Biology subjects

Okada, Y.

Publications and source records attributed to Okada, Y..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patients pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) can potentially recapitulate disease pathology more accurately than existing disease models when differentiated into affected cell types. Thus, successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection procedures, and clonal variations must be overcome. Moreover, their functionality to exhibit muscular contraction has never been reported. Here, we demonstrated that bulk MYOD1- hiPSCs established with puromycin selection, but not with G418 selection, showed high differentiation efficiency, generating more than 80% Myogenin (MyoG)+ and Myosin heavy chain (MHC)+ muscle cells within seven days. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties compared with those of clonally established MYOD1- hiPSCs, suggesting that the bulk method may minimize the effects of clonal variations. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical pulse stimulation, indicating their functionality. Together, the findings indicate that our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and facilitates the generation of muscular disease models. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/468571v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@12c091eorg.highwire.dtl.DTLVardef@ad1ccorg.highwire.dtl.DTLVardef@53ba90org.highwire.dtl.DTLVardef@fd65f5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Cancer/Testis genes are predictive of breast tumor subtypes

Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis and no dedicated therapy, therefore new tools to understand, detect, and treat these tumors are needed. Certain germline genes are re-expressed in tumors, and constitute the Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here, we designed a new approach to examine Cancer/ Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the 2 Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells but not the microenvironment, and that they are not expressed by normal breast progenitors, in other words their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Basal-like tumors expressing both genes have a poorer outcome than tumors expressing either gene alone or neither gene. Therefore, these findings suggest a potential synergistic effect between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worse outcomes.

cancer biology↗

bioRxiv2021

Live cell imaging of metabolic heterogeneity by quantitative fluorescent ATP indicator protein, QUEEN-37C.

Adenosine triphosphate (ATP) is often referred as the energy currency of the cell. Yet, non-invasive, real-time, and quantitative measurement of its concentration in living mammalian cells has been difficult. Here we report an improved fluorescent ATP indicator protein, QUEEN-37C, which is optimized for measuring ATP concentration in living mammalian cells. Absolute value of the ATP concentration can be estimated from the ratiometric fluorescence imaging, and its accuracy was verified by the luciferase assay. Since QUEEN-37C enables the single-cell measurement of ATP concentration, we can not only measure its mean but its distribution in the cell population, which revealed that the ATP concentration is tightly regulated in most cells. We also noted the positive correlations in the ATP concentration among adjacent cells in epithelial cell sheet and mouse embryonic stem cell colonies. Thus, QUEEN-37C would serve as a new tool for the investigation of the single cell heterogeneity of metabolic states.

biophysics↗

bioRxiv2021

Transcriptional states of retroelement-inserted regions and KRAB zinc finger protein association regulate DNA methylation of retroelements in human male germ cells

DNA methylation, repressive histone modifications, and PIWI-interacting RNAs are essential for controlling retroelement silencing in mammalian germ lines. Dysregulation of retroelement silencing is associated with male sterility. Although retroelement silencing mechanisms have been extensively studied in mouse germ cells, little progress has been made in humans. Here, we show that the Kruppel-associated box domain zinc finger proteins (KRAB-ZFPs) are associated with DNA methylation of retroelements in human primordial germ cells (hPGCs), and hominoid-specific retroelement SINE-VNTR-Alus (SVA) is subjected to transcription-directed de novo DNA methylation during human spermatogenesis. Furthermore, we show that the degree of de novo DNA methylation in SVAs varies among human individuals, which confers a significant inter-individual epigenetic variation in sperm. Collectively, our results provide potential molecular mechanisms for the regulation of retroelements in human male germ cells.

molecular biology↗

bioRxiv2021

Multi-Ancestry Meta-Analysis yields novel genetic discoveries and ancestry-specific associations

We present a new method, Multi-Ancestry Meta-Analysis (MAMA), which combines genome-wide association study (GWAS) summary statistics from multiple populations to produce new summary statistics for each population, identifying novel loci that would not have been discovered in either set of GWAS summary statistics alone. In simulations, MAMA increases power with less bias and generally lower type-1 error rate than other multi-ancestry meta-analysis approaches. We apply MAMA to 23 phenotypes in East-Asian- and European-ancestry populations and find substantial gains in power. In an independent sample, novel genetic discoveries from MAMA replicate strongly.

genomics↗

bioRxiv2021

Preparation of "stress-free" concanavalin A-conjugated Dynabeads magnetic beads for CUT&Tag

Epigenome research has employed various methods to identify genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag(R)Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads(R) magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay.

bioinformatics↗

bioRxiv2021

C9ORF72 dipeptide repeat proteins disrupt formation of GEM bodies and induce aberrant accumulation of survival of motor neuron protein

A GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. In the neurons of ALS patients, dipeptide repeat proteins (DPRs) are produced from repeat-containing RNAs by an unconventional form of translation, and some of these proteins, especially those containing poly(glycine-arginine) and poly(proline-arginine), are toxic to neurons. Gemini of coiled bodies (GEMs) are nuclear structures that harbor survival of motor neuron (SMN) protein, and SMN is essential for the assembly of U-rich small nuclear ribonucleoproteins (snRNPs) that are central for splicing. We previously reported that GEMs are lost and that snRNP biogenesis is misregulated in the motor neurons of ALS patients. Here we show that DPRs interfere with GEM formation and proper SMN localization in HeLa cells and iPSC-derived motor neurons from an ALS patient with the C9ORF72 mutation. The accumulation of poly(glycine-arginine) markedly reduced the number of GEMs and caused the formation of aberrant cytoplasmic RNA granules that sequestered SMN. These findings indicate the functional impairment of SMN in motor neurons expressing DPRs and may provide a mechanism to explain the vulnerability of motor neurons of C9ORF72-ALS patients.

neuroscience↗

bioRxiv2021

Genetically independent phenotype analysis identifies LPA and VCAM1 as drug targets for human ageing

The length and quality of life is important to us all, yet identification of promising drug targets for human ageing using genetics has had limited success. Here, we combine six large European-ancestry genome-wide association studies (GWAS) of human ageing traits--healthspan, father and mother lifespan, exceptional longevity, frailty index, and self-rated health--in a principal component framework that maximises their shared genetic architecture. The first principal component (GIP1) is more heritable than the original studies and shows strong genetic correlations with length of life as well as multiple indices of mental and physical wellbeing. We identify 27 genomic regions associated with GIP1, and provide additional, independent evidence for an effect on human ageing for loci near HTT and MAML3 using a study of Finnish and Japanese subject survival. Across the genome, GIP1 associations are enriched in genes involved in haem metabolism and pathways related to transcription, neurogenesis, homeostasis, proteolysis, intracellular signalling, immunity, and the muscle system. Finally, using proteome-wide two-sample Mendelian randomisation and colocalisation, we provide robust evidence for a detrimental effect of blood levels of apolipoprotein(a) (LPA) and vascular cell adhesion molecule 1 (VCAM1) on GIP1. Together, our results demonstrate that combining multiple ageing traits using genetic principal components enhances power to detect biological targets for human ageing.

genomics↗

bioRxiv2021

Compartmentalized three-dimensional human neuromuscular tissue models fabricated on a well-plate-format microdevice

Engineered three-dimensional models of neuromuscular tissues are promising for use in mimicking their disorder states in vitro. Although several models have been developed, it is still challenging to mimic the physically separated structures of motor neurons (MNs) and skeletal muscle (SkM) fibers in the motor units in vivo. In this study, we aimed to develop microdevices for precisely compartmentalized coculturing of MNs and engineered SkM tissues. The developed microdevices, which fit a well of 24 well plates, had a chamber for MNs and chamber for SkM tissues. The two chambers were connected by microtunnels for axons, permissive to axons but not to cell bodies. Human iPSC (hiPSC)-derived MN spheroids in one chamber elongated their axons into microtunnels, which reached the tissue-engineered human SkM in the SkM chamber, and formed functional neuromuscular junctions with the muscle fibers. The cocultured SkM tissues with MNs on the device contracted spontaneously in response to spontaneous firing of MNs. The addition of a neurotransmitter, glutamate, into the MN chamber induced contraction of the cocultured SkM tissues. Selective addition of tetrodotoxin or vecuronium bromide into either chamber induced SkM tissue relaxation, which could be explained by the inhibitory mechanisms. We also demonstrated the application of chemical or mechanical stimuli to the middle of the axons of cocultured tissues on the device. Thus, compartmentalized neuromuscular tissue models fabricated on the device could be used for phenotypic screening to evaluate the cellular type specific efficacy of drug candidates and would be a useful tool in fundamental research and drug development for neuromuscular disorders.

