Proceedings of the 2nd Workshop on Flavor Symmetries and Consequences in Accelerators and Cosmology (FLASY12)

These are the proceedings of the 2nd Workshop on Flavor Symmetries and Consequences in Accelerators and Cosmology, held 30 June 2012 - 4 July 2012, Dortmund, Germany.Comment: Order 400 pages, several figures including the group picture v2: corrected author list and contributio

Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d

Highly multiplexed subcellular RNA sequencing in situ

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ

A novel in vitro assay for assessing efficacy and toxicity of antifungals using human leukemic cells infected with Candida albicans

Aims This study describes a novel in vitro assay that simultaneously determines antifungal efficiency and host cell toxicity using suspensions of human leukemic cells (HL-60) infected with Candida albicans. Methods and Results The effect of Candida infection on host cell viability was evaluated by microscopy of trypan blue stained cells and lactate dehydrogenase (LDH) activity. The in vitro ‘drug potency assay’ utilized the Cell Counting Kit-8 and measured post antifungal treatment viability of Candida-infected HL-60 cells and ability of the antifungal to prevent infection. LDH activity showed that 42% ± 4.0 and 85.3% ± 7.40 of HL-60 cells were killed following Candida infection at multiplicity of infection (MOI) of 1:1 and 1:5, respectively. Using the assay, the antifungal nystatin (0.78-25 μmol l−1) was found to inhibit C. albicans infection as seen by significantly increased viability of HL-60 cells. Using the assay, cytotoxicity of nystatin towards infected HL-60 cells was evident at higher concentrations and this was also confirmed by propidium iodide staining. Conclusions An assay using undisturbed cell suspension conditions was successfully developed for assessing selectivity of antifungal therapy in the host-Candida environment. Significance and Impact of the Study The assay employing Candida infection of host cell suspensions represents a promising method for testing interactions of antifungal compounds with both fungal and host cells

Value-Based File Retention: File Attributes as File Value and Information Waste Indicators

Several file retention policy methods propose that a file retention policy should be based on file value. Though such a retention policy might increase the value of accessible files, the method to arrive at such a policy is underresearched. This article discusses how one can arrive at a method for developing file retention policies based on the use values of files. The method’s applicability is initially assessed through a case study at Capgemini, Netherlands. In the case study, we hypothesize that one can develop a file retention policy by testing causal relations between file attributes (as used by file retention methods) and the use value of files. Unfortunately, most file attributes used by file retention methods have a weak correlation with file value, resulting in the conclusion that these methods do not well select out high- and low-value files. This would imply the ineffectiveness of the used attributes in our study or errors in our conceptualization of file value. We continue with the last possibility and develop indicators for file utility (with low utility being waste). With this approach we were able to detect waste files, in a sample of files, with an accuracy of 80%. We therefore not only suggest further research in information waste detection as part of a file retention policy, but also to further explore other file attributes that could better predict file value and file utility

Joint cell segmentation and cell type annotation for spatial transcriptomics

RNA hybridization‐based spatial transcriptomics provides unparalleled detection sensitivity. However, inaccuracies in segmentation of image volumes into cells cause misassignment of mRNAs which is a major source of errors. Here, we develop JSTA, a computational framework for joint cell segmentation and cell type annotation that utilizes prior knowledge of cell type‐specific gene expression. Simulation results show that leveraging existing cell type taxonomy increases RNA assignment accuracy by more than 45%. Using JSTA, we were able to classify cells in the mouse hippocampus into 133 (sub)types revealing the spatial organization of CA1, CA3, and Sst neuron subtypes. Analysis of within cell subtype spatial differential gene expression of 80 candidate genes identified 63 with statistically significant spatial differential gene expression across 61 (sub)types. Overall, our work demonstrates that known cell type expression patterns can be leveraged to improve the accuracy of RNA hybridization‐based spatial transcriptomics while providing highly granular cell (sub)type information. The large number of newly discovered spatial gene expression patterns substantiates the need for accurate spatial transcriptomic measurements that can provide information beyond cell (sub)type labels

The TeMPO trial (treatment of meniscal tears in osteoarthritis): rationale and design features for a four arm randomized controlled clinical trial

BACKGROUND: Meniscal tears often accompany knee osteoarthritis, a disabling condition affecting 14 million individuals in the United States. While several randomized controlled trials have compared physical therapy to surgery for individuals with knee pain, meniscal tear, and osteoarthritic changes (determined via radiographs or magnetic resonance imaging), no trial has evaluated the efficacy of physical therapy alone in these subjects. METHODS: The Treatment of Meniscal Tear in Osteoarthritis (TeMPO) Trial is a four-arm multi-center randomized controlled clinical trial designed to establish the comparative efficacy of two in-clinic physical therapy interventions (one focused on strengthening and one containing placebo) and two protocolized home exercise programs. DISCUSSION: The goal of this paper is to present the rationale behind TeMPO and describe the study design and implementation strategies, focusing on methodologic and clinical challenges. TRIAL REGISTRATION: The TeMPO Trial was first registered at clinicaltrials.gov with registration No. NCT03059004 . on February 14, 2017