Impact of Disease-Associated Mutations on the Deaminase Activity of ADAR1.

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.

Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species.

BACKGROUND: Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. RESULTS: In this study, the 3-HP ß-alanine pathway consisting of aspartate decarboxylase, ß-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol-1 glucose in the base strain expressing 12 copies of the ß-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol-1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional ß-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol-1 sugars and resulted in a final titer of 36.0 g/L 3-HP. CONCLUSIONS: The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.

Leveraging a Previously Published Population Pharmacokinetic Model to Predict Rivaroxaban Exposure in Real-World Patients.

Population pharmacokinetic (PK)/pharmacodynamic models are commonly used to inform drug dosing; however, often real-world patients are not well represented in the clinical trial population. We sought to determine how well dosing recommended in the rivaroxaban drug label results in exposure for real-world patients within a reference area under the concentration-time curve (AUC) range. To accomplish this, we assessed the utility of a prior published rivaroxaban population PK model to predict exposure in real-world patients. We used the model to predict rivaroxaban exposure for 230 real-world patients using 3 methods: (1) using patient phenotype information only, (2) using individual post hoc estimates of clearance from the prior model based on single PK samples of rivaroxaban collected at steady state without refitting of the prior model, and (3) using individual post hoc estimates of clearance from the prior model based on PK samples of rivaroxaban collected at steady state after refitting of the prior model. We compared the results across 3 software packages (NONMEM, Phoenix NLME, and Monolix). We found that while the average patient-assigned dosing per the drug label will likely result in the AUC falling within the reference range, AUC for most individual patients will be outside the reference range. When comparing post hoc estimates, the average pairwise percentage differences were all <10% when comparing the software packages, but individual pairwise estimates varied as much as 50%. This study demonstrates the use of a prior published rivaroxaban population PK model to predict exposure in real-world patients.

Genetically Engineered Oleaginous Yeast Lipomyces starkeyi for Sesquiterpene α-Zingiberene Production.

Oleaginous yeast, such as Lipomyces starkeyi, are logical organisms for production of higher energy density molecules like lipids and terpenes. We demonstrate that transgenic L. starkeyi strains expressing an α-zingiberene synthase gene from lemon basil or Hall's panicgrass can produce up to 17 mg/L α-zingiberene in yeast extract peptone dextrose (YPD) medium containing 4% glucose. The transgenic strain was further examined in 8% glucose media with C/N ratios of 20 or 100, and YPD. YPD medium resulted in 59 mg/L α-zingiberene accumulation. Overexpression of selected genes from the mevalonate pathway achieved 145% improvement in α-zingiberene synthesis. Optimization of the growth medium for α-zingiberene production led to 15% higher titer than YPD medium. The final transgenic strain produced 700 mg/L α-zingiberene in fed-batch bioreactor culture. This study opens a new synthetic route to produce α-zingiberene or other terpenoids in L. starkeyi and establishes this yeast as a platform for jet fuel biosynthesis.

High-pressure apparatus for monitoring solid-liquid phase transitions.

This work presents a new technique for evaluating the solid-liquid phase transformations in complex diesel fuel blends and diesel surrogates under high-pressure conditions intended to simulate those occurring in vehicle fuel injectors. A high-pressure apparatus based on a visual identification of freezing and thawing has been designed and built to monitor phase behavior and determine the crystallization temperature of complex fuels to predict wax precipitation. The proposed methodology was validated using pure substances-n-hexadecane (C16H34), cyclohexane (C6H12), and a mixture of 0.5848 mol fraction n-hexadecane in cyclohexane. The crystallization temperatures of these compounds were measured from atmospheric pressure to 400 MPa for temperatures varying from 290 K to 363 K and compared to those reported in the literature. The standard error of the estimated temperatures for the experimental data obtained in this work, based on a given pressure, was compared to data from the literature. This methodology will be extended to investigate the properties of more complex fuel mixtures.

Drug-Induced Arrhythmias: A Scientific Statement From the American Heart Association.

