Breadth versus depth: whole transcriptome sequencing has reduced sensitivity for detection of clinically relevant fusions compared to RNA comprehensive genomic profiling.

While there is great potential for unbiased next-generation sequencing (NGS) approaches-eg, whole transcriptome sequencing (WTS)-for exploration, discovery, and clinical application in the realm of oncology, there are limitations that should be considered when relying on these methodologies for clinical decision making. When using WTS for the detection of clinically relevant gene fusions in tumor specimens, a key consideration is whether a limited coverage depth (approximately 30-50X) is sufficient for detecting these events, especially in samples with low tumor purity. We demonstrate the reduced sensitivity of both a commercial WTS assay for the detection of clinically relevant fusions in analytical validation control samples and of a research use only (RUO) WTS assay for the detection of clinically relevant fusions in real-world clinical samples compared to RNA comprehensive genomic profiling (CGP).

Astrocytes in the Optic Nerve Are Heterogeneous in Their Reactivity to Glaucomatous Injury.

The optic nerve head is thought to be the site of initial injury to retinal ganglion cell injury in glaucoma. In the initial segment of the optic nerve directly behind the globe, the ganglion cell axons are unmyelinated and come into direct contact to astrocytes, suggesting that astrocytes may play a role in the pathology of glaucoma. As in other parts of the CNS, optic nerve head astrocytes respond to injury by characteristic changes in cell morphology and gene expression profile.

IRF3 and type I interferons fuel a fatal response to myocardial infarction.

Interferon regulatory factor 3 (IRF3) and type I interferons (IFNs) protect against infections and cancer, but excessive IRF3 activation and type I IFN production cause autoinflammatory conditions such as Aicardi-Goutières syndrome and STING-associated vasculopathy of infancy (SAVI). Myocardial infarction (MI) elicits inflammation, but the dominant molecular drivers of MI-associated inflammation remain unclear. Here we show that ischemic cell death and uptake of cell debris by macrophages in the heart fuel a fatal response to MI by activating IRF3 and type I IFN production.

Genome-wide identification of autosomal genes with allelic imbalance of chromatin state.

In mammals, monoallelic gene expression can result from X-chromosome inactivation, genomic imprinting, and random monoallelic expression (RMAE). Epigenetic regulation of RMAE is not fully understood. Here we analyze allelic imbalance in chromatin state of autosomal genes using ChIP-seq in a clonal cell line.

Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells.

Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential.

Macrophages Facilitate Electrical Conduction in the Heart.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes.

DUSP9 Modulates DNA Hypomethylation in Female Mouse Pluripotent Stem Cells.

Blastocyst-derived embryonic stem cells (ESCs) and gonad-derived embryonic germ cells (EGCs) represent two classic types of pluripotent cell lines, yet their molecular equivalence remains incompletely understood. Here, we compare genome-wide methylation patterns between isogenic ESC and EGC lines to define epigenetic similarities and differences. Surprisingly, we find that sex rather than cell type drives methylation patterns in ESCs and EGCs.

Mutation of a nucleosome compaction region disrupts Polycomb-mediated axial patterning.

Nucleosomes play important structural and regulatory roles by tightly wrapping the DNA that constitutes the metazoan genome. The Polycomb group (PcG) proteins modulate nucleosomes to maintain repression of key developmental genes, including genes whose temporal and spatial expression is tightly regulated to guide patterning of the anterior-posterior body axis. CBX2, a component of the mammalian Polycomb repressive complex 1 (PRC1), contains a compaction region that has the biochemically defined activity of bridging adjacent nucleosomes.

Survival in Quiescence Requires the Euchromatic Deployment of Clr4/SUV39H by Argonaute-Associated Small RNAs.

Quiescence (G0) is a ubiquitous stress response through which cells enter reversible dormancy, acquiring distinct properties including reduced metabolism, resistance to stress, and long life. G0 entry involves dramatic changes to chromatin and transcription of cells, but the mechanisms coordinating these processes remain poorly understood. Using the fission yeast, here, we track G0-associated chromatin and transcriptional changes temporally and show that as cells enter G0, their survival and global gene expression programs become increasingly dependent on Clr4/SUV39H, the sole histone H3 lysine 9 (H3K9) methyltransferase, and RNAi proteins.

Approximating frustration scores in complex networks via perturbed Laplacian spectra.

Systems of many interacting components, as found in physics, biology, infrastructure, and the social sciences, are often modeled by simple networks of nodes and edges. The real-world systems frequently confront outside intervention or internal damage whose impact must be predicted or minimized, and such perturbations are then mimicked in the models by altering nodes or edges. This leads to the broad issue of how to best quantify changes in a model network after some type of perturbation.

Quantifying the Sources of Kinetic Frustration in Folding Simulations of Small Proteins.

Experiments and atomistic simulations of polypeptides have revealed structural intermediates that promote or inhibit conformational transitions to the native state during folding. We invoke a concept of "kinetic frustration" to quantify the prevalence and impact of these behaviors on folding rates within a large set of atomistic simulation data for 10 fast-folding proteins, where each protein's conformational space is represented as a Markov state model of conformational transitions. Our graph theoretic approach addresses what conformational features correlate with folding inhibition and therefore permits comparison among features within a single protein network and also more generally between proteins.

Protein conformational populations and functionally relevant substates.

Functioning proteins do not remain fixed in a unique structure, but instead they sample a range of conformations facilitated by motions within the protein. Even in the native state, a protein exists as a collection of interconverting conformations driven by thermodynamic fluctuations. Motions on the fast time scale allow a protein to sample conformations in the nearby area of its conformational landscape, while motions on slower time scales give it access to conformations in distal areas of the landscape.

Modeling boundary conditions for balanced proliferation in metastatic latency.

Purpose: Nearly half of cancer metastases become clinically evident five or more years after primary tumor treatment; thus, metastatic cells survived without emerging for extended periods. This dormancy has been explained by at least two countervailing scenarios: cellular quiescence and balanced proliferation; these entail dichotomous mechanistic etiologies. To examine the boundary parameters for balanced proliferation, we conducted in silico modeling.

Event detection and sub-state discovery from biomolecular simulations using higher-order statistics: application to enzyme adenylate kinase.

Biomolecular simulations at millisecond and longer time-scales can provide vital insights into functional mechanisms. Because post-simulation analyses of such large trajectory datasets can be a limiting factor in obtaining biological insights, there is an emerging need to identify key dynamical events and relating these events to the biological function online, that is, as simulations are progressing. Recently, we have introduced a novel computational technique, quasi-anharmonic analysis (QAA) (Ramanathan et al.

Quasi-anharmonic analysis reveals intermediate states in the nuclear co-activator receptor binding domain ensemble.

The molten globule nuclear receptor co-activator binding domain (NCBD) of CREB binding protein (CBP) selectively recruits transcription co-activators (TCAs) during the formation of the transcription preinitiation complex. NCBD:TCA interactions have been implicated in several cancers, however, the mechanisms of NCBD:TCA recognition remain uncharacterized. NCBD:TCA intermolecular recognition has challenged traditional investigation as both NCBD and several of its corresponding TCAs are intrinsically disordered.

QAARM: quasi-anharmonic autoregressive model reveals molecular recognition pathways in ubiquitin.

Motivation: Molecular dynamics (MD) simulations have dramatically improved the atomistic understanding of protein motions, energetics and function. These growing datasets have necessitated a corresponding emphasis on trajectory analysis methods for characterizing simulation data, particularly since functional protein motions and transitions are often rare and/or intricate events. Observing that such events give rise to long-tailed spatial distributions, we recently developed a higher-order statistics based dimensionality reduction method, called quasi-anharmonic analysis (QAA), for identifying biophysically-relevant reaction coordinates and substates within MD simulations.

Discovering conformational sub-states relevant to protein function.

Background: Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant.