Biology subjects

Nakamura, K.

Publications and source records attributed to Nakamura, K..

At least 73 records · Page 4Linked to original sources

bioRxiv2020

Prophages integrating into prophages: a mechanism to accumulate type III secretion effector genes and duplicate Shiga toxin-encoding prophages in Escherichia coli

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 PPs (up to 21 PPs), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present prophages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the prophages integrated into prophages are Stx-encoding prophages and have induced the duplication of Stx-encoding phages in a single cell. The identified att sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different att sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These "prophages-in-prophages" represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx-encoding prophages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species. Author summaryMultiple prophages are often integrated in a bacterial host chromosome and some are present at a single locus in tandem. The most striking examples are Shiga toxin (Stx)-producing and enteropathogenic Escherichia coli (STEC and EPEC) strains, which usually contain more than 10 prophages (up to 21). Many of them encode a cluster of type III secretion system (T3SS) effector genes, contributing the acquisition of a large number of effectors (>30) by STEC and EPEC. Here, we describe prophages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Two or three different attachment sequences derived from host chromosomes are present in some prophages, generating prophage clusters in various complex configurations. Of note, some of such prophages-in-prophages are Stx-encoding prophages and have induced the duplication of Stx-encoding prophages. Thus, these "prophages-in-prophages" represent an important inter-phage interaction as they can promote not only the accumulation of T3SS effectors in STEC and EPEC but also the duplication of Stx-encoding prophages and the conversion of EPEC to STEC.

microbiology↗

bioRxiv2020

Defective Repression of OLE3::LUC 1 (DROL1) is specifically required for the splicing of AT-AC-type introns in Arabidopsis

An Arabidopsis mutant named defective repression of OLE3::LUC 1 (drol1) was originally isolated as a mutant with defects in the repression of OLEOSIN3 (OLE3) after seed germination. In this study, we show that DROL1 is an Arabidopsis homolog of yeast DIB1, a subunit of U5 snRNP in the spliceosome. It is also part of a new subfamily that is specific to a certain class of eukaryotes. Comprehensive analysis of the intron splicing using RNA-Seq analysis of the drol1 mutants revealed that most of the minor introns with AT-AC dinucleotide termini had reduced levels of splicing. Only two nucleotide substitutions from AT-AC to GT-AG enabled AT-AC-type introns to be spliced in drol1 mutants. Forty-eight genes, including those having important roles in abiotic stress responses and cell proliferation, exhibited reduced splicing of AT-AC-type introns in the drol1 mutants. Additionally, drol1 mutant seedlings showed retarded growth, similar to that caused by the activation of abscisic acid signaling, possibly as a result of reduced AT-AC-type intron splicing in the endosomal Na+/H+ antiporters and plant-specific histone deacetylases. These results indicate that DROL1 is specifically involved in the splicing of minor introns with AT-AC termini, and that this plays an important role in plant growth and development. Significance statementDefective Repression of OLE3::LUC 1 (DROL1) is a homolog of yeast DIB1, which is a subunit of the U5 snRNP in the spliceosome, but is also part of a new subfamily that is specific to a certain class of eukaryotes. Using RNA-Seq we show that introns with AT-AC dinucleotide termini were specifically retained in the transcriptome of drol1 mutants and that their splicing plays an important role in plant growth and development.

molecular biology↗

bioRxiv2020

Mechanosensitive Ion Channel Piezo1 Regulates Myocyte Fusion during Skeletal Myogenesis

Mechanical stimuli such as stretch and resistance training are essential to regulate growth and function of skeletal muscle. However, the molecular mechanisms involved in sensing mechanical stress during muscle formation remain unclear. Here, we investigate the role of the mechano-sensitive ion channel Piezo1 during myogenic progression. Direct manipulation of Piezo1 in muscle stem cells alters their myogenic progression. Indeed, Piezo1 knockdown suppresses myoblast fusion leading to smaller myotubes. Such event is accompanied by significant downregulation of the fusogenic protein Myomaker. In parallel, while Piezo1 knockdown also lowers Ca2+ influx in response to stretch, Piezo1 activation increases Ca2+ influx in response to stretch and enhances myoblasts fusion. We believe these findings may help understand molecular defects present in some muscle diseases. Altogether our study shows that Piezo1 is essential for terminal muscle differentiation acting on myoblast fusion, suggesting that Piezo1 deregulation may have implications in muscle aging and degenerative diseases including muscular dystrophies.

molecular biology↗

bioRxiv2020

Extracellular DJ-1 induces sterile inflammation in the ischemic brain

Inflammation is implicated in the onset and progression of various diseases, including cerebral pathologies. Here we report that DJ-1, which plays a role within cells as an antioxidant protein, functions as a damage-associated molecular pattern (DAMP), and triggers inflammation if released from dead cells into the extracellular space. We first found that recombinant DJ-1 protein induces the production of various inflammatory cytokines in bone marrow-derived macrophages (BMMs). We further identified a unique peptide sequence in the G and H helices of DJ-1 that activates Toll-like receptor 2 (TLR2) and TLR4. In the ischemic brain, DJ-1 is released into the extracellular space from necrotic neurons within 24 hours after stroke onset and makes direct contact with the surfaces of infiltrating myeloid cells. Administration of an antibody against DJ-1 suppresses the expression of inflammatory cytokines in infiltrating immune cells and attenuates ischemic neuronal damage. Our results demonstrate a previously unknown function of DJ-1 as a DAMP and suggest that extracellular DJ-1 could be a therapeutic target to prevent inflammation in tissue injuries and neurodegenerative diseases. Significance statementDJ-1 has been thoroughly investigated as a cytoprotective antioxidant protein in neurons. However, here we demonstrate that extracellularly released DJ-1 triggers neurotoxic inflammation after ischemic stroke. Intracellular DJ-1 increases in response to oxidative stress in ischemic neurons, but if ischemic stresses result in necrotic cell death, DJ-1 is released extracellularly. Released DJ-1 interacts with TLR2 and TLR4 on the surface of infiltrating myeloid cells and triggers post-ischemic inflammation, leading to the exacerbated pathologies of ischemic stroke. Thus, extracellular DJ-1 is a previously unknown inflammatogenic DAMP, and may be a putative target for therapeutic intervention to prevent progression of inflammatory and neurodegenerative diseases.

immunology↗

bioRxiv2020

SARS-CoV-2 infection of human iPSC-derived cardiac cells predicts novel cytopathic features in hearts of COVID-19 patients

Although COVID-19 causes cardiac dysfunction in up to 25% of patients, its pathogenesis remains unclear. Exposure of human iPSC-derived heart cells to SARS-CoV-2 revealed productive infection and robust transcriptomic and morphological signatures of damage, particularly in cardiomyocytes. Transcriptomic disruption of structural proteins corroborated adverse morphologic features, which included a distinct pattern of myofibrillar fragmentation and numerous iPSC-cardiomyocytes lacking nuclear DNA. Human autopsy specimens from COVID-19 patients displayed similar sarcomeric disruption, as well as cardiomyocytes without DNA staining. These striking cytopathic features provide new insights into SARS-CoV-2 induced cardiac damage, offer a platform for discovery of potential therapeutics, and raise serious concerns about the long-term consequences of COVID-19.

cell biology↗

bioRxiv2020

Resolving the data asynchronicity in high-speed atomic force microscopy measurement via the Kalman Smoother

High-speed atomic force microscopy (HS-AFM) is a scanning probe microscopy that can capture structural dynamics of biomolecules in real time at single molecule level near physiological condition. Albeit much improvement of the instruments, while scanning one frame of HS-AFM movies, biomolecules often change their conformations largely. Thus, the obtained frame images can be hampered by the time-difference, the asynchronicity, in the data acquisition. Here, to resolve this data asynchronicity in the HS-AFM movie, we developed Kalman filter and smoother methods, some of the sequential Bayesian filtering approaches. The Kalman filter/smoother methods use alternative steps of a short time-propagation by a linear dynamical system and a correction by the likelihood of AFM data acquired pixel by pixel. We first tested the method using a toy model of a diffusing cone, showing that the Kalman smoother method outperforms to reproduce the ground-truth movie, compared to that mimics the raw AFM movie, and the Kalman filter result. We then applied the Kalman smoother to a synthetic movie for conformational change dynamics of a motor protein, i.e., dynein, confirming the superiority of the Kalman smoother. Finally, we applied the Kalman smoother to two real HS-AFM movies, FlhAc and centralspindlin, reducing distortion and noise in the AFM movies. The method is general and can be applied to any HS-AFM movies.

biophysics↗

bioRxiv2020

Integrating genotypes and phenotypes improves long-term forecasts of seasonal influenza A/H3N2 evolution

Seasonal influenza virus A/H3N2 is a major cause of death globally. Vaccination remains the most effective preventative. Rapid mutation of hemagglutinin allows viruses to escape adaptive immunity. This antigenic drift necessitates regular vaccine updates. Effective vaccine strains need to represent H3N2 populations circulating one year after strain selection. Experts select strains based on experimental measurements of antigenic drift and predictions made by models from hemagglutinin sequences. We developed a novel influenza forecasting framework that integrates phenotypic measures of antigenic drift and functional constraint with previously published sequence-only fitness estimates. Forecasts informed by phenotypic measures of antigenic drift consistently outperformed previous sequence-only estimates, while sequence-only estimates of functional constraint surpassed more comprehensive experimentally-informed estimates. Importantly, the best models integrated estimates of both functional constraint and either antigenic drift phenotypes or recent population growth.

evolutionary biology↗

bioRxiv2020

Mitochondrial fission is a critical modulator of mutant APP-induced neural toxicity

Alterations in mitochondrial fission may contribute to the pathophysiology of several neurodegenerative diseases, including Alzheimers disease (AD). However, we understand very little about the normal functions of fission, or how fission disruption may interact with AD-associated proteins to modulate pathogenesis. Here we show that loss of the central mitochondrial fission protein dynamin-related 1 (Drp1) in CA1 and other forebrain neurons markedly worsens the learning and memory of mice expressing mutant human amyloid-precursor protein (hAPP) in neurons. In cultured neurons, Drp1KO and hAPP converge to produce mitochondrial Ca2+ (mitoCa2+) overload, despite decreasing mitochondria-associated ER membranes (MAMs) and cytosolic Ca2+. This mitoCa2+ overload occurs independently of ATP levels. These findings reveal a potential mechanism by which mitochondrial fission protects against hAPP-driven pathology.

neuroscience↗

bioRxiv2020

MAPK-induced miR-29 restrains melanoma progression by targeting MAFG

The tumor suppressive miR-29 family of microRNAs is encoded by two clusters, miR-29b1[~]a and miR-29b2[~]c, and is regulated by several oncogenic and tumor suppressive stimuli. Here we investigated whether oncogenic MAPK hyperactivation regulates miR-29 abundance and how this signaling axis impacts melanoma development. Using mouse embryonic fibroblasts and human melanocytes, we found that oncogenic MAPK signaling stimulates p53-independent and p53-dependent transcription of pri-miR-29b1[~]a and pri-miR-29b2[~]c, respectively. Expression analyses revealed that while pri-miR-29a[~]bl remains elevated, pri-miR-29b2[~]c levels decrease during melanoma progression. Using a rapid mouse modeling platform, we showed that inactivation of miR-29 in vivo accelerates melanoma development and decreases overall survival. We identified the transcription factor MAFG as a bona fide miR-29 target that has oncogenic potential in melanocytes and is required for growth of melanoma cells. Our findings suggest that MAPK-induced miR-29 contributes to a tumor suppressive barrier by targeting MAFG, which is overcome by attenuation of miR-29b2[~]c expression.

cancer biology↗

bioRxiv2019

Handling and Packaging of Medical Bags at the Acute Disaster Site Under High Temperature Conditions

The handling of medical bags in high temperature disaster sites has not been fully evaluated. In this study, medical bags were assessed in the closed, semi-opened, and opened state, with and without heat insulating material (HIM) and a coolant. The bags were heated for 300 minutes at 35 or 40{degrees}C, and the internal temperature was measured. closed and semi-opened bags were able to limit temperature increases in the central part of the bag at both 35 and 40{degrees}C to a greater extent than opened bags. When coolant and HIM were used in closed and semi-opened bags, the internal temperature was significantly lower than that seen in the opened state at 40{degrees}C (P < 0.01 and P < 0.001, respectively). In conclusion, in high temperature disaster sites, medical bags should be maintained in a semi-opened or closed state using a HIM and coolant.

scientific communication and education↗

bioRxiv2019

Merger of Betula tatewakiana (Betulaceae) from northern Japan to northeast Asian B. ovalifolia based on ploidy level

It has been controversial whether Betula tatewakiana, a dwarf birch distributed in Hokkaido of northern Japan, is an endemic species or a synonym of B. ovalifolia broadly distributed in northeast Asia. The endemic hypothesis is based on the idea that B. tatewakiana is diploid while B. ovalifolia is tetraploid and that they are separated based on the ploidy level; however no chromosome data have actually been published before. Resolving the taxonomic problem is crucial also in judging the conservation priority of B. tatewakiana in a global perspective. Our chromosome observation revealed that B. tatewakiana is tetraploid as well as B. ovalifolia. Collaterally, we conducted morphological observation and clarified that B. tatewakiana is morphologically identical to B. ovalifolia in white hairs and dense resinous glands respectively on adaxial and abaxial leaf surfaces, based on which they are different from closely related species in the same section Fruticosae. We concluded that the hypothesis that B. tatewakiana is a Hokkaido endemic based on the ploidy level is not supported and that B. tatewakiana should be merged with B. ovalifolia.

plant biology↗

bioRxiv2019

Serpentine mountain uplift in northern Japan triggered the divergence of a narrow endemic from a widespread (sub)arctic Asia-Alaska species complex of Lagotis (Plantaginaceae)

In the circumboreal region, plants often have extremely-wide species ranges. Lagotis minor-glauca species complex widespread from (sub)arctic Asia to Alaska, however, have two allied narrow endemics in northern Japan: a serpentine plant L. takedana endemic to the Yubari Mountains (Mt. Yubari) and a non-serpentine plant L. yesoensis endemic to the Taisetsu Mountains (Mt. Taisetsu). Elucidating their origins sheds light on drivers for secondary-speciation of widespread circumboreal plants. To infer phylogenetic distinctiveness of two narrow endemics with those related taxa, which contained 25 out of all the 29 species of the genus, chloroplast DNA (cpDNA), nuclear ribosomal (nrITS), two low copy nuclear gene (LCN) markers and genome-wide single-nucleotide polymorphism genotyping (MIG-seq) were used. In the result of cpDNA analyses, the Lagotis minor-glauca species complex formed a clade. Within the clade, L. yesoensis and a portion of L. glauca samples formed a subclade. However, monophyly of each of the four species was not supported. In the results of nrITS and two LCN analyses, L. takedana was monophyletic, while monophyly was not recovered for each L. yesoensis, L. glauca, and L. minor. Based on a Bayesian dating analysis using nrITS data, the age of the most recent common ancestor of L. takedana was Ma (95% confidence interval: 0.05-1.75 Ma). Possible scenario is that an ancestral linage being adapted to serpentine soils migrated into the alpine habitat of Mt. Yubari, that was formed with mountain uplift by the early Pleistocene, and subsequently reproductively isolated from non-serpentine populations and speciated. The contrasting result of L. yesoensis, that was phylogenetically indistinct, is possibly explained by incorrect taxonomy, or alternatively, shallow history and incomplete lineage sorting. In Mt. Taisetsu, massive volcanic eruptions had occurred the Early Pleistocene and even after the last glacial period, suggesting that alpine plants have not migrated into and established populations in Mt. Taisetsu until very recently. To fully resolve the phylogeny of the three species L. yesoensis, L. glauca, and L. minor, further analyses using high resolution molecular markers are needed. The present study illustrated that two narrow endemics in northern Japan diverged from the widespread species include phylogenetically distinctive and indistinctive species, owing to historical orogeny and ecological factors.

ecology↗

bioRxiv2019

Two-way migration of Lychnis wilfordii caused by the circular landform of Japan-Korea-northeast China-Russian Far East region and its suggestion for conservation in northeast Asia

In northeast Asia, substantial portion of the floras, including endangered species, are shared among its component countries in the continental, peninsula, and island parts largely through Quaternary migration. To effectively conserve nationally endangered plants in Northeast Asia, transnational conservation studies are vitally needed. Lychnis wilfordii (Caryophyllaceae) has disjunct distribution in Russian Far East (Primorsky Krai), northeast China (Jilin), Korea (Gangwon-do) and Japan (Hokkaido, Aomori, Nagano), surrounding the sea, and this is designated as an endangered species in Japan and Korea. Population genetic and molecular dating analyses were conducted 1) to elucidate geographic genetic structure covering the species range, 2) to test possible scenarios of migration, and 3) to develop logical plans for effective conservation. Population genetic analyses indicated the continent and peninsula parts (north and south Primorsky Krai, Jilin, and Gangwon-do) had higher genetic diversity compared to those in the Japanese Archipelago (Hokkaido and Nagano). Five genetically distinct groups were recognized, namely, Nagano, Gangwon-do, Jilin, north and south Primorsky Krai plus Aomori, and Hokkaido. Genetic distance between Hokkaido and Nagano was larger than between Hokkaido and north Primorsky Krai, and between Nagano and Gangwon-do, crossing national borders and the natural barrier of the sea. Considering these results, L. wilfordii likely migrated from the Asian continent to the Japanese Archipelago using two routes: north route from Russian Far East to Hokkaido and Aomori, and south route from the Korean Peninsula to Nagano. Based on molecular dating, migration from the continent to the islands likely occurred from the middle Pleistocene to the Holocene. For effective conservation of L. wilfordii, Hokkaido and Nagano populations should be distinguished as different evolutionary significant units, although these two regions belong to the same country, because Hokkaido and Nagano populations are at the different ends of the two migratory routes based on the migration scenario.

evolutionary biology↗

bioRxiv2019

Deglycosylation of isoflavone C-glucoside puerarin by combination of two recombinant bacterial enzymes and 3-oxo-glucose.

C-Glucosides are resistant to glycoside hydrolase activity because the anomeric carbon of glucose is directly connected to aglycone via carbon-carbon bonding. A human intestinal bacterium strain PUE related to Dorea species can metabolize the isoflavone C-glucoside puerarin (daidzein 8-C-glucoside) to daidzein and glucose by more than three bacterial enzymes which have not been well-characterized. We previously reported that 3\"-oxo-puerarin is an essential reaction intermediate in enzymatic puerarin degradation and characterized a bacterial enzyme of DgpB-C complex which cleaved the C-glycosidic bond in 3\"-oxo-puerarin. However, the exact enzyme catalyzing the oxidation of C-3\" hydroxyl in puerarin has not been identified, and the other metabolite corresponding to the precursor of D-glucose, derived from the sugar moiety in 3\"-oxo-puerarin in the cleaving reaction catalyzed by the DgpB-C complex, remains unknown.\n\nIn this study, we demonstrated that recombinant DgpA, a Gfo/Idh/MocA family oxidoreductase, catalyzed puerarin oxidation in the presence of 3-oxo-glucose as the hydride accepter. In addition, enzymatic C-deglycosylation of puerarin was achieved by a combination of recombinant DgpA, DgpB-C complex, and 3-oxo-glucose. Furthermore, the metabolite derived from the sugar moiety in 3\"-oxo-puerarin cleaving reaction catalyzed by DgpB-C complex was characterized as 1,5-anhydro-D-erythro -hex-1-en-3-ulose, suggesting that the C-glycosidic linkage is cleaved through a {beta}-elimination like mechanism.\n\nImportanceOne important role of the gut microbiota is to metabolize dietary nutrients and supplements such as flavonoid glycosides. Ingested glycosides are metabolized by intestinal bacteria to more absorbable aglycones and further degradation products which show beneficial effects in humans. Although numerous glycoside hydrolases that catalyze O-deglycosylation have been reported, enzymes responsible for C-deglycosylation are still limited. In this study, we characterized enzymes involved in C-deglycosylation of puerarin from a human intestinal bacterium PUE. To our knowledge, this is the first report of the expression, purification and characterization of an oxidoreductase involved in C-glucoside degradation. This study provides new insights for the elucidation of mechanisms of enzymatic C-deglycosylation.

