Detailed imaging and genetic analysis reveal a secondary BRAFL505H resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three‐dimensional imaging. In all patients, only a subset of lesions progressed. Next‐generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib‐resistant patient‐derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.     

Energy Expenditure and Metabolic Changes of Free-Flying Migrating Northern Bald Ibis

Many migrating birds undertake extraordinary long flights. How birds are able to perform such endurance flights of over 100-hour durations is still poorly understood. We examined energy expenditure and physiological changes in Northern Bald Ibis Geronticus eremite during natural flights using birds trained to follow an ultra-light aircraft. Because these birds were tame, with foster parents, we were able to bleed them immediately prior to and after each flight. Flight duration was experimentally designed ranging between one and almost four hours continuous flights. Energy expenditure during flight was estimated using doubly-labelled-water while physiological properties were assessed through blood chemistry including plasma metabolites, enzymes, electrolytes, blood gases, and reactive oxygen compounds. Instantaneous energy expenditure decreased with flight duration, and the birds appeared to balance aerobic and anaerobic metabolism, using fat, carbohydrate and protein as fuel. This made flight both economic and tolerable. The observed effects resemble classical exercise adaptations that can limit duration of exercise while reducing energetic output. There were also in-flight benefits that enable power output variation from cruising to manoeuvring. These adaptations share characteristics with physiological processes that have facilitated other athletic feats in nature and might enable the extraordinary long flights of migratory birds as well.     

Deficiency of Nuclear Receptor Nur77 Aggravates Mouse Experimental Colitis by Increased NFκB Activity in Macrophages

Nuclear receptor Nur77, also referred to as NR4A1 or TR3, plays an important role in innate and adaptive immunity. Nur77 is crucial in regulating the T helper 1/regulatory T-cell balance, is expressed in macrophages and drives M2 macrophage polarization. In this study we aimed to define the function of Nur77 in inflammatory bowel disease. In wild-type and Nur77^-/- mice, colitis development was studied in dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models. To understand the underlying mechanism, Nur77 was overexpressed in macrophages and gut epithelial cells. Nur77 protein is expressed in colon tissues from Crohn’s disease and Ulcerative colitis patients and colons from colitic mice in inflammatory cells and epithelium. In both mouse colitis models inflammation was increased in Nur77^-/- mice. A higher neutrophil influx and enhanced IL-6, MCP-1 and KC production was observed in Nur77-deficient colons after DSS-treatment. TNBS-induced influx of T-cells and inflammatory monocytes into the colon was higher in Nur77^-/- mice, along with increased expression of MCP-1, TNFα and IL-6, and decreased Foxp3 RNA expression, compared to wild-type mice. Overexpression of Nur77 in lipopolysaccharide activated RAW macrophages resulted in up-regulated IL-10 and downregulated TNFα, MIF-1 and MCP-1 mRNA expression through NFκB repression. Nur77 also strongly decreased expression of MCP-1, CXCL1, IL-8, MIP-1α and TNFα in gut epithelial Caco-2 cells. Nur77 overexpression suppresses the inflammatory status of both macrophages and gut epithelial cells and together with the in vivo mouse data this supports that Nur77 has a protective function in experimental colitis. These findings may have implications for development of novel targeted treatment strategies regarding inflammatory bowel disease and other inflammatory diseases.     

Transient Marker System for Iterative Gene Targeting of a Prototrophic Fungus

Auxotrophic microorganisms are often used for genetic engineering, because their biosynthetic deficiency can be complemented by the transforming DNA and allows selection for transformants that have become prototrophic. However, when complementation is obtained by ectopic expression this may lead to unpredictable side effects on the phenotype and, consequently, misinterpretation of experimental data. There are various ways to overcome the problem of auxotrophy, but the most reliable is to restore the function of the defective biosynthetic gene at the native genomic locus. This can be done by either sexual crossing or further genetic engineering. For fungal species lacking a perfect state or situations in which gene targeting is generally cumbersome we have developed a concept that allows transient disruption of pyrG. When the gene is in the disrupted state, multiple rounds of gene targeting can be performed with the strain. Once the desired genome engineering is completed, pyrG function can be rapidly returned to wild type by a simple selection scheme.     

