Biology subjects

Nakamura, Y.

Publications and source records attributed to Nakamura, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2020

Identification of novel HPFH-like mutations by CRISPR base editing that elevates the expression of fetal hemoglobin

Switching hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin is an effective strategy for the treatment of beta-hemoglobinopathies. Fetal hemoglobin expression is down-regulated in the postnatal period due to the interplay of transcription regulators with the HBG promoters. However, in the hereditary persistence of fetal hemoglobin (HPFH) condition, naturally occurring point mutations in the HBG promoter causes continued expression of fetal globin even during adulthood. Inspired by this natural phenomenon, we screened the proximal promoter of human HBG genes using adenine and cytosine base editors to identify other nucleotide substitutions that could potentially lead to elevated levels of fetal globin. Both the base editors efficiently and precisely edited at the target sites with a minimal generation of indels and no deletion of one of the duplicated HBG genes. Through systematic tiling across the HBG proximal promoter, we identified multiple novel target sites that resulted in a significant increase in fetal globin levels. Further, we individually validated the top eight potential target sites from both the base editors and observed robust elevation in the fetal globin levels up to 47 %, without any detrimental effects on erythroid differentiation. Our screening strategy resulted in the identification of multiple novel point mutations and also validated the known non-deletional HPFH mutations that could elevate the fetal globin expression at therapeutically relevant levels. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter that normally mediates fetal globin silencing and identify additional targets for therapeutic upregulation of fetal hemoglobin.

genomics↗

bioRxiv2020

Effect of leaf temperature on estimating physiological traits of wheat leaves from hyperspectral reflectance

A growing number of leaf traits can be predicted from hyperspectral reflectance data. These include structural and compositional traits, such as leaf mass per area, nitrogen and chlorophyll content, but also physiological traits such a Rubisco carboxylation activity, electron transport rate and respiration rate. Since physiological traits vary with leaf temperature, how does this impact on predictions made from reflectance measurements? We investigated this with two wheat varieties, by repeatedly measuring each leaf through a sequence of temperatures imposed by varying the air temperature in a growth room. The function predicting Rubisco capacity normalised to 25 {degrees}C predicted the same value, regardless of leaf temperatures ranging from 20 to 35{degrees}C. Leaf temperature affected none of the predicted traits: Vcmax25, J, chlorophyll content, LMA, N content per unit leaf area or Vcmax25/N. However, as others have derived models to predict Rubisco activity that includes variation associated with leaf temperature, we discuss whether these functions may include a temperature signal within the reflectance spectra.

plant biology↗

bioRxiv2020

AnAms1.0: A high-quality chromosome-scale assembly of a domestic cat Felis catus of American Shorthair breed

The domestic cat (Felis catus) is one of the most popular companion animals in the world. Comprehensive genomic resources will aid the development and application of veterinary medicine including to improve feline health, in particular, to enable precision medicine which is promising in human application. However, currently available cat genome assemblies were mostly built based on the Abyssinian cat breed which is highly inbred and has limited power in representing the vast diversity of the cat population. Moreover, the current reference assembly remains fragmented with sequences contained in thousands of scaffolds. We constructed a reference-grade chromosome-scale genome assembly of a domestic cat, Felis catus genome of American Shorthair breed, Anicom American shorthair 1.0 (AnAms1.0) with high contiguity (scaffold N50 > 120 Mb), by combining multiple advanced genomic technologies, including PacBio long-read sequencing as well as sequence scaffolding by long-range genomic information obtained from Hi-C and optical mapping data. Homology-based and ab initio gene annotation was performed with the Iso-Seq data. Analyzed data is be publicly accessible on Cats genome informatics (Cats-I, https://cat.annotation.jp/), a cat genome database established as a platform to facilitate the accumulation and sharing of genomic resources to improve veterinary care.

genomics↗

bioRxiv2020

Identification of differential gene expression pattern in lens epithelial cells derived from cataractous and non-cataractous lenses of Shumiya cataract rat

The Shumiya cataract rat (SCR) is a model for hereditary cataract. Two-third of these rats develop lens opacity within 10-11-weeks. Onset of cataract is attributed to the synergetic effect of lanosterol synthase (Lss) and farnesyl-diphosphate farnesyltransferase 1 (Fdft1) mutant alleles that lead to cholesterol deficiency in the lenses, which in turn adversely affects lens biology including the growth and differentiation of lens epithelial cells (LECs). Nevertheless, the molecular events and changes in gene expression associated with the onset of lens opacity in SCR is poorly understood. In the present study, a microarray-based approach was employed to analyze comparative gene expression changes in LECs isolated from the pre-cataractous and cataractous stages of lenses of 5-, 8- and 10-week-old SCRs. The changes in gene expression observed in microarray results in the LECs were further validated using real-time PCR and western blot analyses. Lens opacity was not observed in 5-week-old rats. However, histological analysis revealed mild dysplasia in the anterior suture and poorly differentiated fiber cells at the bow region. Expression of approximately 100 genes, including major intrinsic protein of lens fiber (MIP and Aquaporin0), deoxyribonuclease II beta (Dnase2b), heat shock protein B1 (Hspb1), and crystallin {gamma} ({gamma}Cry) B, C, and F were found to be significantly downregulated (0.07-0.5 fold) in rat LECs derived from cataract lenses compared to that in noncataractous lenses (control). Thus, our study aimed to identify the gene expression patterns during cataract formation in SCRs, which may be responsible for cataractogenesis in SCR. We proposed that mutation in lanosterol synthase was responsible for the downregulation of genes associated with lens fiber differentiation, which in turn leads to the formation of cataract in these rats. Our findings may have wider implications in understanding the effect of cholesterol deficiency and the role of cholesterol-lowering therapeutics on cataractogenesis.

cell biology↗

bioRxiv2020

CHLOROPLAST UNUSUAL POSITIONING 1 is a new type of actin nucleation factor in plants

Plants have evolved unique responses to fluctuating light conditions in their environment. One such response, chloroplast photorelocation movement, optimizes photosynthesis under weak light and prevents photodamage under strong light. CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) plays a pivotal role in the light-responsive chloroplast movements, which are driven by dynamic reorganization of chloroplast actin (cp-actin) filaments. In this study, we demonstrated that fluorescently tagged CHUP1 colocalized and was coordinately reorganized with cp-actin filaments during chloroplast movement in Arabidopsis thaliana. The resulting asymmetric distribution of CHUP1 was reversibly regulated by the blue light receptor phototropin. X-ray crystallography indicated that the CHUP1 C-terminal domain shares structural similarity with the formin homology 2 (FH2) domain, although there is no sequence similarity between the two domains. The CHUP1 C-terminal domain stimulated actin polymerization in the presence of profilin. We conclude that CHUP1 is a novel, plant-specific actin nucleator that functions in cp-actin-based chloroplast movement. HighlightsO_LIBlue light changes the distribution pattern of CHUP1 C_LIO_LIFormin FH2 and CHUP1 C-terminal domains are structurally similar but not homologous C_LIO_LICHUP1 nucleates and severs actin filaments in vitro C_LIO_LICHUP1 is a novel, plant-specific actin nucleator for chloroplast movement C_LI

plant biology↗

bioRxiv2019

RaptRanker: in silico RNA aptamer selection from HT-SELEX experiment based on local sequence and structure information

Aptamers are short single-stranded RNA/DNA molecules that bind to specific target molecules. Aptamers with high binding-affinity and target specificity are identified using an in vitro procedure called high throughput systematic evolution of ligands by exponential enrichment (HT-SELEX). However, the development of aptamer affinity reagents takes a considerable amount of time and is costly because HT-SELEX produces a large dataset of candidate sequences, some of which have insufficient binding-affinity. Here, we present RNA aptamer Ranker (RaptRanker), a novel in silico method for identifying high binding-affinity aptamers from HT-SELEX data by scoring and ranking. RaptRanker analyzes HT-SELEX data by evaluating the nucleotide sequence and secondary structure simultaneously, and by ranking according to scores reflecting local structure and sequence frequencies. To evaluate the performance of RaptRanker, we performed two new HT-SELEX experiments, and evaluated binding affinities of a part of sequences that include aptamers with low binding-affinity. In both datasets, the performance of RaptRanker was superior to Frequency, Enrichment and MPBind. We also confirmed that the consideration of secondary structures is effective in HT-SELEX data analysis, and that RaptRanker successfully predicted the essential subsequence motifs in each identified sequence.

bioinformatics↗

bioRxiv2019

Chromatin organization in early land plants reveals an ancestral association between H3K27me3, transposons, and constitutive heterochromatin

Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes and our knowledge of the evolution of chromatin related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants 400 Mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional organization of the genome of early land plants. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and repeats in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres, and a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that chromatin regions belonging to repressed heterochromatin were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

molecular biology↗

bioRxiv2019

An integrated platform to systematically identify causal variants and genes for polygenic human traits.

