Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning.

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences Single Molecule Real-Time sequencing. By employing reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second strand synthesis. Deletion and insertion rates increase for all RTs during second strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

Development and validation of the Trust in Multidimensional Healthcare Systems Scale (TIMHSS).

CONTEXT: The COVID-19 pandemic has reignited a commitment from the health policy and health services research communities to rebuilding trust in healthcare and created a renewed appetite for measures of trust for system monitoring and evaluation. The aim of the present paper was to develop a multidimensional measure of trust in healthcare that: (1) Is responsive to the conceptual and methodological limitations of existing measures; (2) Can be used to identify systemic explanations for lower levels of trust in equity-deserving populations; (3) Can be used to design and evaluate interventions aiming to (re)build trust. METHODS: We conducted a 2021 review of existing measures of trust in healthcare, 72 qualitative interviews (Aug-Dec 2021; oversampling for equity-deserving populations), an expert review consensus process (Oct 2021), and factor analyses and validation testing based on two waves of survey data (Nov 2021, n = 694; Jan-Feb 2022, n = 740 respectively). FINDINGS: We present the Trust in Multidimensional Healthcare Systems Scale (TIMHSS); a 38-item correlated three-factor measure of trust in doctors, policies, and the system. Measurement of invariance tests suggest that the TIMHSS can also be reliably administered to diverse populations. CONCLUSIONS: This global measure of trust in healthcare can be used to measure trust over time at a population level, or used within specific subpopulations, to inform interventions to (re)build trust. It can also be used within a clinical setting to provide a stronger evidence base for associations between trust and therapeutic outcomes.

The Johns Hopkins Physician-Scientist Training Program to Enhance Institutional Retention and Entry Into Academic Positions: An Evaluation of Program Effectiveness and Outcomes.

PURPOSE: The Johns Hopkins Physician-Scientist Training Program (PSTP) was implemented to overcome well-documented challenges in training and retaining physician-scientists by providing physician-scientist pathway training for residents and clinical fellows. The program's core tenets include monthly seminars, individualized feedback on project proposals, access to mentors, and institutional funding opportunities. This study evaluated the effectiveness and outcomes of the PTSP and provides a framework for replication. METHOD: A query of institutional demographic data and bibliometric variables of the PSTP participants (2017-2020) at a single academic medical center was conducted in 2021. In addition, a voluntary survey collected personal and program evaluation information. RESULTS: Of 145 PSTP scholars, 59 (41%) were women, and 41 (31%), 8 (6%), and 6 (5%) of scholars self-identified as Asian, Hispanic, and Black, respectively. Thirty-three (23%) scholars received PSTP research support or career development microgrants. Of 66 PSTP graduates, 29 (44%) remained at Johns Hopkins as clinical fellows or faculty. Of 48 PSTP graduates in a post-training position, 42 (88%) were in academia, with the majority, 29 (76%), holding the rank of assistant professor. Fifty-nine of 140 available participants responded to the survey (42% response rate). The top-cited reason for joining the PSTP was exposure to mentors and administration (50/58 respondents, 86%), followed by seeking scholarly opportunities (37/58 respondents, 64%). Most scholars intended to continue a career as a physician-scientist. CONCLUSIONS: The PSTP provides internal research support and institutional oversight. Although establishing close mentor-mentee relationships requires individualized approaches, the PSTP provided structured academic pathways that enhanced participating scholars' ability to apply for grants and jobs. The vast majority continued their careers as physician-scientists after training. In light of the national evidence of a "leaky physician-scientist pipeline," programs such as the PSTP can be critical to entry into early academic career positions and institutional retention.

Optimization of Advanced Molecular Genetic Testing Utilization in Hematopathology: A Goldilocks Approach to Bone Marrow Testing.

