Biology subjects

Kita, K.

Publications and source records attributed to Kita, K..

14 recordsLinked to original sources

bioRxiv2026

Structural insights and mechanistic inhibition of Plasmodium falciparum dihydroorotate dehydrogenase by newly identified chemotypes

Resistance to frontline antimalarial drugs continues to threaten malaria control, highlighting the need for new chemotypes. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes a key step in de novo pyrimidine biosynthesis and remains a validated antimalarial target. Although the PfDHODH inhibitor DSM265 advanced to clinical development, resistance-associated mutations highlight the need for structurally distinct inhibitors. Here, we report the discovery and structural characterization of PfDHODH inhibitors identified through target-based high-throughput screening of 432,592 compounds. Primary hits were evaluated by dose-response assays against asexual blood-stage parasites, mammalian mitochondrial toxicity assays, and HepG2 cytotoxicity profiling, yielding 348 hits. Seven representative compounds were prioritized based on efficacy against PfDHODH and orthologs from P. vivax and P. berghei, and their X-ray co-crystal structures revealed distinct binding modes within the active site. Ferulenol exhibited sub-micromolar inhibition of recombinant PfDHODH (rPfDHODH) and rPvDHODH, together with potent blood-stage activity, with its prenylated tail contributing additional hydrophobic interactions. Compound 2 showed the highest potency across the tested DHODH enzymes but reduced selectivity for PfDHODH, potentially due to its distinct binding orientation and furylmethyl substituent. Comparative structural analyses with DSM265 identified binding-site features that may guide the design of next-generation PfDHODH inhibitors with improved potency and selectivity.

microbiology↗

bioRxiv2025

Plasmodium falciparum diacylglycerol acyltransferase maintains phospholipid homeostasis to regulate sexual differentiation, ER stress, and cytoadhesion

Plasmodium falciparum is the causative agent of human malaria, a life-threating infectious disease that imposes a major global health burden. Lipid metabolism is indispensable for this parasites replication and survival, yet most of the molecular components and mechanisms involved remain poorly understood. In eukaryotes, lipid droplets (LDs) serve as dynamic organelles that store neutral lipids (NLs), buffer lipotoxic stress, and regulate signaling pathways, with their biogenesis controlled by diacylglycerol o-acyltransferases (DGATs). Although P. falciparum encodes a putative DGAT (PF3D7_0322300), its role in the parasite life cycle has not been elucidated. We generated conditional PfDGAT-knockout parasites to investigate the enzymes functional significance. PfDGAT deficiency led to parasite death, accompanied by reduced LD formation, elevated phospholipid levels, and induction of ER stress. Moreover, PfDGAT deletion altered protein trafficking, resulting in the decreased cytoadherence of parasite-infected erythrocytes to human brain microvascular endothelial cells, and suppressed parasite sexual differentiation. Thus, PfDGAT deletion affected multiple aspects of the parasites life cycle, highlighting its critical role in parasite survival and pathogenesis. Our findings provide new insights into parasite lipid homeostasis and highlight DGAT as a potential target of antimalarial intervention.

microbiology↗

bioRxiv2024

Accelerating antimalarial drug discovery with a new high-throughput screen for fast-killing compounds

The urgent need for rapidly acting compounds in the development of antimalarial drugs underscores the significance of such compounds in overcoming resistance issues and improving patient adherence to antimalarial treatments. The present study introduces a high-throughput screening (HTS) approach using 1536-well plates, employing Plasmodium falciparum lactate dehydrogenase (PfLDH) combined with nitroreductase (NTR) and fluorescent probes to evaluate inhibition of the growth of the asexual blood stage of malaria parasites. This method was adapted to efficiently measure the parasite reduction ratio (PRR) in a 384-well plate format, streamlining the traditionally time-consuming screening process. By successfully screening numerous compounds, this approach identified fast-killing hits early in the screening process, addressing challenges associated with artemisinin-based combination therapies. The high-throughput PRR method is expected to be of value in continuously monitoring fast-killing properties during structure-activity relationship studies, expediting the identification and development of novel, rapidly acting antimalarial drugs within phenotypic drug discovery campaigns.

microbiology↗

bioRxiv2024

Switching between newly learned motor skills

Studies of cognitive flexibility suggest that switching between different tasks can entail a transient switch cost. Here, we asked whether analogous switch costs exist in the context of switching between different motor skills. We tested whether participants could switch between a newly learned skill associated with a novel visuomotor mapping, and an existing skill associated with an intuitive mapping. Participants showed increased errors in trials immediately following a switch between mappings. These errors were attributable to persisting with the pre-switch policy, rather than imperfect implementation or retrieval of the post-switch policy. A subset of our participants further learned a second new skill. Switching between these two novel skills was initially very challenging, but improved with further training. Our findings suggest that switching between newly learned motor skills can be challenging, and that errors in the context of switching between skills are primarily attributable to perseveration with the wrong control policy.

neuroscience↗

bioRxiv2024

Pivotal roles of Plasmodium falciparum lysophospholipid acyltransferase 1 in cell cycle progression and cytostome internalization

The rapid intraerythrocytic replication of Plasmodium falciparum, a deadly species of malaria parasite, requires a quick but constant supply of phospholipids to support marked cell membrane expansion. In the malarial parasite, many enzymes functioning in phospholipid synthesis pathway have not been identified or characterized. Here, we identified P. falciparum lysophospholipid acyltransferase 1 (PfLPLAT1) and showed that PfLPLAT1 is vital for asexual parasite cell cycle progression and cytostome internalization. Deficiency in PfLPLAT1 resulted in decreased parasitemia and prevented transition to the schizont stage. Parasites lacking PfLPLAT1 also exhibited distinctive omega-shaped vacuoles, indicating disrupted cytostome function. Transcriptomic analyses suggested that this deficiency impacted DNA replication and cell cycle regulation. Mass spectrometry-based enzyme assay and lipidomic analysis demonstrated that recombinant PfLPLAT1 exhibited lysophospholipid acyltransferase activity with a preference for unsaturated fatty acids as its acyl donors and lysophosphatidic acids as an acceptor, with its conditional knockout leading to abnormal lipid composition and marked morphological and developmental changes including stage arrest. These findings highlight PfLPLAT1 as a potential target for antimalarial therapy, particularly due to its unique role and divergence from human orthologs.

microbiology↗

bioRxiv2023

Why do some fungi want to be sterile? The role of dysfunctional Pro1 in the rice blast fungus

Although sexual reproduction is widespread in eukaryotes, some fungal species can only reproduce asexually. Therefore, loss of sexual reproduction may confer survival advantages under certain conditions in certain species. In the rice blast fungus Pyricularia (Magnaporthe) oryzae, several isolates from the region of origin retain mating ability (female fertility), but most isolates are female sterile. Therefore, it is hypothesized that female fertility was lost during its spread from the origin to the rest of the world, and P. oryzae is an ideal biological model for studying the cause of the evolutionary shift in the reproductive mode. Here, we show that functional mutations of Pro1, a global transcriptional regulator of mating-related genes in filamentous fungi, is one cause of loss of female fertility in this fungus. Employing backcrossing between female-fertile and female-sterile field isolates, we identified the putative genomic region involved in female sterility by comparative genomics between the genomes of F4 female-fertile and -sterile progenies. Further genotyping, linkage, and functional analyses revealed that the functional mutation of Pro1 causes the loss of female fertility. RNA sequencing analysis showed that Pro1 regulates global gene expression, including that of several mating-related genes. The dysfunctional Pro1 did not affect the infection processes, such as conidial germination, appressorium formation, and penetration, but conidial release from conidiophores was increased. Furthermore, various types of mutations in Pro1 were detected in geographically distant P. oryzae, including pandemic isolates of wheat blast fungus. These results provide the first evidence that loss of female fertility may be advantageous to the life cycle of some plant pathogenic fungi. SignificanceMany pathogenic and industrial fungi are thought to have abdicated sexual reproduction, but the mechanisms and biological importance have been a long-standing mystery. Discovering why such fungi lost fertility is important to understand their survival strategies. Here, we revealed the genetic basis of how the rice blast fungus lost female fertility in nature and how this affects the life cycle. This has important implications for understanding evolution of blast pathogens and for developing an effective management strategy to control blast disease before a pandemic. Our findings also provide an additional perspective on advantages of asexual reproduction in some eukaryotes.

microbiology↗

bioRxiv2022

Independence and interaction between the control of moving and holding still in post-stroke arm paresis

Moving and holding-still (holding) have been posited to be under separate control regimes for both eye and arm movements. The paretic arm after stroke exhibits different abnormalities during rest vs. movement, providing an opportunity to ask whether control of these behaviors is independently affected in stroke. Here, we quantified resting postural abnormalities in stroke patients by measuring their biases in force production as they held their hand still in various locations in a planar workspace, and then assessed the influence of these resting force biases on reaching in the same workspace. We found that patients had marked resting force biases at each location, even when the arm was supported. However, these biases did not transfer to arm-supported planar reaching movements: not during initial acceleration, not in response to mid-trajectory perturbations, and not during deceleration to a stop. Rather, the abnormal resting forces only appeared to switch on after a movement had fully stopped. These findings suggest that moving and holding are functionally separable modes of control. At the same time, we found that the resting biases mirrored characteristics of abnormal synergies during movement: they markedly decreased when arm support was provided; they were higher in more distant positions that require breaking out of flexion; and they scaled with the Fugl-Meyer score for the upper extremity (a measure of intrusion of abnormal synergies during active movement). These three shared features suggest a common mechanism for resting biases and abnormal synergies, which appears to be a contradiction given the functional separation of moving and holding observed in the same patients. To resolve this paradox, we propose a conceptual model that predicts a breakdown in the functional separation between reaching and holding when patients move in the absence of weight support. This conceptual model posits that synergies are the behavioral manifestation of a spillover of posture into movement. Mapping these functional systems onto anatomical and physiological details of lesioned substrate after stroke may provide implementation-level insight into how normal arm motor control is assembled.

neuroscience↗

bioRxiv2022

Evidence for a common mechanism supporting invigoration of action selection and action execution

The speed, or vigor, of our movements can vary depending on circumstances. For instance, the promise of a reward leads to faster movements. Reward also leads us to move with a lower reaction time, suggesting that the process of action selection can also be invigorated by reward. It has been proposed that invigoration of action selection and of action execution might occur through a common mechanism, and thus these aspects of behavior might be coupled. To test this hypothesis, we asked participants to make reaching movements to "shoot" through a target at varying speeds to assess whether moving more quickly was also associated with more rapid action selection. We found that, when participants were required to move with a lower velocity, the speed of their action selection was also significantly slowed. This finding was recapitulated in a further dataset in which participants determined their own movement speed, but had to move slowly in order to stop their movement inside the target. By re-analyzing a previous dataset, we also found evidence for the converse relationship between action execution and action selection: when pressured to select actions more rapidly, people also executed movements with higher velocity. Our results establish that invigoration of action selection and action execution vary in tandem with one another, supporting the hypothesis of a common underlying mechanism. Significance statementWe show that voluntary increases in the vigor of action execution lead action selection to also occur more rapidly. Conversely, hastening action selection by imposing a deadline to act also leads to increases in movement speed. These findings provide evidence that these two distinct aspects of behavior are modulated by a common underlying mechanism.

neuroscience↗

bioRxiv2021

De novo motor learning of a bimanual control task over multiple days of practice

Although much research on motor learning has focused on how we adapt our movements to maintain performance in the face of imposed perturbations, in many cases we must learn new skills from scratch, or de novo. In comparison to adaptation, relatively little is known about de novo learning. In part, this is because learning a new skill can involve many challenges, including learning to recognize new patterns of sensory input and generate new patterns of motor output. However, even with familiar sensory cues and well-practiced movements, the problem of quickly selecting the appropriate actions in response to the current state is challenging. Here, we devised a bimanual hand-to-cursor mapping which isolates this control problem. We find that participants initially struggled to control the cursor under this bimanual mapping, despite explicit knowledge of the mapping. Performance improved steadily over multiple days of practice, however. Participants exhibited no aftereffects when reverting to a veridical cursor, confirming that participants learned the new task de novo, rather than through adaptation. Corrective responses to mid-movement perturbations of the target were initially weak, but with practice, participants gradually became able to respond rapidly and robustly to these perturbations. After four days of practice, participants behavior under the bimanual mapping almost matched performance with a veridically mapped cursor. However, there remained a small but persistent difference in performance level. Our findings illustrate the dynamics and limitations of learning a novel controller and introduce a promising paradigm for tractably investigating this aspect of motor skill learning.

neuroscience↗

bioRxiv2021

The scaffold-dependent function of RIPK1 in dendritic cells promotes injury-induced colitis

Receptor interacting protein kinase 1 (RIPK1) is a cytosolic multidomain protein that controls cell life and death. While RIPK1 promotes cell death through its kinase activity, it also functions as a scaffold protein to promote cell survival by inhibiting FADD-caspase 8-dependent apoptosis and RIPK3-MLKL-dependent necroptosis. This pro-survival function is highlighted by excess cell death and a perinatal lethality in Ripk1-/- mice. Recently, loss of function mutation of RIPK1 was found in patients with immunodeficiency and inflammatory bowel diseases. Hematopoietic stem cell transplantation restored not only immunodeficiency but also intestinal inflammatory pathology, indicating that RIPK1 in hematopoietic cells is critical to maintain intestinal immune homeostasis. Here, we generated dendritic cell (DC)-specific Ripk1-/- mice in a genetic background with loss of RIPK1 kinase activity and found that the mice developed spontaneous colonic inflammation characterized by increased neutrophil infiltration. In addition, these mice were highly resistant to injury-induced colitis. The increased neutrophil infiltration in the colon and the resistance to colitis were restored by dual inactivation of RIPK3 and FADD, but not by inhibition of RIPK3, MLKL, or ZBP1 alone. Altogether, these results reveal a scaffold activity-dependent role of RIPK1 in protecting colonic DCs from apoptotic insults and maintenance of colonic immune homeostasis.

