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Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling., Graphical Abstract, Yahata et al. refined a previous G13513A-mpTALEN to more efficiently manipulate mtDNA heteroplasmy in MELAS-iPSCs. Mutation-rich MELAS-iPSCs showed unusual differentiation properties, which were recovered by decreasing the heteroplasmy levels with new G13513A-mpTALEN. They further demonstrated that drug-inducible, MYOD-transfected MELAS-iPSCs could efficiently differentiate into MyHC-positive myocytes, with or without mutant mtDNA.

BackgroundMatricellular glycoprotein, SPARC is a secreted molecule, that mediates the interaction between cells and extracellular matrix. SPARC functions as a regulator of matrix organization and modulates cell behavior. In various kinds of cancer, strong SPARC expression was observed in stromal tissues as well as in cancer epithelial cells. The function of SPARC in cancer cells is somewhat controversial and its impact on peritumoral stromal cells remains to be resolved.MethodsWe investigated the effects of SPARC expression in endometrial cancer cells on the surrounding stromal fibroblasts using in vitro co-culture system. Changes in characteristics of fibroblasts were examined by analysis of fibroblast-specific markers and in vitro contraction assay.ResultsSPARC induced AKT phosphorylation and epithelial-to-mesenchymal transition, consistent with previous reports. Cancer-associated fibroblasts of endometrial cancer expressed higher levels of mesenchymal- and fibroblast-associated factors and had a stronger contraction ability. Unexpectedly, cancer-associated fibroblasts expressed comparable levels of SPARC compared with fibroblasts from normal endometrium. However, co-culture of normal fibroblasts with SPARC-expressing Ishikawa cells resulted in activation of the fibroblasts. Immunodepletion of SPARC did not affect the activation of fibroblasts.ConclusionsOur data indicated that SPARC activated fibroblasts only in the presence of fibronectin, which was abundantly secreted from SPARC-expressing endometrial cancer cells. These results suggested that a SPARC-fibronectin-mediated activation of fibroblasts might be involved in enhanced mobility and invasion of cancer cells.

Aflatoxin produced by Aspergillus flavus is known to be strongly related to liver injury (hepatocellular carcinoma) and immune system damage involving leukocytes. This toxin suppresses both the cell-mediated immune system and macrophage function, and decreases the production of complement and interferon molecules. Purpose To evaluate the presence of aflatoxin in infectious lesions as well as how the toxin is taken up by leukocytes. Method Pathological specimens from a patient who died from aspergillosis caused by aflatoxin-producing A. flavus were used. Anti-aflatoxin B1 antibody was reacted with paraffin-embedded lesion specimens from the heart, kidney, and thyroid gland of the patient and observed microscopically. Result Positive reactions were detected in fungal elements and leukocytes (neutrophils and macrophages) in inflammatory lesions. Conclusion Within the patient's body, A. flavus likely produced aflatoxin, which then was taken up by neutrophils and macrophages.These results suggest that leukocyte function and the immune mechanism are locally suppressed by aflatoxin.

Apical periodontitis (AP) is an acute or chronic inflammatory disease caused by complex interactions between infected root canal and host immune system. It results in the induction of inflammatory mediators such as chemokines and cytokines leading to periapical tissue destruction. To understand the molecular pathogenesis of AP, we have investigated inflammatory-related genes that regulate AP development. We found here that macrophage-derived CXCL9, which acts through CXCR3, is recruited by progressed AP. The inhibition of CXCL9 by a CXCR3 antagonist reduced the lesion size in a mouse AP model with decreasing IL-1β, IL-6 and TNFα expression. The treatment of peritoneal macrophages with CXCL9 and LPS induced the transmigration and upregulation of osteoclastogenic cytokines such as IL-1β, IL-6 and matrix metalloprotease 2, a marker of activated macrophages. This suggests that the CXCL9-CXCR3 axis plays a crucial role in the development of AP, mediated by the migration and activation of macrophages for periapical tissue destruction. Our data thus show that CXCL9 regulates the functions of macrophages which contribute to AP pathogenesis, and that blocking CXCL9 suppresses AP progression. Knowledge of the principal factors involved in the progression of AP, and the identification of related inflammatory markers, may help to establish new therapeutic strategies.

In early 2020, Japan repatriated 566 nationals from China. Universal laboratory testing and 14-day monitoring of returnees detected 12 cases of severe acute respiratory syndrome coronavirus 2 infection; initial screening results were negative for 5. Common outcomes were remaining asymptomatic (n = 4) and pneumonia (n = 6). Overall, screening performed poorly.

Alzheimer's disease (AD) is the most common type of dementia, and its pathogenesis is associated with accumulation of β-amyloid (Aβ) peptides. Aβ is produced from amyloid precursor protein (APP) that is sequentially cleaved by β- and γ-secretases. Therefore, APP processing has been a target in therapeutic strategies for managing AD; however, no effective treatment of AD patients is currently available. Here, to identify endogenous factors that modulate Aβ production, we performed a gene microarray–based transcriptome analysis of neuronal cells derived from human induced pluripotent stem cells, because Aβ production in these cells changes during neuronal differentiation. We found that expression of the glycophosphatidylinositol-specific phospholipase D1 (GPLD1) gene is associated with these changes in Aβ production. GPLD1 overexpression in HEK293 cells increased the secretion of galectin 3–binding protein (GAL3BP), which suppressed Aβ production in an AD model, neuroglioma H4 cells. Mechanistically, GAL3BP suppressed Aβ production by directly interacting with APP and thereby inhibiting APP processing by β-secretase. Furthermore, we show that cells take up extracellularly added GAL3BP via endocytosis and that GAL3BP is localized in close proximity to APP in endosomes where amyloidogenic APP processing takes place. Taken together, our results indicate that GAL3BP may be a suitable target of AD-modifying drugs in future therapeutic strategies for managing AD.

Plasmodium, the causative agents of malaria, are obligate intracellular organisms. In humans, pathogenesis is caused by the blood stage parasite, which multiplies within erythrocytes, thus erythrocyte invasion is an essential developmental step. Merozoite form parasites released into the blood stream coordinately secrets a panel of proteins from the microneme secretory organelles for gliding motility, establishment of a tight junction with a target naive erythrocyte, and subsequent internalization. A protein identified in Toxoplasma gondii facilitates microneme fusion with the plasma membrane for exocytosis; namely, acylated pleckstrin homology domain-containing protein (APH). To obtain insight into the differential microneme discharge by malaria parasites, in this study we analyzed the consequences of APH deletion in the rodent malaria model, Plasmodium yoelii, using a DiCre-based inducible knockout method. We found that APH deletion resulted in a reduction in parasite asexual growth and erythrocyte invasion, with some parasites retaining the ability to invade and grow without APH. APH deletion impaired the secretion of microneme proteins, MTRAP and AMA1, and upon contact with erythrocytes the secretion of MTRAP, but not AMA1, was observed. APH-deleted merozoites were able to attach to and deform erythrocytes, consistent with the observed MTRAP secretion. Tight junctions were formed, but echinocytosis after merozoite internalization into erythrocytes was significantly reduced, consistent with the observed absence of AMA1 secretion. Together with our observation that APH largely colocalized with MTRAP, but less with AMA1, we propose that APH is directly involved in MTRAP secretion; whereas any role of APH in AMA1 secretion is indirect in Plasmodium., Parasitology International, 86, art. no. 102479; 2021

