FADD, a novel death domain-containing protein, interacts with the death domain of fas and initiates apoptosis

AbstractUsing the cytoplasmic domain of Fas in the yeast two-hybrid system, we have identified a novel interacting protein, FADD, which binds Fas and Fas-FD5, a mutant of Fas possessing enhanced killing activity, but not the functionally inactive mutants Fas-LPR and Fas-FD8. FADD contains a death domain homologous to the death domains of Fas and TNFR-1. A point mutation in FADD, analogous to the Ipr mutation of Fas, abolishes its ability to bind Fas, suggesting a death domain to death domain interaction. Overexpression of FADD in MCF7 and BJAB cells induces apoptosis, which, like Fas-induced apoptosis, is blocked by CrmA, a specific inhibitor of the interieukin-lβ-converting enzyme. These findings suggest that FADD may play an important role in the proximal signal transduction of Fas

Accuracy, Reproducibility And Bias Of Next Generation Sequencing For Quantitative Small RNA Profiling: A Multiple Protocol Study Across Multiple Laboratories [preprint]

Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols

The Limits of Reductionism in Medicine: Could Systems Biology Offer an Alternative?

In the first of a two part series, Ahn and colleagues discuss the reductionist approach pervading medicine and explain how a systems approach (as advocated by systems biology) may complement reductionism

Identifying climate change information needs for the himalayan region: Results from the GLACINDIA Stakeholder Workshop and Training Program

Here we present results of a workshop designed to bring together stakeholders from different states of the Indian side of the Himalayan arc and an international group of climate scientists in order to discuss how climate change research for this region can be tailored toward the needs of local communities. The stakeholder workshop was jointly organized by the Jawaharlal Nehru University (JNU), New Delhi, India, and the Climate Service Center 2.0, Hamburg, Germany, within the framework of the multidisciplinary international research project GLACINDIA. The project focuses on the water-related effects of changes in glacier mass balance and river runoff in western Himalayas. Given the research focus of the GLACINDIA project, the initial focus of the workshop was on glacier-related hydrological information. During stakeholder interactions the resulting discussion covered a much broader range of urgent climate change information needs for the Himalayan region.publishedVersio

Phospho‐RNA‐seq: a modified small RNA‐seq method that reveals circulating mRNA and lncRNA fragments as potential biomarkers in human plasma

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNAs in blood plasma are extensively characterized, extracellular messenger RNA (mRNA) and long non‐coding RNA (lncRNA) studies are limited. We report that plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA‐seq protocols due to lack of 5′ phosphate or presence of 3′ phosphate. These fragments were revealed using a modified protocol (“phospho‐RNA‐seq”) incorporating RNA treatment with T4‐polynucleotide kinase, which we compared with standard small RNA‐seq for sequencing synthetic RNAs with varied 5′ and 3′ ends, as well as human plasma exRNA. Analyzing phospho‐RNA‐seq data using a custom, high‐stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue‐specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow‐ and liver‐enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof‐of‐concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho‐RNA‐seq opens up new possibilities for plasma transcriptomic biomarker development.SynopsisA modified RNA‐seq method (Phospho‐RNA‐seq) revealed a new population of mRNA/lncRNA fragments in plasma, including ones that track with disease. This opens up new possibilities for disease detection via RNA profiling of plasma and other biofluids.Phospho‐RNA‐seq reveals a large population of mRNA and long non‐coding RNA fragments in human plasma, which are missed by standard small RNA‐seq protocols that depend on target RNAs having a 5′ P and 3′ OH.Accurate detection of plasma mRNA and lncRNA fragments requires a stringent bioinformatic analysis pipeline to avoid false positive alignments to mRNA and lncRNA genes.Phospho‐RNA‐seq identified ensembles of tissue‐specific transcripts in plasma of hematopoietic stem cell transplant patients, which show co‐expression patterns that vary dynamically and track with pathophysiological processes.By enabling access to an unexplored space of extracellular mRNA and lncRNA fragments, phospho‐RNA‐seq opens up new possibilities for monitoring health and disease via transcriptome fragment profiling of plasma and potentially other biofluids.A modified RNA‐seq method reveals a large population of mRNA/lncRNA fragments in plasma that are missed by standard small RNA‐seq protocols including ones that are associated with disease.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/1/embj2019101695_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/2/embj2019101695-sup-0002-EVFigs.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/3/embj2019101695.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/4/embj2019101695-sup-0001-Appendix.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149518/5/embj2019101695.reviewer_comments.pd

Circulating microRNAs and treatment response in the Phase II SWOG S0925 study for patients with new metastatic hormone‐sensitive prostate cancer

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141181/1/pros23452.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141181/2/pros23452_am.pd

Ultra‐Specific Isolation of Circulating Tumor Cells Enables Rare‐Cell RNA Profiling

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134266/1/advs147_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134266/2/advs147-sup-0001-S1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134266/3/advs147.pd

Rapid, ultra low coverage copy number profiling of cell-free DNA as a precision oncology screening strategy.

Current cell-free DNA (cfDNA) next generation sequencing (NGS) precision oncology workflows are typically limited to targeted and/or disease-specific applications. In advanced cancer, disease burden and cfDNA tumor content are often elevated, yielding unique precision oncology opportunities. We sought to demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) as a precision oncology screening strategy via ultra-low coverage (~0.01x) tumor content determination through genome-wide copy number alteration (CNA) profiling. We applied PRINCe to a retrospective cohort of 124 cfDNA samples from 100 patients with advanced cancers, including 76 men with metastatic castration-resistant prostate cancer (mCRPC), enabling cfDNA tumor content approximation and actionable focal CNA detection, while facilitating concordance analyses between cfDNA and tissue-based NGS profiles and assessment of cfDNA alteration associations with mCRPC treatment outcomes. Therapeutically relevant focal CNAs were present in 42 (34%) cfDNA samples, including 36 of 93 (39%) mCRPC patient samples harboring AR amplification. PRINCe identified pre-treatment cfDNA CNA profiles facilitating disease monitoring. Combining PRINCe with routine targeted NGS of cfDNA enabled mutation and CNA assessment with coverages tuned to cfDNA tumor content. In mCRPC, genome-wide PRINCe cfDNA and matched tissue CNA profiles showed high concordance (median Pearson correlation = 0.87), and PRINCe detectable AR amplifications predicted reduced time on therapy, independent of therapy type (Kaplan-Meier log-rank test, chi-square = 24.9, p < 0.0001). Our screening approach enables robust, broadly applicable cfDNA-based precision oncology for patients with advanced cancer through scalable identification of therapeutically relevant CNAs and pre-/post-treatment genomic profiles, enabling cfDNA- or tissue-based precision oncology workflow optimization