Biology subjects

Hashimoto, M.

Publications and source records attributed to Hashimoto, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Rapid enhancement of antibiotic resistance and divergent remodeling of gene expression in single cells of bacteria

Exposing bacterial cell populations to antibiotics often results in the emergence of descendants with enhanced resistance. Phenotypic heterogeneity and global physiological responses are thought to contribute to resistance evolution. However, identifying the specific stages of drug exposure where phenotypic heterogeneity and physiological responses play critical roles remains elusive. Using long-term single-cell lineage tracking of an Escherichia coli resistance model, here we reveal that resistance-enhanced cells emerge rapidly within a few generations in a clonal bacterial cell population under chloramphenicol (Cp) exposure and stably transmit the enhanced resistance for hundreds of generations in the presence of Cp. High expression of a resistance protein was advantageous during the initial period of drug exposure, but conferred only a marginal fitness advantage during the period of resistance enhancement. Transcriptomic profiles of resistance-enhanced cell populations were lineage-specific, suggesting that multiple gene expression states can support enhanced resistance. Altogether, these results suggest that an interplay between initial phenotypic heterogeneity and divergent remodeling of gene expression drives the rapid acquisition of enhanced antibiotic resistance.

microbiology↗

bioRxiv2026

Chemical Boosting of Foldase Condensates Accelerates Oxidative Protein Folding

Dynamic biomolecular condensates play crucial roles in intracellular compartmentalization and physiological functions. While engineering tools for compartmentalization have expanded add-on functionalities, directly amplifying the inherent catalytic machinery within biological phase-separated droplets has remained elusive. Herein, we developed a phase-separated oxidative folding reaction chamber based on protein disulfide isomerase A6 (PDIA6) by chemically targeting its active site CxxC motif to enhance enzymatic activity within PDIA6 droplets. A para-substituted N-methylated pyridinylmethanethiol (pMePySH) enhanced the catalytic oxidative folding of bovine pancreatic trypsin inhibitor, proinsulin, and antibody up to 12-fold within in vitro PDIA6 droplets. Furthermore, pMePySH targeted PDIA6 foci within the endoplasmic reticulum, significantly promoting insulin secretion. These findings offer a powerful platform for the spatiotemporal manipulation of protein folding, with profound implications for the scalable manufacturing of therapeutic antibodies and other complex biopharmaceuticals.

bioengineering↗

bioRxiv2026

Simultaneous TCR and IL-2 agonism selectively enhances epitope-specific CD8 T cell responses during chronic viral infection

Interleukin-2 (IL-2) remains an attractive cytokine for enhancing antigen-specific CD8 T cell responses in cancer immunotherapy, but systemic toxicity hinders its broad clinical application. To address this, various IL-2-based therapeutics have been engineered with altered IL-2 receptor bias or targeted delivery to tumors, the tumor microenvironment, or immune cell populations. Ideally, IL-2 signals should be selectively delivered to antigen-specific CD8 T cells, boosting their responses and promoting effector differentiation while sparing non-targeted populations. Immuno-STATTM (Selective Targeting and Alteration of T cells) is a fusion protein platform comprising a bivalent peptide-MHC class I complex and an affinity-attenuated IL-2 mutein that co-stimulates TCR and IL-2 signaling in epitope-specific CD8 T cells. Here, we investigated whether a DbGP33-41-targeted Immuno-STAT enhances DbGP33-specific CD8 T cell responses in a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. Immuno-STAT treatment selectively expanded DbGP33-specific CD8 T cells with an effector-like phenotype. Non-targeted DbGP276-specific CD8 T cells showed little to no expansion in response to DbGP33-41-targeted Immuno-STAT therapy, underscoring the selectivity of this approach. However, minor changes in phenotypic markers, including increased expression of CD25 and CX3CR1, were observed in non-targeted CD8 T cells, likely reflecting bystander IL-2 signaling. Combining Immuno-STAT with PD-1 blockade augmented DbGP33-specific CD8 T cell responses more effectively than PD-1 blockade alone, with minor effects on the non-targeted DbGP276-specific population. These findings inform the clinical development of Immuno-STAT and other IL-2 therapeutics and highlight the value of coordinated TCR and IL-2 stimulation during chronic antigen exposure, alone or in combination with PD-1 blockade. IMPORTANCEInterleukin-2 (IL-2) is a key cytokine for promoting effector differentiation of antigen-specific CD8 T cells and remains an attractive agent in cancer immunotherapy, but systemic toxicity limits its clinical use. This study addresses a central challenge in IL-2-based immunotherapy: delivering IL-2 to cognate antigen-specific CD8 T cells while minimizing activation of non-targeted populations. Using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection, we show that the Immuno-STAT (Selective Targeting and Alteration of T cells) platform selectively expands targeted virus-specific CD8 T cells and enhances their function while limiting effects on non-targeted populations. We also show that combining Immuno-STAT with PD-1 blockade further enhances targeted virus-specific CD8 T cell responses during chronic LCMV infection. These findings provide mechanistic and preclinical support for integrating T cell receptor (TCR) specificity with IL-2 signaling to advance cancer immunotherapy and guide next-generation IL-2 therapeutics for cancer and chronic infection.

