Biology subjects

Ishikawa, K.

Publications and source records attributed to Ishikawa, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Age-Dependent Effects of Macrophage Derived Cathelicidin Antimicrobial Peptide on Bone Fracture Repair

The pace of fracture repair declines with age, increasing the risk of delayed union and nonunion. Delayed union and nonunion are associated with significant morbidity and even mortality with few effective non-surgical interventions. Here, we identify cathelicidin antimicrobial peptide (CAMP) as an age-dependent regulator of bone repair. Single-cell RNA sequencing and immunohistochemistry demonstrated higher CAMP expression during fracture healing in young compared with aged mice. Global or myeloid lineage-depletion of Camp delayed fracture repair in young mice, whereas Camp depletion in aged mice increased callus bone formation. Heterochronic bone marrow transplantation using the conditional genetic knockout further implicated hematopoietic-lineage derived cells in these effects and demonstrated that myeloid-specific Camp expression is important in the rejuvenation of the pace of fracture healing. CAMP promotes osteogenic differentiation through P2X7 receptor (P2X7R)-dependent signaling, influencing the downstream regulation of NLRP3 expression. NLRP3 expression is also correlated with osteogenesis following Camp modulation in both young and old mice, supporting CAMP-P2X7R-NLRP3 signaling as a regulator of osteogenesis. Together, these findings identify CAMP as a hematopoietic-derived regulator of bone repair whose function is age-dependent, promoting fracture healing in young animals while constraining it in aged animals.

cell biology↗

bioRxiv2026

Height-induced postural threat selectively facilitates spinal reflex in the tibialis anterior muscle during quiet standing

Long-latency stretch reflex and corticospinal excitability in the tibialis anterior muscle (TA) are facilitated when balance is threatened, even without background TA activity, suggesting supraspinal modulation as preparatory tuning for ankle stabilization. However, it remains unclear whether such tuning is evident at the spinal level and specific to the TA among lower-limb muscles. We therefore examined the effects of height-induced postural threat on multi-segmental monosynaptic spinal reflexes (MMR) in lower-limb muscles during quiet standing. Seventeen healthy young males performed 90-s standing tasks under three postural threat conditions, created by combining real and virtual reality (VR) heights: (1) Low-threat (real ground & VR ground), (2) Medium-threat (real table & VR ground), and (3) High-threat (real table & VR bridge). During each condition, transcutaneous spinal cord stimulation (tSCS) was applied to the lumbar spine to elicit MMR in lower-limb muscles. Electromyograms (EMG) were recorded from six muscles of the right leg: vastus medialis (VM), biceps femoris (BF), TA, soleus (SOL), medial (MG), and lateral gastrocnemius (LG). MMR excitability was quantified as peak-to-peak EMG amplitude. Fear ratings and electrodermal activity were higher in High-threat than Low-threat (all p < 0.05), confirming successful threat induction. Peak-to-peak EMG amplitude in the TA was significantly higher in High-threat than Low-threat (17.1% increase, p = 0.0393), whereas background TA activity remained absent across conditions. These results indicate that TA has unique function to facilitate spinal excitability as a preparatory tuning for ankle stabilization. Key pointsO_LIPrevious studies have shown the supraspinal facilitation of the tibialis anterior muscle without background muscle activation as a preparatory tuning for ankle stabilization. C_LIO_LITo test the hypothesis that such tuning is also evident at the spinal level and specific to the tibialis anterior muscle, this study examined whether height-induced postural threat modulates multi-segmental monosynaptic reflex excitability in lower-limb muscles using transcutaneous spinal cord stimulation. C_LIO_LIElectrodermal activity and fear ratings increased under height-induced postural threat, confirming the successful induction of postural threat. C_LIO_LIUnder height-induced postural threat, the multi-segmental monosynaptic reflex was selectively facilitated in the tibialis anterior muscle, while its background activity remained absent. C_LIO_LIOur findings demonstrate selective facilitation of spinal excitability in the tibialis anterior muscle, which may serve as preparatory tuning for ankle stabilization under threat to balance. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/742625v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@19fde78org.highwire.dtl.DTLVardef@c6aa8dorg.highwire.dtl.DTLVardef@e73a76org.highwire.dtl.DTLVardef@af393d_HPS_FORMAT_FIGEXP M_FIG C_FIG Abstract figure legendWhen balance is threatened, corticospinal excitability and long-latency stretch reflex in the tibialis anterior muscle (TA) are facilitated even in the absence of background TA activity, suggesting supraspinal preparatory tuning for ankle stabilization. This study tested the hypothesis that such facilitation is also expressed at the spinal level and is specific to the TA. Participants completed 90-s quiet standing trials under three different height-induced postural threat conditions. During each trial, transcutaneous spinal cord stimulation was delivered over the lumbar spine to elicit multi-segmental monosynaptic reflexes (MMR) in multiple lower-limb muscles. High-threat condition increased fear ratings and electrodermal activity, indicating successful threat induction. Moreover, MMR excitability was selectively increased in the TA under High-threat condition despite the absence of background TA activity. These findings suggest that spinal facilitation is selectively expressed in the TA and may reflect preparatory tuning for ankle stabilization under threat to balance.

