A Fully-Human Antibody Specifically Targeting a Membrane-Bound Fragment of CADM1 Potentiates the T Cell-Mediated Death of Human Small-Cell Lung Cancer Cells

Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer and the leading cause of global cancer-related mortality. Despite the earlier identification of membrane-proximal cleavage of cell adhesion molecule 1 (CADM1) in cancers, the role of the membrane-bound fragment of CAMD1 (MF-CADM1) is yet to be clearly identified. In this study, we first isolated MF-CADM1-specific fully human single-chain variable fragments (scFvs) from the human synthetic scFv antibody library using the phage display technology. Following the selected scFv conversion to human immunoglobulin G1 (IgG1) scFv-Fc antibodies (K103.1–4), multiple characterization studies, including antibody cross-species reactivity, purity, production yield, and binding affinity, were verified. Finally, via intensive in vitro efficacy and toxicity evaluation studies, we identified K103.3 as a lead antibody that potently promotes the death of human SCLC cell lines, including NCI-H69, NCI-H146, and NCI-H187, by activated Jurkat T cells without severe endothelial toxicity. Taken together, these findings suggest that antibody-based targeting of MF-CADM1 may be an effective strategy to potentiate T cell-mediated SCLC death, and MF-CADM1 may be a novel potential therapeutic target in SCLC for antibody therapy.     

Single-Step Fast Tissue Clearing of Thick Mouse Brain Tissue for Multi-Dimensional High-Resolution Imaging

Recent advances in optical clearing techniques have dramatically improved deep tissue imaging by reducing the obscuring effects of light scattering and absorption. However, these optical clearing methods require specialized equipment or a lengthy undertaking with complex protocols that can lead to sample volume changes and distortion. In addition, the imaging of cleared tissues has limitations, such as fluorescence bleaching, harmful and foul-smelling solutions, and the difficulty of handling samples in high-viscosity refractive index (RI) matching solutions. To address the various limitations of thick tissue imaging, we developed an Aqueous high refractive Index matching and tissue Clearing solution for Imaging (termed AICI) with a one-step tissue clearing protocol that was easily made at a reasonable price in our own laboratory without any equipment. AICI can rapidly clear a 1 mm thick brain slice within 90 min with simultaneous RI matching, low viscosity, and a high refractive index (RI = 1.466), allowing the imaging of the sample without additional processing. We compared AICI with commercially available RI matching solutions, including optical clear agents (OCAs), for tissue clearing. The viscosity of AICI is closer to that of water compared with other RI matching solutions, and there was a less than 2.3% expansion in the tissue linear morphology during 24 h exposure to AICI. Moreover, AICI remained fluid over 30 days of air exposure, and the EGFP fluorescence signal was only reduced to ~65% after 10 days. AICI showed a limited clearing of brain tissue >3 mm thick. However, fine neuronal structures, such as dendritic spines and axonal boutons, could still be imaged in thick brain slices treated with AICI. Therefore, AICI is useful not only for the three-dimensional (3D) high-resolution identification of neuronal structures, but also for the examination of multiple structural imaging by neuronal distribution, projection, and gene expression in deep brain tissue. AICI is applicable beyond the imaging of fluorescent antibodies and dyes, and can clear a variety of tissue types, making it broadly useful to researchers for optical imaging applications.     

Characterization of the Secretome of a Specific Cell Expressing Mutant Methionyl-tRNA Synthetase in Co-Culture Using Click Chemistry

Co-culture system, in which two or more distinct cell types are cultured together, is advantageous in that it can mimic the environment of the in vivo niche of the cells. In this study, we presented a strategy to analyze the secretome of a specific cell type under the co-culture condition in serum-supplemented media. For the cell-specific secretome analysis, we expressed the mouse mutant methionyl-tRNA synthetase for the incorporation of the non-canonical amino acid, azidonorleucine into the newly synthesized proteins in cells of which the secretome is targeted. The azidonorleucine-tagged secretome could be enriched, based on click chemistry, and distinguished from any other contaminating proteins, either from the cell culture media or the other cells co-cultured with the cells of interest. In order to have more reliable true-positive identifications of cell-specific secretory bodies, we established criteria to exclude any identified human peptide matched to bovine proteins. As a result, we identified a maximum of 719 secreted proteins in the secretome analysis under this co-culture condition. Last, we applied this platform to profile the secretome of mesenchymal stem cells and predicted its therapeutic potential on osteoarthritis based on secretome analysis.     

Intratracheal Transplantation of Mesenchymal Stem Cells Attenuates Hyperoxia-Induced Microbial Dysbiosis in the Lungs,Brain, and Gut in Newborn Rats

We attempted to determine whether intratracheal (IT) transplantation of mesenchymal stem cells (MSCs) could simultaneously attenuate hyperoxia-induced lung injuries and microbial dysbiosis of the lungs, brain, and gut in newborn rats. Newborn rats were exposed to hyperoxia (90% oxygen) for 14 days. Human umbilical cord blood-derived MSCs (5 × 10⁵) were transplanted via the IT route on postnatal day (P) five. At P14, the lungs were harvested for histological, biochemical, and microbiome analyses. Bacterial 16S ribosomal RNA genes from the lungs, brain, and large intestine were amplified, pyrosequenced, and analyzed. IT transplantation of MSCs simultaneously attenuated hyperoxia-induced lung inflammation and the ensuing injuries, as well as the dysbiosis of the lungs, brain, and gut. In correlation analyses, lung interleukin-6 (IL-6) levels were significantly positively correlated with the abundance of Proteobacteria in the lungs, brain, and gut, and it was significantly inversely correlated with the abundance of Firmicutes in the gut and lungs and that of Bacteroidetes in the lungs. In conclusion, microbial dysbiosis in the lungs, brain, and gut does not cause but is caused by hyperoxic lung inflammation and ensuing injuries, and IT transplantation of MSCs attenuates dysbiosis in the lungs, brain, and gut, primarily by their anti-oxidative and anti-inflammatory effects.     

