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

The RNA Domain Vc1 Regulates Downstream Gene Expression in Response to Cyclic Diguanylate in Vibrio cholerae

In many bacterial species, including the aquatic bacterium and human pathogen Vibrio cholerae, the second messenger cyclic diguanylate (c-di-GMP) modulates processes such as biofilm formation, motility, and virulence factor production. By interacting with various effectors, c-di-GMP regulates gene expression or protein function. One type of c-di-GMP receptor is the class I riboswitch, representatives of which have been shown to bind c-di-GMP in vitro. Herein, we examined the in vitro and in vivo function of the putative class I riboswitch in Vibrio cholerae, Vc1, which lies upstream of the gene encoding GbpA, a colonization factor that contributes to attachment of V. cholerae to environmental and host surfaces containing N-acetylglucosamine moieties. We provide evidence that Vc1 RNA interacts directly with c-di-GMP in vitro, and that nucleotides conserved among this class of riboswitch are important for binding. Yet the mutation of these conserved residues individually in the V. cholerae chromosome inconsistently affects the expression of gbpA and production of the GbpA protein. By isolating the regulatory function of Vc1, we show that the Vc1 element positively regulates downstream gene expression in response to c-di-GMP. Together these data suggest that the Vc1 element responds to c-di-GMP in vivo. Positive regulation of gbpA expression by c-di-GMP via Vc1 may influence the ability of V. cholerae to associate with chitin in the aquatic environment and the host intestinal environment

The Cellular Environment Stabilizes Adenine Riboswitch RNA Structure

There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand-binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute timescale using SHAPE. The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg2+, the approximate Mg2+ concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg2+ concentrations to the buffer used for in vitro analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg2+ alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations

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

The Mrs1 Splicing Factor Binds the bI3 Group I Intron at Each of Two Tetraloop-Receptor Motifs

Most large ribozymes require protein cofactors in order to function efficiently. The yeast mitochondrial bI3 group I intron requires two proteins for efficient splicing, Mrs1 and the bI3 maturase. Mrs1 has evolved from DNA junction resolvases to function as an RNA cofactor for at least two group I introns; however, the RNA binding site and the mechanism by which Mrs1 facilitates splicing were unknown. Here we use high-throughput RNA structure analysis to show that Mrs1 binds a ubiquitous RNA tertiary structure motif, the GNRA tetraloop-receptor interaction, at two sites in the bI3 RNA. Mrs1 also interacts at similar tetraloop-receptor elements, as well as other structures, in the self-folding Azoarcus group I intron and in the RNase P enzyme. Thus, Mrs1 recognizes general features found in the tetraloop-receptor motif. Identification of the two Mrs1 binding sites now makes it possible to create a model of the complete six-component bI3 ribonucleoprotein. All protein cofactors bind at the periphery of the RNA such that every long-range RNA tertiary interaction is stabilized by protein binding, involving either Mrs1 or the bI3 maturase. This work emphasizes the strong evolutionary pressure to bolster RNA tertiary structure with RNA-binding interactions as seen in the ribosome, spliceosome, and other large RNA machines

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

Direct identification of base-paired RNA nucleotides by correlated chemical probing

Many RNA molecules fold into complex secondary and tertiary structures that play critical roles in biological function. Among the best-established methods for examining RNA structure are chemical probing experiments, which can report on local nucleotide structure in a concise and extensible manner. While probing data are highly useful for inferring overall RNA secondary structure, these data do not directly measure through-space base-pairing interactions. We recently introduced an approach for single-molecule correlated chemical probing with dimethyl sulfate (DMS) that measures RNA interaction groups by mutational profiling (RING-MaP). RING-MaP experiments reveal diverse through-space interactions corresponding to both secondary and tertiary structure. Here we develop a framework for using RING-MaP data to directly and robustly identify canonical base pairs in RNA. When applied to three representative RNAs, this framework identified 20%–50% of accepted base pairs with a <10% false discovery rate, allowing detection of 88% of duplexes containing four or more base pairs, including pseudoknotted pairs. We further show that base pairs determined from RING-MaP analysis significantly improve secondary structure modeling. RING-MaP-based correlated chemical probing represents a direct, experimentally concise, and accurate approach for detection of individual base pairs and helices and should greatly facilitate structure modeling for complex RNAs

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