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.     

Spatial Scales of Pollen and Seed-Mediated Gene Flow in Tropical Rain Forest Trees

Gene flow via seed and pollen is a primary determinant of genetic and species diversity in plant communities at different spatial scales. This paper reviews studies of gene flow and population genetic structure in tropical rain forest trees and places them in ecological and biogeographic context. Although much pollination is among nearest neighbors, an increasing number of genetic studies report pollination ranging from 0.5–14 km for canopy tree species, resulting in extensive breeding areas in disturbed and undisturbed rain forest. Direct genetic measures of seed dispersal are still rare; however, studies of fine scale spatial genetic structure (SGS) indicate that the bulk of effective seed dispersal occurs at local scales, and we found no difference in SGS (Sp statistic) between temperate (N?=?24 species) and tropical forest trees (N?=?15). Our analysis did find significantly higher genetic differentiation in tropical trees (F _ST?=?0.177; N?=?42) than in temperate forest trees (F _ST?=?0.116; N?=?82). This may be due to the fact that tropical trees experience low but significant rates of self-fertilization and bi-parental inbreeding, whereas half of the temperate tree species in our survey are wind pollinated and are more strictly allogamous. Genetic drift may also be more pronounced in tropical trees due to the low population densities of most species.     

The Arabidopsis TONNEAU2 gene encodes a putative novel protein phosphatase 2A regulatory subunit essential for the control of the cortical cytoskeleton

In Arabidopsis ton2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B" regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants.     

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.     

A physical map of large segments of pig Chromosome 7q11–q14: comparative analysis with human Chromosome 6p21

The aim of this study was to establish a porcine physical map along the chromosome SSC7q by construction of BAC contigs between microsatellites Sw1409 and S0102. The SLA class II contig, located on SSC7q, was lengthened. Four major BAC contigs and 10 short contigs span a region equivalent to 800 cR measured by IMpRH7000 mapping. The BAC contigs were initiated by PCR screening with primers derived from human orthologous segments, extended by chromosome walking, and controlled and oriented by RH mapping with the two available panels, IMpRH7000Rad and IMNpRH12000Rad. The location of 43 genes was revealed by sequenced segments, either from BAC ends or PCR products from BAC clones. The 220 BAC end sequences (BES) were also used to analyze the different marks of evolution. Comparative mapping analysis between pigs and humans demonstrated that the gene organization on HSA6p21 and on SSC7p11 and q11-q14 segments was conserved during evolution, with the exception of long fragments of HSA6p12 which shuffled and spliced the SLA extended class II region. Additional punctual variations (unique gene insertion/deletion) were observed, even within conserved segments, revealing the evolutionary complexity of this region. In addition, 18 new polymorphic microsatellites have been selected in order to cover the entire SSC7p11-q14 region.     

Characterization of attenuated Renibacterium salmoninarum strains and their use as live vaccines

Two nutritionally mutant strains of Renibacterium salmoninarum (Rs) were isolated that grew on tryticase soy agar (Rs TSA1) or brain heart infusion agar (Rs BHI1). These 2 strains could be continuously cultured on these media, whereas typical R. salmoninarum would only grow on KDM-2 agar. We determined no other phenotypic difference that could be used to distinguish them from wild-type R. salmoninarum. Both strains were found to be avirulent when 5 x 10(6) bacteria were intraperitoneally (i.p.) injected into Atlantic salmon. Rs TSA1, Rs BHI1, and Rs MT-239 (a R. salmoninarum strain previously shown to be attenuated) were tested as live vaccines in 2 separate trials. The best protection was seen with Rs TSA1. Vaccinated Atlantic salmon had relative percent survival (RPS) of 50 at 74 d post-challenge in Trial 1 and 76 at 60 d post-challenge in Trial 2. In both trials, 100% of the control salmon died from bacterial kidney disease (BKD) (within 40 d for Trial 1 and 50 d for Trial 2) after i.p. challenge with 5 x 10(6) live cells of the virulent isolate Rs Margaree.     

The melanin-concentrating hormone (MCH) system modulates behaviors associated with psychiatric disorders

Deficits in sensorimotor gating measured by prepulse inhibition (PPI) of the startle have been known as characteristics of patients with schizophrenia and related neuropsychiatric disorders. PPI disruption is thought to rely on the activity of the mesocorticolimbic dopaminergic system and is inhibited by most antipsychotic drugs. These drugs however act also at the nigrostriatal dopaminergic pathway and exert adverse locomotor responses. Finding a way to inhibit the mesocorticolimbic- without affecting the nigrostriatal-dopaminergic pathway may thus be beneficial to antipsychotic therapies. The melanin-concentrating hormone (MCH) system has been shown to modulate dopamine-related responses. Its receptor (MCH1R) is expressed at high levels in the mesocorticolimbic and not in the nigrostriatal dopaminergic pathways. Interestingly a genomic linkage study revealed significant associations between schizophrenia and markers located in the MCH1R gene locus. We hypothesize that the MCH system can selectively modulate the behavior associated with the mesocorticolimbic dopamine pathway. Using mice, we found that central administration of MCH potentiates apomorphine-induced PPI deficits. Using congenic rat lines that differ in their responses to PPI, we found that the rats that are susceptible to apomorphine (APO-SUS rats) and exhibit PPI deficits display higher MCH mRNA expression in the lateral hypothalamic region and that blocking the MCH system reverses their PPI deficits. On the other hand, in mice and rats, activation or inactivation of the MCH system does not affect stereotyped behaviors, dopamine-related responses that depend on the activity of the nigrostriatal pathway. Furthermore MCH does not affect dizocilpine-induced PPI deficit, a glutamate related response. Thus, our data present the MCH system as a regulator of sensorimotor gating, and provide a new rationale to understand the etiologies of schizophrenia and related psychiatric disorders.     

Automated genome-wide visual profiling of cellular proteins involved in HIV infection

Recent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening. To overcome this, the authors devised a 15-dimensional phenotypic profile to define the viral infection block induced by CD4 silencing in HeLa cells. They demonstrate that this phenotypic profile excludes nonspecific, RNAi-based side effects and viral replication defects mediated by silencing of housekeeping genes. To achieve statistical robustness, the authors used automatically annotated RNAi arrays for seven independent genome-wide RNAi screens. This identified 56 host genes, which reliably reproduced CD4-like phenotypes upon HIV infection. The factors include 11 known HIV interactors and 45 factors previously not associated with HIV infection. As proof of concept, the authors confirmed that silencing of PAK1, Ku70, and RNAseH2A impaired HIV replication in Jurkat cells. In summary, multidimensional, visual profiling can identify genes required for HIV infection.     

Porphyromonas gingivalis participates in pathogenesis of human abdominal aortic aneurysm by neutrophil activation. Proof of concept in rats

Background

Abdominal Aortic Aneurysms (AAAs) represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia.

Methods and Findings

Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET) formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16), and in particular that of Porphyromonas gingivalis (Pg), the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed.

Conclusions

Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression.