Wild type N-ras displays anti-malignant properties, in part by downregulating decorin

Previously, we have shown that wild type N-ras (wt N-ras) harbors an anti-malignant effect against mutated Ras and in tumors without Ras mutations. To investigate the molecular bases of this anti-malignant activity, we have studied the potency of this anti-malignant effect in a model system against SV40 large T antigen (SV40T). We show that wild-type N-ras (wt N-ras) counteracts the effects of SV40T in NIH3T3 cells as seen by a decrease in proliferation, anchorage independence and changes in migration. We also show that wt N-ras elicits the same anti-malignant effects in some human tumor cell lines (HT1080 and MDA-MB-231). Through mRNA and microRNA (miRNAs) expression profiling we have identified genes (decorin) and miRNAs (mir-29A, let-7b) modulated by wt N-ras potentially responsible for the anti-malignant effect. Wt N-ras appears to mediate its anti-malignant effect by downregulating some of the targets of the TGFβ pathway and decorin, which are able to reverse the inhibition of migration induced by wt N-ras. Our experiments show that the molecules that mediate the anti-malignant effect by wt N-ras appear to be different from those modulated by transforming N-ras. The components of the pathways modulated by wt N-ras mediating its anti-malignant effects are potential targets for therapeutic intervention in cancer.     

WWP-1 Is a Novel Modulator of the DAF-2 Insulin-Like Signaling Network Involved in Pore-Forming Toxin Cellular Defenses in Caenorhabditis elegans

Pore-forming toxins (PFTs) are the single largest class of bacterial virulence factors. The DAF-2 insulin/insulin-like growth factor-1 signaling pathway, which regulates lifespan and stress resistance in Caenorhabditis elegans, is known to mutate to resistance to pathogenic bacteria. However, its role in responses against bacterial toxins and PFTs is as yet unexplored. Here we reveal that reduction of the DAF-2 insulin-like pathway confers the resistance of Caenorhabditis elegans to cytolitic crystal (Cry) PFTs produced by Bacillus thuringiensis. In contrast to the canonical DAF-2 insulin-like signaling pathway previously defined for aging and pathogenesis, the PFT response pathway diverges at 3-phosphoinositide-dependent kinase 1 (PDK-1) and appears to feed into a novel insulin-like pathway signal arm defined by the WW domain Protein 1 (WWP-1). In addition, we also find that WWP-1 not only plays an important role in the intrinsic cellular defense (INCED) against PFTs but also is involved in innate immunity against pathogenic bacteria Pseudomonas aeruginosa and in lifespan regulation. Taken together, our data suggest that WWP-1 and DAF-16 function in parallel within the fundamental DAF-2 insulin/IGF-1 signaling network to regulate fundamental cellular responses in C. elegans.     

Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo

Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.     

Bayesian Modeling of the Yeast SH3 Domain Interactome Predicts Spatiotemporal Dynamics of Endocytosis Proteins

SH3 domains are peptide recognition modules that mediate the assembly of diverse biological complexes. We scanned billions of phage-displayed peptides to map the binding specificities of the SH3 domain family in the budding yeast, Saccharomyces cerevisiae. Although most of the SH3 domains fall into the canonical classes I and II, each domain utilizes distinct features of its cognate ligands to achieve binding selectivity. Furthermore, we uncovered several SH3 domains with specificity profiles that clearly deviate from the two canonical classes. In conjunction with phage display, we used yeast two-hybrid and peptide array screening to independently identify SH3 domain binding partners. The results from the three complementary techniques were integrated using a Bayesian algorithm to generate a high-confidence yeast SH3 domain interaction map. The interaction map was enriched for proteins involved in endocytosis, revealing a set of SH3-mediated interactions that underlie formation of protein complexes essential to this biological pathway. We used the SH3 domain interaction network to predict the dynamic localization of several previously uncharacterized endocytic proteins, and our analysis suggests a novel role for the SH3 domains of Lsb3p and Lsb4p as hubs that recruit and assemble several endocytic complexes.     

The New Anthelmintic Tribendimidine is an L-type (Levamisole and Pyrantel) Nicotinic Acetylcholine Receptor Agonist

Background

Intestinal parasitic nematodes such as hookworms, Ascaris lumbricoides, and Trichuris trichiura are amongst most prevalent tropical parasites in the world today. Although these parasites cause a tremendous disease burden, we have very few anthelmintic drugs with which to treat them. In the past three decades only one new anthelmintic, tribendimidine, has been developed and taken into human clinical trials. Studies show that tribendimidine is safe and has good clinical activity against Ascaris and hookworms. However, little is known about its mechanism of action and potential resistance pathway(s). Such information is important for preventing, detecting, and managing resistance, for safety considerations, and for knowing how to combine tribendimidine with other anthelmintics.

Methodology/Principal Findings

To investigate how tribendimidine works and how resistance to it might develop, we turned to the genetically tractable nematode, Caenorhabditis elegans. When exposed to tribendimidine, C. elegans hermaphrodites undergo a near immediate loss of motility; longer exposure results in extensive body damage, developmental arrest, reductions in fecundity, and/or death. We performed a forward genetic screen for tribendimidine-resistant mutants and obtained ten resistant alleles that fall into four complementation groups. Intoxication assays, complementation tests, genetic mapping experiments, and sequencing of nucleic acids indicate tribendimidine-resistant mutants are resistant also to levamisole and pyrantel and alter the same genes that mutate to levamisole resistance. Furthermore, we demonstrate that eleven C. elegans mutants isolated based on their ability to resist levamisole are also resistant to tribendimidine.

Conclusions/Significance

Our results demonstrate that the mechanism of action of tribendimidine against nematodes is the same as levamisole and pyrantel, namely, tribendimidine is an L-subtype nAChR agonist. Thus, tribendimidine may not be a viable anthelmintic where resistance to levamisole or pyrantel already exists but could productively be used where resistance to benzimidazoles exists or could be combined with this class of anthelmintics.     

High-throughput phenotypic assessment of cardiac physiology in four commonly used inbred mouse strains

Mice with genetic alterations are used in heart research as model systems of human diseases. In the last decade there was a marked increase in the recognition of genetic diversity within inbred mouse strains. Increasing numbers of inbred mouse strains and substrains and analytical variation of cardiac phenotyping methods require reproducible, high-throughput methods to standardize murine cardiovascular physiology. We describe methods for non-invasive, reliable, easy and fast to perform echocardiography and electrocardiography on awake mice. This method can be used for primary screening of the murine cardiovascular system in large-scale analysis. We provide insights into the physiological divergence of C57BL/6N, C57BL/6J, C3HeB/FeJ and 129P2/OlaHsd mouse hearts and define the expected normal values. Our report highlights that compared to the other three strains tested C57BL/6N hearts reveal features of heart failure such as hypertrophy and reduced contractile function. We found several features of the mouse ECG to be under genetic control and obtained several strain-specific differences in cardiac structure and function.     

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.     

Integrated stratigraphy and astronomical calibration of the Serravallian/Tortonian boundary section at Monte Gibliscemi (Sicily, Italy)

Results are presented of an integrated stratigraphic (calcareous plankton biostratigraphy, cyclostratigraphy and magnetostratigraphy) study of the Serravallian/Tortonian (S/T) boundary section of Monte Gibliscemi (Sicily, Italy). Astronomical calibration of the sedimentary cycles provides absolute ages for calcareous plankton bio-events in the interval between 9.8 and 12.1 Ma. The first occurrence (FO) of Neogloboquadrina acostaensis, usually taken to delimit the S/T boundary, is dated astronomically at 11.781 Ma, pre-dating the migratory arrival of the species at low latitudes in the Atlantic by almost 2 million years. In contrast to delayed low-latitude arrival of N. acostaensis, Paragloborotalia mayeri shows a delayed low-latitude extinction of slightly more than 0.7 million years with respect to the Mediterranean (last occurrence (LO) at 10.49 Ma at Ceara Rise; LO at 11.205 Ma in the Mediterranean). The Discoaster hamatus FO, dated at 10.150 Ma, is clearly delayed with respect to the open ocean. The ages of D. kugleri first and last common occurrence (FCO and LCO), Catinaster coalitus FO, Coccolithus miopelagicus last (regular) occurrence (L(R)O) and the D. hamatus/neohamatus cross-over, however, are in good to excellent agreement with astronomically tuned ages for the same events at Ceara Rise (tropical Atlantic), suggesting that both independently established timescales are consistent with one another. The lack of a reliable magnetostratigraphy hampers a direct comparison with the geomagnetic polarity timescale of Cande and Kent (1995; CK95), but ages of calcareous nannofossil events suggests that CK95 is significantly younger over the studied time interval. Approximate astronomical ages for the polarity reversals were obtained by exporting astronomical ages of selected nannofossil events from Ceara Rise (and the Mediterranean) to eastern equatorial Pacific ODP Leg 138 Site 845, which has a reliable magnetostratigraphy.Our data from the Rio Mazzapiedi–Castellania section reveal that the base of the Tortonian stratotype corresponds almost exactly with the first regular occurrence (FRO) of N. acostaensis s.s. as defined in the present study, dated at 10.554 Ma. An extrapolated age of 11.8 Ma calculated for the top of the Serravallian stratotype indicates that there is a gap between the top of the Serravallian and the base of the Tortonian stratotype, potentially rendering all bio-events in the interval between 11.8 and 10.554 Ma suitable for delimiting the S/T boundary. Despite the tectonic deformation and the lack of a magnetostratigraphy, Gibliscemi remains a candidate to define the S/T boundary by means of the Tortonian global boundary stratotype section and point (GSSP).