Human myofibroblasts increase the arrhythmogenic potential of human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to remuscularize infarcted hearts but their arrhythmogenicity remains an obstacle to safe transplantation. Myofibroblasts are the predominant cell-type in the infarcted myocardium but their impact on transplanted hiPSC-CMs remains poorly defined. Here, we investigate the effect of myofibroblasts on hiPSC-CMs electrophysiology and Ca2+ handling using optical mapping of advanced human cell coculture systems mimicking cell-cell interaction modalities. Human myofibroblasts altered the electrophysiology and Ca2+ handling of hiPSC-CMs and downregulated mRNAs encoding voltage channels (KV4.3, KV11.1 and Kir6.2) and SERCA2a calcium pump. Interleukin-6 was elevated in the presence of myofibroblasts and direct stimulation of hiPSC-CMs with exogenous interleukin-6 recapitulated the paracrine effects of myofibroblasts. Blocking interleukin-6 reduced the effects of myofibroblasts only in the absence of physical contact between cell-types. Myofibroblast-specific connexin43 knockdown reduced functional changes in contact cocultures only when combined with interleukin-6 blockade. This provides the first in-depth investigation into how human myofibroblasts modulate hiPSC-CMs function, identifying interleukin-6 and connexin43 as paracrine- and contact-mediators respectively, and highlighting their potential as targets for reducing arrhythmic risk in cardiac cell therapy.

A state-based approach to genomics for rare disease and population screening.

PURPOSE: The Alabama Genomic Health Initiative (AGHI) is a state-funded effort to provide genomic testing. AGHI engages two distinct cohorts across the state of Alabama. One cohort includes children and adults with undiagnosed rare disease; a second includes an unselected adult population. Here we describe findings from the first 176 rare disease and 5369 population cohort AGHI participants. METHODS: AGHI participants enroll in one of two arms of a research protocol that provides access to genomic testing results and biobank participation. Rare disease cohort participants receive genome sequencing to identify primary and secondary findings. Population cohort participants receive genotyping to identify pathogenic and likely pathogenic variants for actionable conditions. RESULTS: Within the rare disease cohort, genome sequencing identified likely pathogenic or pathogenic variation in 20% of affected individuals. Within the population cohort, 1.5% of individuals received a positive genotyping result. The rate of genotyping results corroborated by reported personal or family history varied by gene. CONCLUSIONS: AGHI demonstrates the ability to provide useful health information in two contexts: rare undiagnosed disease and population screening. This utility should motivate continued exploration of ways in which emerging genomic technologies might benefit broad populations.

Structure of C60F36: A Gas-Phase Electron Diffraction and Quantum Chemical Computational Study of a Remarkably Distorted Fluorofullerene.

The equilibrium molecular structure of the gaseous fluorofullerene C60F36 has been determined for the first time by the electron diffraction method with the use of quantum chemical calculations up to the RI-MP2/def2-TZVPP level of theory. Vibrational amplitudes and quadratic and cubic force constants were calculated by density functional theory methods. It was found that the sample under study consists of the isomer of C1 symmetry, 81(4)%, with a small amount of the isomer of C3 symmetry, in good accordance with HPLC-MS (atmospheric pressure photoionization), HPLC-UV/vis, and NMR spectroscopic data. The presence of the isomer of T symmetry, up to 5%, cannot be completely excluded. Theoretical structural parameters of the C60F36 molecule were compared with those of the C60F48 molecule. Relative to C60, the C60F36 molecule has a remarkably distorted carbon cage because of steric, electrostatic, and orbital interactions. This results in the longest carbon-carbon bond (1.671 Å) found in free molecules. In particular, about the longest FC-CF bond, the dihedral angle is only around 20°, which leads to the very short nonbonded distance between electronegative vicinal fluorine atoms (2.531 Å) that is much shorter than the sum of van der Waals radii of fluorine atoms (2.94 Å). A natural bond orbital analysis revealed that strong nπ(F) → σ*(FC-CF) interactions delocalize the lone pair of π-type at the fluorine atoms into the antibonding orbital of the FC-CF bond. This hyperconjugation results in additional elongation of FC-CF bonds.

Recruiting diversity where it exists: The Alabama Genomic Health Initiative.

Lack of diversity among genomic research participants results in disparities in benefits from genetic testing. To address this, the Alabama Genomic Health Initiative employed community engagement strategies to recruit diverse populations where they lived. In this paper, we describe our engagement techniques and recruitment strategies, which resulted in significant improvement in representation of African American participants. While African American participation has not reached the representation of this community as a percentage of Alabama's overall population (26%-27%), we have achieved an overall representation exceeding 20% for African Americans. We believe this demonstrates the value of engagement and recruitment where diverse populations reside.

Duration of therapeutic coma and outcome of refractory status epilepticus.

OBJECTIVE: Examine the association of duration of therapeutic coma (TC) with seizure recurrence, morbidity, and mortality in refractory status epilepticus (RSE). Define an optimal window for TC that provides sustained seizure control and minimizes complications. METHODS: Retrospective, observational cohort study involving patients who presented with RSE to the University of Alabama at Birmingham or the University of California at San Francisco from 2010 to 2016. Relationship of duration of TC with primary and secondary outcomes was evaluated using two-sample t tests, simple linear regression, and chi-square tests. Multivariable linear and logistic regression models were used to identify independent predictors. Predictive ability of TC for seizure recurrence was quantified using a receiver-operating characteristic curve. Youden index was used to determine an optimal cutoff value. RESULTS: Multivariable analysis of clinical and treatment characteristics of 182 patients who were treated predominantly with propofol as anesthetic agent showed that longer duration of the first trial of TC (27.2 vs 15.6 hours) was independently associated with a higher chance of seizure recurrence following the first weaning attempt (P = 0.038) but not with poor functional neurologic outcome upon discharge, in-hospital complications, or mortality. Furthermore, higher doses of anesthetic utilized during the first trial of TC were independently associated with fewer in-hospital complications (P = 0.003) and associated with a shorter duration of mechanical ventilation and total length of stay. Duration of TC was identified as an independent predictor of seizure recurrence with an optimal cutoff point at 35 hours. SIGNIFICANCE: This study suggests that a shorter duration yet deeper TC as treatment for RSE may be more effective and safer than the currently recommended TC duration of 24-48 hours. Prospective and randomized trials should be conducted to validate these assertions.

Role of Non-Myocyte Gap Junctions and Connexin Hemichannels in Cardiovascular Health and Disease: Novel Therapeutic Targets?

The heart is a complex organ composed of multiple cell types, including cardiomyocytes and different non-myocyte populations, all working closely together to determine the hearts properties and maintain normal cardiac function. Connexins are abundantly expressed proteins that form plasma membrane hemichannels and gap junctions between cells. Gap junctions are intracellular channels that allow for communication between cells, and in the heart they play a crucial role in cardiac conduction by coupling adjacent cardiomyocytes. Connexins are expressed in both cardiomyocytes and non-myocytes, including cardiac fibroblasts, endothelial cells, and macrophages. Non-myocytes are the largest population of cells in the heart, and therefore it is important to consider what roles connexins, hemichannels, and gap junctions play in these cell types. The aim of this review is to provide insight into connexin-based signalling in non-myocytes during health and disease, and highlight how targeting these proteins could lead to the development of novel therapies. We conclude that connexins in non-myocytes contribute to arrhythmias and adverse ventricular remodelling following myocardial infarction, and are associated with the initiation and development of atherosclerosis. Therefore, therapeutic interventions targeting these connexins represent an exciting new research avenue with great potential.

Traumatic Amputation of Finger From an Alligator Snapping Turtle Bite.

Legend states that the alligator snapping turtle (Macrochelys temminckii) should be handled with extreme caution as it has jaw strength powerful enough to bite a wooden broomstick in half. Tales of bite injuries from what is the largest freshwater turtle in North America exist anecdotally, yet there are few descriptions of medical encounters for such. The risk of infection from reptilian bites to the hand in an aquatic environment warrants thorough antibiotic treatment in conjunction with hand surgery consultation. We present the first case report of a near total amputation of an index finger in an adolescent boy who had been bitten by a wild "gator snapper."

Phomalactone from a Phytopathogenic Fungus Infecting ZINNIA elegans (ASTERACEAE) Leaves.

