ABSTRACT

Introduction Although a relatively young specialty, emergency medicine (EM) is quite popular among medical students and is one of the most competitive large specialties.  Consequently, students increasingly seek more opportunity to differentiate themselves from their colleagues by pursuing more clerkships at the cost of taking out additional loans.  This despite the fact that those who match in emergency medicine typically do so in their top three choices.  We sought to ascertain what factors EM program directors seek in their typical candidate.  Methods Emergency Medicine program directors were recruited via the Council of Residency Directors email listserv to participate in an anonymous survey regarding the United States Medical Licensing Examination (USMLE), the number of Standard Letters of Evaluation (SLOE), and the number of EM rotations during the fourth year.  Results 135 respondents completed the anonymous survey.  59% of respondents stated their program did not have a minimum USMLE Step 1 score, but 39% reported a minimum score of 210 or higher. 95% of programs do not require Step 2 to grant an interview, but 46% require it to place the student on the rank list.  80% require only one EM rotation to grant an interview and none require more than 2.  95% of programs will accept 2 SLOEs for both application and rank list placement.  Conclusion For the typical emergency medicine applicant, there is likely little benefit to performing more than two rotations and obtaining more than two SLOEs. Students can defer USMLE Step 2 but must complete it by the time rank lists are due. Our study was limited by the anonymity of the survey and comments by the respondents revealed the questions did not account for some nuances programs apply to their application review process.

The atrial natriuretic (ANP) and B-type natriuretic peptides (BNP) are important clinical diagnostic markers that, in recent years, have also been leveraged in a therapeutic angle. The release of these peptides is commonly thought to be stimulated by myocardial stretch, whether that be due to a pressure or volume overload. These peptides have several effects in the body, but the main ones involve diuresis and natriuresis at the kidneys, vasodilation, and an inhibition of the sympathetic nervous system. Bulk methods, such as quantitative PCR (qPCR) and bulk RNA-seq, have been able to identify the triggers for the transcription of the genes that encode for these peptides, Nppa and Nppb, in the setting of various cardiac injuries. Limitations of these methods involve not being able to identify who the producers of these peptides are in the injured myocardium, where they might be located, and how quickly the transcription of these genes begins in response to a stressor. Here, using single-cell RNA-seq, I analyze a combination of human and mouse cardiac tissue and identify a small subset of cardiomyocytes that seem to be responsible for professionally producing these peptides. These cardiomyocytes are located directly adjacent to the site of injury and are being robustly produced as soon as four hours following a cardiac injury.

Myocardial infarction (MI) is the leading cause of death in the U.S., affecting elderly populations and more recently, younger populations. Treatments thus far have reduced rates of myocardial infarction (MI), but progress has slowed due to downstream consequences of heart failure, especially involving the immune system response to necrotic cells in the heart, causing subsequent inflammation. To elucidate gene expression and specialized subsets of immune cells that exacerbate or ameliorate inflammation after MI we have analyzed the single-cell transcriptomics of mice bone marrow, the source of inflammation of the heart. We have observed differences in single-cell gene expression between bone marrow in vitro and in vivo of mice four days post-MI, especially in the expression of cardioprotective and proliferative macrophages, marked by genes such as Top2a and Mki67, in vitro. Additionally, we compared single-cell transcriptomic differences of in vitro bone marrow derived macrophages with in vivo bone marrow macrophages. We have also found specialized neutrophils, SiglecFHigh neutrophils, that are suggested to exacerbate inflammation in the heart via the IL6 pathway. Lastly, we reported crosstalk between the IRF3 pathway and the Nrf2 pathway, especially between itaconate, a key Nrf2 component and interferon-beta (IFN-B), a component of the IRF3 pathway. In conclusion, single cell revealed new populations of specialized immune cells that can potentially aid or detriment post-MI recovery. Further investigation into specialized immune cells using in vivo imaging and functional characterization of neutrophils will allow for higher resolution of immune cell function and potential antibody-based therapeutics designed to reduce SiglecFHigh neutrophils.

There is general consensus that immune cells such as macrophages within injured tissue influence remodeling and fibrosis. At the cellular level, this has been attributed to the communication between macrophages and fibroblasts. However, precisely how these communication is mediated at the molecular level remains unknown. We hypothesized fibroblasts are influenced by type I interferon signaling, either directly or indirectly via macrophages, which is a determining factor in the induction of adverse cardiac remodeling. In this study, differential gene expression analysis at a single cell level was done between wild type and knockout mice after myocardial infarction. It was found that a subset population of macrophages produce IRF3-dependent type I Interferon genes. Further, in fibroblasts, it was found matrix metalloproteinases is dominantly expressed in wild type. Given these results, this study proposes that the overexpression of matrix metalloproteinases in fibroblasts is linked to the production of the IRF3-dependent type I interferon genes in macrophages.

Ischemic heart disease (IHD) is the leading cause of morbidity and mortality worldwide. 1It begins with myocardial infarction (MI), which results from obstruction of coronary artery blood flow and leaves dependent cardiomyocytes ischemic due to a mismatch between oxygen supply and demand. Although revascularization and medical management have dramatically reduced acute mortality from MI, surviving patients frequently develop chronic symptomatic heart failure. In adults, cardiomyocyte proliferative potential is limited, and to date, no consequential cardiac regeneration has been demonstrated. Instead, therapeutic efforts have focused on protecting the non-dead but still injured and ischemic borderzone cardiomyocytes (BZ) in the region immediately adjacent to the infarct zone (IZ). BZ are thought to be biologically distinct from the distant well-perfused remote zone cardiomyocytes (RZ), yet surprisingly little is known about their cellular and molecular biology due to technology limitation. In Chapter 1, we developed methods for performing high throughput single nuclei cardiomyocyte transcriptional profiling and use them to transcriptionally define BZ from RZ. We hypothesize that BZ and RZ will exhibit distinct transcriptional signatures that can be revealed by unbiased clustering. To validate the marker genes that best define cardiomyocytes from each territory, we employed a recently developed method for spatially resolved transcriptomics known as Visium 10x, a grid based method ( low resolution) and multiplexed RNA fluorescence in situ hybridization (mFISH) to show that the BZ can be functionally defined by gene expression patterns. Macrophages contribute to diverse physiologic and pathogenic functions across myocardial tissue. They are critical players in the pathophysiological processes induced by myocardial infarction such as removing dead cells near BZ, patrolling local tissue microenvironments, and mounting innate and adaptive anti-pathogen. Morphologic evidence suggests that macrophages are arranged as networks of cells and there is evidence that they communicate with cardiomyocytes to influence cardiac conduction 2. However, existing methods for studying communication between macrophages include transfer of microinjected or scrape loaded membrane impermeant dyes, fluorescence recovery after photobleaching, or measurement of electrical conductance via patch clamping are destructive with low temporal resolution 3. For the next chapter, we developed a transgenic tool to provide a non-destructive assay that infer cell communication with high temporal resolution. We leveraged high resolution imaging with a using tissue-specific genetically encoded calcium indicator (GECI) mice reporter (GCAMP5 fl) animal in which is calcium dependent green-fluorescence protein (GFP) specifically in (Csf1rCre) macrophages. Calcium represents an attractive indicator of cell communication in cells because it is a dynamic second messenger influenced by multiple signaling pathways. In non-communicating populations of cells, calcium dynamics are not necessarily correlated. We reasoned that non-destructive monitoring of calcium dynamics in a population of cells and detection of their spatiotemporal correlations could be used to infer cell communication, even if the molecular stimuli, mediators, and mechanisms were unknown. In order to infer cell communication, we create a computational pipeline called ‘Excess Synchrony’ which automatically preprocess GECI fluorescence time-series measured by time-lapse imaging or intravital microscopy, detect peaks with single cell resolution, and infer cell communication from the synchrony of single cell calcium transients.

