Synthetic Biology Enabled Modification of Bone Cells and Impact on Bone Mineral Resorption
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Hayes, Dulce C.↗
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SYNTHETIC BIOLOGY ENABLED MODIFICATION OF BONE CELLS AND IMPACT ON BONE MINERAL RESORPTION
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Hayes, Dulce C.↗
Nitrene generation and transfer for unnatural biosynthesis in living cells
Abstract Synthetic biology has enabled the production of natural and unnatural products from inexpensive sustainable feedstocks. Yet, the scope of available products has been limited largely to compounds accessible from nature’s chemical reactions. Evolved enzymes can catalyse reactions of unnatural substrates or reactions not found in nature but often require the addition of synthetic reagents to purified enzymes or to resting cells containing those enzymes. Here we show that chemical reactions of metabolic intermediates produced intracellularly in living cells can include intermolecular nitrene transfers. The biosynthesis of N -acetoxyanilines, in combination with the generation and transfer of N -aryl nitrene intermediates from them catalysed by a cytochrome P450, generates amino alcohols, diamines, diarylamines and aminoalkyl arenes from simple carbon feedstocks. These products—common substructures of pharmaceuticals and agrochemicals—are challenging to synthesize by standard organic chemistry and were produced from inexpensive, renewable feedstocks. Evolution of the enzymes in this pathway showed that titres can be increased by engineering and that the products can be synthesized with high enantioselectivity.
Donnell, Isaac↗
Development of a cell surface display system in Chlamydomonas reinhardtii
Cell-surface display systems are biotechnological techniques used to express heterologous proteins on the cell surface. Their application depends directly on the cell system used, as well as on the anchoring point for the surface displayed protein. To meet most application demands an inexpensive, safe, and scalable production platform, that reduces the economic barriers for large scale use is needed. Toward this goal, we screened three possible cell surface anchoring points in the green algae Chlamydomonas by fusing mVenus to prospective anchors moieties. The vectors harboring mVenus:anchor were screened for mVenus fluorescence and tested for cellular localization by confocal laser scanning microscopy. This strategy allowed the identification of two functional anchors, one for the cytoplasmic membrane using the MAW8 GPI-anchor signal, and one for the cell wall using the GP1 protein. We also exploited GP1 chemical and biological traits to release the fused proteins efficiently during cell wall shedding. Our work provides a foundation for surface engineering of C reinhardtii supporting both cell biology studies and biotechnology applications.
59 BASIC BIOLOGICAL SCIENCES↗
Hydrogel-Immobilized Multienzyme Systems for Cell-Free Chemical Bioproduction
Cell-free gene expression systems derived from bacterial lysates enable the expression of biosynthetic pathways from inexpensive and easily prepared DNA templates. These systems hold great promise for modular and on-demand bioproduction of valuable small molecules in resource-limited settings but are constrained in their long-term stability, reusability, and deployability. In this work, we demonstrate that multiple cell-free expressed enzymes can be co-immobilized in biocompatible hydrogels made from poly(ethylene glycol) diacrylate (PEGDA) with added glycerol for enhanced gel integrity. Using small-angle X-ray scattering (SAXS), we show that the mesh size of PEGDA-glycerol hydrogels is comparable to the globular sizes of many proteins and enzymes, which could be used for protein entrapment. We found that the combination between entrapment and chemical ligation of the enzymes was effective to retain proteins. By employing a method for direct fluorescence measurement from hydrogels, we found that proteins can be retained in PEGDA-glycerol for at least a week. By separating the cell-free enzyme expression from the immobilization step, we successfully fabricated enzyme-laden hydrogels with three heterologous cell-free enzymes for the bioconversion of pyruvic acid to malic acid, an industrially valuable and versatile precursor chemical. Both heterologous and endogenous enzymes from the lysate remain functional in photo-cross-linked hydrogels and can be reused for multiple biocatalytic cycles. Moreover, we also found that the immobilized enzymes exhibit up to 1.6-fold higher activity and 2-fold longer lifetimes than free enzymes in liquid reactions. Furthermore, these results could advance the deployment of cell-free synthetic biology because they show that reusable, stable, and durable multienzyme systems can be created using readily available materials and fabrication techniques.
59 BASIC BIOLOGICAL SCIENCES↗
Structure and growth of plant cell walls
Plant cells build nanofibrillar walls that are central to plant growth, morphogenesis and mechanics. Starting from simple sugars, three groups of polysaccharides, namely, cellulose, hemicelluloses and pectins, with very different physical properties are assembled by the cell to make a strong yet extensible wall. Here, this Review describes the physics of wall growth and its regulation by cellular processes such as cellulose production by cellulose synthase, modulation of wall pH by plasma membrane H + -ATPase, wall loosening by expansin and signalling by plant hormones such as auxin and brassinosteroid. In addition, this Review discusses the nuanced roles, properties and interactions of cellulose, matrix polysaccharides and cell wall proteins and describes how wall stress and wall loosening cooperatively result in cell wall growth.
59 BASIC BIOLOGICAL SCIENCES↗
The use of the tyrosine phosphatase antagonist orthovanadate in the study of a cell proliferation inhibitor
Incubation of murine fibroblasts with orthovanadate, a global tyrosine phosphatase inhibitor, was shown to confer a "pseudo-transformed" phenotype with regard to cell morphology and growth characteristics. This alteration was manifested by both an increasing refractile appearance of the cells, consistent with many transformed cell lines, as well as an increase in maximum cell density was attained. Despite the abrogation of cellular tyrosine phosphatase activity, orthovanadate-treated cells remained sensitive to the biological activity of a naturally occurring sialoglycopeptide (SGP) cell surface proliferation inhibitor. The results indicated that tyrosine phosphatase activity, inhibited by orthovanadate, was not involved in the signal transduction pathway of the SGP.
NASA Discipline Number 93-10↗
Toward sustainable, cell-free biomanufacturing
Industrial biotechnology is an attractive approach to address the need for low-cost fuels and products from sustainable resources. Unfortunately, cells impose inherent limitations on the effective synthesis and release of target products. One key constraint is that cellular survival objectives often work against the production objectives of biochemical engineers. Additionally, industrial strains release CO2 and struggle to utilize sustainable, potentially profitable feedstocks. Cell-free biotechnology, which uses biological machinery harvested from cells, can address these challenges with advantages including: (i) shorter development times, (ii) higher volumetric production rates, and (iii) tolerance to otherwise toxic molecules. In this review, we highlight recent advances in cell-free technologies toward the production of non-protein products beyond lab-scale demonstrations and describe guiding principles for designing cell-free systems. Specifically, we discuss carbon and energy sources, reaction homeostasis, and scale-up. Expanding the scope of cell-free biomanufacturing practice could enable innovative approaches for the industrial production of green chemicals.
59 BASIC BIOLOGICAL SCIENCES↗
Cell Extracts from Bacteria and Yeast Retain Metabolic Activity after Extended Storage and Repeated Thawing
Cell-free synthetic biology enables rapid prototyping of biological parts and synthesis of proteins or metabolites in the absence of cell growth constraints. Cell-free systems are frequently made from crude cell extracts, where composition and activity can vary significantly based on source strain, preparation and processing, reagents, and other considerations. This variability can cause extracts to be treated as black boxes for which empirical observations guide practical laboratory practices, including a hesitance to use dated or previously thawed extracts. To better understand the robustness of cell extracts over time, we assessed the activity of cell-free metabolism during storage. As a model, we studied conversion of glucose to 2,3-butanediol. We found that cell extracts from Escherichia coli and Saccharomyces cerevisiae subjected to an 18-month storage period and repeated freeze-thaw cycles retain consistent metabolic activity. Here this work gives users of cell-free systems a better understanding of the impacts of storage on extract behavior.
