Metabolism of new drug modalities research advances - 2023 year in review.

With contributions from colleagues across academia and industry, we have put together the annual reviews of research advances on drug biotransformation and bioactivation since 2016 led by Cyrus Khojasteh. While traditional small molecules and biologics are still predominant in drug discovery, we start to notice a paradigm shift toward new drug modalities (NDMs) including but not limited to peptide and oligonucleotide therapeutics, protein degraders (heterobifunctional degraders and molecule glues), conjugated drugs and covalent inhibitors. The readers can learn more on each new drug modality from several recent comprehensive reviews (Blanco et al. 2022; Hillebrand et al. 2024; Phuna et al. 2024). Based on this trend, we put together this stand-alone review branched from our previous efforts (Baillie et al. 2016; Khojasteh et al. 2023) with a focus on the metabolism of NDMs. We collected 11 articles which exemplify recent discoveries and perspectives in this field.

Biotransformation research advances - 2023 year in review.

This annual review marks the eighth in the series starting with Baillie et al. (2016) Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation. Its format is to highlight important aspects captured in synopsis followed by a commentary with relevant figure and references.

Bioactivation and reactivity research advances - 2023 year in review.

Advances in the field of bioactivation have significantly contributed to our understanding and prediction of drug-induced liver injury (DILI). It has been established that many adverse drug reactions, including DILI, are associated with the formation and reactivity of metabolites. Modern methods allow us to detect and characterize these reactive metabolites in earlier stages of drug development, which helps anticipate and circumvent the potential for DILI. Improved in silico models and experimental techniques that better reflect in vivo environments are enhancing predictive capabilities for DILI risk. Further, studies on the mechanisms of bioactivation, including enzyme interactions and the role of individual genetic differences, have provided valuable insights for drug optimizations. Cumulatively, this progress is continually refining our approaches to drug safety evaluation and personalized medicine.

Four-dimensional flow MRI for quantitative assessment of cerebrospinal fluid dynamics: Status and opportunities.

Neurological disorders can manifest with altered neurofluid dynamics in different compartments of the central nervous system. These include alterations in cerebral blood flow, cerebrospinal fluid (CSF) flow, and tissue biomechanics. Noninvasive quantitative assessment of neurofluid flow and tissue motion is feasible with phase contrast magnetic resonance imaging (PC MRI). While two-dimensional (2D) PC MRI is routinely utilized in research and clinical settings to assess flow dynamics through a single imaging slice, comprehensive neurofluid dynamic assessment can be limited or impractical. Recently, four-dimensional (4D) flow MRI (or time-resolved three-dimensional PC with three-directional velocity encoding) has emerged as a powerful extension of 2D PC, allowing for large volumetric coverage of fluid velocities at high spatiotemporal resolution within clinically reasonable scan times. Yet, most 4D flow studies have focused on blood flow imaging. Characterizing CSF flow dynamics with 4D flow (i.e., 4D CSF flow) is of high interest to understand normal brain and spine physiology, but also to study neurological disorders such as dysfunctional brain metabolite waste clearance, where CSF dynamics appear to play an important role. However, 4D CSF flow imaging is challenged by the long T1 time of CSF and slower velocities compared with blood flow, which can result in longer scan times from low flip angles and extended motion-sensitive gradients, hindering clinical adoption. In this work, we review the state of 4D CSF flow MRI including challenges, novel solutions from current research and ongoing needs, examples of clinical and research applications, and discuss an outlook on the future of 4D CSF flow.

Cotyledon-Specific Flow Evaluation of Rhesus Macaque Placental Injury Using Ferumoxytol Dynamic Contrast-Enhanced MRI.

BACKGROUND: Recently, dynamic contrast-enhanced (DCE) MRI with ferumoxytol as contrast agent has recently been introduced for the noninvasive assessment of placental structure and function throughout. However, it has not been demonstrated under pathological conditions. PURPOSE: To measure cotyledon-specific rhesus macaque maternal placental blood flow using ferumoxytol DCE MRI in a novel animal model for local placental injury. STUDY TYPE: Prospective animal model. SUBJECTS: Placental injections of Tisseel (three with 0.5 mL and two with 1.5 mL), monocyte chemoattractant protein 1 (three with 100 µg), and three with saline as controls were performed in a total of 11 rhesus macaque pregnancies at approximate gestational day (GD 101). DCE MRI scans were performed prior (GD 100) and after (GD 115 and GD 145) the injection (term = GD 165). FIELD STRENGTH/SEQUENCE: 3 T, T1-weighted spoiled gradient echo sequence (product sequence, DISCO). ASSESSMENT: Source images were inspected for motion artefacts from the mother or fetus. Placenta segmentation and DCE processing were performed for the dynamic image series to measure cotyledon specific volume, flow, and normalized flow. Overall placental histopathology was conducted for controls, Tisseel, and MCP-1 animals and regions of tissue infarctions and necrosis were documented. Visual inspections for potential necrotic tissue were conducted for the two Tisseelx3 animals. STATISTICAL TESTS: Wilcoxon rank sum test, significance level P < 0.05. RESULTS: No motion artefacts were observed. For the group treated with 1.5 mL of Tisseel, significantly lower cotyledon volume, flow, and normalized flow per cotyledon were observed for the third gestational time point of imaging (day ~145), with mean normalized flow of 0.53 minute-1 . Preliminary histopathological analysis shows areas of tissue necrosis from a selected cotyledon in one Tisseel-treated (single dose) animal and both Tisseelx3 (triple dose) animals. DATA CONCLUSION: This study demonstrates the feasibility of cotyledon-specific functional analysis at multiple gestational time points and injury detection in a placental rhesus macaque model through ferumoxytol-enhanced DCE MRI. LEVEL OF EVIDENCE: NA TECHNICAL EFFICACY: Stage 2.

False lumen hemodynamics and partial thrombosis in chronic aortic dissection of the descending aorta.

OBJECTIVES: Partial thrombosis of the false lumen (FL) in patients with chronic aortic dissection (AD) of the descending aorta has been associated with poor outcomes. Meanwhile, the fluid dynamic and biomechanical characteristics associated with partial thrombosis remain to be elucidated. This retrospective, single-center study tested the association between FL fluid dynamics and biomechanics and the presence and extent of FL thrombus. METHODS: Patients with chronic non-thrombosed or partially thrombosed FLs in the descending aorta after an aortic dissection underwent computed tomography angiography, cardiovascular magnetic resonance (CMR) angiography, and a 4D flow CMR study. A comprehensive quantitative analysis was performed to test the association between FL thrombus presence and extent (percentage of FL with thrombus) and FL anatomy (diameter, entry tear location and size), fluid dynamics (inflow, rotational flow, wall shear stress, kinetic energy, and flow acceleration and stasis), and biomechanics (pulse wave velocity). RESULTS: Sixty-eight patients were included. In multivariate logistic regression FL kinetic energy (p = 0.038) discriminated the 33 patients with partial FL thrombosis from the 35 patients with no thrombosis. Similarly, in separated multivariate linear correlations kinetic energy (p = 0.006) and FL inflow (p = 0.002) were independently related to the extent of the thrombus. FL vortexes, flow acceleration and stasis, wall shear stress, and pulse wave velocity showed limited associations with thrombus presence and extent. CONCLUSION: In patients with chronic descending aorta dissection, false lumen kinetic energy is related to the presence and extent of false lumen thrombus. CLINICAL RELEVANCE STATEMENT: In patients with chronic aortic dissection of the descending aorta, false lumen hemodynamic parameters are closely linked with the presence and extent of false lumen thrombosis, and these non-invasive measures might be important in patient management. KEY POINTS: • Partial false lumen thrombosis has been associated with aortic growth in patients with chronic descending aortic dissection; therefore, the identification of prothrombotic flow conditions is desirable. • The presence of partial false lumen thrombosis as well as its extent was related with false lumen kinetic energy. • The assessment of false lumen hemodynamics may be important in the management of patients with chronic aortic dissection of the descending aorta.

