Single-cell analysis of human MAIT cell transcriptional, functional and clonal diversity.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize microbial metabolites through a semi-invariant T cell receptor (TCR). Major questions remain regarding the extent of human MAIT cell functional and clonal diversity. To address these, we analyzed the single-cell transcriptome and TCR repertoire of blood and liver MAIT cells and developed functional RNA-sequencing, a method to integrate function and TCR clonotype at single-cell resolution. MAIT cell clonal diversity was comparable to conventional memory T cells, with private TCR repertoires shared across matched tissues. Baseline functional diversity was low and largely related to tissue site. MAIT cells showed stimulus-specific transcriptional responses in vitro, with cells positioned along gradients of activation. Clonal identity influenced resting and activated transcriptional profiles but intriguingly was not associated with the capacity to produce IL-17. Overall, MAIT cells show phenotypic and functional diversity according to tissue localization, stimulation environment and clonotype.

Predation without direction selectivity.

Across the animal kingdom, visual predation relies on motion-sensing neurons in the superior colliculus (SC) and its orthologs. These neurons exhibit complex stimulus preferences, including direction selectivity, which is thought to be critical for tracking the unpredictable escape routes of prey. The source of direction selectivity in the SC is contested, and its contributions to predation have not been tested experimentally. Here, we use type-specific cell removal to show that narrow-field (NF) neurons in the mouse SC guide predation. In vivo recordings demonstrate that direction-selective responses of NF cells are independent of recently reported stimulus-edge effects. Monosynaptic retrograde tracing reveals that NF cells receive synaptic input from direction-selective ganglion cells. When we eliminate direction selectivity in the retina of adult mice, direction-selective responses in the SC, including in NF cells, are lost. However, eliminating retinal direction selectivity does not affect the hunting success or strategies of mice, even when direction selectivity is removed after mice have learned to hunt, and despite abolishing the gaze-stabilizing optokinetic reflex. Thus, our results identify the retinal source of direction selectivity in the SC. They show that NF cells in the SC guide predation, an essential spatial orienting task, independent of their direction selectivity, revealing behavioral multiplexing of complex neural feature preferences and highlighting the importance of feature-selective manipulations for neuroethology.

Quality assurance programs for vitamin A and E in serum: are we doing enough to assess laboratory performance?

OBJECTIVES: Monitoring serum vitamin A (retinol) and vitamin E (α-tocopherol) concentrations is common practice for assessing nutritional status. Measurement of these vitamins can be challenging due to several factors. Whilst the RCPAQAP Vitamins: Serum Program assists participating laboratories in harmonisation, the materials provided do not contain the analogues of retinol and α-tocopherol that may be present in real patient samples. We aimed to assess participants' capacity to accurately report retinol and α-tocopherol in the presence of the vitamin E analogues tocopherol acetate and γ-tocopherol. METHODS: A supplementary series of a control sample and three matched spiked samples were distributed to each laboratory participating in the Program. Retinol and α-tocopherol results for each spiked sample were compared to the results of the control sample submitted by each participant. Acceptability of retinol and α-tocopherol results was determined based on the RCPAQAP allowable performance specifications (APS). RESULTS: Thirteen participants returned results for the supplementary sample series. Interference from α-tocopherol acetate was observed with results below the APS in 30 % (n=4) of laboratories for retinol quantification and in 23 % (n=3) for α-tocopherol quantification. One laboratory returned results above the APS for α-tocopherol when γ-tocopherol was present. CONCLUSIONS: This supplementary sample series has shown that the presence of vitamin E analogues can lead to the over or under estimation of nutritional status by some participants. Affected laboratories are encouraged to review their analytical procedures. To further assess laboratory competence, EQA providers should consider using patient samples or spiked challenge samples.

Text Messages to Improve Medication Adherence and Secondary Prevention After Acute Coronary Syndrome: The TEXTMEDS Randomized Clinical Trial.

