The Role Of Gene Regulation In Cancer: Studies Of Cancer-Related Phenotypes Mediated By Mex3d And By Microrna-618 Implicate Their Potential Oncogenic Role

The control of gene expression is pivotal in the context of molecular pathogenesis of a number of diseases, and thus is of critical relevance to public health. An array of cellular tools exist in controlling gene expression, including epigenetic effects, non-coding RNAs, and RNA-binding proteins. These tools are critical to the modern study of public health, and are used in tandem with population-based studies. This work focuses on specific examples of non-coding RNAs and RNA-binding proteins, describing the effects of microRNA-618, a non-coding RNA, and MEX3D, a post-transcriptional regulator, in cancer. MicroRNAs (miRNAs) form a class of highly conserved endogenous RNAs that inhibit gene expression and may act as oncogenes or as tumor suppressors, regulating extensive cancer-related gene networks. Here, we show the association between a single miRNA, miR-618, and cancer-related pathways in HeLa cells. MiR-618 was identified as a potentially oncogenic microRNA, controlling a number of cancer-related gene networks and pathways. Gain-of-function analysis reveals differential expression of 110 transcripts following miRNA-618 transfection. Notably, three upregulated genes are well-studied oncogenes--KIT, JUN, and FOSB--and three downregulated genes are well-known tumor suppressors--PTPRO, STK11/LKB1, and IGFBP5. Interestingly, investigation using the Ingenuity Pathway Analysis software tool reveals alterations in multiple cancer-related and cell cycle-related networks, including upregulated oncogenes in the top identified network Post-translational modification, cellular development, cellular growth and proliferation following miR-618 transfection. Further, miR-618 expression analysis shows overexpression in HeLa cells compared to normal cervical cells. Our findings present evidence for a novel oncogenic miRNA, miR-618, that is involved in cancer-related gene networks and is overexpressed in cancer. This work also examined the role of a novel post-transcriptional regulator, MEX3D, in cancer. The Oncomine online database reveals that MEX3D is overexpressed in a number of solid tumors, notably in glioma. MEX3D is 3.01-fold overexpressed in glioma cells compared to non-cancerous, normal tissue. Kaplan-Meier survival analysis reveals that higher expression of MEX3D leads to poorer overall survival in overall glioma patients. Lastly, in a pilot case-control study of twelve glioma biopsies, we examined the effects of methylation in CpG sites in the MEX3D gene. The results were unclear, as we found a 3\u27UTR site that was 9.5% hypermethylated compared to normal tissue, a site in the body of the gene that was 24.8% hypermethylated, and second site in the body that was 15.4% hypomethylated compared to normal tissue. Phenotypic studies reveal that MEX3D is responsible for two cancer phenotypes. Knockdown of MEX3D leads to increased cell proliferation and decreased cell invasion, suggesting that overexpression of MEX3D is responsible for increased cell proliferation and decreased cell invasion. This study is the first to describe the effects of miR-618 and of MEX3D in cancer. The findings presented in this work lay the foundation for further mechanistic studies of miR-618 and MEX3D. More work is needed to identify the mechanisms of oncogenesis controlled by these molecules. Our study indicates that miR-618 may be a biomarker for several types of cancer and warrants further investigation

Expanding DARPA’s model of innovation for biopharma: A proposed Advanced Research Projects Agency for Health

This paper proposes approaches for innovation in health to accelerate biomedical innovation and its potential to address public health needs by adopting a mission-oriented approach

Telementoring for remote simulation instructor training and faculty development using telesimulation

Publisher Copyright: © Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Introduction: Simulation-based training is essential for high-quality medical care, but it requires access to equipment and expertise. Technology can facilitate connecting educators to training in simulation. We aimed to explore the use of remote simulation faculty development in Latvia using telesimulation and telementoring with an experienced debriefer located in the USA. Methods: This was a prospective, simulation-based longitudinal study. Over the course of 16 months, a remote simulation instructor (RI) from the USA and a local instructor (LI) in Latvia cofacilitated with teleconferencing. Responsibility gradually transitioned from the RI to the LI. At the end of each session, students completed the Debriefing Assessment for Simulation in Healthcare (DASH) student version form (DASH-SV) and a general feedback form, and the LI completed the instructor version of the DASH form (DASH-IV). Outcome measures were the changes in DASH scores over time. Results: A total of eight simulation sessions were cofacilitated of 16 months. As the role of the LI increased over time, the debrief quality measured with the DASH-IV did not change significantly (from 89 to 87), although the DASH-SV score decreased from a total median score of 89 (IQR 86-98) to 80 (IQR 78-85) (p=0.005). Conclusion: In this study, telementoring with telesimulations resulted in high-quality debriefing. The quality - perceived by the students - was higher with the involvement of the remote instructor and declined during the transition to the LI. This concept requires further investigation and could potentially build local simulation expertise promoting sustainability of high-quality simulation.Peer reviewe

A Collaborative In Situ Simulation-based Pediatric Readiness Improvement Program for Community Emergency Departments

Background More than 30 million children are cared for across 5,000 U.S. emergency departments (EDs) each year. Most of these EDs are not facilities designed and operated solely for children. A Web-based survey provided a national and state-by-state assessment of pediatric readiness and noted a national average score was 69 on a 100-point scale. This survey noted wide variations in ED readiness with scores ranging from 61 in low-pediatric-volume EDs to 90 in the high-pediatric-volume EDs. Additionally, the mean score at the state level ranged from 57 (Wyoming) to 83 (Florida) and for individual EDs ranged from 22 to 100. The majority of prior efforts made to improve pediatric readiness have involved providing Web-based resources and online toolkits. This article reports on the first year of a program that aimed to improve pediatric readiness across community hospitals in our state through in situ simulation-based assessment facilitated by our academic medical center. The primary aim was to improve the pediatric readiness scores in the 10 participating hospitals. The secondary aim was to explore the correlation of simulation-based performance of hospital teams with pediatric readiness scores. Methods This interventional study measured the Pediatric Readiness Survey (PRS) prior to and after implementation of an improvement program. This program consisted of three components: 1) in situ simulations, 2) report-outs, and 3) access to online pediatric readiness resources and content experts. The simulations were conducted in situ (in the ED resuscitation bay) by multiprofessional teams of doctors, nurses, respiratory therapists, and technicians. Simulations and debriefings were facilitated by an expert team from a pediatric academic medical center. Three scenarios were conducted for all teams and include: a 6-month-old with respiratory failure, an 8-year-old with diabetic ketoacidosis (DKA), and a 6-month-old with supraventricular tachycardia (SVT). A performance score was calculated for each scenario. The improvement of PRS was compared before and after the simulation program. The correlation of the simulation performance of each hospital and the PRS was calculated. Results Forty-one multiprofessional teams from 10 EDs in Indiana participated in the study, five were of medium pediatric volume and five were medium- to high-volume EDs. The PRS significantly improved from the first to the second on-site verification assessment (58.4 ± 4.8 to 74.7 ± 2.9, p = 0.009). Total adherence scores to scenario guidelines were 54.7, 56.4, and 62.4% in the respiratory failure, DKA, and SVT scenarios, respectively. We found no correlation between simulation performance and PRS scores. Medium ED pediatric volume significantly predicted higher PRS scores compared to medium-high pediatric ED volume (β = 8.7; confidence interval = 0.72–16.8, p = 0.034). Conclusions Our collaborative improvement program that involved simulation was associated with improvement in pediatric readiness scores in 10 EDs participating statewide. Future work will focus on further expanding of the network and establishing a national model for pediatric readiness improvement

Differences in the Quality of Pediatric Resuscitative Care Across a Spectrum of Emergency Departments

Importance: The quality of pediatric resuscitative care delivered across the spectrum of emergency departments (EDs) in the United States is poorly described. In a recent study, more than 4000 EDs completed the Pediatric Readiness Survey (PRS); however, the correlation of PRS scores with the quality of simulated or real patient care has not been described. Objective: To measure and compare the quality of resuscitative care delivered to simulated pediatric patients across a spectrum of EDs and to examine the correlation of PRS scores with quality measures. Design, Setting, and Participants: This prospective multicenter cohort study evaluated 58 interprofessional teams in their native pediatric or general ED resuscitation bays caring for a series of 3 simulated critically ill patients (sepsis, seizure, and cardiac arrest). Main Outcomes and Measures: A composite quality score (CQS) was measured as the sum of 4 domains: (1) adherence to sepsis guidelines, (2) adherence to cardiac arrest guidelines, (3) performance on seizure resuscitation, and (4) teamwork. Pediatric Readiness Survey scores and health care professional demographics were collected as independent data. Correlations were explored between CQS and individual domain scores with PRS. Results: Overall, 58 teams from 30 hospitals participated (8 pediatric EDs [PEDs], 22 general EDs [GEDs]). The mean CQS was 71 (95% CI, 68-75); PEDs had a higher mean CQS (82; 95% CI, 79-85) vs GEDs (66; 95% CI, 63-69) and outperformed GEDs in all domains. However, when using generalized estimating equations to estimate CQS controlling for clustering of the data, PED status did not explain a higher CQS (beta = 4.28; 95% CI, -4.58 to 13.13) while the log of pediatric patient volume did explain a higher CQS (beta = 9.57; 95% CI, 2.64-16.49). The correlation of CQS to PRS was moderate (r = 0.51; P \u3c .001). The correlation was weak for cardiac arrest (r = 0.24; P = .07), weak for sepsis (rho = 0.45; P \u3c .001) and seizure (rho = 0.43; P = .001), and strong for teamwork (rho = 0.71; P \u3c .001). Conclusions and Relevance: This multicenter study noted significant differences in the quality of simulated pediatric resuscitative care across a spectrum of EDs. The CQS was higher in PEDs compared with GEDs. However, when controlling for pediatric patient volume and other variables in a multivariable model, PED status does not explain a higher CQS while pediatric patient volume does. The correlation of the PRS was moderate for simulation-based measures of quality

Community-based in situ simulation: bringing simulation to the masses

Simulation-based methods are regularly used to train inter-professional groups of healthcare providers at academic medical centers (AMC). These techniques are used less frequently in community hospitals. Bringing in-situ simulation (ISS) from AMCs to community sites is an approach that holds promise for addressing this disparity. This type of programming allows academic center faculty to freely share their expertise with community site providers. By creating meaningful partnerships community-based ISS facilitates the communication of best practices, distribution of up to date policies, and education/training. It also provides an opportunity for system testing at the community sites. In this article, we illustrate the process of implementing an outreach ISS program at community sites by presenting four exemplar programs. Using these exemplars as a springboard for discussion, we outline key lessons learned discuss barriers we encountered, and provide a framework that can be used to create similar simulation programs and partnerships. It is our hope that this discussion will serve as a foundation for those wishing to implement community-based, outreach ISS

Controlling the Growth of the Skin Commensal Staphylococcus epidermidis Using d-Alanine Auxotrophy.

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied d-alanine. The S. epidermidis NRRL B-4268 Δalr1 Δalr2 Δdat strain (SEΔΔΔ) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the d-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in d-alanine-deficient medium, pooled human blood, and skin. In the presence of d-alanine, SEΔΔΔ colonized and increased expression of human β-defensin 2 in cultured human skin models in vitro. SEΔΔΔ showed a low propensity to revert to d-alanine prototrophy and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SEΔΔΔ as a live biotherapeutic strain whose growth can be controlled by d-alanine.IMPORTANCE The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and in vitro evidence of a skin commensal whose growth can be controlled through d-alanine. The basis of this strain will support future clinical studies of this strain in humans

Preparedness for Pediatric Office Emergencies: A Multicenter, Simulation-Based Study

OBJECTIVES Pediatric emergencies can occur in pediatric primary care offices. However, few studies have measured emergency preparedness, or the processes of emergency care, provided in the pediatric office setting. In this study, we aimed to measure emergency preparedness and care in a national cohort of pediatric offices. METHODS This was a multicenter study conducted over 15 months. Emergency preparedness scores were calculated as a percentage adherence to 2 checklists on the basis of the American Academy of Pediatrics guidelines (essential equipment and supplies and policies and protocols checklists). To measure the quality of emergency care, we recruited office teams for simulation sessions consisting of 2 patients: a child with respiratory distress and a child with a seizure. An unweighted percentage of adherence to checklists for each case was calculated. RESULTS Forty-eight teams from 42 offices across 9 states participated. The mean emergency preparedness score was 74.7% (SD: 12.9). The mean essential equipment and supplies subscore was 82.2% (SD: 15.1), and the mean policies and protocols subscore was 57.1% (SD: 25.6). Multivariable analyses revealed that independent practices and smaller total staff size were associated with lower preparedness. The median asthma case performance score was 63.6% (interquartile range: 43.2–81.2), whereas the median seizure case score was 69.2% (interquartile range: 46.2–80.8). Offices that had a standardized process of contacting emergency medical services (EMS) had a higher rate of activating EMS during the simulations. CONCLUSIONS Pediatric office preparedness remains suboptimal in a multicenter cohort, especially in smaller, independent practices. Academic and community partnerships using simulation can help address gaps and implement important processes like contacting EMS

Recoding the metagenome: microbiome engineering in situ.

Synthetic biology has enabled a new generation of tools for engineering the microbiome, including targeted antibiotics, protein delivery, living biosensors and diagnostics, and metabolic factories. Here, we discuss opportunities and limitations in microbiome engineering, focusing on a new generation of tools for in situ genetic modification of a microbiome that hold particular promise in circumventing these limitations

Structures of regulatory machinery reveal novel molecular mechanisms controlling B. subtilis nitrogen homeostasis

In Bacillus subtilis, nitrogen homeostasis is controlled by a unique circuitry composed of the regulator TnrA and the repressor GlnR. Here, Schumacher et al. describe a comprehensive molecular dissection of this network that reveals novel mechanisms, including oligomeric transformations, by which their inducible signal transduction domains are employed to provide a readout of nitrogen levels.All cells must sense and adapt to changing nutrient availability. However, detailed molecular mechanisms coordinating such regulatory pathways remain poorly understood. In Bacillus subtilis, nitrogen homeostasis is controlled by a unique circuitry composed of the regulator TnrA, which is deactivated by feedback-inhibited glutamine synthetase (GS) during nitrogen excess and stabilized by GlnK upon nitrogen depletion, and the repressor GlnR. Here we describe a complete molecular dissection of this network. TnrA and GlnR, the global nitrogen homeostatic transcription regulators, are revealed as founders of a new structural family of dimeric DNA-binding proteins with C-terminal, flexible, effector-binding sensors that modulate their dimerization. Remarkably, the TnrA sensor domains insert into GS intersubunit catalytic pores, destabilizing the TnrA dimer and causing an unprecedented GS dodecamer-to-tetradecamer conversion, which concomitantly deactivates GS. In contrast, each subunit of the GlnK trimer “templates” active TnrA dimers. Unlike TnrA, GlnR sensors mediate an autoinhibitory dimer-destabilizing interaction alleviated by GS, which acts as a GlnR chaperone. Thus, these studies unveil heretofore unseen mechanisms by which inducible sensor domains drive metabolic reprograming in the model Gram-positive bacterium B. subtilis