immunology↗

bioRxiv2026

Molecular basis of collagen triple helix recognition by VWF A-like domain 2 of collagen VII: Implications for interlaced anchoring fibril formation

Anchoring fibrils formed by collagen VII play a critical role in stabilizing the dermal-epidermal junction. The N-terminal non-collagenous (NC1) domain of collagen VII binds firmly to basement membrane components including collagen IV and has also been reported to interact with mesenchymal fibrillar collagens via its von Willebrand factor A-like domain 2 (A2 domain). To elucidate how collagen VII recognizes fibrillar collagen, we performed yeast two-hybrid screening using a triple-helical random peptide library, which resulted in the identification of a Met-Gly-{Phi} ({Phi}; aromatic amino acid residue) motif. Biochemical analysis with synthetic triple-helical peptides revealed a binding preference of Trp > Phe as the {Phi} residue by the A2 domain despite Trp being absent in native collagens. The crystal structure of the A2 domain in complex with the Nle (Met surrogate)-Gly-Trp-containing peptide revealed a unique mechanism by which two distinct hydrophobic pockets of the A2 domain accommodate the Nle and Trp residues corresponding to the Met-Gly-{Phi} motif, engaging all three chains of the triple helix. Subsequent molecular dynamics simulations demonstrated that the A2 domain recognizes the corresponding native Met-Gly-Phe motif in a similar manner, but with lower affinity, implying a transient interaction with mesenchymal collagens. The findings obtained in this work suggest models in which transient A2-triple helix interaction promotes the recruitment of collagen I and III fibrils into the arc-shaped structure of anchoring fibrils. This also provides a foundation for linking structural understanding to skin fragility diseases caused by collagen VII dysfunction.

biochemistry↗

bioRxiv2025

Mycn Reactivates the Cell Cycle in Adult Cardiomyocytes and Promotes Cardioprotection in Myocardial Infarction

BackgroundAdult mammalian cardiomyocytes are terminally differentiated and possess minimal capacity for cell cycle re-entry. However, recent studies have shown that a rare population of cycling cardiomyocytes may confer cardioprotective effects following myocardial infarction (MI). The Myc family--Myc, Mycl, and Mycn--plays central roles in regulating cell cycle progression and cellular plasticity. This raises the possibility that specific Myc isoforms could reactivate the cardiomyocyte cell cycle and promote cardioprotective responses in the ischemic heart. This study aimed to systematically evaluate the effects of Myc, Mycl, and Mycn on cardiomyocyte cell cycle activation and cardiac outcomes following MI. MethodsCardiomyocyte-specific overexpression of Myc, Mycl, or Mycn was achieved in adult mice via adeno-associated virus 9 vectors driven by the cardiac troponin T promoter. Cardiomyocyte cell cycle activity, structural remodeling, and functional outcomes were assessed using RNA sequencing, immunohistochemistry, and transthoracic echocardiography. Cardioprotective effects were evaluated in MI model mice. ResultsWhile all three Myc family members activated cell cycle-related gene expression to varying degrees, Mycn elicited the most robust response. Mycn overexpression significantly enhanced cardiomyocyte cell cycle re-entry, as demonstrated by increased 5-bromo-2-deoxyuridine incorporation and phosphorylation of histone H3. Mycn also induced hypertrophic growth, reflected by increased cardiomyocyte size and heart mass. Transcriptomic analyses revealed that Mycn uniquely upregulated genes associated with extracellular matrix remodeling and paracrine signaling, typically enriched in neonatal cardiomyocytes and previously linked to cardioprotection. Functionally, Mycn expression preserved cardiac contractility after MI, reduced infarct size, and increased capillary density in peri-infarct regions. ConclusionsThese findings identify Mycn as a functionally distinct member of the Myc family in the adult heart, capable of reactivating the cardiomyocyte cell cycle and promoting cardiac protection after ischemic injury. The cardioprotective effects appear to be mediated, in part, by the induction of a neonatal-like gene expression program supporting a stress-adaptive cardiac microenvironment.

molecular biology↗

bioRxiv2024

Fate specification triggers a positive feedback loop of TEAD-YAP and NANOG to promote epiblast formation in preimplantation embryos

In preimplantation embryos, epiblast (EPI) fate specification from the inner cell mass is controlled by the segregation of NANOG and GATA6 expression. TEAD-YAP interaction is activated during EPI formation, and is required for pluripotency factor expression. These events occur asynchronously with similar timing during EPI formation, and their relationship remains elusive. Here, we examined the relationship between NANOG-GATA6 and TEAD-YAP. The nuclear accumulation of YAP takes place only in EPI-specified cells, and a positive feedback loop operates between NANOG and TEAD-YAP. The effects of TEAD-YAP on SOX2 upregulation in EPI-specified cells are likely indirect. EPI fate specification also alters the response of Nanog, Sox2 and Cdx2 to TEAD-YAP. These results suggest that EPI-fate specification alters the transcriptional network from the morula-like to the EPI-specified state and activates TEAD-YAP to trigger a positive feedback loop with NANOG, which stabilizes the EPI fate. The coordinated occurrence of these processes in individual cells likely supports proper EPI formation under the condition of asynchronous EPI-fate specification. Summary statementEpiblast fate specification by segregation of NANOG from GATA6 alters the transcriptional network and activates TEAD-YAP, triggering a positive feedback loop with NANOG to promote epiblast formation.

developmental biology↗

bioRxiv2024

Ca2+-driven PDIA6 phase separation to ensure proinsulin quality control

The endoplasmic reticulum (ER) plays key roles in protein quality control1,2 and dynamic Ca2+ storage3,4 in eukaryotic cells. However, the protein homeostasis (proteostasis) system that regulates these ER functions is still incompletely characterised. Previous study revealed the importance of oligomerization in the function PDIA1, an ER-resident disulfide isomerase and molecular chaperone, regulates oligomeric states in accordance with client folding5. This result suggests that at least some of the 20 members of other PDI family may undergo regulated self-assembly in order to optimally function. Here, we show that Ca2+ triggers the phase separation of PDIA6 into liquid-like condensates. In contrast to the condensation mechanism observed for proteins containing low-complexity domains, our results indicate that transient but specific electrostatic interactions occur between the first and the third folded thioredoxin-like domains of PDIA6. We further show that the Ca2+-driven condensation of PDIA6 recruits PDIA3 and proinsulin, thus increasing their local concentrations. This process results in the 30-fold enhancement of proinsulin folding and in the inhibition of proinsulin aggregation. Our findings shed light on a condensation-driven Ca2+-mediated proteostasis cascade in the ER by revealing how the efficiency of the protein folding process can be enhanced within quality control granules.

biochemistry↗

bioRxiv2024

CENP-C-Mis12 complex establishes a regulatory loop through Aurora B for chromosome segregation

Establishing the correct kinetochore-microtubule attachment is crucial for faithful chromosome segregation. The kinetochore has various regulatory mechanisms for establishing correct bipolar attachment. However, how the regulations are coupled is not fully understood. Here, we demonstrate a regulatory loop between the kinetochore protein CENP-C and Aurora B kinase, which is critical for the error correction of kinetochore-microtubule attachment. This regulatory loop is mediated through the binding of CENP-C to the outer kinetochore Mis12 complex (Mis12C). Although the Mis12C binding region of CENP-C is dispensable for mouse development and proliferation in human RPE-1 cells, those cells lacking this region display increased mitotic defects. The CENP-C-Mis12C interaction facilitates the centromeric recruitment of Aurora B and the mitotic error correction in human cells. Given that Aurora B reinforces the CENP-C-Mis12C interaction, our findings reveal a positive regulatory loop between Aurora B recruitment and the CENP-C-Mis12C interaction, which ensures chromosome bi-orientation for accurate chromosome segregation.

cell biology↗

bioRxiv2023

Organ-on-a-Chip Fabrication using Dynamic Photomask

Organ-on-a-chip (OoC) technology is a powerful tool for creating physiologically relevant microscale models applicable to biomedical studies. Despite the advances in OoC technology, its fabrication method still primarily relies on soft lithography, which (1) lacks the adaptability to accommodate dynamic cell culture (e.g., spheroids and organoid culture) and (2) has a long design-to-prototype cycle that lowers its manufacturability. To overcome these challenges, we developed a system to fabricate OoC (consisting of microchannels and multiple cell types in a well-defined spatial arrangement) dynamically using a digital photomask aligned with a microchamber. Our approach used a pre-defined microfluidic chamber customized by xurography and cell-laden microfluidic channels photopatterned by a digital photomask; the entire design-to-prototype cycle was achieved within two hours. The versatility of our approach offered previously unattainable crucial features in the fabrication of OoC, including a gradual change in the height of the microchannels, and real-time modification of channel designs to trap live tissues (e.g., spheroids). In summary, this work highlights a versatile system to fabricate OoC that can accommodate various design requirements of microenvironments of specific organ tissues. We envision the effectiveness of our system for the rapid fabrication of OoC to contribute to the wide adoption of the technology for therapeutic screening and elucidation of disease mechanisms in both academic and industrial settings.

bioengineering↗

bioRxiv2023

3D Bioprinting Using Poly(ethylene-glycol)-dimethacrylate (PEGDMA) Composite

Recent progress in additive manufacturing has enabled rapid printing of bioinspired structures such as vasculature and alveoli using stereolithography (SLA) bioprinting. Bioinks for SLA often require synthetic polymers as additives to ensure the structural integrity of the printed cell-laden constructs. To this end, high molecular weight (MW) poly(ethylene-glycol)- diacrylate (PEGDA) (MW = 3400) is commonly used to enhance the mechanical property of crosslinked hydrogels, which requires in-house polymer synthesis or the acquisition of costly reagents. This research investigated the use of poly(ethylene-glycol)-dimethacrylate (PEGDMA) (MW = 1000) as a component of a composite bioink to enhance the mechanical properties of the SLA-printed constructs. We successfully demonstrated the fabrication of three-dimensional (3D) constructs with overhang and complex architecture, while human colorectal cancer cells (Caco-2) embedded in the crosslinked bioink exhibited the capability to proliferate on Day 6 of in vitro cell culture. Our study suggested PEGDMA as a viable alternative to high MW PEGDA used in SLA bioprinting. The accessibility to PEGDMA will facilitate the advance in 3D bioprinting to fabricate complex bioinspired structures and tissue surrogates for biomedical applications. Article HighlightsO_LIPoly(ethylene-glycol)-dimethacrylate (PEGDMA) can be used in cell-laden bioprinting to enhance the mechanical property of bioinks. C_LIO_LIPEGDMA-based bioink was non-cytotoxic and conducive to cell proliferation. C_LIO_LIThe facile preparation of PEGDMA composite ink will help to accelerate the research in tissue engineering via bioprinting. C_LI

bioengineering↗

bioRxiv2023

Quinoclamine inhibits Shiga toxin production in enterohemorrhagic Escherichia coli

ObjectivesEnterohemorrhagic Escherichia coli (EHEC) is responsible for the most severe symptoms of E. coli infections, including hemorrhagic colitis and hemorrhagic uremic syndrome. Shiga toxin 2 (Stx2) plays a significant role as a major virulence factor. The genes encoding Stx2 locate in lambda-like prophage on the EHEC genome. Consequently, Stx2 is expressed when production of the phage is induced by the SOS response. Antibiotic treatment is not recommended for curing the bacterial infection, because it is associated with severe hemorrhagic uremic syndrome. If Stx2 production is prevented, EHEC pathogenicity significantly decreases, and antibiotics may be available to treat the infection. MethodsWe conducted two independent screenings to identify Stx2 production inhibitors for libraries from the RIKEN Natural Product Depository (NPDepo); namely, screening of the Authentic Library, and two-round screening of the Pilot and Analog Libraries. ResultsThe screening of Authentic Library identified niclosamide as a Stx2 production inhibitor. Besides, two naphthoquinoids were identified after the two-round of screening of the Pilot and Analog Libraries. Niclosamide, and quinoclamine, which has structure shared in the two naphthoquinoids, prevented cell lysis via the phage production and ceased Stx2 production in EHEC. The SOS reporter assay indicated that quinoclamine prevented the SOS response in E. coli, whereas niclosamide did not. ConclusionsThese findings suggest that quinoclamine inhibited Stx2 production by preventing the SOS response, whereas niclosamide was involved in phage propagation following the SOS response. These compounds can be a potential therapeutic option to treat EHEC infections.

microbiology↗

bioRxiv2021

Biomimetic Vasculatures by 3D-Printed Porous Molds

Anatomically and biologically relevant vascular models are critical to progress our understanding of cardiovascular diseases (CVDs) that can lead to effective therapies. Despite advances in 3D bioprinting, recapitulating complex architectures (i.e., freestanding, branching, multilayered, perfusable) of a cell-laden vascular construct remains technically challenging, and the development of new techniques that can recapitulate both anatomical and biological features of blood vessels is of paramount importance. In this work, we introduce a unique, microfluidics-enabled molding technique that allows us to fabricate anatomically-relevant, cell-laden hydrogel vascular models. Our approach employed 3D-printed porous molds of poly(ethylene glycol) diacrylate (PEGDA) as templates to cast alginate-containing bioinks. Due to the porous and aqueous nature of the PEGDA mold, the calcium ion (Ca2+) was diffusively released to crosslink the bioinks to create hollow structures. Applying this technique, multiscale, multilayered vascular constructs that were freestanding and perfusable were readily fabricated using cell-compatible bioinks (i.e., alginate and gelatin methacryloyl (GelMA)). The bioinks were also readily customizable to either improve the compatibility with specific vascular cells or tune the mechanical modulus to mimic native blood vessels. Importantly, we successfully integrated smooth muscle cells and endothelial cells in a biomimetic organization within our vessel constructs and demonstrated a significant increase in monocyte adhesion upon stimulation with an inflammatory cytokine, tumor necrosis factor-alpha (TNF-). We also demonstrated that the fabricated vessels were amenable for testing percutaneous coronary interventions (i.e., drug-eluting balloons and stents) under physiologically-relevant mechanical states, such as vessel stretching and bending. Overall, we introduce a versatile fabrication technique with multi-faceted possibilities of generating biomimetic vascular models that can benefit future research in mechanistic understanding of CVD progression and the development of therapeutic interventions.

bioengineering↗

bioRxiv2021

Short 5'UTR enables optimal translation of plant virus tricistronic RNA via leaky scanning

Regardless of the general model of translation in eukaryotic cells, a number of studies suggested that many of mRNAs encode multiple proteins. Leaky scanning, which supplies ribosomes to downstream open reading frames (ORFs) by read-through of upstream ORFs, is the most major regulatory mechanism to translate polycistronic mRNAs. However, the general regulatory factors controlling leaky scanning and their biological relevance have rarely been elucidated, with exceptions such as the Kozak sequence. Here, we have analyzed the strategy of a plant RNA virus to translate three movement proteins from a single RNA molecule through leaky scanning. The in planta and in vitro results indicate that significantly shorter 5' UTR of the most upstream ORF promotes leaky scanning, potentially finetuning the translation efficiency of the three proteins in a single RNA molecule to optimize viral propagation. Moreover, in plant endogenous mRNAs, we found that shorter UTRs were more frequently observed in uORFs of polycistronic mRNAs. We propose that the promotion of leaky scanning induced by a short 5' UTR (LISH), together with the Kozak sequence, is a conserved gene regulation mechanism not only in viruses but also in eukaryotes.

plant biology↗

bioRxiv2021

A fungal powdery mildew pathogen induces extensive local and marginal systemic changes in the Arabidopsis thaliana microbiota

O_LIPowdery mildew is a foliar disease caused by epiphytically growing obligate biotrophic ascomycete fungi. How powdery mildew colonization affects host resident microbial communities locally and systemically remains poorly explored. C_LIO_LIWe performed powdery mildew (Golovinomyces orontii) infection experiments with Arabidopsis thaliana grown in either natural soil or a gnotobiotic system and studied the influence of pathogen invasion into standing natural multi-kingdom or synthetic bacterial communities (SynComs). C_LIO_LIWe found that after infection of soil-grown plants, G. orontii outcompetes numerous resident leaf-associated fungi. We further detected a significant shift in foliar but not root-associated bacterial communities in this setup. Pre-colonization of germ-free A. thaliana leaves with a bacterial leaf-SynCom, followed by G. orontii invasion, induced an overall similar shift in the foliar bacterial microbiota and minor changes in the root-associated bacterial assemblage. However, a standing root SynCom in root samples remained robust against foliar infection with G. orontii. Although pathogen growth was unaffected by the leaf SynCom, fungal infection caused a more than two-fold increase in leaf bacterial load. C_LIO_LIOur findings indicate that G. orontii infection affects mainly microbial communities in local plant tissue, possibly driven by pathogen-induced changes in source-sink relationships and host immune status. C_LI

plant biology↗

bioRxiv2020

Membrane-proximal external region is a superior target for mediating effector activity of HIV-1 specific chimeric antigen receptor modified T cells.

The use of chimeric antigen receptor modified-T (CAR-T) cells in adoptive immunotherapy has been popularized through recent success in the field of cancer treatment research. CD4{zeta} CAR, which targets HIV-1-infected cells, has been developed and evaluated in patients. Though well-tolerated for over a decade, efficacy was disappointingly limited. This result encourages us to develop a novel CAR more effective than CD4{zeta} CAR. To quantitatively compare anti-HIV-1 activity of different CAR constructs in a highly sensitive and reproducible manner, we developed a multicolor flow cytometry method for assessing anti-HIV-1 effector T-cell activity. "Target" Jurkat cells inducibly expressing an HIV-1HXBC2 envelope protein and "Non-target" control cells were genetically labeled with red and blue fluorescent protein, respectively, and co-incubated with human primary T cells transduced with anti-HIV-1 "Effector" CARs at various Effector vs Target cell ratios. Absolute cell numbers of each population were collected by MACSQuant Analyzer and used for calculation of relative cytotoxicity. We successfully ranked the cytotoxicity of three previously reported single chain-antibody CARs and six newly developed single-domain antibody CARs in comparison to CD4{zeta} CAR. Interestingly, three CARs--10E8, 2E7, and 2H10--which demonstrate high cytotoxic activity were all known to target the membrane-proximal external region. Use of this novel assay will simplify assessment of new CAR constructs and in turn accelerate the development of new effective CARs against HIV-1. Author SummaryAdoptive immunotherapies that utilize autologous T cells expressing a desired antigen-specific CAR aim to elicit directed immune responses. In recent years, CAR immunotherapies have been promoted extensively in B cell malignancy treatments. The HIV-1-targeting CAR, known as CD4{zeta}, was developed over 20 years ago and has been widely and longitudinally tested in patients. However, its effectiveness was hindered by poor survival and functionality of the transduced cells. To conduct quantitative evaluation of newly designed anti-HIV-1 CARs, we developed a novel multicolor flow-based assay for HIV-1-specific cytotoxicity, enabling sensitive and quantitative assessment in a high-throughput fashion. This assay would be also useful in screening HIV-1-targeting immune receptors--including CARs and T cell receptors--and other immunotherapeutic drugs such as anti-HIV-1 antibodies.

microbiology↗

bioRxiv2019

Pathogenicity of urinary tract infection Escherichia coli in Caenorhabditis elegans

