Biology subjects

Murakami, M.

Publications and source records attributed to Murakami, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Astatine-211 injury induces an immunostimulatory melanoma-cell state and promotes systemic antitumor immunity

Targeted alpha therapy is generally evaluated by absorbed dose and direct tumor-cell killing, but whether alpha-particle-injured tumor cells can induce systemic antitumor immunity remains poorly defined. We investigated the immunogenic consequences of astatine-211 (211At) injury in a prophylactic melanoma vaccination-challenge model. B16F10 cells were exposed ex vivo to 211At, etoposide, or ultraviolet irradiation under conditions that reduced viable-cell counts at 24 hours to approximately 40% of untreated controls. Injured cells were administered subcutaneously to syngeneic C57BL/6J mice before intravenous challenge with viable B16F10 cells. Vaccination with cells exposed to 1 MBq/mL 211At suppressed pulmonary metastasis, reduced regulatory T-cell proportions, and elicited tumor-reactive interferon-{gamma} responses and antibodies. Transcriptomic profiling revealed enrichment of type I interferon, inflammatory, and chemokine-related signatures, together with suppression of cell-cycle, translation, and metabolic programs. At the cellular level, 211At injury was accompanied by {gamma}H2AX accumulation, G2/M arrest, increased reactive oxygen species and cytosolic double-stranded DNA, and senescence-like changes. These features arose without consistent increases in classical damage-associated molecular patterns or prominent induction of senescence-associated secretory phenotype factors at 1 MBq/mL. In vivo pharmacologic inhibition of stimulator of interferon genes (STING) signaling attenuated the antimetastatic effect of vaccination. Together, these findings demonstrate that 211At injury can generate an immunostimulatory tumor-cell state characterized by DNA damage and altered inflammatory signaling. STING contributes to the resulting immune protection, highlighting immunogenic tumor-cell conversion as a consideration in the design of targeted alpha therapies.

immunology↗

bioRxiv2026

High-resolution spatial profiling identifies disease-specific molecular architecture in palmoplantar pustulosis

Palmoplantar pustulosis (PPP) and dyshidrotic eczema (DE) are chronic vesiculopustular dermatoses with overlapping clinical presentations but distinct underlying biology. Although comparative transcriptomic and proteomic analyses between PPP and DE have been reported, they remain limited in number and scope, with no comprehensive understanding of their distinct molecular signatures. Moreover, their molecular mechanisms remain unclear, and currently available therapeutic options are limited. To clarify disease-specific epidermal programs underlying vesicle formation, we conducted Visium HD spatial transcriptomic analysis of FFPE lesional skin samples obtained from patients with PPP and DE, followed by immunohistochemical validation against normal palmoplantar skin controls. Spatial clustering identified a keratinocyte subpopulation adjacent to vesicles that exhibited distinct transcriptional programs in the two diseases. In PPP, vesicle-associated keratinocytes demonstrated marked downregulation of aquaporin-3 (AQP3) and E-cadherin, together with strong, spatially localized activation of JAK-STAT3 signaling. Conversely, DE exhibited diffuse AQP3 expression and more homogeneous activation of JAK-STAT3 signaling throughout the epidermis. These results indicate that, although PPP and DE share inflammatory pathways, they differ substantially in their spatial molecular architecture. Reduced AQP3 expression and localized STAT3 activation may contribute to vesicle formation in PPP, supporting our previous hypothesis that implicates intraepidermal sweat leakage as a pathogenic mechanism in PPP. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/723901v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@72a341org.highwire.dtl.DTLVardef@3c0beaorg.highwire.dtl.DTLVardef@325098org.highwire.dtl.DTLVardef@1493ebf_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

A macrocyclic peptide-based fusion inhibitor targeting SARS-CoV-2 Spike S2 subunit

