Uveal melanoma cells use ameboid and mesenchymal mechanisms of cell motility crossing the endothelium

Uveal melanomas (UMs) are malignant cancers arising from the pigmented layers of the eye. UM cells spread through the bloodstream, and circulating UM cells are detectable in patients before metastases appear. Extravasation of UM cells is necessary for formation of metastases, and transendothelial migration (TEM) is a key step in extravasation. UM cells execute TEM via a stepwise process involving the actin-based processes of ameboid blebbing and mesenchymal lamellipodial protrusion. UM cancers are driven by oncogenic mutations that activate Gαq/11, and this activates TRIO, a guanine nucleotide exchange factor for RhoA and Rac1. We found that pharmacologic inhibition of Gαq/11 in UM cells reduced TEM. Inhibition of the RhoA pathway blocked amoeboid motility but led to enhanced TEM; in contrast, inhibition of the Rac1 pathway decreased mesenchymal motility and reduced TEM. Inhibition of Arp2/3 complex allowed cells to transmigrate without intercalation, a direct mechanism similar to the one often displayed by immune cells. BAP1-deficient (+/–) UM subclones displayed motility behavior and increased levels of TEM, similar to the effects of RhoA inhibitors. We conclude that RhoA and Rac1 signaling pathways, downstream of oncogenic Gαq/11, combine with pathways regulated by BAP1 to control the motility and transmigration of UM cells.     

Identification of PLOD2 as telopeptide lysyl hydroxylase,an important enzyme in fibrosis

The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes.     

Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion

Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-alpha, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-kappaB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn's disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-kappaB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn's disease.     

Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is required for the establishment of neuronal polarity

The development of a polarised morphology with multiple dendrites and a single axon is an essential step in the differentiation of neurons. The establishment of neuronal polarity is directed by the sequential activity of the GTPases Rap1B and Cdc42. Rap1B is initially present in all neurites of unpolarised neurons, but becomes restricted to the tip of a single process during the establishment of neuronal polarity where it specifies axonal identity. Here, we show that the ubiquitin ligases Smad ubiquitination regulatory factor-1 (Smurf1) and Smurf2 are essential for neurite growth and neuronal polarity, respectively, and regulate the GTPases Rho and Rap1B in hippocampal neurons. Smurf2 is required for the restriction of Rap1B to a single neurite. Smurf2 ubiquitinates inactive Rap1B and initiates its degradation through the ubiquitin/proteasome pathway (UPS). Degradation of Rap1B restricts it to a single neurite and thereby ensures that neurons extend a single axon.     

Rates of evolution of avirulence phenotypes and DNA markers in a northwest European population of Puccinia striiformis f. sp. tritici

The effects of evolutionary processes in fungal pathogen populations may occur more rapidly and display larger effects in agricultural systems than in wild ecosystems because of human involvement by plant breeding and crop management. In this study, we analysed the rate of evolution in three lineages of a northwest European population of a biotrophic and asexual reproduced fungal pathogen, Puccinia striiformis f. sp. tritici, causing yellow rust on wheat. Pathogen samples were collected between 1975 and 2002 in the UK and Denmark, and assayed for 14 individual avirulence/virulence alleles and up to 234 amplified fragment length polymorphism (AFLP) primer pairs producing approximately 17,000 AFLP fragments. The large number of fragments and a targeted sampling of isolates allowed a reconstruction of phylogenies in great detail, i.e. no homoplasy and a representation of sequential, evolutionary steps by pathogen samples. A recent, phenotypic loss of avirulence was observed at least once for loci corresponding to P. striiformis f. sp. tritici resistance Yr2, Yr3, Yr4, Yr7, Yr9, and Yr15, whereas Avr6 and Avr17 were lost independently in all three lineages, corresponding to 16 events of loss of avirulence (emergence of virulence). The opposite process, restoration of avirulence, was observed for Yr9 and Yr32. An interpretation of phenotypic changes within lineages as independent mutation events resulted in mutation frequencies from 1.4x10(-6) to 4.1x10(-6) per AFLP fragment (locus) per generation, whereas the effective rate by which a mutation from avirulence to virulence was established in the pathogen population, when subject to selection by host resistance genes, was approximately three orders of magnitude faster.     

High-throughput flow cytometry-based assay to identify apoptosis-inducing proteins

After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry-based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers.     

Monoclonal antibodies for the detection of Puccinia striiformis urediniospores

The fungal pathogen Pst causes yellow rust disease in wheat plants leading to crop losses. The organism spreads by releasing wind-dispersed urediniospores from infected plants. In this study a library of novel monoclonal antibodies (mAbs) was developed against Pst urediniospores. Nine mAb-producing cell lines were cloned and their cross-reactivities characterised against a panel of airborne fungal spores representing genera commonly found in the same environment as Pst. Two specific mAbs were used to develop a competitive ELISA (Pst mAb4) and a subtractive inhibition ELISA (Pst mAb8). Standard curves for both assays had good intra- and interday reproducibility. The subtractive inhibition ELISA had greater sensitivity with a detection limit of 1.5 × 10⁵ spores ml⁻¹. Cross-reactivity studies of Pst mAb8 in the subtractive inhibition ELISA, showed reaction with other Puccinia spores only, suggesting that common epitopes exist within this genus. The biosensor-compatible Pst mAb8 assay principle developed in this study has the potential to be implemented in future ‘label-free’ in-the-field systems for Pst detection.     

Large scale statistical inference of signaling pathways from RNAi and microarray data

Background  

The advent of RNA interference techniques enables the selective silencing of biologically interesting genes in an efficient way. In combination with DNA microarray technology this enables researchers to gain insights into signaling pathways by observing downstream effects of individual knock-downs on gene expression. These secondary effects can be used to computationally reverse engineer features of the upstream signaling pathway.     

Evolutionary divergence in thermal sensitivity and diapause of field and laboratory populations of manduca sexta

The tobacco hornworm Manduca sexta has been an important model system in insect biology for more than half a century. Here we report the evolutionary divergence in thermal sensitivity and diapause initiation between field and laboratory populations that were separated for more than 35 yr (>240 laboratory generations) and that are descendants from the same field populations in central North Carolina. At intermediate rearing temperatures (20 degrees-25 degrees C), mean body size was significantly larger and development time significantly faster in the laboratory than in the field populations. At higher temperatures (30 degrees -35 degrees C), these mean differences between populations were reduced or eliminated, and larval survival at 35 degrees C was significantly lower in the laboratory population than in the field population. F(1) crosses had survival and development time to wandering similar to the field population times at both 25 degrees and 35 degrees C; body mass at wandering for F(1) crosses was intermediate compared with that of the field and laboratory populations. Comparisons with earlier field and laboratory studies suggest evolutionary reductions in thermal tolerance and performance at high temperatures in the laboratory population. The critical photoperiod initiating diapause in field populations in North Carolina did not change detectably between the 1960s and 2005. In contrast, the laboratory population has evolved a reduced tendency to diapause under short-day conditions, relative to the field population.