neuroscience↗

bioRxiv2026

Deletion of the ribosomal protein gene rpmJ activates zntA transcription through a translation-dependent mechanism in Escherichia coli

Bacteria tightly regulate intracellular zinc homeostasis by coordinating zinc uptake and efflux systems. We previously showed that deletion of the ribosomal protein gene rpmJ confers zinc resistance in Escherichia coli in a manner dependent on the zinc efflux transporter zntA. Here, we analyzed the effect of rpmJ deficiency on zntA expression. Under zinc excess conditions, zntA mRNA levels were markedly higher in the rpmJ mutant than in the wild-type strain. Enhanced zntA expression required the native zntA promoter, the native Shine-Dalgarno sequence, and the N-terminal coding region of zntA, indicating that translation initiated from the native Shine-Dalgarno sequence and extending through the N-terminal coding region is required for enhanced transcription initiation from the native zntA promoter. Furthermore, ectopic expression of ykgO, a paralog of rpmJ that is known to replace RpmJ on the ribosome under zinc-limited conditions, abolished the increased zntA expression and zinc resistance conferred by rpmJ deletion. Collectively, these findings suggest that ribosomes lacking RpmJ or YkgO promote transcription initiation from the native zntA promoter through translation of zntA mRNA. IMPORTANCEBacteria must carefully control the amount of zinc inside their cells. Too little zinc prevents essential cellular processes, whereas too much zinc is toxic. We found that removing a small ribosomal protein called RpmJ allows Escherichia coli to survive high zinc levels by increasing production of the zinc exporter zntA. Surprisingly, this increase depends not only on the zntA promoter but also on translation of the beginning of the zntA coding region. Our findings suggest that changes in ribosome composition can stimulate gene transcription through early translation of the same messenger RNA, revealing a previously unrecognized mechanism linking translation and transcription during bacterial adaptation to zinc stress.

molecular biology↗

bioRxiv2026

Muscle-to-Bone CX3CL1 Signaling Promotes Skeletal Repair Through CX3CR1+ Osteoprogenitors

In the context of muscle loss, bone repair is impaired, suggesting that muscle derived signals contribute to bone regeneration. However, how muscle surrounding the injury site communicates with the bone repair niche remains unclear. Here we found that CX3CL1 expression was induced in endothelial cells in muscle surrounding a femoral bone injury site. Deletion of Cx3cl1 impaired bone healing, demonstrating a functional role for CX3CL1 in bone repair. A CX3CL1 receptor, CX3CR1, was expressed by PDGFR stromal progenitors and lineage tracing showed that CX3CR1 expressing osteoprogenitor lineage cells accumulated at the injury site during repair. PDGFR stromal progenitors showed enhanced osteoblastogenesis in response to recombinant CX3CL1. In older mice, local CX3CL1 delivery increased PDGFRCX3CR1 osteoprogenitor accumulation and improved bone repair. These findings identify a muscle bone signaling pathway in which endothelial CX3CL1 promotes bone repair through CX3CR1 expressing osteoprogenitors.

cell biology↗

bioRxiv2025

Overexpression of Escherichia coli yaiX Confers Multidrug Resistance and Enhances Virulence in the Silkworm Infection Model

The emergence of bacteria with both antimicrobial resistance and high virulence has become a global health concern, underscoring the urgent need to elucidate the molecular basis underlying these traits. Here, we employed the silkworm (Bombyx mori) infection model, which is suitable for high-throughput screening, together with an Escherichia coli library containing plasmid clones of all genes from strain W3110, to identify genes whose overexpression enhances virulence. We found that overexpression of the uncharacterized protein YaiX promoted bacterial proliferation in silkworms and increased host lethality. Compared with the empty-vector control, the YaiX-overexpressing strain exhibited resistance to multiple antimicrobial agents with diverse mechanisms of action, including {beta}-lactams, tetracyclines, fluoroquinolones, aminoglycosides, cationic surfactants, and hydrogen peroxide. Sequence analysis revealed that amino acids 18-52 of YaiX contain a transferase hexapeptide domain predicted to form a left-handed parallel {beta}-helix. Overexpression of YaiX mutants lacking regions outside this domain conferred ampicillin resistance, whereas deletion of the hexapeptide domain abolished this phenotype. RNA sequencing and GO enrichment analyses further indicated that YaiX overexpression altered the expression of genes encoding RNA-binding proteins and porins. These findings suggest that YaiX overexpression, through its hexapeptide domain, modulates gene expression and contributes to both multidrug resistance and enhanced virulence in E. coli.

microbiology↗

bioRxiv2025

Autoclaving and long-term storage deplete glutamine sources in complex media and affect bacterial phenotypes

Autoclave sterilization is the most common method for sterilizing reagents and media in biology. However, the effects of heat-induced loss or modification of complex medium components on bacterial growth and phenotypes remain poorly understood. Here, we investigated the impact of autoclaving on glutamine sources in bacterial complex media using an Escherichia coli {Delta}glnA mutant, which requires exogenous glutamine for growth. {Delta}glnA exhibited impaired growth in LB medium after autoclaving compared with non-autoclaved LB, whereas its residual growth indicated the presence of heat-stable glutamine sources. Growth assays and HPLC quantification revealed that free glutamine in LB decreased from 58 {micro}M to 12 {micro}M upon autoclaving, while heat-stable glutamine sources remained at 117 {micro}M. Similar growth defects were observed for {Delta}glnA in autoclaved BHI, TSB, and M17 media compared with their non-autoclaved counterparts. Long-term storage of LB at room temperature for 24 weeks also reduced {Delta}glnA growth regardless of autoclaving, compared with freshly prepared LB. Furthermore, supplementation of glutamine sources into glutamine-deficient MHB medium enhanced biofilm formation by Pseudomonas aeruginosa. Collectively, these results demonstrate that autoclaving and storage reduce glutamine sources in complex media, thereby influencing bacterial growth and phenotypes.

microbiology↗

bioRxiv2025

Paracrine Action of Bone Morphogenetic Protein 3 in Pulmonary Arterial Hypertension

BackgroundDespite recent advancements in the management of pulmonary arterial hypertension (PAH), the disease remains devastating, with limited survival. Although the Bone Morphogenetic Protein (BMP) signaling pathway is known to play an important role in PAH, our current understanding of this pathway remains limited. MethodsWe assessed BMP3 levels in the lungs of mice, rats, and pigs with pulmonary hypertension, and in pulmonary vascular cells from human patients with PAH. We performed in vitro studies on human pulmonary artery smooth muscle cells (hPASMCs) and human pulmonary artery endothelial cells (hPAECs) derived from healthy donors and from patients with PAH. We generated mice with global or SMC-specific deletion of BMP3. Recombinant BMP3 protein and adeno-associated viruses (AAV) were used to overexpress BMP3 in two different models of PAH in rodents. Magnetic resonance imaging, cardiac hemodynamics, morphometric, and histological measurements were performed to evaluate the effects of BMP3 on cardiac function and pulmonary vascular remodeling. ResultsBMP3 is predominantly expressed in PASMCs and is downregulated in PAH. In vitro, conditioned medium from siRNA-BMP3-transfected hPASMCs increased hPAECs migration and proliferation, while PASMC-derived BMP3 inhibited PAH-diseased PAEC dysfunction. In both global and SMC-specific BMP3-deficient mice, exposure to a model of PAH exacerbated cardiac and pulmonary vascular remodeling in middle-aged mice. An intraperitoneally injected recombinant BMP3 prevented and reversed PAH in mice. A lung-targeted overexpression of BMP3, via AAV1-BMP3, reversed pulmonary vascular remodeling and inhibited cardiac dysfunction in mice and rats. Mechanistically, BMP3 activated the BMP/Smad1,5,8 pathway, inhibited the TGF-{beta}/Smad2,3 pathway, and decreased the expression of cell cycle genes in hPAECs and in the lungs of BMP3-treated animals with PAH. ConclusionsOur findings provide evidence that BMP3 overexpression attenuates pulmonary vascular remodeling and inhibits cardiac dysfunction by restoring the balance between the TGF-{beta} and BMP pathways through a cell-cell communication mechanism, offering a novel therapeutic pathway for PAH. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIBMP3 is downregulated in the lungs of mice, rats, and pigs with pulmonary hypertension and in pulmonary artery smooth muscle cells of patients with PAH. C_LIO_LIBMP3 acts as a paracrine factor between pulmonary vascular cells. C_LIO_LIOverexpression of BMP3 decreases pulmonary vascular remodeling and reverses cardiac dysfunction in PAH-diseased rodents. C_LIO_LIBMP3 acts by restoring the balance between the TGF-{beta}/Smad2,3 pathway and the BMP/Smad1,5,8 pathway C_LI What Are the Clinical Implications?O_LIBMP3 represents a promising disease-modifying agent with potential for clinical translation in the treatment of PAH. C_LIO_LIOverexpressing BMP3 with Adeno-Associated Viruses has therapeutic potential to treat PAH. C_LI

pathology↗

bioRxiv2025

Knockout of tusA facilitates flagella formation and cationic antimicrobial resistance by disrupting Fur transcriptional regulation in Escherichia coli

tRNA 2-thiouridine synthesizing protein A (TusA), a sulfur-carrier protein, plays a crucial role in tRNA sulfur modification. Recent studies have reported that tusA deficiency affects iron-sulfur (Fe-S) homeostasis and cluster formation in Escherichia coli; however, its association with this phenotype remains unclear. In this study, we analyzed the phenotype of tusA-deficient E. coli ({Delta}tusA) and its underlying mechanisms using RNA sequencing. We observed that tusA deletion disrupted the expression of genes regulated by the ferric uptake regulator Fur or Fur-regulated transcription factors (flagellar transcriptional regulators D and C [FlhDC] and fumarate and nitrate reduction regulator [Fnr]). Increased expression of flhDC, which is the master regulator of flagellar genes facilitated flagella formation even under conditions in which the wild-type formed few flagella. Additionally, {Delta}tusA was resistant to cationic antibacterial agents, such as cetyltrimethylammonium bromide, cetylpyridinium chloride, and protamine sulfate. This resistance is associated with the increased expression of ompX or ompF, regulated by Fur and Fnr, respectively. Notably, both enhanced flagella formation and resistance to cationic antibacterial agents caused by tusA deletion were abolished in the fur-deficient background. These findings indicate that impaired expression of the fur regulon, possibly because of impaired Fe-S cluster formation, induces multiple phenotypic alterations in {Delta}tusA. IMPORTANCETusA is a sulfur carrier protein involved in tRNA sulfur modification, and its effect on translation has been studied. Recent studies have reported that tusA deficiency affects Fe-S homeostasis and cluster formation in Escherichia coli; however, its association with the phenotype remains unclear. Based on RNA sequencing, we indicated that the altered gene expression in {Delta}tusA resulted from the disruption of fur regulation that is controlled by Fe-S clusters. We further demonstrated that enhanced flagella formation and resistance to cationic drugs were mediated by Fur-dependent gene expression alterations. Our data indicate that the regulation of sulfur allocation for tRNA modification by TusA affects the global gene expression in bacteria.

microbiology↗

bioRxiv2025

Synergistic effects of Cyp51 isozyme-specific azole antifungal agents on fungi with multiple cyp51 isozyme genes

Pathogenic fungi pose significant societal challenges. The limited availability of therapeutic targets due to the eukaryotic nature of fungi emphasizes the importance of available drug targets such as Cyp51, a crucial enzyme in ergosterol biosynthesis, inhibited by azole antifungals. This study explored the susceptibility patterns of azole antifungals against Cyp51 isozyme deletion strains ({Delta}cyp51A and {Delta}cyp51B) in Trichophyton rubrum, the predominant dermatophyte species. Distinct susceptible patterns were observed among azole antifungals for {Delta}cyp51A and {Delta}cyp51B. Although most azole antifungal agents exhibited increased antifungal activity against {Delta}cyp51A, select agents demonstrated increased antifungal activity against {Delta}cyp51B. Remarkably, fluconazole, sulconazole, and imazalil exhibited relatively increased activity against {Delta}cyp51A, whereas prochloraz demonstrated increased activity against {Delta}cyp51B. Combining these isozyme-selective agents exerted synergistic effects against the wild-type strain and the parent ku80-knockout strain but not against individual Cyp51 knockout mutants. Hence, the two Cyp51 isozymes, Cyp51A and Cyp51B, may be inhibited by distinct azole antifungals, exerting a synergistic effect with the dual azole antifungal combination. This synergistic effect was also observed on another fungal species, Aspergillus welwitschiae, which also has two Cyp51 isozymes. These data demonstrate that combining azole antifungals with different Cyp51 isozyme selectivities exerts synergistic effects against fungi possessing multiple Cyp51 isozymes. This study proposes a novel therapeutic approach for addressing fungal infections through the combination of antifungal drugs that inhibit the same enzymatic activity but exhibit different isozyme selectivity. It also emphasizes the potential for developing drugs targeting specific isozymes, a previously underutilized approach in the realm of antifungal drug development.

microbiology↗

bioRxiv2025

Transcranial electrical stimulation for memory enhancement: A systematic review and meta-analysis

BackgroundNon-invasive brain stimulation techniques have received increasing interest for their potential to enhance memory function, a fundamental cognitive aspect of daily life. MethodsThis systematic review and meta-analysis investigated the efficacy of transcranial electrical stimulation (tES) in enhancing memory function in healthy adults, following the pre-registered strategy at PROSPERO (CRD42022353630). ResultsA total of 66 articles (119 trials, 3,786 participants) focusing on transcranial direct current stimulation and transcranial alternating current stimulation were identified. Meta-analysis revealed a significant overall effect of tES on memory function compared with sham stimulation (standardized mean difference [95% confidence interval] = 0.19 [0.12-0.27]), with anodal transcranial direct current stimulation showing the most consistent enhancement. In particular, stimulation of the frontal regions effectively improved working and declarative memories. While the effects remained significant within hours post-stimulation, they diminished after one day or longer. Regarding adverse events, tingling and itching sensations on the scalp occurred more frequently in the active group than in the sham group, but no severe adverse events were reported. Challenges, including publication bias, heterogeneity, and bias toward specific aspects of memory were noted, emphasizing the need for improved experimental rigor and diversification of memory tasks. ConclusionThese findings highlight the potential of tES as a safe and effective tool for memory enhancement while emphasizing areas for future research to develop its applications.

neuroscience↗

bioRxiv2025

Xenopus laevis as an infection model for human pathogenic bacteria

Animal infection models are essential for understanding bacterial pathogenicity and corresponding host immune responses. In this study, we investigated whether juvenile Xenopus laevis could be used as an infection model for human pathogenic bacteria. Xenopus frogs succumbed to intraperitoneal injection containing the human pathogenic bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes. In contrast, non-pathogenic bacteria Bacillus subtilis and Escherichia coli did not induce mortality in Xenopus frogs. The administration of appropriate antibiotics suppressed mortality caused by S. aureus and P. aeruginosa. Strains lacking the agr locus, cvfA (rny) gene, or hemolysin genes in S. aureus, LIPI-1-deleted mutant of L. monocytogenes, which attenuate virulence within mammals, exhibited reduced virulence in Xenopus frogs compared to their respective wild-type counterparts. Bacterial distribution analysis revealed that S. aureus persisted the blood, liver, heart, and muscles of Xenopus frogs until death. These results suggested that intraperitoneal injection of human pathogenic bacteria induces sepsis-like symptoms in Xenopus frogs supporting their use as a valuable animal model for evaluating antimicrobial efficacy and identifying virulence genes in various human pathogenic bacteria.