Continuous emergence of SARS-CoV-2 variants carrying mutations in Spike presents a significant challenge for durable antiviral agents. Here we screen for random 13-amino acid non-mimetic macrocyclic peptides that bind to Spike and identify PA-001 that inhibits SARS-CoV-2 infection with high potency at 0.23-2.9 nM as 50% inhibitory concentration (IC50). PA-001 bound to Spike S2 subunit and inhibited the membrane fusion during virus entry. Through drug-resistant selection, we revealed that PA-001 targeted the fusion peptide proximal region (FPPR) in S2, which has not been recognized as a drug target to date. Consistent with its highly conserved amino acid sequences beyond strains, PA-001 exhibited broad antiviral activity against all tested SARS-CoV-2 variants, in contrast to clinically-approved S1-targeting antibodies that lost activity to Omicron variants. PA-001 suppressed SARS-CoV-2 propagation and disease progression in mouse- and hamster-infection models, both by administration prophylactically and therapeutically. Combination therapy with remdesivir further enhanced antiviral profiles. In clinical phase-I trial, PA-001 was well-tolerated and showed high systemic exposure, with 4,300-10,300-fold concentration of IC50 as maximum plasma concentration by single administration to healthy volunteers. These evidence propose FPPR as an unexpected antiviral drug target accessible by macrocyclic peptides and identify PA-001 as a potent anti-SARS-CoV-2 fusion inhibitor.

microbiology↗

bioRxiv2025

Human TIMELESS, a potential circadian clock regulator, plays an essential role in survival and reawakening of metastasis-initiating cells in bone

Bone metastasis is becoming increasingly common globally. In such cases, cancer cells disseminate from the primary site to bone, subsequently entering a dormant state. Following a certain incubation period, the cells reawaken and grow, forming a metastatic mass. These dormant cells are called metastasis-initiating cells (MICs), but their survival and reawakening are poorly understood. Here, we established an in vivo MIC-reawakening mouse model in bone. Microarray analysis demonstrated enrichment for mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid synthesis in isolated MICs. In addition, transcriptional regulator TIMELESS was identified as an independent poor prognostic factor by using clinical transcriptomic datasets, which was validated by immunostaining on 209 breast cancer cases. TIMELESS-deficient breast, prostate, and bladder cancer cell lines exhibited reduced viability and MIC reawakening in bone. Single-cell analysis on the epigenetic landscape of MICs revealed that motif activities of CLOCK and BMAL1/ARNTL were enhanced in a cluster with increased TIMELESS accessibility scores. As TIMELESS regulates stemness through the OXPHOS metabolic state, an inhibitor targeting the metabolic pathway, MP-A08, was identified to suppress MIC reawakening and growth in bone more efficiently than cisplatin. These results suggest that TIMELESS regulates survival and reawakening in MICs and MP-A08 could contribute to adjuvant therapeutic strategies.

cancer biology↗

bioRxiv2025

Lysyl-phosphatidylglycerol promotes cell-to-cell interaction and biofilm formation of Staphylococcus aureus as a biofilm matrix component

Biofilms formed by Staphylococcus aureus contribute significantly to persistent infections and antibiotic resistance, driven by the unique composition of their extracellular matrix. While previous studies highlighted extracellular DNA, proteins, and polysaccharides as key components, the role of phospholipids in biofilm architecture remains underexplored. This study identifies extracellular phospholipids, including lysyl-phosphatidylglycerol (Lys-PG), phosphatidylglycerol, and cardiolipin (CL), as critical structural elements in S. aureus biofilms. Bacterial phospholipase A1 (PLA1) that cleaves the acyl ester bond at the sn-1 position of phospholipids effectively dispersed pre-formed biofilms and prevented new biofilm formation by hydrolyzing extracellular phospholipids, without affecting bacterial growth or exhibiting cytotoxicity. Microscopy analyses confirmed that PLA1 disrupts membranous nanostructures, including extracellular vesicles and nanofilaments, integral to biofilm stability. Lipidomic analysis revealed an enrichment of Lys-PG and CL in the biofilm matrix. Lys-PG promotes bacterial aggregation by acting as a molecular glue, mediated through electrostatic and hydrophobic interactions. Deletion of the mprF gene, responsible for Lys-PG synthesis, significantly impaired biofilm formation, confirming its essential role. These findings reveal a "moonlighting" function of phospholipids in biofilm architecture, providing insights into biofilm biology and presenting PLA1 as a promising tool for biofilm control. SignificanceBiofilms, dense bacterial communities, pose significant challenges across medical, industrial, and daily life contexts. Understanding their formation mechanisms is crucial for developing effective strategies against them. We investigated biofilm matrix components in S. aureus biofilms, focusing on phospholipids. Our study reveals the presence and significance of Lys-PG in the biofilm matrix, acting as a crucial factor in biofilm formation and maintenance. Through biochemical, lipidomic, and genetic analyses, we demonstrate the role of Lys-PG in facilitating cell-to-cell contacts, contributing to the robustness and thickness of S. aureus biofilms. These findings shed light on the physiological function of extracellular phospholipids in bacterial biofilms and suggest targeting Lys-PG and its synthetic mechanism as a promising strategy for biofilm control.

