Mucosal TLR5 activation controls healthspan and longevity.

Addressing age-related immunological defects through therapeutic interventions is essential for healthy aging, as the immune system plays a crucial role in controlling infections, malignancies, and in supporting tissue homeostasis and repair. In our study, we show that stimulating toll-like receptor 5 (TLR5) via mucosal delivery of a flagellin-containing fusion protein effectively extends the lifespan and enhances the healthspan of mice of both sexes. This enhancement in healthspan is evidenced by diminished hair loss and ocular lens opacity, increased bone mineral density, improved stem cell activity, delayed thymic involution, heightened cognitive capacity, and the prevention of pulmonary lung fibrosis. Additionally, this fusion protein boosts intestinal mucosal integrity by augmenting the surface expression of TLR5 in a certain subset of dendritic cells and increasing interleukin-22 (IL-22) secretion. In this work, we present observations that underscore the benefits of TLR5-dependent stimulation in the mucosal compartment, suggesting a viable strategy for enhancing longevity and healthspan.

Polyporus ulleungus mycelia cultured in MEB medium produce metabolites with anticancer property.

Cancer cells are characterized by apoptosis evasion and uncontrolled cell cycle progression. To combat these characteristics, efforts have been made to find novel natural-source anticancer compounds. The aim of this work is to find new anticancer compounds in Polyporus ulleungus (P. ulleungus) mycelial culture extracts. P. ulleungus mycelium was cultured on four individual media (DYB, MEB, MYB, and PDB) and four extracts were generated from the mycelium culture media. Extracts of P. ulleungus mycelium cultured in MEB medium (pu-MEB) significantly reduced cancer cell growth by triggering apoptosis and S phase arrest. Furthermore, the anticancer effects of pu-MEB were not confined to one type of cancer. Taken together, our results confirmed that P. ulleungus mycelia cultured in MEB medium produce metabolites that exhibit anticancer properties. Development of an optimal medium for P. ulleungus mycelium through optimization of medium components will enable P. ulleungus mycelium to produce metabolites with more anticancer efficacy.

Chemical screening identifies the anticancer properties of Polyporous tuberaster.

Most conventional anticancer drugs cause resistance to chemotherapy, which has emerged as one of the major obstacles to cancer treatment. In order to address this issue, efforts have been made to select new anticancer compounds from natural sources. The aim of this study is to identify novel anticancer compounds from mycelial culture extracts belonging to Polyporus tuberaster (P. tuberaster). Here, we found that mycelial culture extracts of P. tuberaster cultured in PDB medium (pt-PDB) effectively inhibited cancer cell growth. pt-PDB reduced the growth of cancer cells through apoptosis induction and S-phase arrest. The anticancer efficacy of pt-PDB was not to limited to one type of cancer. Furthermore, unlike traditional anticancer medications, pt-PDB did not increase the proportion of side population (SP) cells, which plays a key role in the development of chemoresistance. Taken together, we discovered a novel anticancer drug candidate that has anticancer properties without increasing the proportion of SP cells. This new drug candidate can be used for the treatment of cancer, especially chemoresistant malignancies, and will provide a breakthrough in the treatment of chemoresistant cancer.

Targeting Mitochondrial Oxidative Stress as a Strategy to Treat Aging and Age-Related Diseases.

Mitochondria are one of the organelles undergoing rapid alteration during the senescence process. Senescent cells show an increase in mitochondrial size, which is attributed to the accumulation of defective mitochondria, which causes mitochondrial oxidative stress. Defective mitochondria are also targets of mitochondrial oxidative stress, and the vicious cycle between defective mitochondria and mitochondrial oxidative stress contributes to the onset and development of aging and age-related diseases. Based on the findings, strategies to reduce mitochondrial oxidative stress have been suggested for the effective treatment of aging and age-related diseases. In this article, we discuss mitochondrial alterations and the consequent increase in mitochondrial oxidative stress. Then, the causal role of mitochondrial oxidative stress on aging is investigated by examining how aging and age-related diseases are exacerbated by induced stress. Furthermore, we assess the importance of targeting mitochondrial oxidative stress for the regulation of aging and suggest different therapeutic strategies to reduce mitochondrial oxidative stress. Therefore, this review will not only shed light on a new perspective on the role of mitochondrial oxidative stress in aging but also provide effective therapeutic strategies for the treatment of aging and age-related diseases through the regulation of mitochondrial oxidative stress.

Venadaparib Is a Novel and Selective PARP Inhibitor with Improved Physicochemical Properties, Efficacy, and Safety.

PARP inhibitors have been approved by the FDA for use in the treatment of patients with ovarian, breast, pancreatic, and prostate cancers. PARP inhibitors show diverse suppressive effects on PARP family members and PARP-DNA trapping potency. These properties are associated with distinct safety/efficacy profiles. Here, we report the nonclinical characteristics of venadaparib (also known as IDX-1197 or NOV140101), a novel potent PARP inhibitor. The physiochemical properties of venadaparib were analyzed. Furthermore, the efficacy of venadaparib against PARP enzymes, PAR formation, and PARP trapping activities, and growth inhibition of cell lines with BRCA mutations were evaluated. Ex vivo and in vivo models were also established to study pharmacokinetics/pharmacodynamics, efficacy, and toxicity. Venadaparib specifically inhibits PARP-1 and -2 enzymes. Oral administration of venadaparib HCl at doses above 12.5 mg/kg significantly reduced tumor growth in the OV_065 patient-derived xenograft model. Intratumoral PARP inhibition remained at over 90% until 24 hours after dosing. Venadaparib had wider safety margins than olaparib. Notably, venadaparib showed favorable physicochemical properties and superior anticancer effects in homologous recombination-deficient in vitro and in vivo models with improved safety profiles. Our results suggest the possibility of venadaparib as a next-generation PARP inhibitor. On the basis of these findings, phase Ib/IIa studies on the efficacy and safety of venadaparib have been initiated.

A novel hybrid promoter capable of continuously producing proteins in high yield.

The establishment of cell lines with a high protein production is the most crucial objective in the field of biopharmaceuticals. To this end, efforts have been made to increase transgene expression through promoter improvement, but the efficiency or stability of protein production was insufficient for use in commercial production. Here, we developed a novel strategy to increase the efficiency and stability of protein production by hybridizing a promoter that exhibits higher expression levels at the transient level with a promoter that exhibits higher stability at the stable level. Expression levels of transgenes by each promoter were measured at transient and stable levels for five single promoters: Rous sarcoma virus (RSV), cytomegalovirus (CMV), human phosphoglycerate kinase (hPGK), simian virus 40 (SV40), and zebrafish ubiquitin B (Ubb). The hPGK promoter enabled high-yield transgene expression at transient levels and the SV40 promoter enabled sustained expression at stable levels. Therefore, hPGK and SV40 promoters were selected as candidates for establishing hybrid promoters and two hybrid promoters were constructed; one hybrid promoter in which the SV40 promoter is added before the hPGK promoter (a.k.a. SKYI) and the other hybrid promoter in which the SV40 promoter is added after the hPGK promoter (a.k.a. SKYII). Of the two hybrid promoters, the hybrid promoter SKYII promoted high-yield transgene expression at both transient and stable levels compared to single hPGK and SV40. Together, our findings open new doors in the field of biopharmaceuticals by presenting a novel promoter platform that can be used for high-yield and sustained protein production.

Chemical screening identifies the anticancer properties of Polyporous parvovarius.

One of the biggest obstacles in cancer treatment is the development of chemoresistance. To overcome this, attempts have been made to screen novel anticancer substances derived from natural products. The purpose of this study is to find new anticancer candidates in the mycelium culture extract of mushrooms belonging to Polyporus. Here, we used a high-throughput screening to find agents capable of inhibiting cancer cell proliferation. The culture extract of Polyporus Parvovarius mycelium in DY medium (pp-DY) was effective. pp-DY inhibited cancer cell proliferation by inducing apoptosis and S-phase arrest. The anticancer property of pp-DY was not only effective against one type of cancer, but also against another type of cancer. Compound fractionation was performed, and the active ingredient exhibiting anticancer effects in pp-DY was identified as 3,4-dihydroxybenzaldehyde (Protocatechualdehyde, PCA). PCA, like pp-DY, inhibited the proliferation of cancer cells by inducing apoptosis and S-phase arrest. Furthermore, unlike conventional anticancer drugs, PCA did not increase the proportion of the side population that plays the most important role in the development of chemoresistance. Taken together, our data revealed the novel mycelium culture extract that exhibited anticancer property, and identified active ingredients that did not activate a proportion of the side population. These novel findings may have clinical applications in the treatment of cancer, particularly chemo-resistant cancer.

Functional restoration of lysosomes and mitochondria through modulation of AKT activity ameliorates senescence.

Senescence is a phenomenon defined by alterations in cellular organelles and is the primary cause of aging and aging-related diseases. Recent studies have shown that oncogene-induced senescence is driven by activation of serine/threonine protein kinases (AKT1, AKT2 and AKT3). In this study, we evaluated twelve AKT inhibitors and revealed GDC0068 as a potential agent to ameliorate senescence. Senescence-ameliorating effect was evident from the finding that GDC0068 yielded lysosomal functional recovery as observed by reduction in lysosomal mass and induction in autophagic flux. Furthermore, GDC0068-mediated restoration of lysosomal function activated the removal of dysfunctional mitochondria, resulting in restoration of mitochondrial function. Together, our findings revealed a unique mechanism by which senescence is recovered by functional restoration of lysosomes and mitochondria through modulation of AKT activity.

A Ten-Minute Bioassay to Test Metal Toxicity with the Freshwater Flagellate Euglena agilis.

A chemical analysis of water quality cannot detect some toxicants due to time constraints, high costs, and limited interactions for detection. Bioassays would offer a complementary means to assess pollution levels in water. Euglena is a flagellate green alga and an excellent system for toxicity testing thanks to its ease of culture, rapid growth, and quick response to environmental stresses. Herein, we examined the sensitivity of E. agilis to seven heavy metals by analyzing six end-point parameters: motility, velocity, cell compactness, upward swimming, r-value, and alignment. Notably, the velocity of E. agilis was most sensitive to cadmium (96.28 mg·L-1), copper (6.51 mg·L-1), manganese (103.28 mg·L-1), lead (78.04 mg·L-1), and zinc (101.90 mg·L-1), while r-values were most sensitive to arsenic (12.84 mg·L-1) and mercury (4.26 mg·L-1). In this study, velocity and r-values are presented as useful biomarkers for the assessment of metal toxicity in Euglena. The metals As, Cd, Cu, and Pb were suitable for this test. The advantages of the ecotoxicity test are its rapidity: It takes 10 min to obtain results, as opposed to the typical 3-4 d of exposure time with intensive labor. Moreover, this test can be performed at room temperature under dark conditions.

Restoration of Lysosomal and Mitochondrial Function Through p38 Mitogen-Activated Protein Kinase Inhibition Ameliorates Senescence.

Oncogene-induced senescence (OIS), characterized by irreversible cell cycle arrest by oncogene activation, plays an important role in the pathogenesis of aging and age-related diseases. Recent research indicates that OIS is driven by activation of mitogen-activated protein kinase (MAPK). However, it is not apparent whether MAPK inhibition helps to recover senescence. In our previous study, we uncovered p38 MAPK inhibitor, SB203580, as an effective agent to reduce reactive oxygen species and increase proliferation in premature senescent cells. In this study, we evaluated whether SB203580 could ameliorate senescence in normal senescent cells. The senescence-improving effect was observed in the results that SB203580 treatment restored lysosomal function, as evidenced by a decrease in lysosomal mass and an increase in autophagic vacuoles. Then, SB203580-mediated lysosomal function restoration triggered the clearance of damaged mitochondria, leading to metabolic reprogramming necessary for amelioration of senescence. Indeed, p38 MAPK inhibition by SB203580 improved key senescent phenotypes. Our findings suggest a novel mechanism by which modulation of p38 MAPK activity leads to senescence improvement through functional restoration of lysosome and mitochondria.