Bioactivation and reactivity research advances - 2022 year in review‡.

With the 50th year mark since the launch of Drug Metabolism and Disposition journal, the field of drug metabolism and bioactivation has advanced exponentially in the past decades (Guengerich 2023).This has, in a major part, been due to the continued advances across the whole spectrum of applied technologies in hardware, software, machine learning (ML), and artificial intelligence (AI). LC-MS platforms continue to evolve to support key applications in the field, and automation is also improving the accuracy, precision, and throughput of these supporting assays. In addition, sample generation and processing is being aided by increased diversity and quality of reagents and bio-matrices so that what is being analyzed is more relevant and translatable. The application of in silico platforms (applied software, ML, and AI) is also making great strides, and in tandem with the more traditional approaches mentioned previously, is significantly advancing our understanding of bioactivation pathways and how these play a role in toxicity. All of this continues to allow the area of bioactivation to evolve in parallel with associated fields to help bring novel or improved medicines to patients with urgent or unmet needs.Shuai Wang and Cyrus Khojasteh, on behalf of the authors.

Biotransformation research advances - 2022 year in review.

This annual review is the eighth of its kind since 2016 (Baillie et al. 2016, Khojasteh et al. 2017, Khojasteh et al. 2018, Khojasteh et al. 2019, Khojasteh et al. 2020, Khojasteh et al. 2021, Khojasteh et al. 2022). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation.

Normative Cerebral Hemodynamics in Middle-aged and Older Adults Using 4D Flow MRI: Initial Analysis of Vascular Aging.

Background Characterizing cerebrovascular hemodynamics in older adults is important for identifying disease and understanding normal neurovascular aging. Four-dimensional (4D) flow MRI allows for a comprehensive assessment of cerebral hemodynamics in a single acquisition. Purpose To establish reference intracranial blood flow and pulsatility index values in a large cross-sectional sample of middle-aged (45-65 years) and older (>65 years) adults and characterize the effect of age and sex on blood flow and pulsatility. Materials and Methods In this retrospective study, patients aged 45-93 years (cognitively unimpaired) underwent cranial 4D flow MRI between March 2010 and March 2020. Blood flow rates and pulsatility indexes from 13 major arteries and four venous sinuses and total cerebral blood flow were collected. Intraobserver and interobserver reproducibility of flow and pulsatility measures was assessed in 30 patients. Descriptive statistics (mean ± SD) of blood flow and pulsatility were tabulated for the entire group and by age and sex. Multiple linear regression and linear mixed-effects models were used to assess the effect of age and sex on total cerebral blood flow and vessel-specific flow and pulsatility, respectively. Results There were 759 patients (mean age, 65 years ± 8 [SD]; 506 female patients) analyzed. For intra- and interobserver reproducibility, median intraclass correlation coefficients were greater than 0.90 for flow and pulsatility measures across all vessels. Regression coefficients ß ± standard error from multiple linear regression showed a 4 mL/min decrease in total cerebral blood flow each year (age ß = -3.94 mL/min per year ± 0.44; P < .001). Mixed effects showed a 1 mL/min average annual decrease in blood flow (age ß = -0.95 mL/min per year ± 0.16; P < .001) and 0.01 arbitrary unit (au) average annual increase in pulsatility over all vessels (age ß = 0.011 au per year ± 0.001; P < .001). No evidence of sex differences was observed for flow (ß = -1.60 mL/min per male patient ± 1.77; P = .37), but pulsatility was higher in female patients (sex ß = -0.018 au per male patient ± 0.008; P = .02). Conclusion Normal reference values for blood flow and pulsatility obtained using four-dimensional flow MRI showed correlations with age. © RSNA, 2023 Supplemental material is available for this article. See also the editorial by Steinman in this issue.

Motion compensated self supervised deep learning for highly accelerated 3D ultrashort Echo time pulmonary MRI.

