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.     

An Atypical Tubulin Kinase Mediates Stress-Induced Microtubule Depolymerization in Arabidopsis

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Recruitment of scribble to the synaptic scaffolding complex requires GUK-holder, a novel DLG binding protein

BACKGROUND: Membrane-associated guanylate kinases (MAGUKs), such as Discs-Large (DLG), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between DLG and another PDZ scaffolding protein, SCRIBBLE (SCRIB), during the establishment of cell polarity in developing epithelia. A possible interaction between DLG and SCRIB at synaptic junctions has not yet been addressed. Likewise, the biochemical nature of this interaction remains elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated. RESULTS: Here we report the isolation of a new DLG-interacting protein, GUK-holder, that interacts with the GUK domain of DLG and which is dynamically expressed during synaptic bouton budding. We also show that at Drosophila synapses DLG colocalizes with SCRIB and that this colocalization is likely to be mediated by direct interactions between GUKH and the PDZ2 domain of SCRIB. We show that DLG, GUKH, and SCRIB form a tripartite complex at synapses, in which DLG and GUKH are required for the proper synaptic localization of SCRIB. CONCLUSIONS: Our results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse.     

In vitro release of growth hormone-releasing factor from rat hypothalamus: effect of insulin-like growth factor-1

The release of growth hormone-releasing factor (GHRF) from rat hypothalamus was investigated in vitro. After 60 min preincubation the released GHRF from sliced rat hypothalamic fragments during 60 min incubation was detected by a highly specific and sensitive radioimmunoassay for rat GHRF. The release of GHRF was Ca2+-dependent and enhanced by high concentration of K+. Insulin-like growth factor-1 (IGF-1) significantly decreased GHRF release to 65% and 84% of the control at concentrations of 10(-8) M and 10(-7) M, respectively. These results suggest that this in vitro system is useful for the investigation of the mechanism of GHRF release from the hypothalamus and that IGF-1 is probably involved in the feedback inhibition of growth hormone secretion by attenuating GHRF release from the hypothalamus besides countering the effect of GHRF on the pituitary.     

Consumers and establishment limitations contribute more than competitive interactions in sustaining dominance of the exotic herb garlic mustard in a Wisconsin, USA forest

The understory is a diverse component of temperate forest ecosystems, contributing significantly to forest ecosystem services. Despite their importance, many native understories face stresses from current and past land use, habitat fragmentation, invasive species, and overabundant herbivores. We established a four block, three factor experiment to evaluate the relative contribution of native plant establishment, competitive effects from the invasive herb garlic mustard (Alliaria petiolata), and herbivory from white-tailed deer (Odocoileus virginianus) to better understand the mechanisms promoting low native plant richness and cover and understory dominance by the biennial exotic herb garlic mustard in a NE Wisconsin, USA forest. Four years of garlic mustard removal failed to increase native plant richness or cover in non-restored plots. However, deer access and the introduction of native plants (restoration treatment) both significantly enhanced native plant cover and richness, with restored species cover in fenced plots approximately 216 % that of open-access plots, and the majority of these species flowered at significantly higher proportions inside of fenced areas. In contrast, deer access did not significantly alter the cover, or seed production of garlic mustard. We also found no significant effect of garlic mustard presence on the cover or flowering of restored native species. We conclude that multiple factors, including limited natural establishment by native species and selective herbivory drove low native, high exotic dominance at our site, suggesting that a shift in focus from invasive plant removal to combined native plant restoration and herbivore control is needed to maximize the recovery of this degraded forest understory.     

Phylogenetic relationships among amphisbaenian reptiles based on complete mitochondrial genomic sequences

Complete mitochondrial genomic sequences are reported from 12 members in the four families of the reptile group Amphisbaenia. Analysis of 11,946 aligned nucleotide positions (5797 informative) produces a robust phylogenetic hypothesis. The family Rhineuridae is basal and Bipedidae is the sister taxon to the Amphisbaenidae plus Trogonophidae. Amphisbaenian reptiles are surprisingly old, predating the breakup of Pangaea 200 million years before present, because successive basal taxa (Rhineuridae and Bipedidae) are situated in tectonic regions of Laurasia and nested taxa (Amphisbaenidae and Trogonophidae) are found in Gondwanan regions. Thorough sampling within the Bipedidae shows that it is not tectonic movement of Baja California away from the Mexican mainland that is primary in isolating Bipes species, but rather that primary vicariance occurred between northern and southern groups. Amphisbaenian families show parallel reduction in number of limbs and Bipes species exhibit parallel reduction in number of digits. A measure is developed for comparing the phylogenetic information content of various genes. A synapomorphic trait defining the Bipedidae is a shift from the typical vertebrate mitochondrial gene arrangement to the derived state of trnE and nad6. In addition, a tandem duplication of trnT and trnP is observed in Bipes biporus with a pattern of pseudogene formation that varies among populations. The first case of convergent rearrangement of the mitochondrial genome among animals demonstrated by complete genomic sequences is reported. Relative to most vertebrates, the Rhineuridae has the block nad6, trnE switched in order with the block cob, trnT, trnP, as they are in birds.     

Effect of maternal weight, adipokines, glucose intolerance and lipids on infant birth weight among women without gestational diabetes mellitus

Background:

The delivery of excess maternal nutrients to the fetus is known to increase the risk of macrosomia, even among infants of women without gestational diabetes mellitus. With the current obesity epidemic, maternal adiposity and its associated effects on circulating adipokines and inflammatory proteins may now have a greater impact on fetal growth. We sought to evaluate the independent effects of maternal glycemia, lipids, obesity, adipokines and inflammation on infant birth weight.

Methods:

We included 472 women who underwent an oral glucose tolerance test in late pregnancy and were found not to have gestational diabetes; 104 (22.0%) had gestational impaired glucose tolerance. We also measured fasting levels of insulin, low-and high-density lipoprotein cholesterol, triglycerides, leptin, adiponectin and C-reactive protein. Obstetric outcomes were assessed at delivery.

Results:

The mean birth weight was 3481 g (standard deviation 493 g); 68 of the infants were large for gestational age. On multiple linear regression analysis, positive determinants of birth weight were length of gestation, male infant, weight gain during pregnancy up to the time of the oral glucose tolerance test, body mass index (BMI) before pregnancy and impaired glucose tolerance in pregnancy. Leptin, adiponectin and C-reactive protein levels were each negatively associated with birth weight. On logistic regression analysis, the significant metabolic predictors of having a large-for-gestational-age infant were BMI before pregnancy (odds ratio [OR] 1.16, 95% confidence interval [CI] 1.05–1.27, per 1 kg/m² increase), weight gain during pregnancy up to the time of the oral glucose tolerance test (OR 1.12, 95% CI 1.05–1.19, per 1 kg increase) and leptin level (OR 0.50, 95% CI 0.30–0.82, per 1 standard deviation change).

Interpretation:

Among women without gestational diabetes, maternal adiposity and leptin levels were the strongest metabolic determinants of having a large-for-gestational-age infant rather than glucose intolerance and lipid levels.In 1952, Jørgen Pedersen proposed that delivery of excess maternal glucose to the fetus may be responsible for the increased risk of macrosomia among infants of women with diabetes during pregnancy.¹ He postulated that maternal hyperglycemia leads to fetal hyperglycemia, which in turn stimulates insulin secretion in the fetus, the anabolic effects of which result in excessive fetal growth. Since its introduction, the Pedersen hypothesis has been further extended by other investigators and accepted as the pathophysiologic basis for increased risk of macrosomia among infants of women with diabetes during pregnancy.^2,3 Accordingly, for pregnant women with either pre-existing diabetes or gestational diabetes, modern clinical practice focuses on normalizing blood glucose levels to reduce the risk of fetal hyperglycemia and hence the risk of fetal macrosomia and its associated adverse clinical outcomes (e.g., shoulder dystocia, birth injury, need for cesarean delivery).It is now recognized that the association between maternal nutrients and fetal growth is not restricted solely to women with diabetes. Several studies have shown associations linking maternal blood glucose and triglyceride levels with infant birth weight among women without gestational diabetes.^4–7 This awareness has led to recent recommendations to lower the diagnostic thresholds for gestational diabetes on glucose tolerance testing in pregnancy, to optimize the detection of women who may be at risk of having a large-for-gestational-age infant.⁸Another important factor relevant to the risk of macrosomia is maternal adiposity.⁹ Indeed, the past decade has seen a marked increase in the prevalence of pre-existing obesity among pregnant women.¹⁰ In the context of the current obesity epidemic, we hypothesized that, in women without gestational diabetes, maternal adiposity and its associated effects on circulating levels of adipokines (e.g., adiponectin and leptin) and inflammatory proteins (C-reactive protein) may now have a greater impact than glucose and lipid levels on fetal growth. We conducted this study to evaluate the independent effects of maternal glycemia, lipid levels, obesity, adipokine levels and inflammation on the infant birth weight in a cohort of women without gestational diabetes.     

Signal-to-signal neural networks for improved spike estimation from calcium imaging data

Spiking information of individual neurons is essential for functional and behavioral analysis in neuroscience research. Calcium imaging techniques are generally employed to obtain activities of neuronal populations. However, these techniques result in slowly-varying fluorescence signals with low temporal resolution. Estimating the temporal positions of the neuronal action potentials from these signals is a challenging problem. In the literature, several generative model-based and data-driven algorithms have been studied with varied levels of success. This article proposes a neural network-based signal-to-signal conversion approach, where it takes as input raw-fluorescence signal and learns to estimate the spike information in an end-to-end fashion. Theoretically, the proposed approach formulates the spike estimation as a single channel source separation problem with unknown mixing conditions. The source corresponding to the action potentials at a lower resolution is estimated at the output. Experimental studies on the spikefinder challenge dataset show that the proposed signal-to-signal conversion approach significantly outperforms state-of-the-art-methods in terms of Pearson’s correlation coefficient, Spearman’s rank correlation coefficient and yields comparable performance for the area under the receiver operating characteristics measure. We also show that the resulting system: (a) has low complexity with respect to existing supervised approaches and is reproducible; (b) is layer-wise interpretable, and (c) has the capability to generalize across different calcium indicators.