RESUMO
Nature has inspired the development of biomimetic membrane sensors in which the functionalities of biological molecules, such as proteins and lipids, are harnessed for sensing applications. This review provides an overview of the recent developments for biomembrane sensors compatible with either bulk or planar sensing applications, namely using lipid vesicles or supported lipid bilayers, respectively. We first describe the individual components required for these sensing platforms and the design principles that are considered when constructing them, and we segue into recent applications being implemented across multiple fields. Our goal for this review is to illustrate the versatility of nature's biomembrane toolbox and simultaneously highlight how biosensor platforms can be enhanced by harnessing it.
Assuntos
Técnicas Biossensoriais , Bicamadas Lipídicas , Bicamadas Lipídicas/química , Humanos , Proteínas/análise , Proteínas/químicaRESUMO
Viral mutations frequently outpace technologies used to detect harmful variants. Given the continual emergence of SARS-CoV-2 variants, platforms that can identify the presence of a virus and its propensity for infection are needed. Our electronic biomembrane sensing platform recreates distinct SARS-CoV-2 host cell entry pathways and reports the progression of entry as electrical signals. We focus on two necessary entry processes mediated by the viral Spike protein: virus binding and membrane fusion, which can be distinguished electrically. We find that closely related variants of concern exhibit distinct fusion signatures that correlate with trends in cell-based infectivity assays, allowing us to report quantitative differences in their fusion characteristics and hence their infectivity potentials. We use SARS-CoV-2 as our prototype, but we anticipate that this platform can extend to other enveloped viruses and cell lines to quantifiably assess virus entry.
Assuntos
COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/virologia , Fusão de Membrana , Sistema Livre de Células , Mutação , Ligação ViralRESUMO
Chemical permeation enhancers (CPEs) represent a prevalent and safe strategy to enable noninvasive drug delivery across skin-like biological barriers such as the tympanic membrane (TM). While most existing CPEs interact strongly with the lipid bilayers in the stratum corneum to create defects as diffusion paths, their interactions with the delivery system, such as polymers forming a hydrogel, can compromise gelation, formulation stability, and drug diffusion. To overcome this challenge, differing interactions between CPEs and the hydrogel system are explored, especially those with sodium dodecyl sulfate (SDS), an ionic surfactant and a common CPE, and those with methyl laurate (ML), a nonionic counterpart with a similar length alkyl chain. Notably, the use of ML effectively decouples permeation enhancement from gelation, enabling sustained delivery across TMs to treat acute otitis media (AOM), which is not possible with the use of SDS. Ciprofloxacin and ML are shown to form a pseudo-surfactant that significantly boosts transtympanic permeation. The middle ear ciprofloxacin concentration is increased by 70-fold in vivo in a chinchilla AOM model, yielding superior efficacy and biocompatibility than the previous highest-performing formulation. Beyond improved efficacy and biocompatibility, this single-CPE formulation significantly accelerates its progression toward clinical deployment.
