Pax transactivation domain-interacting protein is required for preserving hematopoietic stem cell quiescence via regulating lysosomal activity

Hematopoietic stem cells (HSC) maintain lifetime whole blood hematopoiesis through self-renewal and differentiation. In order to sustain HSC stemness, most HSC reside in a quiescence state, which is affected by diverse cellular stress and intracellular signal transduction. How HSC accommodate those challenges to preserve lifetime capacity remains elusive. Here we show that Pax transactivation domain-interacting protein (PTIP) is required for preserving HSC quiescence via regulating lysosomal activity. Using a genetic knockout mouse model to specifically delete Ptip in HSC, we find that loss of Ptip promotes HSC exiting quiescence, and results in functional exhaustion of HSC. Mechanistically, Ptip loss increases lysosomal degradative activity of HSC. Restraining lysosomal activity restores the quiescence and repopulation potency of Ptip-/- HSC. Additionally, PTIP interacts with SMAD2/3 and mediates transforming growth factor-β signaling-induced HSC quiescence. Overall, our work uncovers a key role of PTIP in sustaining HSC quiescence via regulating lysosomal activity

In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii) interspecific cloned embryos

The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT) was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming

Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions in both transgenic cells and transgenic mice

In the present study, follistatin (FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the pMAR-FST transgenic mice at F0, F1 and F2. Total muscle growth in F0 mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice

Transcriptome Analysis of Watercore in Pineapple

Watercore is a physiological disorder in pineapples, which is expressed as fluid deposition in intercellular spaces and presents as water soaked. This disorder affects the fruit quality and decreases storage life, resulting in enormous commercial losses to growers and restricting the development of the pineapple industry in China. However, the molecular mechanism of watercore remains unclear. In order to elucidate the molecular mechanism of pineapple watercore, the transcriptome analyses of watercored and normal fruits were carried out in pineapples for the first time using de novo RNA-seq technology. High-quality reads of 46.66 and 43.71 M were obtained in the transcriptomes of normal and mildly watercored fruits, respectively. Clean reads of 45.50 and 42.79 M were obtained after filtering the original data. These genes are useful resources in subsequent pineapple watercore research. Fifty genes in phenylpropanoid biosynthesis, glucose metabolism, calcium transport, and cell wall metabolism were considerably different between normal and watercored fruits. Among them, the expressions of the AcPME, AcBGLU43, Ac4CL5, AcPER1, and AcPOD genes were upregulated by 7–21 times in watercored fruit, while the expressions of AcSUS7 were downregulated by 16.61 times, and the expressions of other differential genes were upregulated or downregulated by more than 2 times. A total of 38 differentially expressed transcription factors were obtained by screening. Among these transcription factors, WRKY was the most abundant, followed by MYB. The acquisition of these genes is important for the first understanding of the molecular mechanism of this physiological disorder

Myostatin Knockout Affects Mitochondrial Function by Inhibiting the AMPK/SIRT1/PGC1α Pathway in Skeletal Muscle

Myostatin (Mstn) is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes. The deletion of the Mstn gene in mice leads to reduced mitochondrial functions. However, the underlying regulatory mechanisms remain unclear. In this study, we used CRISPR/Cas9 to generate myostatin-knockout (Mstn-KO) mice via pronuclear microinjection. Mstn-KO mice exhibited significantly larger skeletal muscles. Meanwhile, Mstn knockout regulated the organ weights of mice. Moreover, we found that Mstn knockout reduced the basal metabolic rate, muscle adenosine triphosphate (ATP) synthesis, activities of mitochondrial respiration chain complexes, tricarboxylic acid cycle (TCA) cycle, and thermogenesis. Mechanistically, expressions of silent information regulator 1 (SIRT1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were down-regulated, while peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) acetylation modification increased in the Mstn-KO mice. Skeletal muscle cells from Mstn-KO and WT were treated with AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR), and the AMPK inhibitor Compound C, respectively. Compared with the wild-type (WT) group, Compound C treatment further down-regulated the expression or activity of pAMPK, SIRT1, citrate synthase (CS), isocitrate dehydrogenase (ICDHm), and α-ketoglutarate acid dehydrogenase (α-KGDH) in Mstn-KO mice, while Mstn knockout inhibited the AICAR activation effect. Therefore, Mstn knockout affects mitochondrial function by inhibiting the AMPK/SIRT1/PGC1α signaling pathway. The present study reveals a new mechanism for Mstn knockout in regulating energy homeostasis

Transcriptome Analysis of Watercore in Pineapple

Watercore is a physiological disorder in pineapples, which is expressed as fluid deposition in intercellular spaces and presents as water soaked. This disorder affects the fruit quality and decreases storage life, resulting in enormous commercial losses to growers and restricting the development of the pineapple industry in China. However, the molecular mechanism of watercore remains unclear. In order to elucidate the molecular mechanism of pineapple watercore, the transcriptome analyses of watercored and normal fruits were carried out in pineapples for the first time using de novo RNA-seq technology. High-quality reads of 46.66 and 43.71 M were obtained in the transcriptomes of normal and mildly watercored fruits, respectively. Clean reads of 45.50 and 42.79 M were obtained after filtering the original data. These genes are useful resources in subsequent pineapple watercore research. Fifty genes in phenylpropanoid biosynthesis, glucose metabolism, calcium transport, and cell wall metabolism were considerably different between normal and watercored fruits. Among them, the expressions of the AcPME, AcBGLU43, Ac4CL5, AcPER1, and AcPOD genes were upregulated by 7–21 times in watercored fruit, while the expressions of AcSUS7 were downregulated by 16.61 times, and the expressions of other differential genes were upregulated or downregulated by more than 2 times. A total of 38 differentially expressed transcription factors were obtained by screening. Among these transcription factors, WRKY was the most abundant, followed by MYB. The acquisition of these genes is important for the first understanding of the molecular mechanism of this physiological disorder

Qingxiangxing Baijiu sensory quality grade classification by 1H NMR and GC combined with multivariate statistical analysis

The traditional uniform artificial sensory evaluation makes it difficult to standardize the classification of different sensory quality grades of Baijiu. In this study, a total of 92 authentic Qingxiangxing Baijiu samples with 3 sensory quality grades were carefully collected. Gas chromatography (GC) was used to determine 46 main flavor components and proton nuclear magnetic resonance (1H NMR) spectroscopy was employed to obtain hydrogen atom characteristic information of organic compounds. The principal component analysis (PCA), k-nearest neighbor (KNN) and linear discriminant analysis (LDA) models were conducted and fully validated by internal leave-one-out cross validation (LOOCV) and external repeated double random cross validation (RDRCV). The sensory quality grades of Qingxiangxing Baijiu were effectively classified by using GC and 1H NMR techniques coupled with PCA/KNN analysis with the averaged accuracy higher than 80%. In addition, synthetic minority oversampling technique (SMOTE) algorithm was successfully used to address the model overfitting problem caused by an unbalanced sample composition. This study demonstrated that 1H NMR and GC combined with multivariate statistical analysis were effective for sensory quality classification of Qingxiangxing Baijiu