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Striated preferentially expressed gene (SPEG), a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle-specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here, we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amount of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1 and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and β1 integrin, both of which are critical components of the focal adhesion complex. Further, β1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.

Striated preferentially expressed protein kinase (SPEG), a myosin light chain kinase, is mutated in centronuclear myopathy (CNM) and/or dilated cardiomyopathy. No precise therapies are available for this disorder, and gene replacement therapy is not a feasible option due to the large size of SPEG. We evaluated the potential of dynamin-2 (DNM2) reduction as a potential therapeutic strategy because it has been shown to revert muscle phenotypes in mouse models of CNM caused by MTM1, DNM2, and BIN1 mutations. We determined that SPEG-β interacted with DNM2, and SPEG deficiency caused an increase in DNM2 levels. The DNM2 reduction strategy in Speg-KO mice was associated with an increase in life span, body weight, and motor performance. Additionally, it normalized the distribution of triadic proteins, triad ultrastructure, and triad number and restored phosphatidylinositol-3-phosphate levels in SPEG-deficient skeletal muscles. Although DNM2 reduction rescued the myopathy phenotype, it did not improve cardiac dysfunction, indicating a differential tissue-specific function. Combining DNM2 reduction with other strategies may be needed to target both the cardiac and skeletal defects associated with SPEG deficiency. DNM2 reduction should be explored as a therapeutic strategy against other genetic myopathies (and dystrophies) associated with a high level of DNM2.

With the rapid development of the global navigation satellite system (GNSS), high-rate GNSS has been widely used for high-precision GNSS coseismic displacement retrieval. In recent decades, relative positioning (RP) and precise point positioning (PPP) are mainly adopted to retrieve coseismic displacements. However, RP can only obtain relative coseismic displacements with respect to a reference station, which might be subject to quaking during a large seismic event. While PPP needs a long (re)convergence period of tens of minutes. There is no convergence time needed in the variometric approach for displacements analysis standalone engine (VADASE) but the derived displacements are accompanied by a drift. Temporal point positioning (TPP) method adopts temporal-differenced ionosphere-free phase measurements between a reference epoch and the current epoch, and there is almost no drift in the displacement derived from TPP method. Nevertheless, the precise orbit and clock products should be applied in the TPP method. The studies in recent years are almost based on the postprocessing precise orbits and clocks or simulated real-time products. Since 2013, international GNSS service (IGS) has been providing an open-access real-time service (RTS), which consists of orbit, clock and other corrections. In this contribution, we evaluated the performance of real-time coseismic displacement retrieval based on TPP method with IGS RTS correction products. At first, the real-time precise orbit and clock offsets are derived from the RTS correction products. Then, the temporal-differenced ionosphere-free (IF) combinations are formed and adopted as the TPP measurements. By applying real-time precise orbit and clock offsets, the coseismic displacement can be real-timely retrieved based on TPP measurements. To evaluate the accuracy, two experiments including a stationary experiment and an application to an earthquake event were carried out. The former gives an accuracy of 1.8 cm in the horizontal direction and 4.1 cm in the vertical direction during the whole period of 15-min. The latter gives an accuracy of 1.2 cm and 2.4 cm in the horizontal and vertical components, respectively.

The monitoring of pregnant women is very important. It plays an important role in reducing fetal mortality, ensuring the safety of perinatal mother and fetus, preventing premature delivery and pregnancy accidents. At present, regular examination is the mainstream method for pregnant women's monitoring, but the means of examination out of hospital is scarce, and the equipment of hospital monitoring is expensive and the operation is complex. Using intelligent information technology (such as machine learning algorithm) can analyze the physiological signals of pregnant women, so as to realize the early detection and accident warning for mother and fetus, and achieve the purpose of high-quality monitoring out of hospital. However, at present, there are not enough public research reports related to the intelligent processing methods of out-of-hospital monitoring for pregnant women, so this paper takes the out-of-hospital monitoring for pregnant women as the research background, summarizes the public research reports of intelligent processing methods, analyzes the advantages and disadvantages of the existing research methods, points out the possible problems, and expounds the future development trend, which could provide reference for future related researches.孕妇监护很重要，在降低胎儿死亡率、保障围产期母胎安全、预防早产和妊娠意外等方面起到极为重要的作用。定期检查是目前孕妇监护的主流方法，但院外检查手段匮乏，而医院监护设备昂贵、操作复杂。利用智能信息技术（如机器学习算法）可以分析孕妇相关生理信号，从而实现对母胎的早期检测和意外预警，达到高质量院外监护的目的。但目前，与孕妇院外监护的智能处理方法相关的公开研究综述报道不足，因此本文以孕妇院外监护为研究背景，对其中智能处理方法的公开研究报道进行综述，分析现有研究方法的优缺点，指出可能存在的问题，阐述未来发展趋势，以期为今后的相关研究提供参考。.

