Focal adhesion kinase protein regulates Wnt3a gene expression to control cell fate specification in the developing neural plate

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase protein localized to regions called focal adhesions, which are contact points between cells and the extracellular matrix. FAK protein acts as a scaffold to transfer adhesion-dependent and growth factor signals into the cell. Increased FAK expression is linked to aggressive metastatic and invasive tumors. However, little is known about its normal embryonic function. FAK protein knockdown during early Xenopus laevis development anteriorizes the embryo. Morphant embryos express increased levels of anterior neural markers, with reciprocally reduced posterior neural marker expression. Posterior neural plate folding and convergence-extension is also inhibited. This anteriorized phenotype resembles that of embryos knocked down zygotically for canonical Wnt signaling. FAK and Wnt3a genes are both expressed in the neural plate, and Wnt3a expression is FAK dependent. Ectopic Wnt expression rescues this FAK morphant anteriorized phenotype. Wnt3a thus acts downstream of FAK to balance anterior-posterior cell fate specification in the developing neural plate. Wnt3a gene expression is also FAK dependent in human breast cancer cells, suggesting that this FAK-Wnt linkage is highly conserved. This unique observation connects the FAK- and Wnt-signaling pathways, both of which act to promote cancer when aberrantly activated in mammalian cells.     

Characterization of nitrogen-fixing bacteria isolated from field-grown barley,oat, and wheat

Diazotrophic bacteria were isolated from the rhizosphere of field-grown Triticum aestivum, Hordeum vulgare, and Avena sativa grown in various regions of Greece. One isolate, with the highest nitrogen-fixation ability from each of the eleven rhizospheres, was selected for further characterisation. Diazotrophic strains were assessed for plant-growth-promoting traits such as indoleacetic acid production and phosphate solubilisation. The phylogenies of 16S rRNA gene of the selected isolates were compared with those based on dnaK and nifH genes. The constructed trees indicated that the isolates were members of the species Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. Furthermore, the ipdC gene was detected in all A. brasilence and one A. zeae isolates. The work presented here provides the first molecular genetic evidence for the presence of culturable nitrogen-fixing P. stutzeri and A. zeae associated with field-grown A. sativa and H. vulgare in Greece.     

Potent and systematic RNAi mediated silencing with single oligonucleotide compounds

RNA interference (RNAi) has been established as an important tool for functional genomics studies and has great promise as a therapeutic intervention for human diseases. In mammalian cells, RNAi is conventionally induced by 19-27-bp RNA duplexes generated by hybridization of two complementary oligonucleotide strands (oligos). Here we describe a novel class of RNAi molecules composed of a single 25-28-nucleotide (nt) oligo. The oligo has a 16-nt mRNA targeting region, followed by an additional 8-10 nt to enable self-dimerization into a partially complementary duplex. Analysis of numerous diverse structures demonstrates that molecules composed of two short helices separated by a loop can efficiently enter and activate the RNA-induced silencing complex (RISC). This finding enables the design of highly effective single-oligo compounds for any mRNA target.     

Enhancement of extracellular purine nucleoside accumulation by <Emphasis Type="Italic">Bacillus</Emphasis> strains through genetic modifications of genes involved in nucleoside export

Using a simple method to introduce genetic modifications into the chromosome of naturally nontransformable Bacillus, a set of marker-free inosine-producing and 5-aminoimidazole-4-carboxamide (AICA) ribonucleoside-producing Bacillus amyloliquefaciens strains has been constructed. These strains differ in expression levels of the genes responsible for nucleoside export. Overexpression of B. amyloliquefaciens pbuE and heterologous expression of Escherichia coli nepI, which encode nucleoside efflux transporters, each notably enhanced inosine production by a B. amyloliquefaciens nucleoside-producing strain. pbuE overexpression was found to increase AICA ribonucleoside accumulation, indicating that the substrate specificity of the PbuE pump extends to this nucleoside. These results demonstrate that identifying genes whose products facilitate transport of a desired nucleoside out of cells and enhancing their expression can improve the performance of strains used for industrial production.     

Combining functional genomics and chemical biology to identify targets of bioactive compounds

Genome sequencing projects have revealed thousands of suspected genes, challenging researchers to develop efficient large-scale functional analysis methodologies. Determining the function of a gene product generally requires a means to alter its function. Genetically tractable model organisms have been widely exploited for the isolation and characterization of activating and inactivating mutations in genes encoding proteins of interest. Chemical genetics represents a complementary approach involving the use of small molecules capable of either inactivating or activating their targets. Saccharomyces cerevisiae has been an important test bed for the development and application of chemical genomic assays aimed at identifying targets and modes of action of known and uncharacterized compounds. Here we review yeast chemical genomic assays strategies for drug target identification.     

Unraveling persistent host cell infection with Coxiella burnetii by quantitative proteomics

The interaction between the immune system and invading bacteria is sufficient to eradicate microorganisms for the majority of bacterial infections, but suppression of the microbicidal response leads to reactivation or chronic evolution of infections and to bacterial persistence. To identify the cellular pathways affected by bacterial persistence, we applied the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique for a comparative study of protein expression in the C. burnetii strains Nine Mile (NM) and its respective strain (NMper) isolated from 18 months persistently infected cell cultures. In total, three different proteome comparisons were performed with the total bacterial proteome, potentially secreted bacterial proteins, and the eukaryotic infected proteome being assessed. Our results revealed that among the 547 identified bacterial proteins, 53 had significantly altered levels of expression and indicated potential metabolic differences between the two strains. Regarding differences in the secreted proteins between both strains and different modulation of the host cell, machineries reflect at least large rearrangements of both bacterial and eukaryotic proteomes during the persistent model of infection when compared to the acute one, which emphasizes that C. burnetii orchestrates a vast number of different bacterial and eukaryotic host cell processes to persist within its host.     

Procoagulant Platelets Form an α-Granule Protein-covered “Cap” on Their Surface That Promotes Their Attachment to Aggregates

Strongly activated “coated” platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin α_IIbβ₃. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein “coat.” Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as “cap,” a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and α_IIbβ₃. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather “capped”) platelets to become incorporated into thrombi despite their lack of active integrins.     

Mechanisms of exclusion: attitudes toward allocation of social rights to out-group population

This study examines the ways in which perceived socioeconomic threat, perceived threat to national identity, and prejudice (as reflected in negative stereotypes and desire for social distance) prompt objection to allocation of rights to out-groups. The paper presents a simultaneous test of three theoretical explanations and delineates the complex interrelationships among all concepts, using data from a representative sample of Israelis in structural equation models. This study finds that: (1) perception of socioeconomic threat is positively associated with perception of threat to national identity; (2) the two types of threat do not directly affect attitudes toward allocation of rights, but instead tend to increase negative stereotyping of the out-group; (3) negative stereotypes are likely to contribute to a desire for social distance from the out-group; and (4) the attribution of negative characteristics and a sense of social distance tend to increase objection to allocation of social rights to the out-group.     

Synthesis and anti-parasitic activity of a novel quinolinone–chalcone series

A series of novel quinolinone–chalcone hybrids and analogues were designed, synthesized and their biological activity against the mammalian stages of Trypanosoma brucei and Leishmania infantum evaluated. Promising molecular scaffolds with significant microbicidal activity and low cytotoxicity were identified. Quinolinone–chalcone 10 exhibited anti-parasitic properties against both organisms, being the most potent anti-L. infantum agent of the entire series (IC₅₀ value of 1.3 ± 0.1 μM). Compounds 4 and 11 showed potency toward the intracellular, amastigote stage of L. infantum (IC₅₀ values of 2.1 ± 0.6 and 3.1 ± 1.05 μM, respectively). Promising trypanocidal compounds include 5 and 10 (IC₅₀ values of 2.6 ± 0.1 and 3.3 ± 0.1 μM, respectively) as well as 6 and 9 (both having IC₅₀ values of <5 μM). Chemical modifications on the quinolinone–chalcone scaffold were performed on selected compounds in order to investigate the influence of these structural features on antiparasitic activity.     

Ovariectomy Reinstates the Infarct Size-Limiting Effect of Postconditioning in Female Rabbits

Gender seems to interfere with the cardioprotective effect of ischemic preconditioning (PreC) and postconditioning (PostC); PreC-conferred protection is weaker or lost in female animals after ovariectomy (Ov), while the role of PostC is still in dispute. We sought to investigate the effect of PostC in female rabbits, its interaction with Ov, and the potential implicated intracellular pathways. Intact or Ov adult female rabbits (n = 46) were subjected to 30 min ischemia and reperfusion with PostC (PostC or OvPostC), which consisted of six cycles of 30-s ischemia/30-s reperfusion at the end of ischemia, or without PostC (Fem or OvFem). Infarct size (I) and area at risk (R) were determined by TTC staining and fluorescent particles, respectively, after 3-h reperfusion in 30 out of 46 animals. Plasma levels of estradiol and nitrite/nitrate (NO _x ) were evaluated. ERKs, p38-MAPK, and Akt assessment was performed in excised hearts 1-min after starting the final reperfusion period in the remaining 16 animals. Infarct size was significantly reduced only in OvPostC group (I/R ratio, 25.3 ± 2.7, vs 48.1 ± 2.0, 43.6 ± 4.2 and 55.1 ± 5.6 % in Fem, OvFem, and PostC groups, p < 0.05). In ovariectomized rabbits, plasma estradiol and NO _x levels were lower than in the normal ones. Akt phosphorylation in ischemic regions was significantly higher in OvPostC group, whereas ERK1/2 and p38-MAPK activation was observed in all ovariectomized animals irrespective of PostC. PostC is not effective in female rabbits, but the protection is reinstated after Ov potentially via the RISK pathway.