Biology subjects

Iwasaki, M.

Publications and source records attributed to Iwasaki, M..

30 records · Page 2Linked to original sources

bioRxiv2022

Resolution of fibrosis in mdx dystrophic mouse after oral consumption of N-163 strain of Aureobasidium pullulans produced biological response modifier β-glucan (BRMG)`

Recent advances in the management of Duchenne Muscular Dystrophy (DMD), such as exon skipping therapy, have reached a clinical stage, and although gene therapy is in clinical trials, the outcome at its best is still considered suboptimal to yield clinically discernible progress. In this study, we evaluated a novel N-163 strain of Aureobasidium pullulans produced biological response modifier {beta}-glucan (BRMG) for its potential as an adjuvant to slow down the progression of the disease by anti-inflammatory and anti-fibrotic effects. This N-163 {beta}-glucan is a safe and orally consumable food supplement with similar effects that have been previously proven in pre-clinical studies of organ fibrosis, and their beneficial effects have been proven in DMD clinical studies through blood parameters as well. In this study, 45 mice in the three groups, 15 each in a group; Gr. 1 normal mice, Gr.2 mdx mice as vehicle, and Gr.3 mdx mice administered the N-163 strain produced {beta}-glucan for 45 days. Blood biochemical parameters, body weight, muscle weight, inflammation score, and fibrosis score were evaluated using H&E and Massons trichrome staining. The N-163 {beta}-glucan group showed a significant decrease in the plasma ALT, AST, and LDH levels (126 {+/-} 69, 634 {+/-} 371, 3335 {+/-} 1258 U/l) compared with the vehicle group (177 {+/-} 27 U/l, 912 {+/-} 126 U/l, 4186 {+/-} 398 U/l). Plasma TGF-{beta} levels increased, and plasma IL-13 levels decreased in the N-163 group. The inflammation score of HE-stained muscle sections in the N-163 group (1.5 {+/-} 0.8) was lower than that in the vehicle group (2.0 {+/-} 0.8). The percentage of centrally nucleated fibres (CNF) evaluated by Massons trichrome staining was 0 in the normal group, while it increased to 80% in the vehicle group and 76.8% in the N-163 group. The N-163 {beta}-glucan group (24.22 {+/-} 4.80) showed a significant decrease in the fibrosis area (Massons trichrome-positive area). The N-163 {beta}-glucan thus, demonstrated its anti-fibrotic effect in this study. Considering their safety and easy oral consumption, this BRMG could be worth large multicentre clinical studies as adjuvant in slowing down the progress of DMD.

genetics↗

bioRxiv2022

IFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation

Activating mutations of KRAS play critical roles in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC). Accumulating evidence indicates that distinct KRAS alleles associate with different prognoses, but the underlying mechanisms are not known. We established isogenic KRAS mutants (KRASG12D, KRASG12V, and KRASWT) using a KRASG12R patient-derived PDAC cell line by CRISPR/Cas9 knock-in. We used these isogenic cell lines, a collection of characterized human PDAC patient-derived cell lines, and murine PDAC models to study the role of these KRAS alleles in vitro and in vivo. We verified that the growth of KRASG12D cells is more aggressive compared to KRASG12V isogenic cells in vitro and in vivo using orthotopic mouse models. Signal transducer and activator of transcription (STAT) activation was the most significant difference between KRASG12D and KRASG12V isogenic PDACs. Furthermore, activation of interferon-alpha (IFNA)/IFNA receptor (IFNAR)1/STAT3 signaling in the cancer cells mediated the more aggressive phenotype of KRASG12D PDACs. Conversely, inhibition of IFNAR1 in patient-derived PDAC cells suppressed tumor growth. Finally, IFNAR1 blockade was also effective in murine PDAC models and induced a significant increase in survival when combined with immune checkpoint blockade therapy. We conclude that the IFNA pathway and IFNAR1/STAT3 axis contribute to a more aggressive tumor progression in human KRASG12D PDACs and that IFNAR1 inhibition is a potential therapeutic target for overcoming resistance to immunotherapy in PDAC. One Sentence SummaryIFNA pathway drives the more aggressive phenotype of KRASG12D-mutant pancreatic ductal adenocarcinomas via IFNAR1/STAT3 activation.

cancer biology↗

bioRxiv2022

Two unique biological response-modifier glucans beneficially regulating gut microbiota and faecal metabolome in a non-alcoholic steatohepatitis animal model, with potential for applications in human health and disease

ObjectiveThe gut microbiome and its metabolites, influenced by age and stress, reflect the metabolism and immune systems health. We assessed the gut microbiota and faecal metabolome in a Stelic Animal Model of non-alcoholic steatohepatitis (NASH). DesignThis model was subjected to the following treatments: reverse osmosis water, AFO-202, N-163, AFO-202+N-163, and telmisartan. Faecal samples were collected at 6 weeks and 9 weeks of age. The gut microbiome was analysed using 16S ribosomal RNA sequence acquired by next-generation sequencing and the faecal metabolome using gas chromatography-mass spectrometry. ResultsThe gut microbial diversity increased greatly in the AFO-202+N-163 group. Post-intervention, the abundance of Firmicutes decreased, while that of Bacteroides increased and was the highest in the AFO-202+N-163 group. The decrease in the Enterobacteria and other Firmicutes abundance and in the Turicibacter and Bilophila abundance was the highest in the AFO-202 and N-163 groups, respectively. The Lactobacillus abundance increased the most in the AFO-202+N-163 group. The faecal metabolites spermidine and tryptophan, beneficial against inflammation and NASH, respectively, were greatly increased in the N-163 group. Succinic acid, beneficial in neurodevelopmental and neurodegenerative diseases, increased in the AFO-202 group. Decrease in fructose was the highest in the AFO-202 group. Leucine and phenylalanine decreased, whereas ornithine, which is beneficial against chronic immune-metabolic-inflammatory pathologies, increased in the AFO-202+N-163 group. ConclusionAFO-202 treatment in mice is beneficial against neurodevelopmental and neurodegenerative diseases and has prophylactic potential against metabolic conditions. N-163 treatment has anti-inflammatory effects against organ fibrosis and neuroinflammatory conditions. In combination, they present anticancer activity. Key messagesO_LIThe influence of gut microbiome on fecal metabolome and their association to several diseases is already known. C_LIO_LIThis study proves the efficacy of 1,3-1,6 beta glucans with pre-biotic potentials, beneficially influencing both gut microbiome and metabolome. C_LIO_LIThese results recommends for an in-depth exploration of relationship among pre-biotics, gut microbiome and gut-multi-organ axes on the fundamentals of disease onset. C_LIO_LIHidden prophylactic and therapeutic solutions to non-contagious diseases with Aureobasidium pullulans produced 1,3-1,6 beta glucans may be unveiled. C_LI

microbiology↗

bioRxiv2022

A novel analgesic pathway from parvocellular oxytocin neurons to the periaqueductal gray

The hypothalamic neuropeptide, oxytocin (OT), exerts prominent analgesic effects via central and peripheral action. Here we discovered a novel subset of OT neurons whose projections preferentially terminate on OT receptor (OTR)-expressing neurons in the ventrolateral periaqueductal gray (vlPAG). Using a newly generated line of transgenic rats (OTR-IRES-Cre), we determined that most of the vlPAG OTR expressing cells being targeted by OT projections are GABAergic in nature. Both optogenetically-evoked axonal OT release in the vlPAG as well as chemogenetic activation of OTR vlPAG neurons results in a long-lasting overall increase of vlPAG neuronal activity. This then leads to an indirect suppression of sensory neuron activity in the spinal cord and strong analgesia. Finally, we describe a novel OT[->]vlPAG[->]spinal cord circuit that seems critical for analgesia in the context of both inflammatory and neuropathic pain. Highlights- We generated a new transgenic knock-in rat line (OTR-IRES-Cre) - A distinct parvOT neuronal population projects to vlPAG but not the SON or spinal cord - OT excites vlPAG OTR neurons which indirectly inhibit SC WDR neurons - This novel parvOT[->]vlPAG[->]SC pathway alleviates nociception but not the affective component of pain

neuroscience↗

bioRxiv2021

Post-transcriptionally regulated genes are essential for pluripotent stem cell survival

The effects of transcription factors on the maintenance and differentiation of pluripotent stem cells (PSCs) have been well studied. However, the importance of post-transcriptional regulatory mechanisms, which cause the quantitative dissociation of mRNA and protein expression, has not been explored in detail. Here, by combining transcriptome and proteome profiling, we identified 228 post-transcriptionally regulated genes with strict upregulation of the protein level in PSCs. Among them, we found that 84 genes were vital for the survival of PSCs and HDFs, including 20 genes that were specifically necessary for the survival of PSCs. These 20 proteins were upregulated only in PSCs and not in differentiated cells derived from the three germ layers. Subcellular fractionation of the mRNA showed that the expression of most of the 20 proteins was regulated at the mRNA localization stage from the nucleus to the cytoplasm, but their translation efficiency was constant. Together, these results revealed that post-transcriptionally regulated genes have a crucial role in PSC survival.

molecular biology↗

bioRxiv2021

A stress-reduced passaging technique improves the viability of human pluripotent cells

Xeno-free culture systems have expanded the clinical and industrial application of human pluripotent stem cells (PSCs). However, yet some problems, such as the reproducibility among the experiments, remain. Here we describe an improved method for the subculture of human PSCs. The revised method significantly enhanced the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications such as gene editing, gene delivery, and directed differentiation. Furthermore, the method did not alter PSC characteristics after long-term culture and attenuated the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and reduces the rate of PSCs failing quality control of human PSC research and application. HighlightsO_LIThe revised passaging method significantly increases the viability of human PSCs. C_LIO_LIThe method triggers less DNA damage and apoptosis signals compared to the conventional method. C_LIO_LIThe stress-reduced method improves the results of downstream applications. C_LIO_LIThe method does not alter PSC characters and attenuates the overgrowth of abnormal subpopulations. C_LI

cell biology↗

bioRxiv2021

An efficient polymer cocktail-based transportation method for cartilage tissue, yielding chondrocytes with enhanced hyaline cartilage expression during in vitro culturing

Chondrocytes are used in cell-based therapies such as autologous chondrocyte implantation (ACI) and matrix-associated cartilage implantation (MACI). To transport the cartilage tissue to the laboratory for in vitro culturing, phosphate-buffered saline (PBS), Euro-Collins solution (ECS) and Dulbeccos Modified Eagles Medium (DMEM) are commonly employed at 4-8 {degrees}C. In this study, eight samples of human cartilage biopsy tissues from elderly patients with severe osteoarthritis undergoing arthroscopy, which would otherwise have been discarded, were used. The cartilage tissue samples were compared to assess the cell yield between two transportation groups: i) a thermo-reversible gelation polymer (TGP) based method without cool preservation (~25 {degrees}C) and ii) ECS transport at 4 {degrees}C. These samples were subjected to in vitro culture in a two-dimensional (2D) monolayer for two weeks and subsequently in a three-dimensional (3D) TGP scaffold for six weeks. The cell count obtained from the tissues transported in TGP was higher (0.2 million cells) than those transported in ECS (0.08 million cells) both after initial processing and after in vitro culturing for 2 weeks in 2D (18 million cells compared with 10 million cells). In addition, mRNA quantification demonstrated significantly higher expression of Col2a1 and SOX-9 in 3D-TGP cultured cells and lower expression of COL1a1 in RT-PCR, characteristic of the hyaline cartilage phenotype, than in 2D culture. This study confirms that the TGP cocktail is suitable for both the transport of human cartilage tissue and for in vitro culturing to yield better-quality cells for use in regenerative therapies.

cell biology↗

bioRxiv2021

Hepatoprotective effects of Aureobasidium pullulans derived Beta 1,3-1,6 biological response modifier glucans in a STAM- animal model of non-alcoholic steatohepatitis

BackgroundNon-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are highly prevalent conditions characterized by inflammation and fibrosis of the liver which can progress to cirrhosis and hepatocellular carcinoma if left untreated. Lifestyle disorders such as obesity, diabetes and dyslipidaemia predispose to and are associated with the disease progression. Conventional modalities are mainly symptomatic, with no definite solution. Beta glucan-based biological response modifiers are a potential strategy in lieu of their beneficial metabolic effects. Aureobasidium pullulans strains AFO-202 and N-163 beta glucans were evaluated for anti-fibrotic and anti-inflammatory hepatoprotective potentials in a NASH animal model in this study. MethodsIn the STAM murine model of NASH, five groups were studied for eight weeks-- (1) vehicle (RO water), (2) AFO-202 beta glucan; (3) N-163 beta glucan, (4) AFO-202+N-163 beta glucan, and (5) telmisartan (standard pharmacological intervention). Evaluation of biochemical parameters in plasma and hepatic histology including Sirius red staining and F4/80 immunostaining were performed. ResultsAFO-202 beta glucan significantly decreased inflammation-associated hepatic cell ballooning and steatosis. N-163 beta glucan decreased fibrosis and inflammation significantly (p value<0.05). The combination of AFO-202 with N-163 significantly decreased the NAFLD Activity Score (NAS) compared with other groups. ConclusionThis preclinical study supports the potential of N-163 and AFO-202 beta glucans alone or in combination as potential preventive and therapeutic agent(s), for NASH. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

pharmacology and toxicology↗

bioRxiv2019

Population-specific and transethnic genome-wide analyses reveal distinct and shared genetic risks of coronary artery disease

To elucidate the genetics of coronary artery disease (CAD) in the Japanese population, we conducted a large-scale genome-wide association study (GWAS) of 168,228 Japanese (25,892 cases and 142,336 controls) with genotype imputation using a newly developed reference panel of Japanese haplotypes including 1,782 CAD cases and 3,148 controls. We detected 9 novel disease-susceptibility loci and Japanese-specific rare variants contributing to disease severity and increased cardiovascular mortality. We then conducted a transethnic meta-analysis and discovered 37 additional novel loci. Using the result of the meta-analysis, we derived a polygenic risk score (PRS) for CAD, which outperformed those derived from either Japanese or European GWAS. The PRS prioritized risk factors among various clinical parameters and segregated individuals with increased risk of long-term cardiovascular mortality. Our data improves clinical characterization of CAD genetics and suggests the utility of transethnic meta-analysis for PRS derivation in non-European populations.

genetics↗

bioRxiv2019

Transethnic meta-analysis of genome-wide association studies identifies three new loci and characterizes population-specific differences for coronary artery disease

BackgroundGenome-wide association studies (GWAS) provided many biological insights into coronary artery disease (CAD), but these studies were mainly performed in Europeans. GWAS in diverse populations have the potential to advance our understanding of CAD.\n\nMethods and ResultsWe conducted two GWAS for CAD in the Japanese population, which included 12,494 cases and 28,879 controls, and 2,808 cases and 7,261 controls, respectively. Then, we performed transethnic meta-analysis using the results of the CARDIoGRAMplusC4D 1000 Genomes meta-analysis with UK Biobank. We identified 3 new loci on chromosome 1q21 (CTSS), 10q26 (WDR11-FGFR2), and 11q22 (RDX-FDX1). Quantitative trait locus analyses suggested the association of CTSS and RDX-FDX1 with atherosclerotic immune cells. Tissue/cell type enrichment analysis showed the involvement of arteries, adrenal glands and fat tissues in the development of CAD. Finally, we performed tissue/cell type enrichment analysis using East Asian-frequent and European-frequent variants according to the risk allele frequencies, and identified significant enrichment of adrenal glands in the East Asian-frequent group while the enrichment of arteries and fat tissues was found in the European-frequent group. These findings indicate biological differences in CAD susceptibility between Japanese and Europeans.\n\nConclusionsWe identified 3 new loci for CAD and highlighted the genetic differences between the Japanese and European populations. Moreover, our transethnic analyses showed both shared and unique genetic architectures between the Japanese and Europeans. While most of the underlying genetic bases for CAD are shared, further analyses in diverse populations will be needed to elucidate variations fully.

genetics↗

bioRxiv2019

GWAS identifies nine nephrolithiasis susceptibility loci related with metabolic and crystallization pathways

Nephrolithiasis is a common urological trait disorder with acute pain. Although previous studies have identified various genetic variations associated with nephrolithiasis, the host genetic factors remain largely unidentified. To identify novel nephrolithiasis loci in the Japanese population, we performed large-scale GWAS (Genome wide association study) using 11,130 cases and 187,639 controls, followed by a replication analysis using 2,289 cases and 3,817 controls. The analysis identified 14 significant loci, including 9 novel loci on 2p23.2-3, 6p21.2, 6p12.3, 6q23.2, 16p12.3, 16q12.2, 17q23.2, 19p13.12, and 20q13.2. Interestingly, 10 of the 14 regions showed a significant association with any of 16 quantitative traits, including metabolic, kidney-related, and electrolyte traits, suggesting a common genetic background among nephrolithiasis patients and these quantitative traits. Four novel loci are related to the metabolic pathway, while the remaining 10 loci are associated with the crystallization pathway. Our findings demonstrate the crucial roles of genetic variations in the development of nephrolithiasis.\n\nSIGNIFICANCE STATEMENTNephrolithiasis is a common urothelial disorders with frequent recurrence rate, but its genetic background is largely remained unidentified. Previous GWAS identified 6 genetic factors in total. Here we performed a GWAS using more than 200,000 samples in the Japanese populations, and identified 14 significant loci and nine of them are novel. We also found that 10 of the 14 loci showed a significant association with any of 16 quantitative traits, including metabolic, kidney-related, and electrolyte traits (BMI, eGFR, UA, Ca etc). All 14 significant loci are associate with either metabolic or crystallization pathways. Thus, our findings elucidated the underlying molecular pathogenesis of nephrolithiasis.

genetics↗

bioRxiv2018

Endogenous oxytocin release in periaqueductal gray eases inflammatory pain through long term suppression of pain perception neurons in the spinal cord

Research has shown that oxytocin (OT) injection to periaqueductal gray (PAG) eases pain. We hypothesized that OT in PAG may eventually suppress the activity of pain perception neurons inside the spinal cord (SC), then block further aversion signals to the brain. Here, we confirmed that PAG receives OT-ergic axons from the paraventricular nucleus in rats. In PAG, optogenetically triggered axonal OT release evoked excitation in some neurons, and inhibition in others. Of these OT-excited neurons, active neurons alternated between one and the other as time passed, but the number of active neurons at each moment was continuously maintained at the same level at least for 300 s after laser beam stimulation. Further, OT release in PAG reduced pain-induced activity of SC neurons, whose effect reached maximum levels at approximately 220 s after the laser beam stimulation. Interestingly, in rats presented with pain but where OT release was not evoked, a similar time course of SC activity reduction was observed, but the magnitude of reduction was minor. Lastly, OT release in PAG raised the threshold of mechanical pain but not heat pain in inflammation model animals.

neuroscience↗