bioengineering↗

bioRxiv2020

Leveraging supervised learning for functionally-informed fine-mapping of cis-eQTLs identifies an additional 20,913 putative causal eQTLs

The large majority of variants identified by GWAS are non-coding, motivating detailed characterization of the function of non-coding variants. Experimental methods to assess variants effect on gene expressions in native chromatin context via direct perturbation are low-throughput. Existing high-throughput computational predictors thus have lacked large gold standard sets of regulatory variants for training and validation. Here, we leverage a set of 14,807 putative causal eQTLs in humans obtained through statistical fine-mapping, and we use 6,121 features to directly train a predictor of whether a variant modifies nearby gene expression. We call the resulting prediction the expression modifier score (EMS). We validate EMS by comparing its ability to prioritize functional variants with other major scores. We then use EMS as a prior for statistical fine-mapping of eQTLs to identify an additional 20,913 putatively causal eQTLs, and we incorporate EMS into co-localization analysis to identify 310 additional candidate genes across UK Biobank phenotypes.

genomics↗

bioRxiv2020

Natural selection reverses the exaggeration of a male sexually selected trait, which increases female fitness.

Theory shows how sexual selection can exaggerate male traits beyond naturally selected optima and also how natural selection can ultimately halt trait elaboration. Empirical evidence supports this theory, but to date, there have been no experimental evolution studies directly testing this logic, and little examination of possible associated effects on female fitness. Here we used experimental evolution of replicate populations of broad-horned flour-beetles to test for evolutionary effects of sex-specific predation on an exaggerated sexually selected male trait, while also testing for effects on female lifetime reproductive success. We found that populations subjected to male-specific predation evolved smaller sexually selected traits and this indirectly increased female fitness, seemingly through intersexual genetic correlations we documented. Predation solely on females had no effects. Our findings support fundamental theory, but also reveal novel outcomes when natural selection targets sex-limited sexually selected characters.

evolutionary biology↗

bioRxiv2020

Individual Ants Do Not Show Active/rest Rhythms in Natural Nest Conditions

Circadian rhythms, which respond to the day/night cycle on the earth, arise from the endogenous timekeeping system within organisms, called the biological clock. For accurate circadian rhythms, endogenous oscillations are synchronized to light and temperature. In social insects, both abiotic and biotic factors (i.e., social interactions) play a significant role in active/rest rhythm regulation. However, it is challenging to monitor individual active-rest rhythms in a colony because of the large group size and small body size. Therefore, it is unclear how social interactions coordinate each individuals active/rest rhythms. This study developed an image-based tracking system using 2D barcodes for Diacamma sp. (a monomorphic ant) and measured the locomotor activities of all colony members under laboratory colony conditions. We also investigated the effect of broods on active/rest rhythms by removing all broods under colony conditions. Active/rest rhythms appeared only in solitary ants, not under colony conditions. In addition, arrhythmic active-rest rhythms were retained after brood removal. Therefore, a mixture of social interactions, not abiotic factors, induces the loss of active/rest rhythms. These results contribute to the knowledge of the diversity pattern of circadian rhythms in social insects.

animal behavior and cognition↗

bioRxiv2020

Tractor: A framework allowing for improved inclusion of admixed individuals in large-scale association studies.

Admixed populations are routinely excluded from medical genomic studies due to concerns over population structure. Here, we present a statistical framework and software package, Tractor, to facilitate the inclusion of admixed individuals in association studies by leveraging local ancestry. We test Tractor with simulations and empirical data focused on admixed African-European individuals. Tractor generates ancestryspecific effect size estimates, can boost GWAS power, and improves the resolution of association signals. Using a local ancestry aware regression model, we replicate known hits for blood lipids in admixed populations, discover novel hits missed by standard GWAS procedures, and localize signals closer to putative causal variants.

genomics↗

bioRxiv2020

Enhancer Remodeling Promotes Tumor-Initiating Activity in NRF2-Activated Non-Small Cell Lung Cancers

Transcriptional dysregulation, which can be caused by genetic and epigenetic alterations, is a fundamental feature of many cancers. A key cytoprotective transcriptional activator, NRF2, is often aberrantly activated in non-small cell lung cancers (NSCLCs) and supports both aggressive tumorigenesis and therapeutic resistance. Herein, we found that persistently activated NRF2 in NSCLCs generates enhancers at gene loci that are not normally regulated by transiently activated NRF2 under physiological conditions. Elevated accumulation of CEBPB in NRF2-activated NSCLCs was found to be one of the prerequisites for establishment of the unique NRF2-dependent enhancers, among which NOTCH3 enhancer was shown to be critical for the promotion of tumor-initiating activity. Enhancer remodeling mediated by NRF2-CEBPB cooperativity promotes tumor-initiating activity and drives malignancy of NRF2-activated NSCLCs via establishment of the NRF2-NOTCH3 regulatory axis.

cancer biology↗

bioRxiv2020

NUDT21 links mitochondrial IPS-1 to RLR-containing stress granules and activates host antiviral defense

Viral RNA in the cytoplasm of mammalian host cells is recognized by retinoic acid- inducible protein-I (RIG-I)-like receptors (RLRs), which localize to cytoplasmic stress granules (SGs). Activated RLRs associate with the mitochondrial adaptor protein IPS-1, which activates antiviral host defense mechanisms including type I interferon (IFN) induction. It has remained unclear, however, how RLRs in SGs and IPS-1 in the mitochondrial outer membrane associate physically and engage in information transfer. Here we show that NUDT21, an RNA binding protein that regulates alternative transcript polyadenylation, physically associates with IPS-1 and mediates its localization to SGs in response to transfection with poly(I:C), a mimic of viral double-stranded RNA. We found that, despite its well-established function in the nucleus, a fraction of NUDT21 localizes to mitochondria in resting cells and becomes localized to SGs in response to poly(I:C) transfection. NUDT21 was also found to be required for efficient type I IFN induction in response to viral infection. Our results together indicate that NUDT21 links RLRs in SGs to mitochondrial IPS-1 and thereby activates host defense responses to viral infection.

immunology↗

bioRxiv2020

Bipartite network analysis of task-ant associations reveals task groups and absence of colonial diurnal activity

Social insects are one of the best examples of complex self-organised systems exhibiting task allocation. How task allocation is achieved is the most fascinating question in behavioural ecology and complex systems science. However, it is difficult to comprehensively characterise task allocation patterns due to behavioural complexity, such as the individual variation, context dependency, and chronological variation. Thus, it is imperative to quantify individual behaviours and integrate them into colony levels. Here, we applied bipartite network analyses to characterise individual-behaviour relationships. We recorded the behaviours of all individuals with verified age in ant colonies and analysed the individual-behaviour relationship at the individual, module, and network levels. Bipartite network analysis successfully detected the module structures, illustrating that certain individuals performed a subset of behaviours (i.e., task groups). We confirmed age polyethism by comparing age between modules. Additionally, to test the daily rhythm of the executed tasks, the data were partitioned between daytime and nighttime, and a bipartite network was re-constructed. This analysis supported that there was no daily rhythm in the tasks performed. These findings suggested that bipartite network analyses could untangle complex task allocation patterns and provide insights into understanding the division of labour.

animal behavior and cognition↗

bioRxiv2020

Structural basis of ethnic-specific variants of PAX4 associated with type 2 diabetes

Recently, we conducted genome-wide association studies of type 2 diabetes (T2D) in a Japanese population, which identified 20 novel T2D loci that were not associated with T2D in Europeans. Moreover, nine novel missense risk variants, such as those of PAX4, were not rare in the Japanese population, but rare in Europeans. We report in silico structural analysis of ethnic-specific variants of PAX4, which suggests the pathogenic effect of these variants.

genetics↗

bioRxiv2020

Genetic analyses identify widespread sex-differential participation bias

Genetic association results are often interpreted with the assumption that study participation does not affect downstream analyses. Understanding the genetic basis of this participation bias is challenging as it requires the genotypes of unseen individuals. However, we demonstrate that it is possible to estimate comparative biases by performing GWAS contrasting one subgroup versus another. For example, we show that sex exhibits autosomal heritability in the presence of sex-differential participation bias. By performing a GWAS of sex in ~3.3 million males and females, we identify over 158 autosomal loci significantly associated with sex and highlight complex traits underpinning differences in study participation between sexes. For example, the body mass index (BMI) increasing allele at the FTO locus was observed at higher frequency in males compared to females (OR 1.02 [1.02-1.03], P=4.4x10-36). Finally, we demonstrate how these biases can potentially lead to incorrect inferences in downstream analyses and propose a conceptual framework for addressing such biases. Our findings highlight a new challenge that genetic studies may face as sample sizes continue to grow.

genetics↗