Integrated transcriptomic and metabolomic analysis reveals the regulation mechanism of early bolting and flowering in two cultivars of Angelica sinensis.

The root of Angelica sinensis is utilized in Traditional Chinese medicine to enhance blood replenishment and facilitate blood circulation. The early bolting and flowering (EBF) of A. sinensis, however, compromises the quality of the roots and restricts the yield of medicinal substances. The study was conducted to compare the transcriptomic and metabolomic profiles between EBF plants and normal plants of two cultivars of A. sinensis, followed by validation of the transcriptome results using qRT-PCR. There were 3677 DEGs in EBF plants compared to normal plants of cultivar 2 (Mingui No.2), and cultivar 4 (Mingui No.4) was 3354. The main differential metabolites in the EBF and normal plants were phenolic acids, flavonoids, lignans, and coumarins. The analysis of 5 EBF-related pathways revealed 28 genes exhibiting differential expression and 5 metabolites showing differential accumulation. The expression of the Lhcb5, Lhcb2, Lhcb6, Lhcb1, Lhca4, ATPG1, EGLC, CELB, AMY, glgA, CYCD3, SnRK2, PYL, AHK2, AUX1, BSK, FabI/K, ACACA and FabV decreased and the expression of the PsbR, PsbA, LHY, FT, CO, malQ, HK, GPI and DELLA increased in EBF plants. In addition, the Abscisic acid, d-Glucose-6P, α-d-Glucose-1P, NADP+, and ADP were more significantly enriched in EBF plants. The findings offer novel perspectives on the EBF mechanisms in A. sinensis and other medicinal plants of the Apiaceae family.

Vitamin D3 supplementation shapes the composition of gut microbiota and improves some obesity parameters induced by high-fat diet in mice.

PURPOSE: Individuals with vitamin D (VD) insufficiency have a greater tendency to develop obesity and have increased systemic inflammation. Gut microbiota are involved in the regulation of host inflammation and energy metabolism, which plays a role in the pathogenesis of obesity. Thus, we aimed to evaluate the effects of different doses of VD3 on body weight, serum lipids, inflammatory factors, and intestinal barrier function in obese mice and to explore the regulatory effect of VD3 on gut microbiota in obese mice. METHODS: Male C57BL/6 J mice received a normal chow diet (NCD, 10% fat) or high-fat diet (HFD, 60% fat) to induce obesity within 10 weeks. Then, HFD mice were supplemented with 5650, 8475, or 11,300 IU VD3/kg diet for 8 weeks. Finally, 16 s rRNA analysis was performed to analyze gut microbiota composition in cecal contents. In addition, body weight, serum lipids, inflammatory factors, and intestinal barrier function were analyzed. RESULTS: VD3 supplementation reduced body weight and the levels of TG, TC, HDL-C, TNF-α, IL-1ß and LPS, and increased ZO-1 in HFD-fed mice. Moreover, it increased α-diversity, reduced F/B ratio and altered microbiota composition by increasing relative abundance of Bacteroidetes, Proteobacteria, Desulfovibrio, Dehalobacterium, Odoribacter, and Parabacteroides and reducing relative abundance of Firmicutes and Ruminococcus. There were significant differences between HFD and NCD groups in several metabolic pathways, including endotoxin biosynthesis, tricarboxylic acid cycle, lipid synthesis and metabolism, and glycolysis. CONCLUSIONS: Low, medium, and high doses of VD3 inhibited weight gain, reduced levels of blood lipids and inflammatory factors, and improved endotoxemia and gut barrier function in obese mice. It also increased the α-diversity of gut microbiota in obese mice and reduced the relative abundance of some intestinal pathogenic bacteria, increased the relative abundance of some beneficial bacteria, and corrected the intestinal flora disorder of obese mice, with the low- and high-dose groups showing better effects than the medium-dose group.

Transcriptomics and metabolomics reveal the primary and secondary metabolism changes in Glycyrrhiza uralensis with different forms of nitrogen utilization.

The roots and rhizomes of Glycyrrhiza uralensis Fisch. represent the oldest and most frequently used herbal medicines in Eastern and Western countries. However, the quality of cultivated G. uralensis has not been adequate to meet the market demand, thereby exerting increased pressure on wild G. uralensis populations. Nitrogen, vital for plant growth, potentially influences the bioactive constituents of plants. Yet, more information is needed regarding the effect of different forms of nitrogen on G. uralensis. G. uralensis seedlings were exposed to a modified Hoagland nutrient solution (HNS), varying concentrations of nitrate (KNO3), or ammonium (NH4)2SO4. We subsequently obtained the roots of G. uralensis for physiology, transcriptomics, and metabolomics analyses. Our results indicated that medium-level ammonium nitrogen was more effective in promoting G. uralensis growth compared to nitrate nitrogen. However, low-level nitrate nitrogen distinctly accelerated the accumulation of flavonoid ingredients. Illumina sequencing of cDNA libraries prepared from four groups-treated independently with low/medium NH4 + or NO3 - identified 364, 96, 103, and 64 differentially expressed genes (DEGs) in each group. Our investigation revealed a general molecular and physiological metabolism stimulation under exclusive NH4 + or NO3 - conditions. This included nitrogen absorption and assimilation, glycolysis, Tricarboxylic acid (TCA) cycle, flavonoid, and triterpenoid metabolism. By creating and combining putative biosynthesis networks of nitrogen metabolism, flavonoids, and triterpenoids with related structural DEGs, we observed a positive correlation between the expression trend of DEGs and flavonoid accumulation. Notably, treatments with low-level NH4 + or medium-level NO3 - positively improved primary metabolism, including amino acids, TCA cycle, and glycolysis metabolism. Meanwhile, low-level NH4 + and NO3 - treatment positively regulated secondary metabolism, especially the biosynthesis of flavonoids in G. uralensis. Our study lays the foundation for a comprehensive analysis of molecular responses to varied nitrogen forms in G. uralensis, which should help understand the relationships between responsive genes and subsequent metabolic reactions. Furthermore, our results provide new insights into the fundamental mechanisms underlying the treatment of G. uralensis and other Glycyrrhiza plants with different nitrogen forms.

Effect of vitamin D3 on lipid droplet growth in adipocytes of mice with HFD-induced obesity.

Vitamin D-regulating action of PPARγ on obesity has been confirmed on adipocyte differentiation. However, it is not clear whether vitamin D affects the morphological size of mature adipocytes by influencing the expression of PPARγ in vivo. Our hypothesis was that Vitamin D3 (VitD3) inhibits the growth of adipocyte size by suppressing PPARγ expression in white adipocytes of obese mice. Five-week-old male C57BL/6J mice were randomly divided into normal diet and high-fat diet groups. After 10 weeks, the body weight between the two groups differed by 26.91%. The obese mice were randomly divided into a high-fat diet, solvent control, low-dose VitD3 (5000 IU/kg·food), medium-dose VitD3 (7500 IU/kg·food), high-dose VitD3 (10,000 IU/kg·food), and PPAR γ antagonist group, and the intervention lasted for 8 weeks. Diet-induced obesity (DIO) mice fed high-dose VitD3 exacerbated markers of adiposity (body weight, fat mass, fat mass rate, size of white and brown adipocytes, mRNA, and protein levels of ATGL and Fsp27), and the protein level of ATGL and Fsp27 decreased in the low-dose group. In conclusion, high-dose VitD3 possibly via inhibiting the ATGL expression, thereby inhibiting lipolysis, increasing the volume of adipocytes, and decreasing their fat-storing ability resulted in decreased Fsp27 expression. Therefore, long-term high-dose oral VitD3 may not necessarily improve obesity, and we need more clinical trials to explore the intervention dose and duration of VitD3 in the treatment of VitD3 deficiency in obese patients.

Dynamin-2 reduction rescues the skeletal myopathy of a SPEG-deficient mouse model.

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-ß 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.

Real-Time Coseismic Displacement Retrieval Based on Temporal Point Positioning with IGS RTS Correction Products.

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.

SPEG binds with desmin and its deficiency causes defects in triad and focal adhesion proteins.

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 ß1 integrin, both of which are critical components of the focal adhesion complex. Further, ß1 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.

[A summary of research progress on intelligent information processing methods for pregnant women's remote monitoring].

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.

A Modified TurboEdit Cycle-Slip Detection and Correction Method for Dual-Frequency Smartphone GNSS Observation.

