Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas 'cutting' combined with homologous recombination of a 'repair' template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains, and rely on relatively low efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an E. coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo, which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic (HIS3, URA3) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a ß-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide for how to use this system.

Natural variation in yeast reveals multiple paths for acquiring higher stress resistance.

BACKGROUND: Organisms frequently experience environmental stresses that occur in predictable patterns and combinations. For wild Saccharomyces cerevisiae yeast growing in natural environments, cells may experience high osmotic stress when they first enter broken fruit, followed by high ethanol levels during fermentation, and then finally high levels of oxidative stress resulting from respiration of ethanol. Yeast have adapted to these patterns by evolving sophisticated "cross protection" mechanisms, where mild 'primary' doses of one stress can enhance tolerance to severe doses of a different 'secondary' stress. For example, in many yeast strains, mild osmotic or mild ethanol stresses cross protect against severe oxidative stress, which likely reflects an anticipatory response important for high fitness in nature. RESULTS: During the course of genetic mapping studies aimed at understanding the mechanisms underlying natural variation in ethanol-induced cross protection against H2O2, we found that a key H2O2 scavenging enzyme, cytosolic catalase T (Ctt1p), was absolutely essential for cross protection in a wild oak strain. This suggested the absence of other compensatory mechanisms for acquiring H2O2 resistance in that strain background under those conditions. In this study, we found surprising heterogeneity across diverse yeast strains in whether CTT1 function was fully necessary for acquired H2O2 resistance. Some strains exhibited partial dispensability of CTT1 when ethanol and/or salt were used as mild stressors, suggesting that compensatory peroxidases may play a role in acquired stress resistance in certain genetic backgrounds. We leveraged global transcriptional responses to ethanol and salt stresses in strains with different levels of CTT1 dispensability, allowing us to identify possible regulators of these alternative peroxidases and acquired stress resistance in general. CONCLUSIONS: Ultimately, this study highlights how superficially similar traits can have different underlying molecular foundations and provides a framework for understanding the diversity and regulation of stress defense mechanisms.

A hypothesis-based hop microbiology laboratory module testing the plausibility of the mythical origin of the India Pale Ale.

As one of the most famous fermented drinks in the world, beer is an especially relatable topic for microbiology courses. Here, we describe a short and easily adaptable module based on the antibacterial properties of hops used in brewing. By the 15th century, beer recipes included hops (the flower of the Humulus lupulus plant) as a bittering agent and antimicrobial. By the 19th century, the highly hopped Indian Pale Ale (IPA) became popular, and a modern myth has emerged that IPAs were invented to survive long ocean voyages such as from Britain to India. With that myth in mind, we designed a hypothesis-driven microbiology lab module that tests the plausibility of this brewing myth-namely that highly hopped beers possess enough antibacterial activity to prevent spoilage, while lowly hopped beers do not. The overall design of the module is to test the antimicrobial properties of hops using petri plates containing varying concentrations of hop extract. The module includes hypothesis generation and testing related to bacterial physiology and cell envelope morphology (hops are not equally effective against Gram-positive and Gram-negative bacteria) and to mechanisms of antimicrobial resistance (as beer spoilage bacteria have repeatedly evolved hop resistance). Pre- and post-assessment showed that students made significant gains in the learning objectives for the module, which encourages critical thinking and hypothesis testing by linking microbial physiology and antimicrobial resistance to an important and topical real-world application.

Foam fractionation for removal of per- and polyfluoroalkyl substances: Towards closing the mass balance.

