Novel Defect Structures in Nematic Liquid Crystal Shells

We use double-emulsion drops to experimentally investigate the defect structures of spherical shells of nematic liquid crystals. We uncover a rich scenario of coexisting defect structures dictated by the unavoidable finite thickness of even the thinnest shell and by the thickness variation around the sphere. These structures are characterized by a varying number of disclination lines and pairs of surface point defects on the inner and outer surfaces of the nematic shell. In the limit of very thick shells the defect structure ultimately merges with that of a bulk nematic liquid crystal drop

Quantitative detection of circulating tumor DNA by droplet-based digital PCR.

Droplet-based digital PCR is used to detect and quantify the seven most frequent KRAS mutations in circulating tumor DNA of patients with advanced colorectal cancer.- The droplet-based digital PCR method is versatile and two modes of analysis are demonstrated: o Duplex analysis enables sensitive detection of wild-type DNA plus one KRAS or BRAF mutation. o Multiplex analysis enables simultaneous detection of wild-type DNA plus 3 or 4 KRAS mutations.- Detection of rare sequences is highly sensitive compared to the same Taqman assay in bulk (10 % LLOD bulk vs 0.0005 % LLOD droplets).- Biomarkers detection is quantitative: the fraction of mutated DNA in patient samples ranges from 0.1 % to 42%.- Results from circulating tumor DNA analysis match the tumor DNA characterization in most cases, and discordant results reveal need for further studies. Copy and paste your text content here, adjusting the font size to fit Background Circulating tumor DNA (ctDNA) is present in plasma of individuals with advanced cancers. 1 ctDNA is a prognostic marker for patients with colorectal cancer (CRC) and it might also be used for predicting the response to targeted therapy. For example, mutations in KRAS indicate which patients will fail to respond to specific therapies (cetuximab, panitunimab). 2 Although ctDNA is characterized by the presence of a somatic mutation, direct quantitative detection through a simple workflow of such mutant DNA is not feasible by current technologies because the ratio of ctDNA to wildtype DNA can be as low as 1/10,000. This study describes the use of droplet-based digital PCR for detection and quantitation of one of the seven most frequent KRAS mutations in ctDNA from plasm

Detection of low prevalence somatic mutations in solid tumors with ultra-deep targeted sequencing

Ultra-deep targeted sequencing (UDT-Seq) can identify subclonal somatic mutations in tumor samples. Early assays' limited breadth and depth restrict their clinical utility. Here, we target 71 kb of mutational hotspots in 42 cancer genes. We present novel methods enhancing both laboratory workflow and mutation detection. We evaluate UDT-Seq true sensitivity and specificity (> 94% and > 99%, respectively) for low prevalence mutations in a mixing experiment and demonstrate its utility using six tumor samples. With an improved performance when run on the Illumina Miseq, the UDT-Seq assay is well suited for clinical applications to guide therapy and study clonal selection in heterogeneous samples

Do Behavioral Foraging Responses of Prey to Predators Function Similarly in Restored and Pristine Foodwebs?

Efforts to restore top predators in human-altered systems raise the question of whether rebounds in predator populations are sufficient to restore pristine foodweb dynamics. Ocean ecosystems provide an ideal system to test this question. Removal of fishing in marine reserves often reverses declines in predator densities and size. However, whether this leads to restoration of key functional characteristics of foodwebs, especially prey foraging behavior, is unclear. The question of whether restored and pristine foodwebs function similarly is nonetheless critically important for management and restoration efforts. We explored this question in light of one important determinant of ecosystem function and structure – herbivorous prey foraging behavior. We compared these responses for two functionally distinct herbivorous prey fishes (the damselfish Plectroglyphidodon dickii and the parrotfish Chlorurus sordidus) within pairs of coral reefs in pristine and restored ecosystems in two regions of these species' biogeographic ranges, allowing us to quantify the magnitude and temporal scale of this key ecosystem variable's recovery. We demonstrate that restoration of top predator abundances also restored prey foraging excursion behaviors to a condition closely resembling those of a pristine ecosystem. Increased understanding of behavioral aspects of ecosystem change will greatly improve our ability to predict the cascading consequences of conservation tools aimed at ecological restoration, such as marine reserves

Pipeline for Large-Scale Microdroplet Bisulfite PCR-Based Sequencing Allows the Tracking of Hepitype Evolution in Tumors

Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into “hepitypes” and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer

Determining lower limits of detection of digital PCR assays for cancer-related gene mutations

Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD) of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR) gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were explored by the same approach. For the EGFR L8585R assay, the assay sensitivity is extremely good, and thus, the LoD is limited by the amount of amplifiable DNA that is analyzed. With 95% confidence limits, the LoD is one mutant in 180,000 wild-type molecules for the evaluation of 3.3 μg of genomic DNA, and detection of one mutant molecule in over 4 million wild-type molecules was achieved when 70 million copies of DNA were processed. The measured false-positive rate for the EGFR L8585R assay is one in 14 million, which indicates the theoretical LoD if an unlimited amount of DNA is evaluated. For the EFGR T790M assay, the LoD is one mutant in 13,000 for analysis of a 3.3 μg sample of genomic DNA, and the dPCR assay limit sensitivity approaches one mutant in 22,000 wild-type molecules