RESUMO
Benthic invertebrates are important trophic links in food webs and useful bioindicators of environmental conditions, but long-term benthic organism abundance data across broad geographic areas are rare and historic datasets are often not readily accessible. This dataset provides densities of benthic macroinvertebrates collected from 1930 to 2019 during surveys in Lake Erie, a Laurentian Great Lake. The surveys were funded by the governments of the United States and Canada to investigate the status and changes in the benthic community. From the total of 21 lake-wide and basin-wide benthic surveys conducted in Lake Erie from 1929 to 2019, we were able to acquire data for 17 surveys, including species-level data for 10 surveys and data by higher taxonomic groups for seven surveys. Our amassed Lake Erie dataset includes data from 11 surveys (including five with species-level data) conducted in the western basin in 1930-2019, seven surveys (six with species-level data) in the central basin, and eight surveys (seven with species-level data) in the eastern basin (1973-2019). This Lake Erie dataset represents the most extensive temporal dataset of benthic invertebrates available for any of the Laurentian Great Lakes. Benthic samples were collected using Ponar or Shipek bottom dredges and taxa densities were calculated as individuals per square meter using the area of the dredge. Density data are provided for taxa in the Annelida, Arthropoda, Mollusca, Cnidaria, Nemertea, and Platyhelminthes phyla. Current taxonomy was used for most groups but, in a few cases, older taxonomic names were used for consistency with historical data. Analysis of this dataset indicates that eutrophication, water quality improvement, and dreissenid introduction were the major drivers of changes in the benthic community in the western basin, while hypoxia was a major factor in the central basin, and dreissenid introduction was the most important driver in the eastern basin. Considering the rarity of high taxonomic resolution long-term benthic data for lake ecosystems, this dataset could be useful to explore broader aspects of ecological theory, including effects of eutrophication, hypoxia, invasive species, and other factors on community organization, phylogenetic and functional diversity, and spatial and temporal scales of variation in community structure. In addition, the dataset could be useful for studies on individual species, including abundance and distribution, species co-occurrence, and how the patterns of dominance and rarity change over space and time. Use of this dataset for academic or educational purposes is encouraged as long as this data paper is properly cited.
Assuntos
Invertebrados , Lagos , Animais , Invertebrados/fisiologia , Invertebrados/classificação , Biodiversidade , Densidade Demográfica , Fatores de Tempo , Monitoramento AmbientalRESUMO
The cell plasma membrane is a two-dimensional, fluid mosaic material composed of lipids and proteins that create a semipermeable barrier defining the cell from its environment. Compared with soluble proteins, the methodologies for the structural and functional characterization of membrane proteins are challenging. An emerging tool for studies of membrane proteins in mammalian systems is a "plasma membrane on a chip," also known as a supported lipid bilayer. Here, we create the "plant-membrane-on-a-chip,â³ a supported bilayer made from the plant plasma membranes of Arabidopsis thaliana, Nicotiana benthamiana, or Zea mays. Membrane vesicles from protoplasts containing transgenic membrane proteins and their native lipids were incorporated into supported membranes in a defined orientation. Membrane vesicles fuse and orient systematically, where the cytoplasmic side of the membrane proteins faces the chip surface and constituents maintain mobility within the membrane plane. We use plant-membrane-on-a-chip to perform fluorescent imaging to examine protein-protein interactions and determine the protein subunit stoichiometry of FLOTILLINs. We report here that like the mammalian FLOTILLINs, FLOTILLINs expressed in Arabidopsis form a tetrameric complex in the plasma membrane. This plant-membrane-on-a-chip approach opens avenues to studies of membrane properties of plants, transport phenomena, biophysical processes, and protein-protein and protein-lipid interactions in a convenient, cell-free platform.
RESUMO
The SARS-CoV-1 spike glycoprotein contains a fusion peptide (FP) segment that mediates the fusion of the viral and host cell membranes. Calcium ions are thought to position the FP optimally for membrane insertion by interacting with negatively charged residues in this segment (E801, D802, D812, E821, D825, and D830); however, which residues bind to calcium and in what combinations supportive of membrane insertion are unknown. Using biological assays and molecular dynamics studies, we have determined the functional configurations of FP-Ca2+ binding that likely promote membrane insertion. We first individually mutated the negatively charged residues in the SARS CoV-1 FP to assay their roles in cell entry and syncytia formation, finding that charge loss in the D802A or D830A mutants greatly reduced syncytia formation and pseudoparticle transduction of VeroE6 cells. Interestingly, one mutation (D812A) led to a modest increase in cell transduction, further indicating that FP function likely depends on calcium binding at specific residues and in specific combinations. To interpret these results mechanistically and identify specific modes of FP-Ca2+ binding that modulate membrane insertion, we performed molecular dynamics simulations of the SARS-CoV-1 FP and Ca2+ions. The preferred residue pairs for Ca2+ binding we identified (E801/D802, E801/D830, and D812/E821) include the two residues found to be essential for S function in our biological studies (D802 and D830). The three preferred Ca2+ binding pairs were also predicted to promote FP membrane insertion. We also identified a Ca2+ binding pair (E821/D825) predicted to inhibit FP membrane insertion. We then carried out simulations in the presence of membranes and found that binding of Ca2+ to SARS-CoV-1 FP residue pairs E801/D802 and D812/E821 facilitates membrane insertion by enabling the peptide to adopt conformations that shield the negative charges of the FP to reduce repulsion by the membrane phospholipid headgroups. This calcium binding mode also optimally positions the hydrophobic LLF region of the FP for membrane penetration. Conversely, Ca2+ binding to the FP E801/D802 and D821/D825 pairs eliminates the negative charge screening and instead creates a repulsive negative charge that hinders membrane penetration of the LLF motif. These computational results, taken together with our biological studies, provide an improved and nuanced mechanistic understanding of the dymanics of SARS-CoV-1 calcium binding and their potential effects on host cell entry.
