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The innate immune system relies on molecular sensors to detect distinctive molecular patterns, including viral double-stranded RNA (dsRNA), which triggers responses resulting in apoptosis and immune infiltration. Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I), serving as a mechanism to distinguish self from non-self RNA and prevent aberrant immune activation. Loss-of-function mutations in the ADAR1 gene are one cause of Aicardi Goutières Syndrome (AGS), a severe autoimmune disorder in children. Although seven out of the eight AGS-associated mutations in ADAR1 occur within the catalytic domain of the ADAR1 protein, their specific effects on the catalysis of adenosine deamination remain poorly understood. In this study, we carried out a biochemical investigation of four AGS-causing mutations (G1007R, R892H, K999N, and Y1112F) in ADAR1 p110 and truncated variants. These studies included adenosine deamination rate measurements with two different RNA substrates derived from human transcripts known to be edited by ADAR1 p110 (glioma-associated oncogene homologue 1 (hGli1), 5-hydroxytryptamine receptor 2C (5-HT2cR)). Our results indicate that AGS-associated mutations at two amino acid positions directly involved in stabilizing the base-flipped conformation of the ADAR-RNA complex (G1007R and R892H) had the most detrimental impact on catalysis. The K999N mutation, positioned near the RNA binding interface, altered catalysis contextually. Finally, the Y1112F mutation had small effects in each of the assays described here. These findings shed light on the differential effects of disease-associated mutations on adenosine deamination by ADAR1, thereby advancing our structural and functional understanding of ADAR1-mediated RNA editing.

Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs' selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs' preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5' direction (5'-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5'-G (-1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5'-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5'-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the -1 position significantly improves editing at 5'-GA sites.

Adenosine Deaminases Acting on RNA (ADARs) are members of a family of RNA editing enzymes that catalyze the conversion of adenosine into inosine in double-stranded RNA (dsRNA). ADARs' selective activity on dsRNA presents the ability to correct mutations at the transcriptome level using guiding oligonucleotides. However, this approach is limited by ADARs' preference for specific sequence contexts to achieve efficient editing. Substrates with a guanosine adjacent to the target adenosine in the 5′ direction (5′-GA) are edited less efficiently compared to substrates with any other canonical nucleotides at this position. Previous studies showed that a G/purine mismatch at this position results in more efficient editing than a canonical G/C pair. Herein, we investigate a series of modified oligonucleotides containing purine or size-expanded nucleoside analogs on guide strands opposite the 5′-G (−1 position). The results demonstrate that modified adenosine and inosine analogs enhance editing at 5′-GA sites. Additionally, the inclusion of a size-expanded cytidine analog at this position improves editing over a control guide bearing cytidine. High-resolution crystal structures of ADAR:/RNA substrate complexes reveal the manner by which both inosine and size-expanded cytidine are capable of activating editing at 5′-GA sites. Further modification of these altered guide sequences for metabolic stability in human cells demonstrates that the incorporation of specific purine analogs at the −1 position significantly improves editing at 5′-GA sites. [ABSTRACT FROM AUTHOR]

For decades, a precept was passed down by academic physicians to trainees and staff as a guideline for inpatient care of patients with diabetes. That bit of wisdom held that [...]

Pharmacists in direct patient care settings are expanding their roles and responsibilities. These changes mandate a deeper and broader understanding of disease states, as well as influencing social, behavioral, and environmental factors. Existing guidelines and accreditation standards related to the training of graduate or cardiovascular pharmacist specialists (ie, Accreditation Council for Graduate Medical Education, American Society of Health‐System Pharmacists, American College of Clinical Pharmacy [ACCP]) provide some guidance on essential competencies. However, they stop short of providing recommendations for how to achieve all the objectives. The purpose of this paper is to build upon existing guidelines/standards, describing our recommendations for pharmacy residency training of a cardiovascular clinical specialist. The paper is broken down into the following sections: (1) Skills and Competencies (Building Clinical Skills, Application of Clinical Knowledge and Skills, Drug Information, Research and Scholarship, Teaching Skills, Interpersonal, Communication, and Presentation Skills), (2) Personal and Professional Growth (Growing Interpersonal Skills, Engaging with the Profession), (3) Program Design (Resident Selection, Preceptors and Mentoring, Expectations for Progress/Milestones, Program and Learning Experience Structure), and (4) Clinical and Therapeutic Content Expertise or Medical Knowledge. After each recommendation, specific details are provided to aid in conceptualizing how each can be achieved. Some recommendations are considered essential whereas others are designated as optional. This paper represents the opinion of the Cardiology Practice and Research Network of the American College of Clinical Pharmacy. It does not necessarily represent an official ACCP commentary, guideline, or statement of policy or position. [ABSTRACT FROM AUTHOR]

Introduction: Non-ambulatory class IV heart failure (HF) patients have not been evaluated in trials of cardiac resynchronization therapy (CRT). There is limited data on the management of class IV HF patients in clinical practice, including CRT eligibility, clinical course, and treatment with advanced HF therapies (left ventricular assist device or transplant). Hypothesis: There are a substantial number of patients with ambulatory and non-ambulatory NYHA Class IV HF who do not have CRT and would be potential candidates for CRT placement. Methods: Patients at a single, academic medical center, referred to the advanced HF clinic or admitted to the hospital for initiation of inotropes from 1/1/08 to 12/31/13 were identified. Manual chart review was completed in order to obtain data on patients' comorbidities and cardiovascular testing. Baseline characteristics of the outpatient and inpatient populations were compared using chi square analyses or T-tests. Descriptive analyses of the two populations were performed, including CRT eligibility; treatment with CRT, left ventricular assist device, and transplant; and mortality. Results: Among 395 patients, 206 were inotrope inpatients and 189 were outpatients. Sixty patients from the inotrope and 62 from the outpatient cohorts were CRT eligible: a predominance of RV pacing (13.3% and 22.5%), presence of a BiV device with inadequate pacing (30% and 19.3%), Class I indications (25.3% and 9.6%), and Class II indications (29.6% and 11.2%), respectively. In the inotropic group 26 patients did not receive LVAD or OHT, compared to 40 patients in the ambulatory group, with only a small proportion of eligible patients receiving CRT (Figure). Conclusions: The majority of patients evaluated for advanced HF therapies met criteria for CRT placement and either died or received LVAD/OHT without CRT implantation. CRT could prove beneficial in those patients who are not LVAD or transplant candidates despite optimal medical management. [ABSTRACT FROM AUTHOR]