The acquisition of drug resistance and virulence by staphylococcal species colonizing humans is a growing public health concern. The present study was conducted to investigate the prevalence, antimicrobial resistance and genetic characteristics of Staphylococcus isolates from the oral cavity and skin (hand) of systemically healthy subjects with dental disease and dental staff in northern Japan. Among a total of 133 subjects (91 patients and 42 staff), 87 coagulase-positive Staphylococcus (83 S. aureus/4 S. argenteus) and 162 coagulase-negative Staphylococcus (CoNS) isolates were recovered from 59 (44.4%) and 95 (71.4%) subjects, respectively. Three oral isolates were methicillin-resistant S. aureus (MRSA) (3.6%, 3/83) that were genotyped as ST8-SCCmec-IVl, ST4775(CC1)-SCCmec-IVa and ST6562(CC8)-SCCmec-IVa. Remarkably, the ST6562 isolate harbored PVL genes on ΦSa2usa and type I ACME (arginine catabolic mobile element). Four methicillin-susceptible isolates were identified as S. argenteus belonging to ST1223 and ST2250, which harbored enterotoxin genes egc-2 and sey, respectively. Among the fourteen CoNS species identified, methicillin-resistant (MR) isolates were detected in five species (11 isolates, 13.3% of CoNS), with S. saprophyticus and S. haemolyticus being the most common. ACME was prevalent in only S. epidermidis and S. capitis. These findings indicated the potential distribution of USA300 clone-like MRSA, toxigenic S. argenteus and MR-CoNS in the oral cavity of dental patients.

Newts are unique salamanders that can regenerate their limbs as postmetamorphic adults. In order to regenerate human limbs as newts do, it is necessary to determine whether the cells homologous to those contributing to the limb regeneration of adult newts also exist in humans. Previous skin manipulation studies in larval amphibians have suggested that stump skin plays a pivotal role in the axial patterning of regenerating limbs. However, in adult newts such studies are limited, though they are informative. Therefore, in this article we have conducted skin manipulation experiments such as rotating the skin 180° around the proximodistal axis of the limb and replacing half of the skin with that of another location on the limb or body. We found that, contrary to our expectations, adult newts robustly regenerated limbs with a normal axial pattern regardless of skin manipulation, and that the appearance of abnormalities was stochastic. Our results suggest that the tissue under the skin, rather than the skin itself, in the intact limb is of primary importance in ensuring the normal axial pattern formation in adult newt limb regeneration. We propose that the important tissues are located in small areas underlying the ventral anterior and ventral posterior skin.

Notch signaling primarily determines T-cell fate. However, the molecular mechanisms underlying the maintenance of T-lineage potential in pre-thymic progenitors remain unclear. Here, we established two murine Ebf1-deficient pro-B cell lines, with and without T-lineage potential. The latter expressed lower levels of Lmo2; their potential was restored via ectopic expression of Lmo2. Conversely, the CRISPR/Cas9-mediated deletion of Lmo2 resulted in the loss of the T-lineage potential. Introduction of Bcl2 rescued massive cell death of Notch-stimulated pro-B cells without efficient LMO2-driven Bcl11a expression but was not sufficient to retain their T-lineage potential. Pro-B cells without T-lineage potential failed to activate Tcf7 due to DNA methylation; Tcf7 transduction restored this capacity. Moreover, direct binding of LMO2 to the Bcl11a and Tcf7 loci was observed. Altogether, our results highlight LMO2 as a crucial player in the survival and maintenance of T-lineage potential in T-cell progenitors via the regulation of the expression of Bcl11a and Tcf7.

Gerbera in vitro shoots were irradiated using three types of ion beams with different line energy transfers (LETs) to investigate the effective LET and absorbed doses for mutagenesis. Furthermore, genomic mutation analyses were conducted on the obtained mutants. Survival rate analysis showed a lower lethal dose 50% (LD50) with ion beams with higher LETs. Trait/morphological mutations exhibited changes in the color and shape of petals and male sterility. Irradiation conditions with the highest growth change and trait/morphological mutation rates in each ion were C irradiation at 10 Gy, Ar irradiation at 5 Gy, and Fe irradiation at 5 Gy, with a range of absorbed dose of around LD50 to about 10 Gy lower. The highest trait/morphological mutation rate was 14.1% with Ar irradiation at 5 Gy, which was one of the criteria for ion beam irradiation of gerbera in vitro shoots. Furthermore, the genomic mutation in the flower color, petal shape, and male sterile mutants were confirmed by genotype analysis using Genotyping by Random Amplicon Sequencing-Direct technology. This is the first study to report the efficient production of gerbera mutants that could be analyzed. Our findings may lead to more efficient gerbera mutant production and analysis technology.

Gα13, a heterotrimeric G-protein of the Gα12/13 subfamily, is associated with aggressive phenotypes in various human cancers. However, the mechanisms by which Gα13 promotes cancer progression have not been fully elucidated. Here, we demonstrate that the activation of Gα13 induces epithelial-mesenchymal transition in ovarian cancer (OvCa) cells through down-regulation of large tumor suppressor kinase (LATS) 1, a critical component of the Hippo signaling pathway. A synthetic biology approach using a mutant GPCR and chimeric G-protein revealed that Gα13-regulated phosphorylation of LATS1 at serine 909 within its activation loop induced recruitment of the itchy E3 ubiquitin protein ligase to trigger LATS1 degradation. Our findings uncover novel mechanisms through which Gα13 activation induces dysregulation of the Hippo signaling pathway, which leads to aggressive cancer phenotypes, and thereby identify a potential target for preventing the metastatic spread of OvCa.-Yagi, H., Onoyama, I., Asanoma, K., Hori, E., Yasunaga, M., Kodama, K., Kijima, M., Ohgami, T., Kaneki, E., Okugawa, K., Yahata, H., Kato, K. Gα13-mediated LATS1 down-regulation contributes to epithelial-mesenchymal transition in ovarian cancer.

Pseudomonas syringae pv. actinidiae (Psa) has been shown to have heterotypic populations including a pandemic highly virulent strain (Psa3 or Psa-V). Although Psa was first recorded in Japan, the virulent strain Psa3 had not been observed until spring of 2014, when the sudden outbreak of Psa3 took place in Japan. Also in Shizuoka Prefecture, we noticed the incidence of bacterial leaf spots in several kiwifruit orchards in June 2014. Isolation of the causal bacterium and identification through PCR and other techniques confirmed that the pathogen was Psa3. In one representative field, the disease was observed on Actinidia deliciosa 'Hayward'. Similar leaf spots were also observed on A. chinensis cultivars planted in the same field, however, the pathogen was identified as a strain of Pseudomonas syringae causing flower-rot (blossom blight) disease of kiwifruit (Ps-fr). The differences between Psa3, Psa1 and Ps-fr were confirmed. Sometimes, Psa3 and Ps-fr were isolated from a single lesion. The transition of the disease incidence from 2014 to 2015 was described. It was demonstrated that Ps-fr inhibited the growth of Psa3 in vitro.

