Knockdown of endogenous myostatin promotes sheep myoblast proliferation

Myostatin (MSTN), is a known negative regulator of myogenesis. Silencing of the function of MSTN could result in increasing muscle mass in mice. To determine the function of endogenous MSTN expression on proliferation of sheep myoblasts, a short-hairpin RNA-targeting sheep MSTN was constructed into lentiviral vector to silence endogenous MSTN expression. We demonstrated that silencing of endogenous MSTN gene with up to approximately 73.3% reduction by short hairpin RNA (shRNA) resulted in significant increase (overall 28.3%) of proliferation of primary ovine myoblasts. The upregulation of proliferation was accompanied by the decrease expression of MyoD (?37.6%, p?=?0.025), myogenin (?33.1%, p?=?0.049), p21 (?49.3%, p?=?0.046), and Smad3 (?50.0%, p?=?0.007). Silencing of myostatin using shRNA may provide a feasible approach to improve meat productivity in farm animals.     

Inhibition of Hypoxia Inducible Factor 1α by siRNA‐Induced Apoptosis in Human Retinoblastoma Cells

Hypoxia, which activates the hypoxia inducible factor 1α (HIF‐1α), is an essential feature of retinoblastoma (RB) and contributes to poor prognosis and resistance to conventional therapy. In this study, the effect of HIF‐1α knockdown by small interfering RNA (siRNA) on cell proliferation, apoptosis, and apoptotic pathways of human Y‐79 RB cells was first investigated. Exposure to hypoxia induced the increased expression of HIF‐1α both in mRNA and protein levels. Then, knockdown of HIF‐1α by siRNA_HIF‐1α resulted in inhibition of cell proliferation and induced cell apoptosis in human Y‐79 RB cells under both normoxic and hypoxic conditions, with hypoxic conditions being more sensitive. Furthermore, knockdown of HIF‐1α could enhance hypoxia‐induced slight increase of Bax/Bcl‐2 ratio and activate caspase‐9 and caspase‐3. These results together indicated that suppression of HIF‐1α expression may be a promising strategy for the treatment of human RB in the future.     

Effects of light intensity on components and topographical structures of extracellular polysaccharides from the cyanobacteria Nostoc sp.

A study on the effects of light intensity (40 and 80 μE/m²/sec) on the components and topographical structures of extracellular polysaccharides (EPS) was carried out in cyanobacteria Nostoc sp.. EPS yield increased with light intensity. However, light intensity did not significantly affect the EPS fractions and monosaccharide composition. Higher light intensity generally resulted in higher protein content of EPS in similar fractions. The topographical structure of EPS, investigated by atomic force microscopy, appeared as spherical lumps, chains and networks. The long chains were observed at higher light intensity. Thus, light intensity affected the yield and nature of EPS.     

Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium

Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca²⁺ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca²⁺ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca²⁺ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.     

CD226 Protein Is Involved in Immune Synapse Formation and Triggers Natural Killer (NK) Cell Activation via Its First Extracellular Domain

CD226, an activating receptor that interacts with the ligands CD155 and CD112, activates natural killer (NK) cells via its immunoreceptor tyrosine-based activatory motif (ITAM). There are two extracellular domains of CD226; however, the comparative functional relevance of these domains remains unknown. In this study, two different deletion mutants, rCD226-ECD1 (the first extracellular domain) and rCD226-ECD (full extracellular domains), were recombinantly expressed. We observed that rCD226-ECD1, similar to rCD226-ECD, specifically bound to ligand-positive cell lines and that this interaction could be competitively blocked by an anti-CD226 mAb. In addition, rCD226-ECD1 was able to block the binding of CD112 mAb to tumor cells in a competitive binding assay. Importantly, based on surface plasmon resonance (SPR), we determined that rCD226-ECD1, similar to rCD226-ECD, directly bound to its ligand CD155 on a protein chip. Functionally, NK cell cytotoxicity against K562 or HeLa cells was blocked by rCD226-ECD1 by reducing the expression of CD69 and granzyme B, indicating the critical role of ECD1 in NK cell activation. We also examined the role of rCD226-ECD1 in effector/target interactions by using rCD226-ECD to block these interactions. Using flow cytometry, we found that the number of conjugates between IL-2-dependent NKL cells and HeLa cells was reduced and observed that the formation of immune synapses was also decreased under confocal microscopy. In addition, we prepared two anti-rCD226-ECD1 agonistic antibodies, 2E6 and 3B9. Both 2E6 and 3B9 antibodies could induce the phosphorylation of ERK in NK-92 cells. Taken together, our results show that CD226 functions via its first extracellular domain.     

