Dynamics of Gene Loss following Ancient Whole-Genome Duplication in the Cryptic Paramecium Complex.

Whole-genome duplications (WGDs) have shaped the gene repertoire of many eukaryotic lineages. The redundancy created by WGDs typically results in a phase of massive gene loss. However, some WGD-derived paralogs are maintained over long evolutionary periods, and the relative contributions of different selective pressures to their maintenance are still debated. Previous studies have revealed a history of three successive WGDs in the lineage of the ciliate Paramecium tetraurelia and two of its sister species from the Paramecium aurelia complex. Here, we report the genome sequence and analysis of 10 additional P. aurelia species and 1 additional out group, revealing aspects of post-WGD evolution in 13 species sharing a common ancestral WGD. Contrary to the morphological radiation of vertebrates that putatively followed two WGD events, members of the cryptic P. aurelia complex have remained morphologically indistinguishable after hundreds of millions of years. Biases in gene retention compatible with dosage constraints appear to play a major role opposing post-WGD gene loss across all 13 species. In addition, post-WGD gene loss has been slower in Paramecium than in other species having experienced genome duplication, suggesting that the selective pressures against post-WGD gene loss are especially strong in Paramecium. A near complete lack of recent single-gene duplications in Paramecium provides additional evidence for strong selective pressures against gene dosage changes. This exceptional data set of 13 species sharing an ancestral WGD and 2 closely related out group species will be a useful resource for future studies on Paramecium as a major model organism in the evolutionary cell biology.

Posttraumatic stress and delay discounting: a meta-analytic review.

Delay discounting-the extent to which individuals show a preference for smaller immediate rewards over larger delayed rewards-has been proposed as a transdiagnostic neurocognitive process across mental health conditions, but its examination in relation to posttraumatic stress disorder (PTSD) is comparatively recent. To assess the aggregated evidence for elevated delay discounting in relation to posttraumatic stress, we conducted a meta-analysis on existing empirical literature. Bibliographic searches identified 209 candidate articles, of which 13 articles with 14 independent effect sizes were eligible for meta-analysis, reflecting a combined sample size of N = 6897. Individual study designs included case-control (e.g. examination of differences in delay discounting between individuals with and without PTSD) and continuous association studies (e.g. relationship between posttraumatic stress symptom severity and delay discounting). In a combined analysis of all studies, the overall relationship was a small but statistically significant positive association between posttraumatic stress and delay discounting (r = .135, p < .0001). The same relationship was statistically significant for continuous association studies (r = .092, p = .027) and case-control designs (r = .179, p < .001). Evidence of publication bias was minimal. The included studies were limited in that many did not concurrently incorporate other psychiatric conditions in the analyses, leaving the specificity of the relationship to posttraumatic stress less clear. Nonetheless, these findings are broadly consistent with previous meta-analyses of delayed reward discounting in relation to other mental health conditions and provide further evidence for the transdiagnostic utility of this construct.

Massive colonization of protein-coding exons by selfish genetic elements in Paramecium germline genomes.

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression.

A brief report on the temporal relations between social functioning and behavioral health among veterans.

Using a four-wave longitudinal design, three competing hypotheses (i.e., social selection, social causation, and reciprocal causation) were tested pertaining to the relation between social functioning and several indices of behavioral health [i.e., post-traumatic stress symptoms (PTSS), distress, and alcohol-related problems] among military veterans exposed to trauma. Across two latent growth curve analyses, data largely supported longitudinal links between improved social functioning and positive behavioral health, often indicating that the improvements in social functioning frequently precede improvements in behavioral health. Overall, findings underscore the merit of directly targeting social functioning as part of mental health treatment among trauma-exposed individuals.

Stamp Out Stigma: a national campaign to decrease stigma and increase behavioral health in fire service.

Firefighters are at risk for behavioral health problems; however, mental health stigma is a barrier to seeking treatment. Although effective treatments exist, many firefighters report that internal stigma prohibits presentation for effective care. This study aimed to decrease stigma about behavioral health in fire service through the development and delivery of a peer-led anti-stigma intervention campaign called Stamp Out Stigma (SOS). SOS consisted of three related phases. During Phase 1 (n = 12), we produced testimonials and piloted the videos with firefighters to determine which were most compelling. During Phase 2 (n = 23), we beta-tested our training curriculum with two departments, resulting in a significant decrease in internalized stigma but not in self-stigma. During Phase 3 (n = 73), we presented the curriculum to two cohorts of firefighters. Participation in the SOS workshop was associated with a significant decrease in firefighters' self-stigma from pre-, M = 22.7, SD = 6.3, to post-workshop, M = 20.8, SD = 5.5, t(66) = 3.2, p = 0.002. Participation in the SOS workshop was associated with a significant decrease in firefighters' internalized stigma, M = 2.0, SD = 0.5, to post-workshop, M = 1.8, SD = 0.4, t(66) = 2.8, p = 0.007. The SOS approach to stigma reduction is promising for modifying negative attitudes toward mental health and treatment seeking among professional firefighters.

The Effectiveness of Online Experiential Learning in a Psychiatry Clerkship.

OBJECTIVE: The Uniformed Services University of the Health Sciences 5-week psychiatry clerkship educates about 180 students a year at sites around the USA. In 2017, weekly in-person experiential learning sessions were implemented for local students and resulted in improved performance in several end-of-clerkship Objective Structured Clinical Examination (OSCE) skills as compared to distant learners who did not receive these sessions. The difference in performance (~ 10%) highlighted a need to provide comparable training for distant learners. Providing in-person, repeated simulated experiential training at multiple distant sites was not practical, requiring development of a novel online approach. METHODS: Students at all four distant sites over 2 years (n = 180) participated in five weekly synchronous online experiential learning sessions, while local students (n = 180) received five weekly in-person experiential learning sessions. Tele-simulation used the same curriculum, centralized faculty, and standardized patients as the in-person iterations. Overall end-of-clerkship OSCE performance was compared for learners receiving online versus in-person experiential learning for non-inferiority. Specific skills were compared to receiving no experiential learning. RESULTS: Overall OSCE performance was non-inferior for students who received synchronous online as compared to in-person experiential learning. Performance on each skill other than communication improved significantly when comparing students who received online versus no experiential learning (p < 0.05). CONCLUSIONS: The use of weekly online experiential learning to enhance clinical skills is comparable to in-person efforts. Virtual, simulated, synchronous experiential learning provides a feasible and scalable platform for training complex clinical skills to clerkship students, a critical capability given the impact the pandemic has had on clinical training.

A vocational rehabilitation partnership to provide transition services to young adults with neurodevelopmental disabilities: The cognitive skills enhancement program.

BACKGROUND: There is an urgent need for services that support a successful transition to postsecondary education and employment for young adults with neurodevelopmental and cognitive disabilities (e.g. autism spectrum disorder, attention-deficit/hyperactivity disorder, traumatic brain injury). OBJECTIVE: The purpose of this expository article is to describe the Cognitive Skills Enhancement Program (CSEP), a comprehensive clinical program designed for young adults with neurodevelopmental and cognitive disabilities transitioning to postsecondary education. METHODS: CSEP was developed through a community-academic partnership between a university and a state vocational rehabilitation program. Young adult participants complete programming that addresses four primary clinical targets: (1) emotion regulation, (2) social skills, (3) work readiness, and (4) community participation with the overall goal to increase awareness and promote successful employment outcomes while they transition to post-secondary education. RESULTS: To date, CSEP has supported 18 years of sustained programming and clinical services to 621 young adults with neurodevelopmental and cognitive disabilities. CONCLUSION: This partnership model allows for flexible responses to participant needs, implementation barriers, and advances in evidence-based practices. CSEP meets the needs of diverse stakeholders (e.g. state vocational rehabilitation, postsecondary training facilities, participants, universities) while providing high-quality and sustainable programming. Future directions include examining the clinical efficacy of current CSEP programming.

Intracardiac MR imaging (ICMRI) guiding-sheath with amplified expandable-tip imaging and MR-tracking for navigation and arrythmia ablation monitoring: Swine testing at 1.5 and 3T.

PURPOSE: Develop a deflectable intracardiac MR imaging (ICMRI) guiding-sheath to accelerate imaging during MR-guided electrophysiological (EP) interventions for radiofrequency (500 kHz) ablation (RFA) of arrythmia. Requirements include imaging at three to five times surface-coil SNR in cardiac chambers, vascular insertion, steerable-active-navigation into cardiac chambers, operation with ablation catheters, and safe levels of MR-induced heating. METHODS: ICMRI's 6 mm outer-diameter (OD) metallic-braided shaft had a 2.6 mm OD internal lumen for ablation-catheter insertion. Miniature-Baluns (MBaluns) on ICMRI's 1 m shaft reduced body-coil-induced heating. Distal section was a folded "star"-shaped imaging-coil mounted on an expandable frame, with an integrated miniature low-noise-amplifier overcoming cable losses. A handle-activated movable-shaft expanded imaging-coil to 35 mm OD for imaging within cardiac-chambers. Four MR-tracking micro-coils enabled navigation and motion-compensation, assuming a tetrahedron-shape when expanded. A second handle-lever enabled distal-tip deflection. ICMRI with a protruding deflectable EP catheter were used for MR-tracked navigation and RFA using a dedicated 3D-slicer user-interface. ICMRI was tested at 3T and 1.5T in swine to evaluate (a) heating, (b) cardiac-chamber access, (c) imaging field-of-view and SNR, and (d) intraprocedural RFA lesion monitoring. RESULTS: The 3T and 1.5T imaging SNR demonstrated >400% SNR boost over a 4 × 4 × 4 cm3 FOV in the heart, relative to body and spine arrays. ICMRI with MBaluns met ASTM/IEC heating limits during navigation. Tip-deflection allowed navigating ICMRI and EP catheter into atria and ventricles. Acute-lesion long-inversion-time-T1-weighted 3D-imaging (TWILITE) ablation-monitoring using ICMRI required 5:30 min, half the time needed with surface arrays alone. CONCLUSION: ICMRI assisted EP-catheter navigation to difficult targets and accelerated RFA monitoring.

Genetics and Epigenetics of Mating Type Determination in Paramecium and Tetrahymena.

While sex is an ancient and highly conserved eukaryotic invention, self-incompatibility systems such as mating types or sexes appear to be derived limitations that show considerable evolutionary plasticity. Within a single class of ciliates, Paramecium and Tetrahymena species have long been known to present a wide variety of mating type numbers and modes of inheritance, but only recently have the genes involved been identified. Although similar transmembrane proteins mediate self/nonself recognition in both ciliates, the mechanisms of mating type determination differ widely, ranging from Mendelian systems to developmental nuclear differentiation, either stochastic or maternally inherited. The non-Mendelian systems rely on programmed editing of the germline genome that occurs during differentiation of the somatic nucleus, and they have co-opted different DNA recombination mechanisms-some previously unknown. Here we review the recent molecular advances and some remaining unsolved questions and discuss the possible implications of these diverse mechanisms for inbreeding/outbreeding balance regulation.

ParameciumDB 2019: integrating genomic data across the genus for functional and evolutionary biology.

ParameciumDB (https://paramecium.i2bc.paris-saclay.fr) is a community model organism database for the genome and genetics of the ciliate Paramecium. ParameciumDB development relies on the GMOD (www.gmod.org) toolkit. The ParameciumDB web site has been publicly available since 2006 when the P. tetraurelia somatic genome sequence was released, revealing that a series of whole genome duplications punctuated the evolutionary history of the species. The genome is linked to available genetic data and stocks. ParameciumDB has undergone major changes in its content and website since the last update published in 2011. Genomes from multiple Paramecium species, especially from the P. aurelia complex, are now included in ParameciumDB. A new modern web interface accompanies this transition to a database for the whole Paramecium genus. Gene pages have been enriched with orthology relationships, among the Paramecium species and with a panel of model organisms across the eukaryotic tree. This update also presents expert curation of Paramecium mitochondrial genomes.

Pre-clerkship EPA assessments: a thematic analysis of rater cognition.

BACKGROUND: Entrustable Professional Activities (EPAs) assessments measure learners' competence with an entrustment or supervisory scale. Designed for workplace-based assessment EPA assessments have also been proposed for undergraduate medical education (UME), where assessments frequently occur outside the workplace and may be less intuitive, raising validity concerns. This study explored how assessors make entrustment determinations in UME, with additional specific comparison based on familiarity with prior performance in the context of longitudinal student-assessor relationships. METHODS: A qualitative approach using think-alouds was employed. Assessors assessed two students (familiar and unfamiliar) completing a history and physical examination using a supervisory scale and then thought-aloud after each assessment. We conducted a thematic analysis of assessors' response processes and compared them based on their familiarity with a student. RESULTS: Four themes and fifteen subthemes were identified. The most prevalent theme related to "student performance." The other three themes included "frame of reference," "assessor uncertainty," and "the patient." "Previous student performance" and "affective reactions" were subthemes more likely to inform scoring when faculty were familiar with a student, while unfamiliar faculty were more likely to reference "self" and "lack confidence in their ability to assess." CONCLUSIONS: Student performance appears to be assessors' main consideration for all students, providing some validity evidence for the response process in EPA assessments. Several problematic themes could be addressed with faculty development while others appear to be inherent to entrustment and may be more challenging to mitigate. Differences based on assessor familiarity with student merits further research on how trust develops over time.

