YBX1 Promotes MSC Osteogenic Differentiation by Activating the PI3K/AKT Pathway.

INTRODUCTION: Bone metabolism has an essential role in bone disease, but its specific mechanism remains unclear. Y-Box Binding Protein 1 (YBX1) is a gene with broad nucleic acid binding properties, which encodes a highly conserved cold shock domain protein. Previous studies have shown that YBX1 is closely related to cell differentiation. However, the function of YBX1 in osteoblast differentiation of bone marrow mesenchymal stem cells (MSCs) was unclear. METHODS: To explore the effect and specific mechanism of YBX1 in osteogenic differentiation of MSCs, we used PCR, Western blot, Alizarin red Staining, alkaline phosphatase (ALP) assays, and siRNA knockdown in our research. We found that YBX1 gradually increased during the process of osteogenic differentiation of MSCs. YBX1 siRNA could negatively regulate the MSCs osteogenic differentiation. Mechanistic studies revealed that YBX1 knockdown could inhibit PI3K/AKT pathway. Furthermore, the specific agonist (SC79) of PI3K/AKT pathway could restore the impaired MSCs osteogenic differentiation which was mediated by YBX1 knockdown. Taken together, we concluded that YBX1 could positively regulate the osteogenic differentiation of MSCs by activating the PI3K/AKT pathway. RESULTS AND DISCUSSION: These results helped us further understand the mechanism of osteogenesis and revealed that YBX1 might be a selectable target in the bone repair field. CONCLUSION: Our study provides a new target and theoretical basis for the treatment of bone diseases.

Correction to: Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication.

In the original publication of this article [1], the author found the brand of vimentin antibody was wrong in Fig. 3. The legend of Fig. 3, 'mouse anti-vimentin mAb (Cell Signaling Technology) at 4 °C overnight' should be 'mouse anti-vimentin mAb (Sigma-Aldrich) at 4 °C overnight'.

Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important globally distributed and highly contagious pathogen that has restricted cell tropism in vivo and in vitro. In the present study, we found that annexin A2 (ANXA2) is upregulated expressed in porcine alveolar macrophages infected with PRRSV. Additionally, PRRSV replication was significantly suppressed after reducing ANXA2 expression in Marc-145 cells using siRNA. Bioinformatics analysis indicated that ANXA2 may be relevant to vimentin, a cellular cytoskeleton component that is thought to be involved in the infectivity and replication of PRRSV. Co-immunoprecipitation assays and confocal analysis confirmed that ANXA2 interacts with vimentin, with further experiments indicating that the B domain (109-174 aa) of ANXA2 contributes to this interaction. Importantly, neither ANXA2 nor vimentin alone could bind to PRRSV and only in the presence of ANXA2 could vimentin interact with the N protein of PRRSV. No binding to the GP2, GP3, GP5, nor M proteins of PRRSV was observed. In conclusion, ANXA2 can interact with vimentin and enhance PRRSV growth. This contributes to the regulation of PRRSV replication in infected cells and may have implications for the future antiviral strategies.

Expression of short peptide by an improved isocaudamer tandem repeat strategy.

An improved isocaudamer tandem repeat strategy for the production of short functional peptide was demonstrated in the study. The coding sequence of short peptide was codon optimized, and two isocaudamers were induced into the end of coding sequence. By re-cutting with isocaudamers and re-ligating, the coding sequence of short peptide in the expression vector was increased in a multiple manner (21, 22, 23, 24, 25 …….). In the present study, an 8 amino-acidresidue peptide of porcine reproductive and respiratory syndrome virus (PRRSV) was effectively expressed in 8 copies and 16 copies by this approach, then the proteins in 8 copies and 16 copies were used to generate antibody against this epitope in rabbits. The results showed that PRRSVs were well recognized by the antibody in indirect immunofluorescence assay. The technology using isocaudamer to insert multiple tandem repeats in the vector provides an important approach for the studies of small molecule peptides.

Production of antisera against porcine haptoglobin: potential for distinguishing haptoglobin subunits.

Haptoglobin (Hp) is one of the acute phase proteins (APPs) that help to alleviate the immune oxidative damage. The present study expressed a truncated porcine Hp in Escherichia coli and produced rabbit and mouse antisera to the recombinant protein, in order to investigate Hp levels in sera from piglets infected with porcine reproduction and respiratory syndrome virus (PRRSV). Antisera prepared revealed both chains of porcine Hp in Western blot, and mouse antisera showed stronger binding activities than the rabbit antisera. With the combination of Hp monoclonal antibodies, this study confirmed that serum Hp was increased in piglets infected with PRRSV and offered a tool to know about subunit levels of Hp in porcine serum. But Hp itself could not be used as a specific biomarker for PRRSV infection, for elevated Hp levels were also obtained from pigs infected with other pathogens.

Highly pathogenic porcine reproductive and respiratory syndrome virus GP5 B antigenic region is not a neutralizing antigenic region.

In 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) caused great economic losses emerged in China and continues to be a threat for the pig industry. B antigenic region (AR) ((37)SHL/FQLIYNL(45)) of GP5 was considered to be a major linear neutralizing AR in PRRSV classical strains. However, peptide-purified antibodies against this AR did not neutralize PRRSV in a recent report. Compared with classical PRRSV, one amino acid mutation (L/F(39)→ I(39)) was found in B AR of HP-PRRSV. To study the ability of B AR of HP-PRRSV to induce neutralizing antibody (NA) in vitro and in vivo, rabbit antisera against B AR with and without the mutation and pig hyperimmune sera with high titer of NAs against HP-PRRSV were prepared. Immunofluorescence assays (IFA) showed that the two rabbit antisera both had reactivity to classical PRRSV CH-1a and HP-PRRSV HuN4 with no observable difference in IFA titer. However, antisera did not have neutralizing activity against classical PRRSV CH-1a and HP-PRRSV HuN4. No correlation was observed between the levels of anti-B AR peptide antibodies and NAs in pig hyperimmune sera that were detected by indirect ELISA and virus neutralization, respectively. B AR peptide-specific serum antibodies had no neutralizing activity and, GST-B fusion protein could not inhibit neutralization of NAs in pig hyperimmune sera. Based on these findings, we conclude that B AR of HP-PRRSV is not a neutralizing AR of HP-PRRSV GP5.

Identification of porcine serum proteins modified in response to HP-PRRSV HuN4 infection by two-dimensional differential gel electrophoresis.

Since 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has become the major pathogen attributed to the prevalent porcine reproductive and respiratory syndrome (PRRS) in China. The present study aims to identify serum proteins modified in response to infection of HuN4, a HP-PRRSV strain isolated from a farm in 2006. 2-D DIGE analysis allowed for the detection of 19 differentially expressed protein spots, of which 18 were identified by MALDI-TOF/TOF MS. These 18 spots represented for a total of 9 proteins (6 up-regulated and 3 down-regulated), most of which belonged to the acute phase proteins in swine and showed a trend of regression in the late phase of the experiment. One of a series of AGP spots was identified for the first time to be decreased in acute phase of PRRSV infection in swine. But the whole level of the protein in the serum did not show significant changes by Western blot. The rising tendency of Hp was confirmed by Western blot and ELISA. These altered proteins were probably involved in the inflammatory process triggered by HuN4 and in alleviating the oxidative damage occurring in the process. In summary, these results may provide new insights into understanding the mechanisms of HP-PRRSV infection.

Cloning and identification of porcine programmed death 1.

Programmed death 1 (PD-1) is a member of the immunoglobulin (Ig) superfamily, which is expressed on activated T cells, B cells and monocytes. Many researches have demonstrated that a high level of PD-1 expression is closely related to persistent infection and immune evasion in some human infections. In order to study the relationship between PD-1 expression and persistent infections caused by some porcine viruses, we first cloned the porcine PD-1 from porcine PBMCs based on the blast result in the EST database using the human PD-1 sequence. Sequence analysis showed that the cloned PD-1 molecule shares 63 and 54% amino acid sequence identity with human and murine PD-1, respectively. Its molecular structure is also similar to that of human and murine PD-1, containing an IgV-like domain in the extracellular region and two immune regulatory motifs in its cytoplasmic tail. The in vitro T cell proliferation assay showed that the cloned PD-1 could inhibit porcine T cell proliferation by 71% and secretion of IFN-gamma and IL-2 by 64 and 53%, respectively. These data suggest that porcine PD-1 negatively regulates the porcine immune response in a similar manner to that of its counterpart in the human and mouse immune system.