Cabozantinib in progressive medullary thyroid cancer

Purpose Cabozantinib, a tyrosine kinase inhibitor (TKI) of hepatocyte growth factor receptor (MET), vascular endothelial growth factor receptor 2, and rearranged during transfection (RET), demonstrated clinical activity in patients with medullary thyroid cancer (MTC) in phase I. Patients and Methods We conducted a double-blind, phase III trial comparing cabozantinib with placebo in 330 patients with documented radiographic progression of metastatic MTC. Patients were randomly assigned (2:1) to cabozantinib (140 mg per day) or placebo. The primary end point was progression-free survival (PFS). Additional outcome measures included tumor response rate, overall survival, and safety. Results The estimated median PFS was 11.2 months for cabozantinib versus 4.0 months for placebo (hazard ratio, 0.28; 95% CI, 0.19 to 0.40; P < .001). Prolonged PFS with cabozantinib was observed across all subgroups including by age, prior TKI treatment, and RET mutation status (hereditary or sporadic). Response rate was 28% for cabozantinib and 0% for placebo; responses were seen regardless of RET mutation status. Kaplan-Meier estimates of patients alive and progression-free at 1 year are 47.3% for cabozantinib and 7.2% for placebo. Common cabozantinib-associated adverse events included diarrhea, palmar-plantar erythrodysesthesia, decreased weight and appetite, nausea, and fatigue and resulted in dose reductions in 79% and holds in 65% of patients. Adverse events led to treatment discontinuation in 16% of cabozantinib-treated patients and in 8% of placebo-treated patients. Conclusion Cabozantinib (140 mg per day) achieved a statistically significant improvement of PFS in patients with progressive metastatic MTC and represents an important new treatment option for patients with this rare disease. This dose of cabozantinib was associated with significant but manageable toxicity

Deciphering interplay between Salmonella invasion effectors

Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a ‘signaling hub’ during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cis binary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen–host interaction

Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images

Fluorescence microscopy is the standard tool for detection and analysis of cellular phenomena. This technique, however, has a number of drawbacks such as the limited number of available fluorescent channels in microscopes, overlapping excitation and emission spectra of the stains, and phototoxicity.We here present and validate a method to automatically detect cell population outlines directly from bright field images. By imaging samples with several focus levels forming a bright field -stack, and by measuring the intensity variations of this stack over the -dimension, we construct a new two dimensional projection image of increased contrast. With additional information for locations of each cell, such as stained nuclei, this bright field projection image can be used instead of whole cell fluorescence to locate borders of individual cells, separating touching cells, and enabling single cell analysis. Using the popular CellProfiler freeware cell image analysis software mainly targeted for fluorescence microscopy, we validate our method by automatically segmenting low contrast and rather complex shaped murine macrophage cells.The proposed approach frees up a fluorescence channel, which can be used for subcellular studies. It also facilitates cell shape measurement in experiments where whole cell fluorescent staining is either not available, or is dependent on a particular experimental condition. We show that whole cell area detection results using our projected bright field images match closely to the standard approach where cell areas are localized using fluorescence, and conclude that the high contrast bright field projection image can directly replace one fluorescent channel in whole cell quantification. Matlab code for calculating the projections can be downloaded from the supplementary site: http://sites.google.com/site/brightfieldorstaining

Enhanced CellClassifier: a multi-class classification tool for microscopy images

BACKGROUND: Light microscopy is of central importance in cell biology. The recent introduction of automated high content screening has expanded this technology towards automation of experiments and performing large scale perturbation assays. Nevertheless, evaluation of microscopy data continues to be a bottleneck in many projects. Currently, among open source software, CellProfiler and its extension Analyst are widely used in automated image processing. Even though revolutionizing image analysis in current biology, some routine and many advanced tasks are either not supported or require programming skills of the researcher. This represents a significant obstacle in many biology laboratories. RESULTS: We have developed a tool, Enhanced CellClassifier, which circumvents this obstacle. Enhanced CellClassifier starts from images analyzed by CellProfiler, and allows multi-class classification using a Support Vector Machine algorithm. Training of objects can be done by clicking directly "on the microscopy image" in several intuitive training modes. Many routine tasks like out-of focus exclusion and well summary are also supported. Classification results can be integrated with other object measurements including inter-object relationships. This makes a detailed interpretation of the image possible, allowing the differentiation of many complex phenotypes. For the generation of the output, image, well and plate data are dynamically extracted and summarized. The output can be generated as graphs, Excel-files, images with projections of the final analysis and exported as variables. CONCLUSION: Here we describe Enhanced CellClassifier which allows multiple class classification, elucidating complex phenotypes. Our tool is designed for the biologist who wants both, simple and flexible analysis of images without requiring programming skills. This should facilitate the implementation of automated high-content screening

The Cost of Virulence: Retarded Growth of Salmonella Typhimurium Cells Expressing Type III Secretion System 1

Virulence factors generally enhance a pathogen's fitness and thereby foster transmission. However, most studies of pathogen fitness have been performed by averaging the phenotypes over large populations. Here, we have analyzed the fitness costs of virulence factor expression by Salmonella enterica subspecies I serovar Typhimurium in simple culture experiments. The type III secretion system ttss-1, a cardinal virulence factor for eliciting Salmonella diarrhea, is expressed by just a fraction of the S. Typhimurium population, yielding a mixture of cells that either express ttss-1 (TTSS-1+ phenotype) or not (TTSS-1− phenotype). Here, we studied in vitro the TTSS-1+ phenotype at the single cell level using fluorescent protein reporters. The regulator hilA controlled the fraction of TTSS-1+ individuals and their ttss-1 expression level. Strikingly, cells of the TTSS-1+ phenotype grew slower than cells of the TTSS-1− phenotype. The growth retardation was at least partially attributable to the expression of TTSS-1 effector and/or translocon proteins. In spite of this growth penalty, the TTSS-1+ subpopulation increased from <10% to approx. 60% during the late logarithmic growth phase of an LB batch culture. This was attributable to an increasing initiation rate of ttss-1 expression, in response to environmental cues accumulating during this growth phase, as shown by experimental data and mathematical modeling. Finally, hilA and hilD mutants, which form only fast-growing TTSS-1− cells, outcompeted wild type S. Typhimurium in mixed cultures. Our data demonstrated that virulence factor expression imposes a growth penalty in a non-host environment. This raises important questions about compensating mechanisms during host infection which ensure successful propagation of the genotype

Divergent Roles of Salmonella Pathogenicity Island 2 and Metabolic Traits during Interaction of S. enterica Serovar Typhimurium with Host Cells

The molecular mechanisms of virulence of the gastrointestinal pathogen Salmonella enterica are commonly studied using cell culture models of infection. In this work, we performed a direct comparison of the interaction of S. enterica serovar Typhimurium (S. Typhimurium) with the non-polarized epithelial cell line HeLa, the polarized cell lines CaCo2, T84 and MDCK, and macrophage-like RAW264.7 cells. The ability of S. Typhimurium wild-type and previously characterized auxotrophic mutant strains to enter host cells, survive and proliferate within mammalian cells and deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) was quantified. We found that the entry of S. Typhimurium into polarized cells was much more efficient than entry into non-polarized cells or phagocytic uptake. While SPI2-T3SS dependent intracellular proliferation was observed in HeLa and RAW cells, the intracellular replication in polarized cells was highly restricted and not affected by defective SPI2-T3SS. The contribution of aromatic amino acid metabolism and purine biosynthesis to intracellular proliferation was distinct in the various cell lines investigated. These observations indicate that the virulence phenotypes of S. Typhimurium are significantly affected by the cell culture model applied

A Model of Salmonella Colitis with Features of Diarrhea in SLC11A1 Wild-Type Mice

Background: Mice do not get diarrhea when orally infected with S. enterica, but pre-treatment with oral aminoglycosides makes them susceptible to Salmonella colitis. However, genetically susceptible ItyS mice (Nramp1 G169D allele) die from systemic infection before they develop diarrhea, so a new model is needed to study the pathogenesis of diarrhea. We pretreated ItyR mice (Nramp1 G169) with oral kanamycin prior to infecting them with virulent S. Typhimurium strain 14028s in order to study Salmonella-induced diarrhea. We used both a visual scoring system and the measurement of fecal water content to measure diarrhea. BALB/c.D2 Nramp1 congenic started losing weight 5 days post-infection and they began to die from colitis 10–14 days after infection. A SPI-1 (invA) mutant caused cecal, but not colonic inflammation and did not cause diarrhea. A phoP- mutant did not cause manifestations of diarrhea in either normal or NADPHdeficient (gp91 phox) mice. However, strain 14028s caused severe colitis and diarrhea in gp91 phox-deficient mice on an ItyR background. pmr A and F mutants, which are less virulent in orally infected BALB/c mice, were fully virulent in this model of colitis. Conclusions: S. enterica must be able to invade the colonic epithelium and to persist in the colon in order to cause colitis with manifestations of diarrhea. The NADPH oxidase is not required for diarrhea in Salmonella colitis. Furthermore,

From North Africa to Latin America and back: comparative findings and theoretical reflections

Taking the different case studies of the book together, one general observation stands out: Key agents of socioeconomic contention, including movements by organized labor and the unemployed that were important in the run-up to the uprisings and that saw their political opportunities open up in the immediate aftermath of the revolutions, have since been effectively marginalized as political actors. The concluding chapter reflects on the causes of this weakness of socioeconomic contention by identifying comparative insights that emerge from the contributions to this volume and by situating them in the context of broader comparative and theoretical debates on the relationship between social movements and political change. More specifically, the chapter first discusses Egypt’s and Tunisia’s post-revolutionary trajectories from a comparative perspective. Second, it discusses these comparative findings in the light of experiences in Latin America. Third, drawing again on comparative scholarship on Latin America, the chapter offers a theoretical interpretation of some of the main dynamics observed in Egypt and Tunisia based on the notion of a popular-sector incorporation crisis. Fourth and finally, the chapter concludes with general implications and an outlook

The Conserved Tarp Actin Binding Domain Is Important for Chlamydial Invasion

The translocated actin recruiting phosphoprotein (Tarp) is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells