Teaching the Law of American Health Care

In writing our casebook, The Law of American Health Care, we started from scratch, rethinking the topics to include and themes around which to organize them. Like many health law professors, we were schooled in and continued to propound the traditional themes of cost, quality, access, and choice. While those concerns certainly pervade many areas of health care law, our casebook\u27s overarching themes emphasize different issues, namely: federalism, individual rights, fiduciary relationships, the modem administrative state, and market regulation. These new themes, we believe, better capture the range of issues and topics essential for the new generation of health lawyers. When we set out to write The Law of American Health Care, our objectives were threefold: (1) simplify; (2) emphasize primary sources; and (3) reorganize the classic state-based law approach to Law and Medicine to reflect the dominance of federal law in the post-Affordable Care Act (ACA) era. In this essay, we will discuss not only how we went about achieving these goals but also how the topical nature of health care law can be addressed through the use of themes, and how themes can facilitate learning on the fly when health care law changes, as it inevitably does

Advances in RNA Secondary and Tertiary Structure Analysis by Chemical Probing

RNA is arguably the most versatile biological macromolecule due to its ability both to encode and to manipulate genetic information. The diverse roles of RNA depend on its ability to fold back on itself to form biologically functional structures that bind small molecules and large protein ligands, to change conformation, and to affect the cellular regulatory state. These features of RNA biology can be structurally interrogated using chemical mapping experiments. The usefulness and applications of RNA chemical probing technologies have expanded dramatically over the past five years due to several critical advances. These innovations include new sequence-independent RNA chemistries, algorithmic tools for high-throughput analysis of complex data sets composed of thousands of measurements, new approaches for interpreting chemical probing data for both secondary and tertiary structure prediction, facile methods for following time-dependent processes, and the willingness of individual research groups to tackle increasingly bold problems in RNA structural biology

Monitoring/Verification using DMS: TATP Example

Field-rugged and field-programmable differential mobility spectrometry (DMS) networks provide highly selective, universal monitoring of vapors and aerosols at detectable levels from persons or areas involved with illicit chemical/biological/explosives (CBE) production. CBE sensor motes used in conjunction with automated fast gas chromatography with DMS detection (GC/DMS) verification instrumentation integrated into situational operations management systems can be readily deployed and optimized for changing application scenarios. The feasibility of developing selective DMS motes for a 'smart dust' sampling approach with guided, highly selective, fast GC/DMS verification analysis is a compelling approach to minimize or prevent the use of explosives or chemical and biological weapons in terrorist activities. Two peroxide-based liquid explosives, triacetone triperoxide (TATP) and hexamethylene triperoxide diamine (HMTD), are synthesized from common chemicals such as hydrogen peroxide, acetone, sulfuric acid, ammonia, and citric acid (Figure 1). Recipes can be readily found on the Internet by anyone seeking to generate sufficient quantities of these highly explosive chemicals to cause considerable collateral damage. Detection of TATP and HMTD by advanced sensing systems can provide the early warning necessary to prevent terror plots from coming to fruition. DMS is currently one of the foremost emerging technologies for the separation and detection of gas-phase chemical species. This is due to trace-level detection limits, high selectivity, and small size. DMS separates and identifies ions at ambient pressures by utilizing the non-linear dependence of an ion's mobility on the radio frequency (rf) electric field strength. GC is widely considered to be one of the leading analytical methods for the separation of chemical species in complex mixtures. Advances in the technique have led to the development of low-thermal-mass fast GC columns. These columns are capable of completing runs in less than 3 minutes. Fast GC columns are also more compact than their traditional counterparts. An earlier collaborative effort involving these authors optimized a handheld, fast GC/DMS, equipped with a non-rad ionization source, for the detection of TATP (Figure 2). The unit combines the separation capabilities of GC with the selectivity of DMS. Analytes are identified both by their elution time from the column and by the characteristic response in the DMS spectrum. Analysis times required to obtain results for these analytes are approximately 80 seconds for TATP and 160 seconds for HMTD (Figure 3). The limit of detection for both TATP and HMTD is approximately 1 ng/{micro}L. Substances that could interfere with the detection of peroxide-based explosives have been studied. Both the GC elution time and the DMS spectral peak locations were unique and do not hinder the detection of either TATP or HMTD