Assuntos
Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Sequência de Aminoácidos , Cálcio/metabolismo , Fusão de Membrana/fisiologia , Peptídeos/química , ÍonsRESUMO
As membrane-mediated antibiotic resistance continues to evolve in Gram-positive bacteria, the development of new approaches to elucidate the membrane properties involved in antibiotic resistance has become critical. Membrane vesicles (MVs) secreted by the cytoplasmic membrane of Gram-positive bacteria contain native components, preserving lipid and protein diversity, nucleic acids, and sometimes virulence factors. Thus, MV-derived membrane platforms present a great model for Gram-positive bacterial membranes. In this work, we report the development of a planar bacterial cytoplasmic membrane-based biosensor using MVs isolated from the Bacillus subtilis WT strain that can be coated on multiple surface types such as glass, quartz crystals, and polymeric electrodes, fostering the multimodal assessment of drug-membrane interactions. Retention of native membrane components such as lipoteichoic acids, lipids, and proteins is verified. This biosensor replicates known interaction patterns of the antimicrobial compound, daptomycin, with the Gram-positive bacterial membrane, establishing the applicability of this platform for carrying out biophysical characterization of the interactions of membrane-acting antibiotic compounds with the bacterial cytoplasmic membrane. We report changes in membrane viscoelasticity and permeability that correspond to partial membrane disruption when calcium ions are present with daptomycin but not when these ions are chelated. This biomembrane biosensing platform enables an assessment of membrane biophysical characteristics during exposure to antibiotic drug candidates to aid in identifying compounds that target membrane disruption as a mechanism of action.
Assuntos
Antibacterianos , Daptomicina , Antibacterianos/farmacologia , Bactérias Gram-Positivas , Membrana Celular , ÍonsRESUMO
Cyclodextrin molecules are increasingly being used in biological research and as therapeutic agents to alter membrane cholesterol content, yet there is much to learn about their interactions with cell membranes. We present a biomembrane-based organic electronic platform capable of detecting interactions of cell membrane constituents with methyl-ß-cyclodextrin (MßCD). This approach enables label-free sensing and quantification of changes in membrane integrity resulting from such interactions. In this work, we employ cholesterol-containing supported lipid bilayers (SLBs) formed on conducting polymer-coated electrodes to investigate how MßCD impacts membrane resistance. By examining the outcomes of MßCD interactions with SLBs of varying cholesterol content, we demonstrate that changes in membrane permeability or resistance can be used as a functional measure for predicting cyclodextrin-mediated cholesterol extraction from cellular membranes. Furthermore, we use the SLB platforms to electronically monitor cholesterol delivery to membranes following exposure to MßCD pre-loaded with cholesterol, observing that cholesterol enrichment is commensurate with an increase in resistance. This biomembrane-based bioelectronic sensing system offers a tool to quantify the modulation of membrane cholesterol content using membrane resistance and provides information regarding MßCD-mediated changes in membrane integrity. Given the importance of membrane integrity for barrier function in cells, such knowledge is essential for our fundamental understanding of MßCD as a membrane cholesterol modulator and therapeutic delivery vehicle.