Foam fractionation has recently attracted attention as a low-cost and environmentally benign treatment technology for water contaminated with per- and polyfluoroalkyl substances (PFAS). However, data on the mass balance over the foam fractionation process are scarce and when available, gaps in the mass balance are often identified. This study verified the high treatment efficiency of a pilot-scale foam fractionation system for removal of PFAS from industrial water contaminated with aqueous film-forming foam. ΣPFAS removal reached up to 84 % and the removal of perfluorooctane sulfonic acid (PFOS) up to 97 %, but the short-chain perfluorobutanoic acid (PFBA) was only removed with a mean efficiency of 1.5 %. In general, mobile short-chain PFAS were removed less efficiently when the perfluorocarbon chain length was below six for carboxylic acids and below five for sulfonic acids. Fluctuations in treatment efficiency due to natural variations in the chemistry of the influent water were minor, confirming the robustness of the technology, but significant positive correlations between PFAS removal and influent metal concentration and conductivity were observed. Over all experiments, the mass balance closure did not differ significantly from 100 %. Nonetheless, PFAS sorption to the walls of the reactor was measured, as well as high PFAS emissions by the air exiting the reactor. PFAS emissions in aerosols correlated positively with mass balance closure. The elevated aerial PFAS concentrations measured in the experimental facility have implications for worker safety and prevention of PFAS-emissions to the atmosphere, and demonstrate the importance of installing appropriate filters on the air outlet of foam fractionation systems.

Discriminating Mild Traumatic Brain Injury and Posttraumatic Stress Disorder Using Latent Neuroimaging and Neuropsychological Profiles in Active-Duty Military Service Members.

OBJECTIVE: Mild traumatic brain injury (mTBI) and posttraumatic stress disorder (PTSD) commonly occur among military Service Members and Veterans and have heterogenous, but also overlapping symptom presentations, which often complicate the diagnoses of underlying impairments and development of effective treatment plans. Thus, we sought to examine whether the combination of whole brain gray matter (GM) and white matter (WM) structural measures with neuropsychological performance can aid in the classification of military personnel with mTBI and PTSD. METHODS: Active-Duty US Service Members ( n = 156; 87.8% male) with a history of mTBI, PTSD, combined mTBI+PTSD, or orthopedic injury completed a neuropsychological battery and T1- and diffusion-weighted structural neuroimaging. Cortical, subcortical, ventricular, and WM volumes and whole brain fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD), and axial diffusivity (AD) were calculated. Latent profile analyses were performed to determine how the GM and WM indicators, together with neuropsychological indicators, classified individuals. RESULTS: For both GM and WM, respectively, a 4-profile model was the best fit. The GM model identified greater ventricular volumes in Service Members with cognitive symptoms, including those with a diagnosis of mTBI, either alone or with PTSD. The WM model identified reduced FA and elevated RD in those with psychological symptoms, including those with PTSD or mTBI and comorbid PTSD. However, contrary to expectation, a global neural signature unique to those with comorbid mTBI and PTSD was not identified. CONCLUSIONS: The findings demonstrate that neuropsychological performance alone is more robust in differentiating Active-Duty Service Members with mTBI and PTSD, whereas global neuroimaging measures do not reliably differentiate between these groups.

The dynamic response to hypo-osmotic stress reveals distinct stages of freshwater acclimation by a euryhaline diatom.

The salinity gradient separating marine and freshwater environments is a major ecological divide, and the mechanisms by which most organisms adapt to new salinity environments are poorly understood. Diatoms are a lineage of ancestrally marine microalgae that have repeatedly colonized and diversified in freshwaters. Cyclotella cryptica is a euryhaline diatom found in salinities ranging from fully freshwater to fully marine, thus providing a powerful system for understanding the genomic mechanisms for mitigating and acclimating to low salinity. To understand how diatoms mitigate acute hypo-osmotic stress, we abruptly shifted C. cryptica from seawater to freshwater and performed transcriptional profiling during the first 10 h. Freshwater shock dramatically remodelled the transcriptome, with ~50% of the genome differentially expressed in at least one time point. The peak response occurred within 1 h, with strong repression of genes involved in cell growth and osmolyte production, and strong induction of specific stress defence genes. Transcripts largely returned to baseline levels within 4-10 h, with growth resuming shortly thereafter, suggesting that gene expression dynamics may be useful for predicting acclimation. Moreover, comparison to a transcriptomics study of C. cryptica following months-long acclimation to freshwater revealed little overlap between the genes and processes differentially expressed in cells exposed to acute stress versus fully acclimated conditions. Altogether, this study highlights the power of time-resolved transcriptomics to reveal fundamental insights into how cells dynamically respond to an acute environmental shift and provides new insights into how diatoms mitigate natural salinity fluctuations and have successfully diversified across freshwater habitats worldwide.