59 BASIC BIOLOGICAL SCIENCES↗
Proteomic Analysis of Exosomes for Discovery of Protein Biomarkers for Prostate and Bladder Cancer
Extracellular vesicles (EVs) are released by nearly all cell types as part of normal cell physiology, transporting biological cargo, including nucleic acids and proteins, across the cell membrane. In pathological states such as cancer, EV-derived cargo may mirror the altered state of the cell of origin. Exosomes are the smaller, 50–150 nanometer-sized EVs released from fusion of multivesicular endosomes with the plasma membrane. Exosomes play important roles in cell-cell communication and participate in multiple cancer processes, including invasion and metastasis. Therefore, proteomic analysis of exosomes is a promising approach to discover potential cancer biomarkers, even though it is still at an early stage. Herein, we critically review the advances in exosome isolation methods and their compatibility with mass spectrometry (MS)-based proteomic analysis, as well as studies of exosomes in pathogenesis and progression of prostate and bladder cancer, two common urologic cancers whose incidence rates continue to rise annually. As urological tumors, both urine and blood samples are feasible for noninvasive or minimally invasive analysis. A better understanding of the biological cargo and functions of exosomes via high-throughput proteomics will help provide new insights into complex alterations in cancer and provide potential therapeutic targets and personalized treatment for patients.
60 APPLIED LIFE SCIENCES↗
Cell-Type-Specific Proteomics Analysis of a Small Number of Plant Cells by Integrating Laser Capture Microdissection with a Nanodroplet Sample Processing Platform
Plant organs and tissues contain multiple cell types, which are well organized in 3-dimensional structure to efficiently perform physiological functions such as homeostasis, response to environmental perturbation, pathogen infection. It is critically important to perform molecular measurements at the cell-type-specific level to discover mechanisms and unique features of cell populations that govern differentiation and respond to external perturbations. Although mass spectrometry-based proteomics has been demonstrated as an enabling discovery tool to study plant physiology, conventional approaches require millions of cells to generate robust biological conclusions. Such requirements mask the cell-to-cell heterogeneities and limit the comprehensive profiling of plant proteins at spatially resolved and cell-type-specific resolutions. This protocol describes a recently-developed proteomics workflow for studying a small number of plant cells by integrating laser capture microdissection, microfluidic nanodroplet-based sample preparation, with ultrasensitive liquid chromatography-mass spectrometry. Using poplar as a model tree species, we provide detailed protocols, including plant tissue harvest, tissue preparation, cryosectioning, laser microdissection, protein digestion, mass spectrometry measurement, and data analysis. We show the workflow enables the precise identification and quantification of thousands of proteins from hundreds of isolated plant root and leaf cells.
59 BASIC BIOLOGICAL SCIENCES↗
ATM signaling modulates cohesin behavior in meiotic prophase and proliferating cells
Cohesins are ancient and ubiquitous regulators of chromosome architecture and function, but their diverse roles and regulation remain poorly understood. During meiosis, chromosomes are reorganized as linear arrays of chromatin loops around a cohesin axis. This unique organization underlies homolog pairing, synapsis, double-stranded break induction, and recombination. We report that axis assembly in Caenorhabditis elegans is promoted by DNA-damage response (DDR) kinases that are activated at meiotic entry, even in the absence of DNA breaks. Downregulation of the cohesin-destabilizing factor WAPL-1 by ATM-1 promotes axis association of cohesins containing the meiotic kleisins COH-3 and COH-4. ECO-1 and PDS-5 also contribute to stabilizing axis-associated meiotic cohesins. Further, our data suggest that cohesin-enriched domains that promote DNA repair in mammalian cells also depend on WAPL inhibition by ATM. Thus, DDR and Wapl seem to play conserved roles in cohesin regulation in meiotic prophase and proliferating cells.