Assuntos
Ciclodextrinas , Impedância Elétrica , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismoRESUMO
A promising new class of biosensors leverages the sensing mechanisms of living cells by incorporating native transmembrane proteins into biomimetic membranes. Conducting polymers (CPs) can further improve the detection of electrochemical signals from these biological recognition elements through their low electrical impedance. Supported lipid bilayers (SLBs) on CPs mimic the structure and biology of the cell membrane to enable such sensing, but their extrapolation to new target analytes and healthcare applications has been difficult due to their poor stability and limited membrane properties. Blending native phospholipids with synthetic block copolymers to create a hybrid SLB (HSLB) may address these challenges by allowing for the tuning of chemical and physical properties during membrane design. We establish the first example of HSLBs on a CP device and show that polymer incorporation enhances bilayer resilience and thus offers important benefits toward bio-hybrid bioelectronics for sensing applications. Importantly, HSLBs outperform traditional phospholipid bilayers in stability by exhibiting strong electrical sealing after exposure to physiologically relevant enzymes that cause phospholipid hydrolysis and membrane degradation. We investigate the impact of HSLB composition on membranes and device performance and demonstrate the ability to finely adjust the lateral diffusivity of HSLBs with modest changes in block copolymer content through a large compositional range. The inclusion of the block copolymer into the bilayer does not disrupt electrical sealing on CP electrodes, an essential metric for electrochemical sensors, or the insertion of a representative transmembrane protein. This work interfacing tunable and stable HSLBs with CPs paves the way for future bioinspired sensors that combine the exciting developments from both bioelectronics and synthetic biology.
Assuntos
Técnicas Biossensoriais , Bicamadas Lipídicas , Bicamadas Lipídicas/química , Membrana Celular/química , Polímeros/química , Proteínas de Membrana/análise , FosfolipídeosRESUMO
Tumor-derived extracellular vesicles (TEVs) induce the epithelial-to-mesenchymal transition (EMT) in nonmalignant cells to promote invasion and cancer metastasis, representing a novel therapeutic target in a field severely lacking in efficacious antimetastasis treatments. However, scalable technologies that allow continuous, multiparametric monitoring for identifying metastasis inhibitors are absent. Here, the development of a functional phenotypic screening platform based on organic electrochemical transistors (OECTs) for real-time, noninvasive monitoring of TEV-induced EMT and screening of antimetastatic drugs is reported. TEVs derived from the triple-negative breast cancer cell line MDA-MB-231 induce EMT in nonmalignant breast epithelial cells (MCF10A) over a nine-day period, recapitulating a model of invasive ductal carcinoma metastasis. Immunoblot analysis and immunofluorescence imaging confirm the EMT status of TEV-treated cells, while dual optical and electrical readouts of cell phenotype are obtained using OECTs. Further, heparin, a competitive inhibitor of cell surface receptors, is identified as an effective blocker of TEV-induced EMT. Together, these results demonstrate the utility of the platform for TEV-targeted drug discovery, allowing for facile modeling of the transient drug response using electrical measurements, and provide proof of concept that inhibitors of TEV function have potential as antimetastatic drug candidates.
Assuntos
Neoplasias da Mama , Vesículas Extracelulares , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Detecção Precoce de Câncer , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Transição Epitelial-Mesenquimal/genética , Movimento Celular , Melanoma Maligno CutâneoRESUMO
Despite advances in membrane protein (MP) structural biology and a growing interest in their applications, these proteins remain challenging to study. Progress has been hindered by the complex nature of MPs and innovative methods will be required to circumvent technical hurdles. Cell-free protein synthesis (CFPS) is a burgeoning technique for synthesizing MPs directly into a membrane environment using reconstituted components of the cellular transcription and translation machinery in vitro. We provide an overview of CFPS and how this technique can be applied to the synthesis and study of MPs. We highlight numerous strategies including synthesis methods and folding environments, each with advantages and limitations, to provide a survey of how CFPS techniques can advance the study of MPs.