Programmed cell death ligand 1 (PD‐L1) on tumor cells suppresses anti‐tumor immunity and has an unfavorable prognostic impact in ovarian cancer patients. We herein report the pathophysiological and therapeutic impacts of PD‐L1 disruption in ovarian cancer. PD‐L1 was genetically disrupted in the murine ovarian cancer cell line ID8 using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated genome editing. PD‐L1 knockout (KO) and control ovarian cancer cells were intraperitoneally inoculated into syngeneic mice, and survival and tumor dissemination were evaluated. Survival times were significantly longer in the PD‐L1‐KO ID8‐inoculated groups than in their control groups, and its therapeutic benefit was enhanced in combination with the cisplatin treatment. Tumor weights and ascites volumes were significantly lower in the PD‐L1‐KO ID8 groups than in their control groups. Immunohistochemical and immunofluorescence analyses showed that intratumoral CD4+ T cells, CD8+ T cells, NK cells and CD11c+ M1 macrophages were significantly increased, whereas regulatory T cells were significantly decreased in the PD‐L1‐KO ID8 groups compared with those in their control groups. The intratumoral mRNA expression of interferon‐γ, tumor‐necrosis factor‐α, interleukin (IL)‐2, IL‐12a, CXCL9 and CXCL10 was significantly stronger, while that of IL‐10, vascular endothelial growth factor, CXCL1 and CXCL2 was significantly weaker in the PD‐L1‐KO ID8 groups. These results indicate that CRISPR/Cas9‐mediated PD‐L1 disruption on tumor cells promotes anti‐tumor immunity by increasing tumor‐infiltrating lymphocytes and modulating cytokine/chemokine profiles within the tumor microenvironment, thereby suppressing ovarian cancer progression. These results suggest that PD‐L1‐targeted therapy by genome editing may be a novel therapeutic strategy for ovarian cancer.

Plasmodium malaria parasites multiply within erythrocytes and possess a repertoire of proteins whose function is to recognize and invade these vertebrate host cells. One such protein involved in erythrocyte invasion is the micronemal protein, Erythrocyte Binding-Like (EBL), which has been studied as a potential target of vaccine development in Plasmodium vivax (PvDBP) and Plasmodium falciparum (EBA-175). In the rodent malaria parasite model Plasmodium yoelii, specific substitutions in the EBL regions responsible for intracellular trafficking (17XL parasite line) or receptor recognition (17X1.1pp. parasite line), paradoxically increase invasion ability and virulence rather than abolish EBL function. Attempts to disrupt the ebl gene locus in the 17XL and 17XNL lines were unsuccessful, suggesting EBL essentiality. To understand the mechanisms behind these potentially conflicting outcomes, we generated 17XL-based transfectants in which ebl expression is suppressed with anhydrotetracycline (ATc) and investigated merozoite behavior during erythrocyte invasion. In the absence of ATc, EBL was secreted to the merozoite surface, whereas following ATc administration parasitemia was negligible in vivo. Merozoites lacking EBL were unable to invade erythrocytes in vitro, indicating that EBL has a critical role for erythrocyte invasion. Quantitative time-lapse imaging revealed that with ATc administration a significant number of merozoites were detached from the erythrocyte after the erythrocyte deformation event and no echinocytosis was observed, indicating that EBL is required for merozoites to establish an irreversible connection with erythrocytes during invasion., Parasitology International, 67(6), pp.706-714; 2018

To interrogate particular neuronal pathways in non-human primates under natural and stress-free conditions, we applied designer receptors exclusively activated by designer drugs (DREADDs) technology to common marmosets. We injected adeno-associated virus vectors expressing the excitatory DREADD hM3Dq into the unilateral substantia nigra in three marmosets. Using multi-tracer positron emission tomography imaging, we detected DREADD expression in vivo, which was confirmed in nigrostriatal dopamine neurons by immunohisto-chemistry, and assessed activation of the substantia nigra and dopamine release following agonist administration. The marmosets rotated in a contralateral direction relative to the activated side 30–90 min after consuming food containing the highly potent DREADD agonist deschloroclozapine (DCZ), but not on the following days without DCZ. These results indicate that non-invasive and reversible DREADD manipulation will extend the utility of marmoset as a primate model for linking neuronal activity and natural behavior in various contexts.

Background We established an in vivo intraradicular biofilm model of apical periodontitis in pigs in which we compared the efficacy of different irrigant activation techniques for biofilm removal. Methods Twenty roots from the deciduous mandibular second premolar of 5 male pigs were used. After pulpectomy, canals were left open for 2 weeks and then sealed for 4 weeks to enable the development of an intracanal biofilm. The intraradicular biofilms was evaluated using SEM and bacterial 16S rRNA gene-sequencing. To investigate the efficacy of biofilm removal, root canal irrigations were performed using conventional needle, passive ultrasonic, subsonic, or laser-activated irrigation. Real-time PCR was conducted to quantitate the remaining biofilm components. Statistical analysis was performed using ANOVA followed by a Tukey kramer post-hoc test with α = 0.05. Results The pulp exposure model was effective in inducing apical periodontitis and SEM analysis revealed a multi-layer biofilm formation inside the root canal. 16S rRNA sequence analysis identified Firmicutes, Bacteroidetes, and Fusobacteria as the predominant bacterial phyla components, which is similar to the microbiome profile seen in humans. None of the tested irrigation techniques completely eradicated the biofilm components from the root canal, but the subsonic and laser-activated irrigation methods produced the lowest bacterial counts (p Conclusions An experimental intraradicular biofilm model has been successfully established in pigs. Within the limitations of the study, subsonic or laser-activated irrigation demonstrated the best biofilm removal results in the pig system.

Simple Summary It is well known that stretching interventions are effective in improving age-related changes in range of motion (ROM) and muscle stiffness. We investigated the effects of various stretching interventions, such as static stretching and hold–relax stretching, on ROM and muscle stiffness in older adults to establish the most effective stretching technique. Our results showed that static stretching and hold–relax stretching increased ROM, which could be contributed by not change in muscle stiffness, but stretch tolerance. Conversely, medial gastrocnemius muscle stiffness decreased only after a static stretching intervention and not after hold–relax stretching. Our results indicated that static stretching intervention improved ROM and muscle stiffness in older adults. Abstract Various stretching techniques are generally recommended to counteract age-related declines in range of motion (ROM) and/or increased muscle stiffness. However, to date, an effective stretching technique has not yet been established for older adults. Consequently, we compared the acute effects of hold relax stretching (HRS) and static stretching (SS) on dorsiflexion (DF) ROM and muscle stiffness among older adults. Overall, 15 elderly men and nine elderly women (70.2 ± 3.9 years, 160.8 ± 7.8 cm, 59.6 ± 9.7 kg) were enrolled, and both legs were randomized to either HRS or SS stretching. We measured DF ROM and muscle stiffness using a dynamometer and ultrasonography before and after 120 s of HRS or SS interventions. Our multivariate analysis indicated no significant interaction effects, but a main effect for DF ROM. Post-hoc tests revealed that DF ROM was increased after both HRS and SS interventions. Moreover, multivariate analysis showed a significant interaction effect for muscle stiffness. Post-hoc tests revealed that muscle stiffness was decreased significantly after only SS intervention. Taken together, our results indicated that both HRS and SS interventions are recommended to increase ROM, and SS is recommended to decrease muscle stiffness in older adults.

