Manu Platt talks about the new NIH Center for Biomedical Engineering Technology Acceleration.

The National Institute of Biomedical Imaging and Bioengineering has established a new Center for Biomedical Engineering Technology Acceleration, dedicated to applying engineering principles to biomedical discovery and therapeutics. We talk to the Center's Director Manu Platt about their plans and the focus on diversity, equity, inclusion and accessibility.

Nebulized lipid nanoparticles.

An article in Nature Biomedical Engineering reports an in vivo workflow for the design of lipid nanoparticles to efficiently deliver mRNA to the lungs via nebulization.

From lipids to lipid nanoparticles to mRNA vaccines.

Lipid nanoparticles are essential to mRNA vaccines. The groundwork for lipid-based drug delivery systems was laid more than 40 years ago in the lab of Pieter Cullis, Professor at the University of British Columbia. Nature Reviews Materials talks to Pieter Cullis about the history and future of lipid nanoparticle-nucleic acid drugs.

Artificially intelligent nanopore for rapid SARS-CoV-2 testing.

An article in Nature Communications reports a method for the rapid detection of SARS-CoV-2 in saliva samples using nanopores and a machine learning algorithm.

Organ chips, organoids and the animal testing conundrum.

Nature Reviews Materials speaks to Donald Ingber, Founding Director of the Wyss Institute for Biologically Inspired Engineering at Harvard University, about the animal testing conundrum and the importance of human-relevant models in biomedical research.

Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate.

Cell size and shape affect cellular processes such as cell survival, growth and differentiation1-4, thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

Scarring vs. functional healing: Matrix-based strategies to regulate tissue repair.

All vertebrates possess mechanisms to restore damaged tissues with outcomes ranging from regeneration to scarring. Unfortunately, the mammalian response to tissue injury most often culminates in scar formation. Accounting for nearly 45% of deaths in the developed world, fibrosis is a process that stands diametrically opposed to functional tissue regeneration. Strategies to improve wound healing outcomes therefore require methods to limit fibrosis. Wound healing is guided by precise spatiotemporal deposition and remodelling of the extracellular matrix (ECM). The ECM, comprising the non-cellular component of tissues, is a signalling depot that is differentially regulated in scarring and regenerative healing. This Review focuses on the importance of the native matrix components during mammalian wound healing alongside a comparison to scar-free healing and then presents an overview of matrix-based strategies that attempt to exploit the role of the ECM to improve wound healing outcomes.

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information.

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin ß1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the ß1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor ß1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Basement membrane fragments in the context of the epithelial-to-mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) enables cells of epithelial phenotype to become motile and change to a migratory mesenchymal phenotype. EMT is known to be a fundamental requisite for tissue morphogenesis, and EMT-related pathways have been described in cancer metastasis and tissue fibrosis. Epithelial structures are marked by the presence of a sheet-like extracellular matrix, the basement membrane, which is assembled from two major proteins, laminin and collagen type IV. This specialized matrix is essential for tissue function and integrity, and provides an important barrier to the potential pathogenic migration of cells. The profound phenotypic transition in EMT involves the epithelial cells disrupting the basement membrane. Matrix metalloproteinases (MMPs) are known to cleave components of basement membranes, but MMP-basement membrane crosstalk during EMT in vivo is poorly understood. However, MMPs have been reported to play a role in EMT-related processes and a variety of basement membrane fragments have been shown to be released by specific MMPs in vitro and in vivo exhibiting distinct biological activities. This review discusses general considerations regarding the basement membrane in the context of EMT, a possible role for specific MMPs in EMT and highlights biologically active basement membrane fragments liberated by MMPs.

Extracellular Stiffness Modulates the Expression of Functional Proteins and Growth Factors in Endothelial Cells.

Angiogenesis, the formation of blood vessels from pre-existing ones, is of vital importance during the early stages of bone healing. Extracellular stiffness plays an important role in regulating endothelial cell behavior and angiogenesis, but how this mechanical cue affects proliferation kinetics, gene regulation, and the expression of proteins implicated in angiogenesis and bone regeneration remains unclear. Using collagen-coated polyacrylamide (PAAm) hydrogels, human umbilical vein endothelial cells (HUVECs) are exposed to an environment that mimics the elastic properties of collagenous bone, and cellular proliferation and gene and protein expressions are assessed. The proliferation and gene expression of HUVECs are not differentially affected by culture on 3 or 30 kPa PAAm hydrogels, henceforth referred to as low and high stiffness gels, respectively. Although the proliferation and gene transcript levels remain unchanged, significant differences are found in the expressions of functional proteins and growth factors implicated both in the angiogenic and osteogenic processes. The down-regulation of the vascular endothelial growth factor receptor-2 protein with concomitant over-expression of caveolin-1, wingless-type 2, bone morphogenic protein 2, and basic fibroblast growth factor on the high stiffness PAAm hydrogel suggests that rigidity has a pro-angiogenic effect with inherent benefits for bone regeneration.

Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration.

Regenerative medicine strategies for restoring articular cartilage face significant challenges to recreate the complex and dynamic biochemical and biomechanical functions of native tissues. As an approach to recapitulate the complexity of the extracellular matrix, collagen-mimetic proteins offer a modular template to incorporate bioactive and biodegradable moieties into a single construct. We modified a Streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate (CS)-binding peptides and then cross-linked with a matrix metalloproteinase 7 (MMP7)-sensitive peptide to form biodegradable hydrogels. Human mesenchymal stem cells (hMSCs) encapsulated in these hydrogels exhibited improved viability and significantly enhanced chondrogenic differentiation compared to controls that were not functionalized with glycosaminoglycan-binding peptides. Hydrogels functionalized with CS-binding peptides also led to significantly higher MMP7 gene expression and activity while the HA-binding peptides significantly increased chondrogenic differentiation of the hMSCs. Our results highlight the potential of this novel biomaterial to modulate cell-mediated processes and create functional tissue engineered constructs for regenerative medicine applications.

A designer peptide as a template for growing Au nanoclusters.

A peptide was designed to generate a sub-nanometric template that guides the growth of fluorescent gold nanoclusters. The peptide was endorsed with nucleating moieties and a three-dimensional structure that arrests the growth of ultrasmall nanoparticles. The nanoclusters are not cytotoxic and can be found in the cytosol of cells.

Model based variable selection as a tool to highlight biological differences in Raman spectra of cells.

In vitro Raman spectroscopy used for non-invasive, non-destructive characterization of single cells and tissues has proven to be a powerful tool for understanding the complex biochemical processes within these biological systems. Additionally it enables the comparison of a wide range of in vitro model systems by discriminating them based on their biomolecular differences. However, one persistent challenge in Raman spectroscopy has been the highly complex structure of cell and tissue spectra, which comprise signals from lipids, proteins, carbohydrates and nucleic acids, which may overlap significantly. This leads to difficulty in discerning which molecular components are responsible for the changes seen between experimental groups. To address this problem, we introduce a technique to highlight the significant biochemical changes between sample groups by applying a novel approach using Partial Least Squares - Discriminant Analysis (PLS-DA) Variable Importance Projection (VIP) scores normally used for variable selection as heat maps combined with group difference spectra to highlight significant differences in Raman band shapes and position. To illustrate this method we analyzed single HeLa cells in their live, fixed, fixed and ethanol dehydrated, to the fixed, dehydrated and then rehydrated states respectively. Fixation, ethanol dehydration and rehydration are known to induce molecular changes in the lipids and proteins within each cell.

Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells.

The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the ß1-chain-derived fragment interacts via α3ß1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs--key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the α3ß1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9.

Nanobiotechnology with S-layer proteins as building blocks.

One of the key challenges in nanobiotechnology is the utilization of self- assembly systems, wherein molecules spontaneously associate into reproducible aggregates and supramolecular structures. In this contribution, we describe the basic principles of crystalline bacterial surface layers (S-layers) and their use as patterning elements. The broad application potential of S-layers in nanobiotechnology is based on the specific intrinsic features of the monomolecular arrays composed of identical protein or glycoprotein subunits. Most important, physicochemical properties and functional groups on the protein lattice are arranged in well-defined positions and orientations. Many applications of S-layers depend on the capability of isolated subunits to recrystallize into monomolecular arrays in suspension or on suitable surfaces (e.g., polymers, metals, silicon wafers) or interfaces (e.g., lipid films, liposomes, emulsomes). S-layers also represent a unique structural basis and patterning element for generating more complex supramolecular structures involving all major classes of biological molecules (e.g., proteins, lipids, glycans, nucleic acids, or combinations of these). Thus, S-layers fulfill key requirements as building blocks for the production of new supramolecular materials and nanoscale devices as required in molecular nanotechnology, nanobiotechnology, biomimetics, and synthetic biology.

Single-molecule force spectroscopy reveals the individual mechanical unfolding pathways of a surface layer protein.

