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Abstract The lasting threat of viral pandemics necessitates the development of tailorable first-response antivirals with specific but adaptive architectures for treatment of novel viral infections. Here, such an antiviral platform has been developed based on a mixture of hetero-peptides self-assembled into functionalized β-sheets capable of specific multivalent binding to viral protein complexes. One domain of each hetero-peptide is designed to specifically bind to certain viral proteins, while another domain self-assembles into fibrils with epitope binding characteristics determined by the types of peptides and their molar fractions. The self-assembled fibrils maintain enhanced binding to viral protein complexes and retain high resilience to viral mutations. This method is experimentally and computationally tested using short peptides that specifically bind to Spike proteins of SARS-CoV-2. This platform is efficacious, inexpensive, and stable with excellent tolerability.

pyXPCSviewer, a Python-based graphical user interface that is deployed at beamline 8-ID-I of the Advanced Photon Source for interactive visualization of XPCS results, is introduced. pyXPCSviewer parses rich X-ray photon correlation spectroscopy (XPCS) results into independent PyQt widgets that are both interactive and easy to maintain. pyXPCSviewer is open-source and is open to customization by the XPCS community for ingestion of diversified data structures and inclusion of novel XPCS techniques, both of which are growing demands particularly with the dawn of near-diffraction-limited synchrotron sources and their dedicated XPCS beamlines.

Abstract Background In the post-GWAS era, there is an unmet need to decode the underpinning genetic etiologies of late-onset Alzheimer’s disease (LOAD) and translate the associations to causation. Methods We conducted ATAC-seq profiling using NeuN sorted-nuclei from 40 frozen brain tissues to determine LOAD-specific changes in chromatin accessibility landscape in a cell-type specific manner. Results We identified 211 LOAD-specific differential chromatin accessibility sites in neuronal-nuclei, four of which overlapped with LOAD-GWAS regions (±100 kb of SNP). While the non-neuronal nuclei did not show LOAD-specific differences, stratification by sex identified 842 LOAD-specific chromatin accessibility sites in females. Seven of these sex-dependent sites in the non-neuronal samples overlapped LOAD-GWAS regions including APOE. LOAD loci were functionally validated using single-nuclei RNA-seq datasets. Conclusions Using brain sorted-nuclei enabled the identification of sex-dependent cell type-specific LOAD alterations in chromatin structure. These findings enhance the interpretation of LOAD-GWAS discoveries, provide potential pathomechanisms, and suggest novel LOAD-loci. Graphical Abstract

Neurophysiological changes that involve activity-dependent neuroplasticity mechanisms via repeated stimulation and locomotor training are not commonly employed in research even though combination of interventions is a common clinical practice. In this randomized clinical trial, we established neurophysiological changes when transcranial magnetic stimulation (TMS) of the motor cortex was paired with transcutaneous thoracolumbar spinal (transspinal) stimulation in human spinal cord injury (SCI) delivered during locomotor training. We hypothesized that TMS delivered before transspinal (TMS-transspinal) stimulation promotes functional reorganization of spinal networks during stepping. In this protocol, TMS-induced corticospinal volleys arrive at the spinal cord at a sufficient time to interact with transspinal stimulation induced depolarization of alpha motoneurons over multiple spinal segments. We further hypothesized that TMS delivered after transspinal (transspinal-TMS) stimulation induces less pronounced effects. In this protocol, transspinal stimulation is delivered at time that allows transspinal stimulation induced action potentials to arrive at the motor cortex and affect descending motor volleys at the site of their origin. Fourteen individuals with motor incomplete and complete SCI participated in at least 25 sessions. Both stimulation protocols were delivered during the stance phase of the less impaired leg. Each training session consisted of 240 paired stimuli delivered over 10-min blocks. In transspinal-TMS, the left soleus H-reflex increased during the stance-phase and the right soleus H-reflex decreased at mid-swing. In TMS-transspinal no significant changes were found. When soleus H-reflexes were grouped based on the TMS-targeted limb, transspinal-TMS and locomotor training promoted H-reflex depression at swing phase, while TMS-transspinal and locomotor training resulted in facilitation of the soleus H-reflex at stance phase of the step cycle. Furthermore, both transspinal-TMS and TMS-transspinal paired-associative stimulation (PAS) and locomotor training promoted a more physiological modulation of motor activity and thus depolarization of motoneurons during assisted stepping. Our findings support that targeted non-invasive stimulation of corticospinal and spinal neuronal pathways coupled with locomotor training produce neurophysiological changes beneficial to stepping in humans with varying deficits of sensorimotor function after SCI.

