Ribosomal protein RPL5 regulates colon cancer cell proliferation and migration through MAPK/ERK signaling pathway.

BACKGROUND: Abnormal expression of ribosomal proteins has an important regulatory effect on the progression of cancer. RPL5 is involved in the progression of various malignancies, however, the role of RPL5 in colon cancer remains is still unclear. METHODS: Data from TCGA and GTEx databases were used to analyze the RPL5 expression in pan-cancer. The expression level of RPL5 in clinical colon cancer tissue samples and human colon cancer cell lines was detected by western blotting; siRNA targeting RPL5 was designed, and its interference efficiency was verified by western blotting and RT-qPCR; CCK8 assay, clone formation assay, cell cycle assay, and cell scratch assay were used to observe the effect of RPL5 on colon cancer cell proliferation and migration; the changes of proteins related to MAPK/ERK signaling pathway were also detected using western blotting. RESULTS: The expression level of RPL5 in colon cancer tissues and cell lines was significantly higher than that in adjacent tissues and NCM460 cells, respectively, and its expression level was higher in HCT116 cells and RKO cells. Knockdown of RPL5 significantly inhibited the proliferation and migration of HCT16 and RKO cells, and arrested the cell cycle in G0/G1 phase. Mechanistic studies revealed that the expression of p-MEK1/2, p-ERK, c-Myc were down-regulated, and the expression of FOXO3 was up-regulated after down-regulation of RPL5, ERK activator (TBHQ) could partially reverse the above-mentioned effects caused by siRPL5. Moreover, TBHQ could partially reverse the inhibitory effect of siRPL5 on the proliferation and migration of colon cancer cells. Collectively, RPL5 promoted colon cell proliferation and migration, at least in part, by activating the MAPK/ERK signaling pathway. CONCLUSION: RPL5 promoted colon cell proliferation and migration, at least in part, by activating the MAPK/ERK signaling pathway, which may serve as a novel therapeutic target for cancers in which MAPK/ERK signaling is a dominant feature.

Correlation between serum ß2-microglobulin level and systemic lupus erythematosus disease activity: A PRISMA-compliant meta-analysis.

BACKGROUND: Numerous studies have explored whether serum beta 2-microglobulin (ß2-MG) can be used as a biomarker for monitoring systemic lupus erythematosus (SLE) disease activity, but the results are conflicting. Therefore, we performed a systematic meta-analysis to further investigate the correlation between serum ß2-MG level and SLE disease activity. METHODS: PubMed, Web of Science, Embase, and CNKI databases were thoroughly searched for eligible studies through April 2022. Standardized mean differences with 95% confidence intervals (95% CIs) were used to depict the differences in serum ß2-MG levels between groups compared in the studies. The correlation between serum ß2-MG level and SLE disease activity was assessed using Fisher z-values. RESULTS: Sixteen articles with combined 1368 SLE patients were included in this meta-analysis. Serum ß2-MG levels were significantly higher in SLE patients than in healthy controls (pooled standardized mean difference: 3.98, 95% CI: 2.50-5.46, P < .01). In addition, patients with active SLE had an increased serum ß2-MG concentration compared to their inactive SLE counterparts. Furthermore, a positive correlation was observed between serum ß2-MG levels and SLE disease activity (pooled Fisher z = 0.78, 95% CI: 0.61-0.96, P < .01). CONCLUSIONS: This study suggests that patients with SLE have higher serum ß2-MG levels than healthy controls and that serum ß2-MG levels are positively correlated with SLE disease activity. Thus, serum ß2-MG level may be a promising biomarker for monitoring SLE disease activity.

A UHPLC-Q-TOF/MS method for the determination of poloxamer 124 and its application in a tissue distribution study in rats.

Poloxamers are commonly used pharmaceutical excipients. They are high molecular weight polymers formed from polypropylene oxide (PPO) and polyethylene oxide (PEO). However, PL124, a low molecular weight example in the poloxamer family, has rarely been reported, and there is no research into its tissue distribution in the body after administration. In this study, rat tissue samples were quantitatively studied via UHPLC-Q-TOF/MS after the intravenous administration of 10 mg kg-1 PL124. The quantitative method showed good sensitivity and selectivity. The linear range of PL124 was 0.1-5 µg mL-1 and the LLOQ was 0.1 µg mL-1. The relative error in terms of the accuracy was no higher than 13.9%, and the relative standard deviation in terms of the precision was no higher than 9.6%. The extraction recovery, matrix effect, and stability results of the established method were also satisfactory. The research showed that PL124 can be quickly distributed to large amounts of tissue, and tissue with higher levels of blood flow has higher concentrations. PL124 could be rapidly eliminated in 4 h from most organs, except the heart and liver. This study can be helpful for the further analysis of PL124.

Ultra-high-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry method for quantifying polymer poloxamer 124 and its application to pharmacokinetic study.

Poloxamer is a commonly used pharmaceutical excipient. It is a high molecular polymer formed using polypropylene oxide and polyethylene oxide units. Specifically, poloxamer 124 is one of the smaller molecular weight in the poloxamer series; however, its pharmacokinetic behaviors in vivo are still unclear. In this study, a method for quantifying poloxamer 124 in rat plasma through ultra-high-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry was developed. The intravenous dosage of PL124 was 10 mg/kg. Plasma was collected at different times. The calibration curve was linear in the range of 0.1-5 µg/mL for the poloxamer 124 (r ≥ 0.9956) with the lower limit of quantitation of 0.1 µg/ml. The relative standard deviation of the intraday and interday precisions was below 8.0%, and the relative error of the accuracy was within ±12.0%. The extraction recovery, matrix effect, and stability were satisfactory in rat plasma. The validated method was successfully applied to a pharmacokinetic study of poloxamer 124 in rats. Results indicated that poloxamer 124 could be rapidly absorbed and eliminated through caudal vein injection. This study is helpful for the further study of poloxamer 124.