Role of Context in RNA Structure: Flanking Sequences Reconfigure CAG Motif Folding in Huntingtin Exon 1 Transcripts

The length of the CAG repeat region in the huntingtin messenger RNA is predictive of Huntington’s disease. Structural studies of CAG repeat-containing RNAs suggest that these sequences form simple hairpin structures; however, in the context of the full-length huntingtin mRNA, CAG repeats may form complex structures that could be targeted for therapeutic intervention. We examined the structures of transcripts spanning the first exon of the huntingtin mRNA with both healthy and disease-prone repeat lengths. In transcripts with 17 to 70 repeats, the CAG sequences base paired extensively with bases in the 5′ UTR and with a conserved region downstream of the CCG repeat region. In huntingtin transcripts with healthy numbers of repeats, the previously observed CAG hairpin was either absent or short. In contrast, in transcripts with disease-associated numbers of repeats, a CAG hairpin was present and extended from a three-helix junction. Our findings demonstrate the profound importance of sequence context in RNA folding and identify specific structural differences between healthy and disease-inducing huntingtin alleles that may be targets for therapeutic intervention

The SL1-SL2 (Stem-Loop) Domain Is the Primary Determinant for Stability of the Gamma Retroviral Genomic RNA Dimer

Retroviral genomes are assembled from two sense-strand RNAs by noncovalent interactions at their 5' ends, forming a dimer. The RNA dimerization domain is a potential target for antiretroviral therapy and represents a compelling RNA folding problem. The fundamental dimerization unit for the Moloney murine sarcoma gamma retrovirus spans a 170-nucleotide minimal dimerization active sequence. In the dimer, two self-complementary sequences, PAL1 and PAL2, form intermolecular duplexes, and an SL1-SL2 (stem-loop) domain forms loop-loop base pairs, mediated by GACG tetraloops, and extensive tertiary interactions. To develop a framework for assembly of the retroviral RNA dimer, we quantified the stability of and established nucleotide resolution secondary structure models for sequence variants in which each motif was compromised. Base pairing and tertiary interactions between SL1-SL2 domains contribute a large free energy increment of -10 kcal/mol. In contrast, even though the PAL1 and PAL2 intermolecular duplexes span 10 and 16 bp in the dimer, respectively, they contribute only -2.5 kcal/mol to stability, roughly equal to a single new base pair. First, these results emphasize that the energetic costs for disrupting interactions in the monomer state nearly balance the PAL1 and PAL2 base pairing interactions that form in the dimer. Second, intermolecular duplex formation plays a biological role distinct from simply stabilizing the structure of the retroviral genomic RNA dimer

Ribosome RNA Assembly Intermediates Visualized in Living Cells

In cells, RNAs likely adopt numerous intermediate conformations prior to formation of functional RNA-protein complexes. We used single-nucleotide resolution selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to probe the structure of Escherichia coli 16S rRNA in healthy growing bacteria. SHAPE-directed modeling indicated that the predominant steady-state RNA conformational ensemble in dividing cells had a base-paired structure different from that expected on the basis of comparative sequence analysis and high-resolution studies of the 30S ribosomal subunit. We identified the major cause of these differences by stopping ongoing in-cell transcription (in essence, an in-cell RNA structure pulse-chase experiment) which caused the RNA to chase into a structure that closely resembled the expected one. Most helices that formed alternate RNA conformations under growth conditions interact directly with tertiary-binding ribosomal proteins and form a C-shape that surrounds the mRNA channel and decoding site. These in-cell experiments lead to a model in which ribosome assembly factors function as molecular struts to preorganize this intermediate and emphasize that the final stages of ribonucleoprotein assembly involve extensive protein-facilitated RNA conformational changes