microbiology↗

bioRxiv2024

Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

Lipogenesis-driven metabolic flux is crucial for differentiation of pathogenic Th17 cells. Although our previous CRISPR-based screening identified PLA2G12A as a key player in this process, it has remained obscure how this secreted phospholipase A2 isoform controls Th17 differentiation. Here we show that global, T cell-specific, or fibroblast-specific deletion of PLA2G12A prevents Th17 differentiation and associated diseases including psoriasis and arthritis. PLA2G12A acts on Th17-derived extracellular vesicles (EVs) to produce lysophospholipids including the ROR{gamma}t activator 1-oleoyl-lysophosphatidylethanolamine. These lysophospholipids are further converted by autotaxin to lysophosphatidic acid (LPA), which assists Th17 differentiation mainly via LPA2 receptor. Moreover, PLA2G12A promotes the secretion and uptake of EVs by Th17 cells and alters their cargo contents. Defective Th17 differentiation by PLA2G12A deficiency is rescued by supplementation with PLA2G12A-modified EVs. Importantly, a PLA2G12A-blocking antibody prevents Th17 differentiation and ameliorates psoriasis and arthritis models. Thus, targeting the PLA2G12A-EV-lysophospholipid axis may be useful for treatment of Th17-related diseases.

immunology↗

bioRxiv2024

Dynamic cognitive differences between internal and external attention are associated with depressive and anxiety disorders

The internal/external attention framework characterises attention focused on internal representations, such as emotions, versus external representations, such as perceptual stimuli. The inability to focus ones attention is considered a critical factor in psychiatric disorders. While these different attentional foci are likely generated by the dynamic interplay of multiple cognitive processes, previous studies have generally examined single cognitive dimensions. We developed a new method, cognitive dynamic similarity analysis (C-DSA), to clarify how cognitive processes differ between experimental conditions. In an MR scanner, participants performed a word-processing task in which they focused on either their own emotions or the number of letters associated with a stimulus. To extract cognitive dynamics at the single-trial level, we applied cognitive dynamics estimation, a recently developed method that generates whole-brain activation maps for four cognitive dimensions (emotion processing, selective attention, self-referential thought, and working memory) using a meta-analytic platform. We then performed C-DSA to calculate the difference between internal and external attention for each cognitive dimension. C-DSA revealed significant differences between internal/external attention in all cognitive dimensions, but especially in emotion processing. Moreover, the difference between attention conditions of selective attention was negatively associated with the severity of depression and state-anxiety, but positively associated with trait-anxiety. Our findings suggest that C-DSA applies to both naturalistic and controlled dynamic processes and may be valuable in clinical settings by linking dynamic cognitive mechanisms with issues like ageing and psychiatric disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC="FIGDIR/small/615274v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1a1bbb4org.highwire.dtl.DTLVardef@12a587forg.highwire.dtl.DTLVardef@1181b45org.highwire.dtl.DTLVardef@1a6716c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

MitoNEET reduces the mitochondrial oxidative phosphorylation during epithelial-mesenchymal transition

Mitochondrial functions range from catabolic to anabolic, which are tightly coordinated to meet cellular demands for proliferation and motility. MitoNEET is a mitochondrial outer membrane protein with a CDGSH domain and is involved in mitochondrial function. Epithelial-to-mesenchymal transition (EMT) is the process in which cells lose their epithelial characteristics and acquire mesenchymal traits, such as motility, which is a vital step for organism development and wound-healing. Cellular motility is associated with high ATP consumption owing to lamellipodia formation, which is supported by upregulated oxidative phosphorylation (OXPHOS) capacity. However, how mitoNEET is involved in the regulation of OXPHOS capacity and subsequent cellular motility remains unclear. Here we show that loss of mitoNEET regulation during EMT impairs both OXPHOS enhancement and cell motility in non-transformed NMuMG mouse mammary gland epithelial cells. We found that mitoNEET is downregulated during EMT, and that the aberrant expression of mitoNEET abolishes the upregulation of OXPHOS, leading to the inhibition of cell motility. Furthermore, we found that mitoNEET topology may be crucial for the regulation of the mitochondrial electron transfer chain, suggesting an additional regulatory pathway for OXPHOS capacity. Our results demonstrate that mitochondrial OXPHOS capacity during EMT is partly regulated by the dynamics of the outer membrane protein. We believe that our findings are the first step towards understanding the mechanisms by which mitochondrial outer membrane protein topology affects organelle functions.

