Dissecting quantitative trait nucleotides by saturation genome editing.

Genome editing technologies have the potential to transform our understanding of how genetic variation gives rise to complex traits through the systematic engineering and phenotypic characterization of genetic variants. However, there has yet to be a system with sufficient efficiency, fidelity, and throughput to comprehensively identify causal variants at the genome scale. Here we explored the ability of templated CRISPR editing systems to install natural variants genome-wide in budding yeast. We optimized several approaches to enhance homology-directed repair (HDR) with donor DNA templates, including donor recruitment to target sites, single-stranded donor production by bacterial retrons, and in vivo plasmid assembly. We uncovered unique advantages of each system that we integrated into a single superior system named MAGESTIC 3.0. We used MAGESTIC 3.0 to dissect causal variants residing in 112 quantitative trait loci across 32 environmental conditions, revealing an enrichment for missense variants and loci with multiple causal variants. MAGESTIC 3.0 will facilitate the functional analysis of the genome at single-nucleotide resolution and provides a roadmap for improving template-based genome editing systems in other organisms.

Genome-scale analysis of interactions between genetic perturbations and natural variation.

Interactions between genetic perturbations and segregating loci can cause perturbations to show different phenotypic effects across genetically distinct individuals. To study these interactions on a genome scale in many individuals, we used combinatorial DNA barcode sequencing to measure the fitness effects of 7,700 CRISPRi perturbations targeting 1,712 distinct genes in 169 yeast cross progeny (or segregants). We identified 460 genes whose perturbation has different effects across segregants. Several factors caused perturbations to show variable effects, including baseline segregant fitness, the mean effect of a perturbation across segregants, and interacting loci. We mapped 234 interacting loci and found four hub loci that interact with many different perturbations. Perturbations that interact with a given hub exhibit similar epistatic relationships with the hub and show enrichment for cellular processes that may mediate these interactions. These results suggest that an individual's response to perturbations is shaped by a network of perturbation-locus interactions that cannot be measured by approaches that examine perturbations or natural variation alone.

Splicing factor Prp18p promotes genome-wide fidelity of consensus 3'-splice sites.

The fidelity of splice site selection is critical for proper gene expression. In particular, proper recognition of 3'-splice site (3'SS) sequences by the spliceosome is challenging considering the low complexity of the 3'SS consensus sequence YAG. Here, we show that absence of the Prp18p splicing factor results in genome-wide activation of alternative 3'SS in S. cerevisiae, including highly unusual non-YAG sequences. Usage of these non-canonical 3'SS in the absence of Prp18p is enhanced by upstream poly(U) tracts and by their potential to interact with the first intronic nucleoside, allowing them to dock in the spliceosome active site instead of the normal 3'SS. The role of Prp18p in 3'SS fidelity is facilitated by interactions with Slu7p and Prp8p, but cannot be fulfilled by Slu7p, identifying a unique role for Prp18p in 3'SS fidelity. This fidelity function is synergized by the downstream proofreading activity of the Prp22p helicase, but is independent from another late splicing helicase, Prp43p. Our results show that spliceosomes exhibit remarkably relaxed 3'SS sequence usage in the absence of Prp18p and identify a network of spliceosomal interactions centered on Prp18p which are required to promote the fidelity of the recognition of consensus 3'SS sequences.

Large scale microfluidic CRISPR screening for increased amylase secretion in yeast.

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

A scalable, GMP-compatible, autologous organotypic cell therapy for Dystrophic Epidermolysis Bullosa.

Background: Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies. Methods: We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing. Findings: iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice. Interpretation: DEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds.

Spliceosomal mutations decouple 3' splice site fidelity from cellular fitness.

