Highly active neurons emerging in vitro.

Mean firing rates vary across neurons in a neuronal network. Although most neurons infrequently emit spikes, a small fraction of neurons exhibit extremely high frequencies of spikes; this fraction of neurons plays a pivotal role in information processing, however, little is known about how these outliers emerge and whether they are maintained over time. In primary cultures of mouse hippocampal neurons, we traced highly active neurons every 24 h for 7 wk by optically observing the fluorescent protein dVenus; the expression of dVenus was controlled by the promoter of Arc, an immediate early gene that is induced by neuronal activity. Under default-mode conditions, 0.3%-0.4% of neurons were spontaneously Arc-dVenus positive, exhibiting high firing rates. These neurons were spatially clustered, exhibited intermittently repeated dVenus expression, and often continued to express Arc-dVenus for approximately 2 wk. Thus, highly active neurons constitute a few select functional subpopulations in the neuronal network.NEW & NOTEWORTHY The overdispersion of neuronal activity levels can often be attributed to very few neurons exhibiting extremely high firing rates, but due to technical difficulty, no studies have examined how these outliers are selected during development and whether they are maintained over time. We optically monitored highly active neurons for as long as 7 wk in vitro and found that they constituted a unique population that was different from other "mediocre" neurons with normal firing rates.

Regeneration of the cell mass in larvae of temnopleurid sea urchins.

Mechanisms of cell mass (CM) formation were analyzed by microsurgery in two temnopleurid sea urchins, Mespilia globulus and Temnopleurus toreumaticus. The CM in temnopleurids is formed at the early larval stage from the left ectodermal invagination, and with the hydrocoel derived from the mesoderm, forms an adult rudiment. After serial removal of the CM, it was strongly regenerated until its attachment to the hydrocoel, with the same timing as in control larvae. Embryos that had the tip of the archenteron or the coelomic pouches removed formed a CM in the normal manner. Removal of the CM plus the left somatocoel or the hydrocoel allowed CM regeneration with and without adult rudiment formation. A transplanted CM enlarged autonomously but did not contribute to adult rudiment formation, and larvae formed a new CM. Our observations suggest that the hydrocoel recognizes its distance from the CM to induce the growth of the CM and controls the normal timing of adult rudiment formation.

Effects of Nodal inhibition on development of temnopleurid sea urchins.

Adult rudiment formation in some temnopleurids begins with the formation of a cell mass that is pinched off the left ectoderm in early larval development. The cell mass forms the adult rudiment with the left coelomic pouch of the mesodermal region. However, details of the mechanisms to establish position of the cell mass are still unknown. We analyzed the inhibiting effect of Nodal, a factor for morphogenesis of the oral region and right side, for location of the cell mass, in four temnopleurids. Pulse inhibition, at least 5 min inhibition, during coelomic pouch formation allowed a cell mass to form on both sides, whereas treatments after that period did not. These results indicate that Nodal signaling controls the oral-aboral axis before gastrulation and then affects the position of the cell mass and adult rudiment up to coelomic pouch formation. They also indicate that the position of the adult rudiment under Nodal signaling pathways is conserved in temnopleurids, as adult rudiment formation is dependent on the cell mass.

Exosomal miRNA Signatures for Late-Onset Acute Graft-Versus-Host Disease in Allogenic Hematopoietic Stem Cell Transplantation.

Recent studies have demonstrated that exosomal microRNAs (miRNAs) have the potential of facilitating molecular diagnosis. Currently, little is known about the underlying mechanism behind late-onset acute graft-versus-host disease (LA GVHD). Identifying differentially expressed miRNAs in exosomes should be useful for understanding the role of miRNAs in this disease. This study was established to investigate the relevance of miRNAs in exosomes derived from patients developing LA GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Plasma samples were collected from patients with LA GVHD (n = 5), non-GVHD (n = 5), and controls (n = 8) for exosomal miRNA expression profiling using a TaqMan low-density array; the results were validated by quantitative reverse transcription polymerase chain reaction (RT-PCR). We analyzed exosomal miRNAs differentially expressed among these three groups. MirTarBase was employed to predict potential target genes of the miRNAs specific for LA GVHD. We detected 55 miRNAs that were differentially expressed with a significant change >2.0-fold between LA GVHD and non-GVHD. Of these, we selected the 10 miRNAs (miR-423-5p, miR-19a, miR-142-3p, miR-128, miR-193b, miR-30c, miR-193a, miR-191, miR-125b, and miR-574-3p) with the most significant differential expression. Using quantitative RT-PCR, we further identified that miR-128 was significantly upregulated at the onset of LA GVHD compared with that in normal controls and is a promising diagnostic marker of LA GVHD, with an area under the curve (AUC) value of 0.975. MirTarBase analysis revealed that the predicted target genes of miR-128 are involved in the immune system and inflammation. Increased expression of miR-128 may serve as a novel, noninvasive biomarker for early LA GVHD diagnosis.

Effect of trelagliptin on vascular endothelial functions and serum adiponectin level in patients with type 2 diabetes: a preliminary single-arm prospective pilot study.

BACKGROUND: Trelagliptin, an oral DPP-4 inhibitor, which is administered once per week and characterized by a long half-life in blood. The effects of trelagliptin on vascular endothelial functions have not been clarified to date. The objective of the present study was to examine the effects of trelagliptin on vascular endothelial functions in patients with type 2 diabetes mellitus (DM) using flow-mediated dilatation (FMD), adiponectin, and asymmetric dimethylarginine (ADMA) as evaluation indicators. METHODS: This study was a preliminary single-arm prospective pilot study. The subjects of this study were type 2 DM patients aged 20-74 years, who visited our outpatient department. The patients were treated with trelagliptin, and their FMD, adiponectin, and ADMA levels were measured at baseline and at 12 weeks after initial treatment to determine the changes during the study period. RESULTS: A total of 27 patients, excluding three dropouts, were included in the population for analysis. Trelagliptin treatment showed no significant changes in FMD (2.42 ± 2.7% at baseline vs. 2.66 ± 3.8% post-treatment, P = 0.785) and ADMA (0.41 ± 0.0 µg/mL at baseline vs. 0.40 ± 0.0 µg/mL post-treatment, P = 0.402). Trelagliptin treatment resulted in a significant increase of serum adiponectin level (7.72 ± 6.9 µg/mL at baseline vs. 8.82 ± 8.3 µg/mL post-treatment, P < 0.002). CONCLUSIONS: In this pilot study, trelagliptin treatment showed no significant changes in FMD. On the other hand, it was believed that trelagliptin treatment may increase serum adiponectin level. Trial Registration http://www.umin.ac.jp (Trial ID UMIN000018311).

Localization of Polystyrene Particles on the Surface of Poly(N-isopropylacrylamide-co-methacrylic acid) Microgels Prepared by Seeded Emulsion Polymerization of Styrene.

Composite microgels with polystyrene nanoparticles were synthesized by seeded emulsion polymerization of styrene in the presence of pH- and temperature-responsive poly(N-isopropylacrylamide-co-methacrylic acid) microgels as seeds. In particular, the core microgels maintained their swelled state as the pH was increased to 10 during seeded emulsion polymerization conducted at an elevated temperature. Furthermore, we tuned the swelling degree of the core microgels at pH 10 by changing the amount of methacrylic acid incorporated during the synthesis of the core microgels. Unlike deswollen microgels, during the seeded emulsion polymerization, the swollen microgels were covered with a monolayer of non-close-packed polystyrene particles on their surface, as confirmed by electron microscopy. A possible mechanism for the seeded emulsion polymerization of styrene in the presence of swollen microgels under alkaline conditions is proposed.

Impact of Spatial Distribution of Charged Groups in Core Poly(N-isopropylacrylamide)-Based Microgels on the Resultant Composite Structures Prepared by Seeded Emulsion Polymerization of Styrene.

A series of raspberry-shaped composite microgels were synthesized by seeded emulsion polymerization of styrene in the presence of hydrogel particles with different distributions of charged groups. Unlike microgels whose charged groups are localized in their center,29 polystyrene nanoparticles were formed inside the core microgels when the microgels whose charged groups were localized on their surface were used as cores for seeded emulsion polymerization. The effects of the surface charge densities of the core microgels and the concentration of styrene monomer during the polymerization on the resultant structures of composite microgels were investigated. The surface structures of obtained composite microgels were mainly evaluated by electron microscopy, and their stimuli responsiveness was evaluated by dynamic light scattering and laser Doppler velocimetry. The internal structures of the composite microgels were visualized from ultrathin cross sections observed by transmission electron microscopy (TEM). Cryo-TEM was used to clarify the microscopic structures of composite microgels when they were in hydrated states. Through a series of characterizations, we summarize the effects of structures of core microgels on the resultant composite structures.

Downregulation of Plasma miR-215 in Chronic Myeloid Leukemia Patients with Successful Discontinuation of Imatinib.