PURPOSE: This study investigated the effectiveness of algorithmic testing in hematopathology at the Brigham and Women's Hospital and Dana-Farber Cancer Institute (DFCI). The algorithm was predicated on test selection after an initial pathologic evaluation to maximize cost-effective testing, especially for expensive molecular and cytogenetic assays. MATERIALS AND METHODS: Standard ordering protocols (SOPs) for 17 disease categories were developed and encoded in a decision support application. Six months of retrospective data from application beta testing was obtained and compared with actual testing practices during that timeframe. In addition, 2 years of prospective data were also obtained from patients at one community satellite site. RESULTS: A total of 460 retrospective cases (before introduction of algorithmic testing) and 109 prospective cases (following introduction) were analyzed. In the retrospective data, 61.7% of tests (509 of 825) were concordant with the SOPs while 38.3% (316 of 825) were overordered and 30.8% (227 of 736) of SOP-recommended tests were omitted. In the prospective data, 98.8% of testing was concordant (244 of 247 total tests) with only 1.2% overordered tests (3 of 247) and 7.6% omitted tests (20 of 264 SOP-recommended tests; overall P < .001). The cost of overordered tests before implementing SOP indicates a potential annualized saving of $1,347,520 in US dollars (USD) in overordered testing at Brigham and Women's Hospital/DFCI. Only two of 316 overordered tests (0.6%) returned any additional information, both for extremely rare clinical circumstances. CONCLUSION: Implementation of SOPs dramatically improved test ordering practices, with a just right number of ancillary tests that minimizes cost and has no significant impact on acquiring key informative test results.

The Role of Nurses in the Management of Adverse Events in Patients Receiving First-Line Axitinib Plus Immuno-Oncology Agents for Advanced Renal Cell Carcinoma.

OBJECTIVES: The recent approval of first-line tyrosine kinase inhibitor plus immuno-oncology agent combination therapy for the treatment of advanced renal cell carcinoma offers substantially improved response rates and survival compared with the previous standard of care. This expansion of treatment options has also led to a greater range and complexity of potential treatment-related adverse events related to overlapping toxicities. The aim of this article is to discuss the management of common treatment-emergent adverse events (AEs) associated with axitinib plus immuno-oncology therapy, highlight the specific roles of oncology nurses in managing these events, and provide AE management resources to aid oncology nurses in their care of patients with advanced renal cell carcinoma. DATA SOURCES: Author experience, journal articles, and treatment guidelines were used. CONCLUSION: The use of oncology nurses and nurse-led innovations to monitor and assess treatments can have a positive impact on the management of AEs in cancer patients by identifying those who are most at risk, providing regular assessment, appropriate patient education, and supporting the monitoring of patient safety. IMPLICATIONS FOR NURSING PRACTICE: Skilled oncology nurses should be a key part of a team that addresses the supportive care needs and management of AEs that are associated with novel cancer treatments. Early and ongoing communication between the patient and oncology nurses regarding the development of adverse events is a critical component of maximizing treatment outcomes and quality of life.

Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Examining vaccine hesitancy among a diverse sample of Canadian adults.

The aim of this study was to explore the sociodemographic and individual-level factors associated with vaccine hesitancy in general, including political affiliation and beliefs in vaccine conspiracy theories, in a diverse group of Canadian adults within the context of the COVID-19 pandemic. 641 responses were included in the analysis, with those self-identifying as Indigenous, Black Canadian, and low-income (household income <$40,000) being sampled to yield data from historically marginalized populations. Demographic variables and responses to questions on vaccine hesitancy, and beliefs in vaccine conspiracy theories were used to explore explanatory variables of vaccine hesitancy. General linear regression models were fit using the method of least squares via PROC GLM and used to examine sociodemographic and individual explanatory variables of vaccine hesitancy. Age, ethnicity, political affiliation, and beliefs in vaccine conspiracies were associated with vaccine hesitancy. Findings are discussed in relation to the critical role of distrust and misinformation in hesitancy. Our data provide insight into how Canadian provincial governments may promote uptake of vaccines in ways that target diverse groups that may differ from those developed in a pre-pandemic context.

LINE-1 retrotransposition and its deregulation in cancers: implications for therapeutic opportunities.

Long interspersed element 1 (LINE-1) is the only protein-coding transposon that is active in humans. LINE-1 propagates in the genome using RNA intermediates via retrotransposition. This activity has resulted in LINE-1 sequences occupying approximately one-fifth of our genome. Although most copies of LINE-1 are immobile, ∼100 copies are retrotransposition-competent. Retrotransposition is normally limited via epigenetic silencing, DNA repair, and other host defense mechanisms. In contrast, LINE-1 overexpression and retrotransposition are hallmarks of cancers. Here, we review mechanisms of LINE-1 regulation and how LINE-1 may promote genetic heterogeneity in tumors. Finally, we discuss therapeutic strategies to exploit LINE-1 biology in cancers.

Meeting report: transposable elements at the crossroads of evolution, health and disease 2023.

The conference "Transposable Elements at the Crossroads of Evolution, Health and Disease" was hosted by Keystone Symposia in Whistler, British Columbia, Canada, on September 3-6, 2023, and was organized by Kathleen Burns, Harmit Malik and Irina Arkhipova. The central theme of the meeting was the incredible diversity of ways in which transposable elements (TEs) interact with the host, from disrupting the existing genes and pathways to creating novel gene products and expression patterns, enhancing the repertoire of host functions, and ultimately driving host evolution. The meeting was organized into six plenary sessions and two afternoon workshops with a total of 50 invited and contributed talks, two poster sessions, and a career roundtable. The topics ranged from TE roles in normal and pathological processes to restricting and harnessing TE activity based on mechanistic insights gained from genetic, structural, and biochemical studies.

Transforming ovarian cancer care by targeting minimal residual disease.

Frontline treatment and resultant cure rates in patients with advanced ovarian cancer have changed little over the past several decades. Here, we outline a multidisciplinary approach aimed at gaining novel therapeutic insights by focusing on the poorly understood minimal residual disease phase of ovarian cancer that leads to eventual incurable recurrences.

Proceedings of the inaugural Dark Genome Symposium: November 2022.

In November 2022 the first Dark Genome Symposium was held in Boston, USA. The meeting was hosted by Rome Therapeutics and Enara Bio, two biotechnology companies working on translating our growing understanding of this vast genetic landscape into therapies for human disease. The spirit and ambition of the meeting was one of shared knowledge, looking to strengthen the network of researchers engaged in the field. The meeting opened with a welcome from Rosana Kapeller and Kevin Pojasek followed by a first session of field defining talks from key academics in the space. A series of panels, bringing together academia and industry views, were then convened covering a wide range of pertinent topics. Finally, Richard Young and David Ting gave their views on the future direction and promise for patient impact inherent in the growing understanding of the Dark Genome.

The landscape of human SVA retrotransposons.

SINE-VNTR-Alu (SVA) retrotransposons are evolutionarily young and still-active transposable elements (TEs) in the human genome. Several pathogenic SVA insertions have been identified that directly mutate host genes to cause neurodegenerative and other types of diseases. However, due to their sequence heterogeneity and complex structures as well as limitations in sequencing techniques and analysis, SVA insertions have been less well studied compared to other mobile element insertions. Here, we identified polymorphic SVA insertions from 3646 whole-genome sequencing (WGS) samples of >150 diverse populations and constructed a polymorphic SVA insertion reference catalog. Using 20 long-read samples, we also assembled reference and polymorphic SVA sequences and characterized the internal hexamer/variable-number-tandem-repeat (VNTR) expansions as well as differing SVA activity for SVA subfamilies and human populations. In addition, we developed a module to annotate both reference and polymorphic SVA copies. By characterizing the landscape of both reference and polymorphic SVA retrotransposons, our study enables more accurate genotyping of these elements and facilitate the discovery of pathogenic SVA insertions.

Examining factors impacting acceptance of COVID-19 countermeasures among structurally marginalised Canadians.

The COVID-19 pandemic highlighted and exacerbated inequities in health for structurally marginalised Canadians. Their location on society's hierarchies constrained their ability to access healthcare and follow recommended health behaviours. The aim of this article is to identify, from the perspective of marginalised populations, factors influencing the acceptance or rejection of COVID-19 countermeasures by structurally marginalised Canadians. Interviews were conducted with Canadians 18 + who identified as Black (n = 8), First Nations, Métis, or Inuit (n = 7) and low-income (<40,000 annual household income) (n = 8) between August and December 2021. Measures were said to impact well-being and interfere with revenue generating activities. Longstanding unfavourable living and environmental conditions as they relate to structural marginalisation was said to fuel anger toward the government and lead to a greater reluctance to accept countermeasures. Participants described concerns about government decisions being made without considering their unique contexts, or knowledge of the experiences of the population for whom these decisions were being made. Effective proactive action from government is important to foster trust with marginalised populations to support acceptance of health information and address growing inequities. Action that demonstrates government competence and commitment to the interests of marginalised populations is critical.

Informing efforts beyond tailored promotional campaigns by understanding contextual factors shaping vaccine hesitancy among equity-deserving populations in Canada: an exploratory qualitative study.

BACKGROUND: Vaccine hesitancy exists on a continuum ranging between complete adherence and complete refusal due to doubts or concerns within a heterogeneous group of individuals. Despite widespread acknowledgement of the contextual factors influencing attitudes and beliefs shaping COVID-19 vaccine hesitancy, qualitative research with equity-deserving groups, accounting for unique lived experiences, remains a gap in the literature. We aim to identify and begin to understand and document the unique contextual factors shaping hesitancy by equity-deserving groups as it relates to relationships with government and health authorities. METHODS: Participants were recruited and interviewed between Aug-Dec 2021. Semi-structured interviews using a convergent interviewing technique were conducted with individuals from the general population, as well as individuals who identify as First Nations, Métis, or Inuit, members of the LGBT2SQ + community, low-income Canadians, Black Canadians, and newcomers. Interviews were audio recorded and transcribed by a team of researchers. Memos were written following interviews and used to complement the thematic analysis of the interview data. Themes are presented in the results section. RESULTS: The rationale for hesitancy among equity-deserving groups is consistent with literature documenting hesitancy in the general population. Contextual factors surrounding equity-deserving groups' attitudes and beliefs, however, are unique and relate to a history of oppression, discrimination, and genocide. We identified factors unique to subgroups; for example, religious or fatalistic beliefs among participant who identify as FNMI, fear associated with lack of testing and speed of vaccines' production among participants who identify as FNMI, Black, and LGBT2SQ + , distrust of the healthcare system for LGBT2SQ + and Black Canadians, and distrust of the government and opposition to vaccine mandates for participating who identify as LGBT2SQ + , low-income, FNMI, or Black Canadian. Newcomers stood out as very trusting of the government and accepting of COVID-19 vaccination. CONCLUSIONS: While our data on vaccine hesitancy largely mirror concerns reported in the vast body of literature citing rationale for COVID-19 hesitancy in high-income countries, the contextual factors identified in our work point to the need for wider systemic change. Our results may be used to support efforts, beyond tailored promotion campaigns, to support the confident acceptance of vaccines for COVID-19 and the acceptance of novel vaccines as future infectious diseases emerge.

Development and validation of the Trust in Government measure (TGM).

BACKGROUND: Trust in government is associated with health behaviours and is an important consideration in population health interventions. While there is a reported decline in public trust in government across OECD countries, the tools used to measure trust are limited in their use for informing action to (re)build trust, and have limitations related to reliability and validity. To address the limitations of existing measures available to track public trust, the aim of the present work was to develop a new measure of trust in government. METHODS: Fifty-six qualitative interviews (Aug-Oct 2021; oversampling for equity-deserving populations) were conducted to design a national survey, including factor analyses and validation testing (N = 878; June 1-14th 2022) in Canada. RESULTS: The measure demonstrated strong internal consistency (α = 0.96) and test validity (CFI = 0.96, RMSEA = 0.09, SRMR = 0.03), suggesting that trust in government can be measured as a single underlying construct. It also demonstrated strong criterion validity, as measured by significant (p < 0.0001) associations of scores with vaccine hesitancy, vaccine conspiracy beliefs, COVID-19 conspiracy beliefs, trust in public health messaging about COVID-19, and trust in public health advice about COVID-19. We present the Trust in Government Measure (TGM); a 13-item unidimensional measure of trust in Federal government. CONCLUSIONS: This measure can be used within high-income countries, particularly member countries within the OECD already in support of using tools to collect, publish and compare statistics. Our measure should be used by researchers and policy makers to measure trust in government as a key indicator of societal and public health.

Vitamin C activates young LINE-1 elements in mouse embryonic stem cells via H3K9me3 demethylation.

BACKGROUND: Vitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone demethylases. The epigenetic remodelling promoted by vitC improves the efficiency of induced pluripotent stem cell derivation, and is required to attain a ground-state of pluripotency in embryonic stem cells (ESCs) that closely mimics the inner cell mass of the early blastocyst. However, genome-wide DNA and histone demethylation can lead to upregulation of transposable elements (TEs), and it is not known how vitC addition in culture media affects TE expression in pluripotent stem cells. RESULTS: Here we show that vitC increases the expression of several TE families, including evolutionarily young LINE-1 (L1) elements, in mouse ESCs. We find that TET activity is dispensable for L1 upregulation, and that instead it occurs largely as a result of H3K9me3 loss mediated by KDM4A/C histone demethylases. Despite increased L1 levels, we did not detect increased somatic insertion rates in vitC-treated cells. Notably, treatment of human ESCs with vitC also increases L1 protein levels, albeit through a distinct, post-transcriptional mechanism. CONCLUSION: VitC directly modulates the expression of mouse L1s and other TEs through epigenetic mechanisms, with potential for downstream effects related to the multiple emerging roles of L1s in cellular function.

Canadians' trust in government in a time of crisis: Does it matter?

The ability of governments and nations to handle crises and protect the lives of citizens is heavily dependent on the public's trust in their governments and related social institutions. The aim of the present research was to understand public trust in government during a time of crisis, drawing on interview data (N = 56) collected during the COVID-19 pandemic (2021). In addition to the general public (n = 11), participants were sampled to obtain diversity as it relates to identifying as First Nations, Métis, and Inuit (n = 7), LGBT2SQ+ (n = 5), low-income (n = 8), Black Canadians (n = 7), young adult (n = 8), and newcomers to Canada (n = 10). Data were coded in consideration of social theories of trust, and specifically the nature of trust between individuals and institutions working with government in pandemic management. Canadians' trust in government was shaped by perceptions of pandemic communication, as well as decision-making and implementation of countermeasures. Data suggest that although participants did not trust government, they were accepting of measures and messages as presented through government channels, pointing to the importance of (re)building trust in government. Perhaps more importantly however, data indicate that resources should be invested in monitoring and evaluating public perception of individuals and institutions generating the evidence-base used to guide government communication and decision-making to ensure trust is maintained. Theoretically, our work adds to our understanding of the nature of trust as it relates to the association between interpersonal and institutional trust, and also the nature of trust across institutions.

Avelumab First-Line Maintenance Therapy: Managing Patients With Advanced Urothelial Carcinoma.

BACKGROUND: The phase 3 of JAVELIN Bladder 100 trial demonstrated that avelumab first-line (1L) maintenance in addition to best supportive care significantly prolonged overall survival compared to best supportive care alone. It is now the standard of care for platinum-eligible patients with locally advanced or metastatic urothelial carcinoma that has not progressed with 1L platinum-containing chemotherapy. OBJECTIVES: This article provides considerations for oncology nurses to effectively implement avelumab 1L maintenance treatment in the clinical setting. METHODS: This article reviews clinical evidence and implications for oncology nurses caring for patients receiving avelumab 1L maintenance treatment. FINDINGS: Oncology nurses can provide comprehensive care for patients with advanced urothelial carcinoma and ensure the safe and appropriate use of avelumab 1L maintenance treatment by educating patients and caregivers, ensuring correct administration, and promptly recognizing and managing immune-related adverse events.