COVID-19 Subunit Vaccine with a Combination of TLR1/2 and TLR3 Agonists Induces Robust and Protective Immunity.

The development of COVID-19 vaccines is critical in controlling global health issues under the COVID-19 pandemic. The subunit vaccines are the safest and most widely used vaccine platform and highly effective against a multitude of infectious diseases. An adjuvant is essential for subunit vaccines to enhance the magnitude and durability of immune responses. In this study, we determined whether a combination of toll-like receptor (TLR)1/2 and TLR3 agonists (L-pampo) can be a potent adjuvant for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine. We measured a neutralizing antibody (nAb) and an angiotensin-converting enzyme 2 (ACE2) receptor-blocking antibody against SARS-CoV-2 receptor-binding domain (RBD). We also detected interferon-gamma (IFN-γ) production by using ELISPOT and ELISA assays. By employing a ferret model, we detected nAbs and IFN-γ producing cells and measured viral load in nasal wash after the challenge of SARS-CoV-2. We found that SARS-CoV-2 antigens with L-pampo stimulated robust humoral and cellular immune responses. The efficacy of L-pampo was higher than the other adjuvants. Furthermore, in the ferret model, SARS-CoV-2 antigens with L-pampo elicited nAb response and antigen-specific cellular immune response against SARS-CoV-2, resulting in substantially decreased viral load in their nasal wash. Our study suggests that SARS-CoV-2 antigens formulated with TLR agonists, L-pampo, can be a potent subunit vaccine to promote sufficient protective immunity against SARS-CoV-2.

A relationship between the surface composition and spectroscopic properties of cesium lead bromide (CsPbBr3) perovskite nanocrystals: focusing on photoluminescence efficiency.

We have previously developed CsPbBr3 NCs exhibiting a tremendously high photoluminescence (PL) and structural stability by adding ZnBr2. However, understanding of these outstanding properties is lacking due to the absence of spectroscopic analyses, such as spectral or dynamical characteristics. In this work, we conducted a comparative analysis of photophysical properties for conventional-CsPbBr3 NCs and ZnBr2-CsPbBr3 NCs. First, we analyzed the blinking traces by comparing the single crystal PL intermittency. It has been found that the PL quantum yield of CsPbBr3 NCs is gradually decreasing at the ensemble level, resulting from a significant activation of the Auger-induced blinking. Furthermore, the time-resolved TA dynamics supports the fact that Auger-type energy transfer accelerates the hot carrier cooling time, and thereby the Auger-induced blinking behavior in the band-edge state becomes dominant over time. Here, ZnBr2-CsPbBr3 NCs showed a low multiexciton Auger amplitude and therefore had a stable PL emission compared with conventional-CsPbBr3 NCs. Finally, we suggest that both NCs differ in intraband spacing possibly due to capping ligands, finally leading to a suppressed Auger process and higher stability for ZnBr2-CsPbBr3 NCs.

Elucidation of Photoluminescence Blinking Mechanism and Multiexciton Dynamics in Hybrid Organic-Inorganic Perovskite Quantum Dots.

Halide perovskites (ABX3 ) have emerged as promising materials in the past decade owing to their superior photophysical properties, rendering them potential candidates as solar cells, light-emitting diode displays, and lasing materials. To optimize their utilization into optoelectronic devices, fundamental understanding of the optical behaviors is necessary. To reveal the comprehensive structure-property relationship, CH3 NH3 PbBr3 (MAPbBr3 ) perovskite quantum dots (PQDs) of three different sizes are prepared by controlling the precipitation temperature. Photoluminescence (PL) blinking, a key process that governs the emission efficiency of the PQD materials, is investigated in detail by the time-resolved spectroscopic measurements of individual dots. The nature of the generated species in the course of blinking events is identified, and the mechanism governing the PL blinking is studied as a function of PQD sizes. Further, the practical applicability of MAPbBr3 PQDs is assessed by studying the multiexciton dynamics under high photoexcitation intensity under which most of the display devices work. Ultrafast transient absorption spectroscopy helped in uncovering the volume-dependent Auger recombination rates, which are further explored by comparing the early-time transitions related to surface trap states and higher band states.

Enhancement of exciton transport in porphyrin aggregate nanostructures by controlling the hierarchical self-assembly.

Exciton transport in meso-tetra(4-sulfonatophenyl) porphyrin (TPPS) J-aggregates was directly imaged using the emission profile analysis method with confocal fluorescence microscopy. By controlling the structural hierarchy of TPPS aggregates, we could comparatively study the exciton transport properties in single nanotubes and bundled structures. Using the one-dimensional diffusion model, the exciton diffusion coefficients of TPPS nanotubes and bundles were estimated as 95 and 393 nm2 ps-1, respectively, showing a dramatic enhancement of exciton transport in bundled structures. To reveal the underlying mechanism of enhanced exciton transport in bundle compared to that in single strands, the spatially resolved measurements of exciton transport images were correlated with the spectral information at each local sites. We have confirmed that nanotube and its bundled form possess different energetic landscapes and exciton migration dynamics. Agglomeration into bundles led to an increase in system-environment coupling and denser distribution of energy states, facilitating longer migration length and accelerated transport. Detailed analysis in this study provides important insights into the structure-dependent exciton transport properties of self-assembled J-aggregate nanostructures.

Investigation and Control of Single Molecular Structures of Meso- Meso Linked Long Porphyrin Arrays.

We have investigated conformational structures of meso- meso linked porphyrin arrays (Z n) by single molecule fluorescence spectroscopy. Modulation depths ( M values) were measured by excitation polarization fluorescence spectroscopy. The M value decreases from 0.85 to 0.46 as the number of porphyrin units increases from 3 to 128, indicating that longer arrays exhibit coiled structures. Such conformational changes depending on the length have been confirmed by coarse-grained simulation. The histograms of M values and traces of centroid position of emitting sites by localization microscopy showed that the structures of longer arrays changed to more stretched after solvent vapor annealing with tetrahydrofuran.

Composition-dependent emission linewidth broadening in lead bromide perovskite (APbBr3, A = Cs and CH3NH3) nanoparticles.

Lead halide perovskite nanoparticles (NPs) are attractive as they exhibit excellent color purity and have a tunable band gap, and can thus be applied in highly efficient photovoltaic and light-emitting diodes. Fundamental studies of emission linewidth broadening due to spectral shifts in perovskite NPs may suggest a way to improve their color purity. However, the carrier-induced Stark shift that causes spectral diffusion still requires investigation. In this study, we explore composition-related emission linewidth broadening by comparing CsPbBr3 and CH3NH3PbBr3 (MAPbBr3) perovskite NPs. We find that the MAPbBr3 NPs are more sensitive to fluctuations in the local electric fields than the CsPbBr3 NPs due to an intrinsic difference in the dipole moment between the two A cations (Cs and MA), which shows a carrier-induced Stark shift. The results indicate that the compositions of perovskite NPs are closely associated with emission linewidth broadening and they also provide insights into the development of NP-based devices with high color purity.

A solution-based single-molecule study of surface-bound PBIs: solvent-mediated environmental effects on molecular flexibility.

We have investigated the fundamental photophysical properties of surface-bound perylene bisimide (PBI) molecules in a solution-phase at the single-molecule level. By efficient immobilization of single PBIs on glass, we were able to simultaneously monitor fluorescence intensity trajectories, fluorescence lifetimes, and emission spectra of individual PBIs in organic and aqueous media using confocal microscopy. We showed that the fluorescence dynamics of single PBIs in the solution phase is highly dependent on their local and chemical environments. Furthermore, we visualized different spatial-fluctuations of surface-bound PBIs using defocused wide-field imaging. While PBIs show more steric flexibility in organic media, the flexible motion of PBI molecules in aqueous solution is relatively prohibited due to a cage effect by a hydrogen bonding network, which is previously unobserved. Our method opens up a new possibility to investigate the photophysical properties of multi-chromophoric systems in various solvents at the single-molecule level for developing optimal molecular devices such as water-proof devices.

