Distinct Roles of Non-Canonical Poly(A) Polymerases in RNA Metabolism

Trf4p and Trf5p are non-canonical poly(A) polymerases and are part of the heteromeric protein complexes TRAMP4 and TRAMP5 that promote the degradation of aberrant and short-lived RNA substrates by interacting with the nuclear exosome. To assess the level of functional redundancy between the paralogous Trf4 and Trf5 proteins and to investigate the role of the Trf4-dependent polyadenylation in vivo, we used DNA microarrays to compare gene expression of the wild-type yeast strain of S. cerevisiae with either that of trf4Δ or trf5Δ mutant strains or the trf4Δ mutant expressing the polyadenylation-defective Trf4(DADA) protein. We found little overlap between the sets of transcripts with altered expression in the trf4Δ or the trf5Δ mutants, suggesting that Trf4p and Trf5p target distinct groups of RNAs for degradation. Surprisingly, most RNAs the expression of which was altered by the trf4 deletion were restored to wild-type levels by overexpression of TRF4(DADA), showing that the polyadenylation activity of Trf4p is dispensable in vivo. Apart from previously reported Trf4p and Trf5p target RNAs, this analysis along with in vivo cross-linking and RNA immunopurification-chip experiments revealed that both the TRAMP4 and the TRAMP5 complexes stimulate the degradation of spliced-out introns via a mechanism that is independent of the polyadenylation activity of Trf4p. In addition, we show that disruption of trf4 causes severe shortening of telomeres suggesting that TRF4 functions in the maintenance of telomere length. Finally, our study demonstrates that TRF4, the exosome, and TRF5 participate in antisense RNA–mediated regulation of genes involved in phosphate metabolism. In conclusion, our results suggest that paralogous TRAMP complexes have distinct RNA selectivities with functional implications in RNA surveillance as well as other RNA–related processes. This indicates widespread and integrative functions of TRAMP complexes for the coordination of different gene expression regulatory processes

Systematic Identification of Combinatorial Drivers and Targets in Cancer Cell Lines

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance

Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

The traditional Chinese medicine component dehydrocostuslactone (DHC) isolated from Saussurea costus (Falc.) Lipschitz, has been shown to have anti-cancer activity. Angiogenesis is an essential process in the growth and progression of cancer. In this study, we demonstrated, for the first time, the anti-angiogenic mechanism of action of DHC to be via the induction of cell cycle progression at the G0/G1 phase due to abrogation of the Akt/glycogen synthase kinase-3β (GSK-3β)/cyclin D1 and mTOR signaling pathway. First, we demonstrated that DHC has an anti-angiogenic effect in the matrigel-plug nude mice model and an inhibitory effect on human umbilical vein endothelial cell (HUVEC) proliferation and capillary-like tube formation in vitro. DHC caused G0/G1 cell cycle arrest, which was associated with the down-regulation of cyclin D1 expression, leading to the suppression of retinoblastoma protein phosphorylation and subsequent inhibition of cyclin A and cdk2 expression. With respect to the molecular mechanisms underlying the DHC-induced cyclin D1 down-regulation, this study demonstrated that DHC significantly inhibits Akt expression, resulting in the suppression of GSK-3β phosphorylation and mTOR expression. These effects are capable of regulating cyclin D1 degradation, but they were significantly reversed by constitutively active myristoylated (myr)-Akt. Furthermore, the abrogation of tube formation induced by DHC was also reversed by overexpression of Akt. And the co-treatment with LiCl and DHC significantly reversed the growth inhibition induced by DHC. Taken together, our study has identified Akt/GSK-3β and mTOR as important targets of DHC and has thus highlighted its potential application in angiogenesis-related diseases, such as cancer

An analysis of single amino acid repeats as use case for application specific background models

Background Sequence analysis aims to identify biologically relevant signals against a backdrop of functionally meaningless variation. Increasingly, it is recognized that the quality of the background model directly affects the performance of analyses. State-of-the-art approaches rely on classical sequence models that are adapted to the studied dataset. Although performing well in the analysis of globular protein domains, these models break down in regions of stronger compositional bias or low complexity. While these regions are typically filtered, there is increasing anecdotal evidence of functional roles. This motivates an exploration of more complex sequence models and application-specific approaches for the investigation of biased regions. Results Traditional Markov-chains and application-specific regression models are compared using the example of predicting runs of single amino acids, a particularly simple class of biased regions. Cross-fold validation experiments reveal that the alternative regression models capture the multi-variate trends well, despite their low dimensionality and in contrast even to higher-order Markov-predictors. We show how the significance of unusual observations can be computed for such empirical models. The power of a dedicated model in the detection of biologically interesting signals is then demonstrated in an analysis identifying the unexpected enrichment of contiguous leucine-repeats in signal-peptides. Considering different reference sets, we show how the question examined actually defines what constitutes the 'background'. Results can thus be highly sensitive to the choice of appropriate model training sets. Conversely, the choice of reference data determines the questions that can be investigated in an analysis. Conclusions Using a specific case of studying biased regions as an example, we have demonstrated that the construction of application-specific background models is both necessary and feasible in a challenging sequence analysis situation