microbiology↗

bioRxiv2024

Escherichia coli growth under low pH, low temperature, and high osmotic stress conditions requires the wzxE flippase for the enterobacterial common antigen intermediate

Colanic acid and enterobacterial common antigen (ECA) are cell surface polysaccharides that are produced by many E. coli isolates. Colanic acid is induced under low pH, low temperature, and hyperosmotic conditions and is important in E. coli resistance to these stresses; however, the role of the ECA is unclear. In this study, we observed that knockout of the flippase wzxE, which converts ECA intermediates from the cytoplasmic side of the inner membrane to the periplasmic side, resulted in low pH sensitivity in E. coli. The wzxE-knockout mutant showed reduced growth potential and viable counts in the extracts of several vegetables (cherry tomatoes, carrots, celery, lettuce, and spinach), which are known to be low pH environments. A double knockout strain of wzxE and wecF, which encodes an enzyme that synthesizes an ECA intermediate, did not show sensitivity to low pH, nor did a double knockout mutant of wzxE and wcaJ, which encodes a colanic acid synthase. The wzxE-knockout mutant was sensitive to low temperature or hyperosmotic conditions, which induced colanic acid synthesis, and these sensitivities were abolished by the additional knockout of wcaJ. These results suggest that ECA intermediates cause E. coli susceptibility to low pH, low temperature, and high osmotic pressure in a colanic acid-dependent manner, and that wzxE suppresses this negative effect. ImportancePolysaccharides covering bacterial cell surfaces, such as colanic acid, confer resistance to various stresses, such as low pH. However, the role of enterobacterial common antigens, carbohydrate antigens that are conserved throughout enterobacteria, in stress resistance is unclear. Our results suggest that lipid III enterobacterial common antigen, a substrate of flippase, causes sensitivity of Escherichia coli to low pH, low temperature, and high osmolarity in dependence on colanic acid synthesis, while wzxE inhibits this negative effect. The wzxE-knockout mutant was sensitive to crude vegetable extracts, suggesting that the creation of WzxE inhibitors could lead to new food poisoning prevention agents.

microbiology↗

bioRxiv2024

Overexpression of Ribosomal Proteins Leads to Zn Resistance in Escherichia coli

Knockout of ribosomal protein bL36 (RpmJ) leads to Zn resistance in Escherichia coli, and the expression of ribosomal protein genes other than RpmJ increase in the rpmJ-knockout strain. In this study, we examined whether the overexpression of ribosomal proteins causes Zn resistance using an E. coli overexpression gene library (ASKA clone library). The overexpression of 48 of the 54 ribosomal proteins led to Zn resistance. However, the overexpression of proteins other than ribosomal proteins did not lead to Zn resistance, suggesting that Zn resistance is a phenomenon specific to the overexpression of ribosomal proteins. In addition, the overexpression of ribosomal proteins did not lead to resistance to metal ions other than Zn (Cu2+, Ni2+, Mn2+, and Ag+), suggesting a Zn-specific resistance mechanism. Deletion of ZntA, a Zn efflux pump, resulted in the loss of Zn resistance in a ribosomal protein-overexpressing strain. Deletion of Lon protease, which is responsible for degrading misfolded proteins, in a ribosomal protein-overexpressing strain resulted in the accumulation of overexpressed ribosomal proteins and loss of Zn resistance. These results suggest that the overexpression of ribosomal proteins leads to Zn resistance in E. coli via ZntA and Lon protease. ImportanceThe ribosome is a complex comprising ribosomal RNAs and more than 50 types of ribosomal proteins. Ribosomal proteins play an important role in ribosomal function responsible for protein translation; however, their involvement in other cellular processes is not fully understood. Based on the finding that ribosomal protein expression increases in a Zn-resistant E. coli mutant, we analyzed 54 ribosomal proteins and found that the overexpression of 48 ribosomal proteins led to Zn resistance. This finding suggests a role for ribosomal proteins in resistance to zinc stress.

microbiology↗

bioRxiv2024

Overexpression of diglucosyldiacylglycerol synthase leads to daptomycin resistance in Bacillus subtilis

The lipopeptide antibiotic daptomycin exhibits bactericidal activity against gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanism of bacterial resistance to daptomycin has gained great importance. In the present study, we found that overexpression of ugtP, which encodes a diglucosyldiacylglycerol synthase, resulted in daptomycin resistance in Bacillus subtilis. The amount of diglucosyldiacylglycerol (Glc2DAG) increased in the ugtP-overexpressed strain, whereas the amounts of the acidic phospholipids cardiolipin (CL) and PG and the basic phospholipid lysylphosphatidylglycerol (Lys-PG) decreased. Moreover, ugtP overexpression did not alter the cell surface charge or the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide (CTAB). Furthermore, under daptomycin exposure, we obtained daptomycin-resistant mutants that carried ugtP mutations and showed increased amounts of Glc2DAG and more than 4-fold increase in the MIC of daptomycin. These results suggest that an increase in the amount of Glc2DAG changes the phospholipid composition and leads to daptomycin resistance without altering the bacterial cell surface charge in B. subtilis. ImportanceDaptomycin is one of the last-resort drugs for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections, and the emergence of daptomycin-resistant bacteria has become a problem. Understanding the mechanism of daptomycin resistance is important for establishing clinical countermeasures against daptomycin-resistant bacteria. In the present study, we found that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, causes daptomycin resistance in B. subtilis, a model organism for gram-positive bacteria. Overexpression of ugtP increased diglucosyldiacylglycerol levels, resulting in altered phospholipid composition and daptomycin resistance. These findings are important for establishing clinical strategies against daptomycin-resistant bacteria, including their detection.

microbiology↗

bioRxiv2024

Contactless metabolism estimation of small animals using high-frequency millimeter-wave radar

Animals flexibly adapt to internal and external environmental changes by utilizing energy produced from oxygen as fuel. By non-invasively monitoring an animals oxygen consumption, it becomes possible to understand an individuals metabolic state. Calorimeters are known for directly measuring oxygen consumption but come with the issue of high initial costs. Despite the development of non-invasive techniques for measuring vital signs--including respiration rate (RR), heart rate (HR), and body temperature (Tb)--as indicators of metabolism, conventional methods encounter difficulties in estimating oxygen consumption rate (VO2). In this study, we developed a system that estimates the oxygen consumption of small animals using signals obtained from millimeter wave (mm-wave) radar technology processed through machine learning. By identifying frequency bands within the mm-wave signals contributing to VO2, our system is capable of predicting oxygen consumption several minutes in advance. Our system enables contactless, low-cost and multiplexed measurements of oxygen consumption, presenting a significant advancement in the field.

physiology↗

bioRxiv2024

Arrayed CRISPRi library to suppress genes required for Schizosaccharomyces pombe viability

The fission yeast, Schizosaccharomyces pombe, is an excellent eukaryote model organism to study essential biological processes. Its genome contains [~]1200 genes essential for cell viability, most of which are evolutionarily conserved. To study these essential genes, resources enabling conditional perturbation of target genes are required. Here, we constructed comprehensive arrayed libraries of plasmids and strains to knock down essential genes in S. pombe, using a dCas9-mediated CRISPRi. These libraries cover [~]98% of all essential genes in fission yeast. We estimate that in [~]60% of the strains in the library, transcription of a target gene was repressed so efficiently that cell proliferation was significantly inhibited. To demonstrate usefulness of these libraries, we performed metabolic analyses with knockdown strains and revealed a flexible interaction among metabolic pathways. Libraries established in this study enable comprehensive functional analyses of essential genes in S. pombe and will facilitate understanding of essential biological processes in eukaryotes.

cell biology↗

bioRxiv2023

Characterization and contribution of RPE senescence to Age-related macular degeneration in Tnfrsf10 knock out mice