Scorpion Venom Heat-Resistant Peptide (SVHRP) Enhances Neurogenesis and Neurite Outgrowth of Immature Neurons in Adult Mice by Up-Regulating Brain-Derived Neurotrophic Factor (BDNF)

Scorpion venom heat-resistant peptide (SVHRP) is a component purified from Buthus martensii Karsch scorpion venom. Although scorpions and their venom have been used in Traditional Chinese Medicine (TCM) to treat chronic neurological disorders, the underlying mechanisms of these treatments remain unknown. We applied SVHRP in vitro and in vivo to understand its effects on the neurogenesis and maturation of adult immature neurons and explore associated molecular mechanisms. SVHRP administration increased the number of 5-bromo-2’-dexoxyuridine (BrdU)-positive cells, BrdU- positive/neuron-specific nuclear protein (NeuN)-positive neurons, and polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive immature neurons in the subventricular zone (SVZ) and subgranular zone (SGZ) of hippocampus. Furthermore immature neurons incubated with SVHRP-pretreated astrocyte-conditioned medium exhibited significantly increased neurite length compared with those incubated with normal astrocyte-conditioned medium. This neurotrophic effect was further confirmed in vivo by detecting an increased average single area and whole area of immature neurons in the SGZ, SVZ and olfactory bulb (OB) in the adult mouse brain. In contrast to normal astrocyte-conditioned medium, higher concentrations of brain-derived neurotrophic factor (BDNF) but not nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) was detected in the conditioned medium of SVHRP-pretreated astrocytes, and blocking BDNF using anti-BDNF antibodies eliminated these SVHRP-dependent neurotrophic effects. In SVHRP treated mouse brain, more glial fibrillary acidic protein (GFAP)-positive cells were detected. Furthermore, immunohistochemistry revealed increased numbers of GFAP/BDNF double-positive cells, which agrees with the observed changes in the culture system. This paper describes novel effects of scorpion venom-originated peptide on the stem cells and suggests the potential therapeutic values of SVHRP.     

Evolutionary history of a widespread tree species Acer mono in East Asia

East Asia has the most diverse temperate flora in the world primarily due to the lack of Pleistocene glaciation and the geographic heterogeneity. Although increasing phylogeography studies in this region provided more proofs in this issue, discrepancies and uncertainty still exist, especially in northern temperate deciduous broad‐leaved and coniferous mixed forest region (II). And a widespread plant species could reduce the complexity to infer the relationship between diversity and physiographical pattern. Hence, we studied the evolution history of a widespread temperate tree, Acer mono, populations in region II and the influence of physiographic patterns on intraspecific genetic diversity. Analyses of chloroplast sequences and nuclear microsatellites indicated high levels of genetic diversity. The diversity distribution was spatially heterogeneous and a latitudinal cline existed in both markers. The spatial distribution pattern between genetic diversity within A. mono and the diversity at species level was generally consistent. Western subtropical evergreen broad‐leaved forest subregion (IVb) had a unique ancient chloroplast clade (CP3) and a nuclear gene pool (GP5) with dominance indicating the critical role of this area in species diversification. Genetic data and ecological niche model results both suggested that populations in region II disappeared during the last glacial maximum (LGM) and recovered from south of Changbai Mt. and the Korean Peninsula. Two distribution centers were likely during the LGM, one in the north edge of warm temperate deciduous broad‐leaved forest region (III) and another in the south edge of region III. This was reflected by the genetic pattern with two spatially independent genetic groups. This study highlights the key role of region III in sustaining genetic diversity in the northern range and connecting diversity between southern and northern range. We elucidated the diversity relationship between vegetation regions which could facilitate the understanding of biodiversity origin and maintenance in East Asia.