Significance of chlorine-dioxide-based oral rinses in preventing SARS-CoV-2 cell entry.

OBJECTIVE: This work aims to determine the efficacy of preprocedural oral rinsing with chlorine dioxide solutions to minimize the risk of coronavirus disease 2019 (COVID-19) transmission during high-risk dental procedures. METHODS: The antiviral activity of chlorine-dioxide-based oral rinse (OR) solutions was tested by pre-incubating with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus in a dosage-dependent manner before transducing to human embryonic kidney epithelial (HEK293T-ACE2) cells, which stably expresses ACE-2 receptor. Viral entry was determined by measuring luciferase activity using a luminescence microplate reader. In the cell-to-cell fusion assay, effector Chinese hamster ovary (CHO-K1) cells co-expressing spike glycoprotein of SARS-CoV-2 and T7 RNA polymerase were pre-incubated with the ORs before co-culturing with the target CHO-K1 cells co-expressing human ACE2 receptor and luciferase gene. The luciferase signal was quantified 24 h after mixing the cells. Surface expression of SARS-CoV-2 spike glycoprotein and ACE-2 receptor was confirmed using direct fluorescent imaging and quantitative cell-ELISA. Finally, dosage-dependent cytotoxic effects of ORs were evaluated at two different time points. RESULTS: A dosage-dependent antiviral effect of the ORs was observed against SARS-CoV-2 cell entry and spike glycoprotein mediated cell-to-cell fusion. This demonstrates that ORs can be useful as a preprocedural step to reduce viral infectivity. CONCLUSIONS: Chlorine-dioxide-based ORs have a potential benefit for reducing SARS-CoV-2 entry and spread.

Glucose-limiting conditions induce an invasive population of MDA-MB-231 breast cancer cells with increased connexin 43 expression and membrane localization.

Gap junctional intercellular communication (GJIC) is a homeostatic process mediated by membrane channels composed of a protein family known as connexins. Alterations to channel activity can modulate suppression or facilitation of cancer progression. These varying roles are influenced by the cancer cell genetic profile and the context-dependent mechanisms of a dynamic extracellular environment that encompasses fluctuations to nutrient availability. To better explore the effects of altered cellular metabolism on GJIC in breast cancer, we generated a derivative of the triple-negative breast cancer cell line MDA-MB-231 optimized for growth in low-glucose. Reduced availability of glucose is commonly encountered during tumor development and leads to metabolic reprogramming in cancer cells. MDA-MB-231 low-glucose adapted cells exhibited a larger size with improved cell-cell contact and upregulation of cadherin-11. Additionally, increased protein levels of connexin 43 and greater plasma membrane localization were observed with a corresponding improvement in GJIC activity compared to the parental cell line. Since GJIC has been shown to affect cellular invasion in multiple cancer cell types, we evaluated the invasive qualities of these cells using multiple three-dimensional Matrigel growth models. Results of these experiments demonstrated a significantly more invasive phenotype. Moreover, a decrease in invasion was noted when GJIC was inhibited. Our results indicate a potential response of triple-negative breast cancer cells to reduced glucose availability that results in changes to GJIC and invasiveness. Delineation of this relationship may help elucidate mechanisms by which altered cancer cell metabolism affects GJIC and how cancer cells respond to nutrient availability in this regard.

Preferential recognition and antagonism of SARS-CoV-2 spike glycoprotein binding to 3-O-sulfated heparan sulfate.

The COVID-19 pandemic caused by SARS-CoV-2 is in immediate need of an effective antidote. Although the Spike glycoprotein (SgP) of SARS-CoV-2 has been shown to bind to heparins, the structural features of this interaction, the role of a plausible heparan sulfate proteoglycan (HSPG) receptor, and the antagonism of this pathway through small molecules remain unaddressed. Using an in vitro cellular assay, we demonstrate HSPGs modified by the 3-O-sulfotransferase isoform-3, but not isoform-5, preferentially increased SgP-mediated cell-to-cell fusion in comparison to control, unmodified, wild-type HSPGs. Computational studies support preferential recognition of the receptor-binding domain of SgP by 3-O-sulfated HS sequences. Competition with either fondaparinux, a 3-O-sulfated HS-binding oligopeptide, or a synthetic, non-sugar small molecule, blocked SgP-mediated cell-to-cell fusion. Finally, the synthetic, sulfated molecule inhibited fusion of GFP-tagged pseudo SARS-CoV-2 with human 293T cells with sub-micromolar potency. Overall, overexpression of 3-O-sulfated HSPGs contribute to fusion of SARS-CoV-2, which could be effectively antagonized by a synthetic, small molecule.

Protein polymer MRI contrast agents: Longitudinal analysis of biomaterials in vivo.

Despite recent advances in tissue engineering to regenerate biological function by combining cells with material supports, development is hindered by inadequate techniques for characterizing biomaterials in vivo. Magnetic resonance imaging is a tomographic technique with high temporal and spatial resolution and represents an excellent imaging modality for longitudinal noninvasive assessment of biomaterials in vivo. To distinguish biomaterials from surrounding tissues for magnetic resonance imaging, protein polymer contrast agents were developed and incorporated into hydrogels. In vitro and in vivo images of protein polymer hydrogels, with and without covalently incorporated protein polymer contrast agents, were acquired by magnetic resonance imaging. T(1) values of the labeled gels were consistently lower when protein polymer contrast agents were included. As a result, the protein polymer contrast agent hydrogels facilitated fate tracking, quantification of degradation, and detection of immune response in vivo. For the duration of the in vivo study, the protein polymer contrast agent-containing hydrogels could be distinguished from adjacent tissues and from the foreign body response surrounding the gels. The hydrogels containing protein polymer contrast agent have a contrast-to-noise ratio 2-fold greater than hydrogels without protein polymer contrast agent. In the absence of the protein polymer contrast agent, hydrogels cannot be distinguished by the end of the gel lifetime.

Synthesis of multimeric MR contrast agents for cellular imaging.

We have prepared a series of molecular multimeric MR contrast agents for cell labeling that are easy to synthesize, relatively low molecular weight, and biocompatible. The relaxivities of the agents range from 17 to 85 mM(-1) s(-1). Cellular uptake is concentration dependent and viability is excellent. MR images of cell pellets reveal a marked increase in observed signal intensity.

Modification of embolic-PVA particles with MR contrast agents.

We report the synthesis and characterization of polyvinyl alcohol (PVA) embolic particles modified with a clinically approved magnetic resonance (MR) contrast agent. PVA particles are used during transcatheter arterial embolization (TAE) procedures and this minimally invasive technique is a widely employed treatment for inoperable tumors. The PVA particles are injected into tumor vessels and prevent blood flow which results in tumor attenuation. An accurate assessment of the endpoint of embolization is critical to successful TAE procedures. Recent reports suggest that 20% of endpoint determination of TAE procedures by angiographic techniques are erroneous. Real time, in vivo imaging of the embolic particles would overcome this limitation. The contrast-modified PVA particles described here show an 80% decrease in T(1) relaxation times compared to unmodified particles. Images of particles in capillary tubes of similar size to catheters used in TAE procedures are clearly visible by MRI.

Optical monitoring of bubble size and shape in a pulsating bubble surfactometer.

The pulsating bubble surfactometer (PBS) is often used for in vitro characterization of exogenous lung surfactant replacements and lung surfactant components. However, the commercially available PBS is not able to dynamically track bubble size and shape. The PBS therefore does not account for bubble growth or elliptical bubble shape that frequently occur during device use. More importantly, the oscillatory volume changes of the pulsating bubble are different than those assumed by the software of the commercial unit. This leads to errors in both surface area and surface tension measurements. We have modified a commercial PBS through the addition of an image-acquisition system, allowing real-time determination of bubble size and shape and hence the accurate tracking of surface area and surface tension. Compression-expansion loops obtained with the commercially available PBS software were compared with those provided by the image-analysis system for dipalmitoylphosphatidylcholine, Infasurf, and Tanaka lipids (dipalmitoylphosphatidylcholine-palmitoyloleoylphosphatidyl-glycerol-palmitic acid, 68:22:9) at concentrations of 0.1 and 1.0 mg/ml and at frequencies of 1 and 20 cycles/min. Whereas minimum surface tension as determined by the image-analysis system is similar to that measured by the commercially available software, the maximum surface tension and the shapes of the interfacial area-surface tension loops are quite different. Differences are attributable to bubble drift, nonsinusoidal volume changes, and variable volume excursions seen with the modified system but neglected by the original system. Image analysis reveals that the extent of loop hysteresis is greatly overestimated by the commercial device and that an apparent, rapid increase in surface tension upon film expansion seen in PBS loops is not observed with the image-analysis system. The modified PBS system reveals new dynamic characteristics of lung surfactant preparations that have not previously been reported.