SHAPE-directed RNA secondary structure prediction

The diverse functional roles of RNA are determined by its underlying structure. Accurate and comprehensive knowledge of RNA structure would inform a broader understanding of RNA biology and facilitate exploiting RNA as a biotechnological tool and therapeutic target. Determining the pattern of base pairing, or secondary structure, of RNA is a first step in these endeavors. Advances in experimental, computational, and comparative analysis approaches for analyzing secondary structure have yielded accurate structures for many small RNAs, but only a few large (>500 nts) RNAs. In addition, most current methods for determining a secondary structure require considerable effort, analytical expertise, and technical ingenuity. In this review, we outline an efficient strategy for developing accurate secondary structure models for RNAs of arbitrary length. This approach melds structural information obtained using SHAPE chemistry with structure prediction using nearest-neighbor rules and the dynamic programming algorithm implemented in the RNAstructure program. Prediction accuracies reach ≥95% for RNAs on the kilobase scale. This approach facilitates both development of new models and refinement of existing RNA structure models, which we illustrate using the Gag-Pol frameshift element in an HIV-1 M-group genome. Most promisingly, integrated experimental and computational refinement brings closer the ultimate goal of efficiently and accurately establishing the secondary structure for any RNA sequence

Developing Fieldable Systems for Chemical Sensing Using Field Asymmetric Ion Mobility Spectrometry and Mass Spectrometry

Currently, there is an urgent need for field-rugged and field-programmable sensor systems that provide highly selective, universal monitoring of vapors and aerosols at detectable levels from persons or areas involved with illicit chemical/biological/explosives (CBE) production. These devices must be portable, low cost, robust, and provide accurate measurements to avoid both false positive and negative results. Furthermore, the information provided by the devices must be received in a timely manner so that informed decisions can be immediately made and the appropriate actions taken. Two technologies that are unparalleled in their sensitivity, selectivity, and trace-level detection capabilities are field asymmetric ion mobility spectrometry (FAIMS) and mass spectrometry. Here, we will show progress that has been made toward developing fieldable FAIMS systems and mass spectrometers. Working in collaboration with Sionex Corporation, the microDMx detector was equipped with a continuous air sampling system to develop selective methods for the analysis of compounds of interest. A microdiaphragm pump (KNF Neuberger, Inc.) is used to pull in gas-phase analytes directly from the air for separation and detection with the FAIMS system. The FAIMS evaluation platform (SVAC) unit currently measures 9.8-inch x 4.6-inch x 3.2-inch, weighs 3.1 lb, and utilizes a {sup 63}Ni source to ionize incoming compounds. Analytes entering the unit are separated and identified by their characteristic response to the compensation voltage (V{sub c}) at a given rf field strength (V{sub rf}). This response has been observed to be unique for a wide range of substances studied. If additional verification were required or a targeted analyte present in a complex chemical matrix, a FAIMS unit equipped with a fast gas chromatography column has been evaluated. The unit combines the separation capabilities of gas chromatography with the selectivity of FAIMS. It measures 9.5-inch x 5.25-inch x 3.5-inch, weighs 3.8 lb, and uses a 10.6 eV photoionization source. Analytes are identified both by their elution time from the column and by the characteristic response in the FAIMS spectrum. Analysis times required to obtain results for most analytes examined are less than three minutes. A fieldable mass spectrometer system is also being developed that includes sampling, ionization, mass selection and detection, vacuum technology, and analytical methodology with remote data transmission. Multiple methods for mass selection are being explored, including both Penning and Paul type ion traps as well as a quadrupole system to determine which is best suited for a portable mass spectrometer. Several ionization sources and ion counting methods will also be evaluated to establish their effectiveness with each system. The intended result of this project is a handheld mass spectrometer system capable of field deployment for the detection and identification of a wide range of gas-phase CBE species