GPCR Inhibitors Have Antiviral Properties against JC Polyomavirus Infection.

JC polyomavirus (JCPyV) infects the majority of the population and initially establishes a persistent but asymptomatic infection of the kidneys. In healthy individuals, the infection remains controlled by the host immune system, but for individuals experiencing prolonged immunosuppression, the infection can reactivate and spread to the brain, where it causes progressive multifocal leukoencephalopathy (PML), which is a fatal neurodegenerative disease. Currently, there are no approved therapies to treat PML, and affected individuals suffer rapid motor weakness and cognitive deterioration.

Conserved sequence features in intracellular domains of viral spike proteins.

Viral spike proteins mutate frequently, but conserved features within these proteins often have functional importance and can inform development of anti-viral therapies which circumvent the effects of viral sequence mutations. Through analysis of large numbers of viral spike protein sequences from several viral families, we found highly (>99%) conserved patterns within their intracellular domains. The patterns generally consist of one or more basic amino acids (arginine or lysine) adjacent to a cysteine, many of which are known to undergo acylation.

Process-Structure-Property Relationship Development in Large-Format Additive Manufacturing: Fiber Alignment and Ultimate Tensile Strength.

Parts made through additive manufacturing (AM) often exhibit mechanical anisotropy due to the time-based deposition of material and processing parameters. In polymer material extrusion (MEX), printed parts have weak points at layer interfaces, perpendicular to the direction of deposition. Poly(lactic acid) with chopped carbon fiber was printed on a large-format pellet printer at various extrusion rates with the same tool pathing to measure the fiber alignment with deposition via two methods and relate it to the ultimate tensile strength (UTS).

Antimicrobial cetylpyridinium chloride causes functional inhibition of mitochondria as potently as canonical mitotoxicants, nanostructural disruption of mitochondria, and mitochondrial Ca efflux in living rodent and primary human cells.

People are exposed to high concentrations of antibacterial agent cetylpyridinium chloride (CPC) via food and personal care products, despite little published information regarding CPC effects on eukaryotes. Here, we show that low-micromolar CPC exposure, which does not cause cell death, inhibits mitochondrial ATP production in primary human keratinocytes, mouse NIH-3T3 fibroblasts, and rat RBL-2H3 immune mast cells. ATP inhibition via CPC (EC 1.

Super-Resolution Imaging Reveals the Nanoscale Distributions of Dystroglycan and Integrin Itga7 in Zebrafish Muscle Fibers.

Cell signaling is determined partially by the localization and abundance of proteins. Dystroglycan and integrin are both transmembrane receptors that connect the cytoskeleton inside muscle cells to the extracellular matrix outside muscle cells, maintaining proper adhesion and function of muscle. The position and abundance of Dystroglycan relative to integrins is thought to be important for muscle adhesion and function.

Dynamics and Patterning of 5-Hydroxytryptamine 2 Subtype Receptors in JC Polyomavirus Entry.

The organization and dynamics of plasma membrane receptors are a critical link in virus-receptor interactions, which finetune signaling efficiency and determine cellular responses during infection. Characterizing the mechanisms responsible for the active rearrangement and clustering of receptors may aid in developing novel strategies for the therapeutic treatment of viruses. Virus-receptor interactions are poorly understood at the nanoscale, yet they present an attractive target for the design of drugs and for the illumination of viral infection and pathogenesis.

Phosphatidylinositol 4,5-Bisphosphate Mediates the Co-Distribution of Influenza A Hemagglutinin and Matrix Protein M1 at the Plasma Membrane.

The fully assembled influenza A virus (IAV) has on its surface the highest density of a single membrane protein found in nature-the glycoprotein hemagglutinin (HA) that mediates viral binding, entry, and assembly. HA clusters at the plasma membrane of infected cells, and the HA density (number of molecules per unit area) of these clusters correlates with the infectivity of the virus. Dense HA clusters are considered to mark the assembly site and ultimately lead to the budding of infectious IAV.

Localization-Based Super-Resolution Microscopy Reveals Relationship between SARS-CoV2 Spike and Phosphatidylinositol (4,5)-bisphosphate.

Localization microscopy circumvents the diffraction limit by identifying and measuring the positions of numerous subsets of individual fluorescent molecules, ultimately producing an image whose resolution depends on the uncertainty and density of localization, and whose capabilities are compatible with imaging living specimens. Spectral resolution can be improved by incorporating a dichroic or dispersive element in the detection path of a localization microscope, which can be useful for separation of multiple probes imaged simultaneously and for detection of changes in emission spectra of fluorophores resulting from changes in their environment. These methodological advances enable new biological applications, which in turn motivate new questions and technical innovations.

Cetylpyridinium chloride (CPC) reduces zebrafish mortality from influenza infection: Super-resolution microscopy reveals CPC interference with multiple protein interactions with phosphatidylinositol 4,5-bisphosphate in immune function.

The COVID-19 pandemic raises significance for a potential influenza therapeutic compound, cetylpyridinium chloride (CPC), which has been extensively used in personal care products as a positively-charged quaternary ammonium antibacterial agent. CPC is currently in clinical trials to assess its effects on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity. Two published studies have provided mouse and human data indicating that CPC may alleviate influenza infection, and here we show that CPC (0.

Triclosan disrupts immune cell function by depressing Ca influx following acidification of the cytoplasm.

Triclosan (TCS) is an antimicrobial agent that was effectively banned by the FDA from hand soaps in 2016, hospital soaps in 2017, and hand sanitizers in 2019; however, TCS can still be found in a few products. At consumer-relevant, non-cytotoxic doses, TCS inhibits the functions of both mitochondria and mast cells, a ubiquitous cell type. Via the store-operated Ca entry mechanism utilized by many immune cells, mast cells undergo antigen-stimulated Ca influx into the cytosol, for proper function.

Detection and Analysis of Uncharged Particles Using Consumer-grade CCDs.

In the current climate of increased global terrorism, the threat of a radiological incident is becoming more realistic than ever, and as such, the necessity of early-warning detection is paramount to national security. To assist with this need, we have investigated the detection of uncharged particle emissions from radiological sources using charged-coupled devices (CCDs), which are contained within a variety of products, including consumer cellphones and traffic cameras. Because the CCD is intrinsically sensitive to charge accumulation as a result of linear energy transfer by the incident particles, each event can be counted and quantified using video-image processing and an estimated energy band assessed by the properties of the pixels.

Influenza Hemagglutinin Modulates Phosphatidylinositol 4,5-Bisphosphate Membrane Clustering.

The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.

QUANTIFICATION OF MITOCHONDRIAL MEMBRANE CURVATURE BY THREE-DIMENSIONAL LOCALIZATION MICROSCOPY.

Mitochondrial membrane organization is important for many biological functions, and is implicated in a number of diseases, but conventional microscopy has insufficient resolution to image biologically relevant structures. We present methods to quantify nanoscale membrane curvature using three-dimensional localization-based super-resolution microscopy. Localizations are analyzed using a cluster algorithm followed by principal component analysis to determine local membrane curvature.

The Role of Probe Photophysics in Localization-Based Superresolution Microscopy.

Fluorescent proteins are used extensively for biological imaging applications; photoactivatable and photoconvertible fluorescent proteins (PAFPs) are used widely in superresolution localization microscopy methods such as fluorescence photoactivation localization microscopy and photoactivated localization microscopy. However, their optimal use depends on knowledge of not only their bulk fluorescence properties, but also their photophysical properties at the single molecule level. We have used fluorescence correlation spectroscopy and cross-correlation spectroscopy to quantify the diffusion, photobleaching, fluorescence intermittency, and photoconversion dynamics of Dendra2, a well-known PAFP used in localization microscopy.

Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations.

Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time.

Antimicrobial agent triclosan is a proton ionophore uncoupler of mitochondria in living rat and human mast cells and in primary human keratinocytes.

Triclosan (TCS) is an antimicrobial used widely in hospitals and personal care products, at ~10 mm. Human skin efficiently absorbs TCS. Mast cells are ubiquitous key players both in physiological processes and in disease, including asthma, cancer and autism.

Dances with Membranes: Breakthroughs from Super-resolution Imaging.

Biological membrane organization mediates numerous cellular functions and has also been connected with an immense number of human diseases. However, until recently, experimental methodologies have been unable to directly visualize the nanoscale details of biological membranes, particularly in intact living cells. Numerous models explaining membrane organization have been proposed, but testing those models has required indirect methods; the desire to directly image proteins and lipids in living cell membranes is a strong motivation for the advancement of technology.

Combining total internal reflection sum frequency spectroscopy spectral imaging and confocal fluorescence microscopy.

Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species.

In cellulo evaluation of phototransformation quantum yields in fluorescent proteins used as markers for single-molecule localization microscopy.

Single-molecule localization microscopy of biological samples requires a precise knowledge of the employed fluorescent labels. Photoactivation, photoblinking and photobleaching of phototransformable fluorescent proteins influence the data acquisition and data processing strategies to be used in (Fluorescence) Photoactivation Localization Microscopy ((F)-PALM), notably for reliable molecular counting. As these parameters might depend on the local environment, they should be measured in cellulo in biologically relevant experimental conditions.

Precisely and accurately localizing single emitters in fluorescence microscopy.

Methods based on single-molecule localization and photophysics have brought nanoscale imaging with visible light into reach. This has enabled single-particle tracking applications for studying the dynamics of molecules and nanoparticles and contributed to the recent revolution in super-resolution localization microscopy techniques. Crucial to the optimization of such methods are the precision and accuracy with which single fluorophores and nanoparticles can be localized.

Simultaneous multicolor imaging of biological structures with fluorescence photoactivation localization microscopy.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm.