A laboratory study of water ice erosion by low-energy ions

Water ice covers the surface of various objects in the outer Solar system.Within the heliopause, surface ice is constantly bombarded and sputtered by energetic particles from the solar wind and magnetospheres. We report a laboratory investigation of the sputtering yield of water ice when irradiated at 10 K by 4 keV singly (13C+, N+, O+, Ar+) and doubly charged ions (13C2+, N2+, O2+). The experimental values for the sputtering yields are in good agreement with the prediction of a theoretical model. There is no significant difference in the yield for singly and doubly charged ions. Using these yields, we estimate the rate of water ice erosion in the outer Solar system objects due to solar wind sputtering. Temperature-programmed desorption of the ice after irradiation with 13C+ and 13C2+ demonstrated the formation of 13CO and 13CO2, with 13CO being the dominant formed species.

Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

AIMS: Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. METHODS: 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. RESULTS: Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p

In Vitro modelling of RSV infection and cytopathogenesis in well-differentiated human primary airway epithelial cells (WD-PAECs).

The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis.

Mapping Protein-DNA Interactions Using ChIP-exo and Illumina-Based Sequencing

Chromatin immunoprecipitation (ChIP) provides a means of enriching DNA associated with transcription factors, histone modifications, and indeed any other proteins for which suitably characterized antibodies are available. Over the years, sequence detection has progressed from quantitative real-time PCR and Southern blotting to microarrays (ChIP-chip) and now high-throughput sequencing (ChIP-seq). This progression has vastly increased the sequence coverage and data volumes generated. This in turn has enabled informaticians to predict the identity of multi-protein complexes on DNA based on the overrepresentation of sequence motifs in DNA enriched by ChIP with a single antibody against a single protein. In the course of the development of high-throughput sequencing, little has changed in the ChIP methodology until recently. In the last three years, a number of modifications have been made to the ChIP protocol with the goal of enhancing the sensitivity of the method and further reducing the levels of nonspecific background sequences in ChIPped samples. In this chapter, we provide a brief commentary on these methodological changes and describe a detailed ChIP-exo method able to generate narrower peaks and greater peak coverage from ChIPped material.

Analysis of Histone Deacetylase 7 (HDAC7) Alternative Splicing and Its Role in Embryonic Stem Cell Differentiation Toward Smooth Muscle Lineage

Histone deacetylases (HDACs) have a central role in the regulation of gene expression, which undergoes alternative splicing during embryonic stem cell (ES) cell differentiation. Alternative splicing gives rise to vast diversity over gene information, arousing public concerns in the last decade. In this chapter, we describe a strategy to detect HDAC7 alternative splicing and analyze its function on ES cell differentiation.

Incomplete DJH rearrangements.

Analysis of Ig genes in B-cell malignancies has become an essential method in molecular diagnosis, and polymerase chain reaction (PCR) amplification of Ig heavy chain gene (IgH) rearrangements is now widely used for detection of clonality and minimal residual disease (MRD). Although several different sensitive protocols are now available for PCR analysis of IgH genes, they are frequently hampered owing to the high rate of somatic hypermutation present in multiple myeloma (MM). We recently described a new approach using incomplete DJH rearrangements as an alternative target. About 60% of MM samples contain an incomplete DJH rearrangement, 90% of them lacking on somatic mutations. This approach allows resolution of problems derived from primer mismatches, making DJH rearrangement a reliable and sensitive target for detection of clonality and MRD investigation in MM.

Subcellular localization of legionella Dot/Icm effectors

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.

Point-and-shoot: rapid quantitative detection methods for on-site food fraud analysis – moving out of the laboratory and into the food supply chain

Major food adulteration and contamination events occur with alarming regularity and are known to be episodic, with the question being not if but when another large-scale food safety/integrity incident will occur. Indeed, the challenges of maintaining food security are now internationally recognised. The ever increasing scale and complexity of food supply networks can lead to them becoming significantly more vulnerable to fraud and contamination, and potentially dysfunctional. This can make the task of deciding which analytical methods are more suitable to collect and analyse (bio)chemical data within complex food supply chains, at targeted points of vulnerability, that much more challenging. It is evident that those working within and associated with the food industry are seeking rapid, user-friendly methods to detect food fraud and contamination, and rapid/high-throughput screening methods for the analysis of food in general. In addition to being robust and reproducible, these methods should be portable and ideally handheld and/or remote sensor devices, that can be taken to or be positioned on/at-line at points of vulnerability along complex food supply networks and require a minimum amount of background training to acquire information rich data rapidly (ergo point-and-shoot). Here we briefly discuss a range of spectrometry and spectroscopy based approaches, many of which are commercially available, as well as other methods currently under development. We discuss a future perspective of how this range of detection methods in the growing sensor portfolio, along with developments in computational and information sciences such as predictive computing and the Internet of Things, will together form systems- and technology-based approaches that significantly reduce the areas of vulnerability to food crime within food supply chains. As food fraud is a problem of systems and therefore requires systems level solutions and thinking.

Inter-Laboratory Evaluation of a Next-Generation Sequencing Panel for Acute Myeloid Leukemia

INTRODUCTION: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder often associated with dismal overall survival. The clinical diversity of AML is reflected in the range of recurrent somatic mutations in several genes, many of which have a prognostic and therapeutic value. Targeted next-generation sequencing (NGS) of these genes has the potential for translation into clinical practice. In order to assess this potential, an inter-laboratory evaluation of a commercially available AML gene panel across three diagnostic centres in the UK and Ireland was performed.

METHODS: DNA from six AML patient samples was distributed to each centre and processed using a standardised workflow, including a common sequencing platform, sequencing chips and bioinformatics pipeline. A duplicate sample in each centre was run to assess inter- and intra-laboratory performance.

RESULTS: An average sample read depth of 2725X (range 629-5600) was achieved using six samples per chip, with some variability observed in the depth of coverage generated for individual samples and between centres. A total of 16 somatic mutations were detected in the six AML samples, with a mean of 2.7 mutations per sample (range 1-4) representing nine genes on the panel. 15/16 mutations were identified by all three centres. Allelic frequencies of the mutations ranged from 5.6 to 53.3 % (median 44.4 %), with a high level of concordance of these frequencies between centres, for mutations detected.

CONCLUSION: In this inter-laboratory comparison, a high concordance, reproducibility and robustness was demonstrated using a commercially available NGS AML gene panel and platform.