Biology subjects

Saito, T.

Publications and source records attributed to Saito, T..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Unraveling factors responsible for pathogenic differences in Lassa virus strains

Lassa virus (LASV) is the etiological agent of Lassa fever (LF), a severe hemorrhagic disease with potential for lethal outcomes. Apart from acute symptoms, LF survivors often endure long-term complications, notably hearing loss, which significantly impacts their quality of life and socioeconomic status in endemic regions of West Africa. Classified as a Risk Group 4 agent, LASV poses a substantial public health threat in affected areas. Our laboratory previously developed a novel lethal guinea pig model of LF utilizing the clinical isolate LASV strain LF2384. However, the specific pathogenic factors underlying LF2384 infection in guinea pigs remained elusive. In this study, we aimed to elucidate the differences in the immunological response induced by LF2384 and LF2350, another LASV isolate from a non-lethal LF case within the same outbreak. Through comprehensive immunological gene profiling, we compared the expression kinetics of key genes in guinea pigs infected with LASV LF2384 and LF2350. Our analysis revealed differential expression patterns for several immunological genes, including CD94, CD19-2, CD23, IL-7, and CIITA, during LF2384 and LF2350 infection. Moreover, through the generation of recombinant LASVs, we sought to identify the specific viral genes responsible for the observed pathogenic differences between LF2384 and LF2350. Our investigations pinpointed the L protein as a crucial determinant of pathogenicity in guinea pigs infected with LASV LF2384. Author summaryLassa virus (LASV) is known to cause lethal hemorrhagic fever, Lassa fever, and is classified as a Risk Group-4 agent, which need to be handled in the highest biocontainment laboratories, biosafety level-4 (BSL-4), due to its high pathogenicity and the lack of preventive or treatment methods. LASV infection has a huge impact on public health and socioeconomics in endemic areas, however, its pathogenic mechanism is still largely unknown. In order to unveil the mechanisms of LASV pathogenesis, we compared the pathogenicity of two LASV isolates, which have opposite phenotypes in guinea pigs. Additionally, we determined the viral factor responsible for pathogenic differences between LASV isolates using reverse genetics. In summary, our study provides valuable insights into the immunological and virological factors underlying the pathogenic differences between LASV strains associated with lethal and non-lethal LF cases. Understanding these distinctions is essential for informing strategies for the diagnosis, treatment, and prevention of LF.

microbiology↗

bioRxiv2024

Selective agonism of GPR34 stimulates microglial uptake and clearance of amyloid β fibrils

Microglia, the primary immune cells of the central nervous system, play a crucial role in maintaining brain homeostasis through phagocytosis of various substrates, including amyloid-{beta} (A{beta}) fibrils, a hallmark of Alzheimer disease (AD) pathology. However, the molecular mechanisms regulating microglial A{beta} uptake remain poorly understood. Here, we identified GPR34, a Gi/o-coupled receptor highly expressed in microglia, as a novel regulator of fibrillar A{beta} phagocytosis. Treatment with a selective GPR34 agonist, M1, specifically enhanced uptake of A{beta} fibrils, but not its monomer or oligomer, in both mouse and human microglia. Mechanistically, M1 reduced intracellular cAMP levels, which inversely correlated with A{beta} uptake activity. Importantly, a single intrahippocampal injection of M1 in an AD mouse model significantly increased microglial A{beta} uptake in vivo. Furthermore, single-nucleus RNA-sequencing analysis of Japanese AD patient samples revealed a significant reduction of GPR34 expression in microglia from AD patients compared to controls. We also observed an age-dependent decline in microglial GPR34 expression in both human and mouse datasets, suggesting a potential contribution of GPR34 downregulation to age-related A{beta} accumulation and AD risk. Collectively, our findings identify GPR34 as a promising target for modulating microglial A{beta} clearance and highlight the therapeutic potential of GPR34 agonists in AD. Significance statementAlzheimer disease (AD) is characterized by amyloid-{beta} (A{beta}) accumulation in the brain. Microglia, the brains immune cells, play a crucial role in the metabolism of A{beta}. We discovered that activating the microglial receptor GPR34 with a selective agonist enhances the phagocytosis of A{beta} fibrils, a key pathogenic form of A{beta}. Importantly, GPR34 expression decreases with aging and AD progression, potentially contributing to impaired A{beta} clearance. Our findings highlight GPR34 as a promising therapeutic target for AD, as boosting its activity could promote A{beta} clearance and slow disease progression. This study provides valuable insights into microglial function in AD and offers a novel strategy for developing disease-modifying therapies.

neuroscience↗

bioRxiv2024

Adenosine deficiency facilitates CA1 synaptic hyperexcitability in the presymptomatic phase of a knock in mouse model of Alzheimer's disease.

The diseases trajectory of Alzheimers disease (AD) is associated with and worsened by hippocampal hyperexcitability. Here we show that during the asymptomatic stage in a knock in mouse model of Alzheimers disease (APPNL-G-F/NL-G-F; APPKI), hippocampal hyperactivity occurs at the synaptic compartment, propagates to the soma and is manifesting at low frequencies of stimulation. We show that this aberrant excitability is associated with a deficient adenosine tone, an inhibitory neuromodulator, driven by reduced levels of CD39/73 enzymes, responsible for the extracellular ATP-to-adenosine conversion. Both pharmacologic (adenosine kinase inhibitor) and non-pharmacologic (ketogenic diet) restorations of the adenosine tone successfully normalize hippocampal neuronal activity. Our results demonstrated that neuronal hyperexcitability during the asymptomatic stage of a KI model of Alzheimers disease originated at the synaptic compartment and is associated with adenosine deficient tone. These results extend our comprehension of the hippocampal vulnerability associated with the asymptomatic stage of Alzheimers disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/590882v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@cd128forg.highwire.dtl.DTLVardef@17d7701org.highwire.dtl.DTLVardef@100337org.highwire.dtl.DTLVardef@14333d0_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LISynaptic hyperexcitability spreads to the soma and manifests at low frequencies of stimulation C_LIO_LISynaptic hyperexcitability is associated with reduced adenosine tone in vitro and in vivo C_LIO_LIReduced adenosine tone is driven by reduced ATP-to-adenosine extracellular conversion C_LIO_LIRestoration of adenosine tone successfully normalizes neuronal excitability in the AD model C_LI

neuroscience↗

bioRxiv2024

Structural and Dynamic Insights into the Biased Signaling Mechanism of the Human Kappa Opioid Receptor

The {kappa}-opioid receptor (KOR) is a member of the G protein-coupled receptor (GPCR) family, responsible for modulating cellular responses through transducers such as G proteins and arrestins. G protein-biased KOR agonists hold promise due to their potential to mitigate side effects such as drug aversion and sedation while preserving analgesic and antipruritic effects. Here, we shed light on the structural dynamics of the human KOR-Gi signaling complex bound with either nalfurafine (a G-protein-biased agonist) or U-50,488H (a balanced agonist) using cryo-electron microscopy (cryo-EM). Cryo-EM structures of the KOR-Gi signaling complexes identify the ligand binding mode in the activated state. Vibrational spectroscopy analysis reveals changes in the ligand-binding pocket upon binding to these ligands. Cell-based mutant experiments pinpoint four amino acids (K2275.40, C2866.47, H2916.52, and Y3127.34; Ballesteros-Weinstein numbering is shown in superscript) that play crucial roles in arrestin recruitment. Among these four amino acids, H2916.52 and Y3127.34 are also implicated in G-protein coupling. Our findings pave the way for targeting specific residues in the KOR ligand-binding pocket to enhance KOR-mediated therapeutic effects while mitigating unwanted side effects.

biophysics↗

bioRxiv2024

Altered spawning seasons of Atlantic salmon broodstock genetically and epigenetically influence cell cycle and lipid-mediated regulations in their offspring