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm towards mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

Facioscapulohumeral dystrophy type 1 (FSHD-1) is the most common autosomal dominant form of muscular dystrophy with a prevalence of ∼1 in 8000 individuals. It is considered a late-onset form of muscular dystrophy and leads to asymmetric muscle weakness in the facial, scapular, trunk and lower extremities. The prevalent hypothesis on disease pathogenesis is explained by misexpression of a germ line, primate-specific transcription factor DUX4-fl (double homeobox 4, full-length isoform) linked to the chromosome 4q35. In vitro and in vivo studies have demonstrated that very low levels of DUX4-fl expression are sufficient to induce an apoptotic and/or lethal phenotype, and therefore modeling of the disease has proved challenging. In this study, we expand upon our previously established injection model of DUX4 misexpression in zebrafish and describe a DUX4-inducible transgenic zebrafish model that better recapitulates the expression pattern and late onset phenotype characteristic of FSHD patients. We show that an induced burst of DUX4 expression during early development results in the onset of FSHD-like phenotypes in adulthood, even when DUX4 is no longer detectable. We also utilize our injection model to study long-term consequences of DUX4 expression in those that fail to show a developmental phenotype. Herein, we introduce a hypothesis that DUX4 expression during developmental stages is sufficient to induce FSHD-like phenotypes in later adulthood. Our findings point to a developmental role of DUX4 misexpression in the pathogenesis of FSHD and should be factored into the design of future therapies.

Wastewater such as digestate from waste biomass anaerobic digestion contains a high concentration of nutrients such as nitrogen and phosphorus. A novel process for simultaneous nitrogen removal and phosphorus recovery was recently developed based on the combination of the anammox process and hydroxyapatite (HAP) crystallization. This study firstly investigated the feasibility of operating this process under low temperature using an anammox-HAP type expanded bed reactor. By gradually increasing nitrogen loading and phosphorus loading rate, a nitrogen removal rate of 8.45 ± 0.49 gN/L/d with a simultaneous phosphorus recovery rate of 0.63 ± 0.06 gP/L/d was obtained at 15 °C. The efficiency for both nitrogen removal and phosphorus recovery was higher than 80%. The addition of Ca2+ was confirmed to be an effective strategy to enhance the efficiency of phosphorus recovery by affecting the supersaturation condition of the solution. However, a high phosphorus load may result in excessively high mineral concentrations in the sludge and affect anammox performance. The granular sludge formed under low temperature conditions in this study has a typical double-layer structure of HAP-core and anammox biofilm. This study firstly demonstrated the feasibility of the anammox-HAP process even under low temperatures and give new options for future digestate treatment.

While anammox is a cost-effective nitrogen treatment process for wastewater with high nutrient strength, phosphorus remains untouched during this process and needs further treatment. In this study, the nitrogen removal and phosphorus recovery were achieved simultaneously at 25 °C using an anammox expanded bed. A stable high nitrogen removal efficiency of 83.7 ± 4.8% at a 1500 mgN/L influent total nitrogen concentration and a phosphorus removal efficiency of 94.2 ± 1.2% at 100 mg P/L influent total phosphorus were obtained during continuous operation. The effects of the nitrogen loading rate, hydraulic retention time (HRT), pH, Ca2+ and PO43- concentration on the phosphorus removal was verified in the long-term operation of the reactor. The sludge produced contained a high content of phosphorus mainly in the form of hydroxyapatite (HAP), and the sludge composition strongly reflected the nitrogen and phosphorus loading. The structure of the anammox-HAP composite granules was illustrated by the use of fluorescence in situ hybridization (FISH) and Raman spectroscopic mapping analysis. The results in this study indicate that by controlling the operation parameters, it is possible to integrate a high efficiency phosphorus recovery with the anammox process, and significantly reduce the nutrient loading for further wastewater treatment.

Derepression of DUX4 in skeletal muscle has emerged as a likely cause of pathology in facioscapulohumeral muscular dystrophy (FSHD). Here we report on the use of antisense phosphorodiamidate morpholino oligonucleotides to suppress DUX4 expression and function in FSHD myotubes and xenografts. The most effective was phosphorodiamidate morpholino oligonucleotide FM10, which targets the polyadenylation signal of DUX4. FM10 had no significant cell toxicity, and RNA-seq analyses of FSHD and control myotubes revealed that FM10 down-regulated many transcriptional targets of DUX4, without overt off-target effects. Electroporation of FM10 into FSHD patient muscle xenografts in mice also down-regulated DUX4 and DUX4 targets. These findings demonstrate the potential of antisense phosphorodiamidate morpholino oligonucleotides as an FSHD therapeutic option.

SummaryDuchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our “chemical-compound-based” strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological “dual-SMAD” inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form “rescued” multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human “DMD-in-a-dish” model using hiPSC-based disease modeling.

The characteristic carmine red color due to heme proteins is always observed in enriched anammox biomass. Heme c is a very important co-factor participating the main metabolic reactions with catalytic and electron-transfer potential in the anammox bacteria, and is possible for use as an indicator to evaluate anammox performance. Knowledge of the relationship between the heme c concentration and the anammox reactor performance is, however, very limited available information is constrained at an operation temperature of 35 °C. In this study, we report the heme c concentration change along with nitrogen removal rate (NRR) in three anammox expanded granular sludge bed reactors operated at different temperatures (15, 25, 35 °C). The response of specific anammox activity (SAA) to temperature was revealed for biomass originating from three reactors. The results indicate a strong relationship between heme c concentration and NRR at different culture temperatures. The possibility of evaluating the anammox performance by combining heme c quantification and the temperature is revealed.

Supplemental Material, Gilbert-Honick_Supplemental_Information for Adipose-derived Stem/Stromal Cells on Electrospun Fibrin Microfiber Bundles Enable Moderate Muscle Reconstruction in a Volumetric Muscle Loss Model by Jordana Gilbert-Honick, Brian Ginn, Yuanfan Zhang, Sara Salehi, Kathryn R. Wagner, Hai-Quan Mao, and Warren L. Grayson in Cell Transplantation

Development of novel therapeutics requires good animal models of disease. Disorders for which good animal models do not exist have very few drugs in development or clinical trial. Even where there are accepted, albeit imperfect models, the leap from promising preclinical drug results to positive clinical trials commonly fails, including in disorders of skeletal muscle. The main alternative model for early drug development, tissue culture, lacks both the architecture and, usually, the metabolic fidelity of the normal tissue in vivo. Herein, we demonstrate the feasibility and validity of human to mouse xenografts as a preclinical model of myopathy. Human skeletal muscle biopsies transplanted into the anterior tibial compartment of the hindlimbs of NOD-Rag1(null) IL2rγ(null) immunodeficient host mice regenerate new vascularized and innervated myofibers from human myogenic precursor cells. The grafts exhibit contractile and calcium release behavior, characteristic of functional muscle tissue. The validity of the human graft as a model of facioscapulohumeral muscular dystrophy is demonstrated in disease biomarker studies, showing that gene expression profiles of xenografts mirror those of the fresh donor biopsies. These findings illustrate the value of a new experimental model of muscle disease, the human muscle xenograft in mice, as a feasible and valid preclinical tool to better investigate the pathogenesis of human genetic myopathies and to more accurately predict their response to novel therapeutics.