cell biology↗

bioRxiv2024

An aging-sensitive compensatory secretory phospholipase that confers neuroprotection and cognitive resilience

Breakdown of lipid homeostasis is thought to contribute to pathological aging, the largest risk factor for neurodegenerative disorders such as Alzheimers Disease (AD). Cognitive reserve theory posits a role for compensatory mechanisms in the aging brain in preserving neuronal circuit functions, staving off cognitive decline, and mitigating risk for AD. However, the identities of such mechanisms have remained elusive. A screen for hippocampal dentate granule cell (DGC) synapse loss-induced factors identified a secreted phospholipase, Pla2g2f, whose expression increases in DGCs during aging. Pla2g2f deletion in DGCs exacerbates aging-associated pathophysiological changes including synapse loss, inflammatory microglia, reactive astrogliosis, impaired neurogenesis, lipid dysregulation and hippocampal-dependent memory loss. Conversely, boosting Pla2g2f in DGCs during aging is sufficient to preserve synapses, reduce inflammatory microglia and reactive gliosis, prevent hippocampal-dependent memory impairment and modify trajectory of cognitive decline. Ex vivo, neuronal-PLA2G2F mediates intercellular signaling to decrease lipid droplet burden in microglia. Boosting Pla2g2f expression in DGCs of an aging-sensitive AD model reduces amyloid load and improves memory. Our findings implicate PLA2G2F as a compensatory neuroprotective factor that maintains lipid homeostasis to counteract aging-associated cognitive decline.

neuroscience↗

bioRxiv2023

Insectivorous birds and bats outperform ants in the top-down regulation of arthropods across strata of a Japanese temperate forest

O_LIBirds, bats, and ants are recognized as significant arthropod predators. However, empirical studies reveal inconsistent trends in their relative roles in top-down control across strata. Here, we describe the differences between forest strata in the separate effects of birds, bats, and ants on arthropod communities and their cascading effects on plant damage. C_LIO_LIWe implemented a factorial design to exclude vertebrates and ants in both the canopy and understory. Additionally, we separately excluded birds and bats from the understory using diurnal and nocturnal exclosures. At the end of the experiments, we collected all arthropods and assessed herbivory damage. C_LIO_LIArthropods responded similarly to predator exclusion across forest strata, with a density increase of 81% on trees without vertebrates and 53% without both vertebrates and ants. Additionally, bird exclusion alone led to an 89% increase in arthropod density, while bat exclusion resulted in a 63% increase. Herbivory increased by 42% when vertebrates were excluded and by 35% when both vertebrates and ants were excluded. Bird exclusion alone increased herbivory damage by 28%, while the exclusion of bats showed a detectable but non-significant increase (by 22%). In contrast, ant exclusion had no significant effect on arthropod density or herbivory damage across strata. C_LIO_LIOur results reveal that the effects of birds and bats on arthropod density and herbivory damage are similar between the forest canopy and understory in this temperate forest. In addition, ants were not found to be significant predators in our system. Furthermore, birds, bats, and ants appeared to exhibit antagonistic relationships in influencing arthropod density. These findings highlight, unprecedentedly, the equal importance of birds and bats in maintaining ecological balance across different strata of a temperate forest. C_LI

ecology↗

bioRxiv2023

Organism-Wide Analysis of Sepsis Reveals Mechanisms of Systemic Inflammation

Sepsis is a systemic response to infection with life-threatening consequences. Our understanding of the impact of sepsis across organs of the body is rudimentary. Here, using mouse models of sepsis, we generate a dynamic, organism-wide map of the pathogenesis of the disease, revealing the spatiotemporal patterns of the effects of sepsis across tissues. These data revealed two interorgan mechanisms key in sepsis. First, we discover a simplifying principle in the systemic behavior of the cytokine network during sepsis, whereby a hierarchical cytokine circuit arising from the pairwise effects of TNF plus IL-18, IFN-{gamma}, or IL-1{beta} explains half of all the cellular effects of sepsis on 195 cell types across 9 organs. Second, we find that the secreted phospholipase PLA2G5 mediates hemolysis in blood, contributing to organ failure during sepsis. These results provide fundamental insights to help build a unifying mechanistic framework for the pathophysiological effects of sepsis on the body.

immunology↗

bioRxiv2022

Cerebellar climbing fibers convey behavioral information of multiplex modalities and form functional modules

Cerebellar climbing fibers (CFs) convey sensorimotor information and their errors, which are used for motor control and learning. Furthermore, they represent reward-related information. Despite such functional diversity of CF signals, it is still unclear whether each CF conveys the information of single or multiple modalities and how the CFs conveying different information are distributed over the cerebellar cortex. We performed two-photon calcium imaging from cerebellar Purkinje cells (PCs) in mice engaged in a voluntary forelimb lever-pull task and demonstrated that CF responses in 93% of PCs could be explained by the combination of multiple behavioral variables, such as lever movement, licking, and reward delivery. Neighboring PCs exhibited similar CF response properties, formed functional clusters, and shared noise fluctuations of responses. Taken together, individual CFs convey behavioral information on multiplex variables and are spatially organized into the functional modules of the cerebellar cortex.

neuroscience↗

bioRxiv2021

Highly concentrated trehalose induces transient senescence-associated secretory phenotype in fibroblasts via CDKN1A/p21

Trehalose is the nonreducing disaccharide of glucose, evolutionarily conserved in invertebrates, but does not exist in vertebrates. The living skin equivalent (LSE) is an organotypic coculture containing keratinocytes cultivated on fibroblast-populated dermal substitutes. We demonstrated that human primary fibroblasts treated with highly concentrated trehalose promote significantly extensive spread of the epidermal layer of LSE without any deleterious effects. The RNA-seq analysis data and Ingenuity pathway analysis of the differentially expressed genes of trehalose-treated 2D and 3D fibroblasts at early time points revealed the involvement of the CDKN1A pathway, which is necessary for the marked upregulation of growth factors including DPT. By contrast, the mRNA-seq data of LSEs 2-weeks after air exposure indicated that gene expression profiles are similar for untreated and trehalose-treated cells in both keratinocytes and fibroblasts. The trehalose-treated fibroblasts were positive for senescence-associated {beta}-galactosidase with the significantly downregulated expressions of LMNB1. Finally, we demonstrated that transplantation of the dermal substitute with trehalose-treated fibroblasts accelerated wound closure and increased capillary formation significantly in the experimental mouse wounds in vivo. These data indicate that high-concentration trehalose can induce the beneficial senescence-associated secretory phenotype in fibroblasts via CDKN1A/p21, which may be therapeutically useful for optimal wound repair.

cell biology↗

bioRxiv2021

Microevolution of Pieris butterfly genes involved in host-plant adaptation along a host-plant community cline

Herbivorous insects have evolved counteradaptations to overcome the chemical defenses of their host plants. Several of these counteradaptations have been elucidated at the molecular level, in particular for insects specialized on cruciferous host plants. While the importance of these counteradaptations for host plant colonization is well established,little is known about their microevolutionary dynamics in the field. In this study, we examine patterns of host plant use and insect counteradaptation in three Pieris butterfly species across Japan. The larvae of these butterflies express nitrile-specifier protein (NSP) and its paralog major allergen (MA) in their gut to overcome the highly diversified glucosinolate-myrosinase defense system of their cruciferous host plants. Pieris napi and Pieris melete colonize wild Brassicaceae whereas Pieris rapae typically uses cultivated Brassica as a host, regardless of the local composition of wild crucifers. As expected, NSP and MA diversity was independent of the local composition of wild Brassicaceae in P. rapae. In contrast, NSP diversity correlated with local host plant diversity in both species that preferred wild Brassicaceae. P. melete and P. napi both revealed two distinct major NSP alleles, which shaped diversity among local populations, albeit with different evolutionary trajectories. In comparison, MA showed no indication for local adaptation. Altogether, MA appeared to be evolutionary more conserved than NSP, suggesting that both genes play different roles in diverting host plant chemical defense.

