Pre-clinical characterization of CX-4945, a potent and selective small molecule inhibitor of CK2 for the treatment of cancer

In this article we describe the preclinical characterization of 5-(3-chlorophenylamino) benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first orally available small molecule inhibitor of protein CK2 in clinical trials for cancer. CX-4945 was optimized as an ATP-competitive inhibitor of the CK2 holoenzyme (Ki = 0.38 nM). Iterative synthesis and screening of analogs, guided by molecular modeling, led to the discovery of orally available CX-4945. CK2 promotes signaling in the Akt pathway and CX-4945 suppresses the phosphorylation of Akt as well as other key downstream mediators of the pathway such as p21. CX-4945 induced apoptosis and caused cell cycle arrest in cancer cells in vitro. CX-4945 exhibited a dose-dependent antitumor activity in a xenograft model of PC3 prostate cancer model and was well tolerated. In vivo time-dependent reduction in the phosphorylation of the biomarker p21 at T145 was observed by immunohistochemistry. Inhibition of the newly validated CK2 target by CX-4945 represents a fresh therapeutic strategy for cancer.     

Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte

A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.     

High-resolution analysis of four efficient yeast replication origins reveals new insights into the ORC and putative MCM binding elements

In budding yeast, the eukaryotic initiator protein ORC (origin recognition complex) binds to a bipartite sequence consisting of an 11 bp ACS element and an adjacent B1 element. However, the genome contains many more matches to this consensus than actually bind ORC or function as origins in vivo. Although ORC-dependent loading of the replicative MCM helicase at origins is enhanced by a distal B2 element, less is known about this element. Here, we analyzed four highly active origins (ARS309, ARS319, ARS606 and ARS607) by linker scanning mutagenesis and found that sequences adjacent to the ACS contributed substantially to origin activity and ORC binding. Using the sequences of four additional B2 elements we generated a B2 multiple sequence alignment and identified a shared, degenerate 8 bp sequence that was enriched within 228 known origins. In addition, our high-resolution analysis revealed that not all origins exist within nucleosome free regions: a class of Sir2-regulated origins has a stably positioned nucleosome overlapping or near B2. This study illustrates the conserved yet flexible nature of yeast origin architecture to promote ORC binding and origin activity, and helps explain why a strong match to the ORC binding site is insufficient to identify origins within the genome.     

Smelling chemosensory signals of males in anxious versus nonanxious condition increases state anxiety of female subjects

The hypothesis of this experiment was that humans in an anxious state compared with a nonanxious state are able to increase anxiety levels in other humans via their body odors. Specifically, we hypothesized that male chemosensory anxiety signals compared with neutral chemosignals increase state anxiety of female subjects. Thirteen male subjects participated in 2 different sweat donation sessions: chemosignals were collected during participation in a high rope course (anxiety condition) and in an ergometer workout (neutral condition). State and trait anxiety were evaluated in 20 female odor recipients using Spielberger's state-trait anxiety inventory in a double-blind design. Comparison of state anxiety of odor donors between control and anxiety condition differed significantly indicating that our model of anxiety induction successfully led to the expected change in emotion. Comparison of state anxiety of odor recipients showed a trend toward higher state anxiety in the anxiety condition compared with the neutral condition after 5 min of odor exposure. After 20 min of odor exposure, state anxiety of female subjects was significantly higher during the perception of sweat collected during the anxiety condition in comparison with the perception of sweat collected during the neutral condition. This experiment gives evidence that male anxiety chemosignals compared with neutral chemosignals are capable of inducing an increased state anxiety in female subjects.     

Contribution of retinoid X receptor signaling to the specification of skeletal muscle lineage

Pluripotent stem cells possess a tremendous potential for the treatment of many diseases because of their capacity to differentiate into a variety of cell lineages. However, they provide little promise for muscle-related diseases, mainly because of the lack of small molecule inducers to efficiently direct myogenic conversion. Retinoic acid, acting through the retinoic acid receptor (RAR) and retinoid X receptor (RXR), affects stem cell fate determination in a concentration-dependent manner, but it only has a modest efficacy on the commitment of ES cells into skeletal muscle lineage. The RXR is very important for embryonic development but is generally considered to act as a silent partner of RAR in a non-permissive mode. In this study, we have examined whether activation of the RXR by rexinoid or RXR-specific signaling play a role in the specification of stem cells into muscle lineage. Our findings demonstrate that mouse ES cells generate skeletal myocytes effectively upon treatment with rexinoid at the early stage of differentiation and that on a molecular level, rexinoid-enhanced myogenesis simulates the sequential events observed in vivo. Moreover, RXR-mediated myogenic conversion requires the function of β-catenin but not RAR. Our studies establish the feasibility of applying the RXR agonist in cell-based therapies to treat muscle-related diseases. The aptitude of mouse ES cells to generate skeletal myocytes following rexinoid induction also provides a model system to study the convergence of different signaling pathways in myogenesis.     

The low resolution structure of ApoA1 in spherical high density lipoprotein revealed by small angle neutron scattering

Spherical high density lipoprotein (sHDL), a key player in reverse cholesterol transport and the most abundant form of HDL, is associated with cardiovascular diseases. Small angle neutron scattering with contrast variation was used to determine the solution structure of protein and lipid components of reconstituted sHDL. Apolipoprotein A1, the major protein of sHDL, forms a hollow structure that cradles a central compact lipid core. Three apoA1 chains are arranged within the low resolution structure of the protein component as one of three possible global architectures: (i) a helical dimer with a hairpin (HdHp), (ii) three hairpins (3Hp), or (iii) an integrated trimer (iT) in which the three apoA1 monomers mutually associate over a portion of the sHDL surface. Cross-linking and mass spectrometry analyses help to discriminate among the three molecular models and are most consistent with the HdHp overall architecture of apoA1 within sHDL.     

The human adenocarcinoma-associated gene, AGR2, induces expression of amphiregulin through Hippo pathway co-activator YAP1 activation

Anterior Gradient Homolog 2 (AGR2) is expressed by the normal intestine and by most human adenocarcinomas, including those derived from the esophagus, pancreas, lung, breast, ovary, and prostate. Xenografts of human adenocarcinoma cell lines in nude mice previously demonstrated that AGR2 supports tumor growth. In addition, AGR2 is able to induce in vitro a transformed phenotype in fibroblast and epithelial cell lines. The mechanism underlying the growth promoting effects of AGR2 is unknown. The present study shows that AGR2 induces expression of amphiregulin (AREG), a growth promoting EGFR ligand. Induced AREG expression in adenocarcinoma cells is able to rescue the transformed phenotype that is lost when AGR2 expression is reduced. Additional experiments demonstrate that AGR2 induction of AREG is mediated by activation of the Hippo signaling pathway co-activator, YAP1. Thus AGR2 promotes growth by regulating the Hippo and EGF receptor signaling pathways.     

Phylogeography of West Nile virus: from the cradle of evolution in Africa to Eurasia, Australia, and the Americas

West Nile virus (WNV) is the most widely distributed of the encephalitic flaviviruses and is a major cause of encephalitis, with isolates obtained from all continents, apart from Antarctica. Subsequent to its divergence from the other members of the Japanese encephalitis virus complex, presumably in Africa, WNV has diverged into individual lineages that mostly correspond with geographic distribution. Here we elucidate the phylogeography and evolutionary history of isolates from lineage 1 of WNV. Interestingly, there are many examples of the same amino acid having evolved independently on multiple occasions. In Africa, WNV exists in an endemic cycle, whereas it is epidemic in Europe, being reintroduced regularly from Africa either directly (in western Europe) or via the Middle East (in eastern Europe). Significantly, introduction into other geographic areas has occurred on one occasion only in each region, leading to subsequent establishment and expansion of the virus in these areas. Only one endemic genotype each is present in India and Australia, suggesting that WNV was successfully introduced into these locations once only. Each introduction occurred many centuries ago, probably due to trade and exploration during the 19th century. Likewise, in the Americas, WNV was successfully introduced in 1999 and subsequently became endemic across most temperate regions of North America (NA). In contrast to previous suggestions, an isolate from the epidemic in Israel in 1998 was not the direct progenitor of the NA epidemic; rather, both epidemics originated from the same (unknown) location.     

Effects of suppressing the DNA mismatch repair system on homeologous recombination in tomato

In plant breeding, the ability to manipulate genetic (meiotic) recombination would be beneficial for facilitating gene transfer from wild relatives of crop plants. The DNA mismatch repair (MMR) system helps maintain genetic integrity by correcting base mismatches that arise via DNA synthesis or damage, and antagonizes recombination between homeologous (divergent) DNA sequences. Previous studies have established that the genomes of cultivated tomato (Solanum lycopersicum) and the wild relative S. lycopersicoides are substantially diverged (homeologous) such that recombination between their chromosomes is strongly reduced. Here, we report the effects on homeologous recombination of suppressing endogenous MMR genes in S. lycopersicum via RNAi-induced silencing of SlMSH2 and SlMSH7 or overexpressing dominant negatives of Arabidopsis MSH2 (AtMSH2-DN) in an alien substitution line (SL-8) of S. lycopersicoides in tomato. We show that certain inhibitions of MMR (RNAi of SlMSH7, AtMSH2-DN) are associated with modest increases in homeologous recombination, ranging from 3.8 to 29.2% (average rate of 17.8%) compared to controls. Unexpectedly, only the AtMSH2-DN proteins but not RNAi-induced silencing of MSH2 was found to increase homeologous recombination. The ratio of single to double crossovers (SCO:DCO ratio) decreased by approximately 50% in progeny of the AtMSH2-DN parents. An increase in the frequency of heterozygous SL-8 plants was also observed in the progeny of the SlMSH7-RNAi parents. Our findings may contribute to acceleration of introgression in cultivated tomato.