Zinnia elegans Jacq. plants are infected by a fungus that causes dark red spots with necrosis on leaves, particularly in late spring to the middle of summer in the Mid-South of the United States. This fungal disease causes the leaves to wilt and eventually kills the plant. The fungus was isolated, cultured in potato dextrose broth, and identified as Nigrospora sphaerica by molecular techniques. Two major lactone metabolites (phomalactone and catenioblin A) were isolated from liquid culture of N. sphaerica isolated from Z. elegans. When injected into leaves of Z. elegans, phomalactone caused lesions similar to those of the fungus. The lesion sizes were proportional to the concentration of the phomalactone. Phomalactone, but not catenioblin A, was phytotoxic to Z. elegans and other plant species by inhibition of seedling growth and by causing electrolyte leakage from photosynthetic tissues of both Z. elegans leaves and cucumber cotyledons. This latter effect may be related to the wilting caused by the fungus in mature Z. elegans plants. Phomalactone was moderately fungicidal to Coletotrichum fragariae and two Phomopsis species, indicating that the compound may keep certain other fungi from encroaching into plant tissue that N. sphaerica has infected. Production of large amounts of phomalactone by N. sphaerica contributes to the pathogenic behavior of this fungus, and may have other ecological functions in the interaction of N. sphaerica with other fungi. This is the first report of isolation of catenioblin A from a plant pathogenic fungus. The function of catenioblin A is unclear, as it was neither significantly phyto- nor fungitoxic.

1,4-Cyclohexanedione. Composition, molecular structures, and internal dynamics of the vapor: an electron diffraction investigation augmented by molecular orbital calculations.

Electron diffraction experiments on the vapor of 1,4-cyclohexanedione have been carried out at a nominal temperature of 435 K. The results are consistent with the presence of a mixture of a chair form of C2h symmetry and a twisted boat form of D2 symmetry. The former has the familiar dynamic properties of a semirigid molecule, but the D2 form undergoes a large-amplitude twisting motion (pseudorotation) that degrades the symmetry to C2. The analysis was designed to elucidate parameter values and internal dynamics of each conformer and the composition of the system. The large-amplitude motion of the twisted boat form was modeled by placement of 10 pseudoconformers at approximately 5° intervals along a pseudorotational coordinate that began at the D2 position and that reflected the angle between the C═O bond vectors. A Gaussian weighting of the pseudeoconformers centered on the (lowest-energy) D2 position was assumed. Differences in the interatomic distances and bond angles of these pseudoconformers were calculated via B3LYP/cc-pVTZ theory and introduced as constraints. The bond length averages over the twisted boat forms followed by values for the chair in square brackets are (rg/Å; [angle]α/deg) r(C-H) = 1.115(11) [1.124(11)], r(C═O) = 1.211(3) [1.233(6)], r(C1-C2) = 1.524(5) [1.526(5)], and r(C2-C3) = 1.533(11) [1.539(11)]. The corresponding ring angle values are [angle](C1C2C3) = 111.1(5) [111.0(4)] and [angle](C6C1C2) = 116.3(8) [115.7(8)]. In the twisted boat form, pseudorotation leads to a weighted average displacement of the angle between the C═O bond vectors, [angle]Δ(CO,CO), equal to 21.3° from the 180° value in the D2 form corresponding to an average angle between the CO bond vectors of 158.7(1)°. The amount of the chair form in the gas at 435 K is 24(10)%. The listed uncertainties are estimated at 2σ.

Hydroxychloroquine and azithromycin alter the contractility of living porcine heart slices.

The cardiotoxicity risk of hydroxychloroquine (HCQ) and azithromycin (AZM) has been the subject of intensive research triggered by safety concerns in COVID-19 patients. HCQ and AZM have been associated with QT interval prolongation and drug-induced arrhythmias, however other cardiotoxicity mechanisms remain largely unexplored. Our group has pioneered the living heart slice preparation, an ex-vivo platform that maintains native cardiac tissue architecture and physiological electrical and contractile properties. Here, we evaluated the cardiotoxic effect of HCQ and AZM applied alone or in combination on cardiac contractility by measuring contractile force and contraction kinetics in heart slices prepared from porcine hearts. Our results show that clinically relevant concentrations of HCQ monotherapy (1-10 µM) reduced contractile force and contraction kinetics in porcine slices in a dose-dependent manner. However, AZM monotherapy decreased contractile force and contraction kinetics only at higher concentrations (30 µM). Combination of HCQ and AZM induced a dose-dependent effect similar to HCQ alone. Furthermore, pre-treating porcine heart slices with the L-type calcium channel agonist Bay K8644 prevented the effect of both drugs, while administration of Bay K8644 after drugs interventions largely reversed the effects, suggesting a mechanism involving inhibition of L-type calcium channels. These findings indicate that HCQ and AZM alter cardiac function beyond QT prolongation with significant contractile dysfunction in intact cardiac tissue. Our porcine heart slices provide a powerful platform to investigate mechanisms of drug cardiotoxicity.

