Fast and accurate mutation detection in whole genome sequences of multiple isogenic samples with IsoMut

Background: Detection of somatic mutations is one of the main goals of next generation DNA sequencing. A wide range of experimental systems are available for the study of spontaneous or environmentally induced mutagenic processes. However, most of the routinely used mutation calling algorithms are not optimised for the simultaneous analysis of multiple samples, or for non-human experimental model systems with no reliable databases of common genetic variations. Most standard tools either require numerous in-house post filtering steps with scarce documentation or take an unpractically long time to run. To overcome these problems, we designed the streamlined IsoMut tool which can be readily adapted to experimental scenarios where the goal is the identification of experimentally induced mutations in multiple isogenic samples. Methods: Using 30 isogenic samples, reliable cohorts of validated mutations were created for testing purposes. Optimal values of the filtering parameters of IsoMut were determined in a thorough and strict optimization procedure based on these test sets. Results: We show that IsoMut, when tuned correctly, decreases the false positive rate compared to conventional tools in a 30 sample experimental setup; and detects not only single nucleotide variations, but short insertions and deletions as well. IsoMut can also be run more than a hundred times faster than the most precise state of art tool, due its straightforward and easily understandable filtering algorithm. Conclusions: IsoMut has already been successfully applied in multiple recent studies to find unique, treatment induced mutations in sets of isogenic samples with very low false positive rates. These types of studies provide an important contribution to determining the mutagenic effect of environmental agents or genetic defects, and IsoMut turned out to be an invaluable tool in the analysis of such data. © 2017 The Author(s)

Rapid Immunomagnetic Negative Enrichment of Neutrophil Granulocytes from Murine Bone Marrow for Functional Studies In Vitro and In Vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo

SWiM: Secure Wildcard Pattern Matching From OT Extension

Suppose a server holds a long text string and a receiver holds a short pattern string. Secure pattern matching allows the receiver to learn the locations in the long text where the pattern appears, while leaking nothing else to either party besides the length of their inputs. In this work we consider secure wildcard pattern matching WPM, where the receiver\u27s pattern is allowed to contain wildcards that match to any character. We present SWiM, a simple and fast protocol for WPM that is heavily based on oblivious transfer (OT) extension. As such, the protocol requires only a small constant number of public-key operations and otherwise uses only very fast symmetric-key primitives. SWiM is secure against semi-honest adversaries. We implemented a prototype of our protocol to demonstrate its practicality. We can perform WPM on a DNA text (4-character alphabet) of length $10^5$ and pattern of length $10^3$ in just over 2 seconds, which is over two orders of magnitude faster than the state-of-the-art scheme of Baron et al. (SCN 2012)

Towards the Establishment of a Porcine Model to Study Human Amebiasis

BACKGROUND: Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis. METHODOLOGY/PRINCIPAL FINDINGS: We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma. CONCLUSIONS: The pig model could help with simultaneously studying intestinal and extraintestinal lesion development

The cystic fibrosis microbiome in an ecological perspective and its impact in antibiotic therapy

The recent focus on the cystic fibrosis (CF) complex microbiome has led to the recognition that the microbes can interact between them and with the host immune system, affecting the disease progression and treatment routes. Although the main focus remains on the interactions between traditional pathogens, growing evidence supports the contribution and the role of emergent species. Understanding the mechanisms and the biological effects involved in polymicrobial interactions may be the key to improve effective therapies and also to define new strategies for disease control. This review focuses on the interactions between microbe-microbe and host-microbe, from an ecological point of view, discussing their impact on CF disease progression. There are increasing indications that these interactions impact the success of antimicrobial therapy. Consequently, a new approach where therapy is personalized to patients by taking into account their individual CF microbiome is suggested.Portuguese Foundation for Science and Technology (FCT), the strategic funding of UID/BIO/04469/2013-CEB and UID/EQU/00511/2013-LEPABE units. This study was also supported by FCT and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects “DNA mimics” PIC/IC/82815/2007, RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), “BioHealth—Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027 and NORTE-07-0124-FEDER-000025—RL2_ Environment and Health, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. The authors also acknowledge the grant of Susana P. Lopes (SFRH/BPD/95616/2013) and of the COST-Action TD1004: Theragnostics for imaging and therapy

The Response of Lemna minor to Mixtures of Pesticides That Are Commonly Used in Thailand

In the field, aquatic organisms are exposed to multiple contaminants rather than to single compounds. It is therefore important to understand the toxic interactions of co-occurring substances in the environment. The aim of the study was to assess the effects of individual herbicides (atrazine, 2,4-D, alachlor and paraquat) that are commonly used in Thailand and their mixtures on Lemna minor. Plants were exposed to individual and binary mixtures for 7 days and the effects on plant growth rate were assesed based on frond area measurements. Experimental observations of mixture toxicity were compared with predictions based on single herbicide exposure data using concentration addition and independent action models. The single compound studies showed that paraquat and alachlor were most toxic to L. minor, followed by atrazine and then 2,4-D. For the mixtures, atrazine with 2,4-D appeared to act antagonistically, whereas alachlor and paraquat showed synergism