immunology↗

bioRxiv2020

GluA4 enables associative memory formation by facilitating cerebellar expansion coding

AMPA receptors (AMPARs) mediate excitatory neurotransmission in the CNS and their subunit composition determines synaptic efficacy. Whereas AMPAR subunits GluA1-GluA3 have been linked to particular forms of synaptic plasticity and learning, the functional role of GluA4 remains elusive. Here we used electrophysiological, computational and behavioral approaches to demonstrate a crucial function of GluA4 for synaptic excitation and associative memory formation in the cerebellum. Notably, GluA4-knockout mice had [~]80% reduced mossy fiber to granule cell synaptic transmission. The fidelity of granule cell spike output was markedly decreased despite attenuated tonic inhibition and increased NMDA receptor-mediated transmission. Computational modeling revealed that GluA4 facilitates pattern separation that is important for associative learning. On a behavioral level, while locomotor coordination was generally spared, GluA4-knockout mice failed to form associative memories during delay eyeblink conditioning. These results demonstrate an essential role for GluA4-containing AMPARs in cerebellar information processing and associative learning.

neuroscience↗

bioRxiv2020

5-amino levulinic acid inhibits SARS-CoV-2 infection in vitro

The current COVID-19 pandemic requires urgent development of effective therapeutics. 5-amino levulinic acid (5-ALA) is a naturally synthesized amino acid and has been used for multiple purposes including as an anticancer therapy and as a dietary supplement due to its high bioavailability. In this study, we demonstrated that 5-ALA treatment potently inhibited infection of SARS-CoV-2, a causative agent of COVID-19. The antiviral effects could be detected in both human and non-human cells, without significant cytotoxicity. Therefore, 5-ALA is a candidate as an oral antiviral drug for COVID-19.

microbiology↗

bioRxiv2019

Imatinib overrides taxane resistance by selective inhibition of novel CLIP1 variant obstructing the microtubule pore

Despite its widespread use, the majority of patients with gastric cancer (GC) will not respond to taxane chemotherapy due to resistance mechanisms. Here, we report the discovery of a novel truncated variant of the microtubule plus-end binding protein (+TIP) CLIP-170, hereafter CLIP-170S, whose expression is enriched in taxane resistant cell lines and patients with GC. To establish causation, we knocked-down (KD) CLIP-170S which completely reversed taxane resistance. Mass-spec proteomics and 5-RACE further showed that CLIP-170S lacked the first 150 amino acids, including the Cap-Gly motif required for microtubule (MT) plus-end localization. Mechanistically, we show that CLIP-170S was mislocalized from the MT plus-end to the MT lattice obstructing the MT pore surface site required for taxane entry into the MT lumen. Computational analysis of RNA-seq data from taxane-sensitive and resistant GC cell lines, predicted imatinib as the top candidate drug to overcome drug resistance. Imatinib treatment completely reversed taxane resistance, as predicted, and did so unexpectedly by selective depletion of CLIP-170S. Importantly, CLIP170S was found to be highly prevalent in tumor biopsies from patients with GC. Taken together, these data identify CLIP-170S as a novel, clinically prevalent +TIP variant that obstructs the MT pore and confers taxane resistance. The discovery of this previously unrecognized variant together with the computational discovery of Imatinib as a selective CLIP-170S inhibitor, implicate the MT pore in clinical taxane resistance and provide new therapeutic opportunities for treatment of GC and beyond.

cancer biology↗

bioRxiv2019

Rapid and Sustained Homeostatic Control of Presynaptic Exocytosis at a Central Synapse

Animal behavior is remarkably robust despite constant changes in neural activity. Homeostatic plasticity stabilizes central nervous system (CNS) function on time scales of hours to days. If and how CNS function is stabilized on more rapid time scales remains unknown. Here we discovered that mossy fiber synapses in the mouse cerebellum homeostatically control synaptic efficacy within minutes after pharmacological glutamate receptor impairment. This rapid form of homeostatic plasticity is expressed presynaptically. We show that modulations of readily-releasable vesicle pool size and release probability normalize synaptic strength in a hierarchical fashion upon acute pharmacological and prolonged genetic receptor perturbation. Presynaptic membrane capacitance measurements directly demonstrate regulation of vesicle pool size upon receptor impairment. Moreover, presynaptic voltage-clamp analysis revealed increased calcium-current density under specific experimental conditions. Thus, homeostatic modulation of presynaptic exocytosis through specific mechanisms stabilizes synaptic transmission in a CNS circuit on time scales ranging from minutes to months. Rapid presynaptic homeostatic plasticity may ensure stable neural circuit function in light of rapid activity-dependent plasticity.

neuroscience↗