Biology subjects

Nakamura, Y.

Publications and source records attributed to Nakamura, Y..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Epigenetic silencing by SMYD3 represses tumor intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck.

Cancers often display immune escape, but the mechanisms and potential for reversibility are incompletely understood. Epigenetic dysregulation has been implicated in the immune escape of various cancer types. We have identified the epigenetic modifier SET and MYND-domain containing protein 3 (SMYD3) as a mediator of immune escape in human papilloma virus (HPV)- negative head and neck squamous cell carcinoma (HNSCC), an aggressive disease with poor prognosis and low response to immunotherapy with pembrolizumab, a programmed-death-1 (PD-1) targeting antibody. SMYD3 loss increased the sensitivity of HNSCC cancer cells to IFN-{beta}, resulting in upregulation of type I IFN response and antigen presentation machinery genes. We found that SMYD3 regulates the transcription of Ubiquitin-Like PHD And RING Finger Domain- Containing Protein 1 (UHRF1), a key epigenetic reader of trimethylated lysine 9 on histone H3 (H3K9me3), which binds to H3K9me3-enriched promoters of key immune-related genes and silences their expression. SMYD3 further maintains the repression of immune-related genes through the deposition of H4K20me3 within the gene body regions of these genes. In an anti-PD-1 immune checkpoint resistant syngeneic mouse model of HPV-negative HNSCC, Smyd3 depletion induced influx of CD8+ T-cells, upregulated PD-L1 and MHC class I molecules, and increased sensitivity to anti-PD-1 therapy. SMYD3 overexpression was associated with decreased CD8 T-cell infiltration in tumor samples from patients with HPV-negative HNSCC, and was associated with poor response to pembrolizumab. Overall, these data highlight a previously unreported function of SMYD3 as a master epigenetic regulator of anti-tumor immune response in HPV-negative HNSCC and provide a rationale for translational approaches combining SMYD3 depletion strategies with checkpoint blockade to overcome anti-PD-1 resistance in this devastating disease.

cancer biology↗

bioRxiv2022

A two-pool mechanism of vesicle release in medial habenula terminals underlies GABAB receptor-mediated potentiation

GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation induces a transition from tonic to phasic release accompanied by a 4-fold increase in readily releasable pool (RRP) size in MHb terminals, mirrored by a similar increase in the docked vesicle number at the presynaptic active zone (AZ). The tonic and phasic release vesicles have distinct coupling distances. We identified two vesicle-associated molecules, synaptoporin and CAPS2, selectively involved in tonic and phasic release, respectively. Synaptoporin mediates augmentation of tonic release and CAPS2 stabilizes readily releasable vesicles during phasic release. A newly developed "Flash and Freeze-fracture" method revealed selective recruitment of CAPS2 to the AZ during phasic release. Thus, we propose a novel two-pool mechanism underlying the GBR-mediated potentiation of release from MHb terminals.

neuroscience↗

bioRxiv2022

Serum amyloid alpha 1-2 are not required for systemic inflammation in the 4T1 murine breast cancer model

Cancers induce the production of acute phase proteins such as serum amyloid alpha (SAA) in the liver and cause systemic inflammation. Despite the well-known coincidence of acute phase response and systemic inflammation, the direct roles of SAA proteins in systemic inflammation in the cancer context remains incompletely characterized, particularly in vivo. Here, we investigate the in vivo significance of SAA proteins in systemic inflammation in the 4T1 murine breast cancer model. 4T1 cancers elevate the expression of SAA1 and SAA2, the two major murine acute phase proteins in the liver. The elevation of Saa1-2 correlates with the up-regulation of immune cell-related genes including neutrophil markers. To examine this correlation in detail, we generate mice that lack Saa1-2 and investigate immune-cell phenotypes. RNA-seq experiments reveal that deletion of Saa1-2 does not strongly affect 4T1-induced activation of immune cell-related genes in the liver and bone marrow. Flow cytometry experiments demonstrate the dispensable roles of SAA1-2 in cancer-dependent neutrophil infiltration to the liver. This study clarifies the negligible contribution of SAA1-2 proteins in systemic inflammation in the 4T1 breast cancer model.

cancer biology↗

bioRxiv2022

A highly contiguous genome assembly of red perilla (Perilla frutescens) domesticated in Japan

Perilla frutescens (Lamiaceae) is an important herbal plant with hundreds of bioactive chemicals, among which perillaldehyde and rosmarinic acid are the two major bioactive compounds in the plant. The leaves of red perilla are used as traditional Kampo medicine or food ingredients. However, the medicinal and nutritional uses of this plant could be improved by enhancing the production of valuable metabolites through the manipulation of key enzymes or regulatory genes using genome editing technology. Here, we generated a high-quality genome assembly of red perilla domesticated in Japan. A near-complete chromosome level assembly of P. frutescens was generated contigs with N50 of 41.5 Mb from PacBio HiFi reads. 99.2% of the assembly was anchored into 20 pseudochromosomes, among which seven pseudochromosomes consisted of one contig, while the rest consisted of less than six contigs. Gene annotation and prediction of the sequences successfully predicted 86,258 gene models, including 76,825 protein-coding genes. Further analysis showed that potential targets of genome editing for the engineering of anthocyanin pathways in P. frutescens are located on the late-stage pathways. Overall, our genome assembly could serve as a valuable reference for selecting target genes for genome editing of P. frutescens.

plant biology↗

bioRxiv2022

Recycling upstream redox enzymes expands the regioselectivity of cycloaddition in pseudo-aspidosperma alkaloid biosynthesis

Nature uses cycloaddition reactions to generate complex natural product scaffolds. Dehydrosecodine is a highly reactive biosynthetic intermediate that undergoes cycloaddition to generate several alkaloid scaffolds that are the precursors to pharmacologically important compounds such as vinblastine and ibogaine. Here we report how dehydrosecodine can be subjected to redox chemistry, which in turn allows cycloaddition reactions with alternative regioselectivity. By incubating dehydrosecodine with reductase and oxidase biosynthetic enzymes that act upstream in the pathway, we can access the rare pseudo-aspidosperma alkaloids, pseudo-tabersonine and pseudo-vincadifformine, both in vitro and by reconstitution in the plant Nicotiana benthamiana from an upstream intermediate. We propose a stepwise mechanism to explain the formation of the pseudo-tabersonine scaffold by structurally characterizing enzyme intermediates, and by monitoring the incorporation of deuterium labels. This discovery highlights how plants use redox enzymes to enantioselectively generate new scaffolds from common precursors.

biochemistry↗

bioRxiv2022

Genome analysis of Parmales, a sister group of diatoms, reveals the evolutionary specialization of diatoms from phago-mixotrophs to photoautotrophs

The order Parmales (Bolidophyceae) is a minor group of pico-sized eukaryotic marine phytoplankton that contains species with cells surrounded by silica plates. Previous studies revealed that Parmales is a member of ochrophytes and sister to diatoms (Bacillariophyta), the most successful phytoplankton group in the modern ocean. Therefore, parmalean genomes can serve as a reference to elucidate both the evolutionary events that differentiated these two lineages and the genomic basis for the ecological success of diatoms vs. the more cryptic lifestyle of parmaleans. Here, we compared the genomes of eight parmaleans and five diatoms to explore their physiological and evolutionary differences. Parmaleans were predicted to be phago-mixotrophs. By contrast, diatoms have undergone loss of genes related to phagocytosis, indicating the ecological specialization from phago-mixotroph to photoautotroph in the early evolution of diatoms. Furthermore, diatoms showed significant enrichment in gene sets involved in silica metabolism, nutrient uptake capacity, carbon concentrating mechanisms, and iron uptake in comparison with parmaleans. Overall, our results suggest a strong evolutionary link between the loss of phago-mixotrophy and specialization to a silicified photoautotrophic life stage early in diatom evolution after diverging from the Parmales lineage.

microbiology↗

bioRxiv2022

Cerebrospinal fluid-contacting neuron tracing reveals structural and functional connectivity for locomotion in the mouse spinal cord

Cerebrospinal fluid-contacting neurons (CSF-cNs) are enigmatic mechano- or chemosensory cells lying along the central canal of the spinal cord. Recent studies in zebrafish larvae and lampreys have shown that CSF-cNs control postures and movements via spinal connections. However, the structures, connectivity, and functions in mammals remain largely unknown. Here we developed a method to genetically target mouse CSF-cNs that highlighted structural connections and functions. We first found that intracerebroventricular injection of adeno-associated virus with a neuron-specific promoter and Pkd2l1-Cre mice specifically labeled CSF-cNs. Single-cell labeling of 71 CSF-cNs revealed rostral axon extensions of over 1800 m in unmyelinated bundles in the ventral funiculus and terminated on CSF-cNs to form a recurrent circuitry, which was further determined by serial electron microscopy and electrophysiology. CSF-cNs were also found to connect with axial motor neurons and premotor interneurons around the central canal and within the axon bundles. Chemogenetic CSF-cNs inactivation reduced speed and step frequency during treadmill locomotion. Our data revealed the basic structures and connections of mouse CSF-cNs to control spinal motor circuits for proper locomotion. The versatile methods developed in this study will contribute to further understanding of CSF-cNs functions in mammals.

neuroscience↗

bioRxiv2022

Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4 reduction of an ,{beta}-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs that catalyse 1,2-aldehyde and 1,2-iminium reductions suggest how the canonical ADH active site can be modified to carry out atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

biochemistry↗

bioRxiv2022

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is the commonly adapted strategy to ameliorate {beta}-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits the HbF tetramer production affecting the therapeutic benefits. Here, we tested various deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified a 11 kb sequence, encompassing Putative Repressor Region (PRR) to {beta}-globin Exon-1 ({beta}E1), as the core deletion that ablates HbA and exhibit superior production of HbF compared to HPFH or other well-established targets. The PRR-{beta}E1 edited hematopoietic stem and progenitor cells (HSPCs) retained engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice generating HbF+ cells in vivo. Importantly, the editing induces therapeutically relevant levels of HbF to reverse the phenotypes of both sickle cell disease and {beta}-thalassemia major. These results indicate that the PRR-{beta}E1 gene editing in patient HSPCs can potentially lead to superior therapeutic outcomes for {beta}-hemoglobinopathies gene therapy.

biochemistry↗

bioRxiv2022

Hydrodynamics shapes self-recruitment in anemonefishes

Many marine species have a pelagic larval phase that undergo dispersal among habitats. Studies on marine larval dispersal have revealed a large variation in the spatial scale of dispersal, and accumulated evidence has shown that seascape patchiness is the major determinant for variation in self-recruitment. However, few studies have investigated the influence of geographic settings on marine larval dispersal. Bays or lagoons generally enhance the retention of larvae, while larvae are more likely to be flushed by strong currents in open coasts. To examine associations between larval dispersal, geographic setting, and hydrodynamics, we compared fin-scale dispersal patterns, self-recruitment, and local retention of two anemonefishes (Amphiprion frenatus and A. perideraion) between a semi-enclosed bay and an open coast in the Philippines combining genetic parentage analysis and biophysical dispersal modelling. Contrary to our expectations, parentage analysis revealed lower estimates of self-recruitment in the semi-closed bay (0-2%) than in the open coast (14-15%). The result was consistent with dispersal simulations predicting lower local retention and self-recruitment in the former (0.4% and 19%) than in the latter (2.9% and 38%). Dispersal modelling also showed that cross-shore currents toward offshore were much stronger around the semi-closed bay and were negatively correlated with local retention and self-recruitment. These results suggest that stronger cross-shore currents around the semi-closed bay transport anemonefish larvae to the offshore and mainly contributed to the lower self-recruitment. Our results highlight difficulty in predicting self-recruitment from geographic setting alone and importance of hydrodynamics on it.

ecology↗

bioRxiv2022

MarpolBase Expression: A Web-based, Comprehensive Platform for Visualization and Analysis of Transcriptomes in the Liverwort Marchantia polymorpha

The liverwort Marchantia polymorpha is equipped with a wide range of molecular and genetic tools and resources that have led to its wide use to explore the evo-devo aspects of land plants. Although its diverse transcriptome data are rapidly accumulating, there is no extensive yet user-friendly tool to exploit such a compilation of data and to summarize results with the latest annotations. Here, we have developed a web-based suite of tools, MarpolBase Expression (MBEX, https://marchantia.info/mbex/), where users can visualize gene expression profiles, identify differentially expressed genes, perform co-expression and functional enrichment analyses, and summarize their comprehensive output in various portable formats. Using oil body biogenesis as an example, we demonstrated that the results generated by MBEX were consistent with the published experimental evidence and also revealed a novel transcriptional network in this process. MBEX should facilitate the exploration and discovery of the genetic and functional networks behind various biological processes in M. polymorpha, and promote our understanding of the evolution of land plants.

plant biology↗

bioRxiv2022

Precise modelling and correction of a spectrum of β-thalassemic mutations in human erythroid cells by base editors

{beta}-thalassemia and HbE result from mutations in the {beta}-globin locus that impedes the production of functional {beta}-hemoglobin and represents one of the most common genetic disorders worldwide. Recent advances in genome engineering have opened up new therapeutic opportunities to directly correct these pathogenic mutations using base editors that install transition mutations (A>G and C>T) in the target region with minimal generation of indels. Herein, for the first time, we demonstrate the usage of base editor in the correction of point mutations spanning multiple regions of the HBB gene, including promoter, intron and exon. To this end, we have engineered human erythroid cells harbouring the diverse HBB mutations, thus eliminating the requirement of patient CD34+ HSPCs with desired mutations for the primary screening by base editors. We further performed precise creation and correction of individual HBB point mutations in human erythroid cells using base editors, which were effectively corrected in the HBB-engineered erythroid model. Intriguingly, most bystander effects produced by the base editor at the target site were reported to exhibit normal hemoglobin variants. Overall, our study provides the proof-of-concept for the precise, efficient and scarless creation and correction of various pathogenic mutations at the coding and non-coding regions of HBB gene in human erythroid cells using base editors and establishes a novel therapeutic platform for the treatment of {beta}-thalassemia/HbE patients. This study can be further explored in correcting the other monogenic disorders caused due to single base substitutions.

genomics↗

bioRxiv2022

Prostaglandin EP3 receptor-expressing preoptic neurons bidirectionally control body temperature via tonic GABAergic signaling

The circuit mechanism of the thermoregulatory center in the preoptic area (POA) is unknown. Using rats, here we show prostaglandin EP3 receptor-expressing POA neurons (POAEP3R neurons) as a pivotal bidirectional controller in the central thermoregulatory mechanism. POAEP3R neurons are activated in response to elevated ambient temperature, but inhibited by prostaglandin E2, a pyrogenic mediator. Chemogenetic stimulation of POAEP3R neurons at room temperature reduces body temperature by enhancing heat dissipation, whereas inhibition of them elicits hyperthermia involving brown fat thermogenesis, mimicking fever. POAEP3R neurons innervate sympathoexcitatory neurons in the dorsomedial hypothalamus (DMH) via tonic inhibitory signaling. Although many POAEP3R neuronal cell bodies express a glutamatergic mRNA marker, paradoxically, their axons in the DMH predominantly contain terminals with GABAergic presynaptic proteins, which are increased by chronic heat exposure. These findings indicate that tonic GABAergic inhibitory signaling from POAEP3R neurons is a fundamental determinant of body temperature for thermal homeostasis and fever.

physiology↗

bioRxiv2022

Mobile elements in human population-specific genome and phenotype divergence

Mobile genetic elements (MEs) are heritable mutagens that contribute to divergence between lineages by recursively generating structural variants. ME variants (MEVs) are difficult to genotype, obscuring their impact on recent genome and trait diversification. We developed a tool that uses short-read sequence data to accurately genotype MEVs, enabling us to study them using statistical genetics methods in global human genomes. We observe population-specific differences in the distribution of Alu insertions that distinguish Japanese from other populations. We integrated MEVs with epigenomic and expression quantitative trait loci (eQTL) maps to determine how they impact traits. This reveals coherent patterns by which specific MEs regulate tissue-specific gene expression, including creating or attenuating enhancers and recruiting post-transcriptional regulators. We pinpoint MEVs as genetic causes of disease risk, including a LINE-1 insertion linked to keloid and other diseases of fibroblast inflammation, by introducing MEVs into the genome-wide association study (GWAS) framework. In addition to nominating previously-hidden MEVs as causes of human diseases, this work highlights MEs as accelerators of human population divergence and begins to decipher the semantics of MEs.

genetics↗

bioRxiv2022

Dynamic interaction of bacterial cellulose synthase subunit D (BcsD) in type I bacterial cellulose synthase

Cellulose is a promising biological material for supporting sustainable human life. This natural polymer is synthesized by cellulose synthase, a protein complex in the cell membrane. Cellulose synthase in bacteria is a hetero-subunit complex, and its subunit organization varies widely depending on the species. In the type I bacterial cellulose synthase complex, the BcsD (bacterial cellulose synthase D) protein is believed to play an important role in producing cellulose with long slender fiber morphology and high crystallinity, given the phenotype of the bcsD-deficient mutant and the specific existence of the type I operon in bacterial species synthesizing crystalline cellulose microfibrils such as Acetobacter. In this study, we successfully established a heterogeneously expressed Bcs protein in Escherichia coli as an experimental model and conducted biochemical studies for the BcsD protein and the other three major subunits of bacterial cellulose synthase, BcsA, BcsB, and BcsC. It has been shown that the BcsD protein interacts with the functionally required minimal subunits of the BcsAB complex, as well as the BcsC protein. Furthermore, it was shown that BcsD interacts with the BcsAB complex in two modes: direct protein-protein interactions and indirect interactions through the product cellulose. The former and latter modes represent the basal and active states of the type I bacterial cellulose synthase, respectively. This dynamic behavior of the BcsD protein is important for the type I bacterial cellulose synthase complex to regulate the crystallization process of cellulose.

biochemistry↗

bioRxiv2022

Plastomes of Garcinia mangostana L. and comparative analysis with other Garcinia species

The two varieties of mangosteen (Garcinia mangostana L.) cultivated in Malaysia are known as Manggis and Mesta. The latter is preferred for its flavor, texture, and seedlessness. Here, we report a complete plastome (156,580 bp) of the Mesta variety which was obtained through a hybrid assembly approach using PacBio and Illumina sequencing reads. It encompasses a large single-copy (LSC) region (85,383 bp) and a small single-copy (SSC) region (17,137 bp) that are separated by 27,230 bp of inverted repeat (IR) regions at both ends. The plastome comprises 128 genes, namely 83 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The plastome of the Manggis variety (156,582 bp) obtained from reference-guided assembly of Illumina reads was found to be nearly identical to Mesta except for two indels and the presence of a single nucleotide polymorphism (SNP). Comparative analyses with other publicly available Garcinia plastomes, including G. anomala, G. gummi-gutta, G. mangostana var. Thailand, G. oblongifolia, G. paucinervis, and G. pedunculata found that the gene content, gene order, and gene orientation were highly conserved among the Garcinia species. Phylogenomic analysis divided the six Garcinia plastomes into three groups with the Mesta and Manggis varieties clustered closer to G. anomala, G. gummi-gutta, and G. oblongifolia, while the Thailand variety clustered with G. pedunculata in another group. These findings serve as future references for the identification of species or varieties and facilitate phylogenomic analysis of lineages from the Garcinia genus to better understand their evolutionary history.

plant biology↗

bioRxiv2022

Mitochondrial Genome of Garcinia mangostana L. variety Mesta

Fruits of Garcinia mangostana L. (mangosteen) are rich in nutrients with xanthones found in the pericarp having great pharmaceutical potential. Mangosteen variety Mesta is only found in Malaysia, which tastes sweeter than the common Manggis variety in Southeast Asia. In this study, we report the complete mitogenome of G. mangostana L. variety Mesta with a total sequence length of 371,235 bp of which 1.7% could be of plastid origin. The overall GC content of the mitogenome is 43.8%, comprising 29 protein-coding genes, 3 rRNA genes, and 21 tRNA genes. Repeat and tandem repeat sequences accounted for 5.8% and 0.15% of the Mesta mitogenome, respectively. There are 333 predicted RNA-editing sites in Mesta mitogenome. These include the RNA-editing events that generated the start codon of nad1 gene and the stop codon of ccmFC gene. Phylogenomic analysis with a maximum likelihood method showed that the mitogenome of mangosteen variety Mesta was grouped under Malpighiales order. This is the first complete mitogenome from the Garcinia genus for future evolutionary studies.

plant biology↗

bioRxiv2022

Prediction of sex-determination mechanisms in avian primordial germ cells using RNA-seq analysis

Avian sex is determined by various factors, such as the dosage of DMRT1 and cell-autonomous mechanisms. While the sex-determination mechanism in gonads is well analyzed, the mechanism in germ cells remains unclear. In this study, we explored the gene expression profiles of male and female primordial germ cells (PGCs) during embryogenesis in chickens to predict the mechanism of sex-determination. Male and female PGCs were isolated from blood and gonads with a purity > 96% using flow cytometry and analyzed using RNA-seq. Prior to settlement in the gonads, female circulating PGCs (cPGCs) obtained from blood displayed sex-biased expression. Gonadal PGCs (gPGCs) also displayed sex-biased expression, and the number of female-biased genes detected was higher than male-biased genes. The female-biased genes in gPGCs were enriched in some metabolic processes. To reveal the mechanisms underlying the transcriptional regulation of female-biased genes in gPGCs, we performed stimulation tests. Stimulation with retinoic acid against cultured gPGCs derived from male embryos resulted in the upregulation of several female-biased genes. Overall, our results suggest that sex determination of avian PGCs possess aspects of both cell-autonomous and somatic cell regulation. Moreover, it appears that sex determination occurs earlier in females than in males.

developmental biology↗