Bone has a complex hierarchical structure that contributes to its superior mechanical properties. Therefore, reproducing the complex hierarchical structure of bone tissue is a promising strategy to construct functional engineered bone tissues. In this study, we aimed to reproduce this complex hierarchical structure by developing a method for the three-dimensional culture of MC3T3-E1 osteoblastic cells in a collagen gel with a multichannel structure (MCCG), which mimics the parallel arrangement of Haversian canals in bone tissue. MCCG was homogeneously calcified via the biomineralization properties of MC3T3-E1s. Confocal laser scanning microscopy revealed that MCCG could support the growth and proliferation of MC3T3-E1 cells in the deeper parts of the engineered bone tissue and that the cells formed a toroidal structure on the channel surface and a network-like structure in the gel matrix region. Furthermore, quasi-quantitative measurement of osteocalcin and dentin matrix protein 1 expression indicated the coexistence of two types of cells with different morphologies and differentiation phenotypes. Thus, three-dimensional culture of MC3T3-E1 cells in MCCG yielded engineered tissues mimicking the hierarchical structures of bone tissues. Engineered bone tissues with a biomimetic hierarchical structure could be used as a model system for investigating bone metabolism and evaluating the efficacy of novel drugs.

Many studies have highlighted the difficulty inherent to the clinical application of fundamental neuroscience knowledge based on machine learning techniques. It is difficult to generalize machine learning brain markers to the data acquired from independent imaging sites, mainly due to large site differences in functional magnetic resonance imaging. We address the difficulty of finding a generalizable marker of major depressive disorder (MDD) that would distinguish patients from healthy controls based on resting-state functional connectivity patterns. For the discovery dataset with 713 participants from 4 imaging sites, we removed site differences using our recently developed harmonization method and developed a machine learning MDD classifier. The classifier achieved an approximately 70% generalization accuracy for an independent validation dataset with 521 participants from 5 different imaging sites. The successful generalization to a perfectly independent dataset acquired from multiple imaging sites is novel and ensures scientific reproducibility and clinical applicability., Biomarkers for psychiatric disorders based on neuroimaging data have yet to be put to practical use. This study overcomes the problems of inter-site differences in fMRI data by using a novel harmonization method, thereby successfully constructing a generalizable brain network marker of major depressive disorder across multiple imaging sites.

Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis., Author summary Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite which causes the most pathogenic form of babesiosis in cattle. Like other apicomplexan parasites, B. bovis-induced modification of host cells is crucial for its survival. However, our knowledge of Babesia surface exposed proteins is limited to variant erythrocyte surface antigen1 (VESA1), which is responsible for fatal cerebral babesiosis. Here we identified two novel exported proteins in B. bovis using red blood cell (RBC) surface biotinylation and mass spectrometry. One of the proteins was determined to be essential for parasite development and pathogenicity. Induced knockdown of this protein resulted in a decreased growth rate, reduced RBC surface protrusions created by the parasite, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells. VESA1 is a ligand for cytoadhesion of iRBCs to capillary endothelial cells which leads to blockage of capillaries and causes cerebral symptoms. The second identified protein is encoded by a large multigene family. The gene was downregulated in blasticidin-S resistant parasites, suggesting that the protein mediates entry of blasticidin-S and likely other solutes across the iRBC membrane. This is the first description of a putative channel or transporter molecule on the surface of Babesia-iRBC. Our results provide new insights into the host cell modifications by B. bovis and their pathogenicity.

Nobiletin, a polymethoxy flavone (PMF), is specific to citrus and has been reported to exhibit important health-supporting properties. Nobiletin has six methoxy groups at the 3′,4′,5,6,7,8-positions, which are catalyzed by O-methyltransferases (OMTs). To date, researches on OMTs in citrus fruit are still limited. In the present study, a novel OMT gene (CitOMT) was isolated from two citrus varieties Satsuma mandarin (Citrus unshiu Marc.) and Ponkan mandarin (Citrus reticulata Blanco), and its function was characterized in vitro. The results showed that the expression of CitOMT in the flavedo of Ponkan mandarin was much higher than that of Satsuma mandarin during maturation, which was consistent with the higher accumulation of nobiletin in Ponkan mandarin. In addition, functional analysis showed that the recombinant protein of CitOMT had methylation activity to transfer a methyl group to 3′-hydroxy group of flavones in vitro. Because methylation at the 3′-position of flavones is vital for the nobiletin biosynthesis, CitOMT may be a key gene responsible for nobiletin biosynthesis in citrus fruit. The results presented in this study will provide new strategies to enhance nobiletin accumulation and improve the nutritional qualities of citrus fruit.

Background The transmembrane glycoprotein podoplanin (PDPN) is upregulated in some tumors and has gained attention as a malignant tumor biomarker. PDPN molecules have platelet aggregation-stimulating domains and, are therefore, suggested to play a role in tumor-induced platelet activation, which in turn triggers epithelial-to-mesenchymal transition (EMT) and enhances the invasive and metastatic activities of tumor cells. In addition, as forced PDPN expression itself can alter the propensity of certain tumor cells in favor of EMT and enhance their invasive ability, it is also considered to be involved in the cell signaling system. Nevertheless, underlying mechanisms of PDPN in tumor cell invasive ability as well as EMT induction, especially by platelets, are still not fully understood. Methods Subclonal TE11A cells were isolated from the human esophageal squamous carcinoma cell line TE11 and the effects of anti-PDPN neutralizing antibody as well as PDPN gene knockout on platelet-induced EMT-related gene expression were measured. Also, the effects of PDPN deficiency on cellular invasive ability and motility were assessed. Results PDPN-null cells were able to provoke platelet aggregation, suggesting that PDPN contribution to platelet activation in these cells is marginal. Nevertheless, expression of platelet-induced EMT-related genes, including vimentin, was impaired by PDPN-neutralizing antibody as well as PDPN deficiency, while their effects on TGF-β-induced gene expression were marginal. Unexpectedly, PDPN gene ablation, at least in either allele, engendered spontaneous N-cadherin upregulation and claudin-1 downregulation. Despite these seemingly EMT-like alterations, PDPN deficiency impaired cellular motility and invasive ability even after TGF-β-induced EMT induction. Conclusions These results suggested that, while PDPN seems to function in favor of maintaining the epithelial state of this cell line, it is indispensable for platelet-mediated induction of particular mesenchymal marker genes as well as the potentiation of motility and invasion capacity.

Plasmodium malaria parasites are obligate intracellular protozoans that use a unique form of locomotion, termed gliding motility, to move through host tissues and invade cells. The process is substrate-dependent and powered by an actomyosin motor that drives the posterior translocation of extracellular adhesins which in turn propel the parasite forward. Gliding motility is essential for tissue translocation in the sporozoite and ookinete stages; however, the short-lived erythrocyte-invading merozoite stage has never been observed to undergo gliding movement. Here we show Plasmodium merozoites possess the ability to undergo gliding motility and that this mechanism is likely an important precursor step for successful parasite invasion. We demonstrate that two human infective species, P. falciparum and P. knowlesi, have distinct merozoite motility profiles which may reflect distinct invasion strategies. Additionally, we develop and validate a higher throughput assay to evaluate the effects of genetic and pharmacological perturbations on both the molecular motor and complex signaling cascade that regulates motility in merozoites. The discovery of merozoite motility provides a new model to study the glideosome and may facilitate the pursuit of new targets for malaria treatment.Significance statementPlasmodium malaria parasites use a unique substrate-dependent locomotion termed gliding motility to translocate through tissues and invade cells. Dogma has suggested that the small labile invasive stages that invade erythrocytes, merozoites, use this motility solely to penetrate target erythrocytes. Here we reveal that merozoites use gliding motility for translocation across host cells prior to invasion. This forms an important pre-invasion step that is powered by a conserved actomyosin motor and is regulated by a complex signaling pathway. This work fundamentally changes our understanding of the role of gliding motility and invasion in the blood and will have a significant impact on our understanding of blood stage host-pathogen interactions, parasite biology, and could have implications for vaccine development.

Contains fulltext : 219632.pdf (Publisher’s version ) (Open Access) Preclinical applications of resting-state functional magnetic resonance imaging (rsfMRI) offer the possibility to non-invasively probe whole-brain network dynamics and to investigate the determinants of altered network signatures observed in human studies. Mouse rsfMRI has been increasingly adopted by numerous laboratories worldwide. Here we describe a multi-centre comparison of 17 mouse rsfMRI datasets via a common image processing and analysis pipeline. Despite prominent cross-laboratory differences in equipment and imaging procedures, we report the reproducible identification of several large-scale resting-state networks (RSN), including a mouse default-mode network, in the majority of datasets. A combination of factors was associated with enhanced reproducibility in functional connectivity parameter estimation, including animal handling procedures and equipment performance. RSN spatial specificity was enhanced in datasets acquired at higher field strength, with cryoprobes, in ventilated animals, and under medetomidine-isoflurane combination sedation. Our work describes a set of representative RSNs in the mouse brain and highlights key experimental parameters that can critically guide the design and analysis of future rodent rsfMRI investigations.

Three Fortunella (kumquat) species, the Meiwa kumquat (F. crassifolia Swingle), the Round kumquat [F. japonica (Thunb.) Swingle] and the Changshou kumquat (F. obovata hort. ex Tanaka), showing tetraploid, which had been induced by treating the seeds with colchicine, were examined for origin and horticultural characteristics (e. g. morphology of leaves, flowers, pollen and fruits). Additionally, these tetraploid kumquats were crossed to produce triploid kumquats. All of the tetraploids were confirmed to be derived from the nucellar embryo of each original kumquat by examining their chromosomal composition with chromomycin A3 staining. All of the tetraploid kumquats had the typical morphological characteristics of tetraploid Citrus plants, such as round and thick leaves, and large flowers and pollen grains. On the other hand, the fruits of these tetraploid plants showed desirable traits for kumquats such as thicker pericarp and higher soluble solids content. Furthermore, when these tetraploids were crossed with some diploid cultivars, triploid progenies were obtained from almost all of the cross combinations.

In the present study, carotenoid metabolism was investigated in the fruits of a novel citrus cultivar, 'Seinannohikari' (Citrus spp.). During the maturation, β,β-xanthophylls were accumulated rapidly with β-cryptoxanthin being the dominant carotenoid compound in the flavedo and juice sacs of 'Seinannohikari'. In the juice sacs of mature fruits, 'Seinannohikari' accumulated high amount of carotenoids, especially β-cryptoxanthin. The content of β-cryptoxanthin in the juice sacs of 'Seinannohikari' was approximately 2.5 times of that in 'Miyagawa-wase' (Citrus unshiu), which is one of its parental cultivars, at the mature stage. Gene expression results showed that the massive accumulation of β-cryptoxanthin might be attributed to the higher expression of carotenoid biosynthetic genes (CitPSY, CitPDS, CitZDS, CitLCYb2, CitHYb, and CitZEP), and lower expression of carotenoid catabolic genes (CitNCED2 and CitNCED3) in the juice sacs of 'Seinannohikari'.

Plasmodium falciparum, the causative agent of malignant malaria, is insensitive to thapsigargin (TG), a well-known inhibitor of the human sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). To understand the key factor causing the difference of the sensitivity, the molecular interaction of TG and each SERCA was analyzed by the fragment molecular orbital (FMO) method. While the major component of the interaction energy was the nonpolar interaction, the major difference in the molecular interaction arose from the polar interaction, namely, the hydrogen bonding interaction with a hydroxyl group of TG. Additionally, we successfully confirmed these FMO calculation results by measuring the inhibitory activity of a synthesized TG derivative. Our calculations and experiments indicated that, by replacing the hydroxyl group of TG with another functional group, the sensitivities of TG to human and P. falciparum SERCAs can be reversed. This study provides important information to develop antimalarial compounds targeting...

The newt, a group of urodele amphibians, has outstanding ability to repeatedly regenerate various body parts, even in the terrestrial life-stage. In this animal, when the limb is amputated, a cell mass named the blastema appears on the stump and eventually gives rise to a new functional limb. Erythrocytes (red blood cells) in most non-mammalian vertebrates, including the newt, preserve their nucleus throughout their life-span, although physiological roles of such nucleated erythrocytes, other than oxygen delivery, are not known. Here we report novel behavior of erythrocytes in the newt. We identified an orphan gene Newtic1, whose transcripts significantly increased in the blastema. Newtic1 was expressed in a subset of erythrocytes that formed a novel clump (EryC). EryC formed a complex with monocytes and was circulating throughout the body. When the limb was amputated, EryCs were newly generated in the stump and accumulated into a distal portion of the growing blastema. Our data suggested that the newt erythrocytes carried multiple secretory molecules including growth factors and matrix metalloproteases, and were capable of delivering these molecules into the blastema as a form of EryCs. This study provides insight into regulations and roles of nucleated erythrocytes, that are independent of oxygen delivery.