Rehospitalizations for chronic diseases such as heart failure are common and costly. However, they are also preventable. In many cases, timely interventions can prevent the patient from reaching a critical point where hospitalization is needed. Since these decompensations occur necessarily in the patient’s home, home health solutions are an ideal choice to give clinicians insight into their patient’s well being and enable timely interventions. However, many home health solutions are limited by requiring adherence or technological literacy, or in some cases, an invasive procedure. We describe here a non-contact adherence-independent system for total body weight and cardiopulmonary monitoring in the home bed (BedScales). We describe the system design and present validation experiments. Additionally, we discovered that nocturnal respiratory rate (NRR) in particular is a very useful, yet understudied biomarker for detecting disease exacerbations. We therefore performed the first large-scale analysis of NRR from over 22,000 sleep studies and found that there is an association between NRR and chronic diseases such as atrial fibrillation, heart failure, and COPD. We installed BedScales devices in the homes of high-risk patients and monitored them for an average of one hundred days each. In doing so, we found instances where NRR rose from baseline in response to diseases such as sepsis, pneumonia, and heart failure. Finally, we present three case reports from patients we monitored with complex physiology, demonstrating how the BedScales platform and NRR would be able to greatly expand the window that clinicians had for performing a timely intervention. We believe that this approach to outpatient management will be able to, in a practical and widely applicable manner, allow clinicians to care for their patients at home and achieve the triple aim of improving care, improving quality of life, and lowering costs.

Cancer is a prevalent disease that has been researched for many years to find a cure. A potential focus is the popular chemotherapeutic, paclitaxel (PTX), which has been known to treat a number of metastatic cancers with high efficacy. However, PTX induces neuropathy that results in acute pain, muscle weakness, among other symptoms. This has caused research to delve into the effects of PTX and the ability to halt these symptoms when using it for treatment. Several studies have shown that PTX influences the activity of TRPV-4, a mechanically gated calcium ion channel, and there has been evidence that suggests that intracellular calcium signals are involved in the pathogenesis of peripheral neuropathy caused by PTX. In coincidence, if TRPV-4 inhibition in dorsal root ganglion cells (DRGs) can be proved with fruition, this can potentially reduce cell death in DRGs. Because of PTX’s potential effect on TRPV-4 channels, it is important to determine if inhibiting these channels would have any effect on calcium dynamics. Pretreatment of wild-type mice DRGs with TRPV-4 inhibitors, GSK-2193874 and HC-067047, was used to determine if specific inhibitors of TRPV-4 have an effect on calcium channels within DRGs in this study.

Despite decades of research, myocardial infarction (MI) and the resulting ischemic heart disease remain one of the leading causes of death. In the hours and days following MI, the ventricle undergoes adverse remodeling, an incompletely understood process in which the composition and architecture of the myocardium dramatically change from functional myocardium to scar tissue. It is well recognized that remodeling depends on the actions of leukocytes (i.e., neutrophils, monocytes, macrophages), yet interventions which attenuate inflammation have yielded disappointing clinical results. This is, in part, because the innate immune response has been historically studied using immunophenotyping and bulk techniques that require candidate targets, limiting biomarker or drug discovery.

The goal of this dissertation is to use next-generation sequencing technologies (NGS) to better understand the cellular and molecular drivers of adverse remodeling after MI. First, I define transcriptional dynamics of leukocytes using single cell RNA-sequencing (scRNA-seq) from their origins in the bone marrow through the blood and into the heart. These data reveal numerous specialized intracardiac neutrophil, monocyte, and macrophage subpopulations that serve as a new ‘vocabulary’ for the next wave of researchers to understand their functional significance.

I focus on a subset of neutrophils and monocytes that both express elevated levels of type I interferon-stimulated genes (ISGs). Originally characterized as host antiviral defense, ablation of interferon (IFN) signaling was recently demonstrated to be cardioprotective after MI. Using NGS tools, I show that myelopoietic IFN signaling begins in the bone marrow, which not only impacts the field of cardiovascular disease, but also has broad implications in autoimmune and sterile inflammatory disease. In the second half of my work, I use spatial transcriptomic technologies to investigate pathological mechanisms in the heart after MI. I test the hypothesis that the ischemic borderzone (BZ), which was historically defined anatomically, could be define transcriptionally. We find that cardiomyocytes (CMs) transcriptomes naturally organize into anatomic and functionally distinct regions and demonstrate that mechanical injury is sufficient to induce BZ transcriptional patterning. These results provide a new framework for researchers to assess cardiac injury and offer mechanistic insights to its propagation beyond ischemia.

Finally, I leverage these findings to explore immune involvement in the healing myocardium. Of the many immune subsets dispersed throughout the infarct, we find that IFN-stimulated cells uniquely localize with BZ CMs due to secondary, intracardiac IFN signaling resulting in characteristic cluster formation, which raises numerous questions regarding initiation and effect. To this extent, we study their role in cases of lethal ventricular rupture and implicate IFN-signaling as a driver of post-MI pathology.