Allele-specific endogenous tagging and quantitative analysis of β-catenin in colorectal cancer cells

Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in β-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of β-catenin are not fully understood. Here, we genome-engineered fluorescently tagged alleles of endogenous β-catenin in a colorectal cancer cell line. Wild-type and oncogenic mutant alleles were tagged with different fluorescent proteins, enabling the analysis of both variants in the same cell. We analyzed the properties of both β-catenin alleles using immunoprecipitation, immunofluorescence, and fluorescence correlation spectroscopy approaches, revealing distinctly different biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3β inhibitor or a truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant β-catenin allele. The one-step tagging strategy demonstrates how genome engineering can be employed for the parallel functional analysis of different genetic variants

Cell Detection with Star-convex Polygons

Automatic detection and segmentation of cells and nuclei in microscopy images is important for many biological applications. Recent successful learning-based approaches include per-pixel cell segmentation with subsequent pixel grouping, or localization of bounding boxes with subsequent shape refinement. In situations of crowded cells, these can be prone to segmentation errors, such as falsely merging bordering cells or suppressing valid cell instances due to the poor approximation with bounding boxes. To overcome these issues, we propose to localize cell nuclei via star-convex polygons, which are a much better shape representation as compared to bounding boxes and thus do not need shape refinement. To that end, we train a convolutional neural network that predicts for every pixel a polygon for the cell instance at that position. We demonstrate the merits of our approach on two synthetic datasets and one challenging dataset of diverse fluorescence microscopy images.Comment: Conference paper at MICCAI 201

EBImage—an R package for image processing with applications to cellular phenotypes

Summary: EBImage provides general purpose functionality for reading, writing, processing and analysis of images. Furthermore, in the context of microscopy-based cellular assays, EBImage offers tools to segment cells and extract quantitative cellular descriptors. This allows the automation of such tasks using the R programming language and use of existing tools in the R environment for signal processing, statistical modeling, machine learning and data visualization

Wearable Positive End-Expiratory Pressure Valve Improves Exercise Performance

Positive end-expiratory pressure (PEEP) provides benefits to pulmonary patients, yet effects in healthy, exercising adults are unknown. PURPOSE: We designed two experiments (EXP) to test a novel PEEP (4.2 cmH2O PEEP) mouthpiece (PMP) on maximal cycling performance of physically active volunteers. METHODS: EXP-1 PMP vs. control (CON) mouthpiece (N=9, Age=30±2 yr, Weight=72.2±3.7 kg, BMI=24.4±1.2, 5♂); and EXP-2 PMP vs. no mouthpiece (NMP) (N=10, Age=27±1 yr, Weight=76.7±3.6 kg, BMI=23.9±0.8, ♂). Exercise test procedures for both experiments were identical. On Day 1, under the first mouthpiece condition assigned at random subjects performed graded exercise cycling testing (GXT) (Corival®) for VO2peak (ml*kg*min-1), oxygen pulse (mlO2*bt) (O2pulse), GXT endurance time (s) (GXT-T), and VO2(ml*kg*min-1)-at-ventilatory-threshold (VO2 @VT). Subjects returned 72 h later (Day 2), to complete an endurance ride timed (s)to exhaustion (VTER) at an intensity equivalent to their VO2 @VT power (W). One week later, subjects repeated exercise testing protocols (Days 3 & 4, time-of-day controlled) under the alternate mouthpiece condition. RESULTS: Selected outcomes were as follows (paired T-test, *PMP vs. CON, respectively: VO2peak=45.2±2.4* vs. 42.4±2.3; VO2@VT=33.7±2.0 vs. 32.3±1.6; GXT-T=521.7±73.4* vs. 495.3±72.8; VTER=846.2±166.0 vs. 743.1±124.7; O2pulse=24.5±1.4* vs. 23.1±1.3. PMP vs. NMP, respectively: VO2peak=43.3±1.6* vs. 41.7±1.6; VO2@AT=31.1±1.2* vs. 29.1±1.3; GXT-T=511.7*±49.6 vs. 486.4±49.6; VTER 872.4±134.0 vs. 792.9 ± 122.4; O2pulse=24.1±0.9* vs. 23.4±0.9. CONCLUSION: These results demonstrate that the novel PEEP mouthpiece we tested confers a significant performance benefit to cyclists completing high intensity exercise. By extension, it is likely to be an advantage in any physical activity having an aerobic component

HTSanalyzeR: an R/Bioconductor package for integrated network analysis of high-throughput screens

Motivation: High-throughput screens (HTS) by RNAi or small molecules are among the most promising tools in functional genomics. They enable researchers to observe detailed reactions to experimental perturbations on a genome-wide scale. While there is a core set of computational approaches used in many publications to analyze these data, a specialized software combining them and making them easily accessible has so far been missing

An integrated gene annotation and transcriptional profiling approach towards the full gene content of the Drosophila genome

BACKGROUND: While the genome sequences for a variety of organisms are now available, the precise number of the genes encoded is still a matter of debate. For the human genome several stringent annotation approaches have resulted in the same number of potential genes, but a careful comparison revealed only limited overlap. This indicates that only the combination of different computational prediction methods and experimental evaluation of such in silico data will provide more complete genome annotations. In order to get a more complete gene content of the Drosophila melanogaster genome, we based our new D. melanogaster whole-transcriptome microarray, the Heidelberg FlyArray, on the combination of the Berkeley Drosophila Genome Project (BDGP) annotation and a novel ab initio gene prediction of lower stringency using the Fgenesh software. RESULTS: Here we provide evidence for the transcription of approximately 2,600 additional genes predicted by Fgenesh. Validation of the developmental profiling data by RT-PCR and in situ hybridization indicates a lower limit of 2,000 novel annotations, thus substantially raising the number of genes that make a fly. CONCLUSIONS: The successful design and application of this novel Drosophila microarray on the basis of our integrated in silico/wet biology approach confirms our expectation that in silico approaches alone will always tend to be incomplete. The identification of at least 2,000 novel genes highlights the importance of gathering experimental evidence to discover all genes within a genome. Moreover, as such an approach is independent of homology criteria, it will allow the discovery of novel genes unrelated to known protein families or those that have not been strictly conserved between species

Robust Wnt signaling is maintained by a Wg protein gradient and Fz2 receptor activity in the developing Drosophila wing

Wnts are secreted proteins that regulate cell fate during development of all metazoans. Wnt proteins were proposed to spread over several cells to activate signaling directly at a distance. In the Drosophila wing epithelium, an extracellular gradient of the Wnt1 homolog Wingless (Wg) was observed extending over several cells away from producing cells. Surprisingly, however, it was also shown that a membrane-tethered Neurotactin-Wg fusion protein (NRT-Wg) can largely replace endogenous Wg, leading to proper patterning of the wing. Therefore, the functional range of Wg and whether Wg spreading is required for correct tissue patterning remains controversial. Here, by capturing secreted Wg on cells away from the source, we show that Wg acts over a distance of up to 11 cell diameters to induce signaling. Furthermore, cells located outside the reach of extracellular Wg depend on the Frizzled2 receptor to maintain signaling. Frizzled2 expression is increased in the absence of Wg secretion and is required to maintain signaling and cell survival in NRT-wg wing discs. Together, these results provide insight into the mechanisms by which robust Wnt signaling is achieved in proliferating tissues