Baptiste Lamarre

Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.

Cicada-inspired cell-instructive nanopatterned arrays

Biocompatible surfaces hold key to a variety of biomedical problems that are directly related to the competition between host-tissue cell integration and bacterial colonisation. A saving solution to this is seen in the ability of cells to uniquely respond to physical cues on such surfaces thus prompting the search for cell-instructive nanoscale patterns. Here we introduce a generic rationale engineered into biocompatible, titanium, substrates to differentiate cell responses. The rationale is inspired by cicada wing surfaces that display bactericidal nanopillar patterns. The surfaces engineered in this study are titania (TiO2) nanowire arrays that are selectively bactericidal against motile bacteria, while capable of guiding mammalian cell proliferation according to the type of the array. The concept holds promise for clinically relevant materials capable of differential physico-mechanical responses to cellular adhesion.

Differentially Instructive Extracellular Protein Micro-nets

An ability to construct biological matter from the molecule up holds promise for applications ranging from smart materials to integrated biophysical models for synthetic biology. Biomolecular self-assembly is an efficient strategy for biomaterial construction which can be programmed to support desired function. A challenge remains in replicating the strategy synthetically, that is at will, and differentially, that is for a specific function at a given length scale. Here we introduce a self-assembly topology enabling a net-like architectural mimetic of native extracellular matrices capable of differential responses to cell adhesion--enhanced mammalian cell attachment and proliferation, and enhanced resistance to bacterial colonization--at the native sub-millimeter length scales. The biological performance of such protein micro-nets directly correlates with their morphological and chemical properties, offering thus an application model for differential extracellular matrices.

Physicochemical and biological assays for quality control of biopharmaceuticals: Interferon alfa-2 case study

A selection of physicochemical and biological assays were investigated for their utility in detecting changes in preparations of Interferon alpha-2a and Interferon alpha-2b (IFN-alpha 2a, IFN-alpha 2b), which had been subjected to stressed conditions, in order to create models of biopharmaceutical products containing product-related impurities. The stress treatments, which included oxidation of methionine residues and storage at elevated temperatures for different periods of time, were designed to induce various degrees of degradation, aggregation or oxidation of the interferon. Biological activity of the stressed preparations was assessed in three different in vitro cell-based bioassay systems: a late-stage anti-proliferative assay and early-stage assays measuring reporter gene activation or endogenous gene expression by quantitative real time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Relevant physicochemical methods such as SDS-PAGE, reverse phase (RP) chromatography, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), proved their complementarity in detecting structural changes in the stressed preparations which were reflected by reductions in biological activity.

Self-assembling viral mimetics: one long journey with short steps.

Recently, the Foresight Institute has pronounced six economic challenges that can be addressed through the progress of nanotechnology. One of these is the health and longevity of human life. Amongst applications anticipated to provide a solution to this challenge, gene therapy appears to be particularly promising. In theory, many diseases that result from genetic disorders can be cured by correcting defective genes. In practice, finding efficient and safe delivery vectors remains the stumbling point on the path of genetic therapies to the clinic. Viruses, otherwise the most efficient transfectors, pose safety concerns over immune reactions, whereas synthetic gene packages greatly lack the structural integrity of viruses. An ideal vector is therefore seen as a compromise between the two: a nanoscale device, which would mimic a virus and act as a virus, but would do this at the designers whim. A strategy to achieve this is offered by the virus architecture itself, the principles of which are translated into the function via exquisitely reproducible self-assembly mechanisms. Thus, to mimic a virus is to mimic the way it is built, i.e., self-assembly. With just a few attempts made so far, the journey to an artificial virus has had a short lifetime, but the promise it holds is not expected to reduce any time soon.

A De Novo Virus-Like Topology for Synthetic Virions

A de novo topology of virus-like assembly is reported. The design is a trifaceted coiled-coil peptide helix, which self-assembles into ultrasmall, monodisperse, anionic virus-like shells that encapsulate and transfer both RNA and DNA into human cells. Unlike existing artificial systems, these shells share the same physical characteristics of viruses being anionic, nonaggregating, abundant, hollow, and uniform in size, while effectively mediating gene silencing and transgene expression. These are the smallest virus-like structures reported to date, both synthetic and native, with the ability to adapt and transfer small and large nucleic acids. The design thus offers a promising solution for engineering bespoke artificial viruses with desired functions.

Anti-antimicrobial Peptides FOLDING-MEDIATED HOST DEFENSE ANTAGONISTS

Background: Direct antagonists of native antimicrobial peptide (AMP) sequences are unknown. Results: Complementary antagonistic sequences can co-fold with AMPs into functionally inert assemblies. Conclusion: Antagonists act as anti-AMPs. Significance: The findings offer a molecular rationale for anti-AMP responses with potential implications for antimicrobial resistance. Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance.

Anti-antimicrobial Peptides

Background: Direct antagonists of native antimicrobial peptide (AMP) sequences are unknown. Results: Complementary antagonistic sequences can co-fold with AMPs into functionally inert assemblies. Conclusion: Antagonists act as anti-AMPs. Significance: The findings offer a molecular rationale for anti-AMP responses with potential implications for antimicrobial resistance. Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance.

Single Molecule Genotyping by TIRF Microscopy

As part of a programme to develop a metrological framework for single molecule measurements in biology, we have investigated the applications of single molecule imaging to genomics. Specifically, we have developed a technique for measuring the frequencies of single nucleotide polymorphisms (SNPs) in complex or pooled samples of DNA. We believe that this technique has applications to statistical genotyping—the identification of correlations between SNP frequencies and particular phenotypes—and other areas where it is desirable to track the frequencies of SNPs in complex DNA populations.

DNA Origami Inside-Out Viruses

A synthetic topology for everted viruses is reported. The topology is a single-stranded virion DNA assembled into a hollow cube with exterior decorated with HIV-Tat transduction domains. The cube incorporates a pH-responsive lid allowing for the controlled encapsulation of functional proteins and their transfer and release into live cells. Unlike viruses, which are protein shells with a [3,5]-fold rotational symmetry that encase nucleic acids, these cubes are [3, 4]-fold DNA boxes encapsulating proteins. Like viruses, such everted DNA-built viruses are monodisperse nanoscale assemblies that infect human cells with a specialist cargo. The design offers a bespoke bottom-up platform for engineering nonpolyhedral, nonprotein synthetic viruses.