Unsupervised, Statistically Based Systems Biology Approach for Unraveling the Genetics of Complex Traits: A Demonstration with Ethanol Metabolism.

BACKGROUND: A statistical pipeline was developed and used for determining candidate genes and candidate gene coexpression networks involved in 2 alcohol (i.e., ethanol [EtOH]) metabolism phenotypes, namely alcohol clearance and acetate area under the curve in a recombinant inbred (RI) (HXB/BXH) rat panel. The approach was also used to provide an indication of how EtOH metabolism can impact the normal function of the identified networks. METHODS: RNA was extracted from alcohol-naïve liver tissue of 30 strains of HXB/BXH RI rats. The reconstructed transcripts were quantitated, and data were used to construct gene coexpression modules and networks. A separate group of rats, comprising the same 30 strains, were injected with EtOH (2 g/kg) for measurement of blood EtOH and acetate levels. These data were used for quantitative trait loci (QTL) analysis of the rate of EtOH disappearance and circulating acetate levels. The analysis pipeline required calculation of the module eigengene values, the correction of these values with EtOH metabolism rates and acetate levels across the rat strains, and the determination of the eigengene QTLs. For a module to be considered a candidate for determining phenotype, the module eigengene values had to have significant correlation with the strain phenotypic values and the module eigengene QTLs had to overlap the phenotypic QTLs. RESULTS: Of the 658 transcript coexpression modules generated from liver RNA sequencing data, a single module satisfied all criteria for being a candidate for determining the alcohol clearance trait. This module contained 2 alcohol dehydrogenase genes, including the gene whose product was previously shown to be responsible for the majority of alcohol elimination in the rat. This module was also the only module identified as a candidate for influencing circulating acetate levels. This module was also linked to the process of generation and utilization of retinoic acid as related to the autonomous immune response. CONCLUSIONS: We propose that our analytical pipeline can successfully identify genetic regions and transcripts which predispose a particular phenotype and our analysis provides functional context for coexpression module components.

Utility of 68Ga-DOTATATE PET-MRI for Gamma Knife® stereotactic radiosurgery treatment planning for meningioma.

OBJECTIVES: To investigate the impact of adding 68Ga-DOTATATE PET/MRI to standard MRI for target volume delineation in Gamma Knife® stereotactic radiosurgery (GKSRS) for meningioma. METHODS: Seventeen patients with 18 lesions undergoing GKSRS for WHO grade 1 meningioma were enrolled in a prospective study. All patients underwent pre-treatment 68Ga-DOTATATE PET/MRI examination in addition to standard procedures. Five clinicians independently contoured the gross tumour volume (GTV) based on standard MRI (GTVMRI) and PET/MRI (GTVPET/MRI) on separate occasions. Interobserver agreement was evaluated using Cohen's Kappa statistic (CKS), Dice similarity coefficient (DC), and Hausdorff distance (HD). Statistical analysis was performed with paired t-test and Wilcoxon signed rank test. RESULTS: The addition of PET/MRI significantly increased GTV contour volume (mean GTVPET/MRI 3.59 cm3 versus mean GTVMRI 3.18 cm3, P = .008). Using the treating clinician's pre-treatment GTVMRI as the reference, median CKS (87.2 vs 77.5, P = .006) and DC (87.2 vs 77.4, P = .006) were significantly lower, and median HD (25.2 vs 31.0, P = .001) was significantly higher with the addition of PET/MRI. No significant difference was observed in interobserver contouring reproducibility between GTVMRI and GTVPET/MRI. CONCLUSION: The addition of 68Ga-DOTATATE PET/MRI for target volume delineation in GKSRS for meningioma is associated with an increase in GTV volume and greater interobserver variation. PET/MRI did not affect interobserver contouring reproducibility. ADVANCES IN KNOWLEDGE: This study provides novel insights into the impact of 68Ga-DOTATATE PET/MRI on GTV delineation and interobserver agreement in meningioma GKSRS, highlighting its potential for improving GKSRS treatment accuracy.

Beyond Genes: Inclusion of Alternative Splicing and Alternative Polyadenylation to Assess the Genetic Architecture of Predisposition to Voluntary Alcohol Consumption in Brain of the HXB/BXH Recombinant Inbred Rat Panel.

