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Objective To construct the eukaryotic expression vectors of human fms related tyrosine kinase 3 (FLT3) gene and FLT3-internal tandem duplication (FLT3-ITD) mutants and purify the native proteins through immunoprecipitation from HEK293T cell lysates. Methods The cDNA fragments of FLT3wt and FLT3-ITD were amplified from bone marrow cells of healthy individuals and FLT3-ITD-mutated acute myeloid leukemia (AML) patients with specific primers, and the PCR products were cloned in CD530A-T2A-GFP expression vectors. FLT3wt and FLT3-ITD plasmids were transfected in HEK293T cells by Polyjet reagent, and the recombinant proteins were purified by immunoprecipitation and competing elution methods. Results FLT3wt and FLT3-ITD-mutated DNA sequences were successfully cloned in CD530A-T2A-GFP expression vectors. FLT3wt and FLT3-ITD mutated proteins were successfully expressed and purified in HEK293T cells as verified by Western blotting and sliver staining. Conclusion FLT3wt and FLT3-ITD expression vectors were successfully constructed, and purified proteins were successfully obtained from HEK293T cells.

This study aims to explore the role of lncRNA TMPO-AS1 in ischemic stroke and corresponding mechanism. Adult male C57BL/6 J mice were subjected to a middle cerebral artery occlusion (MCAO) model of ischemic stroke, then TMPO-AS1 shRNA lentivirus were injected into ipsilateral striatum of mice. The neurological score and cerebral infarction volume were evaluatedHypoxia/glucose deprivation/reoxygenation (OGD/R)-induced BV2 cells were transfected with TMPO-AS1 shRNA (sh-TMPO-AS1) or together with pcDNA-INPP5D, as well as transfected with sh-PU.1 or together with pcDNA-INPP5D, then TMPO-AS1 level, the expression of PU.1 and INPP5D proteins, the secretion of inflammatory factors (TNF-α, IL-6 and IL-1β), the levels of iNOS, CD68,Arg1 and CD206 mRNA were detected. RIP and PNA-pull down assays were used to detect the binding of TMPO-AS1 and PU.1, luciferase reporter gene and chromatin immunoprecipitation (ChIP) assays were used to detect the binding activity of PU.1 and INPP5D. TMPO-AS1 level was increased in peripheral blood of ischemic stroke patients , brain tissues of MCAO/R model mice and OGD/R-induced BV2 cells. TMPO-AS1 interference inhibited the inflammation of OGD/R-induced BV2 cells. TMPO-AS1 also enhanced the nuclear accumulation of PU.1 by binding to the transcription factor PU.1, and promoted the transcriptional activation of INPP5D. The anti-inflammatory effects of TMPO-AS1 interference were reversed by INPP5D overexpression. In addition, TMPO-AS1 interference improved the infarct volume of MCAO mice, and improved sensorimotor and cognitive functions. INPP5D underexpression mediated by TMPO-AS1-PU.1 complex alleviated neuroinflammation after ischemic stroke. [ABSTRACT FROM AUTHOR]

Filamin A (FlnA) is an actin cross-linking protein. Previous studies have demonstrated its role in tumor progression in a wide range of cancer types. It has been reported that FlnA interacts with the DNA damage response protein, breast cancer gene 1 (BRCA1), which is a tumor suppressor gene. However, to the best of our knowledge, there are no studies evaluating the association of these genes in human carcinomas. In the present study, the immunohistochemistry of a tissue microarray was used to investigate the clinical significance of FlnA and BRCA1 expression in pathological specimens collected from 424 patients treated for breast cancer. In addition, FlnA and BRCA1 expression was downregulated in the breast cancer cell line, MCF-7, through FlnA RNA interference. FlnA expression was exhibited by cancer tissues collected from 137 patients with breast cancer, which also exhibited high expression of BRCA1 and were associated with a relatively long survival time. A significant association was identified between FlnA protein expression and tumor size, and between FlnA protein expression and progesterone receptor expression. These results suggest that BRCA1 expression could be regulated by FlnA in the breast cancer cell line, MCF-7. Overall, the present study demonstrates that FlnA expression was associated with BRAC1 expression and tumor size in breast cancer, which provides important implications for future study of FlnA in the progression of human breast cancer. [ABSTRACT FROM AUTHOR]

Both membrane-bound and soluble forms of costimulatory molecules play important roles in immune-regulatory networks. B7-H3, a member of the B7 family, has been found with aberrant expression in tumors and infectious disease. However, the significance of sB7-H3 expression in systemic lupus erythematosus (SLE) has not been investigated. Using the peripheral blood of 78 SLE patients, we established a comprehensive database containing clinical data and relevant laboratory tests. We found that sB7-H3 expression in SLE patients was significantly lower compared with the healthy individuals. In addition, sB7-H3 levels in the patients were positively correlated with the disease activity as indicated by SLE disease activity index score, rashes, fever, and inflammatory cytokines. Moreover, sB7-H3 was associated with the counts of red blood cells and hemoglobin. Our findings suggest that sB7-H3 might counteract the aberrant immune response and potentially serve as a monitoring indicator of disease progression and therapeutic target in SLE treatment. [ABSTRACT FROM AUTHOR]

It has been found that pilose antler peptide has an antidepressant effect on depression. However, the exact molecular mechanism of its antidepressant effect is still unclear. The study sought to determine the impact of monomeric pilose antler peptide (PAP; sequence LVLVEAELRE) on depression as well as investigate potential molecular mechanisms. Chronic unexpected mild stress (CUMS) was used to establish the model, and the effect of PAP on CUMS mice was detected by the behavioral test. The influence of PAP on neuronal cells and dendritic spine density was observed by immunofluorescence and Golgi staining. FGFR3 and the CaMKII-associated pathway were identified using quantitative real-time polymerase chain reaction, and Western blot analysis was utilized to measure their proteins and gene expression levels. Molecular docking and microscale thermophoresis were applied to detect the binding of PAP and FGFR3. Finally, the effect of FGFR3's overexpression on PAP treatment of depression was detected. PAP alleviated the changes in depressive behavior induced by CUMS, promoted the growth of nerve cells, and the density of dendritic spines was increased to its original state. PAP therapy successfully downregulated the expression of FGFR3 and ERK1/2 while upregulating the expression of CREB, BDNF, and CaMKII. Based on the current research, PAP has a therapeutic effect on depression brought on by CUMS by inhibiting FGFR3 expression and enhancing synaptic plasticity. [Display omitted] • PAP (sequence LVLVEAELRE) alleviates depressive behavior in CUMS mice. • PAP enhanced CUMS-induced reduction of neurogenesis and synaptic plasticity. • PAP reduces depressive behavior by affecting FGFR3 and CaMKII-related pathways. • Overexpression of FGFR3 inhibits the therapeutic effects of PAP. [ABSTRACT FROM AUTHOR]