Protocol for Biospecimen Collection and Analysis within the BACPAC Research Program

The Biospecimen Collection and Processing Working Group of the NIH HEAL Initiative BACPAC Research Program was charged with identifying molecular biomarkers of interest to chronic low back pain (cLBP). Having identified biomarkers of interest, the Working Group worked with the New York University Grossman School of Medicine, Center for Biospecimen Research and Development-funded by the Early Phase Pain Investigation Clinical Network Data Coordinating Center-to harmonize consortium-wide and site-specific efforts for biospecimen collection and analysis. Biospecimen collected are saliva, blood (whole, plasma, serum), urine, stool, and spine tissue (paraspinal muscle, ligamentum flavum, vertebral bone, facet cartilage, disc endplate, annulus fibrosus, or nucleus pulposus). The omics data acquisition and analyses derived from the biospecimen include genomics and epigenetics from DNA, proteomics from protein, transcriptomics from RNA, and microbiomics from 16S rRNA. These analyses contribute to the overarching goal of BACPAC to phenotype cLBP and will guide future efforts for precision medicine treatment

O-18 Isotope Effects Support a ConcertedMechanism for Ribonuclease A

Ribonuclease A catalyzes the cleavage of RNA in a two-step process. The phosphodiester bond is cleaved to yield a RNA strand with a free 5′ OH and another RNA strand with a 2′, 3′ cyclic monophosphate at the 3′ end. This cyclic intermediate is then hydrolyzed with enzymatic catalysis.1 Classically, the first step of cleavage of RNA by ribonuclease A has been reported to proceed via a concerted mechanism in which His-12 acts to deprotonate the nucleophilic 2′ OH and His-119 acts to protonate the leaving group.2 Chemical modification studies,3 pH-rate studies,4 and site directed mutagenesis studies of His-12 and His-1195 are consistent with general acid-base catalysis. However, the mechanism has become the subject of much debate as a result of the proposal of a phosphorane intermediate in the catalytic mechanism. This concept was based on data obtained in model studies utilizing cyclodextrin-bis(imidazole) compounds6 or imidazole or morpholine buffers as catalysts.7 We report here a direct study of the enzymatic reaction catalyzed by ribonuclease A to test for the presence of a phosphorane intermediate

The Nature of the Transition State of the Protein-Tyrosine Phosphatase-Catalyzed Reaction

The dephosphorylation of p-nitrophenyl phosphate by Yersinia protein-tyrosine phosphatase (PTPase) and by the rat PTP1 has been examined by measurement of heavy-atom isotope effects at the nonbridge oxygen atoms [18(V/K)nonbridge] at the bridging oxygen atom [18(V/K)bridge] and the nitrogen atom in the leaving group 15(V/K). The effects were measured using an isotope ratio mass spectrometer by the competitive method and thus are effects on V/K. The results for the Yersinia PTPase and rat PTPl, respectively, are 1.0142 ± 0.0004 and 1.0152 ± 0.0006 for 18(V/K)bridge; 0.9981 ± 0.0015 and 0.9998 ± 0.0013 for 18(V/K)nonbridge· and 1.0001 ± 0.0002 and 0.9999 ± 0.0003 for 15(V/K). The magnitudes of the isotope effects are similar to the intrinsic values measured in solution, indicating that the chemical step is rate-limiting for V/K. The transition state for phosphorylation of the enzyme is dissociative in character, as is the case in solution. Binding of the substrate is rapid and reversible, as is the binding-induced conformational change which brings the catalytic general acid into the active site. Cleavage of the P—O bond and proton transfer from the general acid Asp to the leaving group are both far advanced in the transition state, and there is no development of negative charge on the departing leaving group. Experiments with several general acid mutants give values for 18(V/K)bridge of around 1.0280, 15(V/K) of about 1.002, and 18(V/K)nonbridge effects of from 1.0007 to 1.0022. These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion but suggest more nucleophilic participation than in the solution reaction

Descriptive Analysis of an Interdisciplinary Musculoskeletal Program.

BACKGROUND: Interdisciplinary musculoskeletal programs address comorbidities confounding musculoskeletal conditions and serve as an alternative to the single provider model. OBJECTIVE: Descriptive analysis of an interdisciplinary musculoskeletal program. DESIGN: Retrospective descriptive analysis of patients enrolled in an interdisciplinary musculoskeletal program. Retrospective subanalysis: cohort of patients enrolled in interdisciplinary program with low back pain compared to historical cohort of patients in a single provider clinic. SETTING: Academic interdisciplinary musculoskeletal health program. PATIENTS: Patients referred to program with at least one follow-up visit over a 2-year period. INTERVENTIONS: Interdisciplinary musculoskeletal program involving physiatry, pain anesthesia, nutrition, psychology, rheumatology, sleep medicine, nursing, and physical therapy. MAIN OUTCOME MEASUREMENTS: Patient Specific Functional Scale (PSFS), Oswestry Low Back Disability Index (ODI), number of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, opioid prescriptions; Press Ganey scores. RESULTS: One hundred and seventy-three patients were enrolled and had at least one follow-up visit. Twenty-four percent of patients with any musculoskeletal complaint demonstrated clinically significant improvements in total PSFS. Mean improvement in PSFS was + 0.864 (SD 1.94), which was a statistically significant improvement (P = .0005), but not clinically significant. Magnetic resonance imaging was ordered for 5% of patients, and no computed tomography scans were ordered. Six percent of patients received opioid prescriptions. Press Ganey scores: 96% responded favorably in regard to physician communication quality, 86% of patients responded favorably for access to care, and 78% responded favorably for care coordination. 27.8% of patients with low back pain in the interdisciplinary program achieved a significant decrease in their ODI, compared to 26.6% in the single provider clinic (P = .87). CONCLUSIONS: Interdisciplinary musculoskeletal programs are a promising model to improve the functioning of patients with musculoskeletal pain and decrease downstream utilization. These programs may be more appropriate for patients at higher risk of developing chronic pain

Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways

Previous research has identified an association between external radiation and disc degeneration, but the mechanism was poorly understood. This study explores the effects of ionizing radiation (IR) on inducing cellular senescence of annulus fibrosus (AF) in cell culture and in an in vivo mouse model. Exposure of AF cell culture to 10–15 Gy IR for 5 min followed by 5 days of culture incubation resulted in almost complete senescence induction as evidenced by SA-βgal positive staining of cells and elevated mRNA expression of the p16 and p21 senescent markers. IR-induced senescent AF cells exhibited increased matrix catabolism, including elevated matrix metalloproteinase (MMP)-1 and -3 protein expression and aggrecanolysis. Analogous results were seen with whole body IR-exposed mice, demonstrating that genotoxic stress also drives disc cellular senescence and matrix catabolism in vivo. These results have important clinical implications in the potential adverse effects of ionizing radiation on spinal health