Assuntos
Proteínas de Membrana , Biossíntese de Proteínas , Proteínas de Membrana/metabolismo , Sistema Livre de Células/química , Sistema Livre de Células/metabolismoRESUMO
Entry of influenza A viruses (IAVs) into host cells is initiated by binding to sialic acids (Sias), their primary host cell receptor, followed by endocytosis and membrane fusion to release the viral genome into the cytoplasm of the host cell. Host tropism is affected by these entry processes, with a primary factor being receptor specificity. Sias exist in several different chemical forms, including the hydroxylated N-glycolylneuraminic acid (Neu5Gc), which is found in many hosts; however, it has not been clear how modified Sias affect viral binding and entry. Neu5Gc is commonly found in many natural influenza hosts, including pigs and horses, but not in humans or ferrets. Here, we engineered HEK293 cells to express the hydoxylase gene (CMAH) that converts Neu5Ac to Neu5Gc, or knocked out the Sia-CMP transport gene (SLC35A1), resulting in cells that express 95% Neu5Gc or minimal level of Sias, respectively. H3N2 (X-31) showed significantly reduced infectivity in Neu5Gc-rich cells compared to wild-type HEK293 (>95% Neu5Ac). To determine the effects on binding and fusion, we generated supported lipid bilayers (SLBs) derived from the plasma membranes of these cells and carried out single particle microscopy. H3N2 (X-31) exhibited decreased binding to Neu5Gc-containing SLBs, but no significant difference in H3N2 (X-31)'s fusion kinetics to either SLB type, suggesting that reduced receptor binding does not affect subsequent membrane fusion. This finding suggests that for this virus to adapt to host cells rich in Neu5Gc, only receptor affinity changes are required without further adaptation of virus fusion machinery. IMPORTANCE Influenza A virus (IAV) infections continue to threaten human health, causing over 300,000 deaths yearly. IAV infection is initiated by the binding of influenza glycoprotein hemagglutinin (HA) to host cell sialic acids (Sias) and the subsequent viral-host membrane fusion. Generally, human IAVs preferentially bind to the Sia N-acetylneuraminic acid (Neu5Ac). Yet, other mammalian hosts, including pigs, express diverse nonhuman Sias, including N-glycolylneuraminic acid (Neu5Gc). The role of Neu5Gc in human IAV infections in those hosts is not well-understood, and the variant form may play a role in incidents of cross-species transmission and emergence of new epidemic variants. Therefore, it is important to investigate how human IAVs interact with Neu5Ac and Neu5Gc. Here, we use membrane platforms that mimic the host cell surface to examine receptor binding and membrane fusion events of human IAV H3N2. Our findings improve the understanding of viral entry mechanisms that can affect host tropism and virus evolution.
