NMR resonance assignments of RNase P protein from \u3cem\u3eThermotoga maritima\u3c/em\u3e

Ribonuclase P (RNase P) is an essential metallo-endonuclease that catalyzes 5′ precursor-tRNA (ptRNA) processing and exists as an RNA-based enzyme in bacteria, archaea, and eukaryotes. In bacteria, a large catalytic RNA and a small protein component assemble to recognize and accurately cleave ptRNA and tRNA-like molecular scaffolds. Substrate recognition of ptRNA by bacterial RNase P requires RNA-RNA shape complementarity, intermolecular base pairing, and a dynamic protein-ptRNA binding interface. To gain insight into the binding specificity and dynamics of the bacterial protein-ptRNA interface, we report the backbone and side chain 1H, 13C, and 15N resonance assignments of the hyperthermophilic Thermatoga maritima RNase P protein in solution at 318 K. Our data confirm the formation of a stable RNA recognition motif (RRM) with intrinsic heterogeneity at both the N- and C-terminus of the protein, consistent with available structural information. Comprehensive resonance assignments of the bacterial RNase P protein serve as an important first step in understanding how coupled RNA binding and protein-RNA conformational changes give rise to ribonucleoprotein function

NMR-Metabolic Methodology in the Study of GM Foods

The 1H-NMR methodology used in the study of genetically modified (GM) foods is discussed. Transgenic lettuce (Lactuca sativa cv "Luxor") over-expressing the ArabidopsisKNAT1 gene is presented as a case study. Twenty-two water-soluble metabolites (amino acids, organic acids, sugars) present in leaves of conventional and GM lettuce were monitored by NMR and quantified at two developmental stages. The NMR spectra did not reveal any difference in metabolite composition between the GM lettuce and the wild type counterpart. Statistical analyses of metabolite variables highlighted metabolism variation as a function of leaf development as well as the transgene. A main effect of the transgene was in altering sugar metabolism

African Swine Fever Virus DNA polymerase X: biophysical interaction studies and NMR assignments of the polymerase-deoxyguanosine triphosphate complex.

Three constructs of African Swine Fever Virus DNA polymerase X (pol X) were expressed and purified. Buffers containing sodium acetate, sodium cacodylate or 0.5 M NaCl provided good solubility for the enzyme at micromolar concentration, which was required for biophysical studies. The transformation between the oxidized and reduced forms of the enzyme was monitored by nuclear magnetic resonance (NMR). Biochemical assays revealed that the oxidized form of the enzyme showed activity with template-primer substrate, whereas the reduced form was only weakly active. NMR chemical shift assignments for the pol X - deoxyguanosine triphosphate complex were completed. Based on these assignments, chemical shift perturbations of several dNTP binary complexes were mapped to the pol X structure, revealing interactions with the â strands 4, 6, 8-10 and á-helices B and D. Isothermal titration calorimetry (ITC) and NMR measurements suggested a different interaction mode for purines and pyrimidines with pol X which was supported by the difference in dissociation constants (Kd) for purines, 2-10 µM and pyrimidines, 40-50 µM. Dissociation constants in the high nanomolar to low micromolar range were determined for double hairpin and template-primer oligodeoxynucleotide sequences. Binding interactions of oligodeoxynucleotides were mapped to the structure of pol X by analysis of amide chemical shift perturbations, displaying perturbations at the C-terminal end of á-helix E, â-sheet 11 and 12, and at the sub-domain binding interface. Based on these observations, a model analogous to pol â was proposed, where a bent DNA would span the enzyme between the C-terminal side of áE and áC, or áE and the subdomain interface, possibly moving between the two positions, while the enzyme undergoes further conformational changes

OP67 Considerations Of Treatment Novelty In Health Technology Assessment

IntroductionA recent proliferation of value frameworks, as well as the emergence of innovative approaches to treating disease (e.g., cell/gene therapies) have been accompanied by an increased focus on nontraditional elements of value. We sought to understand whether and how health technology assessment (HTA) agencies consider novel aspects of treatment in value assessments.MethodsWe defined treatment novelty as follows: (i) a new mechanism of action or administration; (ii) addresses an unmet need; or (iii) confers a distinct benefit that transforms clinical practice or that is difficult to quantify. We reviewed technical guidance and peer-reviewed literature to investigate how organizations in eight countries (Australia, Canada, England, France, Norway, the Netherlands, Sweden, and the United States) consider aspects of this definition.ResultsAll (n = 8) organizations give special consideration to interventions that address an unmet need, particularly in cancer, rare diseases, and other severe conditions. Nearly all (n = 5) organizations consider whether an intervention produces benefits that may not be adequately quantified. Organizations in England, Norway, and France sometimes recommend drugs with less favorable cost-effectiveness estimates than traditionally considered if the drug addresses rare or severe conditions, or if its quality-of-life benefit is thought to be inadequately quantified. The Institute for Clinical and Economic Review in the United States models cost-effectiveness in rare diseases using both a modified societal and health care system perspective. Importantly, the benefits of novel treatments are frequently considered uncertain, particularly treatments with a new mechanism of action. When uncertainty is high, organizations in Canada, England, France, the Netherlands, and Sweden sometimes issue conditional recommendations until additional evidence is submitted. England and Australia have used risk sharing agreements for drugs determined to be novel but uncertain.ConclusionsThe most widely considered aspects of treatment novelty in HTA are unmet needs and potential benefits that are not easily measured. The willingness to pay for novel treatments is often greater, despite inherent uncertainties about benefit and cost-effectiveness.</jats:sec

The impact of external reference pricing on pharmaceutical costs and market dynamics

Growth in the cost of prescription drugs in the US has generated significant interest in the use of external reference pricing (ERP) to tie prices paid for drugs to those in other countries. We used data from the Pricentric ONE™ database, an international drug pricing database, to examine product launch timing, launch price, and price changes from January 2010 – October 2021 in both ERP and non-ERP settings, with a focus on 100 high-priced drugs of interest to Medicare and Medicaid. We found that ERP policies were associated with a 73% reduction in the likelihood of drug launch within 9 months of regulatory approval relative to non-ERP settings. In addition, while ERP was associated with statistically significant reductions in annual drug price changes, such policies did not impact launch price. In addition, no single ERP feature (e.g., number of countries referenced, ERP calculation) was materially associated with the outcomes of interest. We conclude that ERP policies do not appear to impact drug launch price and may delay access to new therapies, raising questions about the utility of such policies in the US and potential consequences abroad