BackgroundRetinal pigment epithelial cells (RPE) play vital role in the pathogenesis of age-related macular degeneration (AMD). Our laboratory has shown that RPE cellular senescence contributed to the pathophysiology of experimental AMD, and SASP members are involved in this process. Recently, we presented confirmatory evidence to earlier GWAS studies that dysregulation of tumor necrosis factor receptor superfamily 10A (TNFRSF10A) dysregulation leads to AMD development and is linked to RPE dysfunction. This study aims to investigate the contribution of RPE senescence to AMD pathophysiology using TNFRSF10A silenced human RPE (hRPE) cells and Tnfrsf10 KO mice. MethodsSub-confluent primary hRPE cells and TNFRSF10A silenced hRPE were exposed to stress-induced premature senescence with H2O2 (500 M, 48h), and senescence-associated markers ({beta}gal, p16, and p21) were analyzed by RT-PCR and WB analysis. The effect of H2O2-induced senescence in non-silenced and silenced hRPE on OXPHOS and glycolysis was determined using Seahorse XF96 analyzer. Male C57BL/6J Tnfrsf10 KO (Tnfrsf10-/-) mice were used to study the regulation of senescence by TNFRSF10A in vivo. Expression of p16 and p21 in control and KO mice of varying ages were determined by RT-PCR, WB, and immunostaining analysis. ResultsThe senescence-associated p16 and p21 showed a significant (p < 0.01) upregulation with H2O2 induction at the gene (1.8- and 3-fold) and protein (3.2- and 4-fold) levels in hRPE cells. The protein expression of p16 and p21 was further significantly increased by co-treatment with siRNA (p < 0.05 vs. H2O2). Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) (pmol/min/total DNA) increased with senescence induction by H2O2 for 48h in control RPE, and knockdown of TNFRSF10A caused a further increase in OCR and ECAR. In addition, co-treatment with PKC activator significantly improved all parameters. Similarly, in vivo studies showed upregulation of p16 and p21 by RT-PCR, WB, and immunostaining analysis in RPE/choroid of Tnfrsf10 KO mice. When subjected to examination across distinct age groups, namely young (1-3 months), middle (6-9 months), and old (12-15 months) mice, a discernible age-related elevation in the expression of p16 and p21 was observed. ConclusionsOur findings suggest that TNRSF10A is a regulator of regulates in RPE senescence. Further work on elucidating pathways of senescence will facilitate the development of new therapeutic targets for AMD.

pathology↗

bioRxiv2023

A mechanism of melanogenesis mediated by E-cadherin downregulation and its involvement in solar lentigines

BackgroundIntensive studies have revealed pleiotropic melanocytic factors for age spot formation. In particular, dysfunctional keratinocyte differentiation is thought to be an upstream cause of age spot formation. Although keratinocyte differentiation is mediated by a cell-cell contact factor, E-cadherin, its involvement in age spots remains unknown. To find the origin of age spots and an integrated solution, we focused on E-cadherin. MethodsImmunofluorescent staining with cutaneous tissues and cultured cells was performed. Keratinocytes treated with siRNAs were cocultured with melanocytes. With the supernatants of the keratinocyte culture, secretion factors were identified using proteomic analysis. For the activity of melanogenesis and the ingredient screening, a quantitative PCR was performed. For the behavioral analysis of melanocytes, time-lapse imaging of melanocytes was done by confocal laser scanning microscopy. ResultsIn age spots, E-cadherin expression in the epidermis was downregulated, suggesting that E-cadherin is implicated in age spot formation. E-cadherin knockdown (E-cad-KD) keratinocytes not only promoted the secretion of melanocytic/inflammatory factors, but also increased melanogenesis by upregulating the expression of melanogenesis factors. Furthermore, live imaging showed E-cadherin downregulation detained melanocyte dynamics and accelerated melanin-uptake. Finally, we identified Rosa multiflora fruit extract as a solution for upregulating E-cadherin in keratinocytes. ConclusionOur findings showed that E-cadherin downregulation triggers various downstream melanocytic processes such as secretion of melanocytic factors and melanogenesis. Additionally, we showed that Rosa multiflora fruit extract upregulates E-cadherin expression in keratinocytes.

cell biology↗