Assuntos
Interações entre Hospedeiro e Microrganismos , Vírus da Influenza A Subtipo H3N2 , Ácidos Siálicos , Internalização do Vírus , Animais , Humanos , Células HEK293 , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/metabolismo , Fusão de Membrana , Proteínas de Transporte de Nucleotídeos/genética , Proteínas de Transporte de Nucleotídeos/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/farmacologia , Imagem Individual de Molécula , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologiaRESUMO
We address the challenge of understanding how hydrophobic interactions are encoded by fusion peptide (FP) sequences within coronavirus (CoV) spike proteins. Within the FPs of severe acute respiratory syndrome CoV 2 and Middle East respiratory syndrome CoV (MERS-CoV), a largely conserved peptide sequence called FP1 (SFIEDLLFNK and SAIEDLLFDK in SARS-2 and MERS, respectively) has been proposed to play a key role in encoding hydrophobic interactions that drive viral-host cell membrane fusion. Although a non-polar triad (Leu-Leu-Phe (LLF)) is common to both FP1 sequences, and thought to dominate the encoding of hydrophobic interactions, FP1 from SARS-2 and MERS differ in two residues (Phe 2 versus Ala 2 and Asn 9 versus Asp 9, respectively). Here we explore whether single-molecule force measurements can quantify hydrophobic interactions encoded by FP1 sequences, and then ask whether sequence variations between FP1 from SARS-2 and MERS lead to significant differences in hydrophobic interactions. We find that both SARS-2 and MERS wild-type FP1 generate measurable hydrophobic interactions at the single-molecule level, but that SARS-2 FP1 encodes a substantially stronger hydrophobic interaction than its MERS counterpart (1.91 ± 0.03 nN versus 0.68 ± 0.03 nN, respectively). By performing force measurements with FP1 sequences with single amino acid substitutions, we determine that a single-residue mutation (Phe 2 versus Ala 2) causes the almost threefold difference in the hydrophobic interaction strength generated by the FP1 of SARS-2 versus MERS, despite the presence of LLF in both sequences. Infrared spectroscopy and circular dichroism measurements support the proposal that the outsized influence of Phe 2 versus Ala 2 on the hydrophobic interaction arises from variation in the secondary structure adopted by FP1. Overall, these insights reveal how single-residue diversity in viral FPs, including FP1 of SARS-CoV-2 and MERS-CoV, can lead to substantial changes in intermolecular interactions proposed to play a key role in viral fusion, and hint at strategies for regulating hydrophobic interactions of peptides in a range of contexts.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Coronavírus da Síndrome Respiratória do Oriente Médio , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , COVID-19 , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Peptídeos/química , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Internalização do VírusRESUMO
Assembling transmembrane proteins on organic electronic materials is one promising approach to couple biological functions to electrical readouts. A biosensing device produced in such a way would enable both the monitoring and regulation of physiological processes and the development of new analytical tools to identify drug targets and new protein functionalities. While transmembrane proteins can be interfaced with bioelectronics through supported lipid bilayers (SLBs), incorporating functional and oriented transmembrane proteins into these structures remains challenging. Here, we demonstrate that cell-free expression systems allow for the one-step integration of an ion channel into SLBs assembled on an organic conducting polymer, poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). Using the large conductance mechanosensitive channel (MscL) as a model ion channel, we demonstrate that MscL adopts the correct orientation, remains mobile in the SLB, and is active on the polyelectrolyte surface using optical and electrical readouts. This work serves as an important illustration of a rapidly assembled bioelectronic platform with a diverse array of downstream applications, including electrochemical sensing, physiological regulation, and screening of transmembrane protein modulators.
Assuntos
Técnicas Biossensoriais , Bicamadas Lipídicas , Bicamadas Lipídicas/metabolismo , Canais Iônicos , Proteínas de Membrana/metabolismo , Eletrônica , EletrodosRESUMO
The Laurentian Great Lakes have experienced multiple anthropogenic changes in the past century, including cultural eutrophication, phosphorus abatement initiatives, and the introduction of invasive species. Lake Ontario, the most downstream lake in the system, is considered to be among the most impaired. The benthos of Lake Ontario has been studied intensively in the last six decades and can provide insights into the impact of environmental changes over time. We used multivariate community analyses to examine temporal changes in community composition over the last 54 years, and to assess the major drivers of long-term changes in benthos. The benthic community of Lake Ontario underwent significant transformations that correspond with three major periods. The first period, termed the pre/early Dreissena period (1964-1990), was characterized by high densities of Diporeia, Sphaeriidae, and Tubificidae. During the next period defined by zebra mussel dominance (the 1990s) the same groups were still prevalent, but at altered densities. In the most recent period (2000s to present), which is characterized by the dominance and proliferation of quagga mussels deeper into the lake, the community has changed dramatically: Diporeia almost completely disappeared, Sphaeriidae have greatly declined, and densities of quagga mussels, Oligochaeta and Chironomidae have increased. The introduction of invasive dreissenids has changed the Lake Ontario benthic community, historically dominated by Diporeia, Oligochaeta and Sphaeriidae, to a community dominated by quagga mussels and Oligochaeta. Dreissenids, especially the quagga mussel, were the major drivers of these changes over the last half century.