Modified Fluoroquinolones as Antimicrobial Compounds Targeting Chlamydia trachomatis

Chlamydia trachomatis causes the most common sexually transmitted bacterial infection and trachoma, an eye infection. Untreated infections can lead to sequelae, such as infertility and ectopic pregnancy in women and blindness. We previously enhanced the antichlamydial activity of the fluoroquinolone ciprofloxacin by grafting a metal chelating moiety onto it. In the present study, we pursued this pharmacomodulation and obtained nanomolar active molecules (EC₅₀) against this pathogen. This gain in activity prompted us to evaluate the antibacterial activity of this family of molecules against other pathogenic bacteria, such as Neisseria gonorrhoeae and bacteria from the ESKAPE group. The results show that the novel molecules have selectively improved activity against C. trachomatis and demonstrate how the antichlamydial effect of fluoroquinolones can be enhanced.     

Oxidative Stress and Chemoradiation-Induced Oral Mucositis: A Scoping Review of In Vitro,In Vivo and Clinical Studies

Chemoradiation-induced mucositis is a debilitating condition of the gastrointestinal tract eventuating from antineoplastic treatment. It is believed to occur primarily due to oxidative stress mechanisms, which generate Reactive Oxygen Species (ROS). The aim of this scoping review was to assess the role of oxidative stress in the development of Oral Mucositis (OM). Studies from the literature, published in MEDLINE and SCOPUS, that evaluated the oxidative stress pathways or antioxidant interventions for OM, were retrieved to elucidate the current understanding of their relationship. Studies failing inclusion criteria were excluded, and those suitable underwent data extraction, using a predefined data extraction table. Eighty-nine articles fulfilled criteria, and these were sub-stratified into models of study (in vitro, in vivo, or clinical) for evaluation. Thirty-five clinical studies evaluated antioxidant interventions on OM’s severity, duration, and pain, amongst other attributes. A number of clinical studies sought to elucidate the protective or therapeutic effects of compounds that had been pre-determined to have antioxidant properties, without directly assessing oxidative stress parameters (these were deemed “indirect evidence”). Forty-seven in vivo studies assessed the capacity of various compounds to prevent OM. Findings were mostly consistent, reporting reduced OM severity associated with a reduction in ROS, malondialdehyde (MDA), myeloperoxidase (MPO), but higher glutathione (GSH) and superoxide dismutase (SOD) activity or expression. Twenty-one in vitro studies assessed potential OM therapeutic interventions. The majority demonstrated successful a reduction in ROS, and in select studies, secondary molecules were assessed to identify the mechanism. In summary, this review highlighted numerous oxidative stress pathways involved in OM pathogenesis, which may inform the development of novel therapeutic targets.     

VI-116, A Novel Potent Inhibitor of VRAC with Minimal Effect on ANO1

Volume-regulated anion channel (VRAC) is ubiquitously expressed and plays a pivotal role in vertebrate cell volume regulation. A heterologous complex of leucine-rich repeat containing 8A (LRRC8A) and LRRC8B-E constitutes the VRAC, which is involved in various processes such as cell proliferation, migration, differentiation, intercellular communication, and apoptosis. However, the lack of a potent and selective inhibitor of VRAC limits VRAC-related physiological and pathophysiological studies, and most previous VRAC inhibitors strongly blocked the calcium-activated chloride channel, anoctamin 1 (ANO1). In the present study, we performed a cell-based screening for the identification of potent and selective VRAC inhibitors. Screening of 55,000 drug-like small-molecules and subsequent chemical modification revealed 3,3′-((2-hydroxy-3-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (VI-116), a novel potent inhibitor of VRAC. VI-116 fully inhibited VRAC-mediated I⁻ quenching with an IC₅₀ of 1.27 ± 0.18 μM in LN215 cells and potently blocked endogenous VRAC activity in PC3, HT29 and HeLa cells in a dose-dependent manner. Notably, VI-116 had no effect on intracellular calcium signaling up to 10 μM, which completely inhibited VRAC, and showed high selectivity for VRAC compared to ANO1 and ANO2. However, DCPIB, a VRAC inhibitor, significantly affected ATP-induced increases in intracellular calcium levels and Eact-induced ANO1 activation. In addition, VI-116 showed minimal effect on hERG K⁺ channel activity up to 10 μM. These results indicate that VI-116 is a potent and selective VRAC inhibitor and a useful research tool for pharmacological dissection of VRAC.     

Supersonic Nozzle Flow Simulations for Particle Coating Applications: Effects of Shockwaves,Nozzle Geometry,Ambient Pressure,and Substrate Location upon Flow Characteristics

Characteristics of supersonic flow are examined with specific regard to nano-particle thin-film coating. Effects of shockwaves, nozzle geometry, chamber pressure, and substrate location were studied computationally. Shockwaves are minimized to reduce fluctuations in flow properties at the discontinuities across diamond shock structures. Nozzle geometry was adjusted to ensure optimal expansion (i.e., P _exit = P _ambient), where shock formation was significantly reduced and flow kinetic energy maximized. When the ambient pressure was reduced from 1 to 0.01316 bar, the nozzle’s diverging angle must be increased to yield the optimum condition of minimized adversed effects. Beyond some critical distance, substrate location did not seem to be a sensitive parameter on flow characteristics when P _amb = 0.01316 bar; however, overly close proximity to the nozzle exit caused flow disturbances inside the nozzle, thereby adversely affecting coating gas flow.     

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.