59 BASIC BIOLOGICAL SCIENCES↗
Image analysis reveals differences in tumor multinucleations in Black and White patients with human papillomavirus‐associated oropharyngeal squamous cell carcinoma
Background Understanding biological differences between different racial groups of human papillomavirus (HPV)‐associated oropharyngeal squamous cell carcinoma (OPSCC) patients, who have differences in terms of incidence, survival, and tumor morphology, can facilitate accurate prognostic biomarkers, which can help develop personalized treatment strategies. Methods This study evaluated whether there were morphologic differences between HPV‐associated tumors from Black and White patients in terms of multinucleation index (MuNI), an image analysis‐derived metric that measures density of multinucleated tumor cells within epithelial regions on hematoxylin–eosin images and previously has been prognostic in HPV‐associated OPSCC patients. In this study, the authors specifically evaluated whether the same MuNI cutoff that was prognostic of overall survival (OS) and disease‐free survival in their previous study, T TR , is valid for Black and White patients, separately. We also evaluated population‐specific cutoffs, T B for Blacks and T W for Whites, for risk stratification. Results MuNI was statistically significantly different between Black (mean, 3.88e–4; median, 3.67e–04) and White patients (mean, 3.36e–04; median, 2.99e–04), with p = .0078. Using T TR , MuNI was prognostic of OS in the entire population with hazard ratio (HR) of 1.71 ( p = .002; 95% confidence interval [CI], 1.21–2.43) and in White patients with HR of 1.72 ( p = .005; 95% CI, 1.18–2.51). Population‐specific cutoff, T W , yielded improved HR of 1.77 ( p = .003; 95% CI, 1.21–2.58) for White patients, whereas T B did not improve risk‐stratification in Black patients with HR of 0.6 ( p = .3; HR, 0.6; 95% CI, 0.2–1.80). Conclusions Histological difference between White and Black patient tumors in terms of multinucleated tumor cells suggests the need for considering population‐specific prognostic biomarkers for personalized risk stratification strategies for HPV‐associated OPSCC patients.
Koyuncu, Can F.↗
Design challenges for space bioreactors
The design of bioreactors for operation under conditions of microgravity presents problems and challenges. Absence of a significant body force such as gravity can have profound consequences for interfacial phenomena. Marangoni convection can no longer be overlooked. Many speculations on the advantages and benefits of microgravity can be found in the literature. Initial bioreactor research considerations for space applications had little regard for the suitability of the designs for conditions of microgravity. Bioreactors can be classified in terms of their function and type of operation. The complex interaction of parameters leading to optimal design and operation of a bioreactor is illustrated by the JSC mammalian cell culture system. The design of a bioreactor is strongly dependent upon its intended use as a production unit for cell mass and/or biologicals or as a research reactor for the study of cell growth and function. Therefore a variety of bioreactor configurations are presented in rapid summary. Following this, a rationale is presented for not attempting to derive key design parameters such as the oxygen transfer coefficient from ground-based data. A set of themes/objectives for flight experiments to develop the expertise for design of space bioreactors is then proposed for discussion. These experiments, carried out systematically, will provide a database from which engineering tools for space bioreactor design will be derived.
Seshan, P. K.↗
Advances in mass spectrometry-enabled multiomics at single-cell resolution
We report biological organisms are multifaceted, intricate systems where slight perturbations can result in extensive changes in gene expression, protein abundance and/or activity, and metabolic flux. These changes occur at different timescales, spatially across cells of heterogeneous origins, and within single-cells. Hence, multimodal measurements at the smallest biological scales are necessary to capture dynamic changes in heterogeneous biological systems. Of the analytical techniques used to measure biomolecules, mass spectrometry (MS) has proven to be a powerful option due to its sensitivity, robustness, and flexibility with regard to the breadth of biomolecules that can be analyzed. Recently, many studies have coupled MS to other analytical techniques with the goal of measuring multiple modalities from the same single-cell. It is with these concepts in mind that we focus this review on MS-enabled multiomic measurements at single-cell or near-single- cell resolution.