Development and validation of the Durham Risk Score for estimating suicide attempt risk: A prospective cohort analysis.

BACKGROUND: Worldwide, nearly 800,000 individuals die by suicide each year; however, longitudinal prediction of suicide attempts remains a major challenge within the field of psychiatry. The objective of the present research was to develop and evaluate an evidence-based suicide attempt risk checklist [i.e., the Durham Risk Score (DRS)] to aid clinicians in the identification of individuals at risk for attempting suicide in the future. METHODS AND FINDINGS: Three prospective cohort studies, including a population-based study from the United States [i.e., the National Epidemiologic Survey on Alcohol and Related Conditions (NESARC) study] as well as 2 smaller US veteran cohorts [i.e., the Assessing and Reducing Post-Deployment Violence Risk (REHAB) and the Veterans After-Discharge Longitudinal Registry (VALOR) studies], were used to develop and validate the DRS. From a total sample size of 35,654 participants, 17,630 participants were selected to develop the checklist, whereas the remaining participants (N = 18,024) were used to validate it. The main outcome measure was future suicide attempts (i.e., actual suicide attempts that occurred after the baseline assessment during the 1- to 3-year follow-up period). Measure development began with a review of the extant literature to identify potential variables that had substantial empirical support as longitudinal predictors of suicide attempts and deaths. Next, receiver operating characteristic (ROC) curve analysis was utilized to identify variables from the literature review that uniquely contributed to the longitudinal prediction of suicide attempts in the development cohorts. We observed that the DRS was a robust prospective predictor of future suicide attempts in both the combined development (area under the curve [AUC] = 0.91) and validation (AUC = 0.92) cohorts. A concentration of risk analysis found that across all 35,654 participants, 82% of prospective suicide attempts occurred among individuals in the top 15% of DRS scores, whereas 27% occurred in the top 1%. The DRS also performed well among important subgroups, including women (AUC = 0.91), men (AUC = 0.93), Black (AUC = 0.92), White (AUC = 0.93), Hispanic (AUC = 0.89), veterans (AUC = 0.91), lower-income individuals (AUC = 0.90), younger adults (AUC = 0.88), and lesbian, gay, bisexual, transgender, and queer or questioning (LGBTQ) individuals (AUC = 0.88). The primary limitation of the present study was its its reliance on secondary data analyses to develop and validate the risk score. CONCLUSIONS: In this study, we observed that the DRS was a strong predictor of future suicide attempts in both the combined development (AUC = 0.91) and validation (AUC = 0.92) cohorts. It also demonstrated good utility in many important subgroups, including women, men, Black, White, Hispanic, veterans, lower-income individuals, younger adults, and LGBTQ individuals. We further observed that 82% of prospective suicide attempts occurred among individuals in the top 15% of DRS scores, whereas 27% occurred in the top 1%. Taken together, these findings suggest that the DRS represents a significant advancement in suicide risk prediction over traditional clinical assessment approaches. While more work is needed to independently validate the DRS in prospective studies and to identify the optimal methods to assess the constructs used to calculate the score, our findings suggest that the DRS is a promising new tool that has the potential to significantly enhance clinicians' ability to identify individuals at risk for attempting suicide in the future.

Negative Emotionality Interacts with Trauma Exposure to Prospectively Predict Posttraumatic Stress Disorder Symptoms During Firefighters' First 3 Years of Service.

Firefighters (FFs) protect the public despite significant risks to their health and well-being stemming from frequent trauma exposure and other occupational stressors. A minority of FFs develop posttraumatic stress disorder (PTSD) or related mental health problems, whereas most remain remarkably resilient despite enormous stress. This points toward substantial variability in responses to traumatic stress among FFs. Personality, particularly negative emotionality (NEM), has been shown to predict the development of PTSD in other trauma-exposed populations, yet has not been prospectively studied in relation to PTSD in FFs. The aim of this secondary analysis from a broader study of mental health in FFs was to test whether preemployment NEM predicted PTSD symptom severity over time by influencing how FFs respond to traumatic experiences. In this first prospective study of the development of PTSD symptoms in professional FFs, 322 FFs were recruited from seven urban fire academies across the United States and followed over their first 3 years of fire service. We assessed NEM during the fire academy as well as trauma exposure and both self-reported and clinician-rated PTSD symptoms at 1-, 2-, and 3-year follow-ups. Level of trauma exposure and NEM predicted PTSD symptoms over time, and NEM moderated the effect of trauma exposure on clinician-rated PTSD symptoms across both trauma exposure measures at 1- and 3-year follow-ups, f2 = .03-.10, but not at 2-year follow-up nor for self-reported PTSD symptoms. These findings indicate that NEM, assessed upon entry into a high-risk occupation, is useful in predicting PTSD symptom development.

