Generation of mouse iPS cells using an inducible expression of transgenes via the cumate gene-switch.

We applied an inducible gene expression system that utilizes the p-cmt operon, the cumate gene-switch, to generate mouse induced pluripotent stem (iPS) cells. Mouse embryonic fibroblast (MEF) E6E7-MEF cells were transfected with a single cumate gene-switch vector enabling concomitant expression of Oct4, Sox2, c-Myc, Klf4, and Gfp. Then, the cells were cultured with cumate, a monoterpene. An increase in colonies positive for alkaline phosphatase activity was observed dose-dependently with cumate. In the absence of cumate, the expression of GFP, a marker for transgene expression, was undetectable in tightly aggregated iPS cell-like colonies with endogenous expression of NANOG and OCT4. From primary MEFs using the cumate gene-switch, we also isolated iPS cells expressing endogenous NANOG, OCT4, SOX2, KLF4, and SSEA1 with hypo-methylated genomic promoter regions of endogenous Nanog and Oct4. In embryoid bodies with the progression of differentiation, expression of markers for all three germ layers was detected, and contracting cardiomyocytes were observed. Overall, we suggest that the cumate gene-switch is applicable for the generation of mouse iPS cells. The cumate gene-switch in combination with other inducible systems, such as the tet system, may provide useful approaches for analyzing the roles of transgenes underlying the establishment of iPS cells.

Unified Total Synthesis, Stereostructural Elucidation, and Biological Evaluation of Sarcophytonolides.

Sarcophytonolides are cembranolide diterpenes isolated from the soft corals of genus Sarcophyton. Unified total synthesis of sarcophytonolides C, E, F, G, H, and J and isosarcophytonolide D was achieved. The synthetic routes feature NaHMDS- or SmI2-mediated fragment coupling, alkoxycarbonylallylation, macrolactonization, and transannular ring-closing metathesis. These total syntheses led to the absolute configurational confirmation of sarcophytonolide H, elucidation of sarcophytonolides C, E, F, and G, and revision of sarcophytonolide J and isosarcophytonolide D. We also evaluated the antifouling activity and toxicity of the synthetic sarcophytonolides H and J and their analogues as well as the cytotoxicity of the synthetic sarcophytonolides and the key synthetic intermediates.

Late-stage divergent synthesis and antifouling activity of geraniol-butenolide hybrid molecules.

Hybrid molecules consisting of geraniol and butenolide were designed and synthesized by the late-stage divergent strategy. In the synthetic route, ring-closing metathesis was utilized for the construction of a butenolide moiety. A biological evaluation of the eight synthetic hybrid compounds revealed that these molecules exhibit antifouling activity against the cypris larvae of the barnacle Balanus (Amphibalanus) amphitrite with EC50 values of 0.30-1.31 µg mL-1. These results show that hybridization of the geraniol and butenolide structural motifs resulted in the enhancement of the antifouling activity.

Knockdown of gene expression by antisense morpholino oligos in preimplantation mouse embryos cultured in vitro.

Knockdown of gene expression by antisense morpholino oligos (MOs) is a simple and effective method for analyzing the roles of genes in mammalian cells. Here, we demonstrate the efficient delivery of MOs by Endo-Porter (EP), a special transfection reagent for MOs, into preimplantation mouse embryos cultured in vitro. A fluorescein-labeled control MO was applied for monitoring the incorporation of MOs into developing 2-cell embryos in the presence of varying amounts of EP and bovine serum albumin. In optimized conditions, fluorescence was detected in 2-cell embryos within a 3-h incubation period. In order to analyze the validity of the optimized conditions, an antisense Oct4 MO was applied for knockdown of the synthesis of OCT4 protein in developing embryos from the 2-cell stage. In blastocysts, the antisense Oct4 MO induced a decrease in the amount in OCT4 protein to less than half. An almost complete absence of OCT4-positive cells and nearly complete disappearance of the inner cell mass in the outgrowths of blastocysts were also noted. These phenotypes corresponded with those of Oct4-deficient mouse embryos. Overall, we suggest that the delivery of MOs using EP is useful for the knockdown of gene expression in preimplantation mouse embryos cultured in vitro.

Stereodivergent synthesis and relative stereostructure of the C1-C13 fragment of symbiodinolide.

