Nonsense-mediated mRNA decay impacts MSI-driven carcinogenesis and anti-tumor immunity in colorectal cancers

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3epsilon-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2x10(-16)). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs.     

A novel SINE family occurs frequently in both genomic DNA and transcribed sequences in ixodid ticks of the arthropod sub-phylum Chelicerata

Reassociation kinetics and flow cytometry data indicate that ixodid tick genomes are large, relative to most arthropods, containing>or=10(9) base pairs. The molecular basis for this is unknown. We have identified a novel small interspersed element with features of a tRNA-derived SINE, designated Ruka, in genomic sequences of Rhipicephalus appendiculatus and Boophilus (Rhipicephalus) microplus ticks. The SINE was also identified in expressed sequence tag (EST) databases derived from several tissues in four species of ixodid ticks, namely R. appendiculatus, B. (R.) microplus, Amblyomma variegatum and also the more distantly related Ixodes scapularis. Secondary structure predictions indicated that Ruka could adopt a tRNA structure that was, atypically, most similar to a serine tRNA. By extrapolation the frequency of occurrence in the randomly selected BAC clone sequences is consistent with approximately 65,000 copies of Ruka in the R. appendiculatus genome. Real time PCR analyses on genomic DNA indicate copy numbers for specific Ruka subsets between 5800 and 38,000. Several putative conserved Ruka insertion sites were identified in EST sequences of three ixodid tick species based on the flanking sequences associated with the SINEs, indicating that some Ruka transpositions probably occurred prior to speciation within the metastriate division of the Ixodidae. The data strongly suggest that Class I transposable elements form a significant component of tick genomes and may partially account for the large genome sizes observed.     

Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by amyloid deposition of protein-prion (PrPsc), the pathogenic isoform of the host cellular protein PrPc, in the immune and central nervous systems. In the absence of definitive data on the nature of the infectious agent, PrPsc immunohistochemistry (IHC) constitutes one of the main methodologies for pathogenesis studies of these diseases. In situ PrPsc immunolabeling requires formalin fixation and paraffin embedding of tissues, followed by post-embedding antigen retrieval steps such as formic acid and hydrated autoclaving treatments. These procedures result in poor cellular antigen preservation, precluding the phenotyping of cells involved in scrapie pathogenesis. Until now, PrPsc-positive cell phenotyping relied mainly on morphological criteria. To identify these cells under the PrPsc IHC conditions, a new, rapid, and highly sensitive PrPsc double-labeling technique was developed, using a panel of screened antibodies that allow specific labeling of most of the cell subsets and structures using paraffin-embedded lymphoid and neural tissues from sheep, leading to an accurate identification of ovine PrPsc-accumulating cells. This technique constitutes a useful tool for IHC investigation of scrapie pathogenesis and may be applicable to the study of other ovine infectious diseases.     

Variations in levels of clonality among Populus nigra L. stands of different ages

The evolution of genotypic diversity with population age remains poorly explored in clonal plant populations despite the potential for important shifts to occur through the course of time. Woody sprouting species are particularly under-represented in studies investigating intra-specific variations in levels of clonality from one locality to the next and through time. In this study we sought to determine the incidence and frequency of replicate genotypes in natural Populus nigra L. (Salicaceae) stands of different ages. Ten stands of this woody riparian sprouting species were selected in each of three distinct age groups (young, middle-aged and old) along a 30 km stretch of the River Garonne (south-west France). Leaf samples were collected from 15 neighbouring trees in each stand (450 samples in total) and replicate genotypes were identified using five SSR markers. Replicate genotypes were identified in two-thirds of all stands sampled (i.e. 50 of young stands, 100 of middle-aged stands and 50% of old stands). Young stands had significantly fewer replicated genotypes than middle-aged or old stands, while middle-aged stands had the greatest number of replicated genotypes. Replicate genotypes were most often found to occur as nearest neighbours and formed relatively small, discrete units (i.e. 2–4 trees growing in close proximity to one another). This suggests that asexual regeneration frequently occurs through flood-training in this species, although asexual regeneration from translocated fragments also evidently occurs as evidenced by 11 cases of replicate genotypes occurring in widely separated stands (up to 19 km apart). The results of this study highlight the need for a hierarchical sampling strategy in space and across age groups for an accureate understanding of the genotypic structure of woody sprouting species populations. Conservation and management of effective population sizes will benefit from better insight into not only spatial, but also temporal variations in levels of genotypic diversity.Co-ordinating editor: J. Tuomi     

Insights into the composition and assembly of the membrane arm of plant complex I through analysis of subcomplexes in Arabidopsis mutant lines

NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) is the largest complex of the mitochondrial respiratory chain. In eukaryotes, it is composed of more than 40 subunits that are encoded by both the nuclear and mitochondrial genomes. Plant Complex I differs from the enzyme described in other eukaryotes, most notably due to the large number of plant-specific subunits in the membrane arm of the complex. The elucidation of the assembly pathway of Complex I has been a long-standing research aim in cellular biochemistry. We report the study of Arabidopsis mutants in Complex I subunits using a combination of Blue-Native PAGE and immunodetection to identify stable subcomplexes containing Complex I components, along with mass spectrometry analysis of Complex I components in membrane fractions and two-dimensional diagonal Tricine SDS-PAGE to study the composition of the largest subcomplex. Four subcomplexes of the membrane arm of Complex I with apparent molecular masses of 200, 400, 450, and 650 kDa were observed. We propose a working model for the assembly of the membrane arm of Complex I in plants and assign putative roles during the assembly process for two of the subunits studied.     

A STUDY OF GLUCO-CORTICOSTEROID-INDUCED PYKNOSIS IN THE THYMUS AND LYMPH NODE OF THE ADRENALECTOMIZED RAT

Pyknotic nuclei, observed in the thymus of steroid-treated rats, are dense, homogeneous, intensely basophilic and Feulgen positive. Under the electron microscope, the image is that of a complete segregation of the chromatin from the nuclear sap producing a margin or crescent of condensed chromatin. Approximately 30% of all small thymocytes appeared to undergo this type of degeneration within 3–4 hr after administration of the synthetic corticosteroid, dexamethasone. At this time, pyknotic thymocytes were observed in clusters, probably as a result of the activity of dense reticular cells and macrophages. Topographical and experimental data suggest the existence of a select population of steroid-sensitive thymic cells. Furthermore, on the basis of thymidine-³H incorporation studies, it appears that the steroid-sensitive population of thymocytes does not correspond to "aged" cells. In addition, many plasma cells became pyknotic after the same steroid treatment, indicating an unexpected similarity between their nuclei and those of lymphocytes. Finally, steroid failed to induce pyknosis of thymocytes in a variety of in vitro experiments, suggesting that the in vivo effect of steroid is of an indirect nature. The results are discussed in terms of (a) the nature of the nuclear changes characterizing pyknosis, (b) the hypothetical mechanism whereby steroids trigger such changes, and (c) the population of cells susceptible to steroid-induced pyknosis.     

Superinfection prevents recombination of the alphaherpesvirus bovine herpesvirus 1

Homologous recombination between strains of the same alphaherpesvirus species occurs frequently both in vitro and in vivo. This process has been described between strains of herpes simplex virus type 1, herpes simplex virus type 2, pseudorabies virus, feline herpesvirus 1, varicella-zoster virus, and bovine herpesvirus 1 (BoHV-1). In vivo, the rise of recombinant viruses can be modulated by different factors, such as the dose of the inoculated viruses, the distance between inoculation sites, the time interval between inoculation of the first and the second virus, and the genes in which the mutations are located. The effect of the time interval between infections with two distinguishable BoHV-1 on recombination was studied in three ways: (i) recombination at the level of progeny viruses, (ii) interference induced by the first virus infection on β-galactosidase gene expression of a superinfecting virus, and (iii) recombination at the level of concatemeric DNA. A time interval of 2 to 8 h between two successive infections allows the establishment of a barrier, which reduces or prevents any successful superinfection needed to generate recombinant viruses. The dramatic effect of the time interval on the rise of recombinant viruses is particularly important for the risk assessment of recombination between glycoprotein E-negative marker vaccine and field strains that could threaten BoHV-1 control and eradication programs.