ASOptimizer: Optimizing antisense oligonucleotides through deep learning for IDO1 gene regulation.

Recent studies have highlighted the effectiveness of using antisense oligonucleotides (ASOs) for cellular RNA regulation, including targets that are considered undruggable; however, manually designing optimal ASO sequences can be labor intensive and time consuming, which potentially limits their broader application. To address this challenge, we introduce a platform, the ASOptimizer, a deep-learning-based framework that efficiently designs ASOs at a low cost. This platform not only selects the most efficient mRNA target sites but also optimizes the chemical modifications for enhanced performance. Indoleamine 2,3-dioxygenase 1 (IDO1) promotes cancer survival by depleting tryptophan and producing kynurenine, leading to immunosuppression through the aryl-hydrocarbon receptor (Ahr) pathway within the tumor microenvironment. We used ASOptimizer to identify ASOs that target IDO1 mRNA as potential cancer therapeutics. Our methodology consists of two stages: sequence engineering and chemical engineering. During the sequence-engineering stage, we optimized and predicted ASO sequences that could target IDO1 mRNA efficiently. In the chemical-engineering stage, we further refined these ASOs to enhance their inhibitory activity while reducing their potential cytotoxicity. In conclusion, our research demonstrates the potential of ASOptimizer for identifying ASOs with improved efficacy and safety.

Kurarinone induced p53-independent G0/G1 cell cycle arrest by degradation of K-RAS via WDR76 in human colorectal cancer cells.

Kurarinone (KR), a naturally occurring flavonoid in Sophora flavescens Aiton and a traditional herbal medicine, reportedly has anti-cancer activity against various cancer types both in vitro and in vivo. However, the cellular mechanism of KR remains unknown. Therefore, we aimed to elucidate the mechanism of cell cycle arrest induced by KR in human colorectal cancer cells. KR not only reduced cell proliferation but also induced G0/G1 arrest of colorectal cancer cell lines. The results of western blotting analysis showed that KR reduced the protein levels of cyclin D1/D3 and CDK4/6 by downregulating signaling proteins such as K-RAS, c-MYC, and p-extracellular signal-regulated kinase. Additionally, KR arrested the cell cycle in the G0/G1 phase in a p53-independent manner, and decreased the protein level of K-RAS by proteasomal degradation dependent on WDR76, an E3 ubiquitin ligase. From these results, we propose that KR could be a potent anti-cancer agent, acting through the degradation of K-RAS dependent on WDR76, regardless of the p53 status.

New phenalenone derivatives from the Hawaiian volcanic soil-associated fungus Penicillium herquei FT729 and their inhibitory effects on indoleamine 2,3-dioxygenase 1 (IDO1).

Phenalenone derivatives sourced from fungi are polyketides that have attracted significant interest because of their diverse chemical structures and potential bioactivities. As part of our ongoing quest to discover novel natural products with biological properties from diverse natural resources, three unreported phenalenone derivatives (1-3), named ent-12-methoxyisoherqueinone (1), (-)-scleroamide (2), and (+)-scleroamide (3), together with four known phenalenone derivatives, ent-atrovenetinone (4), isoherqueinone (5), herqueinone (6), and ent-peniciherquinone (7) were isolated from the Hawaiian soil fungus Penicillium herquei FT729, collected on the Big Island, Hawaii. Compounds 2 and 3 were enantiomers, which were separated using a chiral-phase HPLC column, which provided optically pure compounds 2 and 3. The structures of the novel compounds were established by extensive spectroscopic analyses, including 1D and 2D NMR and high-resolution ESIMS. Their absolute configurations were determined using quantum chemical electronic circular dichroism (ECD) calculations. The inhibitory activity of the isolated compounds (1-7) against indoleamine 2,3-dioxygenase 1 (IDO1) was assessed. Compounds 1, 5-7 inhibited IDO1, with IC50 values of 32.59, 36.86, 19.05, and 24.18 µM, respectively. These findings demonstrated that the phenalenone derivatives 1 and 5-7, as IDO1 inhibitors, are promising anticancer immunotherapeutic agents.

Angucyclines containing ß-á´ -glucuronic acid from Streptomyces sp. KCB15JA151.

Two angucyclines, pseudonocardones D (1) and E (2), were isolated from Streptomyces sp. KCB15JA151. The planar structure was elucidated by comprehensive spectroscopic analysis. The absolute configuration of the sugar unit was determined based on the basis of coupling constants, ROESY, chemical derivatization and HPLC analysis. The biological activities of compounds 1 and 2 were examined by performing a computational target prediction, which led to tests of the antiestrogenic activity. The result suggested that compound 1 might be an ERα antagonist.

Dutomycin Induces Autophagy and Apoptosis by Targeting the Serine Protease Inhibitor SERPINB6.

Autophagy plays an important role in maintaining tumor cell progression and survival in response to metabolic stress. Thus, the regulation of autophagy can be used as a strategy for anticancer therapy. Here, we report dutomycin (DTM) as a novel autophagy enhancer that eventually induces apoptosis due to excessive autophagy. Also, human serine protease inhibitor B6 (SERPINB6) was identified as a target protein of DTM, and its novel function which is involved in autophagy was studied for the first time. We show that DTM directly binds SERPINB6 and then activates intracellular serine proteases, resulting in autophagy induction. Inhibitory effects of DTM on the function of SERPINB6 were confirmed through enzyme- and cell-based approaches, and SERPINB6 was validated as a target protein using siRNA-mediated knockdown and an overexpression test. In a zebrafish xenograft model, DTM showed a significant decrease in tumor area. Furthermore, the present findings will be expected to contribute to the expansion of novel basic knowledge about the correlation of cancer and autophagy by promoting active further research on SERPINB6, which was not previously considered the subject of cancer biology.

Jejucarbazoles A-C, carbazole glycosides with indoleamine 2,3-dioxygenase 1 inhibitory activity from Streptomyces sp. KCB15JA151.

A bioassay-guided investigation led to the isolation of three new carbazole glycosides, jejucarbazoles A-C (1-3), from Streptomyces sp. KCB15JA151. Their planar structures were elucidated by detailed NMR and MS spectroscopic analysis with a literature study. Their relative and absolute configurations were established by ROESY correlations, coupling constants, LC-MS analysis of thiocarbamoyl-thiazolidine carboxylate derivatives, and ECD calculation. Compounds 1-3 showed indoleamine 2,3-dioxygenase 1 (IDO1) inhibitory activity with IC50 values of 18.38, 9.17, and 8.81 µM. The molecular docking analysis suggested that all compounds act as heme-displacing inhibitors against IDO1 enzyme.

Ent-Peniciherqueinone Suppresses Acetaldehyde-Induced Cytotoxicity and Oxidative Stress by Inducing ALDH and Suppressing MAPK Signaling.

Studies on ethanol-induced stress and acetaldehyde toxicity are actively being conducted, owing to an increase in alcohol consumption in modern society. In this study, ent-peniciherqueinone (EPQ) isolated from a Hawaiian volcanic soil-associated fungus Penicillium herquei FT729 was found to reduce the acetaldehyde-induced cytotoxicity and oxidative stress in PC12 cells. EPQ increased cell viability in the presence of acetaldehyde-induced cytotoxicity in PC12 cells. In addition, EPQ reduced cellular reactive oxygen species (ROS) levels and restored acetaldehyde-mediated disruption of mitochondrial membrane potential. Western blot analyses revealed that EPQ treatment increased protein levels of ROS-scavenging heme oxygenase-1 and superoxide dismutase, as well as the levels of aldehyde dehydrogenase (ALDH) 1, ALDH2, and ALDH3, under acetaldehyde-induced cellular stress. Finally, EPQ reduced acetaldehyde-induced phosphorylation of p38 and c-Jun N-terminal kinase, which are associated with ROS-induced oxidative stress. Therefore, our results demonstrated that EPQ prevents cellular oxidative stress caused by acetaldehyde and functions as a potent agent to suppress hangover symptoms and alcohol-related stress.

Herqueilenone A, a unique rearranged benzoquinone-chromanone from the Hawaiian volcanic soil-associated fungal strain Penicillium herquei FT729.

The study of a Hawaiian volcanic soil-associated fungal strain Penicillium herquei FT729 led to the isolation of one unprecedented benzoquinone-chromanone, herqueilenone A (1) and two phenalenone derivatives (2 and 3). Their structures were determined through extensive analysis of NMR spectroscopic data and gauge-including atomic orbital (GIAO) NMR chemical shifts and ECD calculations. Herqueilenone A (1) contains a chroman-4-one core flanked by a tetrahydrofuran and a benzoquinone with an acetophenone moiety. Plausible pathways for the biosynthesis of 1-3 are proposed. Compounds 2 and 3 inhibited IDO1 activity with IC50 values of 14.38 and 13.69 µM, respectively. Compounds 2 and 3 also demonstrated a protective effect against acetaldehyde-induced damage in PC-12 cells.

Highly oxygenated angucycline from Streptomyces sp. KCB15JA014.

LC/MS-based chemical screening of culture extract led to a new highly oxygenated angucycline derivative, grecocycline D (1), from Streptomyces sp. KCB15JA014, isolated from a soil sample of Oedolgae in Jeju Island, Korea. The planar structure was determined on the basis of spectroscopic analysis, including 1D and 2D NMR techniques as well as HRESIMS and comparison with data from the literature. A relative and absolute configuration of 1 was assigned by ROESY experiment and electronic circular dichroism calculation. Compound 1 showed weak inhibitory activity against indoleamine 2,3-dioxygenase.

Enhanced mating-type switching and sexual hybridization in heterothallic yeast Yarrowia lipolytica.

Yarrowia lipolytica is a non-conventional, heterothallic, oleaginous yeast with wide range of industrial applications. Increasing ploidy can improve advantageous traits for industrial applications including genetic stability, stress resistance, and productivity, but the construction of knockout mutant strains from polyploid cells requires significant effort due to the increased copy numbers of target genes. The goal of this study was to evaluate the effectiveness of a mating-type switching strategy by single-step transformation without a genetic manipulation vestige, and to optimize the conventional method for increasing ploidy (mating) in Y. lipolytica. In this study, mating-type genes in haploid Y. lipolytica cells were scarlessly converted into the opposite type genes by site-specific homologous recombination, and the resulting MATB-type cells were mated at low temperature (22°C) with addition of sodium citrate with each MATA-type haploid cell to yield a MATA/MATB-type diploid strain with genetic information from both parental strains. The results of this study can be used to increase ploidy and for whole genome engineering of a yeast strain with unparalleled versatility for industrial application.

Kushenol E inhibits autophagy and impairs lysosomal positioning via VCP/p97 inhibition.

Autophagy plays a major role in cell survival and has therefore been exploited as an important strategy in cancer therapy. In this study, we evaluated the autophagy-regulatory effects of kushenol E (KE), a bi-prenylated flavonoid isolated from Sophora flavescens and found that KE increased LC3B-II levels while inducing the formation of autophagic vacuoles and immature autophagosomes in HeLa and HCT116 cells. Transmission electron microscopy images revealed that KE treatment generates immature autophagosomes. Furthermore, KE inhibited autophagosome maturation as demonstrated by blocking the degradation of EGFP puncta in HeLa cells stably expressing EGFP-mRFP-LC3B. It also reduced lysosomal activity and cathepsin maturation by disrupting lysosomal positioning, subsequently inducing apoptosis. Further, a combinatorial approach employing cellular thermal shift assays, revealed valosin-containing protein (VCP)/p97 as a potential target protein of KE; the knockdown and overexpression of VCP/p97 confirmed its involvement in regulating lysosomal positioning for autophagy maturation via direct interactions with KE. Thus, KE may possess autophagy-regulating properties mediated by binding to VCP/p97.

Inhibitory effects of flavonoids isolated from Sophora flavescens on indoleamine 2,3-dioxygenase 1 activity.

Indoleamine 2,3-dioxygenase 1 (IDO1), a tryptophan catabolising enzyme, is known as a tumour cell survival factor that causes immune escape in several types of cancer. Flavonoids of Sophora flavescens have a variety of biological benefits for humans; however, cancer immunotherapy effect has not been fully investigated. The flavonoids (1-6) isolated from S. flavescens showed IDO1 inhibitory activities (IC50 4.3-31.4 µM). The representative flavonoids (4-6) of S. flavescens were determined to be non-competitive inhibitors of IDO1 by kinetic analyses. Their binding affinity to IDO1 was confirmed using thermal stability and surface plasmon resonance (SPR) assays. The molecular docking analysis and mutagenesis assay revealed the structural details of the interactions between the flavonoids (1-6) and IDO1. These results suggest that the flavonoids (1-6) of S. flavescens, especially kushenol E (6), as IDO1 inhibitors might be useful in the development of immunotherapeutic agents against cancers.

Smartwatch User Interface Implementation Using CNN-Based Gesture Pattern Recognition.

In recent years, with an increase in the use of smartwatches among wearable devices, various applications for the device have been developed. However, the realization of a user interface is limited by the size and volume of the smartwatch. This study aims to propose a method to classify the user's gestures without the need of an additional input device to improve the user interface. The smartwatch is equipped with an accelerometer, which collects the data and learns and classifies the gesture pattern using a machine learning algorithm. By incorporating the convolution neural network (CNN) model, the proposed pattern recognition system has become more accurate than the existing model. The performance analysis results show that the proposed pattern recognition system can classify 10 gesture patterns at an accuracy rate of 97.3%.

Targeted Delivery of Immunomodulators to Lymph Nodes.

Active-targeted delivery to lymph nodes represents a major advance toward more effective treatment of immune-mediated disease. The MECA79 antibody recognizes peripheral node addressin molecules expressed by high endothelial venules of lymph nodes. By mimicking lymphocyte trafficking to the lymph nodes, we have engineered MECA79-coated microparticles containing an immunosuppressive medication, tacrolimus. Following intravenous administration, MECA79-bearing particles showed marked accumulation in the draining lymph nodes of transplanted animals. Using an allograft heart transplant model, we show that targeted lymph node delivery of microparticles containing tacrolimus can prolong heart allograft survival with negligible changes in tacrolimus serum level. Using MECA79 conjugation, we have demonstrated targeted delivery of tacrolimus to the lymph nodes following systemic administration, with the capacity for immune modulation in vivo.

Etoposide Induces Necrosis Through p53-Mediated Antiapoptosis in Human Kidney Proximal Tubule Cells.

The p53 protein is an important transcription factor that modulates signaling pathways for both cell death and survival. Its antiapoptotic mechanisms that correlate with necrotic and apoptotic cell death are not well understood. Here, we report that etoposide promotes progression of the DNA damage response as well as necrotic morphological changes including plasma membrane rupture using carbon nanotube-tipped/atomic force microscopy (CNT/AFM) probes in human kidney proximal tubule (HK-2) cells. Inhibition of p53 abrogated cell cycle arrest and led to a decrease in the expression levels of repair proteins that were induced by DNA damage. Mitochondrial biogenesis and cytosolic production of reactive oxygen species were also reduced after p53 inhibition; the latter change induced mitochondrial superoxide accumulation and mitochondrial damage, which triggered the activation of caspase 3. Inhibition of p53 also led to a loss of cell adhesion and converted necrotic cell death to apoptotic cell death, with appreciable cell shrinkage and appearance of apoptotic bodies that were observed using CNT/AFM probes. Thus, our study demonstrated that p53 protects against apoptosis, and leads to etoposide-induced necrosis. These results are expected to aid in the understanding of mechanism of antiapoptosis and its relationship to cell death.

Investigating the optimal size of anticancer nanomedicine.

Nanomedicines (NMs) offer new solutions for cancer diagnosis and therapy. However, extension of progression-free interval and overall survival time achieved by Food and Drug Administration-approved NMs remain modest. To develop next generation NMs to achieve superior anticancer activities, it is crucial to investigate and understand the correlation between the physicochemical properties of NMs (particle size in particular) and their interactions with biological systems to establish criteria for NM optimization. Here, we systematically evaluated the size-dependent biological profiles of three monodisperse drug-silica nanoconjugates (NCs; 20, 50, and 200 nm) through both experiments and mathematical modeling and aimed to identify the optimal size for the most effective anticancer drug delivery. Among the three NCs investigated, the 50-nm NC shows the highest tumor tissue retention integrated over time, which is the collective outcome of deep tumor tissue penetration and efficient cancer cell internalization as well as slow tumor clearance, and thus, the highest efficacy against both primary and metastatic tumors in vivo.

Immunosuppressive Activity of Size-Controlled PEG-PLGA Nanoparticles Containing Encapsulated Cyclosporine A.

We encapsulated cyclosporine A (CsA) in poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) (PEG-PLGA) nanoparticles (NPs) by nanoprecipitation of CsA and PEG-PLGA. The resulting CsA/PEG-PLGA-NPs were <100 nm in diameter with a narrow particle size distribution. The NP size could be controlled by tuning the polymer concentration, solvent, or water/solvent ratio during formulation. The PEGylated NPs maintained non-aggregated in salt solution. Solid NPs lyoprotected with bovine serum albumin were prepared for the convenience of storage and transportation. The release kinetics of CsA (55.6% released on Day 1) showed potential for maintaining therapeutic CsA concentrations in vivo. In T-cell assays, both free CsA and CsA/PEG-PLGA-NPs suppressed T-cell proliferation and production of inflammatory cytokines dose dependently. In a mixed lymphocyte reaction assay, the IC(50) values for free CsA and CsA/PEG-PLGA-NPs were found to be 30 and 35 ng/mL, respectively. This nanoparticulate CsA delivery technology constitutes a strong basis for future targeted delivery of immunosuppressive drugs with improved efficiency and potentially reduced toxicity.