Transformation of human umbilical mesenchymal cells into neurons in vitro

Neuronal transplantation has provided a promising approach for treating neurodegenerative diseases. Recently, efforts have been directed at in vitro induction of various stem cells to transform into neurons. We report the first successful quantities in an in vitro attempt at directing the transformation into neurons of human umbilical mesenchymal cells, which are capable of rapid proliferation in vitro and are easily available. When cultured in neuronal conditioned medium, human umbilical mesenchymal cells started to express neuron-specific proteins such as NeuN and neurofilament (NF) on the 3rd day and exhibited retraction of the cell body, elaboration of processes, clustering of cells and expression of functional mRNA responsible for the synthesis of subunits of the kainate receptor and glutamate decarboxylase on the 6th day. Between the 9th and 12th days, the percentage of human umbilical mesenchymal cells expressing NF was as high as 87%, while functionality was demonstrated by glutamate invoking an inward current. At this stage, cells were differentiated into mature neurons in the postmitosis phase.     

Ultrasound combined with SDF‐1α chemotactic microbubbles promotes stem cell homing in an osteoarthritis model

Osteoarthritis (OA) is a common joint disease in the middle and old age group with obvious cartilage damage, and the regeneration of cartilage is the key to alleviating or treating OA. In stem cell therapy, bone marrow stem cell (BMSC) has been confirmed to have cartilage regeneration ability. However, the role of stem cells in promoting articular cartilage regeneration is severely limited by their low homing rate. Stromal cell‐derived factor‐1α (SDF‐1α) plays a vital role in MSC migration and involves activation, mobilization, homing and retention. So, we aim to develop SDF‐1α‐loaded microbubbles MB(SDF‐1α), and to verify the migration of BMSCs with the effect of ultrasound combined with MB(SDF‐1α) in vitro and in vivo. The characteristics of microbubbles and the content of SDF‐1α were examined in vitro. To evaluate the effect of ultrasound combined with chemotactic microbubbles on stem cell migration, BMSCs were injected locally and intravenously into the knee joint of the OA model, and the markers of BMSCs in the cartilage were detected. We successfully prepared MB(SDF‐1α) through covalent bonding with impressive SDF‐1α loading efficacy loading content. In vitro study, ultrasound combined with MB(SDF‐1α) group can promote more stem cell migration with highest migrating cell counts, good cell viability and highest CXCR4 expression. In vivo experiment, more BMSCs surface markers presented in the ultrasound combined with MB(SDF‐1α) group with or without exogenous BMSCs administration. Hence, ultrasound combined with MB(SDF‐1α) could promote the homing of BMSCs to cartilage and provide a novel promising therapeutic approach for OA.     

Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.     

Circular RNA circANKRD36 regulates Casz1 by targeting miR-599 to prevent osteoarthritis chondrocyte apoptosis and inflammation

Osteoarthritis (OA) is an ageing-related disease characterized by articular cartilage degradation and joint inflammation. circRNA has been known to involve in the regulation of multiple inflammatory diseases including OA. However, the mechanism underlying how circRNA regulates OA remains to be elucidated. Here, we report circANKRD36 prevents OA chondrocyte apoptosis and inflammation by targeting miR-599, which specifically degrades Casz1. We performed circRNA sequencing in normal and OA tissues and found the expression of circANKRD36 is decreased in OA tissues. circANKRD36 is also reduced in IL-1β–treated human chondrocytes. FACS analysis and Western blot showed that the knockdown of circANKRD36 promotes the apoptosis and inflammation of chondrocytes in IL-1β stress. We then found miR-599 to be the target of circANKRD36 and correlate well with circANKRD36 both in vitro and in vivo. By database analysis and luciferase assay, Casz1 was found to be the direct target of miR-599. Casz1 helps to prevent apoptosis and inflammation of chondrocytes in response to IL-1β. In conclusion, our results proved circANKRD36 sponge miR-599 to up-regulate the expression of Casz1 and thus prevent apoptosis and inflammation in OA.     

Development and characterization of 70 novel microsatellite markers for the sea cucumber (Apostichopus japonicus)

The sea cucumber (Apostichopus japonicus) is an important item in Asian cuisine. It is currently produced through aquaculture, especially in China, after being overexploited in the wild in the 1990s. We isolated 70 novel polymorphic microsatellite loci using an enrichment-colony hybridization protocol. All loci were characterized in 48 individuals from a natural population in Rongcheng (Shandong, China) using genomic DNA isolated from muscle tissue. The number of alleles ranged from 2 to 17 (mean 7.0), and the observed and expected heterozygosities varied from 0.0010 to 1.0000 and from 0.2125 to 0.9477, respectively. Thirty-one of the 70 loci exhibited departure from Hardy-Weinberg equilibrium. These microsatellite markers should be useful resources for population genetic studies and for molecular marker-assisted breeding of A. japonicus.     

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl‐xL overexpression

Bcl‐x_L, a member of the Bcl‐2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl‐x_L overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐x_L expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) and MS analysis. The consequences of Bcl‐x_L overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐x_L overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif‐containing GTPase‐activating protein 1), cell proliferation (dihydrolipoamide‐S‐acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl‐x_L not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects. Biotechnol. Bioeng. 2010; 105: 358–367. © 2009 Wiley Periodicals, Inc.     

A proteomic approach for identifying cellular proteins interacting with erythropoietin in recombinant Chinese hamster ovary cells

Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull‐down assay was performed with dual‐tagged (N‐terminal GST‐ and C‐terminal hexahistidine‐tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual‐tagged EPO were then resolved by two‐dimensional gel electrophoresis (2DE) and identified by MALDI‐TOF MS/MS. A total of 27 protein spots including glucose‐regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull‐down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Global gene expression profile of Orientia tsutsugamushi

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of Scrub typhus. The control mechanisms for bacterial gene expression are largely unknown. Here, the global gene expression of O. tsutsugamushi within eukaryotic cells was examined using a microarray and proteomic approaches for the first time. These approaches identified 643 genes, corresponding to approximately 30% of the genes encoded in the genome. The majority of expressed genes belonged to several functional categories including protein translation, protein processing/secretion, and replication/repair. We also searched the conserved sequence blocks (CSBs) in the O. tsutsugamushi genome which is unique in that up to 40% of its genome consists of dispersed repeated sequences. Although extensive shuffling of genomic sequences was observed between two different strains, 204 CSBs, covering 48% of the genome, were identified. When combining the data of CSBs and global gene expression, the CSBs correlates well with the location of expressed genes, suggesting the functional conservation between gene expression and genomic location. Finally, we compared the gene expression of the bacteria‐infected fibroblasts and macrophages using microarray analysis. Some major changes were the downregulation of genes involved in translation, protein processing and secretion, which correlated with the reduction in bacterial translation rates and growth within macrophages.     

Solution structure of At3g04780.1-des15, an Arabidopsis thaliana ortholog of the C-terminal domain of human thioredoxin-like protein

The structure of At3g04780.1-des15, an Arabidopsis thaliana ortholog of the C-terminal domain of human thioredoxin-like protein, was determined by NMR spectroscopy. The structure is dominated by a beta-barrel sandwich. A two-stranded anti-parallel beta-sheet, which seals off one end of the beta-barrel, is flanked by two flexible loops rich in acidic amino acids. Although this fold often provides a ligand binding site, the structure did not reveal an appreciable cavity inside the beta-barrel. The three-dimensional structure of At3g04780.1-des15 provides an entry point for understanding its functional role and those of its mammalian homologs.