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 (0.1 µM, 1 h) reduces zebrafish mortality and viral load following influenza infection. However, CPC mechanisms of action upon viral-host cell interaction are currently unknown. We have utilized super-resolution fluorescence photoactivation localization microscopy to probe the mode of CPC action. Reduction in density of influenza viral protein hemagglutinin (HA) clusters is known to reduce influenza infectivity: here, we show that CPC (at non-cytotoxic doses, 5-10 µM) reduces HA density and number of HA molecules per cluster within the plasma membrane of NIH-3T3 mouse fibroblasts. HA is known to colocalize with the negatively-charged mammalian lipid phosphatidylinositol 4,5-bisphosphate (PIP2); here, we show that nanoscale co-localization of HA with the PIP2-binding Pleckstrin homology (PH) reporter in the plasma membrane is diminished by CPC. CPC also dramatically displaces the PIP2-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) from the plasma membrane of rat RBL-2H3 mast cells; this disruption of PIP2 is correlated with inhibition of mast cell degranulation. Together, these findings offer a PIP2-focused mechanism underlying CPC disruption of influenza and suggest potential pharmacological use of this drug as an influenza therapeutic to reduce global deaths from viral disease.

Triclosan disrupts immune cell function by depressing Ca2+ 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 Ca2+ entry mechanism utilized by many immune cells, mast cells undergo antigen-stimulated Ca2+ influx into the cytosol, for proper function. Previous work showed that TCS inhibits Ca2+ dynamics in mast cells, and here we show that TCS also inhibits Ca2+ mobilization in human Jurkat T cells. However, the biochemical mechanism behind the Ca2+ dampening has yet to be elucidated. Three-dimensional super-resolution microscopy reveals that TCS induces mitochondrial swelling, in line with and extending the previous finding of TCS inhibition of mitochondrial membrane potential via its proton ionophoric activity. Inhibition of plasma membrane potential (PMP) by the canonical depolarizer gramicidin can inhibit mast cell function. However, use of the genetically encoded voltage indicators (GEVIs) ArcLight (pH-sensitive) and ASAP2 (pH-insensitive), indicates that TCS does not disrupt PMP. In conjunction with data from a plasma membrane-localized, pH-sensitive reporter, these results indicate that TCS, instead, induces cytosolic acidification in mast cells and T cells. Acidification of the cytosol likely inhibits Ca2+ influx by uncoupling the STIM1/ORAI1 interaction that is required for opening of plasma membrane Ca2+ channels. These results provide a mechanistic explanation of TCS disruption of Ca2+ influx and, thus, of immune cell function.

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 0.079 ± 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.

Disrupted mitochondrial response to nutrients is a presymptomatic event in the cortex of the APP SAA knock-in mouse model of Alzheimer's disease.

Introduction: Reduced brain energy metabolism, mTOR dysregulation, and extracellular amyloid-ß oligomer (xcAßO) buildup characterize AD; how they collectively promote neurodegeneration is poorly understood. We previously reported that xcAßOs inhibit N utrient-induced M itochondrial A ctivity (NiMA) in cultured neurons. We now report NiMA disruption in vivo . Methods: Brain energy metabolism and oxygen consumption were recorded in APP SAA/+ mice using two-photon fluorescence lifetime imaging and multiparametric photoacoustic microscopy. Results: NiMA is inhibited in APP SAA/+ mice before other defects are detected in these amyloid-ß-producing animals that do not overexpress APP or contain foreign DNA inserts into genomic DNA. GSK3ß signals through mTORC1 to regulate NiMA independently of mitochondrial biogenesis. Inhibition of GSK3ß with lithium or TWS119 stimulates NiMA in cultured human neurons, and mitochondrial activity and oxygen consumption in APP SAA mice. Conclusion: NiMA disruption in vivo occurs before histopathological changes and cognitive decline in APP SAA mice, and may represent an early stage in human AD.

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. The subcellular localization and quantification of these receptor proteins can be determined at the nanometer scale by FPALM super-resolution microscopy. We used FPALM to determine localizations of Dystroglycan and integrin proteins in muscle fibers of intact zebrafish (Danio rerio). Results were consistent with confocal imaging data, but illuminate further details at the nanoscale and show the feasibility of using FPALM to quantify interactions of two proteins in a whole organism.

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. As examples, we present fixed-cell imaging of the spike protein SARS-CoV2 (S) and its interactions with host cell components. Results show a relationship between S and the lipid phosphatidylinositol (4,5)-bisphosphate (PIP2). These findings have ramifications for several existing models of plasma membrane organization.

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. The mechanism of spontaneous HA clustering, which occurs with or without other viral components, has not been elucidated. Using super-resolution fluorescence photoactivation localization microscopy (FPALM), we have previously shown that these HA clusters are interdependent on phosphatidylinositol 4,5-biphosphate (PIP2). Here, we show that the IAV matrix protein M1 co-clusters with PIP2, visualized using the pleckstrin homology domain. We find that cetylpyridinium chloride (CPC), which is a positively charged quaternary ammonium compound known for its antibacterial and antiviral properties at millimolar concentrations, disrupts M1 clustering and M1-PIP2 co-clustering at micromolar concentrations well below the critical micelle concentration (CMC). CPC also disrupts the co-clustering of M1 with HA at the plasma membrane, suggesting the role of host cell PIP2 clusters as scaffolds for gathering and concentrating M1 and HA to achieve their unusually high cluster densities in the IAV envelope.

A unified approach for high-throughput quantitative analysis of the residues of multi-class veterinary drugs and pesticides in bovine milk using LC-MS/MS and GC-MS/MS.

This paper reports a unified sample preparation approach for high-throughput multi-residue analysis of veterinary drugs and pesticides in a single sub-sample of bovine milk. The sample (5 g) was deproteinized with acetonitrile before an aliquot (I) was withdrawn, and the remainder was phase-separated using MgSO4 and NaCl. The acetonitrile layer (II) was recovered and the extracts combined, cleaned, and solvent-exchanged before the concentrations of veterinary drugs and pesticides were measured by ultra-fast liquid chromatography tandem mass spectrometry (UFLC-MS/MS). As a unique approach, extract II was analyzed simultaneously using gas chromatography tandem mass spectrometry (GC-MS/MS). Method performance for 78 drugs and 238 pesticides complied with CD 2002/657/EC and SANTE/11813/2017 guidelines, respectively, with significant savings in time and cost. Thus, it would be ideal for regulatory analysis of analytes ranging from non-polar organochlorine pesticides to polar drugs, including penicillins, quinolones, and tetracyclines.