Seasonal encoding by the circadian pacemaker of the SCN

The circadian pacemaker of the suprachiasmatic nucleus (SCN) functions as a seasonal clock through its ability to encode day length [1-6]. To investigate the mechanism by which SCN neurons code for day length, we housed mice under long (LD 16:8) and short (LD 8:16) photoperiods. Electrophysiological recordings of multiunit activity (MUA) in the SCN of freely moving mice revealed broad activity profiles in long days and compressed activity profiles in short days. The patterns remained consistent after release of the mice in constant darkness. Recordings of MUA in acutely prepared hypothalamic slices showed similar differences between the SCN electrical activity patterns in vitro in long and short days. In vitro recordings of neuronal subpopulations revealed that the width of the MUA activity profiles was determined by the distribution of phases of contributing units within the SCN. The subpopulation patterns displayed a significantly broader distribution in long days than in short days. Long-term recordings of single-unit activity revealed short durations of elevated activity in both short and long days (3.48 and 3.85 hr, respectively). The data indicate that coding for day length involves plasticity within SCN neuronal networks in which the phase distribution of oscillating neurons carries information on the photoperiod's duration.     

Dynamics of competitive population abundance of Lactobacillus plantarum ivi gene mutants in faecal samples after passage through the gastrointestinal tract of mice

AIM: This study aims to evaluate the impact of mutation of previously identified in vivo-induced (ivi) genes on the persistence and survival of Lactobacillus plantarum WCFS1 in the gastrointestinal (GI) tract of mice. METHODS AND RESULTS: Nine Lact. plantarum ivi gene replacement mutants were constructed, focussing on ivi genes that encode proteins with a predicted role in cell envelope functionality, stress response and regulation. The in vitro growth characteristics of the mutants appeared identical to those observed for the wild-type strain, which agrees with the recombination-based in vivo expression technology suggestion that these genes are not transcribed in the laboratory. Quantitative PCR experiments demonstrated differences in the relative population dynamics of the Lact. plantarum ivi mutants in faecal samples after passage through the GI tract of mice. CONCLUSIONS: The in situ competition experiments revealed a 100- to 1000-fold reduction of the relative abundance of three of the ivi gene mutants, harbouring deletions of genes predicted to encode a copper transporter, an orphan IIC cellobiose PTS and a cell wall anchored extracellular protein. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments clearly establish that the proteins encoded by these three genes play a key role in Lact. plantarum performance during passage of the GI tract.     

Ligand-induced monomerization of <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis>′ studied using native mass spectrometry and fluorescence resonance energy transfer

Cytochrome c' from Allochromatium vinosum is an attractive model protein to study ligand-induced conformational changes. This homodimeric protein dissociates into monomers upon binding of NO, CO or CN(-) to the iron of its covalently attached heme group. While ligand binding to the heme has been well characterized using a variety of spectroscopic techniques, direct monitoring of the subsequent monomerization has not been reported previously. Here we have explored two biophysical techniques to simultaneously monitor ligand binding and monomerization. Native mass spectrometry allowed the detection of the dimeric and monomeric forms of cytochrome c' and even showed the presence of a CO-bound monomer. The kinetics of the ligand-induced monomerization were found to be significantly enhanced in the gas phase compared with the kinetics in solution, however. Ligand binding to the heme and the dissociation of the dimer in solution were also studied using energy transfer from a fluorescent probe to both heme groups of the protein. Comparison of ligand binding kinetics as observed with UV-vis spectroscopy with changes in fluorescence suggested that binding of one CO molecule per dimer could be sufficient for monomerization.