Genome-wide association studies (GWAS) have identified over 150,000 links between common genetic variants and human traits or complex diseases. Over 80% of these associations map to polymorphisms in non-coding DNA. Therefore, the challenge is to identify disease-causing variants, the genes they affect, and the cells in which these effects occur. We have developed a platform using ATAC-seq, DNaseI footprints, NG Capture-C and machine learning to address this challenge. Applying this approach to red blood cell traits identifies a significant proportion of known causative variants and their effector genes, which we show can be validated by direct in vivo modelling.

genetics↗

bioRxiv2019

fMRI detects bilateral brain network activation following unilateral chemogenetic activation of direct striatal projection neurons

Although abnormal structural and functional connectivity in the striatum during neurological disorders has been reported using functional magnetic resonance imaging (fMRI), the effects of cell-type specific neuronal stimulation on fMRI and related behavioral alterations are not well understood. In this study, we successfully combined DREADD-technology with fMRI (chemo-fMRI) to investigate the alterations of spontaneous neuronal activity induced by the unilateral activation of dopamine D1 receptor-expressing neurons (D1-neurons) in the mouse dorsal striatum (DS). We compared the effects of two different DREADD ligands, clozapine (CLZ) and clozapine-N-oxide (CNO), on behavior and fMRI. We found that the effects of CLZ on behavior were more rapid than those of CNO. In fMRI, both systemic CLZ and CNO administrations, which evoked unilateral activations of D1-neurons in DS, increased the fractional amplitude of low frequency fluctuations (fALFF) in the thalamus and bilateral cortex. In addition, we found the increased gamma-band of local field potentials in DS and bilateral cortex after CLZ-evoked unilateral activation of D1-neuron in the striatum. These results provide bases for better interpretation of cell type-specific activity changes in fMRI.

neuroscience↗

bioRxiv2019

Short-term consumption of sucralose with, but not without, carbohydrate impairs neural and metabolic sensitivity to sugar

There is a general consensus that overconsumption of sugar sweetened beverages contributes to the prevalence of obesity and related comorbidities such as type 2 diabetes (T2D). Whether a similar relationship exists for no, or low-calorie "diet" drinks is a subject of intensive debate and controversy. Here, we show that metabolic dysfunction, coupled with reduced central sensitivity to sweet, but not sour, salty or bitter taste, occurs when sucralose is repeatedly consumed with, but not without, a carbohydrate over a two-week period in healthy humans. A similar exposure to sucralose, with, but not without, a carbohydrate altered substrate utilization in mice. More specifically, greater energy intake was required for the animals to shift from fatty acid to carbohydrate oxidation, indicating a reduced sensitivity to carbohydrate. These findings demonstrate that consumption of sucralose in the presence of a carbohydrate rapidly impairs glucose metabolism and may contribute to the rise in T2D.

physiology↗

bioRxiv2019

Protective role of the deSUMOylating enzyme SENP3 in myocardial ischaemia-reperfusion injury

Interruption of blood supply to the heart is a leading cause of death and disability. However, the molecular events that occur during heart ischaemia, and how these changes prime consequent cell death upon reperfusion, are poorly understood. Protein SUMOylation is a post-translational modification that has been strongly implicated in the protection of cells against a variety of stressors, including ischaemia-reperfusion. In particular, the SUMO2/3-specific protease SENP3 has emerged as an important determinant of cell survival after ischaemic infarct. Here, we used the Langendorff perfusion model to examine changes in the levels and localisation of SUMOylated target proteins and SENP3 in whole heart. We observed a 50% loss of SENP3 from the cytosolic fraction of hearts after preconditioning, a 90% loss after ischaemia and an 80% loss after ischaemia-reperfusion. To examine these effects further, we performed ischaemia and ischaemia-reperfusion experiments in the cardiomyocyte H9C2 cell line. Similar to whole hearts, ischaemia induced a decrease in cytosolic SENP3. Furthermore, shRNA-mediated knockdown of SENP3 led to an increase in the rate of cell death upon reperfusion. Together, our results indicate that cardiac ischaemia dramatically alter levels of SENP3 and suggest that this may a mechanism to promote cell survival after ischaemia-reperfusion in heart.