47 OTHER INSTRUMENTATION↗
Feature-agnostic metabolomics for determining effective subcytotoxic doses of common pesticides in human cells
Although classical molecular biology assays can provide a measure of cellular response to chemical challenges, they rely on a single biological phenomenon to infer a broader measure of cellular metabolic response. These methods do not always afford the necessary sensitivity to answer questions of subcytotoxic effects, nor do they work for all cell types. Likewise, boutique assays such as cardiomyocyte beat rate may indirectly measure cellular metabolic response, but they too, are limited to measuring a specific biological phenomenon and are often limited to a single cell type. For these reasons, toxicological researchers need new approaches to determine metabolic changes across various doses in differing cell types, especially within the low-dose regime. Here, the data collected herein demonstrate that LC-MS/MS-based untargeted metabolomics with a feature-agnostic view of the data, combined with a suite of statistical methods including an adapted environmental threshold analysis, provides a versatile, robust, and holistic approach to directly monitoring the overall cellular metabolomic response to pesticides. When employing this method in investigating two different cell types, human cardiomyocytes and neurons, this approach revealed separate subcytotoxic metabolomic responses at doses of 0.1 and 1 µM of chlorpyrifos and carbaryl. These findings suggest that this agnostic approach to untargeted metabolomics can provide a new tool for determining effective dose by metabolomics of chemical challenges, such as pesticides, in a direct measurement of metabolomic response that is not cell type-specific or observable using traditional assays.
59 BASIC BIOLOGICAL SCIENCES↗
Evaluation of Late Effects of Heavy-Ion Radiation on Mesenchymal Stem Cells
The overall objective of this recently funded study is to utilize well-characterized model test systems to assess the impact of pluripotent stem cell differentiation on biological effects associated with high-energy charged particle radiation. These stem cells, specifically mesenchymal stem cells (MSCs), have the potential for differentiation into bone, cartilage, fat, tendons, and other tissue types. The characterization of the regulation mechanisms of MSC differentiation to the osteoblastic lineage by transcription factors, such as Runx2/Cbfa1 and Osterix, and osteoinductive proteins such as members of the bone morphogenic protein family are well established. More importantly, for late biological effects, MSCs have been shown to contribute to tissue restructuring and repair after tissue injury. The complex regulation of and interactions between inflammation and repair determine the eventual outcome of the responses to tissue injury, for which MSCs play a crucial role. Additionally, MSCs have been shown to respond to reactive oxygen species, a secondary effector of radiation, by differentiating. With this, we hypothesized that differentiation of MSCs can alter or exacerbate the damage initiated by radiation, which can ultimately lead to late biological effects of misrepair/fibrosis which may ultimately lead to carcinogenesis. Currently, studies are underway to examine high-energy X-ray radiation at low and high doses, approximately 20 and 200 Rad, respectively, on cytogenetic damage and gene modulation of isolated MSCs. These cells, positive for MSC surface markers, were obtained from three persons. In vitro cell samples were harvested during cellular proliferation and after both cellular recovery and differentiation. Future work will use established in vitro models of increasing complexity to examine the value of traditional 2D tissue-culture techniques, and utilize 3D in vitro tissue culture techniques that can better assess late effects associated with radiation.
Gonda, S.R.↗
Cell-based composite materials with programmed structures and functions
The present invention is directed to the use of silicic acid to transform biological materials, including cellular architecture into inorganic materials to provide biocomposites (nanomaterials) with stabilized structure and function. In the present invention, there has been discovered a means to stabilize the structure and function of biological materials, including cells, biomolecules, peptides, proteins (especially including enzymes), lipids, lipid vesicles, polysaccharides, cytoskeletal filaments, tissue and organs with silicic acid such that these materials may be used as biocomposites. In many instances, these materials retain their original biological activity and may be used in harsh conditions which would otherwise destroy the integrity of the biological material. In certain instances, these biomaterials may be storage stable for long periods of time and reconstituted after storage to return the biological material back to its original form. In addition, by exposing an entire cell to form CSCs, the CSCs may function to provide a unique system to study enzymes or a cascade of enzymes which are otherwise unavailable.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