Comparing resting-state connectivity of working memory networks in U.S. Service members with mild traumatic brain injury and posttraumatic stress disorder.

Mild traumatic brain injury (mTBI) and posttraumatic stress disorder (PTSD) are prevalent among military populations, and both have been associated with working memory (WM) impairments. Previous resting-state functional connectivity (rsFC) research conducted separately in PTSD and mTBI populations suggests that there may be similar and distinct abnormalities in WM-related networks. However, no studies have compared rsFC of WM brain regions in participants with mTBI versus PTSD. We used resting-state fMRI to investigate rsFC of WM networks in U.S. Service Members (n = 127; ages 18-59) with mTBI only (n = 46), PTSD only (n = 24), and an orthopedically injured (OI) control group (n = 57). We conducted voxelwise rsFC analyses with WM brain regions to test for differences in WM network connectivity in mTBI versus PTSD. Results revealed reduced rsFC between ventrolateral prefrontal cortex (vlPFC), lateral premotor cortex, and dorsolateral prefrontal cortex (dlPFC) WM regions and brain regions in the dorsal attention and somatomotor networks in both mTBI and PTSD groups versus controls. When compared to those with mTBI, individuals with PTSD had lower rsFC between both the lateral premotor WM seed region and middle occipital gyrus as well as between the dlPFC WM seed region and paracentral lobule. Interestingly, only vlPFC connectivity was significantly associated with WM performance across the samples. In conclusion, we found primarily overlapping patterns of reduced rsFC in WM brain regions in both mTBI and PTSD groups. Our finding of decreased vlPFC connectivity associated with WM is consistent with previous clinical and neuroimaging studies. Overall, these results provide support for shared neural substrates of WM in individuals with either mTBI or PTSD.

Latent Neuropsychological Profiles to Discriminate Mild Traumatic Brain Injury and Posttraumatic Stress Disorder in Active-Duty Service Members.

OBJECTIVE: To determine whether cognitive and psychological symptom profiles differentiate clinical diagnostic classifications (eg, history of mild traumatic brain injury [mTBI] and posttraumatic stress disorder [PTSD]) in military personnel. METHODS: US Active-Duty Service Members ( N = 209, 89% male) with a history of mTBI ( n = 56), current PTSD ( n = 23), combined mTBI + PTSD ( n = 70), or orthopedic injury controls ( n = 60) completed a neuropsychological battery assessing cognitive and psychological functioning. Latent profile analysis was performed to determine how neuropsychological outcomes of individuals clustered together. Diagnostic classifications (ie, mTBI, PTSD, mTBI + PTSD, and orthopedic injury controls) within each symptom profile were examined. RESULTS: A 5-profile model had the best fit. The profiles differentiated subgroups with high (34.0%) or normal (21.5%) cognitive and psychological functioning, cognitive symptoms (19.1%), psychological symptoms (15.3%), and combined cognitive and psychological symptoms (10.0%). The symptom profiles differentiated participants as would generally be expected. Participants with PTSD were mainly represented in the psychological symptom subgroup, while orthopedic injury controls were mainly represented in the high-functioning subgroup. Further, approximately 79% of participants with comorbid mTBI and PTSD were represented in a symptomatic group (∼24% = cognitive symptoms, ∼29% = psychological symptoms, and 26% = combined cognitive/psychological symptoms). Our results also showed that approximately 70% of military personnel with a history of mTBI were represented in the high- and normal-functioning groups. CONCLUSIONS: These results demonstrate both overlapping and heterogeneous symptom and performance profiles in military personnel with a history of mTBI, PTSD, and/or mTBI + PTSD. The overlapping profiles may underscore why these diagnoses are often difficult to diagnose and treat, but suggest that advanced statistical models may aid in identifying profiles representing symptom and cognitive performance impairments within patient groups and enable identification of more effective treatment targets.

