In Vivo Knockdown of TAK1 Accelerates Bone Marrow Proliferation/Differentiation and Induces Systemic Inflammation

TAK1 (TGF-β Activated Kinase 1) is a MAPK kinase kinase, which activates the p38- and JNK-MAPK and NF-κB pathways downstream of receptors such as Toll-Like-, cytokine- and T-cell and B-cell receptors. Representing such an important node in the pro-inflammatory signal-transduction network, the function of TAK1 has been studied extensively. TAK1 knock-out mice are embryonic lethal, while conditional knock-out mice demonstrated either a pro- or anti-inflammatory function. To study the function of TAK1 protein in the adult immune system, we generated and characterized a transgenic mouse expressing TAK1 shRNA under the control of a doxycycline-inducible promoter. Following treatment of TAK-1 shRNA transgenic mice with doxycycline an effective knockdown of TAK1 protein levels was observed in lymphoid organs and cells in the peritoneal cavity (>50% down regulation). TAK1 knockdown resulted in significant changes in leukocyte populations in blood, bone marrow, spleen and peritoneal cavity. Upon TAK1 knockdown mice demonstrated splenomegaly, signs of systemic inflammation (increased levels of circulating cytokines and increase in cellularity of the B-cell areas and in germinal center development in the follicles) and degenerative changes in heart, kidneys and liver. Not surprisingly, TAK1-Tg mice treated with LPS or anti-CD3 antibodies showed enhanced cytokine/chemokine secretion. Finally, analysis of progenitor cells in the bone marrow upon doxycycline treatment showed increased proliferation and differentiation of myeloid progenitor cells. Given the similarity of the phenotype with TGF-β genetic models, our data suggest that in our model the function of TAK1 in TGF-β signal-transduction is overruling its function in pro-inflammatory signaling.     

A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility

Background

Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods.

Methodology/Principal Findings

Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC₅₀ values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus.

Conclusions/Significance

Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing.     

Acetic acid increases the phage-encoded enterotoxin A expression in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Background  

The effects of acetic acid, a common food preservative, on the bacteriophage-encoded enterotoxin A (SEA) expression and production in Staphylococcus aureus was investigated in pH-controlled batch cultures carried out at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Also, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea.     

The tumor suppressor DiRas3 forms a complex with H-Ras and C-RAF proteins and regulates localization, dimerization, and kinase activity of C-RAF

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity.     

The reconstruction and biochemical characterization of ancestral genes furnish insights into the evolution of terpene synthase function in the Poaceae

Plant Molecular Biology - Distinct catalytic features of the Poaceae TPS-a subfamily arose early in grass evolution and the reactions catalyzed have become more complex with time. The structural...