Neuronal activity-driven O-GlcNAcylation promotes mitochondrial plasticity.

Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-linked N-acetyl glucosamine (O-GlcNAc) transferase regulates neuronal activity-driven mitochondrial bioenergetics in hippocampal and cortical neurons. We show that neuronal activity upregulates O-GlcNAcylation in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven glucose consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.

Relational Work Is the Work: Virtual Healthcare Transformation for Rural, Remote and First Nations Communities in British Columbia.

The healthcare crisis across unceded First Nations' territories in rural, remote and Indigenous communities in British Columbia (BC) is marked by persistent barriers to accessing care and support close to home. This commentary describes an exceptional story of how technology, trusted partnerships and relationships came together to create an innovative suite of virtual care programs called "Real-Time Virtual Support" (RTVS). We describe key approaches, learnings and future considerations to improve the equity of healthcare delivery for rural, remote and First Nations communities. The key lessons include the following: (1) moving beyond a biomedical model - the collaboration framework for health service design incorporated First Nations' perspective on health and wellness; (2) relational work is the work - the RTVS collaboration was grounded in building connections and relationships to prioritize cultivating trust in the partnership over specific outputs; and (3) aligning to the core values of co-creation - working from a commitment to do things differently and applying an inclusive approach of engagement to integrate perspectives across different sectors and interest groups.

Local and dynamic regulation of neuronal glycolysis in vivo.

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here, we adapted a biosensor for glycolysis, HYlight, for use in Caenorhabditis elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons cell-autonomously perform glycolysis and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function and uncovers unique relationships between neuronal identities and metabolic landscapes in vivo.

Stabilisation of the RirA [4Fe-4S] cluster results in loss of iron-sensing function.

RirA is a global iron regulator in diverse Alphaproteobacteria that belongs to the Rrf2 superfamily of transcriptional regulators, which can contain an iron-sulfur (Fe-S) cluster. Under iron-replete conditions, RirA contains a [4Fe-4S] cluster, enabling high-affinity binding to RirA-regulated operator sequences, thereby causing the repression of cellular iron uptake. Under iron deficiency, one of the cluster irons dissociates, generating an unstable [3Fe-4S] form that subsequently degrades to a [2Fe-2S] form and then to apo RirA, resulting in loss of high-affinity DNA-binding. The cluster is coordinated by three conserved cysteine residues and an unknown fourth ligand. Considering the lability of one of the irons and the resulting cluster fragility, we hypothesized that the fourth ligand may not be an amino acid residue. To investigate this, we considered that the introduction of an amino acid residue that could coordinate the cluster might stabilize it. A structural model of RirA, based on the Rrf2 family nitrosative stress response regulator NsrR, highlighted residue 8, an Asn in the RirA sequence, as being appropriately positioned to coordinate the cluster. Substitution of Asn8 with Asp, the equivalent, cluster-coordinating residue of NsrR, or with Cys, resulted in proteins that contained a [4Fe-4S] cluster, with N8D RirA exhibiting spectroscopic properties very similar to NsrR. The variant proteins retained the ability to bind RirA-regulated DNA, and could still act as repressors of RirA-regulated genes in vivo. However, they were significantly more stable than wild-type RirA when exposed to O2 and/or low iron. Importantly, they exhibited reduced capacity to respond to cellular iron levels, even abolished in the case of the N8D version, and thus were no longer iron sensing. This work demonstrates the importance of cluster fragility for the iron-sensing function of RirA, and more broadly, how a single residue substitution can alter cluster coordination and functional properties in the Rrf2 superfamily of regulators.

Local and dynamic regulation of neuronal glycolysis in vivo.

Energy metabolism supports neuronal function. While it is well established that changes in energy metabolism underpin brain plasticity and function, less is known about how individual neurons modulate their metabolic states to meet varying energy demands. This is because most approaches used to examine metabolism in living organisms lack the resolution to visualize energy metabolism within individual circuits, cells, or subcellular regions. Here we adapted a biosensor for glycolysis, HYlight, for use in C. elegans to image dynamic changes in glycolysis within individual neurons and in vivo. We determined that neurons perform glycolysis cell-autonomously, and modulate glycolytic states upon energy stress. By examining glycolysis in specific neurons, we documented a neuronal energy landscape comprising three general observations: 1) glycolytic states in neurons are diverse across individual cell types; 2) for a given condition, glycolytic states within individual neurons are reproducible across animals; and 3) for varying conditions of energy stress, glycolytic states are plastic and adapt to energy demands. Through genetic analyses, we uncovered roles for regulatory enzymes and mitochondrial localization in the cellular and subcellular dynamic regulation of glycolysis. Our study demonstrates the use of a single-cell glycolytic biosensor to examine how energy metabolism is distributed across cells and coupled to dynamic states of neuronal function, and uncovers new relationships between neuronal identities and metabolic landscapes in vivo.

Developing Antiracist Social Work Practice at a Comprehensive Cancer Center.

The combination of the ongoing violence perpetuated against Black, Brown, and Asian people, and the increased incidence of death of Black, Indigenous, people of color (BIPOC) and Asian Americans and Pacific Islanders (AAPI) at the start of the COVID-19 pandemic, elicited an important response from the field of social work across the nation. This article describes the efforts undertaken by a Social Work Department at a comprehensive cancer center in response to a call to develop antiracist practice. This article recounts the process of creating educational opportunities for oncology social workers to help them identify bias and racism in themselves and throughout the healthcare system, to embrace intentional antiracist practice, and to better advocate for BIPOC/AAPI patients and colleagues. The strategies included the development of an antiracism committee, the use of a social location exercise to influence and disrupt white supremacy, the creation of community guidelines for engaging in conversations about race, and the formulation of a new departmental policy ensuring a commitment to antiracist social work practice. In addition, a forum using multimedia was created to explore racial dynamics and to highlight the narratives of BIPOC and AAPI people. Further, a monthly Antiracist Clinical Case Conference was implemented to explore their role in the context of working with the interdisciplinary team in an oncology setting. This article concludes with recommendations for ongoing antiracist social work practice development that may be applied in various healthcare settings.

Neuronal activity-driven O-GlcNAcylation promotes mitochondrial plasticity.

Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-GlcNAc transferase regulates neuronal activity-driven mitochondrial bioenergetics. We show that neuronal activity upregulates O-GlcNAcylation mainly in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven fuel consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.

A Prescription for Internet: Feasibility of a Tablet Loaner Program to Address Digital Health Inequities.

OBJECTIVE: The coronavirus disease 2019 pandemic accelerated the adoption of telehealth technologies. Persistent disparities in telecommunication devices, internet connectivity, and digital literacy, however, undermine the potential for telemedicine to reduce barriers to health care access. Health systems may have a role in addressing these structural inequities. We describe the operationalization and feasibility of an internet-enabled tablet loaner program at a freestanding children's hospital. METHODS: Between October 2020 and October 2021, pediatricians enrolled families through ambulatory clinics at an academic urban freestanding children's hospital. Eligibility criteria included difficulty accessing virtual care due to lack of stable internet or device. Tablets featured an unlimited data package, access to the patient portal, and virtual visit platform. A private technology company managed device configuration and distribution. To characterize program impact, we compared the proportion of completed clinical encounters during the intervention compared with a preintervention period (March 2020-October 2020) and conducted a qualitative survey with program participants. Participant and visit characteristics were obtained from the electronic medical record and summarized with descriptive statistics. RESULTS: A total of 111 families participated in the tablet loaner program, the majority of whom were Hispanic (51.4%) and black, non-Hispanic (26.1%), and publicly insured (64.9%). Between the preintervention and intervention periods, there was a significant increase in completed video- and phone-based virtual visits (75.3 vs. 79.1%, p = 0.038). The proportion of video-based only visits increased from 82.9 to 88.9%. p < 0.001. Families reported that the tablet improved the patient's ability to receive medical care (93.7%) and was easy to use (93.9%). CONCLUSION: The tablet loaner initiative was associated with an improvement in markers of virtual visit engagement and health care experience. Efforts to expand telemedicine equity must consider technological access and digital literacy as well as broad coalitions across industry, government, and community organizations.

Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate.

Cellular metabolism is regulated over space and time to ensure that energy production is efficiently matched with consumption. Fluorescent biosensors are useful tools for studying metabolism as they enable real-time detection of metabolite abundance with single-cell resolution. For monitoring glycolysis, the intermediate fructose 1,6-bisphosphate (FBP) is a particularly informative signal as its concentration is strongly correlated with flux through the whole pathway. Using GFP insertion into the ligand-binding domain of the Bacillus subtilis transcriptional regulator CggR, we developed a fluorescent biosensor for FBP termed HYlight. We demonstrate that HYlight can reliably report the real-time dynamics of glycolysis in living cells and tissues, driven by various metabolic or pharmacological perturbations, alone or in combination with other physiologically relevant signals. Using this sensor, we uncovered previously unknown aspects of ß-cell glycolytic heterogeneity and dynamics.

COVID-19 and Beyond: A Prototype for Remote/Virtual Social Work Field Placement.

The COVID-19 pandemic necessitated an abrupt conclusion of field placement for social work interns at a comprehensive cancer center. In response to social distancing requirements, social workers, but not interns, were granted access to work remotely. Virtual programming became necessary to meet the interns' remaining educational requirements and provided an opportunity for proper termination from the program. This article will delineate the program redesign for oncology social work interns using remote/virtual modalities. This melded approach involved creating simulated clinical interactions, based on selected points along the illness trajectory targeting specific clinical competencies, which were presented to interns by phone and/or videoconference. Examples will be provided related to developing clinical skills and critical thinking and preparing for professional responsibilities within a broad range of healthcare settings. Guidelines for working with individuals, couples/families, and groups will be included. Issues of individual and group supervision will be explored, with sensitivity to the parallel experience of existential uncertainty and mortality awareness among the interns in the context of the pandemic. Although in-person training is preferable, there are advantages to virtual learning for both supervisors and interns. This creative adaptation of field education provides an innovative programming model that can be used to enhance the experience for social work interns moving forward in various healthcare settings during ordinary or extraordinary circumstances.

A Sort-Seq Approach to the Development of Single Fluorescent Protein Biosensors.

Motivated by the growing importance of single fluorescent protein biosensors (SFPBs) in biological research and the difficulty in rationally engineering these tools, we sought to increase the rate at which SFPB designs can be optimized. SFPBs generally consist of three components: a circularly permuted fluorescent protein, a ligand-binding domain, and linkers connecting the two domains. In the absence of predictive methods for biosensor engineering, most designs combining these three components will fail to produce allosteric coupling between ligand binding and fluorescence emission. While methods to construct diverse libraries with variation in the site of GFP insertion and linker sequences have been developed, the remaining bottleneck is the ability to test these libraries for functional biosensors. We address this challenge by applying a massively parallel assay termed "sort-seq," which combines binned fluorescence-activated cell sorting, next-generation sequencing, and maximum likelihood estimation to quantify the brightness and dynamic range for many biosensor variants in parallel. We applied this method to two common biosensor optimization tasks: the choice of insertion site and optimization of linker sequences. The sort-seq assay applied to a maltose-binding protein domain-insertion library not only identified previously described high-dynamic-range variants but also discovered new functional insertion sites with diverse properties. A sort-seq assay performed on a pyruvate biosensor linker library expressed in mammalian cell culture identified linker variants with substantially improved dynamic range. Machine learning models trained on the resulting data can predict dynamic range from linker sequences. This high-throughput approach will accelerate the design and optimization of SFPBs, expanding the biosensor toolbox.

Assessment of local supply chains and stock management practices for trauma care resources in Ghana: a comparative small sample cross-sectional study.

BACKGROUND: Injuries are a major public health problem globally. With sound planning and organization, essential trauma care can be reliably provided with relatively low-cost equipment and supplies. However, availability of these resources requires an effective and efficient supply chain and good stock management practices. Therefore, this study aimed to assess trauma care resource-related supply management structures and processes at health facilities in Ghana. By doing so, the findings may allow us to identify specific structures and processes that could be improved to facilitate higher quality and more timely care. METHODS: Ten hospitals were purposively selected using results from a previously performed national trauma care capacity assessment of hospitals of all levels in Ghana. Five hospitals with low resource availability and 5 hospitals with high resource availability were assessed using the United States Agency for International Development (USAID) Logistics Indicators Assessment Tool and stock ledger review. Data were described and stock management practices were correlated with resource availability. RESULTS: There were differences in stock management practices between low and high resource availability hospitals, including frequency of reporting and audit, number of stock-outs on day of assessment (median 9 vs 2 stock-outs, range 3-57 vs 0-9 stock-outs, respectively; p = 0.05), duration of stock-outs (median 171 vs 8 days, range 51-1268 vs 0-182 days, respectively; p = 0.02), and fewer of up-to-date stock cards (24 vs 31 up-to-date stock cards, respectively; p = 0.07). Stock-outs were common even among low-cost, essential resources (e.g., nasal cannulas and oxygen masks, endotracheal tubes, syringes, sutures, sterile gloves). Increased adherence to stock management guidelines and higher percentage of up-to-date stock cards were correlated with higher trauma resource availability scores. However, the variance in trauma resource availability scores was poorly explained by these individual factors or when analyzed in a multivariate regression model (r2 = 0.72; p value for each covariate between 0.17-0.34). CONCLUSIONS: Good supply chain and stock management practices are correlated with high trauma care resource availability. The findings from this study demonstrate several opportunities to improve stock management practices, particularly at low resource availability hospitals.

Anatomy of Gun Violence: Contextualized Curriculum to Train Surgical Residents in Both Technical and Non-Technical Skills in the Management of Gun Violence.

BACKGROUND: Gun violence (GV) is a complex public health issue, and the management of GV as a disease engages the surgeon in technical and nontechnical skills. The Anatomy of Gun Violence (AGV) curriculum was developed to teach surgical trainees these seemingly disparate skills, training residents to manage the multiple aspects of firearm injury. STUDY DESIGN: The AGV curriculum was delivered over 6 weeks in the 2017-2018 and 2018-2019 academic years (AY), and used multiple educational methods including didactic lectures, mock oral examinations, a Bleeding Control training session, a GV survivor's personal story, a Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) training session, and the Surgery for Abdominal-thoracic ViolencE (SAVE) simulation lab. As surgical residents were involved over both AYs, components of the curriculum were available every other year to provide variety. As proof of concept, this novel curriculum was objectively evaluated by residents' improvement in knowledge and overall experience using pre- and post-surveys. RESULTS: Sixty surgical residents participated in the AGV curriculum in both AYs, with 41 and 36 residents completing the survey regarding their experiences with the curriculum. The curriculum was well received by residents overall in both AYs (median ± IQR 5 ± 0 and 5 ± 0.1, respectively), with the SAVE simulation lab being the most highly favored portion. Additionally, residents had an average 7.5% improvement in knowledge attributed to the curriculum, with a larger effect seen in the junior residents. CONCLUSIONS: This novel AGV curriculum created a well-received learning experience involving the technical and nontechnical skills necessary to care for GV victims. This comprehensive approach to GV may represent a unique opportunity to engage surgical trainees in both the treatment and prevention of firearm injury.

Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster.

The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae (SvWhiD), which differs from S. coelicolor WhiD (ScWhiD) only at the C terminus. We observed that, like ScWhiD and other Wbl proteins, SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that SvWhiD exists in a monomer-dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB4). Using MS, we determined the dissociation constant (Kd ) for the SvWhiD-σHrdB4 complex as ∼0.7 µm, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8-10 NO molecules per cluster, resulted in dissociation into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB4, consistent with protection of the cluster in the complex.

Mechanisms of iron- and O2-sensing by the [4Fe-4S] cluster of the global iron regulator RirA.

RirA is a global regulator of iron homeostasis in Rhizobium and related α-proteobacteria. In its [4Fe-4S] cluster-bound form it represses iron uptake by binding to IRO Box sequences upstream of RirA-regulated genes. Under low iron and/or aerobic conditions, [4Fe-4S] RirA undergoes cluster conversion/degradation to apo-RirA, which can no longer bind IRO Box sequences. Here, we apply time-resolved mass spectrometry and electron paramagnetic resonance spectroscopy to determine how the RirA cluster senses iron and O2. The data indicate that the key iron-sensing step is the O2-independent, reversible dissociation of Fe2+ from [4Fe-4S]2+ to form [3Fe-4S]0. The dissociation constant for this process was determined as Kd = ~3 µM, which is consistent with the sensing of 'free' iron in the cytoplasm. O2-sensing occurs through enhanced cluster degradation under aerobic conditions, via O2-mediated oxidation of the [3Fe-4S]0 intermediate to form [3Fe-4S]1+. This work provides a detailed mechanistic/functional view of an iron-responsive regulator.

Food in Focus: Youth Exploring Food in Schools Using Photovoice.

OBJECTIVE: As part of a study exploring school food environments, this study aimed to understand youth perspectives of school food. DESIGN: Photovoice, a qualitative visual methodology, was used to engage participants through photo-taking, with goals of enabling reflection, promoting dialogue, and facilitating change. SETTING: Participants were recruited through 2 youth-focused community organizations in Nova Scotia, Canada. PARTICIPANTS: Seven youths took part: 3 from a rural area and 4 from an urban center. PHENOMENON OF INTEREST: Youth perspectives on school food environments. ANALYSIS: The photovoice process of selecting, contextualizing (using the SHOWeD method), and codifying was used for analysis. RESULTS: Four themes were identified. First, spaces and places were important to youth food experiences. Second, key components of food environments were identified as quality, variety, time, and price. Third, the relation between food and social influence was highlighted. Fourth, the importance of amplifying youth voice was discussed. CONCLUSIONS AND IMPLICATIONS: Youth emphasized a desire for greater variety and quality in affordable school food options and the opportunity to be involved in decision-making regarding school food. Future research in other contexts and across larger samples is warranted to extend these findings to help inform stakeholders in school food policy and program implementation.

Understanding System-Level Intervention Points to Support School Food and Nutrition Policy Implementation in Nova Scotia, Canada.

Supporting the implementation of school food and nutrition policies (SFNPs) is an international priority to encourage healthier eating among children and youth. Such policies can improve equitable access, resources, and supports for healthy eating. However, despite the potential impact of SFNPs, several implementation barriers have been reported. This study sought to examine the system-level intervention points within a school food system using a complex systems framework. We conducted semi-structured interviews with various stakeholders working to influence the school food system in Nova Scotia, Canada. We sought to understand their roles and experiences with the SFNP by applying the Intervention Level Framework (ILF), a novel, solutions-oriented approach to better understand how complex systems function. Participants (n = 33) included teachers, parents, cafeteria workers, public health staff and non-profit organizations. Interview transcripts were first coded, then themed and finally analyzed using the ILF, resulting in three intervention points within the school food system. These were defined as: (1) Actors and Elements, (2) System Regulation and Interconnections and (3) Purpose and Values. We concluded that understanding the interactions between these system levels and stakeholder roles can help to inform the development of relevant policy strategies that better support healthier school food environments in this jurisdiction.