Recently, some smartphone manufacturers have subsequently released dual-frequency GNSS smartphones. With dual-frequency observations, the positioning performance is expected to be significantly improved. Cycle-slip detection and correction play an important role in high-precision GNSS positioning, such as precise point positioning (PPP) and real-time kinematic (RTK) positioning. The TurboEdit method utilizes Melbourne-Wübbena (MW) and phase ionospheric residual (PIR) combinations to detect cycle-slips, and it is widely used in the data processing applications for geodetic GNSS receivers. The smartphone pseudorange observations are proved to be much noisier than those collected with geodetic GNSS receivers. Due to the poor pseudorange observation, the MW combination would be difficult to detect small cycle-slips. In addition, some specific cycle-slip combinations, where the ratio of cycle-slip values at different carrier frequencies is close to the frequency ratio, are also difficult to be detected by PIR combination. As a consequence, the traditional TurboEdit method may fail to detect specific small cycle-slip combinations. In this contribution, we develop a modified TurboEdit cycle-slip detection and correction method for dual-frequency smartphone GNSS observations. At first, MW and PIR combinations are adopted to detect cycle-slips by comparing these two combinations with moving-window average values. Then, the epoch-differenced wide-lane combinations are used to estimate the changes of smartphone position and clock bias, and the cycle-slip is identified by checking the largest normalized residual whether it exceeds a predefined threshold value. The process of estimation and cycle-slip identification is implemented in an iterative way until there is no over-limit residual or there is no redundant measurement. At last, the cycle-slip values at each frequency are estimated with the epoch-differenced wide-lane and ionosphere-free combinations, and the least-square ambiguity decorrelation adjustment (LAMBDA) method is adopted to further obtain an integer solution. The proposed method has been verified with 1 Hz dual-frequency smartphone GNSS data. The results show that the modified TurboEdit method can effectively detect and correct even for specific small cycle-slip combinations, e.g., (4, 3), which is difficult to be detected with the traditional TurboEdit method.

Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy.

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 toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

Simultaneous nitrogen removal and phosphorus recovery using an anammox expanded reactor operated at 25 °C.

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.

Relationship of heme c, nitrogen loading capacity and temperature in anammox reactor.

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.

Transgenic zebrafish model of DUX4 misexpression reveals a developmental role in FSHD pathogenesis.

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.

Adipose-derived Stem/Stromal Cells on Electrospun Fibrin Microfiber Bundles Enable Moderate Muscle Reconstruction in a Volumetric Muscle Loss Model.

Current treatment options for volumetric muscle loss (VML) are limited due to donor site morbidity, lack of donor tissue, and insufficient functional recovery. Tissue-engineered skeletal muscle grafts offer the potential to significantly improve functional outcomes. In this study, we assessed the potential pro-myogenic effects of human adipose-derived stem cells (ASCs) seeded onto electrospun uniaxially aligned fibrin hydrogel microfiber bundles. Although both uninduced and 5-azacytidine-induced ASCs exhibited alignment, elongation, and diffuse muscle marker expression when grown on microfiber bundles for 2 months in vitro, both groups failed to fully recapitulate myotube characteristics. To assess the muscle regeneration potential of ASCs in vivo, ASC-seeded fibrin microfiber bundles were implanted in a robust murine VML defect model. Minimal fibrosis was observed surrounding implanted acellular hydrogel fibers at 2 and 4 weeks, and fibers seeded with ASCs exhibited up to 4 times higher volume retention than acellular fibers. We observed increased numbers of cells positive for the regenerating muscle marker embryonic myosin and the mature muscle marker myosin heavy chain in ASC-seeded fibers compared with acellular fibers at 1 and 3 months post-transplantation. Regenerating muscle cells were closely associated with ASC-derived cells and in some cases had potentially fused with them. These findings demonstrate that despite failing to undergo myogenesis in vitro, ASCs combined with electrospun fibrin microfibers moderately increased muscle reconstruction in vivo compared with acellular fibers following a severe VML defect.

Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics.

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.

Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model.

Duchenne 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/TGFß 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.

In Vivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle Gene Therapy.

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, ß-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.

Carnitine and acetylcarnitine modulate mesenchymal differentiation of adult stem cells.

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.

Human skeletal muscle xenograft as a new preclinical model for muscle disorders.

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.

A novel electrochemical biosensor based on dynamic polymerase-extending hybridization for E. coli O157:H7 DNA detection.

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.