Plasmodium malaria parasites are obligate intracellular protozoans that use a unique form of locomotion, termed gliding motility, to move through host tissues and invade cells. The process is substrate dependent and powered by an actomyosin motor that drives the posterior translocation of extracellular adhesins which, in turn, propel the parasite forward. Gliding motility is essential for tissue translocation in the sporozoite and ookinete stages; however, the short-lived erythrocyte-invading merozoite stage has never been observed to undergo gliding movement. Here we show Plasmodium merozoites possess the ability to undergo gliding motility in vitro and that this mechanism is likely an important precursor step for successful parasite invasion. We demonstrate that two human infective species, Plasmodium falciparum and Plasmodium knowlesi, have distinct merozoite motility profiles which may reflect distinct invasion strategies. Additionally, we develop and validate a higher throughput assay to evaluate the effects of genetic and pharmacological perturbations on both the molecular motor and the complex signaling cascade that regulates motility in merozoites. The discovery of merozoite motility provides a model to study the glideosome and adds a dimension for work aiming to develop treatments targeting the blood stage invasion pathways., Proceedings of the National Academy of Sciences of the United States of America, 118(48), art. no. e2114442118; 2021

Combined spraying of gibberellin (GA) and prohydrojasmon (PDJ) was an effective method to prevent the physiological disorder of peel puffing in citrus fruit. However, the GA and PDJ combined treatment inhibited carotenoid biosynthesis during the fruit ripening process, which led to the mature fruit with poor color. In the present study, to improve the coloration of the GA and PDJ-treated fruit, the effects of postharvest treatments of two auxins, indole-3-acetic acid (IAA) and 1-naphthaleneacetic acid (NAA), on carotenoid accumulation were investigated in Satsuma mandarin ‘Aoshima unshiu’ (Citrus unshiu Marc.). The results showed that IAA and NAA treatments induced carotenoid biosynthesis in the GA and PDJ-treated fruit after harvest. With the treatments of IAA and NAA, the contents of β-carotene, β-cryptoxanthin, all-trans-violaxanthin, and 9-cis-violaxanthin were enhanced in both flavedos and juice sacs. The increase in the carotenoid accumulation was accompany with the up-regulation of carotenoid biosynthetic genes and down-regulation of carotenoid catabolic gene in the IAA and NAA treatments. In addition, ethylene production was induced after the IAA and NAA treatments, and the increase of the endogenous ethylene might stimulate carotenoid biosynthesis in citrus fruit. The results presented in this study suggested that the postharvest treatment of auxin was an effective method for improving the coloration of the GA and PDJ-treated fruit.

We previously obtained two intergeneric hybrids with different ploidies, i.e., aneuploid (2n = 28) and eutriploid, from diploid−diploid crosses between ‘Kiyomi’ tangor (Citrus unshiu Marcow. × C. sinensis (L.) Osbeck) and Meiwa kumquat (Fortunella crassifolia Swingle) as novel breeding materials for a seedless kumquat. In this study, we attempted to clarify the construction of the parental genomes of these hybrids by SSR genotyping and genomic in situ hybridization (GISH)−chromomycin A3 (CMA) analysis. SSR genotyping in NSX43 (LG5) and CiBE2227 (LG8) loci revealed that both hybrids inherited one allele from ‘Kiyomi’ tangor and two heterozygous alleles from Meiwa kumquat. The GISH analysis failed due to the high genomic homology between Citrus and Fortunella. At the same time, the CMA karyotype compositions of the two intergeneric hybrids (H15-701: 2A + 1B + 3C + 13D + 7E + 1F + 1Dst, H15-702: 3A + 1B + 2C + 15D + 4E +1F + 1Dst) and both parents (‘Kiyomi’ tangor: 1A + 2B + 2C + 6D + 7E, Meiwa kumquat: 2A + 2C + 12D + 1F + 1Dst) were completely revealed. We identified the parental genome construction and polyploidization processes in both intergeneric hybrids on the basis of these SSR genotypes and CMA karyotype compositions according to the following theory: the SSR genotypes and chromosome compositions were the same as those of the somatic chromosome and two-fold after the first division (even number) in unreduced gametes caused by first-division restitution (FDR) and second-division restitution (SDR), respectively. Consequently, we determined that both intergeneric hybrids may have had two genomes derived from the 2n male unreduced gamete as a result of the FDR of the Meiwa kumquat. In addition, most horticultural traits of the leaves, flowers, and fruits of both hybrids showed intermediate traits of the parents, but the fruit sizes and flowering habits were more like those of the two inherited genomes of Meiwa kumquat.

Limonium sinuatum (L.) Mill. (2n = 2x = 16) is a popular ornamental plant with dimorphism of pollen grains (type A and type B) and stigmas (papilla and cob-like). We applied polyploidy breeding to this species in order to introduce desirable traits. Tetraploid and mixoploid L. sinuatum plants were successfully obtained with oryzalin treatment of L. sinuatum ‘Early Blue’ seeds. All three tetraploids had increased leaf width, stomatal size, flower length, and pollen width compared to those of the diploid, and tetraploids had four germinal pores of pollen grains, whereas the diploid had three. All tetraploids had type A pollen grains and cob-like stigmas. Furthermore, the growth of cultivated tetraploid plants was slow, with later bolting and flowering times. Mixoploids Mixo-1 and Mixo-3 were estimated to be polyploidy periclinal chimeric plants consisting of a tetraploid L1 layer and diploid L2 layer, and Mixo-2 was estimated to be a polyploidy periclinal chimeric plant consisting of the diploid L1 layer and tetraploid L2 layer. Mixo-4 had tetraploid L1 and L2 layers. Mixoploids, except Mixo-4, had type A pollen grains and cob-like stigmas, whereas Mixo-4 had type B pollen grains and papilla stigmas. These polyploids will be useful as polyploidy breeding materials.

Apicomplexa mainly comprises parasitic species and some of them, which infect and cause severe diseases to humans and livestock, have been extensively studied due to the clinical and industrial importance. Besides, apicomplexans are a popular subject of the studies focusing on the evolution initiated by a secondary loss of photosynthesis. By interpreting the position in the tree of eukaryotes and lifestyles of the phylogenetic relatives parsimoniously, the extant apicomplexans are predicted to be the descendants of a parasite bearing a non-photosynthetic (cryptic) plastid. The plastid-bearing characteristic for the ancestral apicomplexan is further strengthened by non-photosynthetic plastids found in the extant apicomplexans. The research on apicomplexan members infecting invertebrates is much less advanced than that on the pathogens to humans and livestock. Gregarines are apicomplexans that infect diverse invertebrates and recent studies based on transcriptome data revealed the presence of cryptic plastids in a subset of the species investigated. In this study, we isolated gregarine-like organisms (GLOs) from three arthropod species and conducted transcriptome analyses on the isolated cells. A transcriptome-based, multi-gene phylogenetic analysis clearly indicated that all of the three GLOs are eugregarines. Significantly, the transcriptome data from the GLO in a centipede appeared to contain the transcripts encoding enzymes involved in the non-mevalonate pathway for isopentenyl diphosphate biosynthesis and C5 pathway for heme biosynthesis. The enzymes involved in the two plastid-localized metabolic pathways circumstantially but strongly suggest that the particular GLO possesses a cryptic plastid. The evolution of cryptic plastids in eugregarines is revised by incorporating the new data obtained from the three GLOs in this study.

Plasmodium falciparum malaria parasites export several hundred proteins to the cytoplasm of infected red blood cells (RBCs) to modify the cell environment suitable for their growth. A Plasmodium translocon of exported proteins (PTEX) is necessary for both soluble and integral membrane proteins to cross the parasitophorous vacuole (PV) membrane surrounding the parasite inside the RBC. However, the molecular composition of the translocation complex for integral membrane proteins is not fully characterized, especially at the parasite plasma membrane. To examine the translocation complex, here we used mini-SURFIN4.1, consisting of a short N-terminal region, a transmembrane region, and a cytoplasmic region of an exported integral membrane protein SURFIN4.1. We found that mini-SURFIN4.1 forms a translocation intermediate complex with core PTEX components, EXP2, HSP101, and PTEX150. We also found that several proteins are exposed to the PV space, including Pf113, an uncharacterized PTEX-associated protein. We determined that Pf113 localizes in dense granules at the merozoite stage and on the parasite periphery after RBC invasion. Using an inducible translocon-clogged mini-SURFIN4.1, we found that a stable translocation intermediate complex forms at the parasite plasma membrane and contains EXP2 and a processed form of Pf113. These results suggest a potential role of Pf113 for the translocation step of mini-SURFIN4.1, providing further insights into the translocation mechanisms for parasite integral membrane proteins., Parasitology International, 83, art. no. 102358; 2021

Hematopoietic stem and progenitor cells (HSPCs) reside in the supportive stromal niche in bone marrow (BM); when needed, however, they are rapidly mobilized into the circulation, suggesting that HSPCs are intrinsically highly motile but usually stay in the niche. We questioned what determines the motility of HSPCs. Here, we show that transforming growth factor (TGF)-β-induced intracellular plasminogen activator inhibitor (PAI)-1 activation is responsible for keeping HSPCs in the BM niche. We found that the expression of PAI-1, a downstream target of TGF-β signaling, was selectively augmented in niche-residing HSPCs. Functional inhibition of the TGF-β-PAI-1 signal increased MT1-MMP-dependent cellular motility, causing a detachment of HSPCs from the TGF-β-expressing niche cells, such as megakaryocytes. Furthermore, consistently high motility in PAI-1-deficient HSPCs was demonstrated by both a transwell migration assay and reciprocal transplantation experiments, indicating that intracellular, not extracellular, PAI-1 suppresses the motility of HSPCs, thereby causing them to stay in the niche. Mechanistically, intracellular PAI-1 inhibited the proteolytic activity of proprotein convertase Furin, diminishing MT1-MMP activity. This reduced expression of MT1-MMP in turn affected the expression levels of several adhesion/deadhesion molecules for determination of HSPC localization, such as CD44, VLA-4, and CXCR4, which then promoted the retention of HSPCs in the niche. Our findings open up a new field for the study of intracellular proteolysis as a regulatory mechanism of stem cell fate, which has the potential to improve clinical HSPC mobilization and transplantation protocols.

Induced pluripotent stem cells (iPSCs) are suitable for studying mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations. Here, we generated iPSCs from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with the m.13513G>A mutation. The patient’s dermal fibroblasts were reprogrammed, and we established two iPSC clones with and without mutant mtDNA. Furthermore, we tried to decrease mutant mtDNA level in iPSCs using transcription activator-like effector nucleases (TALENs). We originally engineered platinum TALENs, which were transported into mitochondria, recognized the mtDNA sequence including the m.13513 position, and preferentially cleaved G13513A mutant mtDNA (G13513A-mpTALEN). The m.13513G>A heteroplasmy level in MELAS-iPSCs was decreased in the short term by transduction of G13513A-mpTALEN. Our data demonstrate that this mtDNA-targeted nuclease would be a powerful tool for changing the heteroplasmy level in heteroplasmic iPSCs, which could contribute to elucidation of the pathological mechanisms of mitochondrial diseases caused by mtDNA mutations.

Kawasaki disease (KD) is a paediatric vasculitis whose pathogenesis remains unclear. Based on experimental studies using a mouse model for KD, we report here that proline-rich protein tyrosine kinase 2 (Pyk2) plays a critical role in the onset of KD-like murine vasculitis. The mouse model for KD was prepared by administrating a Candida albicans water-soluble fraction (CAWS). Unlike CAWS-treated WT mice, CAWS-treated Pyk2-Knockout (Pyk2-KO) mice did not develop apparent vasculitis. A sustained increase in MIG/CXCL9 and IP-10/CXCL10, both of which have potent angiostatic activity, was observed in CAWS-treated Pyk2-KO mice. CAWS-induced activation of STAT3, which negatively regulates the expression of these chemokines, was also attenuated in macrophages derived from Pyk2-KO mice. The present study suggests that defects in Pyk2 suppress KD-like experimental vasculitis, presumably through CXCL9- and CXCL10-dependent interference with neo-angiogenesis. Since Pyk2-KO mice show no life-threatening phenotype, Pyk2 may be a promising therapeutic molecular target for KD.

Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta-like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast-specific manner, we investigated the ligand-specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1-induced Notch signaling was responsible for the expansion of the bone-forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1-Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast-specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast-osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1-mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand-specific activation of Notch signaling in the maintenance of bone homeostasis. J. Cell. Physiol. 232: 2569-2580, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.

To elucidate the regulation of ascorbic acid (AsA) metabolism in response to temperatures, three varieties of citrus juice sacs (Valencia orange, Lisbon lemon, and Satsuma mandarin) in in vitro culture system were subjected to different temperatures (10 °C, 20 °C, and 30 °C). The AsA accumulation was induced at 10 °C, whereas it was not significantly affected at 30 °C as compared with the control at 20 °C in juice sacs of the three citrus varieties. The enhancement of AsA accumulation at 10 °C was regulated by the expression of genes in AsA biosynthetic, oxidation, and regeneration pathway. In Valencia orange and Satsuma mandarin, the higher expression of CitVTC4 gene in biosynthetic pathway, and the higher expression of CitMDAR1 and CitMDAR2 genes in regeneration pathway, together with the lower expression of CitAO gene in oxidation pathway contributed to the higher AsA level at 10 °C. In Lisbon lemon, the higher expressions of CitVTC1, CitVTC2, and CitVTC4 genes in biosynthetic pathway and the lower expression of CitAO gene in oxidation pathway contributed to the higher AsA level at 10 °C.

Riboflavin (vitamin B2) supply to the retina across the inner blood-retinal barrier (BRB) was investigated. In rats, the apparent influx permeability clearance of [3H]riboflavin (62.8 μL/(min·g retina)) was much higher than that of a non-permeable paracellular marker, suggesting the facilitative influx transport of riboflavin across the BRB. The retinal uptake index (RUI) of [3H]riboflavin was 59.0%, and significantly reduced by flavin adenine dinucleotide (FAD), but not by l -ascorbic acid, suggesting the substrate specificity of riboflavin transport. TR-iBRB2 cells, an in vitro model of the inner BRB, showed a temperature- and concentration-dependent [3H]riboflavin uptake with a Km of 113 nM, suggesting that the influx transport of riboflavin across the inner BRB involves a carrier-mediated process. [3H]Riboflavin uptake by TR-iBRB2 cells was slightly altered by Na+- and Cl−-free buffers, suggesting that riboflavin transport at the inner BRB is preferentially Na+- and Cl−-independent. [3H]Riboflavin uptake by TR-iBRB2 cells was significantly reduced by riboflavin analogues while the uptake remained unchanged by other vitamins. The function and inhibition profile suggested the involvement of riboflavin transporters (SLC52A/RFVT) in riboflavin transport at the inner BRB, and this is supported by expression and knockdown analysis of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3) in TR-iBRB2 cells.