[Strong inflammation is essential for expression of articular cartilage-specific citrullinated antigens].

OBJECTIVE: To investigate the expression of citrullinated epitopes in articular cartilage protein and whether its expression is sufficient to induce anti-citrullinated protein antibody (ACPA) response in mice. METHODS: The experimental group was treated with different concentrations of lipopolysaccharide (LPS), heat-inactivated bacteria (Escherichia coli and Staphylococcus aureus) or specific monoclonal antibody against type Ⅱ collagen to induce citrullination of articular cartilage protein, with PBS as the control. Immunohistochemistry with the monoclonal antibody ACC4 (IgG1) that specifically binds to the citrullinated epitope of cartilage protein was performed for detecting the expression of citrullinated protein, with ACC1 (IgG2a) as a positive control antibody and L243 (IgG2a) and Hy2.15 (IgG1) as the negative isotype control. In the in vivo experiment, SD rats were subjected to injection of different doses of LPS in the right knee (with PBS as the controls in the left knee), and 3 days later frozen sections were prepared for immunohistochemical detection of the expression of citrullinated protein. Models of collagen-induced arthritis (CIA) established in different mouse strains were observed for incidence and severity of CIA. Serum samples collected from these models and the sera from rheumatoid arthritis patients were examined for anti-citrullinated protein antibody, and immunohistochemistry was performed to detect the expression of citrullinated protein in the cartilage of the mouse. RESULTS: The citrullinated CII epitope-specific antibody ACC4 did not bind to articular cartilage tissues with different treatments as compared with the positive control antibody ACC1. The ACC4 antibody and the antibodies from patients with rheumatoid arthritis with high titers of anti-citrullinated protein antibody were capable of binding to the synovial tissue around the articular cartilage of the CIA. Luminex analysis showed that the anti-citrullinated protein antibody was lowly expressed in mouse serum, but the anti-type Ⅱ collagen triple helix structure peptide antibody exhibited strong reactivity. CONCLUSIONS: Mild acute inflammatory response is not enough to cause citrullination of articular cartilage protein, and the expression of specific epitope requires a high-intensity inflammatory response. Inflammatory articular cartilage protein can express citrullinated epitopes in type Ⅱ collagen-induced arthritis in mice, but the expression of citrullinated epitopes is not sufficient to induce an immune response to anti-citrullinated antibodies. Stronger stimulation signals are required to induce an immune response for producing anti-citrullinated protein antibodies.

The autoantibody response to cyclic citrullinated collagen type II peptides in rheumatoid arthritis.

OBJECTIVES: The detection of anti-citrullinated peptide antibodies (ACPAs) is a serological hallmark of RA. Autoantibodies reactive with collagen type II (CII) are present in RA sera and synovial fluid and are potentially pathogenic. Here, we investigate the prevalence and specificity of the autoantibody responses to defined citrullinated cyclic peptides derived from CII in a China RA cohort. METHODS: Using bead-based multiplex assay, we examined the presence of autoantibodies binding to 54 cyclic 17-mer citrullinated CII peptides, encompassing all citrullinate epitopes in CII, and the corresponding unmodified peptides in 415 RA patients, in addition to 304 patients with OA. Furthermore, the autoantibody responses to a selected set of 10 cyclic citrullinated peptides were also examined in 203 healthy individuals. RESULTS: Autoantibody responses to cyclic citrullinated CII peptides were higher in RA patients as compared with OA patients or healthy individuals, whereas little or negligible antibody responses to cyclic unmodified CII peptides were observed. Interestingly, several novel citrullinated CII epitopes were identified. Antibodies to these novel citrullinated CII epitopes showed not only substantial overlapping reactivities but also had unique specificities. CONCLUSION: We found a high prevalence of autoantibodies against cyclic citrullinated CII in the sera of patients in a China RA cohort. The present study revealed heterogeneous binding patterns against novel citrullinated CII epitopes, which may help to stratify RA patients into different subgroups.

Purification of an active fragment of Cry1Ie toxin from Bacillus thuringiensis.

The cry1I genes from Bacillus thuringiensis are a class of special genes with unique characteristics; they are silent in B. thuringiensis strains but can be over-expressed in Escherichia coli, resulting in a Cry1I-type protein with a molecular mass of approximately 81kDa. Cry1I-type protein is toxic to Lepidoptera larvae. A truncated Cry1Ie protein, IE648, which corresponds to the first 648 amino acids from the N-terminus of Cry1Ie, was purified from E. coli using Ni-NTA affinity isolation, Q-Sepharose Fast Flow chromatography, and Superdex-200 size-exclusion chromatography. It was determined using laboratory bioassays that the purified IE648 protein has good insecticidal activity. Heterologous competitive binding assays show that IE648 does not compete with Cry1Ac for binding to the brush border membrane vesicles of the Asian corn borer and does not compete with Cry1Ac at concentrations below a 500-fold excess of unlabeled Cry1Ac for binding to the peritrophic matrix of the insect. This result implies that IE648 may be a good candidate as part of a multiple-toxin strategy for the potential control of resistance in insect pests. The method of purification reported here is valuable for further research on the structure and function of IE648 and in evaluating the biosafety of this protein within transgenic plants.