Improved therapeutic approach for spinal muscular atrophy via ubiquitination-resistant survival motor neuron variant.

BACKGROUND: Zolgensma is a gene-replacement therapy that has led to a promising treatment for spinal muscular atrophy (SMA). However, clinical trials of Zolgensma have raised two major concerns: insufficient therapeutic effects and adverse events. In a recent clinical trial, 30% of patients failed to achieve motor milestones despite pre-symptomatic treatment. In addition, more than 20% of patients showed hepatotoxicity due to excessive virus dosage, even after the administration of an immunosuppressant. Here, we aimed to test whether a ubiquitination-resistant variant of survival motor neuron (SMN), SMNK186R, has improved therapeutic effects for SMA compared with wild-type SMN (SMNWT). METHODS: A severe SMA mouse model, SMA type 1.5 (Smn-/-; SMN2+/+; SMN∆7+/-) mice, was used to compare the differences in therapeutic efficacy between AAV9-SMNWT and AAV9-SMNK186R. All animals were injected within Postnatal Day (P) 1 through a facial vein or cerebral ventricle. RESULTS: AAV9-SMNK186R-treated mice showed increased lifespan, body weight, motor neuron number, muscle weight and functional improvement in motor functions as compared with AAV9-SMNWT-treated mice. Lifespan increased by more than 10-fold in AAV9-SMNK186R-treated mice (144.8 ± 26.11 days) as compared with AAV9-SMNWT-treated mice (26.8 ± 1.41 days). AAV9-SMNK186R-treated mice showed an ascending weight pattern, unlike AAV9-SMNWT-treated mice, which only gained weight until P20 up to 5 g on average. Several motor function tests showed the improved therapeutic efficacy of SMNK186R. In the negative geotaxis test, AAV9-SMNK186R-treated mice turned their bodies in an upward direction successfully, unlike AAV9-SMNWT-treated mice, which failed to turn upwards from around P23. Hind limb clasping phenotype was rarely observed in AAV9-SMNK186R-treated mice, unlike AAV9-SMNWT-treated mice that showed clasping phenotype for more than 20 out of 30 s. At this point, the number of motor neurons (1.5-fold) and the size of myofibers (2.1-fold) were significantly increased in AAV9-SMNK186R-treated mice compared with AAV9-SMNWT-treated mice without prominent neurotoxicity. AAV9-SMNK186R had fewer liver defects compared with AAV9-SMNWT, as judged by increased proliferation of hepatocytes (P < 0.0001) and insulin-like growth factor-1 production (P < 0.0001). Especially, low-dose AAV9-SMNK186R (nine-fold) also reduced clasping time compared with SMNWT. CONCLUSIONS: SMNK186R will provide improved therapeutic efficacy in patients with severe SMA with insufficient therapeutic efficacy. Low-dose treatment of SMA patients with AAV9-SMNK186R can reduce the adverse events of Zolgensma. Collectively, SMNK186R has value as a new treatment for SMA that improves treatment effectiveness and reduces adverse events simultaneously.

Depletion of SMN protein in mesenchymal progenitors impairs the development of bone and neuromuscular junction in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the deficiency of the survival motor neuron (SMN) protein, which leads to motor neuron dysfunction and muscle atrophy. In addition to the requirement for SMN in motor neurons, recent studies suggest that SMN deficiency in peripheral tissues plays a key role in the pathogenesis of SMA. Using limb mesenchymal progenitor cell (MPC)-specific SMN-depleted mouse models, we reveal that SMN reduction in limb MPCs causes defects in the development of bone and neuromuscular junction (NMJ). Specifically, these mice exhibited impaired growth plate homeostasis and reduced insulin-like growth factor (IGF) signaling from chondrocytes, rather than from the liver. Furthermore, the reduction of SMN in fibro-adipogenic progenitors (FAPs) resulted in abnormal NMJ maturation, altered release of neurotransmitters, and NMJ morphological defects. Transplantation of healthy FAPs rescued the morphological deterioration. Our findings highlight the significance of mesenchymal SMN in neuromusculoskeletal pathogenesis of SMA and provide insights into potential therapeutic strategies targeting mesenchymal cells for the treatment of SMA.

Association Between Acid-Suppressive Drugs and Risk of Rosacea: Retrospective Study Using the Korean National Health Insurance Service-National Sample Cohort.