Co-inhibition of ATM and ROCK synergistically improves cell proliferation in replicative senescence by activating FOXM1 and E2F1.

The multifaceted nature of senescent cell cycle arrest necessitates the targeting of multiple factors arresting or promoting the cell cycle. We report that co-inhibition of ATM and ROCK by KU-60019 and Y-27632, respectively, synergistically increases the proliferation of human diploid fibroblasts undergoing replicative senescence through activation of the transcription factors E2F1 and FOXM1. Time-course transcriptome analysis identified FOXM1 and E2F1 as crucial factors promoting proliferation. Co-inhibition of the kinases ATM and ROCK first promotes the G2/M transition via FOXM1 activation, leading to accumulation of cells undergoing the G1/S transition via E2F1 activation. The combination of both inhibitors increased this effect more significantly than either inhibitor alone, suggesting synergism. Our results demonstrate a FOXM1- and E2F1-mediated molecular pathway enhancing cell cycle progression in cells with proliferative potential under replicative senescence conditions, and treatment with the inhibitors can be tested for senomorphic effect in vivo.

The translational landscape as regulated by the RNA helicase DDX3.

Continuously renewing the proteome, translation is exquisitely controlled by a number of dedicated factors that interact with the ribosome. The RNA helicase DDX3 belonging to the DEAD box family has emerged as one of the critical regulators of translation, the failure of which is frequently observed in a wide range of proliferative, degenerative, and infectious diseases in humans. DDX3 unwinds double-stranded RNA molecules with coupled ATP hydrolysis and thereby remodels complex RNA structures present in various protein-coding and noncoding RNAs. By interacting with specific features on messenger RNAs (mRNAs) and 18S ribosomal RNA (rRNA), DDX3 facilitates translation, while repressing it under certain conditions. We review recent findings underlying these properties of DDX3 in diverse modes of translation, such as cap-dependent and cap-independent translation initiation, usage of upstream open reading frames, and stress-induced ribonucleoprotein granule formation. We further discuss how disease-associated DDX3 variants alter the translation landscape in the cell. [BMB Reports 2022; 55(3): 125-135].

Targeting regulation of ATP synthase 5 alpha/beta dimerization alleviates senescence.

Senescence is a distinct set of changes in the senescence-associated secretory phenotype (SASP) and leads to aging and age-related diseases. Here, we screened compounds that could ameliorate senescence and identified an oxazoloquinoline analog (KB1541) designed to inhibit IL-33 signaling pathway. To elucidate the mechanism of action of KB1541, the proteins binding to KB1541 were investigated, and an interaction between KB1541 and 14-3-3ζ protein was found. Specifically, KB1541 interacted with 14-3-3ζ protein and phosphorylated of 14-3-3ζ protein at serine 58 residue. This phosphorylation increased ATP synthase 5 alpha/beta dimerization, which in turn promoted ATP production through increased oxidative phosphorylation (OXPHOS) efficiency. Then, the increased OXPHOS efficiency induced the recovery of mitochondrial function, coupled with senescence alleviation. Taken together, our results demonstrate a mechanism by which senescence is regulated by ATP synthase 5 alpha/beta dimerization upon fine-tuning of KB1541-mediated 14-3-3ζ protein activity.

Metabolic reprogramming as a novel therapeutic target for Coxsackievirus B3.

Coxsackievirus B3 (CVB3) is a single-stranded RNA virus that belongs to the Enterovirus genus. CVB3 is a human pathogen associated with serious conditions such as myocarditis, dilated cardiomyopathy, and pancreatitis. However, there are no therapeutic interventions to treat CVB3 infections. In this study, we found that CVB3 induced metabolic alteration in host cells through increasing glycolysis level, as indicated by an increase in the extracellular acidification rate (ECAR). CVB3-mediated metabolic alteration was confirmed by metabolite change analysis using gas chromatography-mass spectrometry (GC-MS). Based on findings, a strategy to inhibit glycolysis has been proposed to treat CVB3 infection. Indeed, glycolysis inhibitors (2-Deoxy-D-glucose, sodium oxide) significantly reduced CVB3 titers after CVB3 infection, indicating that glycolysis inhibitors can be used as effective antiviral agents. Taken together, our results reveal a novel mechanism by which CVB3 infection is controlled by regulation of host cell metabolism.

Targeting Mitochondrial Metabolism as a Strategy to Treat Senescence.

Mitochondria are one of organelles that undergo significant changes associated with senescence. An increase in mitochondrial size is observed in senescent cells, and this increase is ascribed to the accumulation of dysfunctional mitochondria that generate excessive reactive oxygen species (ROS). Such dysfunctional mitochondria are prime targets for ROS-induced damage, which leads to the deterioration of oxidative phosphorylation and increased dependence on glycolysis as an energy source. Based on findings indicating that senescent cells exhibit mitochondrial metabolic alterations, a strategy to induce mitochondrial metabolic reprogramming has been proposed to treat aging and age-related diseases. In this review, we discuss senescence-related mitochondrial changes and consequent mitochondrial metabolic alterations. We assess the significance of mitochondrial metabolic reprogramming for senescence regulation and propose the appropriate control of mitochondrial metabolism to ameliorate senescence. Learning how to regulate mitochondrial metabolism will provide knowledge for the control of aging and age-related pathologies. Further research focusing on mitochondrial metabolic reprogramming will be an important guide for the development of anti-aging therapies, and will provide novel strategies for anti-aging interventions.

Assessment of Various Toxicity Endpoints in Duckweed (Lemna minor) at the Physiological, Biochemical, and Molecular Levels as a Measure of Diuron Stress.

The common, broad-spectrum herbicide diuron poses some risks to the environment due to its long persistence and high toxicity. Therefore, the effective monitoring of diuron residues will inform efforts to assess its impacts on ecosystems. In this study, we evaluated the toxicity targets of diuron in the model aquatic macrophyte Lemna minor at the physiological (growth and photosynthetic efficiency), biochemical (pigment biosynthesis and reactive oxygen species (ROS) levels), and molecular (rbcL transcript) levels. The toxicity of diuron was detectable after 48 h of exposure and the order of sensitivity of toxicity endpoints was gene transcription > maximum electron transport rate (ETRmax) > non-photochemical quenching (NPQ) > maximum quantum yield (Fv/Fm) > ROS > fresh weight > chlorophyll b > chlorophyll a > total frond area > carotenoids. Under diuron stress, pigment, ROS, and gene transcript levels increased while frond area, fresh weight, and photosynthesis (Fv/Fm and ETRmax) gradually decreased with the increasing duration of exposure. Notably, ROS levels, Fv/Fm, frond area, and fresh weight were highly correlated with diuron concentration. The growth endpoints (frond area and fresh weight) showed a strong negative correlation with ROS levels and a positive correlation with Fv/Fm and ETRmax. These findings shed light on the relative sensitivity of different endpoints for the assessment of diuron toxicity.

Disruption of nucleocytoplasmic trafficking as a cellular senescence driver.