The fidelity of splice site selection is thought to be critical for proper gene expression and cellular fitness. In particular, proper recognition of 3'-splice site (3'SS) sequences by the spliceosome is a daunting task considering the low complexity of the 3'SS consensus sequence YAG. Here we show that inactivating the near-essential splicing factor Prp18p results in a global activation of alternative 3'SS, many of which harbor sequences that highly diverge from the YAG consensus, including some highly unusual non-AG 3'SS. We show that the role of Prp18p in 3'SS fidelity is promoted by physical interactions with the essential splicing factors Slu7p and Prp8p and synergized by the proofreading activity of the Prp22p helicase. Strikingly, structure-guided point mutations that disrupt Prp18p-Slu7p and Prp18p-Prp8p interactions mimic the loss of 3'SS fidelity without any impact on cellular growth, suggesting that accumulation of incorrectly spliced transcripts does not have a major deleterious effect on cellular viability. These results show that spliceosomes exhibit remarkably relaxed fidelity in the absence of Prp18p, and that new 3'SS sampling can be achieved genome-wide without a major negative impact on cellular fitness, a feature that could be used during evolution to explore new productive alternative splice sites.

Development and implementation of a pre-tracheostomy multidisciplinary conference: An initiative to improve patient selection.

OBJECTIVE: To describe our institutional experience in implementing a pre-tracheostomy multidisciplinary conference and assess its effects on patient selection and communication between team members and with families. METHODS: Descriptive study and retrospective review of patient outcomes in a period prior to (4/2016-1/2018) and following (2/2018-11/2019) implementation of the conference and conference participant survey. RESULTS: In the 21 months prior to the conference, 53 patients out of 67 consults (79%) went on to have a tracheostomy. After implementation, 96 patients, 42 females and 54 males, between 2 weeks and 22 years of age were discussed. 58 (60%) of patients referred for tracheostomy ultimately underwent surgery. Of those managed without tracheostomy, 16% were extubated, 11% were managed with noninvasive respiratory support, and 13% of families chose to redirect care. There was no difference in time between consultation and surgery (p = 0.9), or post-surgical length of stay after the conference was implemented (p = 0.9). Survey responses were gathered from 34 conference participants. Respondents agreed that the conference was useful in facilitating communication among the care team (91%), promoting understanding of the patient's treatment options (85%), promoting understanding about long-term outcomes and progression of underlying disease process (79%), clarifying risks, benefits, and alternatives of treatment options (82%), and informing discussions with the family (70%). DISCUSSION: Potential benefits of a multidisciplinary pre-tracheostomy conference include improved provider communication and shared decision making between the medical team and family. We found a reduction in the proportion of patients who ultimately underwent tracheostomy as a result of a formal multidisciplinary discussion, but did not find either any delays in care, or reduction in post-operative length of stay. IMPLICATIONS FOR PRACTICE: A multidisciplinary team approach to patient selection can foster communication between team members, identify barriers to discharge and quality care at home, and provide caregivers with information necessary to make an informed decision about their child's care.

Family Health Development: A Theoretical Framework.

In recognition of the family as central to health, the concept of family, rather than individual, health has been an important area of research and, increasingly, clinical practice. There is a need to leverage existing theories of family health to align with our evolving understanding of Life Course Health Development, including the opportunities and constraints of the family context for promoting lifelong individual and population health. The purpose of this article is to propose an integrative model of family health development within a Life Course Health Development lens to facilitate conceptualization, research, and clinical practice. This model provides an organizing heuristic model for understanding the dynamic interactions between family structures, processes, cognitions, and behaviors across development. Potential applications of this model are discussed.

The interplay of additivity, dominance, and epistasis on fitness in a diploid yeast cross.

In diploid species, genetic loci can show additive, dominance, and epistatic effects. To characterize the contributions of these different types of genetic effects to heritable traits, we use a double barcoding system to generate and phenotype a panel of ~200,000 diploid yeast strains that can be partitioned into hundreds of interrelated families. This experiment enables the detection of thousands of epistatic loci, many whose effects vary across families. Here, we show traits are largely specified by a small number of hub loci with major additive and dominance effects, and pervasive epistasis. Genetic background commonly influences both the additive and dominance effects of loci, with multiple modifiers typically involved. The most prominent dominance modifier in our data is the mating locus, which has no effect on its own. Our findings show that the interplay between additivity, dominance, and epistasis underlies a complex genotype-to-phenotype map in diploids.

CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and Bioinformatics Laboratory Module for Undergraduate and High School Students.