Four possible diastereomers of the C1-C13 fragment of symbiodinolide, which were proposed by the stereostructural analysis of the degraded product, were synthesized in a stereodivergent and stereoselective manner. The key transformations were aldol reaction of methyl acetoacetate with the aldehyde, diastereoselective reduction of the resulting ß-hydroxy ketone, and the stereoinversion at the C6 position. Comparison of the (1)H NMR data between the four synthetic products and the degraded product revealed the relative stereostructure of the C1-C13 fragment of symbiodinolide.

Measurement of the Md3+/Md2+ reduction potential studied with flow electrolytic chromatography.

The reduction behavior of mendelevium (Md) was studied using a flow electrolytic chromatography apparatus. By application of the appropriate potentials on the chromatography column, the more stable Md(3+) is reduced to Md(2+). The reduction potential of the Md(3+) + e(-) → Md(2+) couple was determined to be -0.16 ± 0.05 V versus a normal hydrogen electrode.

[Epileptic encephalopathy associated with forced normalization after administration of levetiracetam].

Here we report a case of a 10-year-old female with unclassified epileptic encephalopathy who showed forced normalization after administration of levetiracetam (LEV). She initially presented with intractable tonic and myoclonic seizures that were observed about 10 times a day along with frequent multifocal sharp and slow wave complexes on electroencephalography (EEG). We were forced to decrease the topiramate dose because of the appearance of nystagmus, and her myoclonic seizures became worse. We added LEV (250 mg/day) and her tonic and myoclonic seizures disappeared one day after initiation of LEV administration. However, she showed hyporesponsiveness and akinesia. The disappearance of paroxysmal discharges on EEG confirmed the diagnosis of forced normalization. Despite continuous administration of LEV, tonic and myoclonic seizures relapsed within a month but her psychotic symptoms resolved simultaneously. To the best of our knowledge, this is the first reported case of forced normalization after LEV administration. It should be noted that LEV may cause forced normalization although it can be started at an adequate dosage.

Standardization of Stimulus Location for Functional Electrical Stimulation of Swallowing.

Many nerves and muscles are involved in the swallowing process; hence neuromuscular disorders cause dysphagia resulting in aspiration pneumonia. A critical movement in the pharyngeal phase of swallowing is hyolaryngeal elevation to help protect the airway and open a relaxed upper esophageal sphincter. Functional electrical stimulation (FES) is expected to improve the function of muscles acting on the hyolaryngeal motion, which may contribute to airway protection in dysphagic patients. However, it is difficult to select the stimulus locations that effectively assist laryngeal elevation without the expertise in the anatomy of swallowing-related muscles. Therefore, this study investigated the method to standardize the selection of the stimulus locations based on the dimensions of the larynx. In addition, the effect of stimulus intensity on the amount of laryngeal elevation was evaluated.

[Overview of the infant body part photography condition using 16-rows multidetector CT].

In infant CT scans, it is important to minimize radiation exposure without lowering the quality of the diagnosis. Therefore, appropriate parameters for infant CT scan should be considered at each institute. In order to determine parameters for infant body CT, we measured the physical characteristics of our current CT machine and evaluated scan parameters as S.D. below 10 on the basis of literature recommendations, which say that we should adopt S.D. of the liver when treated with a radiation exposure dose in an adult abdominal CT scan. As a result, the ideal parameters were 90 kV tube voltage, ultra fast detail resolution, B kernel, and Eff.mAs value calculated from patient body width. Infant body CT scans with the mentioned parameters resulted in an S.D. below 10, and this is thought to be applicable to further examinations.

Recovery of motor function of chronic spinal cord injury by extracellular pyruvate kinase isoform M2 and the underlying mechanism.

