Approach to Myelopathy and Myelitis.

Myelopathy can present acutely or more insidiously and has a broad differential diagnosis. In addition to the clinical history and neurologic examination, diagnostic testing, including MRI and cerebrospinal fluid analysis, as well as thorough review of patient comorbidities, risk factors, and potential toxic exposures, can help neurohospitalists distinguish between various causes and potentially start appropriate empiric therapy while awaiting definitive testing. This article focuses on how imaging can help in determining the most likely cause of myelopathy and highlights a range of causes, including compressive, vascular, metabolic and toxic, infectious, autoimmune, neoplastic, and paraneoplastic causes of spinal cord dysfunction.

Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing.

Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity.

A systematic survey of the Cys2His2 zinc finger DNA-binding landscape.

Cys2His2 zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain's well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of >160 000 unique domain-DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain-DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities.

Understanding DNA-binding specificity by bacteria hybrid selection.

Understanding how sequence-specific protein-DNA interactions direct cellular function is of great interest to the research community. High-throughput methods have been developed to determine DNA-binding specificities; one such technique, the bacterial one-hybrid (B1H) system, confers advantages including ease of use, sensitivity and throughput. In this review, we describe the evolution of the B1H system as a tool capable of screening large DNA libraries to investigate protein-DNA interactions of interest. We discuss how DNA-binding specificities produced by the B1H system have been used to predict regulatory targets. Additionally, we examine how this approach has been applied to characterize two common DNA-binding domain families-homeodomains and Cys2His2 zinc fingers-both in organism-wide studies and with synthetic approaches. In the case of the former, the B1H system has produced large catalogs of protein specificity and nuanced information about previously recovered DNA targets, thereby improving our understanding of these proteins' functions in vivo and increasing our capacity to predict similar interactions in other species. In the latter, synthetic screens of the same DNA-binding domains have further refined our models of specificity, through analyzing comprehensive libraries to uncover all proteins able to bind a complete set of targets, and, for instance, exploring how context-in the form of domain position within the parent protein-may affect specificity. Finally, we recognize the limitations of the B1H system and discuss its potential for use in the production of designer proteins and in studies of protein-protein interactions.