Working near a supervised injection facility: A qualitative study of perspectives of firefighter-emergency medical responders.

BACKGROUND: While firefighter-emergency medical responders (FF-EMR) are important stakeholders in cities considering the implementation of a supervised injection facility (SIF), there is little information on perspectives of first responders who serve these communities. The aim of the present study was to identify FF-EMR perspectives on working near a SIF. METHODS: FF-EMRs from Vancouver Fire and Rescue Services completed an online survey that queried participant perspectives on working near a SIF. RESULTS: Four main themes were identified: positive effects, negative effects, duration of assignment, and sense of duty. Similar percentages of first responders reported positive (22.2%) and negative aspects (25.9%) of working near the SIF, while some (18.5%) indicated preference for a short-term assignment to the SIF area. FF-EMRs most commonly described a sense of duty (35.2%). CONCLUSIONS: To our knowledge, our study is the first to identify FF-EMR perspectives related to work near a SIF. Perspectives and concerns of first responders should be considered in policy debates about implementation of new SIFs to guarantee an adequately-prepared first responder workforce.

Dynamics of risk: Recent changes in psychological inflexibility precede subsequent changes in returning US veterans' posttraumatic stress.

OBJECTIVES: As a malleable risk-factor, psychological inflexibility is implicated in the development and maintenance of posttraumatic stress symptoms (PTS). Unfortunately, limited research has addressed whether changes in psychological inflexibility are antecedent to changes in PTS severity over time, or whether such changes are mutually dependent. METHODS: Utilizing bivariate latent difference score modeling, this longitudinal study sequenced intraindividual changes in psychological inflexibility and PTS severity within a sample of 305 returning US veterans. Veterans' self-reported psychological inflexibility and PTS severity were assessed quarterly over 1 year. RESULTS: Results indicated that early reductions in psychological inflexibility potentiated later declines in veterans' PTS severity, accounting for veterans' prior levels of psychological inflexibility and PTS severity. CONCLUSIONS: These findings underscore the unique role of changes in psychological inflexibility as an important mechanism of change in PTS severity and provide empirical support for an antecedent model of the role of psychological inflexibility in PTS recovery.

Experiential Learning Cycles as an Effective Means for Teaching Psychiatric Clinical Skills via Repeated Simulation in the Psychiatry Clerkship.

OBJECTIVE: This retrospective study compares differences in clinical performance on the psychiatry clerkship Objective Structured Clinical Examination (OSCE) between students receiving traditional repeated clinical simulation with those receiving repeated clinical simulation using the Kolb Cycle. METHODS: Psychiatry clerkship OSCE scores from 321 students who completed their psychiatry clerkship in 2016 and 2017 were compared. Specific performance measures included communication skills as determined by the Essential Elements of Communication, gathering a history, documenting a history and mental status exam, defending a differential diagnosis, and proposing a treatment plan. Results were calculated using repeated two-way analysis of variance between students receiving no simulation and traditional repeated simulation training (TRS) as compared to students receiving no simulation and repeated simulation utilizing the Kolb cycle (KRS). RESULTS: Students who received KRS performed significantly better in three of the five components of the clerkship OSCE as compared to students who received TRS. Specifically, students who received KRS performed better on gathering a history (+ 14.1%, p < 0.001), documenting a history (+ 13.4%, p < 0.001), and developing a treatment plan (+ 16.7%, p < 0.001). There were no significant differences in communication skills or in developing and defending a differential diagnosis. CONCLUSIONS: Psychiatry clerkship students engaged in repeated simulations explicitly integrated with the Kolb cycle demonstrate improved clinical skills as measured by OSCE performance. Integration of the Kolb cycle in designing simulation experiences should be carefully considered and may serve as a model for individualized coaching in programs of assessment.

Assessing Health Needs in African American Churches: A Mixed-Methods Study.

Among major racial and ethnic groups in the USA, African Americans are the most religious, and faith-based organizations play an important role in health promotion for African Americans. This study aimed to assess health needs in African American churches using a mixed-methods approach. Based on quantitative and qualitative data collected from eight African American churches in Nebraska in 2017, the most prevalent chronic conditions among participating African American church members (n = 388) included hypertension (60.8%), allergies (41.0%), arthritis (36.4%), high cholesterol (35.8%), and diabetes (28.1%). Significant predictors of fair or poor health were identified as male sex, unemployment, delayed utilization of health care in the past 12 months due to cost, lower frequency of church attendance, and feeling down, depressed, or hopeless in the past 2 weeks. Pastors from participating churches identified cost as one of the primary barriers to providing church-based health services. There were substantial unmet health needs in African American faith communities, especially in the areas of chronic disease prevention and management, and churches would need more support to realize their full potential in faith-based health promotion.

Long-Term Performance of Laboratory-Developed Liquid Chromatography-Tandem Mass Spectrometry Tests and a Food and Drug Administration-Approved Immunoassay for the Therapeutic Drug Monitoring of Everolimus.

BACKGROUND: Laboratory-developed tests (LDTs) are analytical tests developed and validated "in-house" for clinical diagnosis. Regulatory agencies, such as the United States Food and Drug Administration (FDA), encourage using regulatory-approved assays rather than LDTs. In the ongoing Zortracker everolimus study, samples were provided monthly to participating clinical laboratories that conduct therapeutic drug monitoring of everolimus. This allowed for the long-term (6-year) comparison of the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays, which are LDTs, to the FDA-approved everolimus Quantitative Microsphere System (QMS). METHODS: Each laboratory received the same 3 blinded samples. LC-MS/MS and QMS assays were compared using Passing Bablok regression analysis. Data were analyzed in 12-month periods to detect trends over time. RESULTS: The slopes of the Passing Bablok regression curves remained unchanged in 2013 and 2014 (reference LC-MS/MS; test QMS: slope = 0.934 and 1.008). However, by 2016, the slope increased significantly to between 1.111 and 1.320, then dropped to 0.980 in 2017 and 0.912 in 2018, suggestive of changes in QMS bias compared with LC-MS/MS over longer periods. Outliers did not affect these results. The interlaboratory variability of LC-MS/MS and QMS remained unchanged from 2013 to 2015, with coefficients of variation of 15.3%, 18.4%, and 17.2% for LC-MS/MS, and 13.0%, 13.1%, and 15.3% for QMS, respectively, per year. At the end of the observation period, the average coefficients of variation in LC-MS/MS laboratories dropped to 14.3%, 12.6%, and 14.2%, whereas the variability in QMS laboratories was 19.5%, 13.3%, and 19.6% in 2016, 2017, and 2018, respectively. CONCLUSIONS: Initially, QMS everolimus concentrations in patient samples were comparable with those detected in LC-MS/MS laboratories. However, concentration bias of the QMS assay significantly changed within 6 years, emphasizing the need for long-term, independent performance tracking of therapeutic drug monitoring assays, including FDA-approved assays.

Genome-defence small RNAs exapted for epigenetic mating-type inheritance.

In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of the mtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtA expression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.

A mating-type mutagenesis screen identifies a zinc-finger protein required for specific DNA excision events in Paramecium.

In the ciliate Paramecium tetraurelia, functional genes are reconstituted during development of the somatic macronucleus through the precise excision of ∼45 000 single-copy Internal Eliminated Sequences (IESs), thought to be the degenerate remnants of ancient transposon insertions. Like introns, IESs are marked only by a weak consensus at their ends. How such a diverse set of sequences is faithfully recognized and precisely excised remains unclear: specialized small RNAs have been implicated, but in their absence up to ∼60% of IESs are still correctly excised. To get further insight, we designed a mutagenesis screen based on the hypersensitivity of a specific excision event in the mtA gene, which determines mating types. Unlike most IES-containing genes, the active form of mtA is the unexcised one, allowing the recovery of hypomorphic alleles of essential IES recognition/excision factors. Such is the case of one mutation recovered in the Piwi gene PTIWI09, a key player in small RNA-mediated IES recognition. Another mutation identified a novel protein with a C2H2 zinc finger, mtGa, which is required for excision of a small subset of IESs characterized by enrichment in a 5-bp motif. The unexpected implication of a sequence-specific factor establishes a new paradigm for IES recognition and/or excision.