Senescent cells exhibit a reduced response to intrinsic and extrinsic stimuli. This diminished reaction may be explained by the disrupted transmission of nuclear signals. However, this hypothesis requires more evidence before it can be accepted as a mechanism of cellular senescence. A proteomic analysis of the cytoplasmic and nuclear fractions obtained from young and senescent cells revealed disruption of nucleocytoplasmic trafficking (NCT) as an essential feature of replicative senescence (RS) at the global level. Blocking NCT either chemically or genetically induced the acquisition of an RS-like senescence phenotype, named nuclear barrier-induced senescence (NBIS). A transcriptome analysis revealed that, among various types of cellular senescence, NBIS exhibited a gene expression pattern most similar to that of RS. Core proteomic and transcriptomic patterns common to both RS and NBIS included upregulation of the endocytosis-lysosome network and downregulation of NCT in senescent cells, patterns also observed in an aging yeast model. These results imply coordinated aging-dependent reduction in the transmission of extrinsic signals to the nucleus and in the nucleus-to-cytoplasm supply of proteins/RNAs. We further showed that the aging-associated decrease in Sp1 transcription factor expression was critical for the downregulation of NCT. Our results suggest that NBIS is a modality of cellular senescence that may represent the nature of physiological aging in eukaryotes.

Potential Treatment of Lysosomal Storage Disease through Modulation of the Mitochondrial-Lysosomal Axis.

Lysosomal storage disease (LSD) is an inherited metabolic disorder caused by enzyme deficiency in lysosomes. Some treatments for LSD can slow progression, but there are no effective treatments to restore the pathological phenotype to normal levels. Lysosomes and mitochondria interact with each other, and this crosstalk plays a role in the maintenance of cellular homeostasis. Deficiency of lysosome enzymes in LSD impairs the turnover of mitochondrial defects, leading to deterioration of the mitochondrial respiratory chain (MRC). Cells with MRC impairment are associated with reduced lysosomal calcium homeostasis, resulting in impaired autophagic and endolysosomal function. This malicious feedback loop between lysosomes and mitochondria exacerbates LSD. In this review, we assess the interactions between mitochondria and lysosomes and propose the mitochondrial-lysosomal axis as a research target to treat LSD. The importance of the mitochondrial-lysosomal axis has been systematically characterized in several studies, suggesting that proper regulation of this axis represents an important investigative guide for the development of therapeutics for LSD. Therefore, studying the mitochondrial-lysosomal axis will not only add knowledge of the essential physiological processes of LSD, but also provide new strategies for treatment of LSD.

Adiponectin Controls Nutrient Availability in Hypothalamic Astrocytes.

Adiponectin, an adipose tissue-derived hormone, plays integral roles in lipid and glucose metabolism in peripheral tissues, such as the skeletal muscle, adipose tissue, and liver. Moreover, it has also been shown to have an impact on metabolic processes in the central nervous system. Astrocytes comprise the most abundant cell type in the central nervous system and actively participate in metabolic processes between blood vessels and neurons. However, the ability of adiponectin to control nutrient metabolism in astrocytes has not yet been fully elucidated. In this study, we investigated the effects of adiponectin on multiple metabolic processes in hypothalamic astrocytes. Adiponectin enhanced glucose uptake, glycolytic processes and fatty acid oxidation in cultured primary hypothalamic astrocytes. In line with these findings, we also found that adiponectin treatment effectively enhanced synthesis and release of monocarboxylates. Overall, these data suggested that adiponectin triggers catabolic processes in astrocytes, thereby enhancing nutrient availability in the hypothalamus.

ATM mediated-p53 signaling pathway forms a novel axis for senescence control.

Previously, we uncovered a novel mechanism in which senescence is controlled by mitochondrial functional recovery upon Ataxia-telangiectasia mutated (ATM) inhibition. However, it remains elusive how ATM controls signaling pathways to achieve restorative effect. In this study, we performed microarray and found that p53 pathway was differentially expressed upon ATM inhibition. We found that ATM inhibition yields senescence amelioration through p53-dependent manner. The restorative effect was also afforded by direct p53 inhibition. Furthermore, mitochondrial metabolic reprogramming via p53 inhibition was a prerequisite for senescence amelioration. Taken together, our data indicated that p53 pathway functions as potential target for ATM-mediated senescence amelioration.