The flavivirus nonstructural protein 1 (NS1) is secreted from infected cells and contributes to endothelial barrier dysfunction and vascular leak in a tissue-dependent manner. This phenomenon occurs in part via disruption of the endothelial glycocalyx layer (EGL) lining the endothelium. Additionally, we and others have shown that soluble DENV NS1 induces disassembly of intercellular junctions (IJCs), a group of cellular proteins critical for maintaining endothelial homeostasis and regulating vascular permeability; however, the specific mechanisms by which NS1 mediates IJC disruption remain unclear. Here, we investigated the relative contribution of five flavivirus NS1 proteins, from dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses, to the expression and localization of the intercellular junction proteins β-catenin and VE-cadherin in endothelial cells from human umbilical vein and brain tissues. We found that flavivirus NS1 induced the mislocalization of β-catenin and VE-cadherin in a tissue-dependent manner, reflecting flavivirus disease tropism. Mechanistically, we observed that NS1 treatment of cells triggered internalization of VE-cadherin, likely via clathrin-mediated endocytosis, and phosphorylation of β-catenin, part of a canonical IJC remodeling pathway during breakdown of endothelial barriers that activates glycogen synthase kinase-3β (GSK-3β). Supporting this model, we found that a chemical inhibitor of GSK-3β reduced both NS1-induced permeability of human umbilical vein and brain microvascular endothelial cell monolayers in vitro and vascular leakage in a mouse dorsal intradermal model. These findings provide insight into the molecular mechanisms regulating NS1-mediated endothelial dysfunction and identify GSK-3β as a potential therapeutic target for treatment of vascular leakage during severe dengue disease.

Emerging literature suggests that dietary lutein may have important functions in cognitive health, but there is not enough data to substantiate its effects in human cognition. The current study was intended to determine the overall effect of lutein on the main domains of cognition in the adult population based on available placebo randomized-controlled trials. Literature searches were conducted in PubMed, AGRICOLA, Scopus, MEDLINE, and EMBASE on 14 November 2020. The effect of lutein on complex attention, executive function and memory domains of cognition were assessed by using an inverse-variance meta-analysis of standardized mean differences (SMD) (Hedge’s g method). Dietary lutein was associated with slight improvements in cognitive performance in complex attention (SMD 0.02, 95% CI −0.27 to 0.31), executive function (SMD 0.13, 95% CI −0.26 to 0.51) and memory (SMD 0.03, 95% CI −0.26 to 0.32), but its effect was not significant. Change-from-baseline analysis revealed that lutein consumption could have a role in maintaining cognitive performance in memory and executive function. Although dietary lutein did not significantly improve cognitive performance, the evidence across multiple studies suggests that lutein may nonetheless prevent cognitive decline, especially executive function. More intervention studies are needed to validate the role of lutein in preventing cognitive decline and in promoting brain health.

Malakoplakia is a rare granulomatous disease. Its aetiology is unclear but possible theories include infection with microorganisms (especially Escherichia coli), immunosuppression and impaired lysosomal function. It has been commonly documented to affect the genitourinary tract but can affect any organ, with the gastrointestinal system being the next most affected. We present a woman in her 70s, with a 2-week history of right-sided abdominal pain, 13 years following her renal transplant. She was admitted for treatment of an E. coli bacteraemia. CT scan had shown a caecal pole mass, highly suspicious for malignancy. It was surgically resected, and histology revealed findings of malakoplakia within the colon. Surgical intervention was combined with a prolonged course of antibiotics for successful treatment. We highlight the ability of malakoplakia to mimic malignancy and should be considered in the differentials in the context of an immunosuppressed patient with radiological findings of a colonic mass. [ABSTRACT FROM AUTHOR]

Abstract Background Historically, ankle fractures have been treated with open reduction and internal fixation (ORIF) procedures, which are considered safe and effective. Patient characteristics may contribute to postoperative difficulties thereby increasing risk of hospital readmission. The objective of this study was to determine the frequency of and reasons for 30-day readmission and postoperative complications following ORIF for ankle fractures. Methods A retrospective review of the National Surgical Quality Improvement Program (NSQIP) database from 2015 to 2021 identified patients undergoing ORIF for ankle fractures. Patient demographics, complication incidence, and reasons for unplanned hospital readmission were collected. Multivariable analyses identified patient risk factors for any adverse event (AAE) and readmission within 30-days of surgery. Results The 29,905 patients queried who underwent ORIF procedures for ankle fractures between 2015 and 2021 were 49.6 ± 18.40 years of age, 30.9 ± 7.10 kg/m2, and 40.81% male. Of this cohort, 981 (3.30%) experienced 30-day postoperative adverse events, with surgical site infections (SSI; 1.25%) the most common. Unplanned readmission was observed in 2.08% of patients after a mean of 14.64 days. Surgical site related readmissions were 20.55% (n = 128) of reported readmissions with the most common being superficial incisional SSI. Notable risk factors for adverse events included ASA class (OR = 1.579, P

Abstract Introduction Real-world data on ixekizumab utilization in axial spondyloarthritis (axSpA) are limited. We evaluated ixekizumab treatment patterns and health care resource utilization (HCRU) in patients with axSpA using United States Merative L.P. MarketScan® Claims Databases. Methods This retrospective cohort study included adults with axSpA who initiated ixekizumab during the index period (September 2019–December 2021). Index date was the date of the first ixekizumab claim. All patients had continuous medical and pharmacy enrollment during the 12-month pre-index and follow-up periods. Descriptive statistics were used to assess patient demographics (index date); clinical characteristics (pre-index period); treatment patterns (12-month follow-up period); and HCRU (pre-index and 12-month follow-up periods). Results The study included 177 patients (mean age 45.8 years; females 54.8%) with axSpA who initiated ixekizumab. Overall, 79.1% of patients reported prior biologic use; of these, 70.7% received tumor necrosis factor-alpha inhibitors (TNFi) and 49% received secukinumab. The mean (standard deviation [SD]) Charlson Comorbidity Index score was 1.1 (1.3) and ~ 27% of patients reported ≥2 comorbidities. The median (inter-quartile range [IQR]) number of ixekizumab prescription refills was 7 (4–11). The mean (SD) Proportion of Days Covered (PDC) for ixekizumab was 0.6 (0.3) and adherence (PDC ≥80%) was 34.5% (N = 61). Overall, 26.6% (N = 47) of patients switched to a non-index medication and 54.2% (N = 96) of patients discontinued ixekizumab. Among the patients who discontinued ixekizumab (N = 96), 19.8% (N = 19) restarted ixekizumab and 49.0% (N = 47) switched to a non-index medication. The median (IQR) ixekizumab persistence was 268 (120–366) days. Mean axSpA-related outpatient, inpatient, and emergency room visits were similar between the pre-index and follow-up periods. Treatment patterns were largely similar between biologic-experienced patients (N = 140; 79.1%) and the overall population. Conclusions Despite high comorbidity burden and majority of the patients being biologic-experienced, patients initiating ixekizumab for axSpA showed favorable persistence profiles during the 12-month follow-up period.

