Cardiac Sca-1+ cells are not intrinsic stem cells for myocardial development, renewal and repair

Background: For over a decade, Sca-1+ cells within the mouse heart have been widely recognized as a stem cell population with multipotency that can give rise to cardiomyocytes, endothelial cells and smooth muscle cells in vitro and after cardiac grafting. However, the developmental origin and authentic nature of these cells remain elusive. Methods: Here, we used a series of high-fidelity genetic mouse models to characterize the identity and regenerative potential of cardiac resident Sca-1+ cells. Results: With these novel genetic mouse models, we found that Sca-1 does not label cardiac precursor cells during early embryonic heart formation. Postnatal cardiac resident Sca-1+ cells are in fact a pure endothelial cell population. They retain endothelial properties and exhibit minimal cardiomyogenic potential during development, normal aging and upon ischemic injury. Conclusions: Our study provides definitive insights into the nature of cardiac resident Sca-1+ cells. The observations challenge the current dogma that cardiac resident Sca-1+ cells are intrinsic stem cells for myocardial development, renewal and repair and suggest that the mechanisms of transplanted Sca-1+ cells in heart repair need to be reassessed

Novel Myh11 Dual Reporter Mouse Model Provides Definitive Labeling and Identification of Smooth Muscle Cells—Brief Report

Objective: Myh11 encodes a myosin heavy chain protein that is specifically expressed in smooth muscle cells (SMCs) and is important for maintaining vascular wall stability. The goal of this study is to generate a Myh11 dual reporter mouse line for definitive visualization of MYH11+ SMCs in vivo. Approach and Results: We generated a Myh11 knock-in mouse model by inserting LoxP-nlacZ-4XpolyA-LoxP-H2B-GFP-polyA-FRT-Neo-FRT reporter cassette into the Myh11 gene locus. The nuclear (n) lacZ-4XpolyA cassette is flanked by 2 LoxP sites followed by H2B-GFP (histone 2B fused green fluorescent protein). Upon Cre-mediated recombination, nlacZ-stop cassette is removed thereby permitting nucleus localized H2B-GFP expression. Expression of the nuclear localized lacZ or H2B-GFP is under control of the endogenous Myh11 promoter. Nuclear lacZ was expressed specifically in SMCs at embryonic and adult stages. Following germline Cre-mediated deletion of nuclear lacZ, H2B-GFP was specifically expressed in the nuclei of SMCs. Comparison of nuclear lacZ expression with Wnt1Cre and Mef2cCre mediated-H2B-GFP expression revealed heterogenous origins of SMCs from neural crest and second heart field in the great arteries and coronary vessels adjacent to aortic root. Conclusions: The Myh11 knock-in dual reporter mouse model offers an exceptional genetic tool to visualize and trace the origins of SMCs in mice

A one-step incubation ELISA kit for rapid determination of dibutyl phthalate in water, beverage and liquor

A one-step incubation ELISA kit based on monoclonal antibody against dibutyl phthalate (DBP) was developed. After optimizing concentrations of coating antigen, antibody and composition of the assay buffer, an inhibition curve was plotted. The IC50 is 29.6 ng·mL-1, and the detection limit for DBP is 3.6 ng·mL-1. Compared with other ELISA methods, this ELISA kit had a simpler sample preparation, costed less time for detection and could detect more types of sample. The recoveries of DBP in water, beverage and liquor samples were range from 78% to 110.4%, the range of coefficient of variations is 7.7-15.3%. The cross reactivity was very low (&1%) except that for butyl benzyl phthalate (3.9%) and the di-isobutyl phthalate (12.5%). The detection results in liquor showed good correlation with those from GC-MS. All data above indicated that this kit could be used as the fast and high-throughput screening of DBP in water, beverage and liquor

Identification of a β-Arrestin 2 Mutation Related to Autism by Whole-Exome Sequencing