evolutionary biology↗

bioRxiv2021

Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Initially, we show that many of the mutations observed in SARS-CoV-2 during mouse adaptation (at positions 417, 484, 501 of the spike protein) also arise in humans in variants of concern (VOC)2. Their appearance during mouse adaptation indicates that immune pressure is not required for their selection. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, we showed that elevated levels of an eicosanoid, prostaglandin D2 (PGD2) and of a phospholipase, PLA2G2D, contributed to poor outcomes in aged mice3,4. Using our virulent mouse-adapted SARS-CoV-2, we show that infection of middle-aged mice lacking expression of DP1, a PGD2 receptor, or PLA2G2D are protected from severe disease. Further, treatment with a DP1 antagonist, asapiprant, protected aged mice from a lethal infection. DP1 antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, and demonstrates that the PLA2G2D-PGD2/DP1 pathway is a useful target for therapeutic interventions. (Words: 254)

microbiology↗

bioRxiv2021

Reservoir of decision strategies in the mouse brain

In any given situation, the environment can be parsed in different ways to define useful decision variables (DVs) for any task, but the way in which this manifold of potential decision strategies is processed to shape behavioral policies is not known. We recorded neural ensembles in the frontal cortex of mice performing a foraging task admitting multiple DVs. Methods developed to uncover the currently employed DV revealed the use of multiple strategies and latent changes in strategy within sessions. Optogenetic manipulations showed that the secondary motor cortex (M2) is needed for mice to use the different DVs in the task. Surprisingly, we found that, regardless of the DV best explaining the behavior of each mouse, M2 activity reflected a full basis set of computations spanning a repertoire of DVs extending beyond those useful for the present task. This form of multiplexing may confer considerable advantages for learning and adaptive behavior.

neuroscience↗

bioRxiv2021

One pair of motor neurons provokes early spontaneous motor behavior through periodic bursting in the chordate ascidian embryo

Vertebrate rhythmic motor behaviour is generated by the central pattern generator (CPG) located in the spinal cord. However, the development of the CPG has not been elucidated at the single-neuron level. We found that a single pair of motor neurons (A10.64/MN2) constitutes the CPG and regulates rhythmic early motor behaviour in the proto-chordate, Ciona. This pair of cells exhibited Ca2+ oscillation with an 80-sec interval at the mid-tailbud stage and 25 sec at the late tailbud stage. The Ca2+ oscillation occurred independently in a dissociated single cell. In the late tailbud stage, the Ca2+ oscillation began to coincide in phase with the ipsilateral tail muscle contraction, which corresponds to rhythmic early motor behaviour. Interestingly, the number and frequency of tail muscle contractions gradually coincided with spikes in the burst of membrane potential as the embryos developed toward late tailbud stage. Photoablation of A10.64/MN2 abolished the rhythmic early motor behaviour. These findings indicate that the early spontaneous rhythmic motor behaviour of Ciona is directly regulated by A10.64/MN2 as an essential component of the CPG. Our findings shed light on the development and evolution of chordate rhythmic behaviours.

developmental biology↗

bioRxiv2020

A dual prokaryotic (E. coli) expression system (pdMAX)

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. This pdMAX system is a manageable size at 5811 bp. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-{beta}-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. Interestingly, the dual induction plasmid system shows limited protein expression. To analyze the expression of inserted genes, we performed an -complementation assay, in which the -peptide of the lacZ ({beta}-galactosidase) gene is inserted in XL-10 E. coli cells. E. coli expressing the recombinant plasmid form blue colonies when plated on ampicillin/IPTG- or arabinose/X-gal-containing plates if the -peptide was correctly inserted in-frame to produce the tagged protein. This assay system assesses whether the -peptide was inserted at the restriction sites (EcoRV or SmaI), whether the inserted peptide was expressed, and whether the inserted -peptide sequence was ligated in-frame to produce a tagged protein. Frameshifts result in no -complementation and white colonies. With the dual promoter plasmid (pdMAX) system, expressed lacZ activity was significantly decreased comparing with the solo expression (pgMAX) system. Despite this disadvantage, we believe the pdMAX system is still useful for the analysis of distinct genes in E. coli, which will enable different types of expression analysis. Overall, the novel pdMAX system allows for efficient subcloning of two different genes. Furthermore, the pdMAX system could be used to induce and analyze the expression of two distinct genes and adopted to various types of prokaryotic gene expression analyses.

molecular biology↗