Surface layers (S-layers) represent an almost universal feature of archaeal cell envelopes and are probably the most abundant bacterial cell proteins. S-layers are monomolecular crystalline structures of single protein or glycoprotein monomers that completely cover the cell surface during all stages of the cell growth cycle, thereby performing their intrinsic function under a constant intra- and intermolecular mechanical stress. In gram-positive bacteria, the individual S-layer proteins are anchored by a specific binding mechanism to polysaccharides (secondary cell wall polymers) that are linked to the underlying peptidoglycan layer. In this work, atomic force microscopy-based single-molecule force spectroscopy and a polyprotein approach are used to study the individual mechanical unfolding pathways of an S-layer protein. We uncover complex unfolding pathways involving the consecutive unfolding of structural intermediates, where a mechanical stability of 87 pN is revealed. Different initial extensibilities allow the hypothesis that S-layer proteins adapt highly stable, mechanically resilient conformations that are not extensible under the presence of a pulling force. Interestingly, a change of the unfolding pathway is observed when individual S-layer proteins interact with secondary cell wall polymers, which is a direct signature of a conformational change induced by the ligand. Moreover, the mechanical stability increases up to 110 pN. This work demonstrates that single-molecule force spectroscopy offers a powerful tool to detect subtle changes in the structure of an individual protein upon binding of a ligand and constitutes the first conformational study of surface layer proteins at the single-molecule level.

Monte Carlo study of the molecular mechanisms of surface-layer protein self-assembly.

The molecular mechanisms guiding the self-assembly of proteins into functional or pathogenic large-scale structures can be only understood by studying the correlation between the structural details of the monomer and the eventual mesoscopic morphologies. Among the myriad structural details of protein monomers and their manifestations in the self-assembled morphologies, we seek to identify the most crucial set of structural features necessary for the spontaneous selection of desired morphologies. Using a combination of the structural information and a Monte Carlo method with a coarse-grained model, we have studied the functional protein self-assembly into S(surface)-layers, which constitute the crystallized outer most cell envelope of a great variety of bacterial cells. We discover that only few and mainly hydrophobic amino acids, located on the surface of the monomer, are responsible for the formation of a highly ordered anisotropic protein lattice. The coarse-grained model presented here reproduces accurately many experimentally observed features including the pore formation, chemical description of the pore structure, location of specific amino acid residues at the protein-protein interfaces, and surface accessibility of specific amino acid residues. In addition to elucidating the molecular mechanisms and explaining experimental findings in the S-layer assembly, the present work offers a tool, which is chemical enough to capture details of primary sequences and coarse-grained enough to explore morphological structures with thousands of protein monomers, to promulgate design rules for spontaneous formation of specific protein assemblies.

Atomistic structure of monomolecular surface layer self-assemblies: toward functionalized nanostructures.

The concept of self-assembly is one of the most promising strategies for the creation of defined nanostructures and therefore became an essential part of nanotechnology for the controlled bottom-up design of nanoscale structures. Surface layers (S-layers), which represent the cell envelope of a great variety of prokaryotic cells, show outstanding self-assembly features in vitro and have been successfully used as the basic matrix for molecular construction kits. Here we present the three-dimensional structure of an S-layer lattice based on tetrameric unit cells, which will help to facilitate the directed binding of various molecules on the S-layer lattice, thereby creating functional nanoarrays for applications in nanobiotechnology. Our work demonstrates the successful combination of computer simulations, electron microscopy (TEM), and small-angle X-ray scattering (SAXS) as a tool for the investigation of the structure of self-assembling or aggregating proteins, which cannot be determined by X-ray crystallography. To the best of our knowledge, this is the first structural model at an amino acid level of an S-layer unit cell that exhibits p4 lattice symmetry.

Surface layer protein characterization by small angle x-ray scattering and a fractal mean force concept: from protein structure to nanodisk assemblies.

Surface layers (S-layers) are the most commonly observed cell surface structure of prokaryotic organisms. They are made up of proteins that spontaneously self-assemble into functional crystalline lattices in solution, on various solid surfaces, and interfaces. While classical experimental techniques failed to recover a complete structural model of an unmodified S-layer protein, small angle x-ray scattering (SAXS) provides an opportunity to study the structure of S-layer monomers in solution and of self-assembled two-dimensional sheets. For the protein under investigation we recently suggested an atomistic structural model by the use of molecular dynamics simulations. This structural model is now refined on the basis of SAXS data together with a fractal assembly approach. Here we show that a nondiluted critical system of proteins, which crystallize into monomolecular structures, might be analyzed by SAXS if protein-protein interactions are taken into account by relating a fractal local density distribution to a fractal local mean potential, which has to fulfill the Poisson equation. The present work demonstrates an important step into the elucidation of the structure of S-layers and offers a tool to analyze the structure of self-assembling systems in solution by means of SAXS and computer simulations.