BACKGROUND: Rosacea is a common inflammatory skin disease with multiple etiologies. Proton pump inhibitors (PPIs) and histamine-2 receptor antagonists (H2RA) are acid suppressive drugs widely used for gastrointestinal (GI) diseases, and long-term use has been reported to be associated with dysbiosis which is a potential risk for development of rosacea. This study aimed to study the association between rosacea and acid suppressants in the Korean national cohort. METHODS: We used Korean National Health Insurance Service-National Sample Cohort data of 749,166 patients with upper GI diseases between 2001 and 2013. Duration of acid suppressants was compared between patients with and without rosacea together with other sociodemographic characteristics and hazard ratios were estimated. RESULTS: Longer use of acid suppressants was significantly associated with increased risk of rosacea. After adjustment for possible confounders, increased cumulative defined daily dose was significantly associated with risk of rosacea (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.20-2.00; P = 0.001). Other factors significantly associated with risk of rosacea include residing in the rural area (OR, 2.58; 95% CI, 2.18-3.06; P < 0.001), greater Charlson Comorbidity Index score (OR, 1.45; 95% CI, 1.15-1.83; P = 0.002), and comorbidities (malignancy, thyroid disease, and depression). CONCLUSION: Results from our study indicate that H2RA or PPI is associated with the occurrence of rosacea among patients with GI diseases in the Korean population. The risk was increased in dose-dependent manner, even after adjusting for confounding variables. Clinicians should be aware of risks associated with prolonged use of acid suppressive drugs.

Mimicking Bone Extracellular Matrix: From BMP-2-Derived Sequences to Osteogenic-Multifunctional Coatings.

Cell-material interactions are regulated by mimicking bone extracellular matrix on the surface of biomaterials. In this regard, reproducing the extracellular conditions that promote integrin and growth factor (GF) signaling is a major goal to trigger bone regeneration. Thus, the use of synthetic osteogenic domains derived from bone morphogenetic protein 2 (BMP-2) is gaining increasing attention, as this strategy is devoid of the clinical risks associated with this molecule. In this work, the wrist and knuckle epitopes of BMP-2 are screened to identify peptides with potential osteogenic properties. The most active sequences (the DWIVA motif and its cyclic version) are combined with the cell adhesive RGD peptide (linear and cyclic variants), to produce tailor-made biomimetic peptides presenting the bioactive cues in a chemically and geometrically defined manner. Such multifunctional peptides are next used to functionalize titanium surfaces. Biological characterization with mesenchymal stem cells demonstrates the ability of the biointerfaces to synergistically enhance cell adhesion and osteogenic differentiation. Furthermore, in vivo studies in rat calvarial defects prove the capacity of the biomimetic coatings to improve new bone formation and reduce fibrous tissue thickness. These results highlight the potential of mimicking integrin-GF signaling with synthetic peptides, without the need for exogenous GFs.

Notch1 and Notch2 Signaling Exclusively but Cooperatively Maintain Fetal Myogenic Progenitors.

Myogenic progenitors (MPs) generate myocytes that fuse to form myofibers during skeletal muscle development while maintaining the progenitor pool, which is crucial for generating sufficient muscle. Notch signaling has been known to reserve a population of embryonic MPs during primary myogenesis by promoting cell cycle exit and suppressing premature differentiation. However, the roles of individual Notch receptors (Notch1-4) during embryonic/fetal myogenesis are still elusive. In this study, we found that Notch1 and Notch2, which exhibit the highest structural similarity among Notch receptors, maintain the MP population by distinct mechanisms: Notch1 induces cell cycle exit and Notch2 suppresses premature differentiation. Moreover, genetic and cell culture studies showed that Notch1 and Notch2 signaling in MPs are distinctively activated by interacting with Notch ligand-expressing myofibers and MP-lineage cells, respectively. These results suggest that through different activation modes, Notch1 and Notch2 distinctively and cooperatively maintain MP population during fetal myogenesis for proper muscle development.

Bap1/SMN axis in Dpp4+ skeletal muscle mesenchymal cells regulates the neuromuscular system.