CRISPR-Cas9 genome editing technology is widely used in scientific research and biotechnology. As this technology becomes a staple tool in life sciences research, it is increasingly important to incorporate it into biology curricula to train future scientists. To demonstrate the molecular underpinnings and some limitations of CRISPR-based gene editing, we designed a laboratory module to accompany a discussion-based course on genome editing for college and advanced high school biology students. The laboratory module uses CRISPR-Cas9 to target and inactivate the ADE2 gene in Saccharomyces cerevisiae so as to give red colonies, employing an inexpensive yeast model system with a phenotypic readout that is easily detectable without specialized equipment. Students begin by accessing the yeast ADE2 sequence in a genome database, applying their understanding of Cas9 activity to design guide RNA (gRNA) sequences, using a CRISPR analysis tool to compare predicted on- and off-target effects of various gRNAs, and presenting and explaining their choice of an optimal gRNA to disrupt the ADE2 gene. They then conduct yeast transformations using Cas9 and preselected gRNA plasmids with or without donor templates to explore the importance of DNA repair pathways in genome editing. Lastly, they analyze the observed editing rates across different gRNAs targeting ADE2, leading to a discussion of editing efficiency. This module engages students in experimental design, provides hands-on experience with CRISPR-Cas9 gene editing and collaborative data analysis, and stimulates discussion on the uses and limitations of CRISPR-based gene editing technology.

Stress-induced inhibition of mRNA export triggers RNase III-mediated decay of the BDF2 mRNA.

The expression of bromodomain-containing proteins that regulate chromatin structure and accessibility must be tightly controlled to ensure the appropriate regulation of gene expression. In the yeast S. cerevisiae, Bromodomain Factor 2 (BDF2) expression is extensively regulated post-transcriptionally during stress by RNase III-mediated decay (RMD), which is triggered by cleavage of the BDF2 mRNA in the nucleus by the RNase III homolog Rnt1p. Previous studies have shown that RMD-mediated down-regulation of BDF2 is hyperactivated in osmotic stress conditions, yet the mechanisms driving the enhanced nuclear cleavage of BDF2 RNA under these conditions remain unknown. Here, we show that RMD hyperactivation can be detected in multiple stress conditions that inhibit mRNA export, and that Rnt1p remains primarily localized in the nucleus during salt stress. We show that globally inhibiting mRNA nuclear export by anchoring away mRNA biogenesis or export factors out of the nucleus can recapitulate RMD hyperactivation in the absence of stress. RMD hyperactivation requires Rnt1p nuclear localization but does not depend on the BDF2 gene endogenous promoter, and its efficiency is affected by the structure of the stem-loop cleaved by Rnt1p. Because multiple stress conditions have been shown to mediate global inhibition of mRNA export, our results suggest that the hyperactivation of RMD is primarily the result of the increased nuclear retention of the BDF2 mRNA during stress.

Evaluation of the Delaware Contraceptive Access Now (DelCAN) initiative: A qualitative analysis of site leaders' implementation recommendations.

OBJECTIVES: In 2014, Delaware launched a statewide initiative to reduce the rate of unintended pregnancies and increase access to contraception services. Our study objective was to understand the implementation experiences, barriers, and successes across health care practice settings and to provide recommendations for future, similar initiatives. STUDY DESIGN: As part of a larger multicomponent process evaluation, we conducted semistructured interviews with 32 leaders from 26 practice settings implementing the initiative across the state. We analyzed the qualitative data through iterative open, axial, and selective coding using grounded theory methods, employing thematic analysis to identify common themes in implementation experiences. RESULTS: Most practices perceived that patient demand for methods of long-acting reversible contraception (LARC) increased. Many practices had to adapt the intervention to fit the needs and constraints of their settings and patient populations. Primary care practices, smaller practices, and practices that served large numbers of adolescents experienced more barriers compared to obstetrics and gynecology or women's health practices. For current and future iterations of the initiative, leaders emphasized: (1) the need for greater implementation flexibility, (2) the importance of inclusive communication at multiple levels, and (3) attending to logistical challenges, particularly around billing. CONCLUSION: Varied practice settings required significant flexibility and responsiveness to context in order to implement the initiative. Organizations with greater pre-existing capacity were able to offer the full range of contraceptive care, as the initiative intended, in contrast to practices with less pre-existing capacity for providing methods of LARC and other types of contraception. IMPLICATIONS: To meet the specific but heterogenous needs of various practices, it is crucial for future contraceptive access initiatives to conduct a comprehensive pre-implementation assessment. Preceding any training, this assessment should gather input from participants across all roles in a medical practice (e.g., providers, medical assistants, office staff, billing department).

Protocol for High-Resolution Mapping of Splicing Products and Isoforms by RT-PCR Using Fluorescently Labeled Primers.

We describe an RT-PCR protocol that allows high-resolution mapping of splicing products and isoforms using fluorescently labeled primers. Each species contains one fluorescent group allowing a direct comparison of the different isoforms despite size differences. A custom-size ladder enables the precise determination of cDNA lengths and discrimination of isoforms differing by less than five nucleotides on polyacrylamide gels. This protocol also allows the detection of products from in vitro splicing reactions, circumventing the need to use radiolabeled transcripts. For complete details on the use and execution of this protocol, please refer to Gabunilas and Chanfreau (2016).

Robust mapping of polyadenylated and non-polyadenylated RNA 3' ends at nucleotide resolution by 3'-end sequencing.

3'-end poly(A)+ sequencing is an efficient and economical method for global measurement of mRNA levels and alternative poly(A) site usage. A common method involves oligo(dT)19V reverse-transcription (RT)-based library preparation and high-throughput sequencing with a custom primer ending in (dT)19. While the majority of library products have the first sequenced nucleotide reflect the bona fide poly(A) site (pA), a substantial fraction of sequencing reads arise from various mis-priming events. These can result in incorrect pA site calls anywhere from several nucleotides downstream to several kilobases upstream from the bona fide pA site. While these mis-priming events can be mitigated by increasing annealing stringency (e.g. increasing temperature from 37 °C to 42 °C), they still persist at an appreciable level (∼10%) and computational methods must be used to prevent artifactual calls. Here we present a bioinformatics workflow for precise mapping of poly(A)+ 3' ends and handling of artifacts due to oligo(dT) mis-priming and sample polymorphisms. We test pA site calling with three different read mapping programs (STAR, BWA, and BBMap), and show that the way in which each handles terminal mismatches and soft clipping has a substantial impact on identifying correct pA sites, with BWA requiring the least post-processing to correct artifacts. We demonstrate the use of this pipeline for mapping pA sites in the model eukaryote S. cerevisiae, and further apply this technology to non-polyadenylated transcripts by employing in vitro polyadenylation prior to library prep (IVP-seq). As proof of principle, we show that a fraction of tRNAs harbor CCU 3' tails instead of the canonical CCA tail, and globally identify 3' ends of splicing intermediates arising from inefficiently spliced transcripts.

Family Health Leaders: Lessons on Living with Li-Fraumeni Syndrome across Generations.

Li-Fraumeni Syndrome (LFS) is a hereditary disorder that confers an approximately 90% lifetime risk of cancer and requires comprehensive lifetime cancer screening. We explored healthcare roles for managing LFS-related cancer risks and treatments that were assumed by parents, adolescents, and adult children. Semi-structured interviews were conducted with 23 families. Family groupings were comprised of 2-5 members, with the younger generation in each family ranging in age from 7 to 40 years. Using grounded theory methods, we conducted open and focused coding of interview transcript content. Family members described how the role of health leader was implemented in their family, as well as factors such as maturation of a child or death of a member that determined who assumed particular roles and how these roles shifted over time. They often expressed collective responsibility for helping relatives understand LFS and implement appropriate cancer risk management. Members demonstrated their health role by attending others' medical appointments for support or information gathering. The health leader role was intergenerational and provided the family necessary support in navigating complicated healthcare decisions. Our findings provide insight into healthcare providers regarding how LFS patients and their relatives develop unique medical decision-making and caring roles influenced by the hereditary nature of LFS, and how these roles change over time. Providers who are attuned to family role dynamics may be better able to meet relatives' psychosocial and medical needs by understanding how living with LFS influences the family system's functioning and facilitating members' support for each other.

El síndrome de Li-Fraumeni (LFS) es un trastorno hereditario que concede aproximadamente un 90 % de riesgo durante toda la vida de contraer cáncer y exige exámenes completos para la detección del cáncer de por vida. Analizamos los roles sanitarios a la hora de manejar los riesgos y los tratamientos de cáncer relacionados con el LFS que asumieron los padres, los adolescentes y los hijos adultos. Se realizaron entrevistas semiestructuradas con 23 familias. Los agrupamientos familiares estaban compuestos por entre 2 y 5 familiares, donde la edad de la generación más joven de cada familia oscilaba entre 7 y 40 años. Utilizando los métodos de la teoría fundamentada, realizamos una codificación abierta y centrada del contenido de la transcripción de la entrevista. Los miembros de la familia describieron cómo se implementó el rol de jefe de la salud en su familia, así como factores como la maduración de un niño o la muerte de un miembro que determinaron quiénes asumieron roles particulares y cómo estos roles cambiaron con el tiempo. Con frecuencia ellos expresaron la responsabilidad colectiva de ayudar a los familiares a comprender el LFS y a implementar el manejo adecuado del riesgo de contraer cáncer. Los familiares demostraron sus roles sanitarios asistiendo a citas médicas de los demás para recibir apoyo u obtener información. El rol de jefe sanitario fue intergeneracional y proporcionó a la familia el apoyo necesario para manejarse ante decisiones complicadas sobre la asistencia sanitaria. Nuestros resultados brindan información para los prestadores de servicios médicos con respecto a cómo los pacientes de LFS y sus familiares desarrollan roles únicos para la toma de decisiones médicas y el cuidado influenciados por la índole hereditaria del LFS, y cómo estos roles cambian con el tiempo. Es posible que los prestadores que estén acostumbrados a la dinámica de roles familiares sean más capaces de satisfacer las necesidades psicosociales y médicas de los familiares si comprenden cómo vivir con LFS influye en el funcionamiento del sistema familiar y si facilitan el apoyo mutuo de los familiares.

Protein Methylation and Translation: Role of Lysine Modification on the Function of Yeast Elongation Factor 1A.

To date, 12 protein lysine methyltransferases that modify translational elongation factors and ribosomal proteins (Efm1-7 and Rkm 1-5) have been identified in the yeast Saccharomyces cerevisiae. Of these 12, five (Efm1 and Efm4-7) appear to be specific to elongation factor 1A (EF1A), the protein responsible for bringing aminoacyl-tRNAs to the ribosome. In S. cerevisiae, the functional implications of lysine methylation in translation are mostly unknown. In this work, we assessed the physiological impact of disrupting EF1A methylation in a strain where four of the most conserved methylated lysine sites are mutated to arginine residues and in strains lacking either four or five of the Efm lysine methyltransferases specific to EF1A. We found that loss of EF1A methylation was not lethal but resulted in reduced growth rates, particularly under caffeine and rapamycin stress conditions, suggesting EF1A interacts with the TORC1 pathway, as well as altered sensitivities to ribosomal inhibitors. We also detected reduced cellular levels of the EF1A protein, which surprisingly was not reflected in its stability in vivo. We present evidence that these Efm methyltransferases appear to be largely devoted to the modification of EF1A, finding no evidence of the methylation of other substrates in the yeast cell. This work starts to illuminate why one protein can need five different methyltransferases for its functions and highlights the resilience of yeast to alterations in their posttranslational modifications.

Engineered Graphene Oxide Nanocomposite Capable of Preventing the Evolution of Antimicrobial Resistance.

Antimicrobial resistance (AMR) is spreading worldwide and keeps evolving to adapt to antibiotics, causing increasing threats in clinics, which necessitates the exploration of antimicrobial agents for not only killing of resistant cells but also prevention of AMR progression. However, so far, there has been no effective approach. Herein, we designed lanthanum hydroxide and graphene oxide nanocomposites (La@GO) to confer a synergistic bactericidal effect in all tested resistant strains. More importantly, long-term exposure of E. coli (AMR) to subminimum inhibitory concentrations of La@GO does not trigger detectable secondary resistance, while conventional antibiotics and silver nanoparticles lead to a 16- to 64-fold increase in tolerance. The inability of E. coli to evolve resistance to La@GO is likely due to a distinctive extracellular multitarget invasion killing mechanism involving lipid dephosphorylation, lipid peroxidation, and peptidoglycan disruption. Overall, our results highlight La@GO nanocomposites as a promising solution to combating resistant bacteria without inducing the evolution of AMR.

The diagnostic expertise acceleration module (DEAM): promoting the formation of organized knowledge.

Background: Ensuring that learners acquire diagnostic competence in a timely fashion is critical to providing high quality and safe patient care. Resident trainees typically gain experience by undertaking repetitive clinical encounters and receiving feedback from supervising faculty. By critically engaging with the diagnostic process, learners encapsulate medical knowledge into discrete memories that are able to be recollected and refined in subsequent clinical encounters. In the setting of exponentially increasing medical complexity and current duty hour limitations, the opportunities for successful practice in the clinical arena have become limited. Novel educational methods are needed to more efficiently bridge the gap from novice to expert diagnostician. Objective: Using a conceptual framework which incorporates deliberate practice, script theory, and learning curves, we developed an educational module prototype to coach novice learners to formulate organized knowledge (i.e. a repertoire of illness scripts) in an accelerated fashion thereby simulating the ideal experiential learning in a clinical rotation. Design: We developed the Diagnostic Expertise Acceleration Module (DEAM), a web-based module for learning illness scripts of diseases causing pediatric respiratory distress. For each case, the learner selects a diagnosis, receives structured feedback, and then creates an illness script with a subsequent expert script for comparison. Results: We validated the DEAM with seven experts, seven experienced learners and five novice learners. The module data generated meaningful learning curves of diagnostic accuracy. Case performance analysis and self-reported feedback demonstrated that the module improved a learner's ability to diagnose respiratory distress and create high-quality illness scripts. Conclusions: The DEAM allowed novice learners to engage in deliberate practice to diagnose clinical problems without a clinical encounter. The module generated learning curves to visually assess progress towards expertise. Learners acquired organized knowledge through formulation of a comprehensive list of illness scripts.

Reversal of ε-Aminocaproic Acid-Induced Massive Thromboemboli Using Tissue Plasminogen Activator During Cardiac Surgery: A Case Report.

ε-Aminocaproic acid is routinely used in cardiac surgery to prevent excess bleeding. It is rarely associated with thrombotic events. This case report illustrates the formation of intracardiac thrombi leading to massive pulmonary embolism during a coronary artery bypass graft surgery, secondary to the administration of ε-aminocaproic acid as confirmed by intraoperative transesophageal echocardiogram. After a failure of resolution with high-dose heparin, tissue plasminogen activator was used to successfully reverse the patient's hypercoagulable state.

Knowledge Accrual Following Participation in Pediatric Fundamental Critical Care Support Course in Gaborone, Botswana.

OBJECTIVES: To describe provider characteristics, knowledge acquisition, perceived relevance, and instruction quality of the Society of Critical Care Medicine's Pediatric Fundamentals of Critical Care Support course pilot implementation in Botswana. DESIGN: Observational, single center. SETTING: Academic, upper middle-income country. SUBJECTS: Healthcare providers in Botswana. INTERVENTIONS: A cohort of healthcare providers completed the standard 2-day Pediatric Fundamentals of Critical Care Support course and qualitative survey during the course. Cognitive knowledge was assessed prior to and immediately following training using standard Pediatric Fundamentals of Critical Care Support multiple choice questionnaires. Data analysis used Fisher exact, chi-square, paired t test, and Wilcoxon rank-sum where appropriate. MAIN RESULTS: There was a significant increase in overall multiple choice questionnaires scores after training (mean 67% vs 77%; p < 0.001). Early career providers had significantly lower mean baseline scores (56% vs 71%; p < 0.01), greater knowledge acquisition (17% vs 7%; p < 0.02), but no difference in posttraining scores (73% vs 78%; p = 0.13) compared with more senior providers. Recent pediatric resuscitation or emergency training did not significantly impact baseline scores, posttraining scores, or decrease knowledge acquisition. Eighty-eight percent of providers perceived the course was highly relevant to their clinical practice, but only 71% reported the course equipment was similar to their current workplace. CONCLUSIONS: Pediatric Fundamentals of Critical Care Support training significantly increased provider knowledge to care for hospitalized seriously ill or injured children in Botswana. Knowledge accrual is most significant among early career providers and is not limited by previous pediatric resuscitation or emergency training. Further contextualization of the course to use equipment relevant to providers work environment may increase the value of training.