Different expression levels of the TAP peptide transporter lead to recognition of different antigenic peptides by tumor-specific CTL

Decreased antigenicity of cancer cells is a major problem in tumor immunology. This is often acquired by an expression defect in the TAP. However, it has been reported that certain murine Ags appear on the target cell surface upon impairment of TAP expression. In this study, we identified a human CTL epitope belonging to this Ag category. This epitope is derived from preprocalcitonin (ppCT) signal peptide and is generated within the endoplasmic reticulum by signal peptidase and signal peptide peptidase. Lung cancer cells bearing this antigenic peptide displayed low levels of TAP, but restoration of their expression by IFN-γ treatment or TAP1 and TAP2 gene transfer abrogated ppCT Ag presentation. In contrast, TAP upregulation in the same tumor cells increased their recognition by proteasome/TAP-dependent peptide-specific CTLs. Thus, to our knowledge, ppCT(16-25) is the first human tumor epitope whose surface expression requires loss or downregulation of TAP. Lung tumors frequently display low levels of TAP molecules and might thus be ignored by the immune system. Our results suggest that emerging signal peptidase-generated peptides represent alternative T cell targets, which permit CTLs to destroy TAP-impaired tumors and thus overcome tumor escape from CD8(+) T cell immunity.     

Genetic diversity of small eukaryotes in lakes differing by their trophic status

Small eukaryotes, cells with a diameter of less than 5 mum, are fundamental components of lacustrine planktonic systems. In this study, small-eukaryote diversity was determined by sequencing cloned 18S rRNA genes in three libraries from lakes of differing trophic status in the Massif Central, France: the oligotrophic Lake Godivelle, the oligomesotrophic Lake Pavin, and the eutrophic Lake Aydat. This analysis shows that the least diversified library was in the eutrophic lake (12 operational taxonomic units [OTUs]) and the most diversified was in the oligomesotrophic lake (26 OTUs). Certain groups were present in at least two ecosystems, while the others were specific to one lake on the sampling date. Cryptophyta, Chrysophyceae, and the strictly heterotrophic eukaryotes, Ciliophora and fungi, were identified in the three libraries. Among the small eukaryotes found only in two lakes, Choanoflagellida and environmental sequences (LKM11) were not detected in the eutrophic system whereas Cercozoa were confined to the oligomesotrophic and eutrophic lakes. Three OTUs, linked to the Perkinsozoa, were detected only in the Aydat library, where they represented 60% of the clones of the library. Chlorophyta and Haptophyta lineages were represented by a single clone and were present only in Godivelle and Pavin, respectively. Of the 127 clones studied, classical pigmented organisms (autotrophs and mixotrophs) represented only a low proportion regardless of the library's origin. This study shows that the small-eukaryote community composition may differ as a function of trophic status; certain lineages could be detected only in a single ecosystem.     

Receptor-mediated transcytosis of botulinum neurotoxin A through intestinal cell monolayers

Botulism is mainly acquired by the oral route, and botulinum neurotoxin (BoNT) escapes the gastrointestinal tract by crossing the digestive epithelial barrier prior to gaining access to the nerve endings. Here, we show that biologically active BoNT/A crosses intestinal cell monolayers via a receptor-mediated transcytosis, including a transport inhibition at 4°C and a passage at 37°C in a saturable manner within 30–60 min. BoNT/A passage rate was about 10-fold more efficient through the intestinal crypt cell line m-IC_cl2, than through the carcinoma Caco-2 or T84 cells, and was not increased when BoNT/A was associated with the non-toxic proteins (botulinum complex). Like for neuronal cells, BoNT/A binding to intestinal cells was mediated by the half C-terminal domain as tested by fluorescence-activated cytometry and by transcytosis competition assay. A 'double receptor model' has been proposed in which BoNT/A interacts with gangliosides of GD_1b and GT_1b series as well as SV2 protein. Gangliosides of GD_1b and GT_1b series and recombinant intravesicular SV2-C domain partially impaired BoNT/A transcytosis, suggesting a putative role of gangliosides and SV2 or a related protein in BoNT/A transcytosis through Caco-2 and m-IC_cl2 cells.     

Queuosine Biosynthesis Is Required for Sinorhizobium meliloti-Induced Cytoskeletal Modifications on HeLa Cells and Symbiosis with Medicago truncatula

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.     

Lack of Liver X Receptors Leads to Cell Proliferation in a Model of Mouse Dorsal Prostate Epithelial Cell

Recent studies underline the implication of Liver X Receptors (LXRs) in several prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In order to understand the molecular mechanisms involved, we derived epithelial cells from dorsal prostate (MPECs) of wild type (WT) or Lxrαβ−/− mice. In the WT MPECs, our results show that LXR activation reduces proliferation and correlates with the modification of the AKT-survival pathway. Moreover, LXRs regulate lipid homeostasis with the regulation of Abca1, Abcg1 and Idol, and, in a lesser extent, Srebp1, Fas and Acc. Conversely cells derived from Lxrαβ−/− mice show a higher basal phosphorylation and consequently activation of the survival/proliferation transduction pathways AKT and MAPK. Altogether, our data point out that the cell model we developed allows deciphering the molecular mechanisms inducing the cell cycle arrest. Besides, we show that activated LXRs regulate AKT and MAPK transduction pathways and demonstrate that LXRs could be good pharmacological targets in prostate disease such as cancer.     

Assessment of the lipid production potential of six benthic diatom species grown in airlift photobioreactors

Journal of Applied Phycology - In recent years diatoms have emerged as a major algal source for the production of bioactive compounds. Marine diatoms grow quickly and can store high amount of...     

The SSV-Seq 2.0 PCR-Free Method Improves the Sequencing of Adeno-Associated Viral Vector Genomes Containing GC-Rich Regions and Homopolymers

Adeno-associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co-transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high-throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV-Seq 2.0, a PCR-free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR-free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS-based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real-time PCR and are useful methods to improve the safety and efficacy of these viral vectors.     

Effect of 6-Benzoyl-benzothiazol-2-one scaffold on the pharmacological profile of α-alkoxyphenylpropionic acid derived PPAR agonists

Abstract

A series of nitrogen heterocycles containing α–ethoxyphenylpropionic acid derivatives were designed as dual PPARα/γ agonist ligands for the treatment of type 2 diabetes (T2D) and its complications. 6-Benzoyl-benzothiazol-2-one was the most tolerant of the tested heterocycles in which incorporation of O-methyl oxime ether and trifluoroethoxy group followed by enantiomeric resolution led to the (S)-stereoisomer 44?b displaying the best in vitro pharmacological profile. Compound 44?b acted as a very potent full PPARγ agonist and a weak partial agonist on the PPARα receptor subtype. Compound 44?b showed high efficacy in an ob/ob mice model with significant decreases in serum triglyceride, glucose and insulin levels but mostly with limited body-weight gain and could be considered as a selective PPARγ modulator (SPPARγM).