Near-Infrared Photoluminescence of Carbon Nanotubes Powered by Biochemical Reactions of Luciferin/Luciferase.

We report the near-infrared (NIR) photoluminescence of single-wall carbon nanotubes (SWCNTs) generated by chemical energy derived from enzymatic reactions. NIR photoluminescence from SWCNTs has attracted much attention for medical applications, such as bioimaging and biosensors, because of its high transparency and low scattering in biological tissues; however, visible excitation light cannot reach deep tissues. We developed a novel method in which the NIR luminescence of SWCNTs is powered by the biochemical reaction of luciferin/luciferase from fireflies. The luminescence could be detected by a highly sensitive measurement system using an infrared camera, and the optimal conditions for luminescence were investigated. Spectroscopic analysis of the NIR luminescence using chirality-sorted SWCNTs confirmed that the luminescence was derived from SWCNTs. This is the first report achieving NIR photoluminescence of SWCNTs using chemical energy, which does not require external energies, e.g., excitation light or electronic power, and will be applicable to biological imaging and sensing.

Establishment of a Cynomolgus Macaque Model of Human T-Cell Leukemia Virus Type 1 (HTLV-1) Infection by Direct Inoculation of Adult T-Cell Leukemia Patient-Derived Cell Lines for HTLV-1 Infection.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, the precise mechanisms leading to HTLV-1 chronic infection and the onset of the diseases have remained unclear, and effective vaccines for inhibiting the infection and the progression of pathogenesis have therefore not been developed. The use of a nonhuman primate (NHP) model is thought to be important for revealing the mechanisms of the progressive status and for the development of prevention procedures. In this study, we developed a cynomolgus macaque (CM) model of HTLV-1 infection by direct intravenous inoculation of HTLV-1-producing cells derived from ATL patients. The cell line used for infection, ATL-040, was selected as the most infectious one in our cell line library. CMs inoculated intravenously with 1 × 108 ATL-040 cells per animal became persistently infected with HTLV-1, as shown by the HTLV-1 provirus load (PVL) in peripheral blood mononuclear cells and HTLV-1-specific antibodies (2/2 animals). One CM inoculated intravenously with 1 × 107 ATL-040 cells did not have detectable PVLs despite the fact that anti-HTLV-1 antibodies were maintained for more than 2 years. Furthermore, immunological approaches, including CD8+ T cell depletion prior to infection (3/3 animals) and intrathecal inoculation (3/3 animals), led to increased proviral loads in the cynomolgus monkeys. The present method and the cynomolgus monkey model of HTLV-1 infection will be beneficial for immunological and virological studies on HTLV-1 aiming at the development of anti-HTLV-1 prophylactic vaccines and therapy drugs. IMPORTANCE HTLV-1 was discovered in the 1980s as the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, the precise mechanisms leading to HTLV-1 chronic infection and the onset of the diseases still remain unidentified. Thus, no effective vaccines to inhibit the infection and the progressive of pathogenesis have been developed. The use of appropriate animal models is essential for understanding HTLV-1 infection and pathogenesis. In order to establish a new nonhuman primate model for studies on HTLV-1 infection, cynomolgus monkeys were infected with HTLV-1 under a variety of experimental conditions. Our method, using a cell line generated from an ATL patient as a source of HTLV-1, was able to establish HTLV-1 infection in monkeys with a 100% success rate. This cynomolgus macaque model of HTLV-1 infection will contribute to the elucidation of HTLV-1 infection and its associated disease development.

COVID-19 cynomolgus macaque model reflecting human COVID-19 pathological conditions.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health and life. A useful pathological animal model accurately reflecting human pathology is needed to overcome the COVID-19 crisis. In the present study, COVID-19 cynomolgus monkey models including monkeys with underlying diseases causing severe pathogenicity such as metabolic disease and elderly monkeys were examined. Cynomolgus macaques with various clinical conditions were intranasally and/or intratracheally inoculated with SARS-CoV-2. Infection with SARS-CoV-2 was found in mucosal swab samples, and a higher level and longer period of viral RNA was detected in elderly monkeys than in young monkeys. Pneumonia was confirmed in all of the monkeys by computed tomography images. When monkeys were readministrated SARS-CoV-2 at 56 d or later after initial infection all of the animals showed inflammatory responses without virus detection in swab samples. Surprisingly, in elderly monkeys reinfection showed transient severe pneumonia with increased levels of various serum cytokines and chemokines compared with those in primary infection. The results of this study indicated that the COVID-19 cynomolgus monkey model reflects the pathophysiology of humans and would be useful for elucidating the pathophysiology and developing therapeutic agents and vaccines.

Packing density changes of supported lipid bilayers observed by fluorescence microscopy and quartz crystal microbalance-dissipation.

Various properties of supported lipid bilayers such as diffusion and lipid partitioning are well characterized. However, little attention has been paid to their molecular packing density. In this work, the adsorption of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) vesicles on glass and silicon dioxide was investigated using fluorescence microscopy, quartz crystal microbalance-dissipation (QCM-D), and atomic force microscopy. Fluorescence recovery after photobleaching data showed that the adsorption of large unilamellar vesicles (LUVs) on glass yielded supported bilayers with full mobility under alkaline (pH 8.3) and acidic (pH 3-4) conditions. These fluid bilayers exhibited quite different diffusion constants; those at alkaline pH were ~10 times larger than those at acidic pH. The reason for this pH dependence was clarified by investigation of the rupture of giant unilamellar vesicles (GUVs) on glass. Fluorescence data revealed that the area of planar bilayer patches increased at alkaline pH. Thus, we conclude that the rapid diffusion in alkaline solution arises from the decreased molecular density. QCM-D data showed that dissipation increased in a stepwise manner during vesicle fusion on silicon dioxide at alkaline pH. We attribute this behavior to the decrease in packing density of planar bilayers.

Detergent-mediated formation of polymer-supported phospholipid bilayers.

Supported phospholipid bilayers can be formed by established methods such as vesicle fusion and the Langmuir-Blodgett (LB) technique. However, challenges remain in regards to creating supported bilayers from various lipid compositions, using various support surfaces, and incorporating membrane proteins. Here we report a detergent removal method as an alternative means of supported bilayer formation. The process consists of three steps: (1) incubation of phospholipid-poly(ethylene glycol) (PEG)-grafted glass with lipid-detergent micelles; (2) detergent removal by washing the surface with vesicles; and (3) incubation with the vesicles to complete lipid adsorption. These procedures yielded fluid planar bilayers of zwitterionic lipids. Because fluid structures were not obtained by vesicle fusion, the detergent seemed necessary to produce the polymer-supported bilayers. While anionic phospholipids inhibited the attachment of fluid bilayers in the absence of calcium ions, supported bilayers with almost full mobility were obtained from lipid mixtures containing 10-20 mol % anionic lipids in the presence of calcium ions. The incorporation of the anionic lipids in the bulk-facing leaflet was demonstrated by the binding of dye-labeled annexin V.