The Primary Care Management of Headache: Synopsis of the 2020 U.S. Department of Veterans Affairs and U.S. Department of Defense Clinical Practice Guideline.

INTRODUCTION: In June of 2020, the U.S. DVA and DoD approved a new joint clinical practice guideline for assessing and managing patients with headache. This guideline provides a framework to evaluate, treat, and longitudinally manage the individual needs and preferences of patients with headache. METHODS: In October of 2018, the DVA/DoD Evidence-Based Practice Work Group convened a guideline development panel that included clinical stakeholders and conformed to the National Academy of Medicine's tenets for trustworthy clinical practice guidelines. RESULTS: The guideline panel developed key questions, systematically searched and evaluated the literature, created a 1-page algorithm, and advanced 42 recommendations using the Grading of Recommendations Assessment, Development, and Evaluation system. CONCLUSION: This synopsis summarizes the key features of the guideline in three areas: prevention, assessing and treating medication overuse headache, and nonpharmacologic and pharmacologic management of headache.

A Wild Yeast Laboratory Activity: From Isolation to Brewing.

Microbial fermentation is a common form of metabolism that has been exploited by humans to great benefit. Industrial fermentation currently produces a myriad of products ranging from biofuels to pharmaceuticals. About one-third of the world's food is fermented, and the brewing of fermented beverages in particular has an ancient and storied history. Because fermentation is so intertwined with our daily lives, the topic is easily relatable to students interested in real-world applications for microbiology. Here, we describe the curriculum for a guided inquiry-based laboratory course that combines yeast molecular ecology and brewing. The rationale for the course is to compare commercial Saccharomyces cerevisiae yeast strains, which have been domesticated through thousands of generations of selection, with wild yeast, where there is growing interest in their potentially unique brewing characteristics. Because wild yeasts are so easy to isolate, identify, and characterize, this is a great opportunity to present key concepts in molecular ecology and genetics in a way that is relevant and accessible to students. We organized the course around three main modules: isolation and identification of wild yeast, phenotypic characterization of wild and commercial ale yeast strains, and scientific design of a brewing recipe and head-to-head comparison of the performance of a commercial and wild yeast strain in the brewing process. Pre- and postassessment showed that students made significant gains in the learning objectives for the course, and students enjoyed connecting microbiology to a real-world application.

Resting-State Correlations of Fatigue Following Military Deployment.

OBJECTIVE: Persistent fatigue is common among military servicemembers returning from deployment, especially those with a history of mild traumatic brain injury (mTBI). The purpose of this study was to characterize fatigue following deployment using the Multidimensional Fatigue Inventory (MFI), a multidimensional self-report instrument. The study was developed to test the hypothesis that if fatigue involves disrupted effort/reward processing, this should manifest as altered basal ganglia functional connectivity as observed in other amotivational states. METHODS: Twenty-eight current and former servicemembers were recruited and completed the MFI. All 28 participants had a history of at least one mTBI during deployment. Twenty-six participants underwent resting-state functional MRI. To test the hypothesis that fatigue was associated with basal ganglia functional connectivity, the investigators measured correlations between MFI subscale scores and the functional connectivity of the left and right caudate, the putamen, and the globus pallidus with the rest of the brain, adjusting for the presence of depression. RESULTS: The investigators found a significant correlation between functional connectivity of the left putamen and bilateral superior frontal gyri and mental fatigue scores. No correlations with the other MFI subscales survived multiple comparisons correction. CONCLUSIONS: This exploratory study suggests that mental fatigue in military servicemembers with a history of deployment with at least one mTBI may be related to increased striatal-prefrontal functional connectivity, independent of depression. A finding of effort/reward mismatch may guide future treatment approaches.

The genome of a nonphotosynthetic diatom provides insights into the metabolic shift to heterotrophy and constraints on the loss of photosynthesis.

Although most of the tens of thousands of diatom species are photoautotrophs, a small number of heterotrophic species no longer photosynthesize. We sequenced the genome of a nonphotosynthetic diatom, Nitzschia Nitz4, to determine how carbon metabolism was altered in the wake of this trophic shift. Nitzschia Nitz4 has retained its plastid and plastid genome, but changes associated with the transition to heterotrophy were cellular-wide and included losses of photosynthesis-related genes from the nuclear and plastid genomes, elimination of isoprenoid biosynthesis in the plastid, and remodeling of mitochondrial glycolysis to maximize adenosine triphosphte (ATP) yield. The genome contains a ß-ketoadipate pathway that may allow Nitzschia Nitz4 to metabolize lignin-derived compounds. Diatom plastids lack an oxidative pentose phosphate pathway (oPPP), leaving photosynthesis as the primary source of NADPH to support essential biosynthetic pathways in the plastid and, by extension, limiting available sources of NADPH in nonphotosynthetic plastids. The genome revealed similarities between nonphotosynthetic diatoms and apicomplexan parasites for provisioning NADPH in their plastids and highlighted the ancestral absence of a plastid oPPP as a potentially important constraint on loss of photosynthesis, a hypothesis supported by the higher frequency of transitions to parasitism or heterotrophy in lineages that have a plastid oPPP.

Distinct patterns of resting-state connectivity in U.S. service members with mild traumatic brain injury versus posttraumatic stress disorder.

Mild traumatic brain injury (mTBI) is highly prevalent in military populations, with many service members suffering from long-term symptoms. Posttraumatic stress disorder (PTSD) often co-occurs with mTBI and predicts worse clinical outcomes. Functional neuroimaging research suggests there are both overlapping and distinct patterns of resting-state functional connectivity (rsFC) in mTBI versus PTSD. However, few studies have directly compared rsFC of cortical networks in military service members with these two conditions. In the present study, U.S. service members (n = 137; ages 19-59; 120 male) underwent resting-state fMRI scans. Participants were divided into three study groups: mTBI only, PTSD only, and orthopedically injured (OI) controls. Analyses investigated group differences in rsFC for cortical networks: default mode (DMN), frontoparietal (FPN), salience, somatosensory, motor, auditory, and visual. Analyses were family-wise error (FWE) cluster-corrected and Bonferroni-corrected for number of network seeds regions at the whole brain level (pFWE < 0.002). Both mTBI and PTSD groups had reduced rsFC for DMN and FPN regions compared with OI controls. These group differences were largely driven by diminished connectivity in the PTSD group. rsFC with the middle frontal gyrus of the FPN was increased in mTBI, but decreased in PTSD. Overall, these results suggest that PTSD symptoms may have a more consistent signal than mTBI. Our novel findings of opposite patterns of connectivity with lateral prefrontal cortex highlight a potential biomarker that could be used to differentiate between these conditions.

Cingulo-Opercular and Frontoparietal Network Control of Effort and Fatigue in Mild Traumatic Brain Injury.

Neural substrates of fatigue in traumatic brain injury (TBI) are not well understood despite the considerable burden of fatigue on return to productivity. Fatigue is associated with diminishing performance under conditions of high cognitive demand, sense of effort, or need for motivation, all of which are associated with cognitive control brain network integrity. We hypothesize that the pathophysiology of TBI results in damage to diffuse cognitive control networks, disrupting coordination of moment-to-moment monitoring, prediction, and regulation of behavior. We investigate the cingulo-opercular (CO) and frontoparietal (FP) networks, which are engaged to sustain attention for task and maintain performance. A total of 61 individuals with mild TBI and 42 orthopedic control subjects participated in functional MRI during performance of a constant effort task requiring altering the amount of effort (25, 50, or 75% of maximum effort) utilized to manually squeeze a pneumostatic bulb across six 30-s trials. Network-based statistics assessed within-network organization and fluctuation with task manipulations by group. Results demonstrate small group differences in network organization, but considerable group differences in the evolution of task-related modulation of connectivity. The mild TBI group demonstrated elevated CO connectivity throughout the task with little variation in effort level or time on task (TOT), while CO connectivity diminished over time in controls. Several interregional CO connections were predictive of fatigue in the TBI group. In contrast, FP connectivity fluctuated with task manipulations and predicted fatigue in the controls, but connectivity fluctuations were delayed in the mild traumatic brain injury (mTBI) group and did not relate to fatigue. Thus, the mTBI group's hyper-connectivity of the CO irrespective of task demands, along with hypo-connectivity and delayed peak connectivity of the FP, may allow for attainment of task goals, but also contributes to fatigue. Findings are discussed in relation to performance monitoring of prediction error that relies on internal cues from sensorimotor feedback during task performance. Delay or inability to detect and respond to prediction errors in TBI, particularly evident in bilateral insula-temporal CO connectivity, corresponds to day-to-day fatigue and fatigue during task performance.