Cellular metabolic activity, especially mitochondrial metabolism, plays a vital role in regulating cell proliferation and differentiation. Metabolism could therefore be an important factor to consider when using engineering technologies to stimulate tissue development and repair. The small metabolite carnitine and its derivative acetylcarnitine affect the activities of several pathways in mitochondrial metabolism, but their influence on cell differentiation has not yet been thoroughly studied. To elucidate the effects of these two small molecules on mesenchymal tissue engineering, we used adult stem cells as a platform in both monolayer and 3D hydrogel culture systems. We investigated the impact of these two small molecules on the differentiation of adult stem cells and analysed gene expression, cell proliferation and extracellular matrix deposition. We found that the molecules reduced adipogenesis but stimulated osteogenesis and chondrogenesis in both culture systems. Our results suggest that carnitine and acetylcarnitine could affect the differentiation rate of adult stem cells by regulating mitochondrial metabolism. The effects of these two small molecules give rise to the possibility of employing such metabolites in tissue-engineering systems to enhance cell differentiation and tissue development.

A novel biosensor based on single-stranded DNA (ssDNA) probe functionalized aluminum anodized oxide (AAO) nanopore membranes was demonstrated for Escherichia coli O157:H7 DNA detection. An original and dynamic polymerase-extending (PE) DNA hybridization procedure is proposed, where hybridization happens in the existence of Taq DNA polymerase and dNTPs under controlled reaction temperature. The probe strand would be extended as long as the target DNA strand, then the capability to block the ionic flow in the pores has been prominently enhanced by the double strand complex. We have investigated the variation of ionic conductivity during the fabrication of the film and the hybridization using cyclic voltammetry and impedance spectroscopy. The present approach provides low detection limit for DNA (a few hundreds of pmol), rapid label-free and easy-to-use bacteria detection, which holds the potential for future use in various ss-DNA analyses by integrated into a self-contained biochip.

Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, β-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy.

Cellular metabolic activity, especially mitochondrial metabolism, plays a vital role in regulating cell proliferation and differentiation. Metabolism could therefore be an important factor to consider when using engineering technologies to stimulate tissue development and repair. The small metabolite carnitine and its derivative acetylcarnitine affect the activities of several pathways in mitochondrial metabolism, but their influence on cell differentiation has not yet been thoroughly studied. To elucidate the effects of these two small molecules on mesenchymal tissue engineering, we used adult stem cells as a platform in both monolayer and 3D hydrogel culture systems. We investigated the impact of these two small molecules on the differentiation of adult stem cells and analysed gene expression, cell proliferation and extracellular matrix deposition. We found that the molecules reduced adipogenesis but stimulated osteogenesis and chondrogenesis in both culture systems. Our results suggest that carnitine and acetylcarnitine could affect the differentiation rate of adult stem cells by regulating mitochondrial metabolism. The effects of these two small molecules give rise to the possibility of employing such metabolites in tissue-engineering systems to enhance cell differentiation and tissue development.

By means of the specific immuno-recognition and ultra-sensitive mass detection, a quartz crystal microbalance (QCM) biosensor for Escherichia coli O157:H7 detection was developed in this work. As a suitable surfactant, 16-mercaptohexadecanoic acid (MHDA) was introduced onto the Au surface of QCM, and then self-assembled with N-hydroxysuccinimide (NHS) raster as a reactive intermediate to provide an active interface for the specific antibody immobilization. The binding of target bacteria with the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of the electrochemical techniques. Using the immersion-dry-immersion procedure, this QCM biosensor could detect 2.0x10(2) colony forming units (CFU)/ml E. coli O157:H7. In order to reduce the fabrication time, a polyelectrolyte layer-by-layer self-assembly (LBL-SA) method was adopted for fast construction. Finally, the reproducibility of this biosensor was discussed.

The Muthuswamy–Chua system [Formula: see text] describes the simplest electronic circuit which can have chaotic phenomena. In this paper, we first prove the existence of three families of consecutive periodic orbits of the system when α = 0, two of which are located on consecutive invariant surfaces and form two invariant topological cylinders. Then we prove that for α > 0 if the system has a periodic orbit or a chaotic attractor, it must intersect both of the planes z = 0 and z = -1 infinitely many times as t tends to infinity. As a byproduct, we get an example of unstable invariant topological cylinders which are not normally hyperbolic and which are also destroyed under small perturbations.