Modulation of B cell receptor activation by antibody competition.

During repeated virus exposure, pre-existing antibodies can mask viral epitopes by competing with B cell receptors for antigen. Although this phenomenon has the potential to steer B cell responses away from conserved epitopes, the factors that influence epitope masking by competing antibodies remain unclear. Using engineered, influenza-reactive B cells, we investigate how antibodies influence the accessibility of epitopes on the viral surface.

A High-Avidity, Thermostable, and Low-Cost Synthetic Capture for Ultrasensitive Detection and Quantification of Viral Antigens and Aerosols.

Lateral flow assays (LFAs) are currently the most popular point-of-care diagnostics, rapidly transforming disease diagnosis from expensive doctor checkups and laboratory-based tests to potential on-the-shelf commodities. Yet, their sensitive element, a monoclonal antibody, is expensive to formulate, and their long-term storage depends on refrigeration technology that cannot be met in resource-limited areas. In this work, LCB1 affibodies (antibody mimetic miniproteins) were conjugated to bovine serum albumin (BSA) to afford a high-avidity synthetic capture (LCB1-BSA) capable of detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and virus like particles (VLPs).

Macromolecular condensation organizes nucleolar sub-phases to set up a pH gradient.

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass.

Antibody inhibition of influenza A virus assembly and release.

Antibodies are frontline defenders against influenza virus infection, providing protection through multiple complementary mechanisms. Although a subset of monoclonal antibodies (mAbs) has been shown to restrict replication at the level of virus assembly and release, it remains unclear how potent and pervasive this mechanism of protection is, due in part to the challenge of separating this effect from other aspects of antibody function. To address this question, we developed imaging-based assays to determine how effectively a broad range of mAbs against the IAV surface proteins can specifically restrict viral egress.

Antibody Inhibition of Influenza A Virus Assembly and Release.

Antibodies are frontline defenders against influenza virus infection, providing protection through multiple complementary mechanisms. Although a subset of monoclonal antibodies (mAbs) have been shown to restrict replication at the level of virus assembly and release, it remains unclear how potent and pervasive this mechanism of protection is, due in part to the challenge of separating this effect from other aspects of antibody function. To address this question, we developed imaging-based assays to determine how effectively a broad range of mAbs against the IAV surface proteins can specifically restrict viral egress.

Neuraminidase Activity Modulates Cellular Coinfection during Influenza A Virus Multicycle Growth.

Infection of individual cells by multiple virions plays critical roles in the replication and spread of many viruses, but mechanisms that control cellular coinfection during multicycle viral growth remain unclear. Here, we investigate virus-intrinsic factors that control cellular coinfection by influenza A virus (IAV). Using quantitative fluorescence to track the spread of virions from single infected cells, we identify the IAV surface protein neuraminidase (NA) as a key determinant of cellular coinfection.

Six-Dimensional Single-Molecule Imaging with Isotropic Resolution using a Multi-View Reflector Microscope.

Imaging both the positions and orientations of single fluorophores, termed single-molecule orientation-localisation microscopy, is a powerful tool to study biochemical processes. However, the limited photon budget associated with single-molecule fluorescence makes high-dimensional imaging with isotropic, nanoscale spatial resolution a formidable challenge. Here, we realise a radially and azimuthally polarized multi-view reflector (raMVR) microscope for the imaging of the 3D positions and 3D orientations of single molecules, with precision of 10.

A morphological transformation in respiratory syncytial virus leads to enhanced complement deposition.

The complement system is a critical host defense against infection, playing a protective role that can also enhance disease if dysregulated. Although many consequences of complement activation during viral infection are well established, mechanisms that determine the extent to which viruses activate complement remain elusive. Here, we investigate complement activation by human respiratory syncytial virus (RSV), a filamentous respiratory pathogen that causes significant morbidity and mortality.

Forming and loading giant unilamellar vesicles with acoustic jetting.

Giant unilamellar vesicles (GUVs) are a useful platform for reconstituting and studying membrane-bound biological systems, offering reduced complexity compared to living cells. Several techniques exist to form GUVs and populate them with biomolecules of interest. However, a persistent challenge is the ability to efficiently and reliably load solutions of biological macromolecules, organelle-like membranes, and/or micrometer-scale particles with controlled stoichiometry in the encapsulated volume of GUVs.

Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion.

Cholesterol 25-hydroxylase () is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export.

Influenza A virus surface proteins are organized to help penetrate host mucus.

Influenza A virus (IAV) enters cells by binding to sialic acid on the cell surface. To accomplish this while avoiding immobilization by sialic acid in host mucus, viruses rely on a balance between the receptor-binding protein hemagglutinin (HA) and the receptor-cleaving protein neuraminidase (NA). Although genetic aspects of this balance are well-characterized, little is known about how the spatial organization of these proteins in the viral envelope may contribute.

Low-Fidelity Assembly of Influenza A Virus Promotes Escape from Host Cells.

Influenza viruses inhabit a wide range of host environments using a limited repertoire of protein components. Unlike viruses with stereotyped shapes, influenza produces virions with significant morphological variability even within clonal populations. Whether this tendency to form pleiomorphic virions is coupled to compositional heterogeneity and whether it affects replicative fitness remains unclear.

Claudin-4 reconstituted in unilamellar vesicles is sufficient to form tight interfaces that partition membrane proteins.