biochemistry↗

bioRxiv2019

Effective physical properties of cell-cell interaction explain basic structural units of three-dimensional morphogenesis

Mechanical forces are critical for the emergence of diverse three-dimensional morphologies of multicellular systems. However, it remains unclear what kind of mechanical parameters at cellular level substantially contribute to tissue morphologies. This is largely due to technical limitations of live measurements of cellular forces. Here we developed a framework for inferring and modeling mechanical forces of cell-cell interactions. First, by analogy to coarse-grained models in molecular and colloidal sciences, we approximated cells as particles, where mean forces (i.e. effective forces) of pairwise cell-cell interactions are considered. Then, the forces were statistically inferred by fitting the mathematical model to cell tracking data. This method was validated by using synthetic cell tracking data resembling various in vivo situations. Application of our method to the cells in the early embryos of mice and the nematode Caenorhabditis elegans revealed that cell-cell interaction forces can be written as a pairwise potential energy in a manner dependent on cell-cell distances. Importantly, the profiles of the pairwise potentials were quantitatively different among species and embryonic stages, and the quantitative differences correctly described the differences of their morphological features such as spherical vs. distorted cell aggregates, and tightly vs. non-tightly assembled aggregates. We conclude that the effective pairwise potential of cell-cell interactions is a live measurable parameter whose quantitative differences can be a parameter describing three-dimensional tissue morphologies. Author summaryEmergence of diverse three-dimensional morphologies of multicellular organisms is one of the most intriguing phenomena in nature. Due to the complex situations in living systems (e.g. a lot of genes are involved in morphogenesis.), a model for describing the emergent properties of multicellular systems has not been established. To approach this issue, approximation of the complex situations to limited numbers of parameters is required. Here, we searched for mechanical parameters for describing morphologies. We developed a statistical method for inferring mechanical potential energy of cell-cell interactions in three-dimensional tissues; the mechanical potential is an approximation of various mechanical components such as cell-cell adhesive forces, cell surface tensions, etc. Then, we showed that the quantitative differences in the potential is sufficient to reproduce basic three-dimensional morphologies observed during the mouse and C. elegans early embryogenesis, revealing a direct link between cellular level mechanical parameters and three-dimensional morphologies. Our framework provides a noninvasive tool for measuring spatiotemporal cellular forces, which would be useful for studying morphogenesis of larger tissues including organs and their regenerative therapy.

biophysics↗

bioRxiv2019

Fluoxetine-induced dematuration of hippocampal neurons and adult cortical neurogenesis in the common marmoset

The selective serotonin reuptake inhibitor fluoxetine (FLX) is widely used to treat depression and anxiety disorders. Chronic FLX treatment reportedly induces cellular responses in the brain, including increased adult hippocampal and cortical neurogenesis and reversal of neuron maturation in the hippocampus, amygdala, and cortex. However, because most previous studies have used rodent models, it remains unclear whether these FLX-induced changes occur in the primate brain. To evaluate the effects of FLX in the primate brain, we used immunohistological methods to assess neurogenesis and the expression of neuronal maturity markers following chronic FLX treatment (3 mg/kg/day for 4 weeks) in adult marmosets (n = 3 per group). We found increased expression of doublecortin and calretinin, markers of immature neurons, in the hippocampal dentate gyrus of FLX-treated marmosets. Further, FLX treatment reduced parvalbumin expression and the number of neurons with perineuronal nets, which indicate mature fast-spiking interneurons, in the hippocampus, but not in the amygdala or cerebral cortex. We also found that FLX treatment increased the generation of cortical interneurons; however, significant up-regulation of adult hippocampal neurogenesis was not observed in FLX-treated marmosets. These results suggest that dematuration of hippocampal neurons and increased cortical neurogenesis may play roles in FLX-induced effects and/or side effects. Our results are consistent with those of previous studies showing hippocampal dematuration and increased cortical neurogenesis in FLX-treated rodents. In contrast, FLX did not affect hippocampal neurogenesis or dematuration of interneurons in the amygdala and cerebral cortex.

neuroscience↗

bioRxiv2019

A versatile ES cell-based melanoma mouse modeling platform

The cumbersome and time-consuming process of generating new mouse strains and multi-allelic experimental animals often hinders the use of genetically engineered mouse models (GEMM) in cancer research. Here, we describe the development and validation of an embryonic stem cell (ESC)-GEMM platform for rapid modeling of melanoma in mice. Our platform incorporates twelve clinically relevant genotypes composed of combinations of four driver alleles (LSL-BrafV600E, LSL-NrasQ61R, PtenFlox, Cdkn2aFlox) and regulatory alleles to spatiotemporally control the perturbation of genes-of-interest. Our ESCs produce high contribution chimeras, which recapitulate the melanoma phenotypes of conventionally bred mice. Using our ESC-GEMM platform to modulate Pten expression in melanocytes in vivo, we highlight the utility and advantages of gene depletion by CRISPR-Cas9, RNAi, or conditional knockout for melanoma modeling. Moreover, we use complementary genetic methods to demonstrate the impact of Pten restoration on the prevention and maintenance of Pten-deficient melanomas. Finally, we show that chimera-derived melanoma cell lines retain regulatory allele competency and are a powerful resource to complement ESC-GEMM chimera experiments in vitro and in syngeneic grafts in vivo. Thus, when combined with sophisticated genetic tools, our ESC-GEMM platform enables rapid, high-throughput, and versatile studies aimed at addressing outstanding questions in melanoma biology.

cancer biology↗

bioRxiv2019

cyAbrB transcriptional regulators as safety devices to inhibit heterocyst differentiation in Anabaena

Cyanobacteria are monophyletic organisms that perform oxygenic photosynthesis. While they exhibit great diversity, they have a common set of genes. However, the essentiality of them for viability has hampered the elucidation of their functions. One example of the genes is cyabrB1 encoding a transcriptional regulator. In the present study, we investigated the function of cyabrB1 in heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 through CRISPR interference, a method we recently utilized for the photosynthetic production of a useful chemical in the strain. Conditional knockdown of cyabrB1 in the presence of nitrate resulted in formation of heterocysts. Two genes, hetP and hepA, which are required for heterocyst formation, were up-regulated by cyabrB1 knockdown in the presence of combined nitrogen sources. The genes are known to be induced by HetR, a master regulator of heterocyst formation. hetR was not induced by cyabrB1 knockdown. hetP and hepA were repressed by direct binding of cyAbrB1 to their promoter regions in a HetR-independent manner. In addition, the over-expression of cyabrB1 abolished heterocyst formation upon nitrogen depletion. Also, knockout of cyabrB2, a paralogue gene of cyabrB1, in addition to cyabrB1 knockdown, enhanced heterocyst formation in the presence of nitrate, suggesting functional redundancy of cyAbrB proteins. We propose that a balance between amounts of HetR and cyAbrB1 is a key factor influencing heterocyst differentiation during nitrogen step-down. cyAbrB proteins are essential safety devices inhibiting heterocyst differentiation. ImportanceSpore formation in Bacillus subtilis and Streptomyces represents non-terminal differentiation and has been extensively studied as models of prokaryotic cell differentiation. In the two organisms, many cells differentiate simultaneously, and the differentiation is governed by a network in which one regulator stands at the top. Differentiation of heterocysts in Anabaena sp. PCC 7120 has also been extensively studied. The differentiation is unique because it is terminal and only 5-10% vegetative cells differentiate into heterocysts. In the present study, we identified cyAbrB1 as a repressor of two genes that are essential for heterocyst formation, hetP and hepA, independent of HetR, which is a master activator for heterocyst differentiation. The finding is reasonable for unique cell differentiation of Anabaena because cyAbrB1 could suppress heterocyst differentiation tightly in vegetative cells, while only cells in which HetR is over-expressed could differentiate into heterocysts.

microbiology↗