Testosterone and Resting State Connectivity of the Parahippocampal Gyrus in Men With History of Deployment-Related Mild Traumatic Brain Injury.

INTRODUCTION: The purpose of this study was to explore the effect of low testosterone level on whole-brain resting state (RS) connectivity in male veterans with symptoms such as sleep disturbance, fatiguability, pain, anxiety, irritability, or aggressiveness persisting after mild traumatic brain injury (mTBI). Follow-up analyses were performed to determine if sleep scores affected the results. MATERIALS AND METHODS: In our cross-sectional design study, RS magnetic resonance imaging scans on 28 veterans were performed, and testosterone, sleep quality, mood, and post-traumatic stress symptoms were measured. For each participant, we computed the average correlation of each voxel's time-series with the rest of the voxels in the brain, then used AFNI's 3dttest++ on the group data to determine whether the effects of testosterone level on whole-brain connectivity were significant. We then performed follow-up region of interest-based RS analyses of testosterone, with and without sleep quality as a covariate. The study protocol was approved by the National Institute of Health's Combined Neuroscience Institutional Review Board. RESULTS: Sixteen participants reported repeated blast exposure in theater, leading to symptoms; the rest reported exposure to a single blast or a nonblast TBI. Thirty-three percent had testosterone levels <300 ng/dL. Testosterone level was lower in participants who screened positive for post-traumatic stress disorder compared to those who screened negative, but it did not reach statistical significance. Whole-brain connectivity and testosterone level were positively correlated in the left parahippocampal gyrus (LPhG), especially in its connectivity with frontal areas, the lingual gyrus, cingulate, insula, caudate, and right parahippocampal gyrus. Further analysis revealed that the effect of testosterone on LPhG connectivity is only partially mediated by sleep quality. Sleep quality by itself had an effect on connectivity of the thalamus, cerebellum, precuneus, and posterior cingulate. CONCLUSION: Lower testosterone levels were correlated with lower connectivity of the LPhG. Weaknesses of this study include a retrospective design based on self-report of mTBI and the lack of a control group without TBI. Without a control group or pre-injury testosterone measures, we were not able to attribute the rate of low testosterone in our participants to TBI per se. Also testosterone levels were checked only once. The high rate of low testosterone level that we found suggests there may be an association between low testosterone level and greater post-traumatic stress disorder symptoms following deployment, but the causality of the relationships between TBI and deployment stress, testosterone level, behavioral symptomatology, and LPhG connectivity remains to be determined. Our study on men with persistent symptoms postdeployment and post-mTBI may help us understand the role of low testosterone and sleep quality in persistent symptoms and may be important in developing therapeutic interventions. Our results highlight the role of the LPhG, as we found that whole-brain connectivity in that region was positively associated with testosterone level, with only a limited portion of that effect attributable to sleep quality.

Barriers and facilitators to implementation of epilepsy self-management programs: a systematic review using qualitative evidence synthesis methods.