PURPOSE: To investigate motion compensated, self-supervised, model based deep learning (MBDL) as a method to reconstruct free breathing, 3D pulmonary UTE acquisitions. THEORY AND METHODS: A self-supervised eXtra dimension MBDL architecture (XD-MBDL) was developed that combined respiratory states to reconstruct a single high-quality 3D image. Non-rigid motion fields were incorporated into this architecture by estimating motion fields from a lower resolution motion resolved (XD-GRASP) reconstruction. Motion compensated XD-MBDL was evaluated on lung UTE datasets with and without contrast and compared to constrained reconstructions and variants of self-supervised MBDL that do not account for dynamic respiratory states or leverage motion correction. RESULTS: Images reconstructed using XD-MBDL demonstrate improved image quality as measured by apparent SNR (aSNR), contrast to noise ratio (CNR), and visual assessment relative to self-supervised MBDL approaches that do not account for dynamic respiratory states, XD-GRASP and a recently proposed motion compensated iterative reconstruction strategy (iMoCo). Additionally, XD-MBDL reduced reconstruction time relative to both XD-GRASP and iMoCo. CONCLUSION: A method was developed to allow self-supervised MBDL to combine multiple respiratory states to reconstruct a single image. This method was combined with graphics processing unit (GPU)-based image registration to further improve reconstruction quality. This approach showed promising results reconstructing a user-selected respiratory phase from free breathing 3D pulmonary UTE acquisitions.

Accelerated 4D-flow MRI with 3-point encoding enabled by machine learning.

PURPOSE: To investigate the acceleration of 4D-flow MRI using a convolutional neural network (CNN) that produces three directional velocities from three flow encodings, without requiring a fourth reference scan measuring background phase. METHODS: A fully 3D CNN using a U-net architecture was trained in a block-wise fashion to take complex images from three flow encodings and to produce three real-valued images for each velocity component. Using neurovascular 4D-flow scans (n = 144), the CNN was trained to predict velocities computed from four flow encodings by standard reconstruction including correction for residual background phase offsets. Methods to optimize loss functions were investigated, including magnitude, complex difference, and uniform velocity weightings. Subsequently, 3-point encoding was evaluated using cross validation of pixelwise correlation, flow measurements in major arteries, and in experiments with data at differing acceleration rates than the training data. RESULTS: The CNN-produced 3-point velocities showed excellent agreements with the 4-point velocities, both qualitatively in velocity images, in flow rate measures, and quantitatively in regression analysis (slope = 0.96, R2  = 0.992). Optimizing the training to focus on vessel velocities rather than the global velocity error and improved the correlation of velocity within vessels themselves. The lowest error was observed when the loss function used uniform velocity weighting, in which the magnitude-weighted inverse of the velocity frequency uniformly distributed weighting across all velocity ranges. When applied to highly accelerated data, the 3-point network maintained a high correlation with ground truth data and demonstrated a denoising effect. CONCLUSION: The 4D-flow MRI can be accelerated using machine learning requiring only three flow encodings to produce three-directional velocity maps with small errors.

Fat mitigation strategies to improve image quality of radial 4D flow MRI in obese subjects.

PURPOSE: This study addresses the challenges in obtaining abdominal 4D flow MRI of obese patients. We aimed to evaluate spectral saturation and inner volume excitation as methods to mitigating artifacts originating from adipose signals, with the goal of enhancing image quality and improving quantification. METHODS: Radial 4D flow MRI acquisitions with fat mitigation (inner volume excitation [IVE] and intermittent fat saturation [FS]) were compared to a standard slab selective excitation (SSE) in a test-retest study of 15 obese participants. IVE selectively excited a cylindrical region of interest, avoiding contamination from peripheral adipose tissue, while FS globally suppressed fat based on spectral selection. Acquisitions were evaluated qualitatively based on expert ratings and quantitatively based on conservation of mass, test-retest repeatability, and a divergence free quality metric. Errors were evaluated statistically using the absolute and relative errors, regression, and Bland-Altman analysis. RESULTS: IVE demonstrated superior performance quantitatively in the conservation of mass analysis in the portal vein, with higher correlation and lower bias in regression analysis. IVE also produced flow fields with the lowest divergence error and was rated best in overall image quality, delineating small vessels, and producing the least streaking artifacts. Evaluation results did not differ significantly between FS and SSE. Test-retest reproducibility was similarly high for all sequences, with data suggesting biological variations dominate the technical variability. CONCLUSION: IVE improved hemodynamic assessment of radial 4D flow MRI in the abdomen of obese participants while FS did not lead to significant improvements in image quality or flow metrics.

