Exploration of the visual system: Part 1: Dissection of the mouse eye for RNA, protein, and histological analyses

Due to the power of genetics, the mouse has become a widely used animal model in vision research. However, its eyeball has an axial length of only about 2 mm. The present protocol describes how to easily dissect the small rodent eye post mortem. This allows collecting different tissues of the eye, i.e., cornea, lens, iris, retina, optic nerve, retinal pigment epithelium (RPE), and sclera. We further describe in detail how to process these eye samples in order to obtain high‐quality RNA for RNA expression profiling studies. Depending on the eye tissue to be analyzed, we present appropriate lysis buffers to prepare total protein lysates for immunoblot and immuno‐precipitation analyses. Fixation, inclusion, embedding, and cryosectioning of the globe for routine histological analyses (HE staining, DAPI staining, immunohistochemistry, in situ hybridization) is further presented. These basic protocols should allow novice investigators to obtain eye tissue samples rapidly for their experiments.

Actinobaculum schaalii: clinical observation of 20 cases.

Actinobaculum schaalii is a new species that has so far been isolated from human blood, urine and pus. Its importance has probably been underestimated and other Actinobaculum spp. may also have been underdiagnosed. This retrospective study comprises all known cases of A. schaalii infections identified since 2004 in the canton of Neuchâtel (170,000 inhabitants), Switzerland. Strains were cultivated and isolated in the bacteriology laboratory using its routine procedure. Identification included a Rapid ID 32 A strip (bioMérieux) and 16S rRNA gene sequencing. Twenty-one positive samples were found in 19 patients (11 male, 8 female) of all ages (range 16-91 years): 10 from urine (50%), six from blood (30%), one from both blood and urine (5%), and three from pus (15%). Thirteen out of 17 (76%) cases with either blood or urine specimens had underlying genitourinary tract pathologies. When urine cultures were positive for A. schaalii, leucocytes were found in all samples (10/10, 100%) but all nitrite tests were negative (10/10, 100%). The onset of appropriate treatment was delayed due to the diminished sensitivity of A. schaalii to the antibiotics commonly used for UTIs (i.e. ciprofloxacin and trimethoprim/sulfamethoxazole) and to the delay in microbiological diagnosis. A. schaalii should specifically be searched in all cases of leukocyturia with a negative nitrite test but with Gram-positive rods in the Gram stain, in patients with underlying genitourinary tract pathology, instead of dismissing these findings as clinically irrelevant colonization by coryneform bacteria. This infection may be much more common than previously thought.

Low current and nadir CD4+ T-cell counts are associated with higher hepatitis C virus RNA levels in the Swiss HIV cohort study.

BACKGROUND: The aim of this study was to evaluate the effect of CD4+ T-cell counts and other characteristics of HIV-infected individuals on hepatitis C virus (HCV) RNA levels. METHODS: All HIV-HCV-coinfected Swiss HIV Cohort Study participants with available HCV RNA levels and concurrent CD4+ T-cell counts before starting HCV therapy were included. Potential predictors of HCV RNA levels were assessed by multivariate censored linear regression models that adjust for censored values. RESULTS: The study included 1,031 individuals. Low current and nadir CD4+ T-cell counts were significantly associated with higher HCV RNA levels (P = 0.004 and 0.001, respectively). In individuals with current CD4+ T-cell counts < 200/microl, median HCV RNA levels (6.22 log10 IU/ml) were +0.14 and +0.24 log10 IU/ml higher than those with CD4+ T-cell counts of 200-500/microl and > 500/microl. Based on nadir CD4+ T-cell counts, median HCV RNA levels (6.12 log10 IU/ml) in individuals with < 200/microl CD4+ T-cells were +0.06 and +0.44 log10 IU/ml higher than those with nadir T-cell counts of 200-500/microl and > 500/microl. Median HCV RNA levels were also significantly associated with HCV genotype: lower values were associated with genotype 4 and higher values with genotype 2, as compared with genotype 1. Additional significant predictors of lower HCV RNA levels were female gender and HIV transmission through male homosexual contacts. In multivariate analyses, only CD4+ T-cell counts and HCV genotype remained significant predictors of HCV RNA levels. Conclusions: Higher HCV RNA levels were associated with CD4+ T-cell depletion. This finding is in line with the crucial role of CD4+ T-cells in the control of HCV infection.

Maf1, a new player in the regulation of human RNA polymerase III transcription.

BACKGROUND: Human RNA polymerase III (pol III) transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and is broadly conserved in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We show that human endogenous Maf1 can be co-immunoprecipitated with pol III and associates in vitro with two pol III subunits, the largest subunit RPC1 and the alpha-like subunit RPAC2. Maf1 represses pol III transcription in vitro and in vivo and is required for maximal pol III repression after exposure to MMS or rapamycin, treatments that both lead to Maf1 dephosphorylation. CONCLUSIONS/SIGNIFICANCE: These data suggest that Maf1 is a major regulator of pol III transcription in human cells.

Using digital RNA counting and flow cytometry to compare mRNA with protein expression in acute leukemias.

BACKGROUND: The diagnosis of malignant hematologic diseases has become increasingly complex during the last decade. It is based on the interpretation of results from different laboratory analyses, which range from microscopy to gene expression profiling. Recently, a method for the analysis of RNA phenotypes has been developed, the nCounter technology (Nanostring® Technologies), which allows for simultaneous quantification of hundreds of RNA molecules in biological samples. We evaluated this technique in a Swiss multi-center study on eighty-six samples from acute leukemia patients. METHODS: mRNA and protein profiles were established for normal peripheral blood and bone marrow samples. Signal intensities of the various tested antigens with surface expression were similar to those found in previously performed Affymetrix microarray analyses. Acute leukemia samples were analyzed for a set of twenty-two validated antigens and the Pearson Correlation Coefficient for nCounter and flow cytometry results was calculated. RESULTS: Highly significant values between 0.40 and 0.97 were found for the twenty-two antigens tested. A second correlation analysis performed on a per sample basis resulted in concordant results between flow cytometry and nCounter in 44-100% of the antigens tested (mean = 76%), depending on the number of blasts present in a sample, the homogeneity of the blast population, and the type of leukemia (AML or ALL). CONCLUSIONS: The nCounter technology allows for fast and easy depiction of a mRNA profile from hematologic samples. This technology has the potential to become a valuable tool for the diagnosis of acute leukemias, in addition to multi-color flow cytometry.

Role of mutations in the phosphoprotein on RNA polymerase activity of canine distemper virus following cell culture adaptation

