A Novel Neurofilament Light Chain ELISA Validated in Patients with Alzheimer’s Disease,Frontotemporal Dementia,and Subjective Cognitive Decline,and the Evaluation of Candidate Proteins for Immunoassay Calibration

Neurofilament light chain (Nf-L) is a well-known biomarker for axonal damage; however, the corresponding circulating Nf-L analyte in cerebrospinal fluid (CSF) is poorly characterized. We therefore isolated new monoclonal antibodies against synthetic peptides, and these monoclonals were characterized for their specificity on brain-specific intermediate filament proteins. Two highly specific antibodies, ADx206 and ADx209, were analytically validated for CSF applications according to well-established criteria. Interestingly, using three different sources of purified Nf-L proteins, a significant impact on interpolated concentrations was observed. With a lower limit of analytical sensitivity of 100 pg/mL using bovine Nf-L as the calibrator, we were able to quantify the Nf-L analyte in each sample, and these Nf-L concentrations were highly correlated to the Uman diagnostics assay (Spearman rho = 0.97, p < 0.001). In the clinical diagnostic groups, the new Nf-L ELISA could discriminate patients with Alzheimer’s disease (AD, n = 20) from those with frontotemporal lobe dementia (FTD, n = 20) and control samples with subjective cognitive decline (SCD, n = 20). Henceforth, this novel Nf-L ELISA with well-defined specificity and epitopes can be used to enhance our understanding of harmonizing the use of Nf-L as a clinically relevant marker for neurodegeneration in CSF.     

HDAC8-Selective Inhibition by PCI-34051 Enhances the Anticancer Effects of ACY-241 in Ovarian Cancer Cells

HDAC6 is overexpressed in ovarian cancer and is known to be correlated with tumorigenesis. Accordingly, ACY-241, a selective HDAC6 inhibitor, is currently under clinical trial and has been tested in combination with various drugs. HDAC8, another member of the HDAC family, has recently gained attention as a novel target for cancer therapy. Here, we evaluated the synergistic anticancer effects of PCI-34051 and ACY-241 in ovarian cancer. Among various ovarian cancer cells, PCI-34051 effectively suppresses cell proliferation in wild-type p53 ovarian cancer cells compared with mutant p53 ovarian cancer cells. In ovarian cancer cells harboring wild-type p53, PCI-34051 in combination with ACY-241 synergistically represses cell proliferation, enhances apoptosis, and suppresses cell migration. The expression of pro-apoptotic proteins is synergistically upregulated, whereas the expressions of anti-apoptotic proteins and metastasis-associated proteins are significantly downregulated in combination treatment. Furthermore, the level of acetyl-p53 at K381 is synergistically upregulated upon combination treatment. Overall, co-inhibition of HDAC6 and HDAC8 through selective inhibitors synergistically suppresses cancer cell proliferation and metastasis in p53 wild-type ovarian cancer cells. These results suggest a novel approach to treating ovarian cancer patients and the therapeutic potential in developing HDAC6/8 dual inhibitors.     

Generalized Approach towards Secretion-Based Protein Production via Neutralization of Secretion-Preventing Cationic Substrate Residues

Many heterologous proteins can be secreted by bacterial ATP-binding cassette (ABC) transporters, provided that they are fused with the C-terminal signal sequence, but some proteins are not secretable even though they carry the right signal sequence. The invention of a method to secrete these non-secretable proteins would be valuable both for understanding the secretory physiology of ABC transporters and for industrial applications. Herein, we postulate that cationic “supercharged” regions within the target substrate protein block the secretion by ABC transporters. We also suggest that the secretion of such substrate proteins can be rescued by neutralizing those cationic supercharged regions via structure-preserving point mutageneses. Surface-protruding, non-structural cationic amino acids within the cationic supercharged regions were replaced by anionic or neutral hydrophilic amino acids, reducing the cationic charge density. The examples of rescued secretions we provide include the spike protein of SARS-CoV-2, glutathione-S-transferase, streptavidin, lipase, tyrosinase, cutinase, growth factors, etc. In summary, our study provides a method to predict the secretability and a tool to rescue the secretion by correcting the secretion-blocking regions, making a significant step in understanding the physiological properties of ABC transporter-dependent protein secretion and laying the foundation for the development of a secretion-based protein-producing platform.     

Topical Capsaicin in Poly(lactic-co-glycolic)acid (PLGA) Nanoparticles Decreases Acute Itch and Heat Pain

Background: Capsaicin, the hot pepper agent, produces burning followed by desensitization. To treat localized itch or pain with minimal burning, low capsaicin concentrations can be repeatedly applied. We hypothesized that alternatively controlled release of capsaicin from poly(lactic-co-glycolic acid) (PLGA) nanoparticles desensitizes superficially terminating nociceptors, reducing burning. Methods: Capsaicin-loaded PLGA nanoparticles were prepared (single-emulsion solvent evaporation) and characterized (size, morphology, capsaicin loading, encapsulation efficiency, in vitro release profile). Capsaicin-PLGA nanoparticles were applied to murine skin and evaluated in healthy human participants (n = 21) for 4 days under blinded conditions, and itch and nociceptive sensations evoked by mechanical, heat stimuli and pruritogens cowhage, β-alanine, BAM8-22 and histamine were evaluated. Results: Nanoparticles (loading: 58 µg capsaicin/mg) released in vitro 23% capsaicin within the first hour and had complete release at 72 h. In mice, 24 h post-application Capsaicin-PLGA nanoparticles penetrated the dermis and led to decreased nociceptive behavioral responses to heat and mechanical stimulation (desensitization). Application in humans produced a weak to moderate burning, dissipating after 3 h. A loss of heat pain up to 2 weeks was observed. After capsaicin nanoparticles, itch and nociceptive sensations were reduced in response to pruritogens cowhage, β-alanine or BAM8-22, but were normal to histamine. Conclusions: Capsaicin nanoparticles could be useful in reducing pain and itch associated with pruritic diseases that are histamine-independent.     

Dimerization Tendency of 3CLpros of Human Coronaviruses Based on the X-ray Crystal Structure of the Catalytic Domain of SARS-CoV-2 3CLpro

3CLpro of SARS-CoV-2 is a promising target for developing anti-COVID19 agents. In order to evaluate the catalytic activity of 3CLpros according to the presence or absence of the dimerization domain, two forms had been purified and tested. Enzyme kinetic studies with a FRET method revealed that the catalytic domain alone presents enzymatic activity, despite it being approximately 8.6 times less than that in the full domain. The catalytic domain was crystallized and its X-ray crystal structure has been determined to 2.3 Å resolution. There are four protomers in the asymmetric unit. Intriguingly, they were packed as a dimer though the dimerization domain was absent. The RMSD of superimposed two catalytic domains was 0.190 for 182 Cα atoms. A part of the long hinge loop (LH-loop) from Gln189 to Asp197 was not built in the model due to its flexibility. The crystal structure indicates that the decreased proteolytic activity of the catalytic domain was due to the incomplete construction of the substrate binding part built by the LH-loop. A structural survey with other 3CLpros showed that SARS-CoV families do not have interactions between DM-loop due to the conformational difference at the last turn of helix α7 compared with others. Therefore, we can conclude that the monomeric form contains nascent enzyme activity and that its efficiency increases by dimerization. This new insight may contribute to understanding the behavior of SARS-CoV-2 3CLpro and thus be useful in developing anti-COVID-19 agents.     

Bias-error reduction in nanometer resolution microscopic motion tracking vision systems

Microscopic Motion Tracking Systems utilize sub-pixel algorithms to achieve close to nanometer precision motion measurement. This paper characterizes the bias-error of template matching image registration techniques which are commonly used to achieve this level of sub-pixel accuracy. An accurate functional expression for such bias error is derived, which shows how the error depends on image content. Furthermore, this expression is directly utilized in a bias reduction scheme that is shown to significantly reduce the bias error. The newly derived expressions and reduction schemes for bias are applied to a Microscopic Vision System that measures micro-object motion with close to nanometer resolution.     

Mechanical and moisture-dependent swelling properties of compressed Japanese cedar

In this research, five groups of compressed wood specimens were investigated, four made from compressed Japanese cedar (Cryptomeria japonica D. Don) with compression ratios (CR) of 33%, 50%, 67% and 70%, and one without compressing for control purpose. The measurements of moisture-dependent swelling of the specimens were carried out in the R, T and L directions, with typical strain values of 10%, 1.5% and 0.1% respectively corresponding to the CR = 67%. Mechanical property tests of the compressed wood were also undertaken. Material properties obtained cover radial and tangential swelling strain rates, shear moduli in LR, LT and RT planes, Young’s moduli in the L, R, T directions and Poisson ratios in all planes. The Young’s moduli along the longitudinal and radial directions have been increased by over 300% in relation to the CR of 70%.     

A Composite Formation Route to Well‐Crystalline Manganese Oxide Nanocrystals: High Catalytic Activity of Manganate–Alumina Nanocomposites

Manganese oxide nanocrystals are combined with aluminum oxide nanocrystals to improve their crystallinity via calcination without a significant increase of crystal size. A nanocomposite, consisting of two metal oxides, can be synthesized by the reaction between permanganate anions and aluminum oxyhydroxide keggin cations. The as‐prepared manganese oxide–aluminum oxide nanocomposite is X‐ray amorphous whereas heat‐treatment gives rise to the crystallization of an α‐MnO₂ phase at 600 °C and Mn₃O₄/Mn₂O₃ and γ‐Al₂O₃ phases at 800 °C. Electron microscopy and N₂ adsorption‐desorption‐isotherm analysis clearly demonstrate that the as‐prepared nanocomposite is composed of a porous assembly of monodisperse primary particles with a size of ～20 nm and a surface area of >410 m² g^?1. Of particular interest is that the small particle size of the as‐prepared nanocomposite is well‐maintained up to 600 °C, a result of the prevention of the growth of manganate grains through nanoscale mixing with alumina grains. The calcined nanocomposite shows very‐high catalytic activity for the oxidation of cyclohexene with an extremely high conversion efficiency of >95% within 15 min. The present results show that the improvement of the crystallinity without significant crystal growth is very crucial for optimizing the catalytic activity of manganese oxide nanocrystals.