Post transcriptional modifications of RNA are powerful mechanisms by which eukaryotes expand their genetic diversity. For instance, researchers estimate that most transcripts in humans undergo alternative splicing and alternative polyadenylation. These splicing events produce distinct RNA molecules, which in turn yield distinct protein isoforms and/or influence RNA stability, translation, nuclear export, and RNA/protein cellular localization. Due to their pervasiveness and impact, we hypothesized that alternative splicing and alternative polyadenylation in brain can contribute to a predisposition for voluntary alcohol consumption. Using the HXB/BXH recombinant inbred rat panel (a subset of the Hybrid Rat Diversity Panel), we generated over one terabyte of brain RNA sequencing data (total RNA) and identified novel splice variants (via StringTie) and alternative polyadenylation sites (via aptardi) to determine the transcriptional landscape in the brains of these animals. After establishing an analysis pipeline to ascertain high quality transcripts, we quantitated transcripts and integrated genotype data to identify candidate transcript coexpression networks and individual candidate transcripts associated with predisposition to voluntary alcohol consumption in the two-bottle choice paradigm. For genes that were previously associated with this trait (e.g., Lrap, Ift81, and P2rx4) (Saba et al., Febs. J., 282, 3556-3578, Saba et al., Genes. Brain. Behav., 20, e12698), we were able to distinguish between transcript variants to provide further information about the specific isoforms related to the trait. We also identified additional candidate transcripts associated with the trait of voluntary alcohol consumption (i.e., isoforms of Mapkapk5, Aldh1a7, and Map3k7). Consistent with our previous work, our results indicate that transcripts and networks related to inflammation and the immune system in brain can be linked to voluntary alcohol consumption. Overall, we have established a pipeline for including the quantitation of alternative splicing and alternative polyadenylation variants in the transcriptome in the analysis of the relationship between the transcriptome and complex traits.

Aptardi predicts polyadenylation sites in sample-specific transcriptomes using high-throughput RNA sequencing and DNA sequence.

Annotation of polyadenylation sites from short-read RNA sequencing alone is a challenging computational task. Other algorithms rooted in DNA sequence predict potential polyadenylation sites; however, in vivo expression of a particular site varies based on a myriad of conditions. Here, we introduce aptardi (alternative polyadenylation transcriptome analysis from RNA-Seq data and DNA sequence information), which leverages both DNA sequence and RNA sequencing in a machine learning paradigm to predict expressed polyadenylation sites. Specifically, as input aptardi takes DNA nucleotide sequence, genome-aligned RNA-Seq data, and an initial transcriptome. The program evaluates these initial transcripts to identify expressed polyadenylation sites in the biological sample and refines transcript 3'-ends accordingly. The average precision of the aptardi model is twice that of a standard transcriptome assembler. In particular, the recall of the aptardi model (the proportion of true polyadenylation sites detected by the algorithm) is improved by over three-fold. Also, the model-trained using the Human Brain Reference RNA commercial standard-performs well when applied to RNA-sequencing samples from different tissues and different mammalian species. Finally, aptardi's input is simple to compile and its output is easily amenable to downstream analyses such as quantitation and differential expression.

Clinical impact of magnetic resonance imaging distortions on gamma knife radiosurgery.

INTRODUCTION: Magnetic resonance imaging (MRI) is the preferred imaging modality for Leksell Gamma Knife® (LGK) stereotactic radiosurgery (SRS) treatment planning (TP) due to superior soft tissue definition compared to computed tomography (CT). However, inherent distortions in MRI can affect treatment accuracy. The aim of this study was to develop a model to visualise the effect of MRI distortion on LGK SRS target coverage. METHODS: A model was developed using MR images of a QUASARTM GRID3D QA phantom. One hundred and twenty-five points were compared against known phantom geometry. Using linear interpolation, the model was applied retrospectively to 10 brain metastases patient data sets treated with LGK. The model estimated the corrected shot position accounting for distortion. A total of 44 metastases were investigated regarding the effects of MRI distortion on target coverage. RESULTS: The model indicated significantly reduced mean error by 0.30 mm and variance by 0.09 mm (P = 0.008). After model application, 23 (53%) metastases showed reduced coverage. Six of the 23 metastases were deemed to be potentially clinically significant changes. Results indicated MRI distortion had a greater effect on smaller targets (mean 0.06cc) located further away from the image isocentre (mean 64.88 mm). CONCLUSION: This study developed a model to visualise the effect of MRI distortion on LGK SRS target coverage. Results suggest that MRI distortion can affect target coverage and the developed model may be one method to assess its impact. These results indicate that MRI distortion may have a greater effect on smaller targets located at the image periphery.

