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Heme is a ubiquitous and essential iron containing metallo-organic cofactor required for virtually all aerobic life. Heme synthesis is initiated and completed in mitochondria, followed by certain covalent modifications and/or its delivery to apo-hemoproteins residing throughout the cell. While the biochemical aspects of heme biosynthetic reactions are well understood, the trafficking of newly synthesized heme—a highly reactive and inherently toxic compound—and its subsequent delivery to target proteins remain far from clear. In this review, we summarize current knowledge about heme biosynthesis and trafficking within and outside of the mitochondria.

Effective stakeholder engagement increases research relevance and utility. Though published principles of community-based participatory research and patient-centered outcomes research offer guidance, few resources offer effective techniques to engage stakeholders and translate their engagement into improvements in research process and outcomes. The Indiana Clinical and Translational Sciences Institute (Indiana CTSI) is home to Research Jam (RJ), an interdisciplinary team of researchers, project management professionals, and design experts, that employs human-centered design (HCD) to engage stakeholders in the research process. Establishing HCD services at the Indiana CTSI has allowed for accessible and innovative stakeholder-engaged research. RJ offers services for stakeholder-informed study design, measurement, implementation, and dissemination. RJ’s services are in demand to address research barriers pertaining to a diverse array of health topics and stakeholder groups. As a result, the RJ team has grown significantly with both institutional and extramural support. Researchers involved in RJ projects report that working with RJ helped them learn how to better engage with stakeholders in research and changed the way they approach working with stakeholders. RJ can serve as a potential model for effectively engaging stakeholders through HCD to improve translational research.

The rise in pediatric obesity and its accompanying condition, type 2 diabetes (T2D), is a serious public health concern. T2D in adolescents is associated with poor health outcomes and decreased life expectancy. Effective diabetes prevention strategies for high-risk adolescents and their families are urgently needed. The aim of this study was to co-design a diabetes prevention program for adolescents by using human-centered design methodologies. We partnered with at-risk adolescents, parents, and professionals with expertise in diabetes prevention or those working with adolescents to conduct a series of human-centered design research sessions to co-design a diabetes prevention intervention for youth and their families. In order to do so, we needed to (1) better understand environmental factors that inhibit/promote recommended lifestyle changes to decrease T2D risk, (2) elucidate desired program characteristics, and (3) explore improved activation in diabetes prevention programs. Financial resources, limited access to healthy foods, safe places for physical activity, and competing priorities pose barriers to adopting lifestyle changes. Adolescents and their parents desire interactive, hands-on learning experiences that incorporate a sense of fun, play, and community in diabetes prevention programs. The findings of this study highlight important insights of 3 specific stakeholder groups regarding diabetes prevention and lifestyle changes. The findings of this study demonstrate that, with appropriate methods and facilitation, adolescents, parents, and professionals can be empowered to co-design diabetes prevention programs.

Adolescents with type 1 diabetes mellitus have difficulty achieving optimal glycemic control, partly due to competing priorities that interfere with diabetes self-care. Often, significant diabetes-related family conflict occurs, and adolescents' thoughts and feelings about diabetes management may be disregarded. Patient-centered diabetes outcomes may be better when adolescents feel engaged in the decision-making process. The objective of our study was to codesign a clinic intervention using shared decision making for addressing diabetes self-care with an adolescent patient and parent advisory board. The patient and parent advisory board consisted of 6 adolescents (teens) between the ages 12 and 18 years with type 1 diabetes mellitus and their parents recruited through our institution's Pediatric Diabetes Program. Teens and parents provided informed consent and participated in 1 or both of 2 patient and parent advisory board sessions, lasting 3 to 4 hours each. Session 1 topics were (1) patient-centered outcomes related to quality of life, parent-teen shared diabetes management, and shared family experiences; and (2) implementation and acceptability of a patient-centered diabetes care plan intervention where shared decision making was used. We analyzed audio recordings, notes, and other materials to identify and extract ideas relevant to the development of a patient-centered diabetes management plan. These data were visually coded into similar themes. We used the information to develop a prototype for a diabetes management plan tool that we pilot tested during session 2. Session 1 identified 6 principal patient-centered quality-of-life measurement domains: stress, fear and worry, mealtime struggles, assumptions and judgments, feeling abnormal, and conflict. We determined 2 objectives to be principally important for a diabetes management plan intervention: (1) focusing the intervention on diabetes distress and conflict resolution strategies, and (2) working toward a verbalized common goal. In session 2, we created the diabetes management plan tool according to these findings and will use it in a clinical trial with the aim of assisting with patient-centered goal setting. Patients with type 1 diabetes mellitus can be effectively engaged and involved in patient-centered research design. Teens with type 1 diabetes mellitus prioritize reducing family conflict and fitting into their social milieu over health outcomes at this time in their lives. It is important to acknowledge this when designing interventions to improve health outcomes in teens with type 1 diabetes mellitus.

