Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.     

Occurrence of VIP and peptide HM in human pancreas and their influence on pancreatic endocrine secretion in man

By immunohistochemistry it was found that VIP- and peptide HI/peptide HM (PHI/PHM)-like immunoreactivity occurred in autonomic neurons in the human pancreas. Antisera against both VIP and PHI/PHM reacted with neuronal cells in local ganglia and these ganglia also contained PHI/PHM- and VIP-immunoreactive fibre plexuses. VIP- and PHI/PHM-positive fibres were also seen close to the Langerhans' islets. In addition, PHI/PHM- but not VIP-like immunoreactivity was observed in the endocrine cells often located in the periphery of the islets. The nature of these PHI/PHM-positive cells remains to be established. I.v. infusion of VIP at constant rates of 300 and 900 pmol/kg X h for 30 min in 6 healthy volunteers resulted in plateau values of 102 +/- 26 and 291 +/- 25 pM, respectively. These levels of VIP which are above those found in the circulation under physiological conditions stimulated secretion of insulin, C-peptide and pancreatic glucagon dose-dependently. On the contrary prolonged (60 min) infusion of PHM in doses resulting in plasma levels up to 1340 +/- 405 pM had no effect on pancreatic hormone secretion. These findings suggest that VIP is a likely neurotransmitter in the control of endocrine pancreatic secretion while PHM has a less prominent role, if any.     

Ras activation in T cells determines the development of antigen-induced airway hyperresponsiveness and eosinophilic inflammation

The central role for Th2 cells in the development of Ag-induced airway hyperresponsiveness and eosinophilic inflammation is well documented. We have reported a crucial role for TCR-induced activation of the Ras/extracellular signal-regulated kinase mitogen-activated protein kinase cascade in Th2 cell differentiation. Here, we show that the development of both OVA-induced airway hyperresponsiveness and eosinophilic airway inflammation in a mouse asthma model are attenuated in transgenic mice by the overexpression of enzymatically inactive Ras molecules in T cells. In addition, reduced levels of IL-5 production and eosinophilic inflammation induced by nematode infection (Nippostrongylus brasiliensis or Heligmosomoides polygyrus) were detected. Thus, the level of Ras activation in T cells appears to determine Th2-dependent eosinophilic inflammation and Ag-induced airway hyperresponsiveness.     

Possible role of prostaglandin F in blastocyst implantation

The synthesis and release of Prostaglandin F (PGF) by the rabbit blastocyst and endometrium were investigated on Day 6 and Day 7, using radioimmunoassay, autoradiography and conversion experiments. The following results were obtained: The content of PGF in the blastocyst increased significantly (P less than 0.01) from Day 6 to Day 7. The content of PGF in the endometrium was significantly higher (P less than 0.05) on Day 7 implantation sites compared to the other areas. The in vitro synthesis and release of PGF by Day 6 blastocysts sharply increased after one and two hours of culture, respectively. Thereafter both values declined with time. The in vitro synthesis and release of PGF by Day 6 endometria increased continuously with time. 14C-arachidonic acid (14C-AA) was incorporated into Day 6 blastocysts in vitro and converted to PGF2 alpha. These results suggest that both the endometrium and the blastocyst are the sources of the PGs involved in implantation, and that PGF derived from the blastocysts may act as the trigger of implantation.     

Changes in cerebral level of monoamines by Japanese encephalitis virus infection

The changes in monoamine levels of different brain regions following Japanese encephalitis virus (JEV) intraperitoneal inoculation were examined in experimentally JEV-infected mice. In addition, virus distribution was studied using infectivity assay and immuno-histochemistry of viral antigen. 1) The level of monoamines in brain tissues was not affected by 48 hours after viral inoculation, but marked effects were elicited at 96 hours after the inoculation. The cerebral concentration of 5-hydroxyindole-3-acetic acid (5 HIAA) was increased, while that of dopamine (DA) showed a decrease. Especially these alteration were observed in the cerebral cortex, but not in the cerebellum. 2) The viral growth in the brain was observed at 48 hours after the inoculation. The growth in the cerebellum, however, was found to be lower than those in other cerebral regions. 3) The viral antigen was detected in the cerebral cortex, hippocampus, mesencephalon and diencephalon in addition to the substantia nigra and striatum. From these results, it is presumed that clinical manifestation of JEV infection may involve the changes in the metabolism of neurotransmitter, especially those of DA and serotonin in the brain.     