BACKGROUND: TEXTMEDS (Text Messages to Improve Medication Adherence and Secondary Prevention After Acute Coronary Syndrome) examined the effects of text message-delivered cardiac education and support on medication adherence after an acute coronary syndrome. METHODS: TEXTMEDS was a single-blind, multicenter, randomized controlled trial of patients after acute coronary syndrome. The control group received usual care (secondary prevention as determined by the treating clinician); the intervention group also received multiple motivational and supportive weekly text messages on medications and healthy lifestyle with the opportunity for 2-way communication (text or telephone). The primary end point of self-reported medication adherence was the percentage of patients who were adherent, defined as >80% adherence to each of up to 5 indicated cardioprotective medications, at both 6 and 12 months. RESULTS: A total of 1424 patients (mean age, 58 years [SD, 11]; 79% male) were randomized from 18 Australian public teaching hospitals. There was no significant difference in the primary end point of self-reported medication adherence between the intervention and control groups (relative risk, 0.93 [95% CI, 0.84-1.03]; P=0.15). There was no difference between intervention and control groups at 12 months in adherence to individual medications (aspirin, 96% vs 96%; ß-blocker, 84% vs 84%; angiotensin-converting enzyme inhibitor/angiotensin receptor blocker, 77% vs 80%; statin, 95% vs 95%; second antiplatelet, 84% vs 84% [all P>0.05]), systolic blood pressure (130 vs 129 mm Hg; P=0.26), low-density lipoprotein cholesterol (2.0 vs 1.9 mmol/L; P=0.34), smoking (P=0.59), or exercising regularly (71% vs 68%; P=0.52). There were small differences in lifestyle risk factors in favor of intervention on body mass index <25 kg/m2 (21% vs 18%; P=0.01), eating ≥5 servings per day of vegetables (9% vs 5%; P=0.03), and eating ≥2 servings per day of fruit (44% vs 39%; P=0.01). CONCLUSIONS: A text message-based program had no effect on medical adherence but small effects on lifestyle risk factors. REGISTRATION: URL: https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=364448; Unique identifier: ANZCTR ACTRN12613000793718.

Adenovirus vectors activate Vδ2+ γδT cells in a type I interferon-, TNF-, and IL-18-dependent manner.

Vδ2+ γδT cells are unconventional T cells that can be activated by cytokines without TCR signaling. Adenovirus vaccine vectors activated Vδ2+ γδT cells in an interleukin 18-, TNF-, and type I interferon-dependent manner. This stimulatory capacity was associated with adenovirus vectors of non-species C origin, including the ChAdOx1 vaccine platform.

A liquid chromatography tandem mass spectrometry method for plasma glucagon.

Glucagon is a peptide involved in controlling the body's blood glucose levels. The majority of analytical methods used for its quantitation are based on immunoassays that suffer from cross-reactivity with other peptides. For accurate routine analysis a liquid chromatography tandem mass spectrometry (LC-MSMS) was developed. Glucagon was extracted from plasma samples by a combination of protein precipitation using ethanol and mixed anion exchange solid phase extraction. Linearity for glucagon was above 0.99 (r2) up to a concentration range of 771 ng/L with a lower limit of quantification established at 19 ng/L. Precision of the method was below 9% (coefficient of variation). Recovery was 93%. Correlations with the existing immunoassay displayed a significant negative bias.

Integrated psychological care services within seizure settings: Key components and implementation factors among example services in four ILAE regions: A report by the ILAE Psychiatry Commission.

Mental health comorbidities are prevalent and problematic in patients with seizures but often suboptimally managed. To address common gaps in care, the Integrated Mental Health Care Pathways Task Force of the International League Against Epilepsy (ILAE) Psychiatry Commission was tasked with providing education and guidance on the integration of mental health management (e.g., screening, referral, treatment) into routine seizure care. This report aims to describe a variety of established services in this area, with a specific focus on psychological care models. Services were identified by members of the ILAE Psychiatry Commission and authors of psychological intervention trials in epilepsy. A total of eight services met inclusion criteria and agreed to be showcased. They include three pediatric and five adult services located across four distinct ILAE regions (Europe, North America, Africa, Asia Oceania). The report describes the core operations, known outcomes, and implementation factors (i.e., barriers and facilitators) of these services. The report concludes with a set of practical tips for building successful psychological care services within seizure settings, including the importance of having local champions, clearly defining the scope of the service, and establishing sustainable funding models. The breadth of exemplars demonstrates how models tailored to the local environment and resources can be implemented. This report is an initial step to disseminate information regarding integrated mental health care within seizure care settings. Future work is needed to systematically examine both psychological and pharmacological care models and to further establish the evidence base in this area, especially around clinical impact, and cost-effectiveness.