Manipulating spawning seasons of Atlantic salmon (Salmo salar) is a common practice to facilitate year-round harvesting in salmon aquaculture. This process involves adjusting water temperature and light regime to control female broodstock maturation. However, recent studies have indicated that altered spawning seasons can significantly affect the nutritional status and growth performance of the offspring. Therefore, gaining a deeper understanding of the biological regulations influenced by these alterations is crucial to enhance the growth performance of fish over multiple generations. In this study, we investigated omics data from four different spawning seasons achieved through recirculating aquaculture systems (RAS) and sea-pen-based approaches. In addition to the normal spawning season in November (sea-pen), three altered seasons were designated: off-season (five-month advance, RAS), early season (two-month advance, sea-pen), and late season (two-month delay, sea-pen). We conducted comprehensive gene expression and DNA methylation analysis on liver samples collected from the start-feeding larvae of the next generation. Our results revealed distinct gene expression and DNA methylation patterns associated with the altered spawning seasons. Specifically, offspring from RAS-based off-season exhibited altered lipid-mediated regulation, while those from sea-pen-based early and late seasons showed changes in cellular processes, particularly in cell cycle regulation when compared to the normal season. The consequences of our findings are significant for growth and health, potentially providing information for developing valuable tools for assessing growth potential and optimizing production strategies in aquaculture. Author SummaryThis study examines the impact of manipulating Atlantic salmon spawning seasons in aquaculture on genetic and epigenetic regulations in their offspring. Manipulating water temperature and light cycles during broodstock rearing allows for year-round harvesting. In aquaculture, altering spawning seasons is a common practice; however, recent research suggests that these changes affect the nutritional status and growth of offspring. To understand these effects at the molecular level, we analysed transcriptomic and epigenetic data from salmon offspring born to broodstock with four different spawning seasons. Our results reveal distinct transcriptomic and epigenetic patterns associated with altered seasons, influencing lipid metabolism and cell cycle regulation. These findings hold significant implications for improving aquaculture practices, potentially enhancing growth performance and nutritional quality of farmed salmon across generations as temperature and light are crucial abiotic factors influencing the next generation. Furthermore, our study potentially contributes to understanding the impact of climate change on the spawning behaviour of aquatic vertebrates, particularly in relation to increasing global ocean temperatures and the resulting changes in photoperiod as species migrate northward in the Northern Hemisphere, experiencing longer daylight in summer and shorter daylight in winter.

genomics↗

bioRxiv2024

Metabolic resistance of Abeta3pE-42, epitope of the anti-Alzheimer therapeutic antibody, donanemab.

The amyloid {beta} peptide (A{beta}) starting with pyroglutamate (pE) at position 3 and ending at position 42 (A{beta}3pE-42) is a dominant species that accumulates in the Alzheimers disease (AD) brain. Consistently, a therapeutic antibody raised against this species, donanemab, has been shown to be effective in recent clinical trials. While the primary A{beta} species produced physiologically is A{beta}1-40/42, an explanation for how and why this physiological A{beta} is converted to the pathological form has remained elusive. The conversion of A{beta}1-42 to A{beta}3pE-42 is likely to take place after deposition of A{beta}1-42 given that A{beta}3pE-42 plaques arise significantly later than A{beta}1-42 deposition in the brains of single App knock-in and APP-transgenic mice. Here, we present experimental evidence that accounts for the aging-associated A{beta}3pE-42 deposition: [1] A{beta}3pE-42 is metabolically more stable than other A{beta}X-42 species; [2] Deficiency of neprilysin (NEP), the major A{beta}-degrading enzyme, induces a relatively selective deposition of A{beta}3pE-42 in APP-Tg mice. [3] A{beta}3pE-42 deposition always colocalizes with cored plaques in both APP-Tg and App knock-in mouse brains; [4] A{beta}3E-42, an immediate precursor of A{beta}3pE-42, as well as A{beta}2A-42 and A{beta}4F-42 are more short-lived than A{beta}1-42 in vivo, indicating that simple N-terminal truncation that can arise enzymatically or spontaneously makes A{beta}X-42 easier to catabolize. Consistently, newly generated knock-in mice, AppNL-({Delta}DA)-F and AppNL-({Delta}DA)-Q-F, showed no detectable A{beta} pathology even after aging, indicating that the A{beta}3E-42 and A{beta}3Q-42 species are extremely labile to the in vivo catabolic system and that the E/Q cyclase activity present in mouse brain is insufficient for A{beta}3pE-42 generation. In addition, a deficiency of NEP facilitated A{beta}3pE-42 deposition. Of note, we identified a trace amount of A{beta}3pE-42 and its immediate precursor, A{beta}3E-42, in the insoluble fraction of NEP-deficient APP-Tg mouse brains. A{beta}3pE-42 is thus likely to be a probabilistic by-product of A{beta}1-42 metabolism that selectively accumulates over a long-time range of brain aging. It is likely produced in the solid state or at the solid-liquid interface. Our findings suggest that anti-A{beta} therapies will probably be most effective if given before A{beta}3pE-42 deposition takes place.