In-Situ Formed Type I Nanocrystalline Perovskite Film for Highly Efficient Light-Emitting Diode.

Excellent color purity with a tunable band gap renders organic-inorganic halide perovskite highly capable of performing as light-emitting diodes (LEDs). Perovskite nanocrystals show a photoluminescence quantum yield exceeding 90%, which, however, decreases to lower than 20% upon formation of a thin film. The limited photoluminescence quantum yield of a perovskite thin film has been a formidable obstacle for development of highly efficient perovskite LEDs. Here, we report a method for highly luminescent MAPbBr3 (MA = CH3NH3) nanocrystals formed in situ in a thin film based on nonstoichiometric adduct and solvent-vacuum drying approaches. Excess MABr with respect to PbBr2 in precursor solution plays a critical role in inhibiting crystal growth of MAPbBr3, thereby forming nanocrystals and creating type I band alignment with core MAPbBr3 by embedding MAPbBr3 nanocrystals in the unreacted wider band gap MABr. A solvent-vacuum drying process was developed to preserve nanocrystals in the film, which realizes a fast photoluminescence lifetime of 3.9 ns along with negligible trapping processes. Based on a highly luminescent nanocrystalline MAPbBr3 thin film, a highly efficient green LED with a maximum external quantum efficiency of 8.21% and a current efficiency of 34.46 cd/A was demonstrated.

Structure-property relationships in two-dimensionally extended benzoporphyrin molecules probed using single-molecule fluorescence spectroscopy.

The photophysical properties of a series of highly π-conjugated benzoporphyrin molecules (s) with different shapes were investigated in the condensed phase using single-molecule fluorescence spectroscopy. The fluorescence properties of single s were found to be affected by the number of porphyrin units and their molecular shapes. Notably, the single-molecule fluorescence dynamics of the s revealed an increase in the fluorescence lifetimes and blue shifts of the fluorescence spectra indicative of decreasing π-conjugation pathways in the molecules. The distributions of the spectroscopic parameters and the photostability for the molecules also suggest conformational complexities and heterogeneities. Specifically, as the number of constituent porphyrin units increased, the one-step photobleaching behavior ratio and photostability decreased, and the spectroscopic parameter distributions broadened. The structural properties of the s were also directly determined using defocused wide-field imaging and linear dichroism analyses. In particular, molecules with the same number of constituent porphyrins but different molecular shapes exhibited distinct photophysical properties. In summary, these observations provide guidance for the design of molecular systems that can enhance the performance of molecular electronic devices.

Direct observation of structural properties and fluorescent trapping sites in macrocyclic porphyrin arrays at the single-molecule level.

By utilizing single-molecule defocused wide-field fluorescence microscopy, we have investigated the molecular structural properties such as transition dipole moment orientations and the angular relationship among chromophores, as well as structural distortions and flexibilities depending on the ring size, in a series of cyclic porphyrin arrays bearing close likeness in overall architectures to the LH2 complexes in purple bacterial photosynthetic systems. Furthermore, comparing the experimental results with molecular dynamics simulations, we ascertained site selection for fluorescent trapping sites. Collectively, these experimental and computational results provide the basis for structure-property relationships and energy hopping/emitting processes in an important class of artificial light-harvesting molecular systems widely used in molecular electronics technology.

Dysregulation of Parkin-mediated mitophagy in thyroid Hürthle cell tumors.

Abnormal accumulation of defective mitochondria is the hallmark of oncocytes, which are frequently observed in thyroid Hürthle cell lesions. Autophagy is an essential cellular catabolic mechanism for the degradation of dysfunctional organelles and has been implicated in several human diseases. It is yet unknown how autophagic turnover of defective mitochondria in Hürthle cell tumors is regulated. We characterized the expression patterns of molecular markers including Beclin1, LC3, PINK1 and Parkin, which are required for autophagy or mitophagy, in human oncocytic lesions of the thyroid. To undertake mechanistic studies, we investigated autophagy and mitophagy using XTC.UC1 cells, the only in vitro model of Hürthle cell tumors. Beclin1 and LC3 were highly expressed in oncocytes of Hürthle cell tumors. XTC.UC1 showed autophagic responses to starvation and rapamycin treatment, whereas they displayed ineffective activation of mitophagy, which is triggered by the coordinated action of PINK1 and Parkin in response to CCCP. This resulted in a decreased turnover of abnormal mitochondria. The mechanisms underlying defective mitophagy and mitochondrial turnover were investigated by genetic analysis of the PARK2 gene in XTC.UC1 and Hürthle cell tumor tissues. XTC.UC1 and several tumors harbored the V380L mutation, resulting in dysfunctional autoubiquitination and decreased E3 ligase activity. Consistently, oncocytes in Hürthle cell tumors displayed comparable expression of PINK1 but decreased Parkin expression in comparison to normal thyrocytes. The introduction of wild-type Parkin sensitized XTC.UC1 to death induced by CCCP. This study provides a possible etiological basis for oncocytic formation in heterogeneous Hürthle cell tumors through insufficient mitophagy leading to ineffective turnover of aberrant mitochondria caused by dysfunctional Parkin-mediated pathways of mitochondria quality control.

Inhomogeneity in the excited-state torsional disorder of a conjugated macrocycle.

The photophysics of conjugated polymers has generally been explained based on the interactions between the component conjugated chromophores in a tangled chain. However, conjugated chromophores are entities with static and dynamic structural disorder, which directly affects the conjugated polymer photophysics. Here we demonstrate the impact of chain structure torsional disorder on the spectral characteristics for a macrocyclic oligothiophene 1, which is obscured in conventional linear conjugated chromophores by diverse structural disorders such as those in chromophore size and shape. We used simultaneous multiple fluorescence parameter measurement for a single molecule and quantum-mechanical calculations to show that within the fixed conjugation length across the entire ring an inhomogeneity from torsional disorder in the structure of 1 plays a crucial role in causing its energetic disorder, which affords the spectral broadening of ∼220 meV. The torsional disorder in 1 fluctuated on the time scale of hundreds of milliseconds, typically accompanied by spectral drifts on the order of ∼10 meV. The fluctuations could generate torsional defects and change the electronic structure of 1 associated with the ring symmetry. These findings disclose the fundamental nature of conjugated chromophore that is the most elementary spectroscopic unit in conjugated polymers and suggest the importance of engineering structural disorder to optimize polymer-based device photophysics. Additionally, we combined defocused wide-field fluorescence microscopy and linear dichroism obtained from the simultaneous measurements to show that 1 emits polarized light with a changing polarization direction based on the torsional disorder fluctuations.

Reconstruction of the Molecular Structure of a Multichromophoric System Using Single-Molecule Defocused Wide-Field Imaging.

Single-molecule defocused wide-field imaging (DWFI) has been demonstrated to be useful to determine molecular structure parameters, such as the orientations of transition dipole moments and the angular relationships between chromophores in multichromophoric molecular systems. For a series of acetylene-linked perylene bisimide (PBI) macrocycles with different ring size comprising three to six PBI dyes, we reconstructed the molecular structure of the multichromophoric system using DWFI method. Furthermore, we revealed that the structural heterogeneities and distortions depend on the ring size. Our findings illustrate the use of DWFI to gain deeper insight into the structure-property relationships of artificial light-harvesting molecular systems.

A 1,3-phenylene-bridged hexameric porphyrin wheel and efficient excitation energy transfer along the wheel.

A 1,3-phenylene-bridged hexameric Zn(II) porphyrin wheel was synthesized by a Suzuki-Miyaura coupling reaction through a one-pot or a stepwise route. The hexameric wheel structure was revealed by using X-ray diffraction analysis. The porphyrin wheel exhibits a split Soret band due to effective exciton coupling and displays efficient excitation energy transfer along the wheel. Measurements of fluorescence anisotropy decay and pump-power-dependent decay reveal a rapid excitation energy hopping along the wheel with a rate of 1.4 ps.

Shape-dependent two-photon absorption in two-dimensionally extended benzoporphyrin arrays.

In this study, we have investigated the shape-dependence of TPA cross-section values of two-dimensionally extended benzoporphyrin arrays to determine the relationship between the directionality of π-conjugation pathways and TPA properties.

Novel modular endo-ß-1,4-xylanase with transglycosylation activity from Cellulosimicrobium sp. strain HY-13 that is homologous to inverting GH family 6 enzymes.

The gene (2304-bp) encoding a novel xylanolytic enzyme (XylK2) with a catalytic domain, which is 70% identical to that of Cellulomonas flavigena DSM 20109 GH6 ß-1,4-cellobiohydrolase, was identified from an earthworm (Eisenia fetida)-symbiotic bacterium, Cellulosimicrobium sp. strain HY-13. The enzyme consisted of an N-terminal catalytic GH6-like domain, a fibronectin type 3 (Fn3) domain, and a C-terminal carbohydrate-binding module 2 (CBM 2). XylK2ΔFn3-CBM 2 displayed high transferase activity (788.3 IU mg(-1)) toward p-nitrophenyl (PNP) cellobioside, but did not degrade xylobiose, glucose-based materials, or other PNP-sugar derivatives. Birchwood xylan was degraded by XylK2ΔFn3-CBM 2 to xylobiose (59.2%) and xylotriose (40.8%). The transglycosylation activity of the enzyme, which enabled the formation of xylobiose (33.6%) and xylotriose (66.4%) from the hydrolysis of xylotriose, indicates that it is not an inverting enzyme but a retaining enzyme. The endo-ß-1,4-xylanase activity of XylK2ΔFn3-CBM 2 increased significantly by approximately 2.0-fold in the presence of 50mM xylobiose.

A highly active endo-ß-1,4-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of Eisenia fetida.

A xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum, guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManK, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50°C and pH 7.0, ManK exhibited extraordinary high specific activities of 7109 IU/mg and 5158 IU/mg toward locust bean gum and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. Thin layer chromatography revealed that the enzyme degraded locust bean gum to mannobiose and mannotetraose. No detectable amount of mannose was produced from hydrolytic reactions with the substrates. ManK strongly attached to Avicel, ß-cyclodextrin, lignin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManK suggest that it is a unique endo-ß-1,4-mannanase without additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.

Cloning and characterization of a modular GH5 ß-1,4-mannanase with high specific activity from the fibrolytic bacterium Cellulosimicrobium sp. strain HY-13.

The gene (1272-bp) encoding a ß-1,4-mannanase from a gut bacterium of Eisenia fetida, Cellulosimicrobium sp. strain HY-13 was cloned and expressed in Escherichia coli. The recombinant ß-1,4-mannanase (rManH) was approximately 44.0 kDa and has a catalytic GH5 domain that is 65% identical to that of the Micromonospora sp. ß-1,4-mannosidase. The enzyme exhibited the highest catalytic activity toward mannans at 50 °C and pH 6.0. rManH displayed a high specific activity of 14,711 and 8498 IU mg⁻¹ towards ivory nut mannan and locust bean gum, respectively; however it could not degrade the structurally unrelated polysaccharides, mannobiose, or p-nitrophenyl sugar derivatives. rManH was strongly bound to ivory nut mannan, Avicel, chitosan, and chitin but did not attach to curdlan, insoluble oat spelt xylan, lignin, or poly(3-hydroxybutyrate). The superior biocatalytic properties of rManH suggest that the enzyme can be exploited as an effective additive in the animal feed industry.

Cyclodipeptide-bridged porphyrin dimer supramolecular assemblies.

A cyclodipeptide-bridged porphyrin dimer has formed fibrous and toroidal multi-porphyrin array systems by hydrogen bonding-mediated self-assembly.