Biology subjects

Kawai, K.

Publications and source records attributed to Kawai, K..

10 recordsLinked to original sources

bioRxiv2026

Dual-site specificity of the archaeal tRNA m2G methyltransferase Trm14

N2-methylguanosine (m2G) is widely found at multiple positions in tRNAs across the three domains of life. Tryptophan tRNA from Thermococcus kodakarensis contains m2G at position 67. We previously proposed that the tRNA m2G methyltransferase Trm14 is responsible for m2G67 formation in tRNATrp from T. kodakarensis, although Trm14 was originally identified as the enzyme catalyzing m2G6 formation in tRNACys in Methanocaldococcus jannaschii. Thus, it remained unclear whether Trm14 could also methylate G67. Here, we characterized archaeal Trm14. Biochemical analyses using recombinant T. kodakarensis Trm14 revealed that the enzyme catalyzes m2G formation at positions 6 and 67 in T. kodakarensis tRNACys and tRNATrp transcripts, respectively. Mass spectrometric analyses demonstrated the loss of m2G6 and m2G67 in native tRNACys and tRNATrp, respectively, from a T. kodakarensis trm14 gene disruptant strain, providing direct evidence for the dual-site specificity of T. kodakarensis Trm14. The growth phenotype of the trm14 gene disruptant strain was comparable to that of the wild-type strain. In contrast, a trm14/trm11 double disruptant, in which trm11 encodes the tRNA m2G10/m22G10 methyltransferase, exhibited severe growth retardation at 95 {degrees}C. This suggests that m2G6/m2G67 and m2G10/m22G10 cooperatively contribute to cellular fitness at high temperatures. Biochemical analyses revealed that Trm14 methylates all 46 T. kodakarensis tRNA transcripts. Furthermore, we found that recombinant M. jannaschii Trm14 methylated both positions. In contrast, the bacterial ortholog TrmN modified only position 6 in tRNA. Overall, this study expands our understanding of archaeal Trm14 by demonstrating its broader substrate specificity and the physiological significance of these modifications under hyperthermophilic conditions.

molecular biology↗

bioRxiv2026

Pathway-Centric Integration of CRISPR Fitness with Molecular Features Draws Cancer State Maps

Cancer cells display heterogeneous pathway activity that shapes therapeutic vulnerability, but mapping it remains challenging. Transcriptomic scores do not directly measure functional activity, and CRISPR knockout data alone lack molecular interpretability. We introduce StateMap, a pathway-centric framework integrating gene expression and genome-wide CRISPR knockout fitness data from the Cancer Dependency Map. For a given pathway, StateMap selects features by co-dependency and mutual information, then projects cell lines into a low-dimensional space reflecting pathway activity and molecular state. Applied to the Hippo pathway, it resolved five functional states refining the YAP-on/YAP-off dichotomy. Notably, the Hippo-strong state showed selective dependence on integrin V{beta}5; ITGAV depletion triggered Hippo-dependent cell aggregation and G1 arrest via enhanced cell-cell adhesion. Machine learning transfer to TCGA identified a matching subtype with poor prognosis, nominated NNMT as a biomarker, and predicted sensitivity to the V inhibitor Cilengitide. StateMap enables pathway-specific state mapping and discovery of state-selective therapeutic vulnerabilities.

cancer biology↗

bioRxiv2025

Vagus nerve stimulation modulates information representation of sustained activity in layer specific manner in the rat auditory cortex

The brain of a living organism enables stable information processing in response to constantly changing external environments and internal states. As one of such cortical modulation, the present study focused on the effect of vagus nerve stimulation (VNS) therapy on information representation of the auditory cortex. By quantifying sound representation using machine learning, we investigated whether VNS alters cortical information representation in a layer-specific and frequency band-specific manner. A microelectrode array meticulously mapped the band-specific power and phase-locking value of sustained activities in every layer of the rat auditory cortex. Sparse logistic regression was used to decode the test frequency from these neural characteristics. The comparison of decoding accuracy before and after the application of VNS indicated that sound representation of the high-gamma band activity was impaired in the deeper layers, i.e., layers 5 and 6, while it was slightly improved in the superficial layers, i.e., layers 2, 3, and 4. Moreover, there was an improvement of sound representation in theta band activity in the deeper layers, demonstrating the layer-specific and frequency band-specific effect of VNS. Given that the cortical laminar structure and oscillatory activity in multiple frequency bands helps the auditory cortex to act as a hub for feed-forward and feed-back pathways in various information processing, the current findings support the possibility that VNS provide complex effects on brain function by altering the balance of cortical activity between layers and frequency bands.

neuroscience↗

bioRxiv2024

Systemic identification of functionally conserved lncRNA metabolic regulators in human and mouse livers

BACKGROUND & AIMSUnlike protein-coding genes, the majority of human long non-coding RNAs (lncRNAs) lack conservation based on their sequences, posing a challenge for investigating their role in a pathophysiological context for clinical translation. This study explores the hypothesis that non-conserved lncRNAs in human and mouse livers may share similar metabolic functions, giving rise to functionally conserved lncRNA metabolic regulators (fcLMRs). METHODSWe developed a sequence-independent strategy to select putative fcLMRs, and performed extensive analysis to determine the functional similarities of putative human and mouse LMR pairs (h/mLMRs). RESULTSWe found that several pairs of putative fcLMRs share similar functions in regulating gene expression. We further demonstrated that a pair of fcLMRs, h/mLMR1, robustly regulated triglyceride levels by modulating the expression of a similar set of lipogenic genes. Mechanistically, h/mLMR1 binds to PABPC1, a regulator of protein translation, via short motifs on either lncRNA with divergent sequences but similar structures. This interaction inhibits protein translation, activating an amino acid-mTOR-SREBP1 axis to regulate lipogenic gene expression. Intriguingly, PABPC1-binding motifs on each lncRNA fully rescued the functions of their corresponding LMRs in the opposite species. Given the elevated expression of h/mLMR1 in humans and mice with hepatic steatosis, the PABPC1-binding motif on hLMR1 emerges as a potential non-conserved human drug target whose functions can be fully validated in a physiologically relevant setting before clinical studies. CONCLUSIONSOur study supports that fcLMRs represent a novel and prevalent biological phenomenon, and deep phenotyping of genetic mLMR mouse models constitutes a powerful approach to understand the pathophysiological role of lncRNAs in the human liver.

physiology↗

bioRxiv2024

Live-cell imaging and CLEM reveal the existence of ACTN4-dependent ruffle-edge lamellipodia acting as a novel mode of cell migration

-Actinin-4 (ACTN4) expression levels are correlated with the invasive and metastatic potential of cancer cells; however, the underlying mechanism remains unclear. Here, we identified ACTN4-localized ruffle-edge lamellipodia using live-cell imaging and correlative light and electron microscopy (CLEM). BSC-1 cells expressing EGFP-ACTN4 showed that ACTN4 was most abundant in the leading edges of lamellipodia, although it was also present in stress fibers and focal adhesions. ACTN4 localization in lamellipodia was markedly diminished by phosphoinositide 3-kinase inhibition, whereas its localization in stress fibers and focal adhesions remained. Furthermore, overexpression of ACTN4, but not ACTN1, promoted lamellipodial formation. Live-cell analysis demonstrated that ACTN4-enriched lamellipodia are highly dynamic and associated with cell migration. CLEM revealed that ACTN4-enriched lamellipodia exhibit a characteristic morphology of multilayered ruffle-edges that differs from canonical flat lamellipodia. Similar ruffle-edge lamellipodia were observed in A549 and MBA-MD-231 invasive cancer cells. ACTN4 knockdown suppressed the formation of ruffle-edge lamellipodia and cell migration during wound healing in A549 monolayer cultures. Additionally, membrane-type 1 matrix metalloproteinase was observed in the membrane ruffles, suggesting that ruffle-edge lamellipodia have the ability to degrade the extracellular matrix and may contribute to active cell migration/invasion in certain cancer cell types.

cell biology↗

bioRxiv2023

Polygenic Risk Score Associates with Atherosclerotic Plaque Characteristics at Autopsy

BackgroundPolygenic risk scores (PRS) for coronary artery disease (CAD) potentially improve cardiovascular risk prediction. However, their relationship with histopathologic features of CAD has never been examined systematically. MethodsFrom 4,327 subjects referred to CVPath by the State of Maryland Office Chief Medical Examiner (OCME) for sudden death between 1994 and 2015, 2,455 cases were randomly selected for genotyping. We generated PRS from 291 known CAD risk loci. Detailed histopathologic examination of the coronary arteries was performed in all subjects. The primary study outcome measurements were histopathologic plaque features determining severity of atherosclerosis, including %stenosis, calcification, thin-cap fibroatheromas (TCFA), and thrombotic CAD. ResultsAfter exclusion of cases with insufficient DNA sample quality or with missing data, 954 cases (mean age 48.8{+/-}14.7; 75.7% men) remained in the final study cohort. Subjects in the highest PRS quintile exhibited more severe atherosclerosis compared to subjects in the lowest quintile, with greater %stenosis (80.3%{+/-}27.0% vs. 50.4%{+/-}38.7%; adjusted p<0.001) and a higher frequency of calcification (69.6% vs. 35.8%; adjusted p=0.004) and TCFAs (26.7% vs. 9.5%; adjusted p=0.007). Even after adjustment for traditional CAD risk factors subjects within the highest PRS quintile had higher odds of severe atherosclerosis (i.e., [≥]75% stenosis; adjusted OR 3.77; 95%CI 2.10-6.78; p<0.001) and plaque rupture (adjusted OR 4.05; 95%CI 2.26-7.24; p<0.001). Moreover, subjects within the highest quintile had higher odds of CAD-associated cause of death, especially among those aged 50 years and younger (adjusted OR 4.08; 95%CI 2.01-8.30; p<0.001). No associations were observed with plaque erosion. ConclusionsThis is the first autopsy study investigating associations between PRS and atherosclerosis severity at the histopathologic level in subjects with sudden death. Our pathological analysis suggests PRS correlates with plaque burden and features of advanced atherosclerosis and may be useful as a method for CAD risk stratification, especially in younger subjects. HighlightsO_LIIn this autopsy study including 954 subjects within the CVPath Sudden Death Registry, high PRS correlated with plaque burden and atherosclerosis severity. C_LIO_LIThe PRS showed differential associations with plaque rupture and plaque erosion, suggesting different etiologies to these two causes of thrombotic CAD. C_LIO_LIPRS may be useful for risk stratification, particularly in the young. Further examination of individual risk loci and their association with plaque morphology may help understand molecular mechanisms of atherosclerosis, potentially revealing new therapy targets of CAD. C_LI Graphic AbstractA polygenic risk score, generated from 291 known CAD risk loci, was assessed in 954 subjects within the CVPath Sudden Death Registry. Histopathologic examination of the coronary arteries was performed in all subjects. Subjects in the highest PRS quintile exhibited more severe atherosclerosis as compared to subjects in the lowest quintile, with a greater plaque burden, more calcification, and a higher frequency of plaque rupture.

genetics↗

bioRxiv2022

Predictive coding during action observation - an Electrocorticographic study

Predictive coding is a theoretical framework that has received much attention for its ability to generate testable hypotheses on how multiple brain regions integrate information during cognitive functions. Given relatively large sensorimotor delays, during social interactions, predicting the behavior of others is crucial to enable joint actions or provide competitive advantages. The action observation network (AON) has been extensively studied, but how information is integrated across its main nodes remains poorly understood. Here we leverage the high spatial and temporal resolution of intracranial Electrocorticography (ECoG), to characterize how the key nodes of the AON - including precentral, supramarginal and visual areas - exchange information. We found more top-down beta oscillation from precentral to supramarginal contacts during the observation of predictable actions while more bottom-up gamma oscillation from visual to supramarginal contacts were measured for unpredictable actions. These results, in line with predictive coding, provide critical evidence towards an understanding of how nodes of the AON integrate information to process the actions of others.