neuroscience↗

bioRxiv2024

Aβ-driven nuclear pore complex dysfunction alters activation of necroptosis proteins in a mouse model of Alzheimer's Disease

The emergence of A{beta} pathology is one of the hallmarks of Alzheimers disease (AD), but the mechanisms and impact of A{beta} in progression of the disease is unclear. The nuclear pore complex (NPC) is a multi-protein assembly in mammalian cells that regulates movement of macromolecules across the nuclear envelope and its function is shown to undergo age-dependent decline during normal aging and is also impaired in multiple neurodegenerative disorders. Yet not much is known about the impact of A{beta} on NPC function in neurons. Here, we examined NPC and nucleoporin (NUP) distribution and nucleocytoplasmic transport using a mouse model of AD (AppNL-G-F/NL-G-F) that expresses A{beta} in young animals. Our studies revealed that a time-dependent accumulation of intracellular A{beta} corresponded with a reduction of NPCs and NUPs in the nuclear envelope which resulted in the degradation of the permeability barrier and inefficient segregation of nucleocytoplasmic proteins, and active transport. As a result of the NPC dysfunction App KI neurons become more vulnerable to inflammation-induced necroptosis - a programmed cell death pathway where the core components are activated via phosphorylation through nucleocytoplasmic shutting. Collectively, our data implicates A{beta} in progressive impairment of nuclear pore function and further confirms that the protein complex is vulnerable to disruption in various neurodegenerative diseases and is a potential therapeutic target.

neuroscience↗

bioRxiv2023

Oligodendrocytes and neurons contribute to amyloid-β deposition in Alzheimer's disease

In Alzheimers disease (AD), amyloid-{beta} (A{beta}) is thought to be of neuronal origin. However, in single-cell RNAseq datasets from mouse and human, we found transcripts of amyloid precursor protein (APP) and the amyloidogenic-processing machinery equally abundant in oligodendrocytes (OLs). By cell-type-specific deletion of Bace1 in a humanized knock-in AD model, APPNLGF, we demonstrate that almost a third of cortical A{beta} deposited in plaques is derived from OLs. However, excitatory projection neurons must provide a threshold level of A{beta} production for plaque deposition to occur and for oligodendroglial A{beta} to co-aggregate. Indeed, very few plaques are deposited in the absence of neuronally-derived A{beta}, although soluble A{beta} species are readily detected, especially in subcortical white matter. Our data identify OLs as a source of A{beta} in vivo and further underscore a non-linear relationship between cellular A{beta} production and resulting plaque formation. Ultimately, our observations are relevant for therapeutic strategies aimed at disease prevention in AD.

neuroscience↗

bioRxiv2023

Can Colpoda travel across oceans? Salinity tolerance of resting cysts may enable global dispersal of the species.

Protist species are distributed worldwide. The processes that enabled this global distribution are unclear. One possible means is through the oceanic dispersal of freshwater protists, although this has not been investigated in detail to date. Here, the ability of resting cysts to tolerate saline conditions is examined as a possible mechanism that enables the oceanic dispersal of protists. Resting cysts of Colpoda cucullus, a freshwater soil protist, can tolerate saline conditions of at least 3.5% NaCl for more than one week. A transcriptome analysis showed that the relative levels of expression of genes associated with membrane function are increased in resting cysts, indicating that salinity tolerance is associated with reconstruction of the cell membrane. Additionally, the outer layer of the cyst wall, a shell-like ectocyst, includes chitin. This may function as a form of "biological armor" that protects the cell from physical stress during oceanic dispersal. Teaser: Fresh water protists may be dispersed across oceans by formation of resting cysts that can tolerate saline conditions.

zoology↗

bioRxiv2023

Divergent age-dependent conformational rearrangement within Aβ-amyloid deposits in APP23, APPPS1, and AppNL-F mice

Amyloid plaques composed of fibrils of misfolded A{beta} peptides are pathological hallmarks of Alzheimers disease (AD). A{beta} fibrils are polymorphic in their tertiary and quaternary molecular structures. This structural polymorphism may carry different pathologic potency and can putatively contribute to clinical phenotypes of AD. Therefore, mapping of structural polymorphism of A{beta} fibrils is valuable to understand disease mechanisms. Here, we investigated how A{beta} fibril morphology in situ differs in A{beta} plaque of different mouse models expressing familial mutations in the A{beta}PP gene. We used a combination of conformation-sensitive luminescent conjugated oligothiophene (LCO) ligands, A{beta}-specific antibodies, and different fluorescence microscopy techniques. LCO fluorescence mapping revealed that mouse models APP23, APPPS1, and AppNL-F have different fibril structures depending on A{beta}PP-processing genotype. Co-staining of A{beta}-specific antibodies showed that individual plaques from APP23 mice, expressing Swedish mutations (NL) have two distinct fibril polymorph regions of core and corona. The plaque core is predominantly composed of compact A{beta}40 fibrils and the corona region is dominated by diffusely packed A{beta}40 fibrils. On the other hand, the APP knock-in mouse AppNL-F, expressing Iberian mutation (F) along with Swedish mutation has tiny, cored plaques consisting mainly of compact A{beta}42 fibrils, vastly different from APP23 even at elevated age up to 21 months. Age dependent polymorph maturation of plaque cores observed for APP23 and APPPS1 mice >12 months, was minuscule in AppNL-F. These structural studies of amyloid plaques in situ can map disease-relevant fibril polymorph distributions to guide the design of diagnostic and therapeutic molecules. SignificanceAlzheimers disease (AD) is associated with the formation of deposits in the brain known as A{beta}-amyloid plaques. AD can emerge as a sporadic disease or due to familial mutations in genes encoding for A{beta} precursor and processing proteins. The A{beta}-amyloid found in plaques displays different structures in sporadic AD and in various types of familial AD. We hypothesize that understanding plaque morphology and development is crucial for understanding the initiation and progression of AD. We here compared amyloid structures in three of the most used mouse models of human A{beta}-plaque formation. Our findings suggest significant differences in plaque morphologies and structural maturation processes during aging. Our results emphasize that strain-like differences of A{beta}-amyloids develop as a function of A{beta} precursor protein-processing genetics and age.