RESUMO
The rise of antibiotic resistance is a growing worldwide human health issue, with major socioeconomic implications. An understanding of the interactions occurring at the bacterial membrane is crucial for the generation of new antibiotics. Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles have been used to mimic these membranes, but their utility has been restricted by the simplistic nature of these systems. A breakthrough in the field has come with the use of outer membrane vesicles derived from Gram-negative bacteria to form SLBs, thus providing a more physiologically relevant system. These complex bilayer systems hold promise but have not yet been fully characterized in terms of their composition, ratio of natural to synthetic components, and membrane protein content. Here, we use correlative atomic force microscopy (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the high resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the enhanced resolution that AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, and we can quantify the amount of bacterial membrane incorporated into the bilayer. We prove the system's tunability by generating bilayers made using OMVs engineered to contain a green fluorescent protein (GFP) binding nanobody fused with the porin OmpA. We are able to directly visualize protein-protein interactions between GFP and the nanobody complex. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs to generate a high-resolution platform for investigating bacterial membrane interactions for the development of next-generation antibiotics.
Assuntos
Membrana Externa Bacteriana , Bicamadas Lipídicas , Antibacterianos , Escherichia coli , Proteínas de Fluorescência Verde , Humanos , Bicamadas Lipídicas/química , Microscopia de Força AtômicaRESUMO
Based on its predicted ability to affect transmissibility and pathogenesis, surveillance studies have highlighted the role of a specific mutation (P681R) in the S1/S2 furin cleavage site of the SARS-CoV-2 spike protein. Here we analyzed A.23.1, first identified in Uganda, as a P681R-containing virus several months prior to the emergence of B.1.617.2 (Delta variant). We performed assays using peptides mimicking the S1/S2 from A.23.1 and B.1.617 and observed significantly increased cleavability with furin compared to both an original B lineage (Wuhan-Hu1) and B.1.1.7 (Alpha variant). We also performed cell-cell fusion and functional infectivity assays using pseudotyped particles and observed an increase in activity for A.23.1 compared to an original B lineage spike. However, these changes in activity were not reproduced in the B lineage spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution, this substitution needs to occur on the background of other spike protein changes to enable its functional consequences. IMPORTANCE During the course of the SARS-CoV-2 pandemic, viral variants have emerged that often contain notable mutations in the spike gene. Mutations that encode changes in the spike S1/S2 (furin) activation site have been considered especially impactful. The S1/S2 change from proline to arginine at position 681 (P681R) first emerged in the A.23.1 variant in Uganda, and subsequently occurred in the more widely transmitted Delta variant. We show that the A.23.1 spike is more readily activated by the host cell protease furin, but that this is not reproduced in an original SARS-CoV-2 spike containing the P681R mutation. Changes to the S1/S2 (furin) activation site play a role in SARS-CoV-2 infection and spread, but successful viruses combine these mutations with other less well identified changes, occurring as part of natural selection.
