The naturally occurring flavonoid nobiletin reverses methotrexate resistance via inhibition of P-glycoprotein synthesis

Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA). However, after long-term treatment, some patients develop resistance. P-glycoprotein (P-gp), as an indispensable drug transporter, is essential for mediating this MTX resistance. In addition, nobiletin (NOB), a naturally occurring polymethoxylated flavonoid, has also been shown to reverse P-gp–mediated MTX resistance in RA groups; however, the precise role of NOB in this process is still unclear. Here, we administered MTX and NOB alone or in combination to collagen II-induced arthritic (CIA) mice and evaluated disease severity using the arthritis index, synovial histopathological changes, immunohistochemistry, and P-gp expression. In addition, we used conventional RNA-seq to identify targets and possible pathways through which NOB reverses MTX-induced drug resistance. We found that NOB in combination with MTX could enhance its performance in synovial tissue and decrease P-gp expression in CIA mice compared to MTX treatment alone. In vitro, in MTX-resistant fibroblast-like synoviocytes from CIA cells (CIA-FLS/MTX), we show that NOB treatment downregulated the PI3K/AKT/HIF-1α pathway, thereby reducing the synthesis of the P-gp protein. In addition, NOB significantly inhibited glycolysis and metabolic activity of CIA-FLS/MTX cells, which could reduce the production of ATP and block P-gp, ultimately decreasing the efflux of MTX and maintaining its anti-RA effects. In conclusion, this study shows that NOB overcomes MTX resistance in CIA-FLS/MTX cells through the PI3K/AKT/HIF-1α pathway, simultaneously influencing metabolic processes and inhibiting P-gp–induced drug efflux.     

Down-regulation of the expression of beta1,4-galactosyltransferase V promotes integrin beta1 maturation

In previous study, we have shown that beta1,4-galactosyltransferase V (GalT V) functions as a positive growth regulator in glioma. Here, we reported that down-regulation of the expression of GalT V in SHG44 cells by transfection with antisense cDNA specifically up-regulated the expression of cell surface integrin beta1 without the change of its mRNA, and with integrin beta1 125 kDa mature form increased and 105 kDa precursor form decreased. It is well known that the N-glycans of integrins modulate the location and functions of integrins. The SHG44 cells transfected with antisense cDNA of GalT V demonstrated decreased Golgi localization of integrin beta1, strengthened the interaction between integrin alpha5 and beta1 subunit, and enhanced the adhesion ability to fibronectin and the level of focal adhesion kinase phosphorylation. Our results suggested that the down-regulation of the expression of GalT V could promote the expression of cell surface integrin beta1 and subsequently inhibit glioma malignant phenotype.     

Diversity of the O-superfamily conotoxins from Conus miles.

Conopeptides display prominent features of hypervariability and high selectivity of large gene families that mediate interactions between organisms. Remarkable sequence diversity of O-superfamily conotoxins was found in a worm-hunting cone snail Conus miles. Five novel cDNA sequences encoding O-superfamily precursor peptides were identified in C. miles native to Hainan by RT-PCR and 3'-RACE. They share the common cysteine pattern of the O-superfamily conotoxin (C-C-CC-C-C, with three disulfide bridges). The predicted peptides consist of 27-33 amino acids. We then performed a phylogenetic analysis of the new and published homologue sequences from C. miles and the other Conus species. Sequence divergence (%) and residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were analyzed. Percentage divergence of the amino acid sequences of the prepro region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical with to highly divergent from inter- and intra-species. Despite the O-superfamily being a large and diverse group of peptides, widely distributed in the venom ducts of all major feeding types of Conus and discovered in several Conus species, it was for the first time that the newly found five O-superfamily peptides in this research came from the vermivorous C. miles. So far, conotoxins of the O-superfamily whose properties have been characterized are from piscivorous and molluscivorous Conus species, and their amino acid sequences and mode of action have been discussed in detail. The elucidated cDNAs of the five toxins are new and of importance and should attract the interest of researchers in the field, which would pave the way for a better understanding of the relationship of their structure and function.     

Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells

A recently developed strategy of sequencing alternative polyadenylation (APA) sites (SAPAS) with second-generation sequencing technology can be used to explore complete genome-wide patterns of tandem APA sites and global gene expression profiles. spermatogonial stem cells (SSCs) maintain long-term reproductive abilities in male mammals. The detailed mechanisms by which SSCs self-renew and generate mature spermatozoa are not clear. To understand the specific alternative polyadenylation pattern and global gene expression profile of male germline stem cells (GSCs, mainly referred to SSCs here), we isolated and purified mouse Thy1⁺ cells from testis by magnetic-activated cell sorting (MACS) and then used the SAPAS method for analysis, using pluripotent embryonic stem cells (ESCs) and differentiated mouse embryonic fibroblast cells (MEFs) as controls. As a result, we obtained 99,944 poly(A) sites, approximately 40% of which were newly detected in our experiments. These poly(A) sites originated from three mouse cell types and covered 17,499 genes, including 831 long non-coding RNA (lncRNA) genes. We observed that GSCs tend to have shorter 3''UTR lengths while MEFs tend towards longer 3''UTR lengths. We also identified 1337 genes that were highly expressed in GSCs, and these genes were highly consistent with the functional characteristics of GSCs. Our detailed bioinformatics analysis identified APA site-switching events at 3''UTRs and many new specifically expressed genes in GSCs, which we experimentally confirmed. Furthermore, qRT-PCR was performed to validate several events of the 334 genes with distal-to-proximal poly(A) switch in GSCs. Consistently APA reporter assay confirmed the total 3''UTR shortening in GSCs compared to MEFs. We also analyzed the cis elements around the proximal poly(A) site preferentially used in GSCs and found C-rich elements may contribute to this regulation. Overall, our results identified the expression level and polyadenylation site profiles and these data provide new insights into the processes potentially involved in the GSC life cycle and spermatogenesis.     

N-糖基化在肿瘤细胞逃逸CD8+T细胞中的意义和机制

肿瘤在发生发展过程中会产生异常糖基化结构,改变细胞功能,使其能逃逸机体细胞的免疫识别和免疫攻击,实现免疫逃逸。细胞膜表面蛋白的N-糖基化参与调控肿瘤细胞的增殖、迁移、侵袭、干细胞特性等。近年来,一系列研究进一步证实,肿瘤细胞免疫检查点分子的N-糖基化,以及识别N-糖基化的动物凝集素在肿瘤免疫逃逸CD8⁺T细胞中发挥了重要作用,如PD-L1的N-糖基化能增强其自身蛋白质的稳定性,促进与其受体PD-1的相互作用,并且靶向PD-1、PD-L1蛋白N-糖基化的抗体,能够有效抑制肿瘤免疫逃逸和肿瘤发展。