The survival of motor neuron (SMN) protein is a major component of the pre-mRNA splicing machinery and is required for RNA metabolism. Although SMN has been considered a fundamental gene for the central nervous system, due to its relationship with neuromuscular diseases, such as spinal muscular atrophy, recent studies have also revealed the requirement of SMN in non-neuronal cells in the peripheral regions. Here, we report that the fibro-adipogenic progenitor subpopulation expressing Dpp4 (Dpp4+ FAPs) is required for the neuromuscular system. Furthermore, we also reveal that BRCA1-associated protein-1 (Bap1) is crucial for the stabilization of SMN in FAPs by preventing its ubiquitination-dependent degradation. Inactivation of Bap1 in FAPs decreased SMN levels and accompanied degeneration of the neuromuscular junction, leading to loss of motor neurons and muscle atrophy. Overexpression of the ubiquitination-resistant SMN variant, SMNK186R, in Bap1-null FAPs completely prevented neuromuscular degeneration. In addition, transplantation of Dpp4+ FAPs, but not Dpp4- FAPs, completely rescued neuromuscular defects. Our data reveal the crucial role of Bap1-mediated SMN stabilization in Dpp4+ FAPs for the neuromuscular system and provide the possibility of cell-based therapeutics to treat neuromuscular diseases.

Maintenance of type 2 glycolytic myofibers with age by Mib1-Actn3 axis.

Age-associated muscle atrophy is a debilitating condition associated with loss of muscle mass and function with age that contributes to limitation of mobility and locomotion. However, the underlying mechanisms of how intrinsic muscle changes with age are largely unknown. Here we report that, with age, Mind bomb-1 (Mib1) plays important role in skeletal muscle maintenance via proteasomal degradation-dependent regulation of α-actinin 3 (Actn3). The disruption of Mib1 in myofibers (Mib1ΔMF) results in alteration of type 2 glycolytic myofibers, muscle atrophy, impaired muscle function, and Actn3 accumulation. After chronic exercise, Mib1ΔMF mice show muscle atrophy even at young age. However, when Actn3 level is downregulated, chronic exercise-induced muscle atrophy is ameliorated. Importantly, the Mib1 and Actn3 levels show clinical relevance in human skeletal muscles accompanied by decrease in skeletal muscle function with age. Together, these findings reveal the significance of the Mib1-Actn3 axis in skeletal muscle maintenance with age and suggest the therapeutic potential for the treatment or amelioration of age-related muscle atrophy.

The hypothalamic-pituitary-gonadal axis controls muscle stem cell senescence through autophagosome clearance.

BACKGROUND: With organismal aging, the hypothalamic-pituitary-gonadal (HPG) activity gradually decreases, resulting in the systemic functional declines of the target tissues including skeletal muscles. Although the HPG axis plays an important role in health span, how the HPG axis systemically prevents functional aging is largely unknown. METHODS: We generated muscle stem cell (MuSC)-specific androgen receptor (Ar) and oestrogen receptor 2 (Esr2) double knockout (dKO) mice and pharmacologically inhibited (Antide) the HPG axis to mimic decreased serum levels of sex steroid hormones in aged mice. After short-term and long-term sex hormone signalling ablation, the MuSCs were functionally analysed, and their aging phenotypes were compared with those of geriatric mice (30-month-old). To investigate pathways associated with sex hormone signalling disruption, RNA sequencing and bioinformatic analyses were performed. RESULTS: Disrupting the HPG axis results in impaired muscle regeneration [wild-type (WT) vs. dKO, P < 0.0001; Veh vs. Antide, P = 0.004]. The expression of DNA damage marker (in WT = 7.0 ± 1.6%, dKO = 32.5 ± 2.6%, P < 0.01; in Veh = 13.4 ± 4.5%, Antide = 29.7 ± 5.5%, P = 0.028) and senescence-associated ß-galactosidase activity (in WT = 3.8 ± 1.2%, dKO = 10.3 ± 1.6%, P < 0.01; in Veh = 2.1 ± 0.4%, Antide = 9.6 ± 0.8%, P = 0.005), as well as the expression levels of senescence-associated genes, p16Ink4a and p21Cip1 , was significantly increased in the MuSCs, indicating that genetic and pharmacological inhibition of the HPG axis recapitulates the progressive aging process of MuSCs. Mechanistically, the ablation of sex hormone signalling reduced the expression of transcription factor EB (Tfeb) and Tfeb target gene in MuSCs, suggesting that sex hormones directly induce the expression of Tfeb, a master regulator of the autophagy-lysosome pathway, and consequently autophagosome clearance. Transduction of the Tfeb in naturally aged MuSCs increased muscle mass [control geriatric MuSC transplanted tibialis anterior (TA) muscle = 34.3 ± 2.9 mg, Tfeb-transducing geriatric MuSC transplanted TA muscle = 44.7 ± 6.7 mg, P = 0.015] and regenerating myofibre size [eMyHC+ tdTomato+ myofibre cross-section area (CSA) in control vs. Tfeb, P = 0.002] after muscle injury. CONCLUSIONS: Our data show that the HPG axis systemically controls autophagosome clearance in MuSCs through Tfeb and prevents MuSCs from senescence, suggesting that sustained HPG activity throughout life regulates autophagosome clearance to maintain the quiescence of MuSCs by preventing senescence until advanced age.

Performance of AlGaN/GaN Nanowire Omega-Shaped-Gate Fin-Shaped Field-Effect Transistor.

The AlGaN/GaN nanowire omega-shaped-gate FinFET have been successfully fabricated demonstrating much improved performance compared to conventional AlGaN/GaN MISHFET. The AlGaN/GaN omega-shaped-gate FinFET exhibited the remarkable on-state performances, such as maximum drain current of 1.1 A/mm, low on-resistance, and low current collapse compared to that of the conventional device structure. In addition, the excellent off-state performances were measured: low off-state leakage current as low as -10(-10) mA, the theoretical SS value of -62 mV/dec, and high I(ON)/I(OFF) ratio (-10(9)). Improved dc performances were obtained for omega-shaped-gate structure due to the fully depletion of the active fin body and perfectly separation of the depleted fin from the underlying thick GaN buffer layer. Furthermore, the additional reason for the enhanced device performance of the proposed device is the improved gate controllability compared to the conventional MISHFET. The proposed nano-structure device is very promising candidate for the steep switching device applications.

AIMP2 Controls Intestinal Stem Cell Compartments and Tumorigenesis by Modulating Wnt/ß-Catenin Signaling.

Wnt/ß-catenin (CTNNB1) signaling is crucial for the proliferation and maintenance of intestinal stem cells (ISC), but excessive activation leads to ISC expansion and eventually colorectal cancer. Thus, negative regulators are required to maintain optimal levels of Wnt/ß-catenin signaling. Aminoacyl-tRNA synthetase-interacting multifunctional proteins (AIMP) function in protein synthesis, but have also been implicated in signaling cascades affecting angiogenesis, immunity, and apoptosis. In this study, we investigated the relationship between AIMP2 and Wnt/ß-catenin signaling in a murine model of intestinal homeostasis and tumorigenesis. Hemizygous deletion of Aimp2 resulted in enhanced Wnt/ß-catenin signaling, increased proliferation of cryptic epithelial cells, and expansion of ISC compartments. In an Apc(Min/+) background, Aimp2 hemizygosity increased adenoma formation. Mechanistically, AIMP2 disrupted the interaction between AXIN and Dishevelled-1 (DVL1) to inhibit Wnt/ß-catenin signaling by competing with AXIN. Furthermore, AIMP2 inhibited intestinal organoid formation and growth by suppressing Wnt/ß-catenin signaling in an Aimp2 gene dosage-dependent manner. Collectively, our results showed that AIMP2 acts as a haploinsufficient tumor suppressor that fine-tunes Wnt/ß-catenin signaling in the intestine, illuminating the regulation of ISC abundance and activity. Cancer Res; 76(15); 4559-68. ©2016 AACR.

Design and analysis of vertical-channel gallium nitride (GaN) junctionless nanowire transistors (JNT).

Vertical-channel gallium nitride (GaN) junctionless nanowire transistor (JNT) has been designed and characterized by technology computer-aided design (TCAD) simulations. Various characteristics such as wide bandgap, strong polariztion field, and high electron velocity make GaN one of the attractive materials in advanced electronics in recent times. Nanowire-structured GaN can be applicable to various transistors for enhanced electrical performances by its geometrical feature. In this paper, we analyze the direct-current (DC) characteristics depending on various channel doping concentrations (N(ch)) and nanowire radii (R(NW)). Furthermore, the radio-frequency (RF) characteristics under optimized conditions are extracted by small-signal equivalent circuit modeling. For the optimally designed vertical GaN JNT demonstrated on-state current (I(on)) of 345 µA/µm and off-state current (I(off)) of 3.7 x 10(-18) A/µm with a threshold voltage (V(t)) of 0.22 V, and subthreshold swing (S) of 68 mV/dec. Besides, f(T) and f(max) under different operating conditions (gate voltage, V(GS)) have been obtained.

Probing graphene grain boundaries with optical microscopy.