cell biology↗

bioRxiv2018

The C-terminal Helix 9 motif regulates cannabinoid receptor type 1 trafficking and surface expression

Cannabinoid type 1 receptor (CB1R) is only stably surface expressed in axons, where it downregulates neurotransmitter release. How this tightly regulated axonal surface polarity is established and maintained is unclear. To address this question, we used time-resolved imaging to determine the trafficking of CB1R from biosynthesis to mature polarised localisation. We show that the secretory pathway delivery of CB1R is axonally biased and that surface expressed CB1R is more stable in axons than in dendrites. This dual mechanism is mediated by the CB1R C-terminal and involves the Helix 9 (H9) domain. Removal of the H9 domain increases dendrite secretory pathway delivery and decreases in surface stability. Furthermore, CB1R{Delta}H9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1RWT. Together, these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process.

neuroscience↗

bioRxiv2018

Age of onset in genetic prion disease and the design of preventive clinical trials

Regulatory agencies worldwide have adopted programs to facilitate drug development for diseases where the traditional approach of a randomized trial with a clinical endpoint is expected to be prohibitively lengthy or difficult. Here we provide quantitative evidence that this criterion is met for the prevention of genetic prion disease. We assemble age of onset or death data from N=1,094 individuals with high penetrance mutations in the prion protein gene (PRNP), generate survival and hazard curves, and estimate statistical power for clinical trials. We show that, due to dramatic and unexplained variability in age of onset, randomized preventive trials would require hundreds or thousands of at-risk individuals in order to be statistically powered for an endpoint of clinical onset, posing prohibitive cost and delay and likely exceeding the number of individuals available for such trials. Instead, the characterization of biomarkers suitable to serve as surrogate endpoints will be essential for the prevention of genetic prion disease. Biomarker-based trials may require post-marketing studies to confirm clinical benefit. Parameters such as longer trial duration, increased enrollment, and the use of historical controls in a post-marketing study could provide opportunities for subsequent determination of clinical benefit.

neuroscience↗

bioRxiv2018

BAP1 deubiquitinase is a potent repressor of fetal hemoglobin biosynthesis

Human globin gene production transcriptionally \"switches\" from fetal to adult synthesis shortly after birth, and is controlled by macromolecular complexes that enhance or suppress transcription by cis-elements scattered throughout the locus. The DRED repressor is recruited to the {varepsilon}- and {gamma}-globin promoters by the orphan nuclear receptors TR2 (NR2C1) and TR4 (NR2C2) to engender their silencing in adult erythroid cells. Here we found that nuclear receptor corepressor-1 (NCoR1) is a critical component of DRED that acts as a scaffold to unite the DNA binding and epigenetic enzyme components (e.g. DNMT1 and LSD1) that elicit DRED function. We also describe a potent new regulator of {gamma}-globin repression: the deubiquitinase BAP1 is a component of the repressor complex whose activity maintains NCoR1 at sites in the {beta}-globin locus, and BAP1 inhibition in erythroid cells massively induces {gamma}-globin synthesis. These data provide new mechanistic insights through the discovery of novel epigenetic enzymes that mediate {gamma}-globin gene repression.

developmental biology↗

bioRxiv2018

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells.

Sickle Cell Disease and {beta}-thalassemia, which are caused by defective or deficient adult {beta}-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Expression of the fetal {beta}-like globin, also known as {gamma}-globin, can ameliorate both disorders by serving in place of the adult {beta}-globin. Here we use CRISPR-Cas9 gene editing to explore a putative {gamma}-globin silencer region identified by comparison of naturally-occurring deletion mutations associated with up-regulated {gamma}-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of fetal hemoglobin (HbF) or {gamma}-globin. Screening of individual sgRNAs in one sub-region revealed three single guides that caused mild increases in {gamma}-globin expression. However, clonal cell lines with the 1.7 kb region deleted did not up-regulate {gamma}-globin and neither did lines with either of two of sub-regions identified in the screen deleted. These data suggest that the region is not an autonomous {gamma}-globin silencer, and thus by itself is not a suitable therapeutic target in the {beta}-hemoglobinopathies.

