Transcriptional Profiling Uncovers a Network of Cholesterol-Responsive Atherosclerosis Target Genes

Despite the well-documented effects of plasma lipid lowering regimes halting atherosclerosis lesion development and reducing morbidity and mortality of coronary artery disease and stroke, the transcriptional response in the atherosclerotic lesion mediating these beneficial effects has not yet been carefully investigated. We performed transcriptional profiling at 10-week intervals in atherosclerosis-prone mice with human-like hypercholesterolemia and a genetic switch to lower plasma lipoproteins (Ldlr−/−Apo100/100 Mttpflox/flox Mx1-Cre). Atherosclerotic lesions progressed slowly at first, then expanded rapidly, and plateaued after advanced lesions formed. Analysis of lesion expression profiles indicated that accumulation of lipid-poor macrophages reached a point that led to the rapid expansion phase with accelerated foam-cell formation and inflammation, an interpretation supported by lesion histology. Genetic lowering of plasma cholesterol (e.g., lipoproteins) at this point all together prevented the formation of advanced plaques and parallel transcriptional profiling of the atherosclerotic arterial wall identified 37 cholesterol-responsive genes mediating this effect. Validation by siRNA-inhibition in macrophages incubated with acetylated-LDL revealed a network of eight cholesterol-responsive atherosclerosis genes regulating cholesterol-ester accumulation. Taken together, we have identified a network of atherosclerosis genes that in response to plasma cholesterol-lowering prevents the formation of advanced plaques. This network should be of interest for the development of novel atherosclerosis therapies

Alternative mRNA Editing in Trypanosomes Is Extensive and May Contribute to Mitochondrial Protein Diversity

The editing of trypanosome mitochondrial mRNAs produces transcripts necessary for mitochondrial functions including electron transport and oxidative phosphorylation. Precursor-mRNAs are often extensively edited by specific uridine insertion or deletion that is directed by small guide RNAs (gRNAs). Recently, it has been shown that cytochrome c oxidase subunit III (COXIII) mRNAs can be alternatively edited to encode a novel mitochondrial membrane protein composed of a unique hydrophilic N-terminal sequence of unknown function and the C-terminal hydrophobic segment of COXIII. To extend the analysis of alternative editing in Trypanosoma brucei we have constructed libraries with over 1100 full-length mitochondrial cDNAs and the sequences of over 1200 gRNA genes. Using this data, we show that alternative editing of COXIII, ATPase subunit 6 (A6), and NADH dehydrogenase subunits 7, 8 and 9 (ND7, 8, 9) mRNAs can produce novel open reading frames (ORFs). Several gRNAs potentially responsible for the alternative editing of these mRNAs were also identified. These findings show that alternative editing of mitochondrial mRNAs is common in T. brucei and expands the diversity of mitochondrial proteins in these organisms

Fiber Mediated Receptor Masking in Non-Infected Bystander Cells Restricts Adenovirus Cell Killing Effect but Promotes Adenovirus Host Co-Existence

The basic concept of conditionally replicating adenoviruses (CRAD) as oncolytic agents is that progenies generated from each round of infection will disperse, infect and kill new cancer cells. However, CRAD has only inhibited, but not eradicated tumor growth in xenograft tumor therapy, and CRAD therapy has had only marginal clinical benefit to cancer patients. Here, we found that CRAD propagation and cancer cell survival co-existed for long periods of time when infection was initiated at low multiplicity of infection (MOI), and cancer cell killing was inefficient and slow compared to the assumed cell killing effect upon infection at high MOI. Excessive production of fiber molecules from initial CRAD infection of only 1 to 2% cancer cells and their release prior to the viral particle itself caused a tropism-specific receptor masking in both infected and non-infected bystander cells. Consequently, the non-infected bystander cells were inefficiently bound and infected by CRAD progenies. Further, fiber overproduction with concomitant restriction of adenovirus spread was observed in xenograft cancer therapy models. Besides the CAR-binding Ad4, Ad5, and Ad37, infection with CD46-binding Ad35 and Ad11 also caused receptor masking. Fiber overproduction and its resulting receptor masking thus play a key role in limiting CRAD functionality, but potentially promote adenovirus and host cell co-existence. These findings also give important clues for understanding mechanisms underlying the natural infection course of various adenoviruses

The Mitochondrial Genome and Transcriptome of the Basal Dinoflagellate Hematodinium sp.: Character Evolution within the Highly Derived Mitochondrial Genomes of Dinoflagellates

The sister phyla dinoflagellates and apicomplexans inherited a drastically reduced mitochondrial genome (mitochondrial DNA, mtDNA) containing only three protein-coding (cob, cox1, and cox3) genes and two ribosomal RNA (rRNA) genes. In apicomplexans, single copies of these genes are encoded on the smallest known mtDNA chromosome (6 kb). In dinoflagellates, however, the genome has undergone further substantial modifications, including massive genome amplification and recombination resulting in multiple copies of each gene and gene fragments linked in numerous combinations. Furthermore, protein-encoding genes have lost standard stop codons, trans-splicing of messenger RNAs (mRNAs) is required to generate complete cox3 transcripts, and extensive RNA editing recodes most genes. From taxa investigated to date, it is unclear when many of these unusual dinoflagellate mtDNA characters evolved. To address this question, we investigated the mitochondrial genome and transcriptome character states of the deep branching dinoflagellate Hematodinium sp. Genomic data show that like later-branching dinoflagellates Hematodinium sp. also contains an inflated, heavily recombined genome of multicopy genes and gene fragments. Although stop codons are also lacking for cox1 and cob, cox3 still encodes a conventional stop codon. Extensive editing of mRNAs also occurs in Hematodinium sp. The mtDNA of basal dinoflagellate Hematodinium sp. indicates that much of the mtDNA modification in dinoflagellates occurred early in this lineage, including genome amplification and recombination, and decreased use of standard stop codons. Trans-splicing, on the other hand, occurred after Hematodinium sp. diverged. Only RNA editing presents a nonlinear pattern of evolution in dinoflagellates as this process occurs in Hematodinium sp. but is absent in some later-branching taxa indicating that this process was either lost in some lineages or developed more than once during the evolution of the highly unusual dinoflagellate mtDNA