Community-acquired methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections affect many healthy children. A significant number of these children are hospitalized and require surgical incision and drainage (I&D). Once sent home, these children and families are asked to complete burdensome home decolonization and hygiene procedures in an effort to prevent the high rate of recurrent infections. This component of the Methicillin-resistant Staphylococcus aureus Eradication and Decolonization in Children (MEDiC) study aimed to develop a toolkit to assist MEDiC study participants in completing MRSA decolonization and hygiene procedures at home (the MEDiC kit). In all, 5 adolescents (aged 10-18 years) who had undergone an I&D procedure for a skin infection and 11 parents of children who had undergone an I&D procedure for a skin infection were engaged in a 4-hour group workshop using a human-centered design approach. The topics covered in this workshop and analyzed for this paper were (1) attitudes about MRSA decolonization procedures and (2) barriers to the implementation of MRSA decolonization and hygiene procedures. The team analyzed the audio and artifacts created during the workshop and synthesized their findings to inform the creation of the MEDiC kit. The workshop activities uncovered barriers to successful completion of the decolonization and hygiene procedures: lack of step-by-step instruction, lack of proper tools in the home, concerns about adverse events, lack of control over some aspects of the hygiene procedures, and general difficulty coordinating all the procedures. Many of these could be addressed as part of the MEDiC kit. In addition, the workshop revealed that effective communication about decolonization would have to address concerns about the effects of bleach, provide detailed information, give reasons for the specific decolonization and hygiene protocol steps, and include step-by-step instructions (preferably through video). Through direct engagement with patients and families, we were able to better understand how to support families in implementing MRSA decolonization and hygiene protocols. In addition, we were able to better understand how to communicate about MRSA decolonization and hygiene protocols. With this knowledge, we created a robust toolkit that uses patient-driven language and visuals to help support patients and families through the implementation of these protocols. ClinicalTrials.gov NCT02127658; https://clinicaltrials.gov/ct2/show/NCT02127658.

Skin and soft tissue infections (SSTIs) due to community-acquired methicillin-resistant Staphylococcus aureus (MRSA) can lead to a number of significant known medical outcomes including hospitalization, surgical procedures such as incision and drainage (I&D), and the need for decolonization procedures to remove the bacteria from the skin and nose and prevent recurrent infection. Little research has been done to understand patient and caregiver-centered outcomes associated with the successful treatment of MRSA infection. This study aimed to uncover MRSA decolonization outcomes that are important to patients and their parents in order to create a set of prototype measures for use in the MRSA Eradication and Decolonization in Children (MEDiC) study. A 4-hour, human-centered design (HCD) workshop was held with 5 adolescents (aged 10-18 years) who had experienced an I&D procedure and 11 parents of children who had experienced an I&D procedure. The workshop explored the patient and family experience with skin infection to uncover patient-centered outcomes of MRSA treatment. The research team analyzed the audio and artifacts created during the workshop and coded for thematic similarity. The final themes represent patient-centered outcome domains to be measured in the MEDiC comparative effectiveness trial. The workshop identified 9 outcomes of importance to patients and their parents: fewer MRSA outbreaks, improved emotional health, improved self-perception, decreased social stigma, increased amount of free time, increased control over free time, fewer days of school or work missed, decreased physical pain and discomfort, and decreased financial burden. This study represents an innovative HCD approach to engaging patients and families with lived experience with MRSA SSTIs in the study design and trial development to determine meaningful patient-centered outcomes. We were able to identify 9 major recurrent themes. These themes were used to develop the primary and secondary outcome measures for MEDiC, a prospectively enrolling comparative effectiveness trial. ClinicalTrials.gov NCT02127658; https://clinicaltrials.gov/ct2/show/NCT02127658.

Background The majority of those aged 65 and older will visit the emergency department (ED) in the last six months of life. Knowing a patient's goals of care is important, and existing medical records do not always represent them well. We set out to determine the baseline availability of advance directives and goals of treatment in those ED patients at increased risk for mortality. Methods This prospective cohort study included a sample of adult ED patients who had a mortality predictor by an End-of-Life (EOL) Deterioration Index-guided electronic best practice advisory (BPA) or admission to any of the network's intensive care units (ICU). Electronic medical record (EMR) abstraction was used to evaluate for documentation of healthcare proxy, healthcare power of attorney (POA), living will, advance care plans, or physician orders for life-sustaining treatment (POLST). Results A total of 9,321 patient encounters, representing 7,204 unique patients, were included in the analyzed sample. Most patients' charts lacked advance care planning documentation such as healthcare proxy (98.7%, N=9200), healthcare POA (93.0%, N=8665), living will (94.6%, N=8816), advance care planning status (66.8%, N=6226), and POLST (95.8%, N=8928). Overall, urban sites had a larger percentage of encounters in which a high-risk patient might benefit from advance care planning discussions than rural sites. Females had a higher percentage of documentation across all variables of goals of care, with significant differences in healthcare POA (p < 0.001), advance directives (p < 0.001), and POLST (p = 0.008). Conclusions The majority of patients with a higher risk for mortality, as indicated by an EOL Deterioration Index-guided BPA or hospital ICU admission, do not have documentation in the EMR across all variables of goals of care.

Long considered to be a static protein cofactor, a growing body of evidence suggests that heme may function as a dynamic signaling molecule. Heme is an essential yet cytotoxic signaling molecule1 . Consequently, cells must regulate heme bioavailability in a manner that allows for essential function while mitigating heme’s toxic effects. Many human diseases including, cancers2 , neurodegenerative disorders3, and cardiovascular diseases4 , stem from defects in heme homeostatic mechanisms. Unfortunately, these mechanisms are poorly understood, in part due to the difficulty of imaging heme within live cells. Genetically encoded ratiometric heme sensors have been developed to probe bioavailable heme and utilized to identify heme trafficking factors and physiological processes that dynamically mobilize subcellular heme pools. These processes include cell catabolic mechanisms; labile heme has been implicated to control the heme catabolic enzymes and the Ubiquitin-Proteasome system.To elucidate heme-based signal transduction, my thesis work focused on: the development of a library of genetically encoded fluorescent heme sensors with a wide range of heme binding affinities, characterizing new targets of heme signaling, and the discovery of a new role for heme oxygenase-2 (HO-2) in regulating heme availability independent of its role in catalyzing heme degradation. The first chapter will cover the importance of heme in cell biology, including its roles as a signaling molecule and in human disease. In the second chapter, I will discuss the development and characterization of a library of heme sensors with differing heme affinities for broader applications. For example, the sensor library can be used to measure heme in a variety of compartments and cell types and distinguish between ferric and ferrous heme species. Chapter Three will cover a newly discovered role for heme in regulating the Ubiquitin-Proteasome system. Specifically, this chapter will focus on heme’s ability to regulate the activity of the yeast E1-ligase Uba1 and regulation of proteasome activity. The fourth chapter is focused on understanding the physiological role of the heme catabolic enzyme HO-2 under conditions in which heme is limiting. To this end, I found that HO-2 acts to bind and buffer heme, under basal conditions, rather than enzymatically degrade it. The final chapter will be a discussion and summary of this work.To elucidate the mechanisms underlying heme trafficking and signaling, the Reddi lab previously developed a pair of genetically encoded heme sensors, a high affinity sensor, HS1, which tightly binds both oxidation states of hem, KDII= 1 pM and KDIII= 3 nM, and a moderate affinity heme sensor, HS1-M7A, which exhibits a KDIIvalue of 25 nM and KDIII= 1 µM.5In yeast, HS1 quantitatively saturates with heme in the cytosol, nucleus, and mitochondria. In contrast, HS1-M7A is ~50% bound in the cytosol and < 30% bound in the nucleus and mitochondrial matrix. Since heme levels vary between different cell types and subcellular compartments, I sought to develop an expanded library of heme sensors with varying affinities for ferric and ferrous heme to measure and image labile heme in many different organisms, cell types, and physiological contexts. After screening 40 variants, results indicate that heme loading of the sensor is under both kinetic and thermodynamic control, and different subcellular locales exhibit microenvironments that favor rapid equilibration of heme with the sensor whereas others do not. In addition, my results thus far have identified at least one heme sensor variant that selectively binds ferrous heme.

Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin and finally elution and identification by mass spectrometry. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in a SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization and vesicular trafficking, many of which have been associated with heme through complimentary studies published recently. Taken together, these results provide support for the emerging role for heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond. Competing Interest Statement The authors have declared no competing interest.

The in vitro formation of stable G-quadruplexes (G4s) in human ribosomal RNA (rRNA) was recently reported. However, their formation in cells and their cellular roles have not been resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest ribosomes are central hubs of heme metabolism. Competing Interest Statement The authors have declared no competing interest.