Leksell Gamma Knife® - The first 1000 patients from the radiation therapist's perspective.

The Princess Alexandra Hospital (PAH) Gamma Knife® Centre of Queensland (GKCoQ) began operations in October of 2015 as a sub-specialty located within a larger radiation oncology service at PAH. It is uniquely positioned as the only Leksell Gamma Knife® (LGK) treatment unit available in the public hospital system in Australia, and the first and only service in Queensland. The GKCoQ treated the 1000th patient on 23 January 2019. LGK is a non-invasive alternative to neurosurgery which uses radioactive cobalt sources to treat a variety of intracranial conditions ranging from tumours and metastases to functional disorders. It is a platform for stereotactic radiosurgery, a highly precise form of radiotherapy utilising very high doses to the target while maximally sparing surrounding normal brain. LGK enables patient planning and treatment to be done in one day as an outpatient procedure. This paper will outline our LGK service and provide insight into the expanded role that radiation therapists have within the multidisciplinary team required to deliver radiosurgery in a timely manner. The training programme and radiation licensing pathway that have been established for radiation therapists will also be described.

Alcohol Sensitivity as an Endophenotype of Alcohol Use Disorder: Exploring Its Translational Utility between Rodents and Humans.

Alcohol use disorder (AUD) is a complex, chronic, relapsing disorder with multiple interacting genetic and environmental influences. Numerous studies have verified the influence of genetics on AUD, yet the underlying biological pathways remain unknown. One strategy to interrogate complex diseases is the use of endophenotypes, which deconstruct current diagnostic categories into component traits that may be more amenable to genetic research. In this review, we explore how an endophenotype such as sensitivity to alcohol can be used in conjunction with rodent models to provide mechanistic insights into AUD. We evaluate three alcohol sensitivity endophenotypes (stimulation, intoxication, and aversion) for their translatability across human and rodent research by examining the underlying neurobiology and its relationship to consumption and AUD. We show examples in which results gleaned from rodents are successfully integrated with information from human studies to gain insight in the genetic underpinnings of AUD and AUD-related endophenotypes. Finally, we identify areas for future translational research that could greatly expand our knowledge of the biological and molecular aspects of the transition to AUD with the broad hope of finding better ways to treat this devastating disorder.

Mutational Analysis of Neuropeptide Y Reveals Unusual Thermal Stability Linked to Higher-Order Self-Association.

Neuropeptide Y (NPY) is a 36-residue peptide, abundant in the central and peripheral nervous system. The peptide interacts with membrane-bound receptors to control processes such as food intake, vasoconstriction, and memory retention. The N-terminal polyproline sequence of NPY folds back onto a C-terminal α-helix to form a hairpin structure. The hairpin undergoes transient unfolding to allow the monomer to interact with its target membranes and receptors and to form reversible dimers in solution. Using computational, functional, and biophysical approaches, we characterized the role of two conserved tyrosines (Y20 and Y27) located within the hydrophobic core of the hairpin fold. Successive mutation of the tyrosines to more hydrophobic phenylalanines increased the thermal stability of NPY and reduced functional activity, consistent with computational studies predicting a more stable hairpin structure. However, mutant stability was high relative to wild-type: melting temperatures increased by approximately 20 °C for the single mutants (Y20F and Y27F) and by 30 °C for the double mutant (Y20F + Y27F). These findings suggested that the mutations were not just simply enhancing hairpin structure stability, but might also be driving self-association to dimer. Using analytical ultracentrifugation, we determined that the mutations indeed increased self-association, but shifted the equilibrium toward hexamer-like species. Notably, these latter species were not unique to the NPY mutants, but were found to preexist at low levels in the wild-type population. Collectively, the findings indicate that NPY self-association is more complex than previously recognized and that the ensemble of NPY quaternary states is tunable by modulating hairpin hydrophobicity.