In our previous study, we found that pyruvate kinase isoform M2 (PKM2) was secreted from the skeletal muscle and extended axons in the cultured neuron. Indirect evidence suggested that secreted PKM2 might relate to the recovery of motor function in spinal cord injured (SCI) mice. However, in vivo direct evidence has not been obtained, showing that extracellular PKM2 improved axonal density and motor function in SCI mice. In addition, the signal pathway of extracellular PKM2 underlying the increase in axons remained unknown. Therefore, this study aimed to identify a target molecule of extracellular PKM2 in neurons and investigate the critical involvement of extracellular PKM2 in functional recovery in the chronic phase of SCI. Recombinant PKM2 infusion to the lateral ventricle recovered motor function in the chronic phase of SCI mice. The improvement of motor function was associated with axonal increase, at least of raphespinal tracts connecting to the motor neurons directly or indirectly. Target molecules of extracellular PKM2 in neurons were identified as valosin-containing protein (VCP) by the drug affinity responsive target stability method. ATPase activation of VCP mediated the PKM2-induced axonal increase and recovery of motor function in chronic SCI related to the increase in axonal density. It is a novel finding that axonal increase and motor recovery are mediated by extracellular PKM2-VCP-driven ATPase activity.

Two regioisomers of condensed thioheterocyclic triazine synthesized from 6-phenyl-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-5-one.

In the crystal structure of 6-phenyl-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-5-one, C(9)H(7)N(3)OS, (I), the 1,2,4-triazine moieties are connected by face-to-face contacts through two kinds of double hydrogen bonds (N-H...O and N-H...S), which form planar ribbons along the a axis. The ribbons are crosslinked through C-H...pi interactions between the phenyl rings. The molecular structures of two regioisomeric compounds, namely 6-phenyl-2,3-dihydro-7H-1,3-thiazolo[3,2-b][1,2,4]triazin-7-one, C(11)H(9)N(3)OS, (II), and 3-phenyl-6,7-dihydro-4H-1,3-thiazolo[2,3-c][1,2,4]triazin-4-one, C(11)H(9)N(3)OS, (III), which were prepared by the condensation reaction of (I) with 1,2-dibromoethane, have been characterized by X-ray crystallography and spectroscopic studies. The crystal structures of (II) and (III) both show two crystallographically independent molecules. While the two compounds are isomers, the unit-cell parameters and crystal packing are quite different and (II) has a chiral crystal structure.

Acteoside Improves Muscle Atrophy and Motor Function by Inducing New Myokine Secretion in Chronic Spinal Cord Injury.

Chronic spinal cord injury (SCI) is difficult to cure, even by several approaches effective at the acute or subacute phase. We focused on skeletal muscle atrophy as a detrimental factor in chronic SCI and explored drugs that protect against muscle atrophy and activate secretion of axonal growth factors from skeletal muscle. We found that acteoside induced the secretion of axonal growth factors from skeletal muscle cells and proliferation of these cells. Intramuscular injection of acteoside in mice with chronic SCI recovered skeletal muscle weight reduction and motor function impairment. We also identified pyruvate kinase isoform M2 (PKM2) as a secreted factor from skeletal muscle cells, stimulated by acteoside. Extracellular PKM2 enhanced proliferation of skeletal muscle cells and axonal growth in cultured neurons. Further, we showed that PKM2 might cross the blood-brain barrier. These results indicate that effects of acteoside on chronic SCI might be mediated by PKM2 secretion from skeletal muscles. This study proposes that the candidate drug acteoside and a new myokine, PKM2, could be used for the treatment of chronic SCI.

Estimation of Muscle Activity Change under Different Bolus Conditions using Musculoskeletal Model of Swallowing.

Swallowing, deglutition, is realized by highly coordinated activities of many nerves and muscles, but it is hard to observe directly due to intracorporal movement, and there is a limitation to the number of muscles that can be percutaneously measured. In addition, since there are few studies on the mechanical analysis of the swallowing movement, the detailed muscle activity pattern during swallowing has not yet been clarified. To tackle this problem from the viewpoint of biomechanics, we have been developing the musculoskeletal model of swallowing which can estimate the activities of swallowing-related muscles based on the movements of hyoid bone and thyroid cartilage. In this paper, we analyzed the activities of swallowing-related muscles under two different bolus conditions: bolus of water and nectar thickened liquid to investigate the effect of physical property of bolus in the activities of swallowing-related muscles.

Numerical simulation of interaction between organs and food bolus during swallowing and aspiration.

The mechanism of swallowing is still not fully understood, because the process of swallowing is a rapid and complex interaction among several involved organs and the food bolus. In this work, with the aim of studying swallowing and aspiration processes noninvasively and systematically, a computer simulation method for analyzing the involved organs and water (considered as the food bolus) is proposed. The shape and motion of the organs involved in swallowing are modeled in the same way as in our previous study, by using the Hamiltonian moving particle simulation (MPS) method and forced displacements on the basis of motion in a healthy volunteer. The bolus flow is simulated using the explicit MPS method for fluid analysis. The interaction between the organs and the bolus is analyzed using a fluid-structure coupling scheme. To validate the proposed method, the behavior of the simulated bolus flow is compared qualitatively and quantitatively with corresponding medical images. In addition to the healthy motion model, disorder motion models are constructed for reproducing the aspiration phenomenon by computer simulation. The behaviors of the organs and the bolus considered as the food bolus in the healthy and disorder motion models are compared for evaluating the mechanism of aspiration.

Repair of segmental bone defects in rabbit tibiae using a complex of beta-tricalcium phosphate, type I collagen, and fibroblast growth factor-2.

The objective of this study was to evaluate the effects of a complex of beta-tricalcium phosphate (beta-TCP) granules, collagen, and fibroblast growth factor-2 (FGF-2) on cortical bone repair in rabbits. Segmental bone defects of 5 mm in length were created in the middle of the tibial shaft. The defect was stabilized with a plate and screws, and was filled with 0.3 ml of a complex of beta-TCP granules and 5% collagen, with or without 200 microg of recombinant human fibroblast growth factor-2 (rhFGF-2). Bone regeneration and beta-TCP resorption were assessed by X-ray and micro-CT scanner. A three-point bending test was also performed. The results showed that the segmental bone defect was not only radiologically, but also mechanically healed with cortical bone 12 weeks after implantation of the complex with rhFGF-2. In contrast, after implantation of the complex without rhFGF-2, most of the defect was filled with beta-TCP and only a small amount of bone formation was found. These results suggest that resorption of beta-TCP is important for bone formation and may be promoted by FGF-2 in the beta-TCP implantation site. In addition, the complex of beta-TCP granules and collagen combined with rhFGF-2 provides a paste-like material that is easy to handle. This material may be of considerable use in the treatment of cortical bone defects.

Total Synthesis of Sarcophytonolide H and Isosarcophytonolide D: Structural Revision of Isosarcophytonolide D and Structure-Antifouling Activity Relationship of Sarcophytonolide H.

The first total syntheses of sarcophytonolide H and the originally proposed and correct structures of isosarcophytonolide D have been achieved via transannular ring-closing metathesis (RCM). These total syntheses culminated in the stereostructural confirmation of sarcophytonolide H and the reassignment of isosarcophytonolide D, respectively. The antifouling activity of the synthetic sarcophytonolide H and its analogues was also evaluated.

Exogenous expression of homeoprotein EGAM1N prevents in vitro cardiomyogenesis by impairing expression of T and Nkx2.5, but not Mef2c, in mouse embryonic stem cells.

Generation of multiple cell types from embryonic stem (ES) cells and induced pluripotent stem cells is crucial to provide materials for regenerative medicine. EGAM1N has been found in preimplantation mouse embryos and mouse ES cells as a functionally unclassified homeoprotein. Recently, we reported that expression of EGAM1N suppressed the in vitro differentiation of ES cells into progenitor cells that arise in early embryogenesis. To clarify the effect of EGAM1N on terminal differentiation, embryoid bodies (EBs) were prepared from ES cells expressing exogenous Egam1n. In EBs expressing Egam1n, cardiomyogenesis was inhibited by impairing the expression of crucial transcription factors Brachyury T and Nkx2.5 in the generation of mesoderm and cardiomyocytes, respectively. Expression levels of Mef2c, another crucial gene for cardiomyogenesis, were unaffected. Conversely, the expression levels of Gata6 and Plat, markers for the primitive endoderm lineage, and Cdx2, a marker for the trophectoderm lineage, were increased. These results suggested that certain cell populations in EBs expressing Egam1n preferentially differentiated to such cell lineages. Our results suggest that EGAM1N not only affects the generation of progenitor cells during early embryogenesis, but also the progression of terminal differentiation, such as cardiomyogenesis, in mouse ES cells.

Partial inhibition of differentiation associated with elevated protein levels of pluripotency factors in mouse embryonic stem cells expressing exogenous EGAM1N homeoprotein.

We previously reported that transcripts encoding the homeoprotein EGAM1N are expressed in preimplantation mouse embryos and embryonic stem (ES) cells, and the exogenous expression of EGAM1N inhibits the differentiation of ES cells. In order to clarify the relationship between the inhibition of differentiation and EGAM1N, we generated mouse MG1.19 ES cells stably expressing EGAM1N. Control transfectants with an empty vector formed relatively flattened cell colonies similar to those observed in parental MG1.19 cells. In contrast, Egam1n transfectants formed tightly aggregated cell colonies with increased localization of CDH1 at cell-to-cell interfaces. The protein levels of pluripotency factors, including TBX3 and SOX2, were also increased. The expression of Tbx3 transcripts was induced, although the level of Sox2 transcripts was almost unchanged. The expression of EGAM1N resulted in no obvious changes in the expression of genes encoding receptors, protein kinases, transcription factors, and their encoded proteins involved in the LIF-STAT3 signaling pathway. Alkaline phosphatase activity, a marker for the undifferentiated state, in Egam1n transfectants was exhibited in a clonal proliferation assay. When differentiation of Egam1n transfectants was induced, progression was prevented with increases in transcript levels of Pou5f1, Sox2, Nanog, Klf4, Tbx3, and their encoded proteins. However, Egam1n transfectants formed relatively flattened-cell layers as observed in the control, indicating that the expression of EGAM1N could not maintain LIF-independent self-renewal of ES cells. Overall, we suggest that expression of EGAM1N could inhibit differentiation, at least in part, by elevating the protein levels of pluripotency factors in MG1.19 ES cells.

Pial arteries respond earlier than penetrating arterioles to neural activation in the somatosensory cortex in awake mice exposed to chronic hypoxia: an additional mechanism to proximal integration signaling?

The pial and penetrating arteries have a crucial role in regulating cerebral blood flow (CBF) to meet neural demand in the cortex. Here, we examined the longitudinal effects of chronic hypoxia on the arterial diameter responses to single whisker stimulation in the awake mouse cortex, where activity-induced responses of CBF were gradually attenuated. The vasodilation responses to whisker stimulation under prehypoxia normal conditions were 8.1% and 12% relative to their baselines in the pial arteries and penetrating arterioles, respectively. After 3 weeks of hypoxia, however, these responses were significantly reduced to 5.5% and 4.1%, respectively. The CBF response, measured using laser-Doppler flowmetry (LDF), induced by the same whisker stimulation was also attenuated (14% to 2.6%). A close linear correlation was found for the responses between the penetrating arteriolar diameter and LDF, and their temporal dynamics. After 3 weeks of chronic hypoxia, the initiation of vasodilation in the penetrating arterioles was significantly extended, but the pial artery responses remained unchanged. These results show that vasodilation of the penetrating arterioles followed the pial artery responses, which are not explainable in terms of proximal integration signaling. The findings therefore indicate an additional mechanism for triggering pial artery dilation in the neurovascular coupling.

Development of a numerical simulator of human swallowing using a particle method (Part 2. Evaluation of the accuracy of a swallowing simulation using the 3D MPS method).

The aim of this study was to develop and evaluate the accuracy of a three-dimensional (3D) numerical simulator of the swallowing action using the 3D moving particle simulation (MPS) method, which can simulate splashes and rapid changes in the free surfaces of food materials. The 3D numerical simulator of the swallowing action using the MPS method was developed based on accurate organ models, which contains forced transformation by elapsed time. The validity of the simulation results were evaluated qualitatively based on comparisons with videofluorography (VF) images. To evaluate the validity of the simulation results quantitatively, the normalized brightness around the vallecula was used as the evaluation parameter. The positions and configurations of the food bolus during each time step were compared in the simulated and VF images. The simulation results corresponded to the VF images during each time step in the visual evaluations, which suggested that the simulation was qualitatively correct. The normalized brightness of the simulated and VF images corresponded exactly at all time steps. This showed that the simulation results, which contained information on changes in the organs and the food bolus, were numerically correct. Based on these results, the accuracy of this simulator was high and it could be used to study the mechanism of disorders that cause dysphasia. This simulator also calculated the shear rate at a specific point and the timing with Newtonian and non-Newtonian fluids. We think that the information provided by this simulator could be useful for development of food products, medicines, and in rehabilitation facilities.