neuroscience↗

bioRxiv2023

NEDD4-binding protein 1 suppresses HBV replication by degrading pgRNA

Chronic infection with hepatitis B virus (HBV) places patients at increased risk for liver cirrhosis and hepatocellular carcinoma. Although nucleos(t)ide analogs are mainly used for the treatment of HBV, they require long-term administration and may lead to the emergence of drug resistant mutants. Therefore, to identify targets for the development of novel anti-HBV drugs, we screened for HBV-suppressive host factors using a plasmid expression library of RNA-binding proteins (RBPs). We screened 132 RBPs using an expression plasmid library by measuring HBV relaxed circular DNA (rcDNA) levels in hepatocellular carcinoma. Our screen identified NEDD4-binding protein 1 (N4BP1) as having an anti-HBV effect. In hepatocellular carcinoma cell lines transfected or infected with HBV, overexpression of N4BP1 decreased rcDNA levels while knockdown or knockout of the gene encoding N4BP1 rescued rcDNA levels. N4BP1 possesses the KH-like and RNase domains and both were required for the anti-HBV effect of N4BP1. Additionally, we measured levels of HBV pregenomic RNA (pgRNA) and covalently closed circular DNA (cccDNA) in the RBP-transfected cells and confirmed that N4BP1 binds pgRNA directly and degraded both the 3.5 kb and 2.4/2.1 kb HBV RNA. In summary, N4BP1 is a newly identified host factor able to counteract HBV production by promoting the degradation of 3.5 kb and 2.1/2.4 kb HBV RNA. ImportanceThere is still a large number of HBV-infected people in the world today because of no curative treatment for HBV infection. In this study, we focused on and screened RNA-binding proteins to identify new host factors which inhibit HBV replication. As a result, we found that NEDD4-binding protein 1 (N4BP1) expression suppresses rcDNA production by promoting the degradation of pregenomic RNA, 2.4kb and 2.1kb HBV RNA. Furthermore, KH-like domain or RNase domain of N4BP1 were involved in this anti-HBV effect. In addition, the N4BP1 levels were lower in HCC resection samples of exacerbated patients, suggesting that individual N4BP1 levels might be related to HCC progression. This novel factor can potentially become a key to new HBV treatments.

microbiology↗

bioRxiv2023

One-carbon metabolism nutrients impact the interplay between DNA methylation and gene expression in liver, enhancing protein synthesis in Atlantic Salmon

Supplementation of one-carbon (1C) metabolism micronutrients, which include B-vitamins and methionine, is essential for the healthy growth and development of Atlantic salmon (Salmo salar). However, the recent shift towards non-fish meal diets in salmon aquaculture has led to the need for reassessments of recommended micronutrient levels. Despite the importance of 1C metabolism in growth performance and various cellular regulations, the molecular mechanisms affected by these dietary alterations are less understood. To investigate the molecular effect of 1C nutrients, we analysed gene expression and DNA methylation using two types of omics data: RNA sequencing (RNA-seq) and reduced-representation bisulfite sequencing (RRBS). We collected liver samples at the end of a feeding trial that lasted 220 days through the smoltification stage, where fish were fed three different levels of four key 1C nutrients: methionine, vitamin B6, B9, and B12. Our results indicate that the dosage of 1C nutrients significantly impacts genetic and epigenetic regulations in the liver of Atlantic salmon, particularly in biological pathways related to protein synthesis. The interplay between DNA methylation and gene expression in these pathways may play an important role in the mechanisms underlying growth performance affected by 1C metabolism. Author SummaryAtlantic salmon rely on one-carbon (1C) metabolism micronutrients like B-vitamins and methionine, which they acquire through their diets. Small pelagic fish are the primary source in the wild, but finding sustainable alternatives such as plants, insects, and algae is challenging as salmon aquaculture expands. Adjusting nutrient levels when changing base ingredients further complicates the task. Understanding the molecular mechanisms affected by these micronutrients is crucial for aquaculture sustainability. In this study, we investigated the molecular effects of 1C metabolism micronutrients on Atlantic salmon. Liver samples from salmon fed varying levels of key 1C nutrients over a 220-day trial were analysed using RNA sequencing (RNA-seq) and reduced-representation bisulfite sequencing (RRBS) to assess gene expression and DNA methylation, respectively. Our results revealed significant impacts of 1C nutrient dosage on genetic and epigenetic regulations in the salmon liver, particularly in protein synthesis pathways. The interplay between DNA methylation and gene expression in these pathways influences growth performance under 1C metabolism. Uncovering molecular changes resulting from dietary alterations provides valuable insights to optimize nutritional requirements in salmon aquaculture, supporting sustainable production and welfare of this important species.

genetics↗

bioRxiv2023

Glucose stockpile in the intestinal apical brush border in C. elegans

Since understanding the mechanisms of glucose transport is a crucial approach for pathological diseases induced by glucose toxicities such as diabetes, numerous studies have unveiled molecular functions involved in glucose transport in the nematode Caenorhabditis elegans, a commonly used model organism. However, physicochemical behaviors of glucose in intestinal lumen-to-cell are still elusive. To address that, we here evaluated a diffusion coefficient of glucose in the intestinal apical brush border in C. elegans by fluorescence recovery after photobleaching (FRAP) with fluorescent glucose. Our results indicate that the fluorescent glucose is accumulated in the intestinal brush border, where the diffusion coefficient of glucose is [~]10-8 cm2/s, two orders of magnitude slower than that in bulk. Namely, the intestinal brush border is considered to be a highly viscous layer composed of intestinal microvilli and more dominantly glycocalyx. These findings imply that this high concentration of glucose in the viscous layer acts as a stockpile.

biophysics↗

bioRxiv2023

Effects of psychogenic stress on oxidative stress and antioxidant capacity at different growth stages.

This study examined the psychogenic stress (PS) effects on changes in oxidative stress and the antioxidant capacity of an organism at different growth stages. The experimental animals were male Wistar rats of five different ages from growth periods (GPs) to old age. The growth stages were randomly classified into control (C) and experimental (PS) groups. PS was performed using restraint and water immersion once daily for 3 h for 4 weeks. Reactive oxygen metabolites (d-ROMs) and the biological antioxidant potential (BAP) were measured before and after the experiment. In addition, the liver and adrenal glands were removed, and the wet weight was measured. The d-ROM and BAP of all growth stages given PS increased significantly. The d-ROM in the C group without PS increased significantly in GPs while decreased significantly in old-aged. In addition, the BAP of the C group in GP and early adulthood were all significantly elevated. There were significant differences in organ weights between the C and PS groups at all growth stages. Oxidative stress and antioxidant capacity differed depending on the organisms developmental status and growth stage, and PS also showed different effects. In particular, the variability in oxidative stress was remarkable, suggesting that the effect of PS was more significant in the organisms immature organs.

developmental biology↗

bioRxiv2023

Eosinophils exert direct and indirect anti-tumorigenic effects in the development of esophageal squamous cell carcinoma

Background/AimsEosinophils are present in several solid tumors and have context-dependent function. Our aim is to define the contribution of eosinophils in esophageal squamous cell carcinoma (ESCC), since their role in ESCC is unknown. MethodsEosinophils were enumerated in tissues from two ESCC cohorts. Mice were treated with 4-nitroquinolone-1-oxide (4-NQO) for 8 weeks to induce pre-cancer or 16 weeks to induce carcinoma. Eosinophil number was modified by monoclonal antibody to IL-5 (IL5mAb), recombinant IL-5 (rIL-5), or genetically with eosinophil-deficient ({Delta}dblGATA) mice or mice deficient in eosinophil chemoattractant eotaxin-1 (Ccl11-/-). Esophageal tissue and eosinophil specific RNA-sequencing was performed to understand eosinophil function. 3-D co-culturing of eosinophils with pre-cancer or cancer cells was done to ascertain direct effects of eosinophils. ResultsActivated eosinophils are present in higher numbers in early stage versus late stage ESCC. Mice treated with 4-NQO exhibit more esophageal eosinophils in pre-cancer versus cancer. Correspondingly, epithelial cell Ccl11 expression is higher in mice with pre-cancer. Eosinophil depletion using three mouse models (Ccl11-/- mice, {Delta}dblGATA mice, IL5mAb treatment) all display exacerbated 4-NQO tumorigenesis. Conversely, treatment with rIL-5 increases esophageal eosinophilia and protects against pre-cancer and carcinoma. Tissue and eosinophil RNA-sequencing revealed eosinophils drive oxidative stress in pre-cancer. In vitro co-culturing of eosinophils with pre-cancer or cancer cells resulted in increased apoptosis in the presence of a degranulating agent, which is reversed with N-acetylcysteine, a reactive oxygen species (ROS) scavenger. {Delta}dblGATA mice exhibited increased CD4 T cell infiltration, IL-17, and enrichment of IL-17 pro-tumorigenic pathways. ConclusionEosinophils likely protect against ESCC through ROS release during degranulation and suppression of IL-17.