Assuntos
COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , COVID-19/virologia , Furina/genética , Furina/metabolismo , Humanos , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , UgandaRESUMO
Photosynthetic semiconductor biohybrids (PSBs) convert light energy to chemical energy through photo-driven charge transfer from nanocrystals to microorganisms that perform bioreactions of interest. Initial proof-of-concept PSB studies with an emphasis on enhanced CO2 conversion have been encouraging; however, bringing the broad prospects of PSBs to fruition is contingent on establishing a firm fundamental understanding of underlying interfacial charge transfer processes. We introduce a bioelectronic platform that reduces the complexity of PSBs by focusing explicitly on interactions between colloidal quantum dots (QDs), microbial outer membranes, and native, small-molecule redox mediators. Our model platform employs a standard three-electrode electrochemical cell with supported outer membranes of Pseudomonas aeruginosa, pyocyanin redox mediators, and semiconducting CdSe QDs dispersed in an aqueous electrolyte. We present a comprehensive electrochemical analysis of this platform via electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and chronoamperometry (CA). EIS reveals the formation and electronic properties of supported outer membrane films. CV reveals the electrochemically active surface area of P. aeruginosa outer membranes and that pyocyanin is the sole species that performs redox with these outer membranes under sweeping applied potential. CA demonstrates that photoexcited charge transfer in this system is driven by the reduction of pyocyanin at the QD surface followed by diffusion of reduced pyocyanin through the outer membrane. The broad applicability of this platform across many bacterial species, QD architectures, and controlled environmental conditions affords the possibility to define design principles for future PSB systems to synergistically integrate concurrent advances in genetically engineered organisms and inorganic nanomaterials.
Assuntos
Nanoestruturas , Pontos Quânticos , Eletrodos , Oxirredução , SemicondutoresRESUMO
Dynamic wetting phenomena are typically described by a constitutive law relating the dynamic contact angle θ to contact-line velocity UCL. The so-called Davis-Hocking model is noteworthy for its simplicity and relates θ to UCL through a contact-line mobility parameter M, which has historically been used as a fitting parameter for the particular solid-liquid-gas system. The recent experimental discovery of Xia & Steen (2018) has led to the first direct measurement of M for inertial-capillary motions. This opens up exciting possibilities for anticipating rapid wetting and dewetting behaviors, as M is believed to be a material parameter that can be measured in one context and successfully applied in another. Here, we investigate the extent to which M is a material parameter through a combined experimental and numerical study of binary sessile drop coalescence. Experiments are performed using water droplets on multiple surfaces with varying wetting properties (static contact angle and hysteresis) and compared with numerical simulations that employ the Davis-Hocking condition with the mobility M a fixed parameter, as measured by the cyclically dynamic contact angle goniometer, i.e. no fitting parameter. Side-view coalescence dynamics and time traces of the projected swept areas are used as metrics to compare experiments with numerical simulation. Our results show that the Davis-Hocking model with measured mobility parameter captures the essential coalescence dynamics and outperforms the widely used Kistler dynamic contact angle model in many cases. These observations provide insights in that the mobility is indeed a material parameter.
RESUMO
Glycans, including oligosaccharides and glycoconjugates, play an integral role in modulating the biological functions of macromolecules. Many physiological and pathological processes are mediated by interactions between glycans, which has led to the use of glycans as biosensors for pathogen and biomarker detection. Elucidating the relationship between glycan structure and biological function is critical for advancing our understanding of the impact glycans have on human health and disease and for expanding the repertoire of glycans available for bioanalysis, especially for diagnostics. Such efforts have been limited by the difficulty in obtaining sufficient quantities of homogenous glycan samples needed to resolve the exact relationships between glycan structure and their structural or modulatory functions on a given glycoconjugate. Synthetic strategies offer a viable route for overcoming these technical hurdles. In recent years, microfluidics have emerged as powerful tools for realizing high-throughput and reproducible syntheses of homogenous glycans for the potential use in functional studies. This critical review provides readers with an overview of the microfluidic technologies that have been developed for chemical and enzymatic glycan synthesis. The advantages and limitations associated with using microreactor platforms to improve the scalability, productivity, and selectivity of glycosylation reactions will be discussed, as well as suggested future work that can address certain pitfalls.