Abstract: The canine distemper virus A75/17 wild-type strain, which is unable to replicate in cell lines, was adapted to growth in Vero cells. Sequence comparison between the A75/17 and the Vero cell-adapted A75/17-V virus revealed 7 amino acid differences between the 2 viruses. Three of these were located in the matrix protein, three in the phosphoprotein also changing the V protein but not the C protein and one in the large protein. The phosphoprotein and the large protein constituted the viral RNA polymerase whose activity was studied by transfection experiments using a reverse genetic system with a plasmid encoding a minireplicon and expression plasmids encoding the nucleocapsid protein and the viral RNA polymerase subunits. Surprinsingly, the enzyme of A75/17 CDV was significantly more active in cell lines compared to the polymerase of A75/17-V CDV. The decrease in overall enzyme activity was found to be due to both decreased replication and transcription activity. This polymerase attenuation was confirmed in CHO cells infection stably expressing the dog SLAM receptor mainly found in dog's lymphoid organs and allowing both virus strains to enter these cells at the same efficiency. A75/17-V CDV replicated more slowly in CHODogSLAM cells than A75/17 CDV and syncytium formation was significantly decreased compared to A75/17 infected CHODogSLAM cells.. Cell culture adaptation lead to an attenuated virus strain both in vitro and in vivo with decreased polymerase activity and syncytium forming capability showing an important role of the polymerase in determining the phenoytpe of the virus. In addition, this reduced phenotype of A75/17-V CDV was shown to be due to the P mutations in the P protein only, showing an important function of the polycistronic P gene in the adaptation process. The role of the matrix protein was found not to have any effect on polymerase activity, however its participation in the adaptation process still needs to be elucidated. The accessory proteins V and C were shown to act on polymerase activity, but their functions in virus pathogenicity and in inhibiting the interferon system have not been studied in this thesis. The V proteins have an activating effect on the polymerase of both the A75/17 and the A75/17-V CDV strains. Although the C protein amino acid sequence was not changed during adaptation of wild-type canine distemper virus in Vero cells, the C protein was demonstrated to have opposite effects on polymerase activity of both virus strains suggesting a different interaction of the C protein with the proteins forming the polymerase complex, which could modulate polymeras activity. These effects were demonstrated by transfection experiments and studying recombinant viruses not expressing the C protein. Thus, the abrogation of the C protein decrease the activity of the wild-type polymerase. In contrast, the polymerase activity of the Vero cell- adapted virus is enhanced in the absence of the C protein and this has also been demonstrated with a recombinant virus, which grew faster in the first 48 hours of infection. Future studies will focus on the generation of recombinant wild-type viruses, which should be very helpful in understanding the molecular mechanisms underlying the adaptation process and the loss of pathogenicity.

Loss of the RNA polymerase III repressor MAF1 confers obesity resistance.

MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

The striatal long noncoding RNA Abhd11os is neuroprotective against an N-terminal fragment of mutant huntingtin in vivo.

A large number of gene products that are enriched in the striatum have ill-defined functions, although they may have key roles in age-dependent neurodegenerative diseases affecting the striatum, especially Huntington disease (HD). In the present study, we focused on Abhd11os, (called ABHD11-AS1 in human) which is a putative long noncoding RNA (lncRNA) whose expression is enriched in the mouse striatum. We confirm that despite the presence of 2 small open reading frames (ORFs) in its sequence, Abhd11os is not translated into a detectable peptide in living cells. We demonstrate that Abhd11os levels are markedly reduced in different mouse models of HD. We performed in vivo experiments in mice using lentiviral vectors encoding either Abhd11os or a small hairpin RNA targeting Abhd11os. Results show that Abhd11os overexpression produces neuroprotection against an N-terminal fragment of mutant huntingtin, whereas Abhd11os knockdown is protoxic. These novel results indicate that the loss lncRNA Abhd11os likely contribute to striatal vulnerability in HD. Our study emphasizes that lncRNA may play crucial roles in neurodegenerative diseases.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

Le coeur des ARN non codants - Un long chemin à découvrir [In the heart of noncoding RNA: a long way to go].

The identification and characterization of long noncoding RNA in a variety of tissues represent major achievements that contribute to our understanding of the molecular mechanisms controlling gene expression. In particular, long noncoding RNA play crucial roles in the epigenetic regulation of the adaptive response to environmental cues via their capacity to target chromatin modifiers to specific locus. In addition, these transcripts have been implicated in controlling splicing, translation and degradation of messenger RNA. Long noncoding RNA have also been shown to act as decoy molecules for microRNA. In the heart, a few long noncoding RNA have been demonstrated to regulate cardiac commitment and differentiation during development. Furthermore, recent findings suggest their involvement as regulators of the pathophysiological response to injury in the adult heart. Their high cellular specificity makes them attractive target molecules for innovative therapies and ideal biomarkers.

Assessing Recent Selection and Functionality at Long Noncoding RNA Loci in the Mouse Genome.

Long noncoding RNAs (lncRNAs) are one of the most intensively studied groups of noncoding elements. Debate continues over what proportion of lncRNAs are functional or merely represent transcriptional noise. Although characterization of individual lncRNAs has identified approximately 200 functional loci across the Eukarya, general surveys have found only modest or no evidence of long-term evolutionary conservation. Although this lack of conservation suggests that most lncRNAs are nonfunctional, the possibility remains that some represent recent evolutionary innovations. We examine recent selection pressures acting on lncRNAs in mouse populations. We compare patterns of within-species nucleotide variation at approximately 10,000 lncRNA loci in a cohort of the wild house mouse, Mus musculus castaneus, with between-species nucleotide divergence from the rat (Rattus norvegicus). Loci under selective constraint are expected to show reduced nucleotide diversity and divergence. We find limited evidence of sequence conservation compared with putatively neutrally evolving ancestral repeats (ARs). Comparisons of sequence diversity and divergence between ARs, protein-coding (PC) exons and lncRNAs, and the associated flanking regions, show weak, but significantly lower levels of sequence diversity and divergence at lncRNAs compared with ARs. lncRNAs conserved deep in the vertebrate phylogeny show lower within-species sequence diversity than lncRNAs in general. A set of 74 functionally characterized lncRNAs show levels of diversity and divergence comparable to PC exons, suggesting that these lncRNAs are under substantial selective constraints. Our results suggest that, in mouse populations, most lncRNA loci evolve at rates similar to ARs, whereas older lncRNAs tend to show signals of selection similar to PC genes.

Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.

RNA enrichment method for quantitative transcriptional analysis of pathogens in vivo applied to the fungus Candida albicans.

UNLABELLED: In vivo transcriptional analyses of microbial pathogens are often hampered by low proportions of pathogen biomass in host organs, hindering the coverage of full pathogen transcriptome. We aimed to address the transcriptome profiles of Candida albicans, the most prevalent fungal pathogen in systemically infected immunocompromised patients, during systemic infection in different hosts. We developed a strategy for high-resolution quantitative analysis of the C. albicans transcriptome directly from early and late stages of systemic infection in two different host models, mouse and the insect Galleria mellonella. Our results show that transcriptome sequencing (RNA-seq) libraries were enriched for fungal transcripts up to 1,600-fold using biotinylated bait probes to capture C. albicans sequences. This enrichment biased the read counts of only ~3% of the genes, which can be identified and removed based on a priori criteria. This allowed an unprecedented resolution of C. albicans transcriptome in vivo, with detection of over 86% of its genes. The transcriptional response of the fungus was surprisingly similar during infection of the two hosts and at the two time points, although some host- and time point-specific genes could be identified. Genes that were highly induced during infection were involved, for instance, in stress response, adhesion, iron acquisition, and biofilm formation. Of the in vivo-regulated genes, 10% are still of unknown function, and their future study will be of great interest. The fungal RNA enrichment procedure used here will help a better characterization of the C. albicans response in infected hosts and may be applied to other microbial pathogens. IMPORTANCE: Understanding the mechanisms utilized by pathogens to infect and cause disease in their hosts is crucial for rational drug development. Transcriptomic studies may help investigations of these mechanisms by determining which genes are expressed specifically during infection. This task has been difficult so far, since the proportion of microbial biomass in infected tissues is often extremely low, thus limiting the depth of sequencing and comprehensive transcriptome analysis. Here, we adapted a technology to capture and enrich C. albicans RNA, which was next used for deep RNA sequencing directly from infected tissues from two different host organisms. The high-resolution transcriptome revealed a large number of genes that were so far unknown to participate in infection, which will likely constitute a focus of study in the future. More importantly, this method may be adapted to perform transcript profiling of any other microbes during host infection or colonization.

Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2.

TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.

Prevalence and Distribution of Leishmania RNA Virus 1 in Leishmania Parasites from French Guiana.

In South America, the presence of the Leishmania RNA virus type 1 (LRV1) was described in Leishmania guyanensis and Leishmania braziliensis strains. The aim of this study was to determine the prevalence distribution of LRV1 in Leishmania isolates in French Guiana given that, in this French overseas department, most Leishmania infections are due to these parasite species. The presence of the virus was observed in 74% of Leishmania spp. isolates, with a highest presence in the internal areas of the country.