Interferon-Gamma-Producing CD8+ Tissue Resident Memory T Cells Are a Targetable Hallmark of Immune Checkpoint Inhibitor-Colitis.

BACKGROUND & AIMS: The pathogenesis of immune checkpoint inhibitor (ICI)-colitis remains incompletely understood. We sought to identify key cellular drivers of ICI-colitis and their similarities to idiopathic ulcerative colitis, and to determine potential novel therapeutic targets. METHODS: We used a cross-sectional approach to study patients with ICI-colitis, those receiving ICI without the development of colitis, idiopathic ulcerative colitis, and healthy controls. A subset of patients with ICI-colitis were studied longitudinally. We applied a range of methods, including multiparameter and spectral flow cytometry, spectral immunofluorescence microscopy, targeted gene panels, and bulk and single-cell RNA sequencing. RESULTS: We demonstrate CD8+ tissue resident memory T (TRM) cells are the dominant activated T cell subset in ICI-colitis. The pattern of gastrointestinal immunopathology is distinct from ulcerative colitis at both the immune and epithelial-signaling levels. CD8+ TRM cell activation correlates with clinical and endoscopic ICI-colitis severity. Single-cell RNA sequencing analysis confirms activated CD8+ TRM cells express high levels of transcripts for checkpoint inhibitors and interferon-gamma in ICI-colitis. We demonstrate similar findings in both anti-CTLA-4/PD-1 combination therapy and in anti-PD-1 inhibitor-associated colitis. On the basis of our data, we successfully targeted this pathway in a patient with refractory ICI-colitis, using the JAK inhibitor tofacitinib. CONCLUSIONS: Interferon gamma-producing CD8+ TRM cells are a pathological hallmark of ICI-colitis and a novel target for therapy.

Classification of intestinal T-cell receptor repertoires using machine learning methods can identify patients with coeliac disease regardless of dietary gluten status.

In coeliac disease (CeD), immune-mediated small intestinal damage is precipitated by gluten, leading to variable symptoms and complications, occasionally including aggressive T-cell lymphoma. Diagnosis, based primarily on histopathological examination of duodenal biopsies, is confounded by poor concordance between pathologists and minimal histological abnormality if insufficient gluten is consumed. CeD pathogenesis involves both CD4+ T-cell-mediated gluten recognition and CD8+ and γδ T-cell-mediated inflammation, with a previous study demonstrating a permanent change in γδ T-cell populations in CeD. We leveraged this understanding and explored the diagnostic utility of bulk T-cell receptor (TCR) sequencing in assessing duodenal biopsies in CeD. Genomic DNA extracted from duodenal biopsies underwent sequencing for TCR-δ (TRD) (CeD, n = 11; non-CeD, n = 11) and TCR-γ (TRG) (CeD, n = 33; non-CeD, n = 21). We developed a novel machine learning-based analysis of the TCR repertoire, clustering samples by diagnosis. Leave-one-out cross-validation (LOOCV) was performed to validate the classification algorithm. Using TRD repertoire, 100% (22/22) of duodenal biopsies were correctly classified, with a LOOCV accuracy of 91%. Using TCR-γ (TRG) repertoire, 94.4% (51/54) of duodenal biopsies were correctly classified, with LOOCV of 87%. Duodenal biopsy TRG repertoire analysis permitted accurate classification of biopsies from patients with CeD following a strict gluten-free diet for at least 6 months, who would be misclassified by current tests. This result reflects permanent changes to the duodenal γδ TCR repertoire in CeD, even in the absence of gluten consumption. Our method could complement or replace histopathological diagnosis in CeD and might have particular clinical utility in the diagnostic testing of patients unable to tolerate dietary gluten, and for assessing duodenal biopsies with equivocal features. This approach is generalisable to any TCR/BCR locus and any sequencing platform, with potential to predict diagnosis or prognosis in conditions mediated or modulated by the adaptive immune response. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Civilian walking blood bank emergency preparedness plan.

BACKGROUND: The current global pandemic has created unprecedented challenges in the blood supply network. Given the recent shortages, there must be a civilian plan for massively bleeding patients when there are no blood products on the shelf. Recognizing that the time to death in bleeding patients is less than 2 h, timely resupply from unaffected locations is not possible. One solution is to transfuse emergency untested whole blood (EUWB), similar to the extensive military experience fine-tuned over the last 19 years. While this concept is anathema in current civilian transfusion practice, it seems prudent to have a vetted plan in place. METHODS AND MATERIALS: During the early stages of the 2020 global pandemic, a multidisciplinary and international group of clinicians with broad experience in transfusion medicine communicated routinely. The result is a planning document that provides both background information and a high-level guide on how to emergently deliver EUWB for patients who would otherwise die of hemorrhage. RESULTS AND CONCLUSIONS: Similar plans have been utilized in remote locations, both on the battlefield and in civilian practice. The proposed recommendations are designed to provide high-level guidance for experienced blood bankers, transfusion experts, clinicians, and health authorities. Like with all emergency preparedness, it is always better to have a well-thought-out and trained plan in place, rather than trying to develop a hasty plan in the midst of a disaster. We need to prevent the potential for empty shelves and bleeding patients dying for lack of blood.

Streamlined three step total vitamin C analysis by HILIC-UV for laboratory testing.

OBJECTIVES: In the clinical setting, the analysis and quantification of vitamin C (ascorbic acid) poses several challenges including analyte instability and poor retention by reverse phase HPLC systems. In this article we describe a rapid hydrophilic interaction chromatography ultraviolet method for the measurement of total vitamin C in plasma which overcomes these issues. METHODS: Ascorbic acid and the internal standard were separated under isocratic conditions using a Waters BEH-Amide column and a mobile phase containing 0.005 M potassium phosphate in 80% acetonitrile. RESULTS: The proposed method was validated and showed good precision (coefficient of variation <5%), accuracy (>99%), and analyte stability after extraction (>24 h). CONCLUSIONS: The simple sample preparation allows full automation and rapid analytical run times of the assay and is therefore suitable for a high-throughput clinical chromatography laboratory.

Incidence of Undiagnosed Celiac Disease Presenting as Bone Stress Injuries to a Sport and Exercise Medicine Clinic.

OBJECTIVE: To evaluate the incidence of undiagnosed celiac disease (CD) in patients presenting with bone stress injuries (BSI) to a NHS Sport and Exercise Medicine (SEM) clinic. DESIGN: Retrospective cohort study. SETTING: Single tertiary-level SEM clinic. PATIENT/PARTICIPANTS: One hundred consecutive patients with radiologically proven BSIs. INTERVENTIONS: Laboratory blood tests (LBT) can unmask underlying metabolic bone disorders. Anti-tissue transglutaminase antibody (TTG) testing has a high sensitivity and specificity for CD. In this SEM clinic, clinicians were encouraged to perform LBT including TTG, at time of diagnosis of BSI. A retrospective analysis of age, sex, fracture site, co-morbidities, TTG result, and subsequent investigations was performed. MAIN OUTCOME MEASURES: The primary outcome was the number and percentage of patients with BSIs and either positive TTG (CD seropositivity) or a diagnosis of CD. RESULTS: Of the 100 patients with radiologically proven BSIs, 70% were female, and the mean age was 37 years (range 16-69). Eighty-five percent had the appropriate LBTs, of which 70% (60/85) were female, and the mean age was 37(16-69). Metatarsal (35%) and tibial (21%) were the most common BSIs. Anti-tissue transglutaminase antibody was performed in 85 patients. Two patients (2/85) had pre-existing CD and were excluded from incidence calculations. Five patients [5/83 (6%), mean age 38 years (28-57), 80% female] had a positive TTG, of whom 3 have subsequently had CD confirmed by endoscopic biopsy. Four patients with a positive TTG underwent dual-energy X-ray absorptiometry with osteopenia found in 3 (75%) cases. CONCLUSIONS: In this cohort, the incidence of CD seropositivity was 6%, and the prevalence of biopsy-confirmed CD was 5%, approximately 5-fold higher than UK population estimates. Anti-tissue transglutaminase antibody screening for CD should be considered in all patients presenting with BSIs.

An Educational Experiment Resulting from COVID-19: The Use of At-Home Waxing and Webinars for Teaching a 3-Week Intensive Course in Tooth Morphology to First Year Dental Students.