Autism spectrum disorder (ASD) is a complex neurological disease characterized by impaired social communication and interaction skills, rigid behavior, decreased interest, and repetitive activities. The disease has a high degree of genetic heterogeneity, and the genetic cause of ASD in many autistic individuals is currently unclear. In this study, we report a patient with ASD whose clinical features included social interaction disorder, communication disorder, and repetitive behavior. We examined the patient’s genetic variation using whole-exome sequencing technology and found new de novo mutations. After analysis and evaluation, ARRB2 was identified as a candidate gene. To study the potential contribution of the ARRB2 gene to the human brain development and function, we first evaluated the expression profile of this gene in different brain regions and developmental stages. Then, we used weighted gene coexpression network analysis to analyze the associations between ARRB2 and ASD risk genes. Additionally, the spatial conformation and stability of the ARRB2 wild type and mutant proteins were examined by simulations. Then, we further established a mouse model of ASD. The results showed abnormal ARRB2 expression in the mouse ASD model. Our study showed that ARRB2 may be a risk gene for ASD, but the contribution of de novo ARRB2 mutations to ASD is unclear. This information will provide references for the etiology of ASD and aid in the mechanism-based drug development and treatment

In Vitro Neurotrophic Properties and Structural Characterization of a New Polysaccharide LTC-1 from <i>Pyrola corbieri</i> Levl (Luticao)

Pyrola corbieri Levl has been used to strengthen bones and nourish the kidney (the kidney governs the bone and is beneficial to the brain) by the local Miao people in China. However, the functional components and neurotrophic activity have not been reported. A new acidic homogeneous heteropolysaccharide named LTC-1 was obtained and characterized by periodate oxidation, Smith degradation, partial acid hydrolysis, GC–MS spectrometry, methylation analysis, and Fourier transform infrared spectroscopy, and its molecular weight was 3239 Da. The content of mannuronic acid (Man A) in LTC-1 was 46%, and the neutral sugar was composed of L-rhamnose (L-Rha), L-arabinose (L-Ara), D-xylose (D-Xyl), D-mannose (D-Man), D-glucose (D-Glc) and D-galactose (D-Gal) with a molar ratio of 1.00:3.63:0.86:1.30:6.97:1.30. The main chain of LTC-1 was composed of Glc, Gal, Man, Man A and the branched chain Ara, Glc, Gal. The terminal residues were composed of Glc and Gal. The main chain and branched chains were linked by (1→5)-linked-Ara, (1→3)-linked-Glc, (1→4)-linked-Glc, (1→6)-linked-Glc, (1→3)-linked-Gal, (1→6)-linked-Gal, (1→3, 6)-linked-Man and ManA. Meanwhile, neurotrophic activity was evaluated through PC12 and primary hippocampal neuronal cell models. LTC-1 exhibited neurotrophic activity in a concentration-dependent manner, which significantly induced the differentiation of PC12 cells, promoted the neurite outgrowth of PC12 cells, enhanced the formation of the web architecture of dendrites, and increased the density of dendritic spines in hippocampal neurons and the expression of PSD-95. These results displayed significant neurotrophic factor-like activity of LTC-1, which suggests that LTC-1 is a potential treatment option for neurodegenerative diseases

Overexpression of NaV1.6 in the rostral ventrolateral medulla in rats mediates stress-induced hypertension via glutamate regulation

Background The rostral ventrolateral medulla (RVLM) plays a key role in mediating the development of stress-induced hypertension (SIH). Furthermore, enhanced glutamate transport within glutamatergic neurons in the RVLM mediates pressor responses. Data from our previous studies suggest that the voltage-gated sodium channel NaV1.6 is overexpressed in neurons in the RVLM in SIH model rats and participates in the resulting elevation of blood pressure. However, previous studies have not investigated the relationship between NaV1.6 expression and glutamatergic neurons. Methods Here, we constructed an SIH rat model by knocking down NaV1.6 via microinjection of clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA into the RVLM. Glutamate-related markers were quantified by Western blotting and immunofluorescence, and blood pressure was measured in the rats. Results Our findings showed that vesicular glutamate transporter 1 (VGluT1) protein expression in the RVLM was higher in SIH rats than in Control rats, and GAD67 protein expression in SIH rats was lower than that in Control rats. Therefore, the number of VGluT1-positive neurons increased, while the number of GAD67-labeled neurons decreased after stress. After knocking down NaV1.6 expression in the RVLM, VGluT1 expression and the number of VGluT1-positive neurons decreased relative to those in SIH rats, while GAD67 protein expression and the number of GAD67-labeled neurons increased relative to those in SIH rats. Conclusions These results indicate that overexpression of NaV1.6 in the RVLM may mediate the transport and transformation of glutamate in neurons, and NaV1.6 may participate in SIH