Search Results (4)

The Cystic Fibrosis Conductance Transmembrane Regulator gene encodes for the CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result in impaired ion transport, subsequently leading to perturbed secretion in all exocrine glands and, therefore, the multi-organ disease cystic fibrosis (CF). In recent years, several studies have reported on CFTR expression in immune cells as demonstrated by immunofluorescence, flow cytometry, and immunoblotting. However, these data are mainly restricted to single-cell populations and show significant variation depending on the methodology used. Here, we investigated CFTR transcription and protein expression using standardized protocols in a comprehensive panel of immune cells. Methods: We applied a high-resolution Western blot protocol using a combination of highly specific monoclonal CFTR antibodies that have been optimized for the detection of CFTR in epithelial cells and healthy primary immune cell subpopulations sorted by flow cytometry and used immortalized cell lines as controls. The specificity of CFTR protein detection was controlled by peptide competition and enzymatic Peptide-N-Glycosidase-F (PNGase) digest. CFTR transcripts were analyzed using quantitative real-time PCR and normalized to the level of epithelial T84 cells as a reference. Results: CFTR mRNA expression could be shown for primary CD4⁺ T cells, NK cells, as well as differentiated THP-1 and Jurkat T cells. In contrast, we failed to detect CFTR transcripts for CD14⁺ monocytes and undifferentiated THP-1 cells, as well as for B cells and CD8⁺ T cells. Prominent immunoreactive bands were detectable by immunoblotting with the combination of four CFTR antibodies targeting different epitopes of the CFTR protein. However, in biosamples of non-epithelial origin, these CFTR-like protein bands could be unmasked as false positives through peptide competition or PNGase digest, meaning that the observed mRNA transcripts were not necessarily translated into CFTR proteins, which could be detected via immunoblotting. Our results confirm that mRNA expression in immune cells is many times lower than in that cells of epithelial origin. The immunoreactive signals in immune cells turned out to be false positives, and may be provoked by the presence of a high-affinity protein with a similar epitope. Non-specific binding (e.g., Fab-interaction with glycosyl branches) might also contribute to false positive signals. Our findings highlight the necessity of accurate controls, such as CFTR-negative cells, as well as peptide competition and glycolytic digest in order to identify genuine CFTR protein by immunoblotting. Our data suggest, furthermore, that CFTR protein expression data from techniques such as histology, for which the absence of a molecular weight or other independent control prevents the unmasking of false positive immunoreactive signals, must be interpreted carefully as well. Full article

Maize breeding is greatly affected by hybrid vigor, a phenomenon that hybrids exhibit superior performance than parental lines. The immortalized F₂ population (IMF₂) is ideal for the genetic dissection and prediction of hybrid performance. Here, in this study, we conducted the QTL mapping and genomic prediction of six traits related to plant architecture using an IMF₂ population. Broad-sense heritability of these traits ranged from 0.85 to 0.94. Analysis of genetic effects showed that additive variance was the main contributor to phenotypic variations. The mapping of quantitative trait loci (QTLs) revealed 10 to 16 QTLs (including pleiotropic loci and epistatic QTLs) for the six traits. Additionally, we identified 15 fine-tuning QTLs for plant height (PH). For genomic prediction (GP), the model of additive and dominance (AD) exhibited higher prediction accuracy than those fitting general combining ability (GCA) and its combination with special combining ability (SCA) effects for all tested traits. And adding the epistasis (E) effect into the AD model did not significantly increase its prediction accuracy. Moreover, the identified 15 fine-tuning QTLs of PH, which exerted large genomic prediction effects, were verified by the marker effect of GP. Our results not only provide an approach for the fine-mapping of fine-tuning QTLs but also serve as references for GP breeding in crops. Full article

Corneal wound healing is a complex biological process that integrates a host of different signals to coordinate cell behavior. Upon wounding, there is the generation of an endogenous wound electric field that serves as a powerful cue to guide cell migration. Concurrently, the corneal epithelium reduces sialylated glycoforms, suggesting that sialylation plays an important role during electrotaxis. Here, we show that pretreating human telomerase-immortalized corneal epithelial (hTCEpi) cells with a sialyltransferase inhibitor, P-3FAX-Neu5Ac (3F-Neu5Ac), improves electrotaxis by enhancing directionality, but not speed. This was recapitulated using Kifunensine, which inhibits cleavage of mannoses and therefore precludes sialylation on N-glycans. We also identified that 3F-Neu5Ac enhanced the responsiveness of the hTCEpi cell population to the electric field and that pretreated hTCEpi cells showed increased directionality even at low voltages. Furthermore, when we increased sialylation using N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), hTCEpi cells showed a decrease in both speed and directionality. Importantly, pretreating enucleated eyes with 3F-Neu5Ac significantly improved re-epithelialization in an ex vivo model of a corneal injury. Finally, we show that in hTCEpi cells, sialylation is increased by growth factor deprivation and reduced by PDGF-BB. Taken together, our results suggest that during corneal wound healing, reduced sialylated glycoforms enhance electrotaxis and re-epithelialization, potentially opening new avenues to promote corneal wound healing. Full article

Epstein–Barr virus (EBV) infects 95% of the world’s population and persists latently in the body. It immortalizes B-cells and is associated with lymphomas. LCLs (lymphoblastoid cell lines, EBV latency III B-cells) inhibit anti-tumoral T-cell response following PD-L1 overexpression (programmed death-ligand 1 immune checkpoint). Many cancer cells, including some DLBCLs (diffuse large B-cell lymphomas), also overexpress PD-L1. Immunotherapies are based on inhibition of PD-L1/PD-1 interactions but present some dose-dependent toxicities. We aim to find new strategies to improve their efficiency by decreasing PD-L1 expression. Fucoidan, a polysaccharide extracted from brown seaweed, exhibits immunomodulatory and anti-tumor activities depending on its polymerization degree, but data are scarce on lymphoma cells or immune checkpoints. LCLs and DLBCLs cells were treated with native fucoidan (Fucus vesiculosus) or original very-low-molecular-weight fucoidan formulas (vLMW-F). We observed cell proliferation decrease and apoptosis induction increase with vLMW-F and no toxicity on normal B- and T-cells. We highlighted a decrease in transcriptional and PD-L1 surface expression, even more efficient for vLMW than native fucoidan. This can be explained by actin network alteration, suggesting lower fusion of secretory vesicles carrying PD-L1 with the plasma membrane. We propose vLMW-F as potential adjuvants to immunotherapy due to their anti-proliferative and proapoptotic effects and ability to decrease PD-L1 membrane expression. Full article