GABA Coordinates with Insulin in Regulating Secretory Function in Pancreatic INS-1 β-Cells

Pancreatic islet β-cells produce large amounts of γ-aminobutyric acid (GABA), which is co-released with insulin. GABA inhibits glucagon secretion by hyperpolarizing α-cells via type-A GABA receptors (GABAARs). We and others recently reported that islet β-cells also express GABAARs and that activation of GABAARs increases insulin release. Here we investigate the effects of insulin on the GABA-GABAAR system in the pancreatic INS-1 cells using perforated-patch recording. The results showed that GABA produces a rapid inward current and depolarizes INS-1 cells. However, pre-treatment of the cell with regular insulin (1 µM) suppressed the GABA-induced current (IGABA) by 43%. Zinc-free insulin also suppressed IGABA to the same extent of inhibition by regular insulin. The inhibition of IGABA occurs within 30 seconds after application of insulin. The insulin-induced inhibition of IGABA persisted in the presence of PI3-kinase inhibitor, but was abolished upon inhibition of ERK, indicating that insulin suppresses GABAARs through a mechanism that involves ERK activation. Radioimmunoassay revealed that the secretion of C-peptide was enhanced by GABA, which was blocked by pre-incubating the cells with picrotoxin (50 µM, p<0.01) and insulin (1 µM, p<0.01), respectively. Together, these data suggest that autocrine GABA, via activation of GABAARs, depolarizes the pancreatic β-cells and enhances insulin secretion. On the other hand, insulin down-regulates GABA-GABAAR signaling presenting a feedback mechanism for fine-tuning β-cell secretion

Clinical and Non-Clinical Aspects of Distal Radioulnar Joint Instability

Untreated distal radioulnar joint (DRUJ) injuries can give rise to long lasting complaints. Although common, diagnosis and treatment of DRUJ injuries remains a challenge. The articulating anatomy of the distal radius and ulna, among others, enables an extensive range of forearm pronosupination movements. Stabilization of this joint is provided by both intrinsic and extrinsic stabilizers and the joint capsule. These structures transmit the load and prevent the DRUJ from luxation during movement. Several clinical tests have been suggested to determine static or dynamic DRUJ stability, but their predictive value is unclear. Radiologic evaluation of DRUJ instability begins with conventional radiographs in anterioposterior and true lateral view. If not conclusive, CT-scan seems to be the best additional modality to evaluate the osseous structures. MRI has proven to be more sensitive and specific for TFCC tears, potentially causing DRUJ instability. DRUJ instability may remain asymptomatic. Symptomatic DRUJ injuries treatment can be conservative or operative. Operative treatment should consist of restoration of osseous and ligamenteous anatomy. If not successful, salvage procedures can be performed to regain stability

Active Suppression of Early Immune Response in Tobacco by the Human Pathogen Salmonella Typhimurium

The persistence of enteric pathogens on plants has been studied extensively, mainly due to the potential hazard of human pathogens such as Salmonella enterica being able to invade and survive in/on plants. Factors involved in the interactions between enteric bacteria and plants have been identified and consequently it was hypothesized that plants may be vectors or alternative hosts for enteric pathogens. To survive, endophytic bacteria have to escape the plant immune systems, which function at different levels through the plant-bacteria interactions. To understand how S. enterica survives endophyticaly we conducted a detailed analysis on its ability to elicit or evade the plant immune response. The models of this study were Nicotiana tabacum plants and cells suspension exposed to S. enterica serovar Typhimurium. The plant immune response was analyzed by looking at tissue damage and by testing oxidative burst and pH changes. It was found that S. Typhimurium did not promote disease symptoms in the contaminated plants. Live S. Typhimurium did not trigger the production of an oxidative burst and pH changes by the plant cells, while heat killed or chloramphenicol treated S. Typhimurium and purified LPS of Salmonella were significant elicitors, indicating that S. Typhimurium actively suppress the plant response. By looking at the plant response to mutants defective in virulence factors we showed that the suppression depends on secreted factors. Deletion of invA reduced the ability of S. Typhimurium to suppress oxidative burst and pH changes, indicating that a functional SPI1 TTSS is required for the suppression. This study demonstrates that plant colonization by S. Typhimurium is indeed an active process. S. Typhimurium utilizes adaptive strategies of altering innate plant perception systems to improve its fitness in the plant habitat. All together these results suggest a complex mechanism for perception of S. Typhimurium by plants

Assessment of acute myocardial infarction: current status and recommendations from the North American society for cardiovascular imaging and the European society of cardiac radiology

There are a number of imaging tests that are used in the setting of acute myocardial infarction and acute coronary syndrome. Each has their strengths and limitations. Experts from the European Society of Cardiac Radiology and the North American Society for Cardiovascular Imaging together with other prominent imagers reviewed the literature. It is clear that there is a definite role for imaging in these patients. While comparative accuracy, convenience and cost have largely guided test decisions in the past, the introduction of newer tests is being held to a higher standard which compares patient outcomes. Multicenter randomized comparative effectiveness trials with outcome measures are required

Transcriptional control in the prereplicative phase of T4 development

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two α subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of σ70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of σ70, which then allows the T4 activator MotA to also interact with σ70. In addition, AsiA restructuring of σ70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity