Reconstituting human somitogenesis in vitro.

The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms; however, the details of this process in humans remain largely unknown owing to ethical and technical limitations. Despite recent advances with pluripotent stem cell-based approaches1-5, models that robustly recapitulate human somitogenesis in both space and time remain scarce. Here we introduce a pluripotent stem cell-derived mesoderm-based 3D model of human segmentation and somitogenesis-which we termed 'axioloid'-that captures accurately the oscillatory dynamics of the segmentation clock and the morphological and molecular characteristics of sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF-WNT signalling gradients and retinoic acid signalling components. We identify an unexpected critical role of retinoic acid signalling in the stabilization of forming segments, indicating distinct, but also synergistic effects of retinoic acid and extracellular matrix on the formation and epithelialization of somites. Comparative analysis demonstrates marked similarities of axioloids to the human embryo, further validated by the presence of a Hox code in axioloids. Finally, we demonstrate the utility of axioloids for studying the pathogenesis of human congenital spine diseases using induced pluripotent stem cells with mutations in HES7 and MESP2. Our results indicate that axioloids represent a promising platform for the study of axial development and disease in humans.

Differentiation of Hypertrophic Chondrocytes from Human iPSCs for the In Vitro Modeling of Chondrodysplasias.

Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, we developed a three-dimensional culture protocol to differentiate hypertrophic chondrocytes from induced pluripotent stem cells (iPSCs) and examine the phenotype caused by MATN3 and COL10A1 mutations. Intracellular MATN3 or COL10 retention resulted in increased ER stress markers and ER size in most mutants, but activation of the UPR was dependent on the mutation. Transcriptome analysis confirmed a UPR with wide-ranging changes in bone homeostasis, extracellular matrix composition, and lipid metabolism in the MATN3 T120M mutant, which further showed altered cellular morphology in iPSC-derived growth-plate-like structures in vivo. We then applied our in vitro model to drug testing, whereby trimethylamine N-oxide led to a reduction of ER stress and intracellular MATN3.

Species-specific segmentation clock periods are due to differential biochemical reaction speeds.

Although mechanisms of embryonic development are similar between mice and humans, the time scale is generally slower in humans. To investigate these interspecies differences in development, we recapitulate murine and human segmentation clocks that display 2- to 3-hour and 5- to 6-hour oscillation periods, respectively. Our interspecies genome-swapping analyses indicate that the period difference is not due to sequence differences in the HES7 locus, the core gene of the segmentation clock. Instead, we demonstrate that multiple biochemical reactions of HES7, including the degradation and expression delays, are slower in human cells than they are in mouse cells. With the measured biochemical parameters, our mathematical model accounts for the two- to threefold period difference between the species. We propose that cell-autonomous differences in biochemical reaction speeds underlie temporal differences in development between species.

Recapitulating the human segmentation clock with pluripotent stem cells.

Pluripotent stem cells are increasingly used to model different aspects of embryogenesis and organ formation1. Despite recent advances in in vitro induction of major mesodermal lineages and cell types2,3, experimental model systems that can recapitulate more complex features of human mesoderm development and patterning are largely missing. Here we used induced pluripotent stem cells for the stepwise in vitro induction of presomitic mesoderm and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modelling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We observed oscillatory expression of core segmentation clock genes, including HES7 and DKK1, determined the period of the human segmentation clock to be around five hours, and demonstrated the presence of dynamic travelling-wave-like gene expression in in vitro-induced human presomitic mesoderm. Furthermore, we identified and compared oscillatory genes in human and mouse presomitic mesoderm derived from pluripotent stem cells, which revealed species-specific and shared molecular components and pathways associated with the putative mouse and human segmentation clocks. Using CRISPR-Cas9-based genome editing technology, we then targeted genes for which mutations in patients with segmentation defects of the vertebrae, such as spondylocostal dysostosis, have been reported (HES7, LFNG, DLL3 and MESP2). Subsequent analysis of patient-like and patient-derived induced pluripotent stem cells revealed gene-specific alterations in oscillation, synchronization or differentiation properties. Our findings provide insights into the human segmentation clock as well as diseases associated with human axial skeletogenesis.

Hyponatremia with Loss of High Signal Intensity in the Posterior Pituitary Lobe on T1-weighted Magnetic Resonance Imaging.

Herein, we report on an 82-year-old woman who presented with anorexia. The patient had hyponatremia with preserved urinary osmotic pressure. T1-weighted magnetic resonance imaging (MRI) showed a lack of high signal intensity (SI) in the posterior pituitary lobe. Based on the patient's high levels of N-terminal prohormone of brain natriuretic peptide (NT-proBNP), heart failure was suspected. The heart failure may have caused arginine vasopressin (AVP) secretion. The depletion of AVP secretory granules may therefore cause the posterior pituitary gland to disappear on T1-weighted MRI.

Diabetic Hemichorea-hemiballism after Prompt Improvement in Hyperglycemia.

We herein report a case of hemichorea-hemiballism in an 85-year-old man diagnosed with diabetes at 76 years of age. After a one-year interruption in treatment, he was treated with a low-calorie diet, linagliptin, and nateglinide. Over 51 days, his HbA1c level decreased from 15.8% to 7.7%. After a prompt improvement in his hyperglycemia, he began experiencing involuntary movements in the right upper and lower extremities. T1-weighted magnetic resonance imaging showed a high signal intensity in the left lens nucleus. The patient was diagnosed with diabetic hemichorea-hemiballism and received haloperidol (1 mg/day) as treatment.

Effects of the selective KACh channel blocker NTC-801 on atrial fibrillation in a canine model of atrial tachypacing: comparison with class Ic and III drugs.

The present study examines the effects of NTC-801, a highly selective acetylcholine (ACh) receptor-activated potassium (KACh) channel blocker, on atrial fibrillation (AF) in a canine model with electrical remodeling. An experimental substrate for AF was created in dogs via left atrial (LA) tachypacing (400 bpm, 3-5 weeks). NTC-801, dofetilide, and flecainide were intravenously infused for 15 minutes, and the effects on AF inducibility, atrial effective refractory period (ERP), and atrial conduction velocity were examined. The effect of NTC-801 on AF termination was also evaluated. Atrial ERP was shortened and AF inducibility was increased after LA tachypacing. NTC-801 (0.3-3 µg·kg⁻¹·min⁻¹) prolonged atrial ERP irrespective of stimulation frequency and dose-dependently decreased AF inducibility. Dofetilide (5.3 µg·kg⁻¹·min⁻¹) and flecainide (0.13 mg·kg⁻¹·min⁻¹) did not significantly inhibit AF inducibility and minimally affected atrial ERP. Flecainide decreased atrial conduction velocity, whereas NTC-801 and dofetilide did not. NTC-801 (0.1 mg/kg) converted AF to normal sinus rhythm. In summary, NTC-801 exerted more effective antiarrhythmic effects than dofetilide and flecainide in a canine LA-tachypacing AF model. The antiarrhythmic activity of NTC-801 was probably due to prolonging atrial ERP independently of stimulation frequency. These results suggest that NTC-801 could prevent AF more effectively in the setting of atrial electrical remodeling.

Xanthine oxidase inhibition by febuxostat attenuates experimental atherosclerosis in mice.

Atherosclerosis is a chronic inflammatory disease due to lipid deposition in the arterial wall. Multiple mechanisms participate in the inflammatory process, including oxidative stress. Xanthine oxidase (XO) is a major source of reactive oxygen species (ROS) and has been linked to the pathogenesis of atherosclerosis, but the underlying mechanisms remain unclear. Here, we show enhanced XO expression in macrophages in the atherosclerotic plaque and in aortic endothelial cells in ApoE(-/-) mice, and that febuxostat, a highly potent XO inhibitor, suppressed plaque formation, reduced arterial ROS levels and improved endothelial dysfunction in ApoE(-/-) mice without affecting plasma cholesterol levels. In vitro, febuxostat inhibited cholesterol crystal-induced ROS formation and inflammatory cytokine release in murine macrophages. These results demonstrate that in the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XO-inhibition by febuxostat is a potential therapy for atherosclerosis.

Febuxostat, an inhibitor of xanthine oxidase, suppresses lipopolysaccharide-induced MCP-1 production via MAPK phosphatase-1-mediated inactivation of JNK.

Excess reactive oxygen species (ROS) formation can trigger various pathological conditions such as inflammation, in which xanthine oxidase (XO) is one major enzymatic source of ROS. Although XO has been reported to play essential roles in inflammatory conditions, the molecular mechanisms underlying the involvement of XO in inflammatory pathways remain unclear. Febuxostat, a selective and potent inhibitor of XO, effectively inhibits not only the generation of uric acid but also the formation of ROS. In this study, therefore, we examined the effects of febuxostat on lipopolysaccharide (LPS)-mediated inflammatory responses. Here we show that febuxostat suppresses LPS-induced MCP-1 production and mRNA expression via activating MAPK phosphatase-1 (MKP-1) which, in turn, leads to dephosphorylation and inactivation of JNK in macrophages. Moreover, these effects of febuxostat are mediated by inhibiting XO-mediated intracellular ROS production. Taken together, our data suggest that XO mediates LPS-induced phosphorylation of JNK through ROS production and MKP-1 inactivation, leading to MCP-1 production in macrophages. These studies may bring new insights into the novel role of XO in regulating inflammatory process through MAPK phosphatase, and demonstrate the potential use of XO inhibitor in modulating the inflammatory processes.

Microcalorimeter-type energy dispersive X-ray spectrometer for a transmission electron microscope.

A new energy dispersive X-ray spectrometer (EDS) with a microcalorimeter detector equipped with a transmission electron microscope (TEM) has been developed for high- accuracy compositional analysis in the nanoscale. A superconducting transition-edge-sensor-type microcalorimeter is applied as the detector. A cryogen-free cooling system, which consists of a mechanical and a dilution refrigerator, is selected to achieve long-term temperature stability. In order to mount these detector and refrigerators on a TEM, the cooling system is specially designed such that these two refrigerators are separated. Also, the detector position and arrangement are carefully designed to avoid adverse affects between the superconductor detector and the TEM lens system. Using the developed EDS system, at present, an energy resolution of 21.92 eV full-width-at-half maximum has been achieved at the Cr K alpha line. This value is about seven times better than that of the current typical commercial Si(Li) detector, which is usually around 140 eV. The developed microcalorimeter EDS system can measure a wide energy range, 1-20 keV, at one time with this high energy resolution that can resolve peaks from most of the elements. Although several further developments will be needed to enable practical use, highly accurate compositional analysis with high energy resolution will be realized by this microcalorimeter EDS system.

Susceptibility and resistance genes to fluoroquinolones in methicillin-resistant Staphylococcus aureus isolated in 2002.

The activity of six fluoroquinolones (FQs) was determined against 100 methicillin-resistant Staphylococcus aureus (MRSA) isolated in 2002 along with mutations in the grlA and gyrA genes and in the norA promoter of these isolates. Of the isolates tested, 97% had mutations in grlA and gyrA. A single mutation in grlA and gyrA resulted in a decrease of susceptibility to old generation FQs (norfloxacin, enoxacin, ciprofloxacin, fleroxacin, sparfloxacin and levofloxacin) but not to new generation FQs (gatifloxacin and moxifloxacin). Double mutations of both grlA and gyrA resulted in high-level resistance to all FQs tested. All norA mutants (15%) contained double mutations in grlA and gyrA and showed no decrease of MIC in the presence of reserpine, which is known to inhibit the drug-efflux pump. Our results showed that double mutations in grlA and gyrA were necessary for the expression of high-level resistance to new generation FQs. As different FQ-resistant mutants occur in the same PFGE type, FQ-resistant MRSA may well develop individually.