Complete Mitochondrial Genome Sequence of Three Tetrahymena Species Reveals Mutation Hot Spots and Accelerated Nonsynonymous Substitutions in Ymf Genes

The ciliate Tetrahymena, a model organism, contains divergent mitochondrial (Mt) genome with unusual properties, where half of its 44 genes still remain without a definitive function. These genes could be categorized into two major groups of KPC (known protein coding) and Ymf (genes without an identified function). To gain insights into the mechanisms underlying gene divergence and molecular evolution of Tetrahymena (T.) Mt genomes, we sequenced three Mt genomes of T.paravorax, T.pigmentosa, and T.malaccensis. These genomes were aligned and the analyses were carried out using several programs that calculate distance, nucleotide substitution (dn/ds), and their rate ratios (ω) on individual codon sites and via a sliding window approach. Comparative genomic analysis indicated a conserved putative transcription control sequence, a GC box, in a region where presumably transcription and replication initiate. We also found distinct features in Mt genome of T.paravorax despite similar genome organization among these ∼47 kb long linear genomes. Another significant finding was the presence of at least one or more highly variable regions in Ymf genes where majority of substitutions were concentrated. These regions were mutation hotspots where elevated distances and the dn/ds ratios were primarily due to an increase in the number of nonsynonymous substitutions, suggesting relaxed selective constraint. However, in a few Ymf genes, accelerated rates of nonsynonymous substitutions may be due to positive selection. Similarly, on protein level the majority of amino acid replacements occurred in these regions. Ymf genes comprise half of the genes in Tetrahymena Mt genomes, so understanding why they have not been assigned definitive functions is an important aspect of molecular evolution. Importantly, nucleotide substitution types and rates suggest possible reasons for not being able to find homologues for Ymf genes. Additionally, comparative genomic analysis of complete Mt genomes is essential in identifying biologically significant motifs such as control regions

Crystal Structure of the Heteromolecular Chaperone, AscE-AscG, from the Type III Secretion System in Aeromonas hydrophila

10.1371/journal.pone.0019208PLoS ONE64

A systematic search strategy identifies cubilin as independent prognostic marker for renal cell carcinoma

$\textbf{Background:}$ There is an unmet clinical need for better prognostic and diagnostic tools for renal cell carcinoma (RCC). $\textbf{Methods:}$ Human Protein Atlas data resources, including the transcriptomes and proteomes of normal and malignant human tissues, were searched for RCC-specific proteins and cubilin (CUBN) identified as a candidate. Patient tissue representing various cancer types was constructed into a tissue microarray ($n$ = 940) and immunohistochemistry used to investigate the specificity of CUBN expression in RCC as compared to other cancers. Two independent RCC cohorts ($n$ = 181; $n$ = 114) were analyzed to further establish the sensitivity of CUBN as RCC-specific marker and to explore if the fraction of RCCs lacking CUBN expression could predict differences in patient survival. $\textbf{Results:}$ CUBN was identified as highly RCC-specific protein with 58% of all primary RCCs staining positive for CUBN using immunohistochemistry. In venous tumor thrombi and metastatic lesions, the frequency of CUBN expression was increasingly lost. Clear cell RCC (ccRCC) patients with CUBN positive tumors had a significantly better prognosis compared to patients with CUBN negative tumors, independent of T-stage, Fuhrman grade and nodal status (HR 0.382, CI 0.203–0.719, $P$ = 0.003). $\textbf{Conclusions:}$ CUBN expression is highly specific to RCC and loss of the protein is significantly and independently associated with poor prognosis. CUBN expression in ccRCC provides a promising positive prognostic indicator for patients with ccRCC. The high specificity of CUBN expression in RCC also suggests a role as a new diagnostic marker in clinical cancer differential diagnostics to confirm or rule out RCC.This work was supported by the Swedish Cancer Society and the Knut and Alice Wallenberg Foundation. The work of DJH and GDS was funded by the Chief Scientist Office (grant number ETM37), Renal Cancer Research Fund and Kidney Cancer Scotland

Characterization and intracellular localization of putative Chlamydia pneumoniae effector proteins

We here describe four proteins of Chlamydia pneumoniae, which might play a role in host-pathogen interaction. The hypothetical bacterial proteins CPn0708 and CPn0712 were detected in Chlamydia pneumoniae-infected host cells by indirect immunofluorescence tests with polyclonal antisera raised against the respective proteins. While CPn0708 was localized within the inclusion body, CPn0712 was identified in the inclusion membrane and in the surrounding host cell cytosol. CPn0712 colocalizes with actin, indicating its possible interaction with components of the cytoskeleton. Investigations on CPn0809 and CPn1020, two Chlamydia pneumoniae proteins previously described to be secreted into the host cell cytosol, revealed colocalization with calnexin, a marker for the ER. Neither CPn0712, CPn0809 nor CPn1020 were able to inhibit host cell apoptosis. Furthermore, transient expression of CPn0712, CPn0809 and CPn1020 by the host cell itself had no effect on subsequent infection with Chlamydia pneumoniae. However, microarray analysis of CPn0712-expressing host cells revealed six host cell genes which were regulated as in host cells infected with Chlamydia pneumoniae, indicating the principal usefulness of heterologous expression to study the effect of Chlamydia pneumoniae proteins on host cell modulation