Small-Molecule-Directed Endogenous Regeneration of Visual Function in a Mammalian Retinal Degeneration Model.

Degenerative retinal diseases associated with photoreceptor loss are a leading cause of visual impairment worldwide, with limited treatment options. Phenotypic profiling coupled with medicinal chemistry were used to develop a small molecule with proliferative effects on retinal stem/progenitor cells, as assessed in vitro in a neurosphere assay and in vivo by measuring Msx1-positive ciliary body cell proliferation. The compound was identified as having kinase inhibitory activity and was subjected to cellular pathway analysis in non-retinal human primary cell systems. When tested in a disease-relevant murine model of adult retinal degeneration (MNU-induced retinal degeneration), we observed that four repeat intravitreal injections of the compound improved the thickness of the outer nuclear layer along with the regeneration of the visual function, as measured with ERG, visual acuity, and contrast sensitivity tests. This serves as a proof of concept for the use of a small molecule to promote endogenous regeneration in the eye.

RhoGTPases - A novel link between cytoskeleton organization and cisplatin resistance.

For more than three decades, platinum compounds have been the first line treatment for a wide spectrum of solid tumors. Yet, cisplatin resistance is a major impediment in cancer therapy, and deciphering the mechanisms underlying chemoresistance is crucial for the development of novel therapies with enhanced efficacy. The Rho subfamily of small GTPases plays a significant role in cancer progression, and a growing body of evidence points toward the involvement of these proteins in anticancer drug resistance, including cisplatin resistance. The cycling between active and inactive states, governed by the balance between their GEFs, GAPs and GDIs, RhoGTPases, acts as molecular switches with a pivotal role in actin cytoskeleton organization. The Rho subfamily of proteins is involved in many key cellular processes including adhesion, vesicular trafficking, proliferation, survival, cell morphology and cell-matrix interactions. Although RhoA, RhoB and RhoC are highly homologous and share some upstream regulators and downstream effectors, they each have different roles in cancer progression and chemoresistance. While RhoA and RhoC are upregulated in many tumors and can stimulate transformation, RhoB appears to exhibit tumor suppressor characteristics with proapoptotic effects. In the current review, we discuss the role of Rho subfamily of proteins in cancer, and focus on their involvement in intrinsic and acquired drug resistance.

Mechanistic insight into GPCR-mediated activation of the microtubule-associated RhoA exchange factor GEF-H1.

The RhoGEF GEF-H1 can be sequestered in an inactive state on polymerized microtubules by the dynein motor light-chain Tctex-1. Phosphorylation of GEF-H1 Ser885 by PKA or PAK kinases creates an inhibitory 14-3-3-binding site. Here we show a new mode of GEF-H1 activation in response to the G-protein-coupled receptor (GPCR) ligands lysophosphatidic acid (LPA) or thrombin that is independent of microtubule depolymerization. LPA/thrombin stimulates disassembly of the GEF-H1:dynein multi-protein complex through the concerted action of Gα and Gßγ. Gα binds directly to GEF-H1 and displaces it from Tctex-1, while Gßγ binds to Tctex-1 and disrupts its interaction with the dynein intermediate chain, resulting in the release of GEF-H1. Full activation of GEF-H1 requires dephosphorylation of Ser885 by PP2A, which is induced by thrombin. The coordinated displacement of GEF-H1 from microtubules by G-proteins and its dephosphorylation by PP2A demonstrate a multistep GEF-H1 activation and present a unique mechanism coupling GPCR signalling to Rho activation.

The RhoGEF GEF-H1 is required for oncogenic RAS signaling via KSR-1.

Cellular transformation by oncogenic RAS engages the MAPK pathway under strict regulation by the scaffold protein KSR-1. Here, we report that the guanine nucleotide exchange factor GEF-H1 plays a critical role in a positive feedback loop for the RAS/MAPK pathway independent of its RhoGEF activity. GEF-H1 acts as an adaptor protein linking the PP2A B' subunits to KSR-1, thereby mediating the dephosphorylation of KSR-1 S392 and activation of MAPK signaling. GEF-H1 is important for the growth and survival of HRAS(V12)-transformed cells and pancreatic tumor xenografts. GEF-H1 expression is induced by oncogenic RAS and is correlated with pancreatic neoplastic progression. Our results, therefore, identify GEF-H1 as an amplifier of MAPK signaling and provide mechanistic insight into the progression of RAS mutant tumors.

Virulence factors of septicemic Escherichia coli strains.

Extraintestinal pathogenic Escherichia coli strains (ExPEC) are the cause of a diverse spectrum of invasive human and animal infections, often leading to septicemia. This review deals with the virulence genes of septicemic ExPEC strains. We discuss the meaning of a virulence gene and survey the genomic, genetic and physiological studies on these strains. Apparently, there are a few virulence factors, which are conserved in the septicemic strains, implying that they are essential for the infection. For the other virulence-related genes a high level of diversity is observed, demonstrating that all stages of the infection can be mediated by a number of alternative virulence factors. The variable profile of virulence genes in septicemic E. coli strains, as well as a prevalence of mobility-related sequences point out the existence of a "mix and match" combinatorial system.

Extensive gene diversity in septicemic Escherichia coli strains.

Extraintestinal pathogenic Escherichia coli strains (ExPEC) are the cause of a diverse spectrum of invasive infections in humans and animals, and these infections often lead to septicemia. Strains of serogroups O2 and O78 of E. coli are involved in human urinary tract infections and newborn meningitis and also constitute the major serotypes involved in avian colisepticemia. In the present study we compared the unique genomic sequences of two such septicemic strains, strains O2-1772 and O78-9, obtained by suppression subtractive hybridization. Evaluation of the degree of similarity between these two strains, which cause the same disease, revealed a high degree of diversity, with only a few shared genes. Subsequently, additional strains of each serogroup of human and animal origin were screened by PCR, and the results provided further evidence for the existence of a high degree of genome plasticity. These results were unexpected, in view of data showing that the two O157:H7 strains that have been sequenced are nearly identical in terms of virulence factors. Furthermore, the data obtained for the septicemic strains suggest that each step in the infection can be mediated by a number of alternative virulence factors, indicating the existence of a mix-and-match combinatorial system. Although whole-genome comparisons of E. coli strains causing different diseases have shown great differences in gene contents, we show that such differences exist even within strains that cause the same disease and that target the same host tissues. Moreover, in addition to the high level of genome plasticity, we show that the large pool of virulence genes in the septicemic strains is independent of the host, implying a high degree of zoonotic risk.