Grain boundaries in graphene are formed by the joining of islands during the initial growth stage, and these boundaries govern transport properties and related device performance. Although information on the atomic rearrangement at graphene grain boundaries can be obtained using transmission electron microscopy and scanning tunnelling microscopy, large-scale information regarding the distribution of graphene grain boundaries is not easily accessible. Here we use optical microscopy to observe the grain boundaries of large-area graphene (grown on copper foil) directly, without transfer of the graphene. This imaging technique was realized by selectively oxidizing the underlying copper foil through graphene grain boundaries functionalized with O and OH radicals generated by ultraviolet irradiation under moisture-rich ambient conditions: selective diffusion of oxygen radicals through OH-functionalized defect sites was demonstrated by density functional calculations. The sheet resistance of large-area graphene decreased as the graphene grain sizes increased, but no strong correlation with the grain size of the copper was revealed, in contrast to a previous report. Furthermore, the influence of graphene grain boundaries on crack propagation (initialized by bending) and termination was clearly visualized using our technique. Our approach can be used as a simple protocol for evaluating the grain boundaries of other two-dimensional layered structures, such as boron nitride and exfoliated clays.

Scalable complementary logic gates with chemically doped semiconducting carbon nanotube transistors.

Use of random network carbon nanotube (CNT) transistors and their applications to complementary logic gates have been limited by several factors such as control of CNT density, existence of metallic CNTs producing a poor yield of devices, absence of stable n-dopant and control of precise position of the dopant, and absence of a scalable and cost-effective fabrication process. Here, we report a scalable and cost-effective fabrication of complementary logic gates by precisely positioning an air-stable n-type dopant, viologen, by inkjet printing on a separated semiconducting CNTs network. The obtained CNT transistors showed a high yield of nearly 100% with an on/off ratio of greater than 10(3) in an optimized channel length (∼9 µm). The n-doped semiconducting carbon nanotube transistors showed a nearly symmetric behavior in the on/off current and threshold voltage with p-type transistors. CMOS inverter, NAND, and NOR logic gates were integrated on a HfO2/Si substrate using the n/p transistor arrays. The gain of inverter is extraordinarily high, which is around 45, and NAND and NOR logic gates revealed excellent output on and off voltages. These series of whole processes were conducted under ambient conditions, which can be used for large-area and flexible thin film technology.

Role of anions in the AuCl3-doping of carbon nanotubes.

The doping/dedoping mechanism of carbon nanotubes (CNTs) with AuCl(3) has been investigated with regard to the roles of cations and anions. Contrary to the general belief that CNTs are p-doped through the reduction of cationic Au(3+) to Au(0), we observed that chlorine anions play a more important role than Au cations in doping. To estimate the effects of Cl and Au on CNTs, the CNT film was dedoped as a function of the annealing temperature (100-700 °C) under an Ar ambient and was confirmed by the sheet resistance change and the presence of a G-band in the Raman spectra. The X-ray photoelectron spectroscopy (XPS) analysis revealed that the doping level of the CNT film was strongly related to the amount of adsorbed chlorine atoms. Annealing at temperatures up to 200 °C did not change the amount of adsorbed Cl atoms on the CNTs, and the CNT film was stable under ambient conditions. Alternatively, Cl atoms started to dissociate from CNTs at 300 °C, and the stability of the film was degraded. Furthermore, the change in the amount of Cl atoms in CNTs was inversely proportional to the change in the sheet resistance. Our observations of the Cl adsorption, either directly or mediated by an Au precursor on the CNT surface, are congruent with the previous theoretical prediction.

Transparent organic p-dopant in carbon nanotubes: bis(trifluoromethanesulfonyl)imide.

We propose bis(trifluoromethanesulfonyl)imide [(CF(3)SO(2))(2)N](-) (TFSI) as a transparent strong electron-withdrawing p-type dopant in carbon nanotubes (CNTs). The conventional p-dopant, AuCl(3), has several drawbacks, such as hygroscopic effect, formation of Au clusters, decrease in transmittance, and high cost in spite of the significant increase in conductivity. TFSI is converted from bis(trifluoromethanesulfonyl)amine (TFSA) by accepting electrons from CNTs, subsequently losing a proton as a characteristic of a Brønsted acid, and has an inductive effect from atoms with high electronegativity, such as halogen, oxygen, and nitrogen. TFSI produced a similar improvement in conductivity to AuCl(3), while maintaining high thermal stability, and no appreciable change in transmittance with no cluster formation. The effectiveness of TFSI was compared with that of other derivatives.