Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking ...immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.
In the aftermath of a terrorist attack political stakes are high: legislators fear being seen as lenient or indifferent and often grant the executive broader authorities without thorough debate. The ...judiciary's role, too, is restricted: constitutional structure and cultural norms narrow the courts' ability to check the executive at all but the margins. The dominant 'Security or Freedom' framework for evaluating counterterrorist law thus fails to capture an important characteristic: increased executive power that shifts the balance between branches of government. This book re-calculates the cost of counterterrorist law to the United Kingdom and the United States, arguing that the damage caused is significantly greater than first appears. Donohue warns that the proliferation of biological and nuclear materials, together with willingness on the part of extremists to sacrifice themselves, may drive each country to take increasingly drastic measures with a resultant shift in the basic structure of both states.
Transcription factors bind DNA sequence motif vocabularies in cis-regulatory elements (CREs) to modulate chromatin state and gene expression during cell state transitions. A quantitative ...understanding of how motif lexicons influence dynamic regulatory activity has been elusive due to the combinatorial nature of the cis-regulatory code. To address this, we undertook multiomic data profiling of chromatin and expression dynamics across epidermal differentiation to identify 40,103 dynamic CREs associated with 3,609 dynamically expressed genes, then applied an interpretable deep-learning framework to model the cis-regulatory logic of chromatin accessibility. This analysis framework identified cooperative DNA sequence rules in dynamic CREs regulating synchronous gene modules with diverse roles in skin differentiation. Massively parallel reporter assay analysis validated temporal dynamics and cooperative cis-regulatory logic. Variants linked to human polygenic skin disease were enriched in these time-dependent combinatorial motif rules. This integrative approach shows the combinatorial cis-regulatory lexicon of epidermal differentiation and represents a general framework for deciphering the organizational principles of the cis-regulatory code of dynamic gene regulation.
We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair ...to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.
The Original Fourth Amendment Donohue, Laura K.
The University of Chicago law review,
06/2016, Letnik:
83, Številka:
3
Journal Article
Recenzirano
The meaning of the rights enshrined in the Constitution provides a critical baseline for understanding the limits of government action—perhaps nowhere more so than in regard to the Fourth Amendment. ...At the time it was adopted, the Fourth Amendment prohibited the government from entering into any home, warehouse, or place of business against the owner's wishes to search for or to seize persons, papers, or effects, absent a specific warrant. Consistent with English common law, the notable exception was when law enforcement or citizens were pursuing a known felon. Outside of such circumstances, search and seizure required government officials to approach a magistrate and, under oath, to provide evidence of the suspected offense and to particularly describe the place to be searched and persons or things to be seized. Scholars' insistence that the Fourth Amendment does not entail a general protection against government entry into the home without a warrant does more than just fail to appreciate the context. It contradicts the meaning of the text itself, which carefully lays out the conditions that must be met before the government may intrude. Reclaiming this meaning is essential for understanding the scope of the original Fourth Amendment and for ensuring a doctrine that reflects fidelity to the founding principles.
We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. ...This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control ...glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show that GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors. In this state, GSCs upregulate primitive developmental programs and are dependent upon Notch signaling. This transition is accompanied by widespread redistribution of repressive histone methylation. Accordingly, persister GSCs upregulate, and are dependent on, the histone demethylases KDM6A/B. Slow-cycling cells with high Notch activity and histone demethylase expression are present in primary glioblastomas before treatment, potentially contributing to relapse. Our findings illustrate how cancer cells may hijack aspects of native developmental programs for deranged proliferation, adaptation, and tolerance. They also suggest strategies for eliminating refractory tumor cells by targeting epigenetic and developmental pathways.
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•GSCs can become drug-tolerant and persister-like following RTK inhibitor exposure•Persister GSCs exhibit NSC-like programs and are sustained by Notch signaling•Notch-high, slow-cycling GSCs are found in primary GBM tumors•Persister GSCs reorganize their repressive chromatin and are dependent on KDM6
Liau et al. find that glioblastoma stem cells (GSCs) reversibly transition to a slow-cycling, persister-like state following RTK inhibitor treatment. The persister state is marked by redistribution of repressive chromatin, upregulation of neurodevelopmental programs, and dependency on KDM6. Thus, cancer cells may hijack chromatin reorganization for proliferation, adaptation, and tolerance.
Noncoding variants of presumed regulatory function contribute to the heritability of neuropsychiatric disease. A total of 2,221 noncoding variants connected to risk for ten neuropsychiatric ...disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, bipolar disorder, borderline personality disorder, major depression, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder and schizophrenia, were studied in developing human neural cells. Integrating epigenomic and transcriptomic data with massively parallel reporter assays identified differentially-active single-nucleotide variants (daSNVs) in specific neural cell types. Expression-gene mapping, network analyses and chromatin looping nominated candidate disease-relevant target genes modulated by these daSNVs. Follow-up integration of daSNV gene editing with clinical cohort analyses suggested that magnesium transport dysfunction may increase neuropsychiatric disease risk and indicated that common genetic pathomechanisms may mediate specific symptoms that are shared across multiple neuropsychiatric diseases.
Part I begins with a historical treatment of the common law presumed right of access to judicial records, laying out the contours of the current test. Part II turns to the First Amendment qualified ...right of access, drawing out the test applied by the courts and providing examples of procedures and muniments thus covered, before examining, in Part III, interests jointly protected by the common law and First Amendment framings. Part IV focuses on three parallel First Amendment right of access cases brought before the Foreign Intelligence Surveillance Court (FISC) and Foreign Intelligence Surveillance Court of Review (FISCR) in 2013 and the months that followed, in which litigants sought access to FISC opinions containing significant interpretations of law. Part V tackles the argument that the sensitive nature of matters before the FISC/R and their institutional design foreclose public access. The article concludes in Part V by noting that while certain information may need to be withheld in relation to particularly sensitive intelligence, insofar as matters of law and the logic employed to reach the courts’ conclusions are concerned, the public has a right to seek access to the associated judicial muniments.
Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute ...to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC, which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.
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•Genome-wide identification of Notch target enhancers and genes in B cell lymphomas•Target genes regulate critical B cell pathways•B cell cancer growth relies on lineage-restricted Notch-activated MYC enhancers•Provides a rationale for targeting Notch in lymphoma
Ryan et al. reveal targets of Notch signaling in B cell cancers associated with Notch gain-of-function mutations. Many Notch-responsive genes are part of pathways implicated in B cell cancer pathogenesis. These findings provide insights into the role of Notch and a rationale for targeting Notch in B cell cancers.
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