Uropathogenic Escherichia coli (UPEC) is a major bacterial pathogen that causes urinary tract infections (UTIs). Several virulence factors (VFs) in the bacteria have been associated with the pathogenicity. The mouse is an available UTI model for studying the pathogenicity; however, Caenorhabditis elegans represents as an alternative surrogate host for studying UPEC with the capacity for high-throughput analysis. Therefore, we established a simple assay for a UPEC infection model with C. elegans for large-scale screening. An E. coli culture to be tested and synchronized C. elegans were mixed in 96-well plates, and the pathogenicity was determined by comparison of the turbidity before and after incubation. A total of 133 clinically isolated E. coli strains, which included UTI-associated and fecal isolates, were applied to demonstrate the liquid pathogenicity assay. The E. coli isolates associated with UTIs showed higher pathogenicity in C. elegans than the fecal isolates, suggesting that the simple assay with C. elegans is useful as a UPEC infectious model. From the screening, VFs involved with iron acquisition (chuA, fyuA, and irp2) were significantly associated with high pathogenicity. C. elegans is a heme auxotroph, and iron homeostasis also serves innate immunity in C. elegans. We then evaluated whether the VFs in UPEC were involved in the pathogenicity. Mutants of E. coli UTI89 with defective iron acquisition systems were applied to a solid killing assay with C. elegans. As a result, the survival rate of C. elegans fed with the mutants significantly increased compared to when fed with the parent strain. To our knowledge, this is the first report of the involvement of iron acquisition in the pathogenicity of UPEC in a C. elegans model.

microbiology↗

bioRxiv2019

Self-Organization of Whole Gene Expression through Coordinated Chromatin Structural Transition: Validation of Self-Organized Critical Control of Genome Expression

Through our studies on whole genome regulation, we have demonstrated the existence of self-organized critical control (SOC) of whole gene expression - genomic self-organization mechanism through the emergence of a critical point (CP) at both the cell population and single cell level. In this paper, based on HRG and EGF-stimulated MCF-7 breast-cancer cell line, we shed light on the origin of critical transitions stemming from coordinated chromatin remodeling. In so doing, we validated the core of the SOC control mechanism through the application of a non-linear signal analysis technique (Recurrence Quantification Analysis: RQA), and of Principal Component Analysis (PCA). The main findings were: O_LITranscriptional co-regulation follows a strong and invariant exponential decay as between gene spacing along the chromosome is increased. This shows that the co-regulation occurs on a mainly positional basis reflecting local chromatin organization. C_LIO_LIThere are two main fluctuation modes on the top of the cell-kind specific gene expression values spanning the entire genome expression. These modes establish an autonomous genomic critical control system (genome-engine) through the activation of the CP for cell-fate guiding critical transitions revealed by SOC analysis. C_LI The elucidation of the link between spatial position on chromosome and co-regulation together with the identification of specific locations on the genome devoted to the generalization of perturbation stimuli, give a molecular basis to the self-organization dynamics of genome expression and cell-fate decision.

genomics↗

bioRxiv2019

Field evaluation of a quantitative, and rapid malaria diagnostic system using a fluorescent Blue-ray optical device

We improved a previously developed quantitative malaria diagnostic system based on fluorescent Blue-ray optical device. Here, we first improved the diagnostic system to enable fully automated operation and the field application was evaluated in Kenya. We detected Plasmodium falciparum in blood samples collected from 288 individuals aged 1-16 years using nested polymerase chain reaction (nPCR), rapid diagnostic test (RDT), and automated system. Compared to RDT, the automated system exhibited a higher sensitivity (100%; 95% confidence interval [CI], 93.3-100%) and specificity (92.8%; 95%CI, 88.5-95.8%). The limit of detection was 0.0061%. Linear regression analysis revealed a correlation between the automated system and microscopic examination for detecting parasitemia (adjusted R2 value=0.63, P=1.13x10-12). The automated system exhibited a stable quantification of parasitemia and a higher diagnostic accuracy for parasitemia than RDT. This indicates the potential of this system as a valid alternative to conventional methods used at local health facilities, which lack basic infrastructure.

physiology↗