Biology subjects

Adachi, K.

Publications and source records attributed to Adachi, K..

14 recordsLinked to original sources

bioRxiv2026

A red fluorescent sensor for intensity-and lifetime imaging of cAMP in living cells

cAMP (cyclic adenosine monophosphate) is a crucial intracellular messenger involved in neuromodulation and plasticity, but more information on its spatiotemporal regulation is desirable. Here we constructed a red fluorescent indicator based on a truncated cAMP binding site derived from a regulatory PKA subunit of protozoan origin. It contains a single binding site for cAMP highly specific for cAMP over cGMP. chILI (cAMP highly-responsive Intensity and Lifetime Indicator) can be imaged as an intensity-based probe, but alternatively also offers options for fluorescence lifetime imaging. When expressed in the mouse brain using AAV-vector mediated gene transfer, it reports spontaneous and evoked cAMP transients from CA1 neurons in the hippocampus of awake mice. chILI will add valuable options for monitoring cAMP in vitro and in vivo.

neuroscience↗

bioRxiv2026

Dynamic visualization of physiological CaMKII activity using sensitive FRET biosensors

Calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key mediator of complex physiological processes throughout the body, from the brain to the reproductive system, where CaMKII translates spatiotemporally dynamic calcium elevations into specific biological functions. Directly visualizing CaMKII activity dynamics in living cells using genetically encoded fluorescent biosensors can thus provide crucial insights into the molecular regulation of health and disease. Yet the ability to sensitively and specifically monitor endogenous CaMKII activity in physiologically relevant contexts is limited by the lack of sensors that can achieve robust, quantitative visualization of CaMKII responses. Here, we leveraged a recent serine/threonine kinome-wide substrate atlas to rationally engineer a powerful suite of Forster resonance energy transfer (FRET)-based CaMKII kinase activity reporters with high specificity, sensitivity, and signal-to-noise ratio. Using these biosensors, we were able to sensitively and robustly visualize endogenous CaMKII activity dynamics in both cultured cell lines and primary cells, including cardiomyocytes, oocytes, and neurons. We further utilized 2pFLIM imaging of organotypic hippocampal slices to quantitatively track LTP-induced CaMKII activity within single dendritic spines, highlighting a major advance in the study of physiological CaMKII signaling.

bioengineering↗

bioRxiv2026

Efficient generation of hematopoietic progenitor cells from human pluripotent stem cells by robotic automation

Differentiation of human pluripotent stem cells (hPSCs) into therapeutic cell types requires stringent control of developmental signals, but conventional manual operations introduce substantial intra- and inter-operator variability. While automation can reduce such variability, fixed systems often lack compatibility with the dynamic, stage-specific, and format-diverse manipulations required for hPSC culture and differentiation. Here, we establish a flexible robotic platform to standardize a stroma- and xeno-free embryoid body (EB)-based hematopoietic progenitor cell (HPC) differentiation process and integrate machine learning (ML) to optimize key process parameters including the concentrations of multiple signaling molecules. Despite intrinsic biological stochasticity and technical complexity associated with EB formation, the robotic platform substantially reduced experimental variation. ML-guided, unbiased exploration not only identified combinations of signaling inputs that markedly improved the efficiency and reproducibility of HPC differentiation and subsequent natural killer cell generation, but also illuminated key signaling logic underlying early human hematopoietic development. Together, these results demonstrate that data-driven robotic automation can uncover optimal culture conditions that are difficult to identify through conventional human-driven experimentation.

developmental biology↗

bioRxiv2025

A thermodynamic chromatin polymer model characterizes the epigenetic conditions for Hox collinearity.

Hox genes consist of four clusters, A, B, C, and D, each containing up to 13 paralogous genes. They are linearly arrayed on cluster regions in the same order as their expression timings along the anterior-posterior (A-P) axis development. This correspondence is known as temporal collinearity, and when the expression order of Hox genes is disrupted, the A-P axis is not accurately established. Although essential molecules and genomic regulatory elements for Hox collinearity have been identified, it remains unclear whether they act on individual genes or instead coordinate the cluster-wide sequential activation along the genomic order. To address this question, we developed a theoretical framework based on the statistical thermodynamics of chromatin modeled as a polymer, interpreting collinearity as a sequential opening of nucleosomes along the gene cluster. The model incorporates interactions between nucleosomes and DNA-binding factors, including transcription factors, represented by energy terms for binding and chromatin bridging. This formulation links the thermodynamic stability of chromatin states with the sequential opening process along the Hox clusters. Our model characterized key quantities that determine the order of chromatin opening and revealed intrinsic constraints that limit sequential opening of chromatin, underlying Hox collinearity. Significance statementHox collinearity, a remarkable correspondence between the genomic order and expression timing of Hox genes, provides cells with positional information on the anterior-posterior axis during development. Although essential molecules and genomic regulatory elements have been identified, the mechanism of sequential activation along the genomic order remains elusive. To address this question, we theoretically investigated the epigenetic states of chromatin using a chromatin polymer model in statistical thermodynamics. Our model provides a possible explanation for the sequential activation of the chromatin underlying Hox collinearity. This work bridges developmental genetics and statistical thermodynamics, offering a quantitative framework for understanding gene activation order along the genome.

biophysics↗

bioRxiv2025

Focused acoustic-radiation-force microscopy for live-cell selective nucleus deformation and nucleus viscoelasticity measurement

Acoustic-radiation-force (ARF) microscopy was developed for non-invasive, selective mechanical stimulation to a single live-cell nucleus and long-term measurement of its mechanical properties. High-frequency focused ultrasound wave achieved a spatially confined ARF field around target nucleus, and its cyclic deformation was induced by amplitude modulation on the ultrasound wave, which was captured by a dark-field microscopy. This method was applied to human mesenchymal stem cells, revealing their extremely high mechanical homeostasis.

biophysics↗

bioRxiv2024

Serine chirality guides metabolic flow between one-carbon metabolism and neuromodulator synthesis

O_SCPLOWLC_SCPLOW-serine serves as a central metabolic node that integrates glycolytic flux, lipid metabolism, and one-carbon metabolism. In the mature central nervous system, O_SCPLOWLC_SCPLOW-serine is actively stereo-converted to O_SCPLOWDC_SCPLOW-serine, which functions as a neurotransmitter. However, the role of O_SCPLOWDC_SCPLOW-serine in cellular metabolism remains unclear. Here, we show that O_SCPLOWDC_SCPLOW-serine competes with mitochondrial O_SCPLOWLC_SCPLOW-serine transport, thereby suppressing one-carbon metabolism. Metabolomic analysis revealed that O_SCPLOWDC_SCPLOW-serine reduces intracellular glycine and formate levels, indicating inhibition of the initial step of the one-carbon pathway. Molecular dynamics simulations and enzymatic assays revealed that O_SCPLOWDC_SCPLOW-serine has low affinity for serine hydroxymethyltransferase 2 (Shmt2), which catalyzes the first step in mitochondrial one-carbon metabolism, and does not directly inhibit its activity. Instead, membrane transport assays demonstrated that O_SCPLOWDC_SCPLOW-serine competes with mitochondrial O_SCPLOWLC_SCPLOW-serine transport, depleting the substrate of Shmt2. Functionally, under O_SCPLOWLC_SCPLOW-serine poor conditions in vitro and ex vivo, O_SCPLOWDC_SCPLOW-serine inhibited the proliferation of immature and undifferentiated neural cells including glioblastoma stem cells, which depend highly on one-carbon metabolism. Notably, endogenous O_SCPLOWDC_SCPLOW-serine levels were low during early neurodevelopment, but increased with maturation, coinciding with a shift in the transcriptional profiles of serine metabolic enzymes at the cellular level. Given that O_SCPLOWLC_SCPLOW-serine supports neurodevelopment and O_SCPLOWDC_SCPLOW-serine modulates neurotransmission, this developmental shift in serine enantiomer metabolism appears to align with the functional transitions of the maturing nervous system. Thus, our findings reveal that serine chirality can influence mitochondrial substrate availability and one-carbon flux, offering previously unappreciated insight into the stereoselective regulation of cellular metabolism.

developmental biology↗

bioRxiv2024

AAV Capsid Screening for Translational Pig Research Using a Mouse Xenograft Liver Model

In gene therapy, delivery vectors are a key component for successful gene delivery and safety, based on which adeno-associated viruses (AAVs) gained popularity in particular for the liver, but also for other organs. Traditionally, rodents have been used as animal models to develop and optimize treatments, but species and organ specific tropism of AAV desire large animal models more closely related to humans for preclinical in-depth studies. Relevant AAV variants with the potential for clinical translation in liver gene therapy were previously evolved in vivo in a xenogeneic mouse model transplanted with human hepatocytes. Here, we selected and evaluated efficient AAV capsids using chimeric mice with a >90% xenografted pig hepatocytes. The pig is a valuable preclinical model for therapy studies due to its anatomic and immunological similarities to humans. Using a DNA-barcoded recombinant AAV library containing 47 different capsids and subsequent Illumina sequencing of barcodes in the AAV vector genome DNA and transcripts in the porcine hepatocytes, we found the AAVLK03 and AAVrh20 capsid to be the most efficient delivery vectors regarding transgene expression in porcine hepatocytes. In attempting to validate these findings with primary porcine hepatocytes, we observed capsid-specific differences in cell entry and transgene expression efficiency where the AAV2, AAVAnc80, and AAVDJ capsids showed superior efficiency to AAVLK03 and AAVrh20. This work highlights intricacies of in vitro testing with primary hepatocytes and the requirements for suitable pre-clinical animal models but suggests the chimeric mouse to be a valuable model to predict AAV capsids to transduce porcine hepatocytes efficiently.

molecular biology↗

bioRxiv2024

Slow running benefits: Boosts in mood and facilitation of prefrontal cognition even at very light intensity

Although running upright has been reported to have positive effects on both physical and mental health, the minimum running intensity/speed that would benefit mood and prefrontal cognition is not yet clear. For this reason, we aimed to investigate the acute effect of very slow running, which is classified as a very light intensity exercise, on mood, executive function (EF), and their neural substrates in the prefrontal cortex (PFC). Twenty-four healthy participants completed a 10-minute very slow running session on a treadmill at 35% [Formula] and a resting control session in randomized order. EF was measured using the Stroop task and the mood state was measured using the Two-Dimensional Mood Scale (TDMS) before and after both sessions. Cortical hemodynamic changes while performing the task were monitored using functional near-infrared spectroscopy (fNIRS). The results show that ten minutes of very slow running significantly enhanced mood, reduced Stroop interference time (i.e., enhanced EF), and elicited left lateral PFC activation. Moreover, head acceleration, the magnitude of up-and-down oscillations, was measured during running, and a significant positive correlation with pleasant mood was found. Head acceleration is a remarkable characteristic of running and may be one of the factors related to a pleasant mood induced by very slow running. In conclusion, the current study reveals that a single bout of running, even at very slow speed, elicits a pleasant mood and improved executive function with enhancing activation in prefrontal subregions. This shed light on the slow running benefits to brain health.

neuroscience↗

bioRxiv2023

Emerging cooperativity between Oct4 and Sox2 governs the pluripotency network in mouse embryos

During the first lineage segregation, mammalian embryos generate the inner cell mass (ICM) and trophectoderm (TE). ICM gives rise to the epiblast (EPI) that forms all cell types of the body, an ability referred to as pluripotency. The molecular mechanisms that induce pluripotency in embryos remain incompletely elucidated. Using knockout (KO) mouse models in conjunction with low-input ATAC-seq and RNA-seq, we found that Oct4 and Sox2 gradually come into play in the early ICM, coinciding with the initiation of Sox2 expression. Oct4 and Sox2 activate the pluripotency-related genes through the putative OCT-SOX enhancers in the early ICM. Furthermore, we observed a substantial reorganization of chromatin landscape and transcriptome from the morula to the early ICM stages, which was partially driven by Oct4 and Sox2, highlighting their pivotal role in promoting the developmental trajectory towards the ICM. Our study provides new insights into the establishment of the pluripotency network in mouse preimplantation embryos.

developmental biology↗

bioRxiv2023

Dendritic, delayed, and stochastic CaMKII activation underlies behavioral time scale plasticity in CA1 synapses

Behavioral time scale plasticity (BTSP), is a form of non-Hebbian plasticity induced by integrating pre- and postsynaptic components separated by behavioral time scale (seconds). BTSP in the hippocampal CA1 neurons underlies place cell formation. However, the molecular mechanisms underlying this behavioral time scale (eligibility trace) and synapse specificity are unknown. CaMKII can be activated in a synapse-specific manner and remain active for a few seconds, making it a compelling candidate for the eligibility trace during BTSP. Here, we show that BTSP can be induced in a single dendritic spine using 2-photon glutamate uncaging paired with postsynaptic current injection temporally separated by behavioral time scale. Using an improved CaMKII sensor, we saw no detectable CaMKII activation during this BTSP induction. Instead, we observed a dendritic, delayed, and stochastic CaMKII activation (DDSC) associated with Ca2+ influx and plateau 20-40 s after BTSP induction. DDSC requires both pre-and postsynaptic activity, suggesting that CaMKII can integrate these two signals. Also, optogenetically blocking CaMKII 30 s after the BTSP protocol inhibited synaptic potentiation, indicating that DDSC is an essential mechanism of BTSP. IP3-dependent intracellular Ca2+ release facilitates both DDSC and BTSP. Thus, our study suggests that the non-synapse specific CaMKII activation provides an instructive signal with an extensive time window over tens of seconds during BTSP.

neuroscience↗

bioRxiv2023

Enhancing gene transfer to renal tubules and podocytes by context-dependent selection of AAV capsids

Despite recent remarkable advancements in adeno-associated virus (AAV) vector technologies, effective gene delivery to the kidney remains a significant challenge. Here we show that AAV vector transduction in proximal tubules and podocytes, the crucial targets for renal gene therapy, can be enhanced remarkably through a meticulous selection of both AAV capsids and route of administration, tailored to the condition of the kidney. In this study, we performed a side-by-side comparison of 47 AAV capsids using AAV Barcode-Seq and identified six AAV capsids, including AAV-KP1, that exhibit remarkable enhancement of renal transduction in mice when delivered locally via the renal vein or the renal pelvis. Individual capsid validation analyses revealed that local delivery of AAV-KP1, but not AAV9, enables remarkably enhanced proximal tubule transduction while minimizing off-target liver transduction. In a mouse model of chronic kidney disease, intravenous administration of AAV9, not AAV-KP1, showed efficient renal tubule and podocyte transduction, which was not observed in the control wild-type mice. We also provide evidence that these contrasting observations between AAV-KP1 and AAV9 are attributed to their distinct pharmacokinetic profiles. Thus, this study highlights the importance of context-dependent capsid selection and engineering for successful renal gene therapy.

cell biology↗

bioRxiv2022

Exome-wide benchmark of difficult-to-sequence regions using short-read next-generation DNA sequencing

Next-generation DNA sequencing (NGS) in short-read mode has been recently used for genetic testing in various clinical settings. NGS data accuracy is crucial in clinical settings, and several reports regarding quality control of NGS data, focusing mostly on establishing NGS sequence read accuracy, have been published thus far. Variant calling is another critical source of NGS errors that remains mostly unexplored despite its established significance. In this study, we used a machine-learning-based method to establish an exome-wide benchmark of difficult-to-sequence regions using 10 genome sequence features on the basis of real-world NGS data accumulated in The Genome Aggregation Database (gnomAD) of the human reference genome sequence (GRCh38/hg38). We used the obtained metrics, designated "UNMET score," along with other lines of structural information of the human genome to identify difficult-to-sequence genomic regions using conventional NGS. Thus, the UNMET score could provide appropriate caveats to address potential sequential errors in protein-coding exons of the human reference genome sequence GRCh38/hg38 in clinical sequencing.

genomics↗

bioRxiv2021

Functional characterization of the biogenic amine transporter system on human macrophages

Monocyte-derived macrophages are key players in tissue homeostasis and disease regulated by a variety of signaling molecules. Recent literature has highlighted the ability for biogenic amines to regulate macrophage functions, but the mechanisms governing biogenic amine signaling on and around immune cells remains nebulous. In the central nervous system, biogenic amine transporters are regarded as the master regulators of neurotransmitter signaling. While we and others have shown macrophages express these transporters, relatively little is known of their function on these cells. To address these knowledge gaps, we interrogated the function of norepinephrine (NET) and dopamine (DAT) transporters on human monocyte-derived macrophages. We found that both NET and DAT are present and can uptake substrate from the extracellular space at baseline. Not only was DAT expressed in cultured macrophages, but it was also detected in a subset of intestinal macrophages in situ. Surprisingly, we discovered a NET-independent, DAT-mediated immuno-modulatory mechanism in response to lipopolysaccharide (LPS). LPS induced reverse transport of dopamine through DAT, engaging autocrine/paracrine signaling loop that regulated the macrophage response. Removing this signaling loop enhanced the pro-inflammatory response to LPS. Finally, we found that this DAT-immune axis was disrupted in disease. Collectively, our data introduce a novel role for DAT in the regulation of innate immunity during health and disease.

neuroscience↗

bioRxiv2021

Oct4 primarily controls enhancer activity rather than accessibility

The transcription factor Oct4 is essential for maintaining stem cell pluripotency and for efficient cell reprogramming, but its functional roles are far from being understood. Here, we investigate the functions of Oct4 by rapidly depleting Oct4 from mouse embryonic stem cells and conducting a time-resolved multiomics analysis. Oct4 depletion leads to an immediate loss of its binding to putative enhancers that are accessible in chromatin. Loss of Oct4 is accompanied by a concomitant decrease in mRNA synthesis from putative target genes that are part of the transcriptional network that maintains pluripotency. Oct4 binding to enhancers does not correlate with chromatin accessibility, whereas Sox2 can apparently retain accessibility after Oct4 depletion even in the absence of eRNA synthesis. These results are consistent with the model that Sox2 primarily acts as a pioneer factor that renders enhancers accessible, whereas Oct4 acts primarily as a transcriptional activator that stimulates transcription of pluripotency enhancers and their target genes.

genomics↗