PURPOSE: To report the outcomes of presenting a 3-week intensive course in tooth morphology to first year dental students using "at-home" waxing projects and webinars. MATERIALS AND METHODS: Students were provided with the instrumentation and materials required to complete 5 waxing projects at home during the 3-week course. In the same time period, the didactic content was presented via 11 webinar sessions. A postcourse survey provided student perspectives regarding this new experience. RESULTS: Students were able to effectively complete high-quality waxing projects at home by using step-by-step images and videos but the survey indicated an overwhelming preference for in-person faculty feedback. Webinars based on the students having studied the 3D Tooth Atlas and an instructor reviewing content in the Atlas was effective in teaching the didactic aspect of tooth morphology as evidenced by the student grades and survey results. However, most of the students indicated a preference for physically going to class and being able to interact with faculty and classmates as opposed to online webinars. The condensed 3-week version of the course was part of an expanded 4-week student orientation and worked well, allowing other clinical precursor courses to occur before their usual time in the curriculum, thereby allowing students to begin earlier patient treatment. CONCLUSIONS: The at-home waxing exercises produced very good results by having the students use step-by-step images and videos in the 3D Tooth Atlas. However, the students indicated a strong preference for personal faculty feedback that was not available at home. Students effectively learned the didactic aspects of tooth morphology through the webinars with accompanying use of the 3D Tooth Atlas but again most students prefer physically going to class and being able to interact with faculty and classmates. These preferences for contact with classmates and faculty supports the natural human desire for personal interactions with other human beings.

An innovation procurement clinical framework: A qualitative study.

Innovation Procurement Strategies (IPS) strive for purchasing healthcare solutions that do not yet exist on the market and are increasingly being advocated to improve health outcomes while managing escalating healthcare costs. Due to the newness of IPS, there are limited resources available to healthcare organizations and professionals looking to engage in IPS. The purpose of this study was to develop an evidence-based clinical framework to guide healthcare organizations and professionals. Adopting a qualitative grounded theory approach, we interviewed participants with experience in innovation procurement to understand the skills, resources, and supports needed to initiate and oversee an IPS project. Using thematic design and open coding, three overarching themes emerged from the data and formed the basis of our IPS clinical framework. By describing the components, skills, and supports and resources necessary for engaging in IPS, our framework addresses the knowledge gap in healthcare organizations and professionals wishing to implement IPS.

An automated workflow approach for the analysis of flavin adenine dinucleotide by HPLC with internal standard.

Flavin adenine dinucleotide (FAD) is an active coenzyme of vitamin B2 involved in oxidation and reduction reactions. In this study we have developed a fully automated method for the analysis of FAD by fluorescence detection. FAD is extracted from whole blood samples by trichloroacetic acid, with diethyl ribityl isoalloxazine used as an internal standard. Linearity for FAD was above 0.99 (r2) up to a concentration range of 1000 nmol/L. Precision of the method (intra-day and inter-day) compared against commercial quality control material was below 8% (coefficient of variation) with recovery of FAD exceeding 90%. Accuracy of the protocol was compared against a previous cycle from an external quality assurance program (RCPAQAP) (n = 12) with satisfactory agreement. Overall, this method has increased laboratory workflow and reduced manual labour required in performing FAD analysis.

Omics AnalySIs System for PRecision Oncology (OASISPRO): a web-based omics analysis tool for clinical phenotype prediction.

SUMMARY: Precision oncology is an approach that accounts for individual differences to guide cancer management. Omics signatures have been shown to predict clinical traits for cancer patients. However, the vast amount of omics information poses an informatics challenge in systematically identifying patterns associated with health outcomes, and no general purpose data mining tool exists for physicians, medical researchers and citizen scientists without significant training in programming and bioinformatics. To bridge this gap, we built the Omics AnalySIs System for PRecision Oncology (OASISPRO), a web-based system to mine the quantitative omics information from The Cancer Genome Atlas (TCGA). This system effectively visualizes patients' clinical profiles, executes machine-learning algorithms of choice on the omics data and evaluates the prediction performance using held-out test sets. With this tool, we successfully identified genes strongly associated with tumor stage, and accurately predicted patients' survival outcomes in many cancer types, including adrenocortical carcinoma. By identifying the links between omics and clinical phenotypes, this system will facilitate omics studies on precision cancer medicine and contribute to establishing personalized cancer treatment plans. AVAILABILITY AND IMPLEMENTATION: This web-based tool is available at http://tinyurl.com/oasispro; source codes are available at http://tinyurl.com/oasisproSourceCode. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Evaluation of a lyophilized platelet-derived hemostatic product.

BACKGROUND: Current limitations of platelet shelf life to 5 days have led to an increasingly greater demand for hemostatic agents with greater longevity. The objective of this study was to evaluate the function of a lyophilized platelet-derived hemostatic product (thrombosome [TS]) as a potential alternative to fresh platelets. METHODS: Platelets were collected from whole blood from healthy donors. TSs were reconstituted with water and added to various configurations of reassembled whole blood (platelets, plasma, and RBCs); measures included rotational thromboelastometry (ROTEM), optical aggregometry, mitochondrial function, calibrated automated thrombogram, collagen adhesion under flow (shear flow assay), and flow cytometry. RESULTS: In ROTEM, no differences were observed between maximum clot formation values for contact pathway activation thromboelastometry tests with TSs or platelet samples. Significantly decreased aggregation was observed in the TSs versus platelets (p < 0.001 for all agonists). Flow cytometry measures demonstrated significant decreases in glycoprotein Ib expression and increases in phosphatidylserine expression in the TS group (p < 0.01). The calibrated automated thrombogram assay was suggestive (lag time and peak thrombin) that the TSs might have some thrombogenic properties. Measurements of mitochondrial function revealed that TSs had no functional mitochondria. CONCLUSION: In this study, TSs were shown to have nonfunctional mitochondria. ROTEM measures revealed that the TSs had no impact on clot strength. Likewise, compared to platelets, the TSs displayed minimal aggregation, had significantly more phosphatidylserine (measure of activation status), but had the ability to adhere to a collagen surface under flow conditions and contribute to clot formation and induced greater thrombin generation.

Should phosphatidylethanol be currently analysed using whole blood, dried blood spots or both?

Phosphatidylethanol (PEth) are phospholipids produced through non-oxidative ethanol metabolism. They accumulate in red blood cells and have been traditionally analysed in whole blood as potential biomarkers for moderate to long-term alcohol consumption. More recently, their analysis in dried blood spots has been gaining favour, namely, due to the ease in sampling, transport and storage conditions required. This paper aims at providing a short comparative review between analysing PEth in whole blood and dried blood spots and the potential pitfalls that researchers may face when setting up PEth testing for clinical use.

Differential Presynaptic ATP Supply for Basal and High-Demand Transmission.

The relative contributions of glycolysis and oxidative phosphorylation to neuronal presynaptic energy demands are unclear. In rat hippocampal neurons, ATP production by either glycolysis or oxidative phosphorylation alone sustained basal evoked synaptic transmission for up to 20 min. However, combined inhibition of both ATP sources abolished evoked transmission. Neither action potential propagation failure nor depressed Ca2+ influx explained loss of evoked synaptic transmission. Rather, inhibition of ATP synthesis caused massive spontaneous vesicle exocytosis, followed by arrested endocytosis, accounting for the disappearance of evoked postsynaptic currents. In contrast to its weak effects on basal transmission, inhibition of oxidative phosphorylation alone depressed recovery from vesicle depletion. Local astrocytic lactate shuttling was not required. Instead, either ambient monocarboxylates or neuronal glycolysis was sufficient to supply requisite substrate. In summary, basal transmission can be sustained by glycolysis, but strong presynaptic demands are met preferentially by oxidative phosphorylation, which can be maintained by bulk but not local monocarboxylates or by neuronal glycolysis.SIGNIFICANCE STATEMENT Neuronal energy levels are critical for proper CNS function, but the relative roles for the two main sources of ATP production, glycolysis and oxidative phosphorylation, in fueling presynaptic function in unclear. Either glycolysis or oxidative phosphorylation can fuel low-frequency synaptic function and inhibiting both underlies loss of synaptic transmission via massive vesicle release and subsequent failure to endocytose lost vesicles. Oxidative phosphorylation, fueled by either glycolysis or endogenously released monocarboxylates, can fuel more metabolically demanding tasks such as vesicle recovery after depletion. Our work demonstrates the flexible nature of fueling presynaptic function to maintain synaptic function.