Background Approximately 10-20% of Kawasaki disease (KD) patients are resistant to intravenous immunoglobulin (IVIG) treatment. Further, these patients are at a particularly high risk of having coronary artery abnormalities. The mechanisms of IVIG resistance in KD have been analyzed using patient leukocytes, but not patient vascular endothelial cells (ECs). The present study clarifies the mechanisms of IVIG resistance in KD using an induced pluripotent stem cell (iPSC) disease model.Methods and Results:Dermal fibroblasts or peripheral blood mononuclear cells from 2 IVIG-resistant and 2 IVIG-responsive KD patients were reprogrammed by the episomal vector-mediated transduction of 6 reprogramming factors. KD patient-derived iPSCs were differentiated into ECs (iPSC-ECs). The gene expression profiles of iPSC-ECs generated from IVIG-resistant and IVIG-responsive KD patients were compared by RNA-sequencing analyses. We found that the expression ofCXCL12was significantly upregulated in iPSC-ECs from IVIG-resistant KD patients. Additionally, Gene Set Enrichment Analysis (GSEA) revealed that gene sets involved in interleukin (IL)-6 signaling were also upregulated. Conclusions The first iPSC-based model for KD is reported here. Our mechanistic analyses suggest thatCXCL12, which plays a role in leukocyte transmigration, is a key molecule candidate for IVIG resistance and KD severity. They also indicate that an upregulation of IL-6-related genes may be involved in this pathogenesis.

OBJECTIVE Extracorporeal shock wave therapy (ESWT) is widely used to treat various human diseases. Low-energy ESWT increases expression of vascular endothelial growth factor (VEGF) in cultured endothelial cells. The VEGF stimulates not only endothelial cells to promote angiogenesis but also neural cells to induce neuroprotective effects. A previous study by these authors demonstrated that low-energy ESWT promoted expression of VEGF in damaged neural tissue and improved locomotor function after spinal cord injury (SCI). However, the neuroprotective mechanisms in the injured spinal cord produced by low-energy ESWT are still unknown. In the present study, the authors investigated the cell specificity of VEGF expression in injured spinal cords and angiogenesis induced by low-energy ESWT. They also examined the neuroprotective effects of low-energy ESWT on cell death, axonal damage, and white matter sparing as well as the therapeutic effect for improvement of sensory function following SCI. METHODS Adult female Sprague-Dawley rats were divided into the SCI group (SCI only) and SCI-SW group (low-energy ESWT applied after SCI). Thoracic SCI was produced using a New York University Impactor. Low-energy ESWT was applied to the injured spinal cord 3 times a week for 3 weeks after SCI. Locomotor function was evaluated using the Basso, Beattie, and Bresnahan open-field locomotor score for 42 days after SCI. Mechanical and thermal allodynia in the hindpaw were evaluated for 42 days. Double staining for VEGF and various cell-type markers (NeuN, GFAP, and Olig2) was performed at Day 7; TUNEL staining was also performed at Day 7. Immunohistochemical staining for CD31, α-SMA, and 5-HT was performed on spinal cord sections taken 42 days after SCI. Luxol fast blue staining was performed at Day 42. RESULTS Low-energy ESWT significantly improved not only locomotion but also mechanical and thermal allodynia following SCI. In the double staining, expression of VEGF was observed in NeuN-, GFAP-, and Olig2-labeled cells. Low-energy ESWT significantly promoted CD31 and α-SMA expressions in the injured spinal cords. In addition, low-energy ESWT significantly reduced the TUNEL-positive cells in the injured spinal cords. Furthermore, the immunodensity of 5-HT–positive axons was significantly higher in the animals treated by low-energy ESWT. The areas of spared white matter were obviously larger in the SCI-SW group than in the SCI group, as indicated by Luxol fast blue staining. CONCLUSIONS The results of this study suggested that low-energy ESWT promotes VEGF expression in various neural cells and enhances angiogenesis in damaged neural tissue after SCI. Furthermore, the neuroprotective effect of VEGF induced by low-energy ESWT can suppress cell death and axonal damage and consequently improve locomotor and sensory functions after SCI. Thus, low-energy ESWT can be a novel therapeutic strategy for treatment of SCI.

A ploidy chimera of the Meiwa kumquat (Fortunella crassifolia Swingle), which had been induced by treating the nucellar embryos with colchicine, and had diploid (2n = 2x = 18) and tetraploid (2n = 4x = 36) cells, was examined for its ploidy level, morphological characteristics, and sizes of its cells in its leaves, flowers, and fruits to reveal the ploidy level of each histogenic layer. Furthermore, the chimera was crossed with the diploid kumquat to evaluate the ploidy level of its reproductive organs. The morphological characteristics and the sizes of the cells in the leaves, flowers, and fruits of the chimera were similar to those of the tetraploid Meiwa kumquat and the ploidy periclinal chimera known as “Yubeni,” with diploids in the histogenic layer I (L1) and tetraploids in the histogenic layer II (L2) and III (L3). However, the epidermis derived from the L1 of the chimera showed the same result as the diploid Meiwa kumquat in all organs and cells. The sexual organs derived from the L2 of the chimera were significantly larger than those of the diploid. Moreover, the ploidy level of the seedlings obtained from the chimera was mostly tetraploid. In the midrib derived from the L3, the chimera displayed the fluorescence intensity of a tetraploid by flow cytometric analysis and had the same size of the cells as the tetraploid and the Yubeni. According to these results, the chimera is thought to be a ploidy periclinal chimera with diploid cells in the outermost layer (L1) and tetraploid cells in the inner layers (L2 and L3) of the shoot apical meristem. The chimera had desirable fruit traits for a kumquat such as a thick pericarp, a high sugar content, and a small number of developed seeds. Furthermore, triploid progenies were obtained from reciprocal crosses between the chimera and diploid kumquat.

Epstein-Barr virus (EBV) is associated with several disorders. EBV is known to modulate the proliferation and survival of hematopoietic cells such as B cells and T cells in human. However, the effects of EBV on hematopoiesis itself have not been investigated. To study EBV infection in murine models, their hematopoiesis must be humanized, since EBV infection is limited only in primates. To engraft the human hematopoiesis, NOD/Shi-scid-IL2rγnull (NOG) mice were used. Usually, the hematopoiesis humanized mice reconstitute only lymphoid cells, but myeloid cells are not. However, we revealed human macrophages (hMφ) and their precursor monocytes were increased in peripheral tissues of EBV-infected mice. Furthermore, our previous report indicated Mφ accumulation in spleen was essential for development of EBV-positive tumors, suggesting that EBV modulates human hematopoiesis in order to thrive. Interestingly, we revealed a dramatic increase of immature granulocytes only in bone marrow of EBV-infected mice. In addition, GM-CSF, a cytokine that is essential for differentiation of the myeloid lineage, was significantly increased in EBV-infected mice. These results were also reproduced in patients with EBV-related disorders. We suggest that the hematopoietic alterations during EBV-infection might contribute immune suppression to the development and exacerbation of EBV-related disorders.