Porcine Organotypic Epicardial Slice Protocol: A Tool for the Study of Epicardium in Cardiovascular Research.

The epicardium has recently gained interest in the cardiovascular field due to its capacity to support heart regeneration after ischemic injury. Models to study the epicardium of large animals in vitro are limited and mainly based on epicardial cell isolation/differentiation from stem cells, followed by 2D cells culture. In this method paper, we describe the procedure to obtain and culture 3D organotypic heart slices presenting an intact epicardium, as a novel model to study the epicardial physiology and activation. Epicardial slices are obtained from porcine hearts using a high-precision vibratome and retain a healthy epicardial layer embedded in its native extracellular environment and connected with other cardiac cells (cardiomyocytes, fibroblasts, vascular cells etc.). Epicardial slices can be cultured for 72 h, providing an ideal model for studying the epicardium physiology or perform pharmacological interventions/gene therapy approaches. We also report on methods to assesses the viability and composition of the epicardial slices, and evaluate their architecture in 3D through tissue decoloration. Finally, we present a potential application for a nanomaterial-based gene transfer method for tracking of epicardial cells within the slice. Crucially, given the similarity in morphology and physiology of porcine heart with its human counterpart, our system provides a platform for translational research while providing a clinically relevant and ethical alternative to the use of small animals in this type of research.

Associations of Obesity and Neighborhood Factors With Urinary Stone Parameters.

INTRODUCTION: Obesity is associated with kidney stone disease, but it is unknown whether this association differs by SES. This study assessed the extent to which obesity and neighborhood characteristics jointly contribute to urinary risk factors for kidney stone disease. METHODS: This was a retrospective analysis of adult patients with kidney stone disease evaluated with 24-hour urine collection (2001-2020). Neighborhood-level socioeconomic data were obtained for a principal component analysis, which identified 3 linearly independent factors. Associations between these factors and 24-hour urine measurements were assessed using linear regression as well as groupings of 24-hour urine results using multivariable logistic regression. Finally, multiplicative interactions were assessed testing effect modification by obesity, and analyses stratified by obesity were performed. Analyses were performed in 2021. RESULTS: In total, 1,264 patients met the study criteria. Factors retained on principal component analysis represented SES, family structure, and housing characteristics. On linear regression, there was a significant inverse correlation between SES and 24-hour urine sodium (p=0.0002). On multivariable logistic regression, obesity was associated with increased odds of multiple stone risk factors (OR=1.61; 95% CI=1.15, 2.26) and multiple dietary factors (OR=1.33; 95% CI=1.06, 1.67). No significant and consistent multiplicative interactions were observed between obesity and quartiles of neighborhood SES, family structure, or housing characteristics. CONCLUSIONS: Obesity was associated with the presence of multiple stone risk factors and multiple dietary factors; however, the strength and magnitude of these associations did not vary significantly by neighborhood SES, family structure, and housing characteristics.

Furanocoumarin with Phytotoxic Activity from the Leaves of Amyris elemifera (Rutaceae).

Bioassay-guided fractionation of the ethyl acetate extract of Amyris elemifera leaves was carried out to identify phytotoxic and antifungal constituents. A novel phytotoxic furanocoumarin 8-(3-methylbut-2-enyloxy)-marmesin acetate (1) and its deacyl analog 8-(3-methylbut-2-enyloxy)-marmesin (2) were isolated. The X-ray crystal structure determination is reported for the first time for 1. Both 1 and 2 have the S configuration at C-2' based on X-ray crystallographic data. Both these compounds inhibited the growth of the dicot Lactuca sativa (lettuce) and the monocot Agrostis stolonifera with a more pronounced inhibitory effect on the monocots at 330 µM by 1. In Lemna paucicostata Hegelm phytotoxicity bioassay, the IC50 value for 1 was 26 µM, whereas 2 had an IC50 value of 102 µM. Compounds 1 and 2 were weakly antifungal against Colletotrichum fragariae Brooks in TLC bioautography.

Myocardial Viability Imaging using Manganese-Enhanced MRI in the First Hours after Myocardial Infarction.

Early measurements of tissue viability after myocardial infarction (MI) are essential for accurate diagnosis and treatment planning but are challenging to obtain. Here, manganese, a calcium analogue and clinically approved magnetic resonance imaging (MRI) contrast agent, is used as an imaging biomarker of myocardial viability in the first hours after experimental MI. Safe Mn2+ dosing is confirmed by measuring in vitro beating rates, calcium transients, and action potentials in cardiomyocytes, and in vivo heart rates and cardiac contractility in mice. Quantitative T1 mapping-manganese-enhanced MRI (MEMRI) reveals elevated and increasing Mn2+ uptake in viable myocardium remote from the infarct, suggesting MEMRI offers a quantitative biomarker of cardiac inotropy. MEMRI evaluation of infarct size at 1 h, 1 and 14 days after MI quantifies myocardial viability earlier than the current gold-standard technique, late-gadolinium-enhanced MRI. These data, coupled with the re-emergence of clinical Mn2+ -based contrast agents open the possibility of using MEMRI for direct evaluation of myocardial viability early after ischemic onset in patients.

Development of a COVID-19 Application Ontology for the ACT Network.

Clinical data networks that leverage large volumes of data in electronic health records (EHRs) are significant resources for research on coronavirus disease 2019 (COVID-19). Data harmonization is a key challenge in seamless use of multisite EHRs for COVID-19 research. We developed a COVID-19 application ontology in the national Accrual to Clinical Trials (ACT) network that enables harmonization of data elements that that are critical to COVID-19 research. The ontology contains over 50,000 concepts in the domains of diagnosis, procedures, medications, and laboratory tests. In particular, it has computational phenotypes to characterize the course of illness and outcomes, derived terms, and harmonized value sets for SARS-CoV-2 laboratory tests. The ontology was deployed and validated on the ACT COVID-19 network that consists of nine academic health centers with data on 14.5M patients. This ontology, which is freely available to the entire research community on GitHub at https://github.com/shyamvis/ACT-COVID-Ontology, will be useful for harmonizing EHRs for COVID-19 research beyond the ACT network.

Development of a Coronavirus Disease 2019 (COVID-19) Application Ontology for the Accrual to Clinical Trials (ACT) network.

Clinical data networks that leverage large volumes of data in electronic health records (EHRs) are significant resources for research on coronavirus disease 2019 (COVID-19). Data harmonization is a key challenge in seamless use of multisite EHRs for COVID-19 research. We developed a COVID-19 application ontology in the national Accrual to Clinical Trials (ACT) network that enables harmonization of data elements that are critical to COVID-19 research. The ontology contains over 50 000 concepts in the domains of diagnosis, procedures, medications, and laboratory tests. In particular, it has computational phenotypes to characterize the course of illness and outcomes, derived terms, and harmonized value sets for severe acute respiratory syndrome coronavirus 2 laboratory tests. The ontology was deployed and validated on the ACT COVID-19 network that consists of 9 academic health centers with data on 14.5M patients. This ontology, which is freely available to the entire research community on GitHub at https://github.com/shyamvis/ACT-COVID-Ontology, will be useful for harmonizing EHRs for COVID-19 research beyond the ACT network.

Introduction to the D-SPECT for Technologists: Workflow Using a Dedicated Digital Cardiac Camera.

The D-SPECT is a dedicated cardiac camera that incorporates a solid-state semiconductor detector. This camera differs greatly from conventional SPECT/CT systems, resulting in significant differences in patient imaging. This continuing education article focuses on the specifications of both SPECT/CT and D-SPECT systems, radiopharmaceutical dosing requirements, imaging workflows, and some disadvantages of using each camera system. When used properly, the D-SPECT system can provide high-quality cardiac images with lower doses and faster exam times than conventional SPECT/CT systems.

Is (-)-catechin a novel weapon of spotted knapweed (Centaurea stoebe)?

The novel weapons hypothesis states that some invasive weed species owe part of their success as invaders to allelopathy mediated by allelochemicals that are new to the native species. Presumably, no resistance has evolved among the native species to this new allelochemical (i.e., the novel weapon). In their native habitat, however, the plants that co-evolved with these invasive species have theoretically evolved defenses that obviate the allelochemical advantage. Previous studies have claimed that catechin is such a novel weapon of spotted knapweed (Centaurea stoebe = C. maculosa), an invasive species in the non-native habitat of North America. These studies indicated that (-)-catechin is more phytotoxic than (+)-catechin. Other studies have not found sufficient catechin in field soils to support this theory. We report that (-)-catechin and (+)-catechin are essentially equal, but poorly phytotoxic to a variety of plant species in bioassays without soil. In a dose/response experiment with Montana soils, we found the lowest dose for a growth reduction of two native Montana grasses (Koeleria macrantha and Festuca idahoensis) by a racemic mixture of (+/-)-catechin that ranged from about 25 to 50 mM, concentrations, orders of magnitude higher than expected in nature. Autoclaving the soil before adding the catechin did not affect the activity of catechin. We found (-)-catechin to be a potent antioxidant, in contrast to a previous claim that it acts as an allelochemical by causing oxidative stress. Our findings suggest that catechin is not a novel weapon of spotted knapweed and that other allelochemical(s) or alternative mechanisms must be found to explain the success of this species as an invader in North America.

Civilian aviation fatalities involving pilot ethanol and a previous record of substance abuse.

INTRODUCTION: Pilots are subject to the same temptations as the general population, but due to the demands of flying, the use of ethanol or other impairing substances are more likely to have severe consequences. The Federal Aviation Administration (FAA) requires pilots to report all adverse legal actions involving their use of ethanol and/or other drugs. The purpose of this study was to evaluate fatal civil aviation accidents between the years 2000 and 2007 in which ethanol was present in the pilot and the pilot had a record of previous drug and/or alcohol offenses. METHODS: Case histories and accident information for the period under study were obtained from the National Transportation Safety Board (NTSB). Toxicological information was obtained from the Civil Aerospace Medical Institute's Forensic Toxicology Research Laboratory. RESULTS: During the examined period 215 of the 2391 pilot specimens received for toxicological analysis had documented alcohol or drug related offenses. Of the 215 pilot specimens received, 23 showed evidence of ethanol consumption prior to the fatal incident and 16 of those had ethanol concentrations above the FAA's legal limit of 40 mg x dl(-1). DISCUSSION: These results confirm earlier reports that pilots with previous alcohol and/or drug offenses are more likely than others to engage in repeated substance abuse in association with flying with resultant fatal accidents. These findings support the NTSB's recommendation that the FAA implement more thorough verification of alcohol and drug offenses as a means of identifying pilots at increased risk of causing accidents.

Antiemetic and sedative levels found together in 26 civil aviation pilot fatalities, 2000-2006.

INTRODUCTION: Antiemetics and sedatives are two drug classes that may have harmful side effects when mixed. This is especially dangerous for pilots. Although many of these compounds are considered disqualifying by the FAA, their use occurs. Some pilots may be unaware of the danger of combining compounds from these two drug classes. Our laboratory was interested in evaluating the circumstances surrounding accidents in which the pilot was found positive for drugs from each of these two classes. METHODS: Epidemiological, toxicological, and aeromedical findings from pilots involved in such accidents were collected for the 7-yr period from 2000 through 2006. Case histories, accident information, and the probable cause of the aviation accidents were obtained from the National Transportation Safety Board (NTSB). Toxicological information was obtained from the Civil Aerospace Medical Institute's (CAMI's) Forensic Toxicology Research Laboratory. RESULTS: Of the 2184 fatal aviation accidents over this time period, 26 were found positive for compounds from both the antiemetic and the sedative drug classes. All 26 aircraft were operated under 14 CFR Part 91 as general aviation. All pilots were male; 21 tested positive for a disqualifying substance that may have affected their ability to control the aircraft. CONCLUSION: Although the percentage of accidents in which the pilot tests positive for a compound from each class is small, it is important for all pilots to understand the dangers of self-medicating and concomitant use of such substances. Under-reporting of medications by pilots during their certification process occurs; education is the key to preventing inadvertent drug-drug interactions.