritin is a protein that stores and releases iron to prevent diseases associated with iron dysregulation in plants, animals, and bacteria. The conversion between iron-loaded holo-ferritin and empty apo-ferritin is an important process for iron regulation. To date, studies of ferritin have used either ensemble measurements to quantify the characteristics of a large number of proteins or single-molecule approaches to interrogate labeled or modified proteins. Here we demonstrate the first real-time study of the dynamics of iron ion loading and biomineralization within a single, unlabeled ferritin protein. Using optical nanotweezers, we trapped single apo- and holo-ferritins indefinitely, distinguished one from the other, and monitored their structural dynamics in real time. The study presented here deepens the understanding of the iron uptake mechanism of ferritin proteins, which may lead to new therapeutics for iron-related diseases.

Triphasic 3D In Vitro Model of Bone-Tendon-Muscle Interfaces to Study Their Regeneration.

The transition areas between different tissues, known as tissue interfaces, have limited ability to regenerate after damage, which can lead to incomplete healing. Previous studies focussed on single interfaces, most commonly bone-tendon and bone-cartilage interfaces. Herein, we develop a 3D in vitro model to study the regeneration of the bone-tendon-muscle interface. The 3D model was prepared from collagen and agarose, with different concentrations of hydroxyapatite to graduate the tissues from bones to muscles, resulting in a stiffness gradient. This graduated structure was fabricated using indirect 3D printing to provide biologically relevant surface topographies. MG-63, human dermal fibroblasts, and Sket.4U cells were found suitable cell models for bones, tendons, and muscles, respectively. The biphasic and triphasic hydrogels composing the 3D model were shown to be suitable for cell growth. Cells were co-cultured on the 3D model for over 21 days before assessing cell proliferation, metabolic activity, viability, cytotoxicity, tissue-specific markers, and matrix deposition to determine interface formations. The studies were conducted in a newly developed growth chamber that allowed cell communication while the cell culture media was compartmentalised. The 3D model promoted cell viability, tissue-specific marker expression, and new matrix deposition over 21 days, thereby showing promise for the development of new interfaces.

Functional bioinspired nanocomposites for anticancer activity with generation of reactive oxygen species.

Cancer is a debilitating and deadly disease caused by the uncontrolled growth of aberrant cell populations. This disease cannot always be controlled with traditional therapies and medicines. Different medicines are being used for this purpose, however these medicines have their side effects and are harmful to healthy cells. A better way to cure cancer disease is by limiting the agglomeration of cancer cells, minimizing their growth and their population by destroying these harmful cells. This could be achieved by controlling the function of mitochondria and DNA in cancer cells with the use of biocompatible materials with tuneable physical properties. Accordingly, research is ongoing as to the use of nanomaterials and nanotechnology in medicine. Zinc oxide semiconductor nanoparticles have displayed good anticancer behaviour. They have unique properties such as biocompatibility, good stability, and are environmentally friendly. Owing to these characteristics, they are focused on biological applications such as drug delivery and cancer therapy. In the present research work, zinc oxide, titanium dioxide nanoparticles and titanium oxide-zinc oxide nanocomposites were successfully trailed for anti-cancer activity. Pure zinc oxide nanoparticles (ZnO NPs), titanium dioxide nanoparticles (TiO2 NPs) and their nanocomposites (TiO2+ZnO NPs) were prepared by the co-precipitation technique. The structural properties were investigated by X-ray diffraction, which confirmed the Wurtzite structure of pure ZnO NPs. The morphology of the NPs was checked by scanning electron microscopy. For incident light having a higher energy band gap of nanomaterials, the electrons are excited to the conduction band and these electrons generate reactive oxygen species (ROS). The efficacy of these nanomaterials was checked by exposing the NPs to the human liver cancer cell HepG2. The MTT assay describes anticancer activity via cell viability. The cell viability of composites was observed to be greater than pure ZnO NPs. Their results showed that the structure of ZnO NPs remains the same with composites of TiO2 NPs, but the band gap of the composite was intermediate for individual samples. It also showed that the anticancer activity of composites was also less than pure ZnO NPs which is due to the reduction of ROS generation. This is observed that nanocomposites of ZnO and TiO2 could be effective in the development of a treatment of human liver cancer cells.

Materials-Based Approach for Interrogating Human Prostate Cancer Cell Adhesion and Migratory Potential Using a Fluoroalkylsilica Culture Surface.

OPCT-1 is a heterogeneous prostate cancer cell line derived from primary (rather than metastatic) disease which contains epithelial, mesenchymal, and CD44high/CD24low cancer stem cell (CSC) subpopulations and from which we have previously generated and characterized stable mesenchymal (P4B6B) and epithelial (P5B3) cell subpopulations. In this contribution, we explore the effect of tissue culture surface chemistry (standard tissue culture plastic (TCP) and a fluoroalkylsilica (FS) culture surface with inherently low surface energy) on the phenotype and adherent capacity of mesenchymal and epithelial cell populations. We demonstrate that OPCT-1 cells adherent to FS surfaces comprise both epithelial- and mesenchymal-like populations; a mesenchymal subpopulation derived from OPCT1 (P4B6B) poorly adheres to FS and formed spheroids, whereas an epithelial subpopulation derived from OPCT1 (P5B3) forms an adherent monolayer. In contrast, P4B6B cells do adhere to FS when cocultured with P5B3 cells. Taken together, these findings demonstrate that EMT/cell differentiation status dictates cell adhesive capacity and provide a novel insight into the relationship between epithelial and mesenchymal cell populations in metastasis. Importantly, the differences in adherence capacity between P4B6B and P5B3 are not apparent using standard TCP-based culture, thereby highlighting the value of using alternative culture surfaces for studying cell surface interaction/adhesion phenomena and interrogating mechanisms involved in adhesion and detachment of metastatic tumor cells.

Distributions of Silica and Biopolymer Structural Components in the Spore Elater of Equisetum arvense, an Ancient Silicifying Plant.

Equisetum species are primitive vascular plants that benefit from the biogenesis of silica bio-organic inclusions in their tissues and participate in the annual biosilica turnover in local eco-systems. As means of Equisetum reproduction and propagation, spores are expected to reflect the evolutionary adaptation of the plants to the climatic conditions at different times of the year. Combining methods of Raman and scanning electron microscopy and assisted with density functional theory, we conducted material spatial-spectral correlations to characterize the distribution of biopolymers and silica based structural elements that contribute to the bio-mineral content of the elater. The elater tip has underlying skeletal-like structural elements where cellulose fibers provide strength and flexibility, both of which are necessary for locomotion. The surface of the elater tips is rich with less ordered pectin like polysaccharide and shows a ridged, folded character. At the surface we observe silica of amorphous, colloidal form in nearly spherical structures where the silica is only a few layers thick. We propose the observed expansion of elater tips upon germination and the form of silica including encapsulated biopolymers are designed for ready dispersion, release of the polysaccharide-arginine rich content and to facilitate silica uptake to the developing plant. This behavior would help to condition local soil chemistry to facilitate competitive rooting potential and stem propagation.

Traditional materials from new sources - conflicts in analytical methods for calcium carbonate.

Calcium carbonate (E170) is a common food and pharmaceutical additive/ingredient. In addition to a source of calcium, the carbonate has uses including as a colour, acidity regulator and bulking agent. Globally, a range of regulatory agencies and pharmacopoeia control the analyses and specification of additives in food, supplements, pharmaceutical substances and excipients. Accordingly, a range of specifications and analyses exist for calcium carbonate depending on the application and market of the product. In this contribution, we analyse calcium carbonates from geological, synthetic and biogenic sources, focussing on acid insoluble impurities, a test required by current monographs. Analysis of calcium carbonate from different origins may require modification of existing tests to comply with regulatory bodies, due to the variation of impurities specific to the source of the material. We suggest an analytical approach involving centrifugation that improves analytical efficiency (up to 85% time reduction), especially for calcium carbonate of biological origin.

Biogenic porous silica and silicon sourced from Mexican Giant Horsetail (Equisetum myriochaetum) and their application as supports for enzyme immobilization.

Porous silica-based materials are attractive for biomedical applications due to their biocompatibility and biodegradable character. In addition, inorganic supports such as porous silicon are being developed due to integrated circuit chip compatibility and tunable properties leading to a wide range of multidisciplinary applications. In this contribution, biosilica extracted from a rarely studied plant material (Equisetum Myriochaetum), its conversion to silicon and the potential for both materials to be used as supports for enzyme immobilization are investigated. E. myriochaetum was subject to conventional acid digestion to extract biogenic silica with a% yield remarkably higher (up to 3 times) than for other Equisetum sp. (i.e. E. Arvense). The surface area of the isolated silica was ∼400 m2/g, suitable for biotechnological applications. Biogenic silicon was obtained by magnesiothermic reduction. The materials were characterized by SEM-EDX, XRD, FT-IR, ICP-OES, TGA and BET analysis and did not contain significant levels of class 1 heavy elements (such as Pb, Cd, Hg and As). Two commercial peroxidases, horseradish peroxidase (HRP) and Coprinus cinereus peroxidase (CiP) were immobilized onto the biogenic materials using three different functionalization routes: (A) carbodiimide, (B) amine + glutaraldehyde and (C) amine + carbodiimide. Although both biogenic silica and porous silicon could be used as supports differences in behaviour were observed for the two enzymes. For HRP, loading onto biogenic silica via the glutaraldehyde immobilization technique (route B) was most effective. The loading of CiP showed a much higher peroxidase activity onto porous silicon than silica functionalized by the carbodiimide method (route A). From the properties of the extracted materials obtained from Equisetum Myriochaetum and the immobilization results observed, these materials appear to be promising for industrial and biomedical applications.

Fluorescently-tagged polyamines for the staining of siliceous materials.

Siliceous frustules of diatom algae contain unique long-chain polyamines, including those having more than six nitrogen atoms. These polyamines participate in the formation of the siliceous frustules of the diatoms but their precise physiological role is not clear. The main hypotheses include formation of a polyamine and polyphosphate supramolecular matrix. We have synthesized novel fluorescent dyes from a synthetic oligomeric mixture of polyamines and the fluorophore 7-nitro-2,1,3-benzoxadiazole. The long polyamine chain ensures the high affinity of these dyes to silica, which allows their application in the staining of siliceous materials, such as valves of diatom algae and fossilized samples from sediments. The fluorescently stained diatom valves were found to be promising liquid flow tracers in hydrodynamic tests. Furthermore, complexation of the polyamine component of the dyes with carbonic polymeric acids results in changes to the visible spectrum of the fluorophore, which allows study of the stability of the complex vs the length of the polyamine chain. Using poly (vinyl phosphonic acid) as a model for phosphate functionality in silaffins (a potential matrix in the formation of biogenic silica) little complexation with the polyamine fluorophores was observed, bringing into question the role of a polyamine - polymeric phosphate matrix in biosilicification.

The Importance and Clinical Relevance of Surfaces in Tissue Culture.

Cell and tissue culture has evolved from the use of simple glassware for the propagation of cells and tissues into a comprehensive platform for interrogating complex biological systems, directing cell fate, and deriving products with clinical and therapeutic value. However, despite significant advances, current in vitro culture approaches remain limited in their capacity to model the clinical/biological complexities of disease, in part at least, due to the deficiencies of existing culture materials. The challenge is therefore to identify innovative materials-based solutions that have greater control over cells in vitro, while better representing biological systems in vivo. Such platforms would be suitable for biomarker discovery and tissue engineering applications. This review examines the development of tissue culture materials, advances in our understanding of cell-surface interactions, and the application of this knowledge toward the development of new approaches for better examining biological events.

Controlling the dynamics of cell transition in heterogeneous cultures using surface chemistry.

Developing materials that can preferentially select defined cancer cell populations for biological characterization will greatly enhance our understanding of cancer cell growth, differentiation, and invasion. The transitional events between epithelial and mesenchymal phenotypes are particularly crucial, as primary tumors and secondary metastasis are generally epithelial in nature, whereas circulating mesenchymal cells derived from primary epithelial cells appear to facilitate the spread of disease and its resistance to therapy. This study describes an amino-functionalized material, which promotes the enrichment of an epithelial phenotype from a single cell line containing both epithelial and mesenchymal subpopulations of cancer cells. The isolation and transitional control of such subpopulations using functional materials will advance understanding of the disease process, have a significant impact on the downstream development of new targeted cancer therapeutics, and also be applicable to tissue engineering and regenerative medicine.

Inference of gene regulatory networks using boolean-network inference methods.

The modeling of genetic networks especially from microarray and related data has become an important aspect of the biosciences. This review takes a fresh look at a specific family of models used for constructing genetic networks, the so-called Boolean networks. The review outlines the various different types of Boolean network developed to date, from the original Random Boolean Network to the current Probabilistic Boolean Network. In addition, some of the different inference methods available to infer these genetic networks are also examined. Where possible, particular attention is paid to input requirements as well as the efficiency, advantages and drawbacks of each method. Though the Boolean network model is one of many models available for network inference today, it is well established and remains a topic of considerable interest in the field of genetic network inference. Hybrids of Boolean networks with other approaches may well be the way forward in inferring the most informative networks.