neuroscience↗

bioRxiv2021

Stress-impaired reward pathway promotes distinct feeding behavior patterns

Psychosocial stress can impact feeding behavior outcomes. Although many studies have examined alterations to food intake, little is known about how stress affects feeding behavior patterns. To determine the impact of psychological stress on feeding behavior patterns, mice were subjected to various psychosocial stressors (social isolation, intermittent high-fat-diet, or physical restraint) prior to timed observations in a feeding arena that incorporated multiple bait loci. In addition, in vivo microdialysis was used to assess the effects of stressors on the reward system by measuring dopamine levels in the nucleus accumbens (NAcc) shell. Impaired feeding behavior patterns characterized by significant deviations in bait selection (i.e. fixated feeding) and prolonged periods of eating (i.e. protracted feeding) were observed in stressed mice relative to non-stressed controls. In addition to clear behavioral effects, the stressors also negatively impacted dopamine levels at the nucleus accumbens shell. Normalization of dopamine reversed the fixated feeding behavior, whereas specifically inhibiting neuronal activity in the dopaminergic neurons of the ventral tegmental area that project to the nucleus accumbens shell caused similar impairments in feeding. Given that the deviations were not consistently accompanied by changes in the amount of bait consumed, body weight, or metabolic factors, the qualitative effects of psychosocial stressors on feeding behavior likely reflect perturbations to a critical pathway in the mesolimbic dopamine system. These findings provide compelling evidence that aberrations in feeding behavior patterns can be developed as sensitive biomarkers of psychosocial stress and possibly a prodromal state of neuropsychiatric diseases. Significance StatementFeeding behavior can be affected by neuropsychiatric disorders including psychosocial stressors, and the evaluation of eating behavior was mainly based on food intake. However, it is speculated that not only food intake but also feeding behavior patterns can be affected in such disorders. The biological processes underlying the feeding behavior patterns have not been clarified yet. We found that aberrant feeding behaviors in mice characterized by fixated feeding were provoked by psychosocial stressors. The qualitative effects of psychosocial stressors on feeding behavior reflect perturbations in the mesolimbic dopamine system. These findings provide compelling evidence that aberrations in feeding behavior patterns can be developed as sensitive biomarkers of psychosocial stress and possibly a prodromal state of neuropsychiatric diseases.

neuroscience↗

bioRxiv2021

Rab10-positive tubular structures represent a novel endocytic pathway that diverges from canonical macropinocytosis in RAW264 macrophages

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules belonged to a novel, non-degradative, endocytic pathway. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel, non-degradative, endocytic pathway that diverges from canonical macropinocytosis.

cell biology↗

bioRxiv2020

Population genetic structure in the insular Ryukyu flying fox, Pteropus dasymallus

Small isolated populations are vulnerable to both stochastic events and the negative consequences of genetic drift. For threatened species, the genetic management of such populations has therefore become a crucial aspect of conservation. Flying foxes (Pteropus spp, Chiroptera) are keystone species with essential roles in pollination and seed dispersal in tropical and subtropical ecosystems. Yet many flying fox species are also of conservation concern, having experienced dramatic population declines driven by habitat loss and hunting. The Ryukyu flying fox (Pteropus dasymallus) ranges from Japan and Taiwan to the northern Philippines, and has undergone precipitous population crashes on several islands in recent decades. To assess population genetic structure and diversity in P. dasymallus, and its likely causes, we analyzed mitochondrial and microsatellite DNA. Both markers showed significant genetic differentiation among most island populations with patterns of isolation-by-distance. However, while mitochondrial haplotypes showed some mixing across the region, likely reflecting historical colonization and/or dispersal events, microsatellites markers showed clear subdivisions corresponding to the position of deep ocean trenches. The current distribution of P. dasymallus and its subspecific diversity therefore appears to have arisen through vicariance coupled with a long history of restricted gene flow across oceanic barriers. We conclude that isolated island subgroups should be managed separately, with efforts directed at reducing further declines.

ecology↗