Many widely used medications may cause or exacerbate a variety of arrhythmias. Numerous antiarrhythmic agents, antimicrobial drugs, psychotropic medications, and methadone, as well as a growing list of drugs from other therapeutic classes (neurological drugs, anticancer agents, and many others), can prolong the QT interval and provoke torsades de pointes. Perhaps less familiar to clinicians is the fact that drugs can also trigger other arrhythmias, including bradyarrhythmias, atrial fibrillation/atrial flutter, atrial tachycardia, atrioventricular nodal reentrant tachycardia, monomorphic ventricular tachycardia, and Brugada syndrome. Some drug-induced arrhythmias (bradyarrhythmias, atrial tachycardia, atrioventricular node reentrant tachycardia) are significant predominantly because of their symptoms; others (monomorphic ventricular tachycardia, Brugada syndrome, torsades de pointes) may result in serious consequences, including sudden cardiac death. Mechanisms of arrhythmias are well known for some medications but, in other instances, remain poorly understood. For some drug-induced arrhythmias, particularly torsades de pointes, risk factors are well defined. Modification of risk factors, when possible, is important for prevention and risk reduction. In patients with nonmodifiable risk factors who require a potentially arrhythmia-inducing drug, enhanced electrocardiographic and other monitoring strategies may be beneficial for early detection and treatment. Management of drug-induced arrhythmias includes discontinuation of the offending medication and following treatment guidelines for the specific arrhythmia. In overdose situations, targeted detoxification strategies may be needed. Awareness of drugs that may cause arrhythmias and knowledge of distinct arrhythmias that may be drug-induced are essential for clinicians. Consideration of the possibility that a patient's arrythmia could be drug-induced is important.

Catheter ablation of atrial fibrillation in patients with diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation (AF). Few studies have compared clinical outcomes after catheter ablation between patients with and those without DM. OBJECTIVE: The purpose of this study was to compare AF ablation outcomes in patients with and those without DM. METHODS: We performed a retrospective analysis of 351 consecutive patients who underwent first-time AF ablation. Clinical outcomes included freedom from recurrent atrial arrhythmia, symptom burden (Mayo AF Symptom Inventory score), cardiovascular and all-cause hospitalizations, and periprocedural complications. RESULTS: Patients with DM (n = 65) were older, had a higher body mass index, more persistent AF, more hypertension, and larger left atrial diameter (P <.05 for all). Median (Q1, Q3) total radiofrequency duration [64.0 (43.6, 81.4) minutes vs 54.3 (39.2, 76.4) minutes; P = .132] and periprocedural complications (P = .868) did not differ between patients with and those without DM. After a median follow-up of 29.5 months, arrhythmia recurrence was significantly higher in the DM group compared to the no-DM group after adjustment for baseline differences (adjusted hazard ratio [HR] 2.24; 95% confidence [CI] 1.42-3.55; P = .001). There was a nonsignificant trend toward higher AF recurrence with worse glycemic levels (HR 1.29; 95% CI 0.99-1.69; P = .064). CONCLUSION: Although safety outcomes associated with AF ablation were similar between patients with and those without DM, arrhythmia-free survival was significantly lower among patients with DM. Poor glycemic control seems to an important risk factor for AF recurrence.

Complications involving the subcutaneous implantable cardioverter-defibrillator: Lessons learned from MAUDE.

BACKGROUND: Reports on the subcutaneous implantable cardioverter-defibrillator (S-ICD) cumulatively demonstrate a low rate of complications, but clinical experience with this technology is limited compared with transvenous devices. OBJECTIVE: The purpose of this study was to describe and analyze S-ICD complications reported to the Food and Drug Administration's Manufacturer and User Facility Device Experience database. METHODS: We reviewed all S-ICD events reported to the Manufacturer and User Facility Device Experience submitted over 24 months (from February 2016 through February 2018) through a prospective and standardized approach at a time when an estimated 15,000 S-ICDs were in service. RESULTS: After removing duplicate entries and nonclinical events (n = 493), 1604 events remained. A total of 542 instances of infection were reported with system removal in 414/542 (77.5%). Inappropriate shocks occurred in 550 patients, and 382 (69%) were attributed to oversensing; in response, 254 (56%), 147 (33%), and 80 (18%) patients underwent system reprogramming, removal, or revision, respectively. There were 15 deaths, and causes included defibrillation failure during follow-up (n = 2), ventricular fibrillation induced by the device (n = 4), device-device interaction resulting in undersensing (n = 1), procedure-related complications (n = 4), and uncertain etiology (n = 4). There were 137 reports of system migration, and in 57 (42%) of these, there were associated inappropriate shocks. System migration events were managed with a combination of system revision (69 [51%]), reprogramming (25 [18%]), and system removal (44 [32%]). CONCLUSION: Several S-ICD complications have been reported that appear to be related to the ICD's design and function over time. A better understanding of these complications may help inform patient selection, implant technique, and postimplantation management.

Dofetilide dose reductions and discontinuations in women compared with men.

BACKGROUND: Compared with men, women have longer corrected QT (QTc) intervals, lower clearance of dofetilide, and higher rates of drug-induced torsades de pointes, but the dofetilide dosing algorithm is the same for men and women. OBJECTIVE: The purpose of this study was to evaluate the tolerability of the 500 µg twice daily dose of dofetilide for men and women. METHODS: Men and women admitted to Duke University Medical Center (January 1, 2006, to October 19, 2012) for the initiation of dofetilide 500 µg twice daily were matched 1:1 on age and estimated creatinine clearance. Electrocardiograms throughout dosing were analyzed, and rates of dofetilide discontinuations and dose reductions were compared in unadjusted and adjusted analyses. RESULTS: For 220 matched men and women, the median age was 62.5 years (interquartile range 55-69 years) and the median eCrCl was 98.1 mL/min (interquartile range 77.6-126.2 mL/min). Women were less likely than men to have hypertension and interventricular conduction delay but were otherwise similar. During dofetilide initiation, women were more likely than men to have their dofetilide dose discontinued or reduced (55% vs 32%; P < .001). In most women (82%) and men (69%), the reason for dose adjustment was significant QTc prolongation. In the adjusted analysis, female sex was associated with higher rates of dofetilide dose discontinuations or reductions (odds ratio 3.01; 95% confidence interval 1.58-5.71; P < .01). CONCLUSION: More than half of women who initiated on 500 µg twice daily of dofetilide required medication discontinuations or dose reductions, mostly because of QTc prolongation. Additional studies are needed to evaluate the optimal dosing algorithm of dofetilide in women.

Incidence and Predictors of Left Atrial Appendage Thrombus in Patients Treated With Nonvitamin K Oral Anticoagulants Versus Warfarin Before Catheter Ablation for Atrial Fibrillation.

The utility of routine transesophageal echocardiography (TEE) to exclude left atrial appendage (LAA) thrombus before atrial fibrillation (AF) ablation in patients treated with nonvitamin K oral anticoagulant (NOAC) therapy is unclear. This single-center retrospective study sought to investigate the incidence of LAA thrombus in patients undergoing routine TEE before AF ablation treated with warfarin or NOAC therapy. We included 937 routine pre-AF ablation TEE procedures performed in patients treated with warfarin (n = 517) or NOAC (n = 420). Patients were anticoagulated without interruption for at least 4 consecutive weeks before the TEE. Patients treated with warfarin had lower LAA velocity and underwent TEE earlier in the study period than those treated with NOAC (p <0.05). The incidence of LAA thrombus was higher in patients treated with warfarin (1.55%, 8 of 517) compared with patients treated with NOAC (0.24%, 1 of 420, p = 0.0473 for difference). No LAA thrombus was identified in NOAC-treated patients with a CHA2DS2-VASC score <5 and in warfarin-treated patients with a CHA2DS2-VASC score <2. TEE-related complications occurred in 3 of 937 procedures (0.3%). In conclusion, LAA thrombus is detected rarely during pre-AF ablation TEE. Treatment with an NOAC is associated with a lower incidence of pre-AF ablation LAA thrombus compared with warfarin.

Positive Impact of Continuous-Flow Left Ventricular Assist Device Implantation on Glycemic Control in Patients with Type 2 Diabetes Mellitus and Advanced Chronic Systolic Heart Failure.

STUDY OBJECTIVE: To evaluate the impact of continuous-flow left ventricular assist device (LVAD) implantation on glycemic control in patients with type 2 diabetes mellitus and advanced chronic systolic heart failure. DESIGN: Retrospective medical record review. SETTING: Large academic tertiary and quaternary care hospital. PATIENTS: Eighty-three adults with type 2 diabetes mellitus and advanced chronic systolic heart failure who underwent implantation of a continuous-flow LVAD between July 1, 2008, and June 30, 2013. MEASUREMENTS AND MAIN RESULTS: Baseline demographic data and laboratory values pertinent to glycemic control (hemoglobin A1c [A1C], total daily insulin requirements, noninsulin antidiabetic medication use, and body mass index [BMI]) were collected for each patient. Pre-LVAD data were compared with data obtained during the 24 months after LVAD implantation. The mean age of the study population was 61.3 years, 70% were men, and 63% had ischemic cardiomyopathy. The first available mean ± SD A1C after LVAD implantation was 6.21 ± 1.5% at a median of 4.8 months (interquartile range 3.3-8.9), which represented a significant decrease from the pre-LVAD A1C of 7.46 ± 1.5% (p<0.001). Average daily insulin requirements decreased by 22.9 units at the end of 24 months (p<0.001). Over half of patients with prescriptions for noninsulin antidiabetic medications were able to discontinue therapy by the end of the study. Of note, BMI increased in the second year after LVAD implantation from a baseline of 32.3 kg/m2 to 34.9 kg/m2 (p=0.004). Regression analysis revealed that baseline A1C was the only independent predictor of change in A1C. CONCLUSION: LVAD implantation was associated with a significant improvement in glycemic control. Further prospective studies are needed to evaluate the long-term impact of LVAD implantation on the clinical course of diabetes.

Statin intolerance: more questions than answers.

The dramatic effectiveness of statins in improving the course of atherosclerotic cardiovascular disease tends to overshadow questions of statin intolerance. Thus after more than 25 years of clinical statin use, intolerance remains a poorly understood, frustrating issue for patients and providers. It has been extraordinarily difficult to define statin intolerance and its implications for clinical practice. Here, we briefly summarize current knowledge and raise questions that need to be addressed.

Elevation of fasting morning glucose relative to hemoglobin A1c in normoglycemic patients treated with niacin and with statins.

BACKGROUND: Niacin increases fasting glucose levels, and statins modestly increase the rate of new-onset diabetes. The clinical importance and mechanisms of these effects are not fully explored. OBJECTIVE: On the basis of anecdotal observations, we hypothesized that elevated morning fasting glucose may be accompanied by relatively normal hemoglobin A1c (HbA1c) in patients treated with niacin and other lipid-modifying drugs. We conducted a retrospective cohort analysis to test this hypothesis. METHODS: The Duke Lipid Clinic database (1994-2007) was screened for simultaneous determinations of fasting morning glucose and HbA1c, yielding 1483 data pairs among 554 subjects. Subjects with diabetes, by clinical diagnosis, medication, or any HbA1c ≥6.5%, or nondiabetes were analyzed separately. Repeated-measures linear regression featured glucose as dependent variable and included terms for HbA1c, drug(s), and their interaction. RESULTS: Regression lines for glucose on HbA1c had altered slopes in the presence of niacin and/or statin use in normoglycemic subjects. The corresponding interaction terms (drug and HbA1c) were significant (niacin P = .026, statin P = .013). Fibrate use had no effect (interaction P = .49). When modeled together, niacin and statin effects were independent. Regression curves in diabetic patients were not affected by lipid medications. CONCLUSION: Elevated fasting glucose may be accompanied by relatively normal HbA1c in niacin- and statin-treated patients. HbA1c reflects average daily glucose levels and is likely a better measure of the glycemic effect of lipid medications. Because our data were retrospective, confirmation from randomized trials is needed.