BACKGROUND: Epilepsy affects nearly 50 million people worldwide. Self-management is critical for individuals with epilepsy in order to maintain optimal physical, cognitive, and emotional health. Implementing and adopting a self-management program requires considering many factors at the person, program, and systems levels. We conducted a systematic review of qualitative and mixed-methods studies to identify facilitators and barriers that impact implementation and adoption of self-management programs for adults with epilepsy. METHODS: We used established systematic review methodologies for qualitative and mixed-methods studies. We included studies addressing facilitators (i.e., factors that aided) or barriers (i.e., factors that impeded) to implementation and adoption of self-management interventions for adults with epilepsy. We conducted a narrative thematic synthesis to identify facilitators and barriers. RESULTS: The literature search identified 2700 citations; 13 studies met eligibility criteria. Our synthesis identified five themes that categorize facilitators and barriers to successful implementation epilepsy self-management: (1) relevance, intervention content that facilitates acquisition of self-management skills; (2) personalization, intervention components that account for the individual's social, physical, and environmental characteristics; (3) intervention components, components and dosing of the intervention; (4) technology considerations, considerations that account for individual's use, familiarity with, and ownership of technology; and (5) clinician interventionist, role and preparation of the individual who leads intervention. We identified facilitators in 11 of the 13 studies and barriers in 11 of the 13 studies and classified these by social-ecological level (i.e., patient/caregiver, program, site/system). CONCLUSION: Identification of facilitators and barriers at multiple levels provides insight into disease-specific factors that influence implementation and adoption of self-management programs for individuals with epilepsy. Our findings indicate that involving individuals with epilepsy and their caregivers in intervention development, and then tailoring intervention content during the intervention, can help ensure the content is relevant to intervention participants. Our findings also indicate the role of the clinician (i.e., the individual who provides self-management education) is important to intervention implementation, and key issues with clinicians were identified as barriers and opportunities for improvement. Overall, our findings have practical value for those seeking to implement and adopt self-management interventions for epilepsy and other chronic illnesses. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration number is CRD42018098604.

Comparison of RNA isolation methods on RNA-Seq: implications for differential expression and meta-analyses.

BACKGROUND: The increasing number of transcriptomic datasets has allowed for meta-analyses, which can be valuable due to their increased statistical power. However, meta-analyses can be confounded by so-called "batch effects," where technical variation across different batches of RNA-seq experiments can clearly produce spurious signals of differential expression and reduce our power to detect true differences. While batch effects can sometimes be accounted for, albeit with caveats, a better strategy is to understand their sources to better avoid them. In this study, we examined the effects of RNA isolation method as a possible source of batch effects in RNA-seq design. RESULTS: Based on the different chemistries of "classic" hot phenol extraction of RNA compared to common commercial RNA isolation kits, we hypothesized that specific mRNAs may be preferentially extracted depending upon method, which could masquerade as differential expression in downstream RNA-seq analyses. We tested this hypothesis using the Saccharomyces cerevisiae heat shock response as a well-validated environmental response. Comparing technical replicates that only differed in RNA isolation method, we found over one thousand transcripts that appeared "differentially" expressed when comparing hot phenol extraction with the two kits. Strikingly, transcripts with higher abundance in the phenol-extracted samples were enriched for membrane proteins, suggesting that indeed the chemistry of hot phenol extraction better solubilizes those species of mRNA. CONCLUSIONS: Within a self-contained experimental batch (e.g. control versus treatment), the method of RNA isolation had little effect on the ability to identify differentially expressed transcripts. However, we suggest that researchers performing meta-analyses across different experimental batches strongly consider the RNA isolation methods for each experiment.

Accurate and Sensitive Quantitation of the Dynamic Heat Shock Proteome Using Tandem Mass Tags.

Cells respond to environmental perturbations and insults through modulating protein abundance and function. However, the majority of studies have focused on changes in RNA abundance because quantitative transcriptomics has historically been more facile than quantitative proteomics. Modern Orbitrap mass spectrometers now provide sensitive and deep proteome coverage, allowing direct, global quantification of not only protein abundance but also post-translational modifications (PTMs) that regulate protein activity. We implemented and validated using the well-characterized heat shock response of budding yeast, a tandem mass tagging (TMT), triple-stage mass spectrometry (MS3) strategy to measure global changes in the proteome during the yeast heat shock response over nine time points. We report that basic-pH, ultra-high performance liquid chromatography (UPLC) fractionation of tryptic peptides yields superfractions of minimal redundancy, a crucial requirement for deep coverage and quantification by subsequent LC-MS3. We quantified 2275 proteins across three biological replicates and found that differential expression peaked near 90 min following heat shock (with 868 differentially expressed proteins at 5% false discovery rate). The sensitivity of the approach also allowed us to detect changes in the relative abundance of ubiquitination and phosphorylation PTMs over time. Remarkably, relative quantification of post-translationally modified peptides revealed striking evidence of regulation of the heat shock response by protein PTMs. These data demonstrate that the high precision of TMT-MS3 enables peptide-level quantification of samples, which can reveal important regulation of protein abundance and regulatory PTMs under various experimental conditions.

Self-management of Epilepsy: A Systematic Review.

Background: Although self-management is recommended for persons with epilepsy, its optimal strategies and effects are uncertain. Purpose: To evaluate the components and efficacy of self-management interventions in the treatment of epilepsy in community-dwelling persons. Data Sources: English-language searches of MEDLINE, Cochrane Central Register of Controlled Trials, PsycINFO, and CINAHL in April 2018; the MEDLINE search was updated in March 2019. Study Selection: Randomized and nonrandomized comparative studies of self-management interventions for adults with epilepsy. Data Extraction: An investigator assessed study characteristics; intervention details, including 6 components of self-management; and outcomes, which were verified by a second reviewer. Risk of bias (ROB) was assessed independently by 2 investigators. Data Synthesis: 13 randomized and 2 nonrandomized studies (2514 patients) evaluated self-management interventions. Interventions were delivered primarily in group settings, used a median of 4 components, and followed 2 general strategies: 1 based on education and the other on psychosocial therapy. Education-based approaches improved self-management behaviors (standardized mean difference, 0.52 [95% CI, 0.0 to 1.04]), and psychosocial therapy-based approaches improved quality of life (mean difference, 6.64 [CI, 2.51 to 10.77]). Overall, self-management interventions did not reduce seizure rates, but 1 educational intervention decreased a composite of seizures, emergency department visits, and hospitalizations. Limitation: High ROB in most studies, incomplete intervention descriptions, and studies limited to English-language publications. Conclusion: There is limited evidence that self-management strategies modestly improve some patient outcomes that are important to persons with epilepsy. Overall, self-management research in epilepsy is limited by the range of interventions tested, the small number of studies using self-monitoring technology, and uncertainty about components and strategies associated with benefit. Primary Funding Source: U.S. Department of Veterans Affairs. (PROSPERO: CRD42018098604).

Subcortical shape and neuropsychological function among U.S. service members with mild traumatic brain injury.

In a recent manuscript, our group demonstrated shape differences in the thalamus, nucleus accumbens, and amygdala in a cohort of U.S. Service Members with mild traumatic brain injury (mTBI). Given the significant role these structures play in cognitive function, this study directly examined the relationship between shape metrics and neuropsychological performance. The imaging and neuropsychological data from 135 post-deployed United States Service Members from two groups (mTBI and orthopedic injured) were examined. Two shape features modeling local deformations in thickness (RD) and surface area (JD) were defined vertex-wise on parametric mesh-representations of 7 bilateral subcortical gray matter structures. Linear regression was used to model associations between subcortical morphometry and neuropsychological performance as a function of either TBI status or, among TBI patients, subjective reporting of initial concussion severity (CS). Results demonstrated several significant group-by-cognition relationships with shape metrics across multiple cognitive domains including processing speed, memory, and executive function. Higher processing speed was robustly associated with more dilation of caudate surface area among patients with mTBI who reported more than one CS variables (loss of consciousness (LOC), alteration of consciousness (AOC), and/or post-traumatic amnesia (PTA)). These significant patterns indicate the importance of subcortical structures in cognitive performance and support a growing functional neuroanatomical literature in TBI and other neurologic disorders. However, prospective research will be required before exact directional evolution and progression of shape can be understood and utilized in predicting or tracking cognitive outcomes in this patient population.