genetics↗

bioRxiv2018

A long-chain fatty acid elongase Elovl6 regulates mechanical damage-induced keratinocyte death and skin inflammation

Mechanical damage on the skin not only affect the barrier function but also induce various immune responses, which trigger or exacerbate the inflammation in healthy individuals and patients with inflammatory skin diseases. However, how mechanical damage-induced skin inflammation is regulated remains largely unknown. Here, we show that mechanical damage due to tape stripping triggered keratinocyte death and release of danger-associated molecular patterns (DAMPs) such as high-mobility group box 1 protein (HMGB-1) and IL-1, which induced production of proinflammatory cytokines and chemokines IL-1{beta} and CXCL-1 by keratinocytes in mice. We also show that a long-chain fatty acid elongase Elovl6 is expressed in keratinocytes. Mice deficient in Elovl6 had increased epidermal levels of cis-vaccenic acid (CVA); this accelerated keratinocyte death triggered by tape stripping and release of DAMPs and exacerbated skin inflammation. Our results demonstrate that Elovl6 regulates mechanical damage-triggered keratinocyte death and skin inflammation.

immunology↗

bioRxiv2017

A randomized comparison of different hormone replacement protocols for thawed blastocyst transfer

BackgroundThere are no randomized controlled trials evaluating the pregnancy rates after thawed blastocyst transfers in patients treated with various hormone replacement regimens.\n\nMethodsA prospective randomized controlled trial was conducted to evaluate the outcomes in three different hormone replacement protocols for thawed blastocyst transfer. A total of 330 women (median age 38.2 years) who were undergoing IVF at our clinic were enrolled.\n\nResultsSerum estradiol (E2) levels were 267.71 pg/ml in Premarin group, 391.22 pg/ml in Estrogel group and 495.12 pg/ml in Estrana tape group. Therefore, serum E2 levels in Estrana tape group were higher than those of the other two groups (P<0.01). The pregnancy rate in the Estrogel group was higher than that in the Premarin group (30.0% versus 17.3%, P=0.026, odds ratio 2.05, 95% confidence interval: 1.09-3.87). Furthermore, the pregnancy rate in the Estrana tape group was higher than that in the Estrogel group (43.6% versus 30.0%, P=0.036, odds ratio 1.81, 95% confidence interval: 1.04-3.14).\n\nConclusionThe serum E2 levels contributed to differences observed in the pregnancy rate among the three different protocols. Thus, Estrana tape has an advantage as a hormone replacement protocol for thawed blastocyst transfer.

clinical trials↗

bioRxiv2017

Abnormally high levels of serum α-klotho result in a poor outcome for clinical pregnancy-A prospective cohort study-

BackgroundThe klotho protein has been extensively studied. However, there are no studies examining the association between serum alpha klotho levels and the clinical outcome of post-clinical pregnancy.\n\nMethodsWe conducted a prospective cohort study in 42 patients (median age 37.4 years) to evaluate the association between serum alpha klotho levels during the follicular phase of preimplantation and the clinical outcome data of post-clinical pregnancy. The patients provided informed consent at our clinic. The serum alpha klotho levels were evaluated using a human soluble alpha klotho assay kit. The fetal chromosomal abnormalities were investigated at our clinic. We also assessed the clinical outcomes of post-clinical pregnancies.\n\nResultsThe serum alpha klotho level during the follicular phase of preimplantation for non-pregnant women was 544.31 pg/ml (mean). The clinical pregnancy rate was 38.1%. There were chromosomal abnormalities observed in four unborn children (9.5%; Down syndrome, etc). The serum alpha klotho levels during the follicular phase of preimplantation in the chromosomal abnormalities group were higher than in the group without chromosomal abnormalities (P=0.029, abnormalities group 659.26 pg/ml [mean] versus control 530.23 pg/ml [mean]). A multiple logistic regression analysis showed the chromosomal abnormalities rates in unborn children were positively influenced by serum alpha klotho levels during the follicular phase of preimplantation (p=0.0008) and the patients age (p=0.008).\n\nConclusionPrevious studies have demonstrated that increased alpha klotho levels in human serum are positively correlated with health. However, abnormally high levels of serum alpha klotho during the follicular phase of preimplantation may predict a poor outcome for clinical pregnancy.

genetics↗