Approximately 40% of chronic myeloid leukemia (CML) patients who discontinue imatinib (IM) therapy maintain undetectable minimal residual disease (UMRD) for more than one year (stopping IM (STOP-IM)). To determine a possible biomarker for STOP-IM CML, we examined plasma miRNA expression in CML patients who were able to discontinue IM. We first screened candidate miRNAs in unselected STOP-IM patients, who had sustained UMRD after discontinuing IM for more than six months, in comparison with healthy volunteers, by using a TaqMan low-density array for plasma or exosomes. Exosomal miR-215 and plasma miR-215 were downregulated in the STOP-IM group compared to the control, indicating that the biological relevance of the plasma miR-215 level is equivalent to that of the exosomal level. Next, we performed real-time quantitative RT-PCR in 20 STOP-IM patients, 32 patients with UMRD on continued IM therapy (IM group) and 28 healthy volunteers. The plasma miRNA-215 level was significantly downregulated in the STOP-IM group (p < 0.0001); we determined the cut-off level and divided the IM group patients into two groups according to whether the plasma miR-215 was downregulated or not. The IM group patients with a low plasma miR-215 level had a significantly higher total IM intake, compared to the patients with elevated miR-215 levels (p = 0.0229). Functional annotation of miR-215 target genes estimated by the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools involved cell cycle, mitosis, DNA repair and cell cycle checkpoint. Our study suggests a possible role of miR-215 in successful IM discontinuation.

Layer III neurons control synchronized waves in the immature cerebral cortex.

Correlated spiking activity prevails in immature cortical networks and is believed to contribute to neuronal circuit maturation; however, its spatiotemporal organization is not fully understood. Using wide-field calcium imaging from acute whole-brain slices of rat pups on postnatal days 1-6, we found that correlated spikes were initiated in the anterior part of the lateral entorhinal cortex and propagated anteriorly to the frontal cortex and posteriorly to the medial entorhinal cortex, forming traveling waves that engaged almost the entire cortex. The waves were blocked by ionotropic glutamatergic receptor antagonists but not by GABAergic receptor antagonists. During wave events, glutamatergic and GABAergic synaptic inputs were balanced and induced UP state-like depolarization. Magnified monitoring with cellular resolution revealed that the layer III neurons were first activated when the waves were initiated. Consistent with this finding, layer III contained a larger number of neurons that were autonomously active, even under a blockade of synaptic transmission. During wave propagation, the layer III neurons constituted a leading front of the wave. The waves did not enter the parasubiculum; however, in some cases, they were reflected at the parasubicular border and propagated back in the opposite direction. During this reflection process, the layer III neurons in the medial entorhinal cortex maintained persistent activity. Thus, our data emphasize the role of layer III in early network behaviors and provide insight into the circuit mechanisms through which cerebral cortical networks maturate.

Raspberry-shaped composite microgel synthesis by seeded emulsion polymerization with hydrogel particles.

A series of raspberry-shaped composite microgels were synthesized by the seeded emulsion polymerization of styrene with hydrogel particles. Thermoresponsive microgels of poly(N-isopropylacrylamide) cross-linked with N,N'-methylenebis(acrylamide) acted as cores for the polymerization. During the surfactant-free polymerization, the core microgels shrank at 70 °C to provide thermoresponsive composite microgels, and the polystyrene particles attached to core microgels became bigger with increasing styrene concentration. Conversely, composite microgels synthesized with sodium dodecyl sulfate (SDS) ([SDS] > 6.5 mM) did not exhibit thermoresponsive deswelling behavior because polystyrene particles covered the core microgels. In particular, polystyrene particles formed composites on the microgel surface as well as inside the microgels when the SDS concentration exceeded a critical value for core microgel swelling at 70 °C. A mechanism is proposed based on these results for the seeded emulsion polymerization of water-immiscible monomers with microgels.

Magnesium Oxide as an Unexpected Bronchial Foreign Body in an Elderly Patient.

As cases of magnesium oxide pill aspiration are rare, the associated airway proinflammatory properties and appropriate analytic strategies remain unclear. An 81-year-old woman presenting with dyspnea was diagnosed with magnesium oxide pill aspiration. Computed tomography, a "mixing test" with levodopa, and a magnesium content analysis revealed a similar density between the foreign body and her prescribed magnesium oxide pill. The patient recovered without airway complications after foreign body removal. Clinicians should be aware of magnesium oxide tablets as potential bronchial foreign bodies in elderly patients because they may not dissolve without exposure to gastric juices.

Effective generation mechanisms of tropical instability waves as represented by high-resolution coupled atmosphere-ocean prediction experiments.

Cusp-shaped fluctuations of the sea surface temperature (SST) front in the tropical Pacific, now known as tropical instability waves (TIWs), were discovered by remote sensing in the 1970s. Their discovery was followed by both theoretical and analytical studies, which, along with in situ observations, identified several possible generation mechanisms. Although modeling studies have shown that TIWs strongly influence the heat budget, their influence on local variations of realistically initialized predictions is not yet understood. We here evaluate a series of medium-range (up to ~ 10 days) coupled atmosphere-ocean predictions by a coupled model with different horizontal resolutions. Observational SST, surface wind stress, heat flux, and pressure data showed that representation of temporally and spatially local variations was improved by resolving fine-scale SST variations around the initialized coarse-scale SST front fluctuations of TIWs. Our study thus demonstrates the advantage of using high-resolution coupled models for medium-range predictions. In addition, analysis of TIW energetics showed two dominant sources of energy to anticyclonic eddies: barotropic instability between equatorial zonal currents and baroclinic instability due to intense density fronts. In turn, the eddy circulation strengthened both instabilities in the resolved simulations. This revealed feedback process refines our understanding of the generation mechanisms of TIWs.

Synchronized spike waves in immature dentate gyrus networks.

At early developmental stages, immature neuronal networks of the neocortex and hippocampus spontaneously exhibit synchronously oscillating activities, which are believed to play roles in normal circuit maturation. The tissue development of the dentate gyrus (DG) in the hippocampal formation is exceptionally late compared with other brain regions and persists until postnatal periods. Using patch-clamp recording and functional multineuron calcium imaging, we found that the DG networks of postnatal day (P)3-7 mice spontaneously generated traveling waves of action potentials, which were initiated at the upper blade of the granule cell layer and propagated to the lower blade. The propagation was dependent on glutamatergic and electrical synapses, but not on GABAergic receptor activity. Remarkably, the DG waves were almost completely abolished in offspring born to female rats exposed to restraint stress during pregnancy. In the prenatally stressed offspring, DG granule cell dendrites developed normally until P3 and showed atrophy by P9. Thus, the DG waves may be required for the maturation of DG granule cells.

Assessing qualitative changes in simulated periodontal ligament and alveolar bone using a non-contact electromagnetic vibration device.

The objective of this study is to investigate the ability of a non-contact electromagnetic vibration device to assess a simulated periodontal ligament and alveolar bone conditions in experimental tooth models by applying mechanical parameters (resonant frequency, elastic modulus, and coefficient of viscosity). The non-contact electromagnetic vibration device was made up of three components: vibrator, detector, and analyzer. The experimental tooth model consisted of a cylindrical rod made of polyacetal, a tissue conditioner for soft lining material, and urethane or urethane foam to simulate the tooth, periodontal ligament, and alveolar bone, respectively. The tissue conditioner was prepared by mixing various volumes of liquid with powder. Periotest values (PTVs) were also measured under the same conditions as those of the non-contact electromagnetic vibration device. All of the mechanical parameters derived from the non-contact electromagnetic vibration device significantly decreased as the proportion of liquid increased. Values for the three parameters of the urethane models were significantly larger than those of the urethane foam models. In contrast, PTVs increased significantly as the proportion of liquid increased; however, no significant difference was observed between the urethane and urethane foam models. The non-contact electromagnetic vibration device may be capable of evaluating not only periodontal ligament conditions but also bone quality. Mechanical parameters may be useful for assessing qualitative changes in the periodontal ligament and alveolar bone.

Age-related decrease of miRNA-92a levels in human CD8+ T-cells correlates with a reduction of naïve T lymphocytes.

MicroRNA (miR)-17-92a expression plays a crucial role in lymphocyte ontogeny. We therefore set out to determine miR-92a expression levels in peripheral blood lymphocytes from healthy subjects to ascertain any association between these levels and ageing. We found a positive correlation between the miR-92a expression level and the percentages of RO-CD8+CD27+ (P = 0.0046) and CD3+CD8+CD62L+ (P = 0.0011). This suggests that the majority of miR-92a of CD8+ T cells is derived from naïve cells, and the miR-92a expression level in CD8+ T cells declines progressively with age. These results indicate that the age-related attrition of naïve T cells is linked to a reduction of miR-92a in human T -lymphocytes. Therefore, we should careful attention when evaluating human miRNA levels in T lymphocytes to use normal control values.

A no-contact vibration device for measuring implant stability.

OBJECTIVES: Monitoring implant stability is an important factor in determining the long-term success rate of implants. Periotest values and resonance frequency analysis have been widely used for this purpose, but these indicators mainly reflect the mobility and/or stability of implants. Thus, a no-contact electromagnetic vibration device was developed and tested for monitoring both tooth mobility and periodontal tissue conditions. The aim of this study was to evaluate the ability of a no-contact electromagnetic vibration device to measure implant stability under various peri-implant conditions using mechanical parameters. MATERIAL AND METHODS: The device consisted of three components: the vibrator, detector, and analyzer. The mechanical parameters resonant frequency, elastic modulus, and coefficient of viscosity were used to measure simulated atrophic bone defects in periodontal tissues. RESULTS: The resonant frequency and the elastic modulus increased with an increase in supporting bone height. In contrast, the coefficient of viscosity decreased with bone height. Values for the three parameters for the formed urethane models were lower than those for the urethane models. CONCLUSIONS: A no-contact electromagnetic vibration device may be capable of monitoring implant stability, and mechanical parameters may be useful for assessing the condition of periodontal tissues around implants.

Functional Multiple-Spine Calcium Imaging from Brain Slices.

Most excitatory inputs arrive at dendritic spines in a postsynaptic neuron. To understand dendritic information processing, it is critical to scrutinize the spatiotemporal dynamics of synaptic inputs along dendrites. This protocol combines spinning-disk confocal imaging with whole-cell patch-clamp recording to perform wide-field, high-speed optical recording of synaptic inputs in a neuron loaded with a calcium indicator in ex vivo cultured networks. Our protocol enables simultaneous detection of synaptic inputs as calcium signals from hundreds of spines in multiple dendritic branches. For complete details on the use and execution of this protocol, please refer to Takahashi et al. (2012, 2016), Kobayashi et al. (2019), and Ishikawa and Ikegaya (2020).

The oral iron chelator deferasirox represses signaling through the mTOR in myeloid leukemia cells by enhancing expression of REDD1.

To evaluate the effect of deferasirox in human myeloid leukemia cells, and to identify the molecular pathways responsible for antiproliferative effects on leukemia cells during chelation therapy, we performed gene expression profiling to focus on the pathway involved in the anticancer effect of deferasirox. The inhibitory concentration (IC50) of deferasirox was 17-50 microM in three human myeloid cell lines (K562, U937, and HL60), while those in fresh leukemia cells obtained from four patients it varied from 88 to 172 microM. Gene expression profiling using Affymerix GeneChips (U133 Plus 2.0) revealed up-regulation of cyclin-dependent kinase inhibitor 1A (CDKN1A) encoding p21CIP, genes regulating interferon (i.e. IFIT1). Pathways related to iron metabolism and hypoxia such as growth differentiation factor 15 (GDF-15) and Regulated in development and DNA damage response (REDD1) were also prominent. Based on the results obtained from gene expression profiling, we particularly focused on the REDD1/mTOR (mammalian target of rapamycin) pathway in deferasirox-treated K562 cells, and found an enhanced expression of REDD1 and its down-stream protein, tuberin (TSC2). Notably, S6 ribosomal protein as well as phosphorylated S6, which is known to be a target of mTOR, was significantly repressed in deferasirox-treated K562 cells, and REDD1 small interfering RNA restored phosphorylation of S6. Although iron chelation may affect multiple signaling pathways related to cell survival, our data support the conclusion that REDD1 functions up-stream of tuberin to down-regulate the mTOR pathway in response to deferasirox. Deferasirox might not only have benefit for iron chelation but also may be an antiproliferative agent in some myeloid leukemias, especially patients who need both iron chelation and reduction of leukemia cells.

Effect of dietary fish oil intake on ubiquitin ligase expression during muscle atrophy induced by sciatic nerve denervation in mice.

Dietary fish oil intake improves muscle atrophy in several atrophy models however the effect on denervation-induced muscle atrophy is not clear. Thus, the aim of this study was to investigate the effects of dietary fish oil intake on muscle atrophy and the expression of muscle atrophy markers induced by sciatic nerve denervation in mice. We performed histological and quantitative mRNA expression analysis of muscle atrophy markers in mice fed with fish oil with sciatic nerve denervation. Histological analysis indicated that dietary fish oil intake slightly prevented the decrease of muscle fiber diameter induced by denervation treatment. In addition, dietary fish oil intake suppressed the MuRF1 (tripartite motif-containing 63) expression up-regulated by denervation treatment, and this was due to decreased tumor necrosis factor-alpha (TNF-α) production in skeletal muscle. We concluded that dietary fish oil intake suppressed MuRF1 expression by decreasing TNF-α production during muscle atrophy induced by sciatic nerve denervation in mice.