Tight junctions have been hypothesized to act as molecular fences in the plasma membrane of epithelial cells, helping to form differentiated apical and basolateral domains. While this fence function is believed to arise from the interaction of four-pass transmembrane claudins, the complexity of tight junctions has made direct evidence of their role as a putative diffusion barrier difficult to obtain. Here, we address this challenge by reconstituting claudin-4 into giant unilamellar vesicles using microfluidic jetting.

Accurate computational design of multipass transmembrane proteins.

The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable.

Miniaturized Impedance Flow Cytometer: Design Rules and Integrated Readout.

A dual-channel credit-card-sized impedance cell counter featuring a throughput of 2000 cell/s and detection of single yeast cells (5 μm) with a signal-to-noise ratio of 20 dB is presented. Its compactness is achieved by a CMOS ASIC combining a lock-in impedance demodulator with an oversampling 20-bit ΣΔ ADC and real-time peak detection embedded in field-programmable gate array. The module is coupled to a dielectrophoretic cell-sorting microfluidic device, offering compact and label-free electrical readout that replaces the need for a fluorescence microscope and, thus, is suitable for point-of-care diagnostics.

The biology of boundary conditions: cellular reconstitution in one, two, and three dimensions.

Reconstituting cellular behavior outside the complex environment of the cell allows the study of biological processes in simplified and controlled settings. Making the leap from cells to test tubes, however, carries the inevitable risk of removing too much context and therefore sacrificing the important biochemical, mechanical, or geometrical constraints that guide the system's behavior. In response to this challenge, reconstitution experiments have recently begun to focus not only on including the right molecules but also on faithfully recapitulating the constraints that are present within a cell.

Cytoplasmic volume modulates spindle size during embryogenesis.

Rapid and reductive cell divisions during embryogenesis require that intracellular structures adapt to a wide range of cell sizes. The mitotic spindle presents a central example of this flexibility, scaling with the dimensions of the cell to mediate accurate chromosome segregation. To determine whether spindle size regulation is achieved through a developmental program or is intrinsically specified by cell size or shape, we developed a system to encapsulate cytoplasm from Xenopus eggs and embryos inside cell-like compartments of defined sizes.

Microfluidic genome-wide profiling of intrinsic electrical properties in Saccharomyces cerevisiae.

Methods to analyze the intrinsic physical properties of cells - for example, size, density, rigidity, or electrical properties - are an active area of interest in the microfluidics community. Although the physical properties of cells are determined at a fundamental level by gene expression, the relationship between the two remains exceptionally complex and poorly characterized, limiting the adoption of intrinsic separation technologies. To improve our current understanding of how a cell's genotype maps to a measurable physical characteristic and quantitatively investigate the potential of using these characteristics as biomarkers, we have developed a novel screen that combines microfluidic cell sorting with high-throughput sequencing and the haploid yeast deletion library to identify genes whose functions modulate one such characteristic - intrinsic electrical properties.

Isodielectric separation and analysis of cells.

Measuring the electrical properties of a cell provides a fast and accessible means of identifying or characterizing cells whose biological state differs from the population as a whole. This chapter describes a microfluidic method for characterizing the electrical properties of cells based upon their convergence to equilibrium in an electrical conductivity gradient. The method, called isodielectric separation, uses the dielectrophoretic force induced on polarizable objects in spatially nonuniform electric fields to deflect cells to the point in the conductivity gradient where their polarization charge vanishes.

Emergent behavior in particle-laden microfluidic systems informs strategies for improving cell and particle separations.

Colloidal particles placed in an energy landscape interact with each other, giving rise to complex dynamic behavior that affects the ability to process and manipulate suspensions of these particles. Propagating across scales ranging from the local behavior of 10's of particles to non-local behavior encompassing >10(6) particles, these particle interactions are pervasive and challenging to describe quantitatively, especially in the confined environments typical of microfluidic devices. To better understand the effects of particle interactions in this context, we have performed experiments and simulations involving a simple microfluidic device in which hydrodynamic and electrostatic forces are leveraged to concentrate and separate particle mixtures.

Electrically addressable vesicles: tools for dielectrophoresis metrology.

Dielectrophoresis (DEP) has emerged as an important tool for the manipulation of bioparticles ranging from the submicron to the tens of microns in size. Here we show the use of phospholipid vesicle electroformation techniques to develop a new class of test particles with specifically engineered electrical propserties to enable identifiable dielectrophoretic responses in microfabricated systems. These electrically addressable vesicles (EAVs) enable the creation of electrically distinct populations of test particles for DEP.

Assembly of metal nanoparticles into nanogaps.

The directed assembly of nanoparticles and nanoscale materials onto specific locations of a surface is one of the major challenges in nanotechnology. Here we present a simple and scalable method and model for the assembly of nanoparticles in between electrical leads. Gold nanoparticles, 20 nm in diameter, were assembled inside electrical gaps ranging from 15 to 150 nm with the use of positive ac dielectrophoresis.

Microfluidic arrays for logarithmically perfused embryonic stem cell culture.

We present a microfluidic device for culturing adherent cells over a logarithmic range of flow rates. The device sets flow rates through four separate cell-culture chambers using syringe-driven flow and a network of fluidic resistances. The design is easy to fabricate with no on-chip valves and is scalable both in the number of culture chambers as well as in the range of applied flow rates.