The development and implementation of a performance appraisal framework for radiation therapists in planning and simulation.

It is a challenge for radiation therapists (RTs) to keep pace with changing planning technology and techniques while maintaining appropriate skills levels. The ability of individual RTs to meet the demands of this constantly changing practice can only be assured through establishing clearly defined standards for practice and a systematic process for providing feedback on performance. Investigation into existing models for performance appraisal produced minimal results so a radiation therapy-specific framework was developed. The goal for this initiative was to establish a framework that would reflect the complexity of practice and provide a clear measure of performance against them. This paper outlines the implementation of this framework into practice and discusses some lessons learned in the process. The framework was developed and implemented in six stages: (1) project team, (2) scope, (3) dosimetry pilot, (4) staff consultation, (5) finalisation and implementation and (6) future development and evaluation. Both cultural and organisational obstacles needed to be addressed before this framework could be successfully introduced. Even though this slowed progress, addressing these obstacles during the development process was essential to the success of this framework. The incremental approach provided the opportunity for each aspect to be tested and the development of subsequent stages to be informed by lessons learned during the previous one. This approach may be beneficial when developing and implementing projects involving performance appraisal to promote consistency, fairness and quality.

Evaluation of a performance appraisal framework for radiation therapists in planning and simulation.

INTRODUCTION: Constantly evolving technology and techniques within radiation therapy require practitioners to maintain a continuous approach to professional development and training. Systems of performance appraisal and adoption of regular feedback mechanisms are vital to support this development yet frequently lack structure and rely on informal peer support. METHODS: A Radiation Therapy Performance Appraisal Framework (RT-PAF) for radiation therapists in planning and simulation was developed to define expectations of practice and promote a supportive and objective culture of performance and skills appraisal. Evaluation of the framework was conducted via an anonymous online survey tool. Nine peer reviewers and fourteen recipients provided feedback on its effectiveness and the challenges and limitations of the approach. RESULTS: Findings from the evaluation were positive and suggested that both groups gained benefit from and expressed a strong interest in embedding the approach more routinely. Respondents identified common challenges related to the limited ability to implement suggested development strategies; this was strongly associated with time and rostering issues. CONCLUSIONS: This framework successfully defined expectations for practice and provided a fair and objective feedback process that focussed on skills development. It empowered staff to maintain their skills and reach their professional potential. Management support, particularly in regard to provision of protected time was highlighted as critical to the framework's ongoing success. The demonstrated benefits arising in terms of staff satisfaction and development highlight the importance of this commitment to the modern radiation therapy workforce.

Effect of the thermal environment on the efficiency of packed columns in supercritical fluid chromatography.

When a packed column is operated at temperatures and pressures near the critical point in supercritical fluid chromatography, the thermal environment in which it is placed has a significant impact on retention and efficiency. We measured the retention factors, plate heights, and related parameters for elution of a test mixture of alkylbenzenes with 5% methanol/95% carbon dioxide mobile phase on a 250 mm × 4.6 mm i.d. column packed with 5-micron Luna-C18 particles. Separations were performed at outlet pressures from 100 to 150 bar and a column oven temperature of 323K. For a bare column thermostated with convective air, significant efficiency losses were observed for outlet pressures equal to or less than 120 bar. These large efficiency losses are attributed to radial temperature gradients. Addition of foam insulation resulted in significant improvements in efficiency. Operating the column in still air using a commercially available column heater provided the best overall performance, with no measurable efficiency loss over the entire range of pressures studied. A reduced plate height of 1.88 was obtained at an optimum flow rate of 3.0 mL/min at 100 bar outlet pressure and with the temperature of the incoming mobile phase set approximately 2.3K above the temperature of the column oven. Retention time repeatability for all three thermal conditions was equal to or less than 0.5% RSD. These results demonstrate that it is possible to perform fast, efficient separations with excellent repeatability using SFC under near-critical conditions if the thermal environment is optimized to minimize the generation of radial temperature gradients.