Enzymatic bypass of an N6-deoxyadenosine DNA-ethylene dibromide-peptide cross-link by translesion DNA polymerases.

Unrepaired DNA-protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA-protein cross-links can be cleaved into DNA-peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide-DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (kcat/Km) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended.

Motion-corrected 4D-Flow MRI for neurovascular applications.

Neurovascular 4D-Flow MRI has emerged as a powerful tool for comprehensive cerebrovascular hemodynamic characterization. Clinical studies in at risk populations such as aging adults indicate hemodynamic markers can be confounded by motion-induced bias. This study develops and characterizes a high fidelity 3D self-navigation approach for retrospective rigid motion correction of neurovascular 4D-Flow data. A 3D radial trajectory with pseudorandom ordering was combined with a multi-resolution low rank regularization approach to enable high spatiotemporal resolution self-navigators from extremely undersampled data. Phantom and volunteer experiments were performed at 3.0T to evaluate the ability to correct for different amounts of induced motions. In addition, the approach was applied to clinical-research exams from ongoing aging studies to characterize performance in the clinical setting. Simulations, phantom and volunteer experiments with motion correction produced images with increased vessel conspicuity, reduced image blurring, and decreased variability in quantitative measures. Clinical exams revealed significant changes in hemodynamic parameters including blood flow rates, flow pulsatility index, and lumen areas after motion correction in probed cerebral arteries (Flow: P<0.001 Lt ICA, P=0.002 Rt ICA, P=0.004 Lt MCA, P=0.004 Rt MCA; Area: P<0.001 Lt ICA, P<0.001 Rt ICA, P=0.004 Lt MCA, P=0.004 Rt MCA; flow pulsatility index: P=0.042 Rt ICA, P=0.002 Lt MCA). Motion induced bias can lead to significant overestimation of hemodynamic markers in cerebral arteries. The proposed method reduces measurement bias from rigid motion in neurovascular 4D-Flow MRI in challenging populations such as aging adults.

Biotransformation novel advances - 2021 year in review.

Biotransformation field is constantly evolving with new molecular structures and discoveries of metabolic pathways that impact efficacy and safety. Recent review by Kramlinger et al. (2022) nicely captures the future (and the past) of highly impactful science of biotransformation (see the first article). Based on the selected articles, this review was categorized into three sections: (1) new modalities biotransformation, (2) drug discovery biotransformation, and (3) drug development biotransformation (Table 1).

Ferumoxytol dynamic contrast enhanced magnetic resonance imaging identifies altered placental cotyledon perfusion in rhesus macaques†.

Identification of placental dysfunction in early pregnancy with noninvasive imaging could be a valuable tool for assessing maternal and fetal risk. Dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) can be a powerful tool for interrogating placenta health. After inoculation with Zika virus or sham inoculation at gestation age (GA) 45 or 55 days, animals were imaged up to three times at GA65, GA100, and GA145. DCE MRI images were acquired at all imaging sessions using ferumoxytol, an iron nanoparticle-based contrast agent, and analyzed for placental intervillous blood flow, number of perfusion domains, and perfusion domain volume. Cesarean section was performed at GA155, and the placenta was photographed and dissected for histopathology. Photographs were used to align cotyledons with estimated perfusion domains from MRI, allowing comparison of estimated cotyledon volume to pathology. Monkeys were separated into high and low pathology groups based on the average number of pathologies present in the placenta. Perfusion domain flow, volume, and number increased through gestation, and total blood flow increased with gestation for both low pathology and high pathology groups. A statistically significant decrease in perfusion domain volume associated with pathology was detected at all gestational ages. Individual perfusion domain flow comparisons demonstrated a statistically significant decrease with pathology at GA100 and GA145, but not GA65. Since ferumoxytol is currently used to treat anemia during human pregnancy and as an off-label MRI contrast agent, future transition of this work to human pregnancy may be possible.

Simultaneous 3D-TOF angiography and 4D-flow MRI with enhanced flow signal using multiple overlapping thin slab acquisition and magnetization transfer.

PURPOSE: To investigate the fusion of 3D time-of-flight principles into 4D-flow MRI to enhance vessel contrast and signal without an exogenous contrast agent, enabling simultaneous in-flow based angiograms. METHODS: A 4D-flow MRI technique was developed consisting of multiple overlapping slabs with intermittent magnetization transfer preparation. The scan time penalty associated with multiple slab acquisitions was mitigated by using undersampled distributed spiral trajectories and compressed sensing reconstruction. A flow phantom was used to characterize in-flow enhancement, velocity noise improvement, and flow rate measurements against the single-slab 4D-flow MRI. In a patient-volunteer cohort (n = 15), magnitude-based angiograms were radiologically evaluated against 3D time-of-flight, and velocity measurements were compared pixel-wise against single-slab and contrast-enhanced 4D-flow MRI. RESULTS: Multiple-slab acquisitions, together with magnetization transfer preparation, substantially improved vessel signal, contrast, and vessel conspicuity in magnitude angiograms. Both clinical 3D time-of-flight and the proposed technique produced equivalent vessel depictions with no statistically significant difference (p < .1). Both techniques also produced clear depictions of brain aneurysms in all patients; however, very small vessels tended to show reduced conspicuity in the proposed technique. Velocity measurements agreed with contrast-enhanced and single-slab scans with high correlations (R2 = 0.941-0.974) and agreements (slopes = 0.994-1.071). Slab boundary and magnetization transfer-related artifacts were not observed in velocity measurements, and velocity noise was reduced with in-flow enhancement over single-slab scans (phantom). CONCLUSION: The vessel signal and contrast can be improved in 4D-flow MRI without exogenous contrast agents by utilizing in-flow enhancement, efficient sampling, and compressed sensing. The in-flow enhancement also enables simultaneous 3D time-of-flight angiograms useful for flow quantification and diagnosis.

Virtual injections using 4D flow MRI with displacement corrections and constrained probabilistic streamlines.

PURPOSE: Streamlines from 4D-flow MRI have been used clinically for intracranial blood-flow tracking. However, deterministic and stochastic errors degrade streamline quality. The purpose of this study is to integrate displacement corrections, probabilistic streamlines, and novel fluid constraints to improve selective blood-flow tracking and emulate "virtual bolus injections." METHODS: Both displacement artifacts (deterministic) and velocity noise (stochastic) inherently occur during phase-contrast MRI acquisitions. Here, two displacement correction methods, single-step and iterative, were tested in silico with simulated displacements and were compared with ground-truth velocity fields. Next, the effects of combining displacement corrections and constrained probabilistic streamlines were performed in 10 healthy volunteers using time-averaged 4D-flow data. Measures of streamline length and depth into vasculature were then compared with streamlines generated with no corrections and displacement correction alone using one-way repeated-measures analysis of variance and Friedman's tests. Finally, virtual injections with improved streamlines were generated for three intracranial pathology cases. RESULTS: Iterative displacement correction outperformed the single-step method in silico. In volunteers, the combination of displacement corrections and constrained probabilistic streamlines allowed for significant improvements in streamline length and increased the number of streamlines entering the circle of Willis relative to streamlines with no corrections and displacement correction alone. In the pathology cases, virtual injections with improved streamlines were qualitatively similar to dynamic arterial spin labeling images and allowed for forward/reverse selective flow tracking to characterize cerebrovascular malformations. CONCLUSION: Virtual injections with improved streamlines from 4D-flow MRI allow for flexible, robust, intracranial flow tracking.

Improved free-breathing liver fat and iron quantification using a 2D chemical shift-encoded MRI with flip angle modulation and motion-corrected averaging.

OBJECTIVES: 3D chemical shift-encoded (CSE) MRI enables accurate and precise quantification of proton density fat fraction (PDFF) and R2*, biomarkers of hepatic fat and iron deposition. Unfortunately, 3D CSE-MRI requires reliable breath-holding. Free-breathing 2D CSE-MRI with sequential radiofrequency excitation is a motion-robust alternative but suffers from low signal-to-noise-ratio (SNR). To overcome this limitation, this work explores the combination of flip angle-modulated (FAM) 2D CSE imaging with a non-local means (NLM) motion-corrected averaging technique. METHODS: In this prospective study, 35 healthy subjects (27 children/8 adults) were imaged on a 3T MRI-system. Multi-echo 3D CSE ("3D") and 2D CSE FAM ("FAM") images were acquired during breath-hold and free-breathing, respectively, to obtain PDFF and R2* maps of the liver. Multi-repetition FAM was postprocessed with direct averaging (DA)- and NLM-based averaging and compared to 3D CSE using Bland-Altmann and regression analysis. Image qualities of PDFF and R2* maps were reviewed by two radiologists using a Likert-like scale (score 1-5, 5 = best). RESULTS: Compared to 3D CSE, multi-repetition FAM-NLM showed excellent agreement (regression slope = 1.0, R2 = 0.996) for PDFF and good agreement (regression slope 1.08-1.15, R2 ≥ 0.899) for R2*. Further, multi-repetition FAM-NLM PDFF and R2* maps had fewer artifacts (score 3.8 vs. 3.2, p < 0.0001 for PDFF; score 3.2 vs. 2.6, p < 0.001 for R2*) and better overall image quality (score 4.0 vs. 3.5, p < 0.0001 for PDFF; score 3.4 vs. 2.7, p < 0.0001 for R2*). CONCLUSIONS: Free-breathing FAM-NLM provides superior image quality of the liver compared to the conventional breath-hold 3D CSE-MRI, while minimizing bias for PDFF and R2* quantification. KEY POINTS: • 2D CSE FAM-NLM is a free-breathing method for liver fat and iron quantification and viable alternative for patients unable to hold their breath. • 2D CSE FAM-NLM is a feasible alternative to breath-hold 3D CSE methods, with low bias in proton density fat fraction (PDFF) and no clinically significant bias in R2*. • Quantitatively, multiple repetitions in 2D CSE FAM-NLM lead to improved SNR.

Differential DNA methylation across environments has no effect on gene expression in the eastern oyster.

It has been hypothesized that environmentally induced changes to gene body methylation could facilitate adaptive transgenerational responses to changing environments. We compared patterns of global gene expression (Tag-seq) and gene body methylation (reduced representation bisulfite sequencing) in 80 eastern oysters Crassostrea virginica from six full-sib families, common gardened for 14 months at two sites in the northern Gulf of Mexico that differed in mean salinity. At the time of sampling, oysters from the two sites differed in mass by 60% and in parasite loads by nearly two orders of magnitude. They also differentially expressed 35% of measured transcripts. However, we observed differential methylation at only 1.4% of potentially methylated loci in comparisons between individuals from these different environments, and little correspondence between differential methylation and differential gene expression. Instead, methylation patterns were largely driven by genetic differences among families, with a PERMANOVA analysis indicating nearly a two orders of magnitude greater number of genes differentially methylated between families than between environments. An analysis of CpG observed/expected values (CpG O/E) across the C. virginica genome showed a distinct bimodal distribution, with genes from the first cluster showing the lower CpG O/E values, greater methylation and higher and more stable gene expression, while genes from the second cluster showed lower methylation, and lower and more variable gene expression. Taken together, the differential methylation results suggest that only a small portion of the C. virginica genome is affected by environmentally induced changes in methylation. At this point, there is little evidence to suggest that environmentally induced methylation states would play a leading role in regulating gene expression responses to new environments.