cancer biology↗

bioRxiv2023

Subchronic Toxicity Assessment of Perfluoroalkyl Substances by Mixed Exposure of Nine Substances at Daily Intake Relevant Concentration

Per- and poly-fluoroalkyl substances (PFAS) have been utilized extensively for various applications owing to their distinctive chemical properties. They exhibit high persistence in the environment and accumulate within the human body, necessitating toxicity assessments. However, the consequences of prolonged, low-level exposure to PFAS and concurrent exposure to multiple PFAS have not been explored. In this study, male C57BL/6J mice (aged 8 weeks) were exposed to a composite of nine PFAS, which include long-chain PFAS (e.g. perfluorooctanoic acid and perfluorooctanesulfonic acid) and short-chain PFAS (e.g. perfluorobutanoic acid and perfluorobutanesulfonic acid) at concentrations equivalent to the estimated daily human intake in the composition reported (1 {micro}g/L [sum of the nine compounds], the maximum reported exposure concentration) via drinking water. Histological examination revealed vacuolization of hepatocytes and irregular arrangement of hepatocyte cords, suggesting that exposure to low levels of the PFAS mixture causes morphological changes in liver tissues. Transcriptome analysis revealed that PFAS exposure mainly altered a group of genes related to metabolism and chemical carcinogenesis. Machine learning analysis of the liver metabolome showed a typical concentration-independent alteration upon PFAS exposure, and in addition to known substances such as glutathione, a compound with unknown biological function; 2,5-dihydro-2,4-dimethyloxazole was found. This study demonstrates that daily exposure to PFAS leads to morphological changes in liver tissues and alters the expression of metabolism-and cancer-related genes as well as phospholipid metabolism. Future studies are required to evaluate the chronic toxicity of prolonged, low-level exposure to PFAS mixtures and to investigate the health effects of PFAS.

pharmacology and toxicology↗

bioRxiv2023

Exon 44 skipping in Duchenne muscular dystrophy: NS-089/NCNP-02, a dual-targeting antisense oligonucleotide

Exon-skipping therapy mediated by antisense oligonucleotides (ASOs) is expected to provide a therapeutic option for Duchenne muscular dystrophy (DMD). ASOs for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated ASOs for exon 44 skipping (applicable to approximately 6% of all DMD patients) to improve activity by using a novel ASO design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to simultaneously target two splicing regulators in exon 44, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a DMD patient to whom exon 44 skipping is applicable. We also assessed the in vivo activity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an ASO with a novel connected-sequence design, showed both in vitro and in vivo exon-skipping activity.

cell biology↗

bioRxiv2023

Nutrient-regulated dynamics of chondroprogenitors in the postnatal murine growth plate

Longitudinal bone growth relies on endochondral ossification in the cartilaginous growth plate where chondrocytes accumulate and synthesize the matrix scaffold that is replaced by bone. The chondroprogenitors in the resting zone maintain the continuous turnover of chondrocytes in the growth plate. Malnutrition is a leading cause of growth retardation in children; however, after recovery from nutrient deprivation, bone growth is accelerated beyond the normal rate, a phenomenon termed catch-up growth. Though nutritional status is a known regulator of long bone growth, it is largely unknown if and how chondroprogenitor cells respond to deviations in nutrient availability. Here, using fate-mapping analysis in Axin2CreERT2 mice, we showed that dietary restriction increased the number of Axin2+ chondroprogenitors in the resting zone and simultaneously inhibited their differentiation. Once nutrient deficiency was resolved, the accumulated chondroprogenitor cells immediately restarted differentiation and formed chondrocyte columns, contributing to accelerated growth. Furthermore, we showed that nutrient deprivation reduced the level of phosphorylated Akt in the resting zone, and that exogenous IGF-1 canceled this reduction and stimulated differentiation of the pooled chondroprogenitors, decreasing their numbers. Our study of Axin2CreERT2 revealed that nutrient availability regulates the balance between accumulation and differentiation of chondroprogenitors in the growth plate, and further demonstrated that IGF-1 partially mediates this regulation by promoting the committed differentiation of the chondroprogenitor cells.

developmental biology↗