Thresholds of color attribute differences in detecting camouflaged textures

We examined the threshold at which a camouflaged color texture pattern (target) embedded in a surrounding colored texture pattern (background) was discriminated by making the difference between their color distributions serve as a cue. The texture consisted of 900 colored disks. The color applied to the disk was chosen from a normal distribution with the mean and the standard deviation set beforehand. The mean of the background's distribution was a standard achromatic color set at L*=40, u*=0, and v*=0 of CIELUV. In experiment 1, the mean of the target's color distribution was shifted from the background's one. The threshold for the mean of the target's color distribution depended on the standard deviation and increased as the standard deviation became bigger. In experiment 2, the standard deviation of the target's color distribution was shifted. There was the slight dependence of threshold of the standard deviation of the target's distribution on that of the background's distribution. In experiment 3, both of the mean and the standard deviation of the target's color distribution were shifted at the same time. The threshold was not determined by each of the mean and the standard deviation independently. There seemed to be some compensating contribution between them to each other. The threshold could be characterized by Doyle metric or modified Doyle metric.     

Effects of Adding Alkaline Material on the Heavy Metal Chemical Fractions in Soil under Flooded and Non-Flooded Conditions

We investigated the effect of adding an alkaline material (containing calcium carbonate and gypsum) on the immobilization of heavy metals (Cd, Cu, Pb, and Zn) in a paddy soil slightly contaminated with Cd and Zn under flooded and non-flooded conditions in the laboratory. Adding the alkaline material increased the soil pH and significantly decreased the exchangeable fraction of all of the metals, especially for Cd (>75% decrease) and Zn (ca. 90% decrease), under both flooded and non-flooded conditions. Drying the flooded soil samples increased the ratio of exchangeable fraction to the total fraction, particularly for Cd. The exchangeable fraction ratio was lower in the dried, previously flooded samples that contained the alkaline material than in the samples that did not contain the alkaline material, indicating that adding the alkaline material would be an effective way of immobilizing heavy metals during the oxidation of anoxic soils. These results show that the alkaline material can be used to immobilize heavy metals under both anoxic and oxic conditions, and that the effects of flooding and amending a paddy soil with alkaline material on the chemical forms will be different between heavy metals.     

Vertical Distribution and Estimated Doses from Artificial Radionuclides in Soil Samples around the Chernobyl Nuclear Power Plant and the Semipalatinsk Nuclear Testing Site

For the current on-site evaluation of the environmental contamination and contributory external exposure after the accident at the Chernobyl Nuclear Power Plant (CNPP) and the nuclear tests at the Semipalatinsk Nuclear Testing Site (SNTS), the concentrations of artificial radionuclides in soil samples from each area were analyzed by gamma spectrometry. Four artificial radionuclides (²⁴¹Am, ¹³⁴Cs, ¹³⁷Cs, and ⁶⁰Co) were detected in surface soil around CNPP, whereas seven artificial radionuclides (²⁴¹Am, ⁵⁷Co, ¹³⁷Cs, ⁹⁵Zr, ⁹⁵Nb, ⁵⁸Co, and ⁶⁰Co) were detected in surface soil around SNTS. Effective doses around CNPP were over the public dose limit of 1 mSv/y (International Commission on Radiological Protection, 1991). These levels in a contaminated area 12 km from Unit 4 were high, whereas levels in a decontaminated area 12 km from Unit 4 and another contaminated area 15 km from Unit 4 were comparatively low. On the other hand, the effective doses around SNTS were below the public dose limit. These findings suggest that the environmental contamination and effective doses on the ground definitely decrease with decontamination such as removing surface soil, although the effective doses of the sampling points around CNPP in the present study were all over the public dose limit. Thus, the remediation of soil as a countermeasure could be an extremely effective method not only for areas around CNPP and SNTS but also for areas around the Fukushima Dai-ichi Nuclear Power Plant (FNPP), and external exposure levels will be certainly reduced. Long-term follow-up of environmental monitoring around CNPP, SNTS, and FNPP, as well as evaluation of the health effects in the population residing around these areas, could contribute to radiation safety and reduce unnecessary exposure to the public.     

Relationship between endothelin and thromboxane A2 in rat liver microcirculation.

In our previous study, we determined changes in hepatic blood flow using a Laser Doppler blood flow meter after i.v. injection of endothelin-1 (ET-1) or endothelin-3 (ET-3) at 2 nmol/kg in rats and found that ET-3 caused greater decreases in blood flow than ET-1. In the present study, we determined how the arachidonic acid cascade, mainly thromboxane A2 (TXA2), is related to ET-1 and ET-3 using indomethacin (INDO), which inhibits the biosynthesis of prostaglandin (PG), and OKY-046, a selective inhibitor of TXA2 synthesis. In the first series of experiments, ET-1 and ET-3 were administered after inhibiting the biosynthesis of PG by s.c. injection of 2 mg/kg of INDO. While INDO failed to inhibit the slight decrease in hepatic blood flow induced by ET-1, it significantly inhibited the marked decrease in hepatic blood flow elicited by ET-3. In the next series of experiments, ET-1 and ET-3 were administered after administration of 20 mg/kg of OKY-046. OKY-046 showed no effects in animals treated with ET-1, as in those pre-treated with INDO, while it significantly inhibited the decreases in hepatic blood flow induced by ET-3. These findings suggest that ET-1 decreases hepatic blood flow due to its direct effects although to a lesser extent than ET-3, while ET-3 does so due not only to its direct effects but also to TXA2-mediated effects. It is therefore likely that in addition to ET family peptides, PG-mediated mechanisms are involved in the regulation of hepatic microcirculation by ETs.     

Phosphorus-31 magnetic resonance spectroscopy of forearm flexor muscles in student rowers using an exercise protocol adjusted for differences in cross-sectional muscle area

To assess exercise energy metabolism of forearm flexor muscles in rowers, six male student rowers and six control subjects matched for age and sex were studied using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Firstly, to adjust for the effect of differences in cross-sectional muscle area, the maximal cross-sectional area (CSAmax) of the forearm flexor muscles was estimated in each individual using magnetic resonance imaging. Multistage exercise was then carried out with an initial energy production of 1 J.cm-2 CSAmax for 1 min and an increment of 1 J.cm-2 CSAmax every minute to the point of muscle exhaustion. A series of measurements of 31P-MRS were performed every minute. The CSAmax was significantly greater in the student rowers than in the control subjects [19.8 (SD 2.2) vs 17.1 (SD 1.2) cm2, P less than 0.05]. The absolute maximal exercise intensity (J.min-1) was greater in the rowers than in the control subjects. However, the maximal exercise intensity per unit of muscle cross sectional area (J.min-1.cm-2) was not significantly different between the two groups. During mild to moderate exercise intensities, a decrease in phosphocreatine and an increase in inorganic phosphate before the onset of acidosis were significantly less in the rowers, indicating a requirement of less adenosine 5'-diphosphate to drive adenosine 5'-triphosphate production. The onset of acidosis was also significantly delayed in the rowers. No difference was observed in forearm blood flow between the two groups at the same exercise intensity (J.min-1.cm-2).(ABSTRACT TRUNCATED AT 250 WORDS)