Background: Spina bifida cystica (SB) is one of the most common neural tube defects affecting 1 in 2,500 newborns worldwide. Due to the neurological deficits associated with SB, injuries from thermal exposure, trauma, or other etiology may be more severe rendering their treatment a challenge for a traditional approach to burn and wound management. Despite this relatively high disease incidence rate, few studies exist to evaluate wound development or the natural history of wound healing in patients with SB. Case presentation: We present a case of a pediatric patient with a history of SB who sustained a full thickness burn to the right plantar foot in February of 2023. The 8-year-old was riding an all-terrain vehicle when his right foot made contact with the hot tailpipe. The insensate neuropathy associated with the patient’s SB contributed to the extreme severity of thermal contact injury. We report the burn, treatments, the usage of a novel dermal replacement, and outcomes of this case. Conclusion: This case demonstrates successful limb salvage in a pediatric patient with severe plantar foot burns utilizing a novel collagen-elastin dermal matrix for tissue reconstruction. Limb salvage was the preferred treatment option over amputation and a prosthetic considering the patient’s age and need for future prostheses. Given the severity of the burn, the impaired wound healing due to his baseline neuropathy, and his baseline ambulatory status, the optimal outcome in this case was limb salvage, which was successfully achieved.

Abstract Background Sepsis is a life-threatening condition which may arise from infection in any organ system and requires early recognition and management. Healthcare professionals working in any specialty may need to manage patients with sepsis. Educating medical students about this condition may be an effective way to ensure all future doctors have sufficient ability to diagnose and treat septic patients. However, there is currently no consensus on what competencies medical students should achieve regarding sepsis recognition and treatment. This study aims to outline what sepsis-related competencies medical students should achieve by the end of their medical student training in both high or upper-middle incomes countries/regions and in low or lower-middle income countries/regions. Methods Two separate panels from high or upper-middle income and low or lower-middle income countries/regions participated in a Delphi method to suggest and rank sepsis competencies for medical students. Each panel consisted of 13–18 key stakeholders of medical education and doctors in specialties where sepsis is a common problem (both specialists and trainees). Panelists came from all continents, except Antarctica. Results The panels reached consensus on 38 essential sepsis competencies in low or lower-middle income countries/regions and 33 in high or upper-middle incomes countries/regions. These include competencies such as definition of sepsis and septic shock and urgency of antibiotic treatment. In the low or lower-middle income countries/regions group, consensus was also achieved for competencies ranked as very important, and was achieved in 4/5 competencies rated as moderately important. In the high or upper-middle incomes countries/regions group, consensus was achieved in 41/57 competencies rated as very important but only 6/11 competencies rated as moderately important. Conclusion Medical schools should consider developing curricula to address essential competencies, as a minimum, but also consider addressing competencies rated as very or moderately important.

IntroductionThe present two-wave longitudinal study examined the relation between White U.S. parents' color-conscious racial socialization for African Americans and adolescents' racial attitudes, and explored how parental psychological control might moderate this relation.MethodParticipants included 412 White U.S. adolescents (42% girls; Mage = 15.63 years, SD = 1.24) and their primary caregivers (52% mothers). They completed online questionnaires, with parents reporting on their color-conscious racial socialization at Time 1, and adolescents providing measures of their perception of parents' psychological control at Time 1, color-blind attitudes, and racial prejudice at both time points, ~16 months apart.ResultsOur findings revealed that parents' color-conscious racial socialization for African Americans was negatively associated with adolescents' negative racial attitudes. Additionally, parental psychological control moderated this relation: the negative association was evident only when parents exhibited low or average levels of psychological control.DiscussionThis study highlights the crucial role parents play in shaping their adolescents' racial attitudes and underscores the necessity of an autonomy-supportive environment to enhance the success of parents' color-conscious socialization practices. Understanding these dynamics is essential for developing effective interventions to reduce racial biases and foster inclusive attitudes among adolescents.

Abstract Introduction The objective of this analysis is to evaluate the improvement in spinal pain with ixekizumab, placebo, and adalimumab based on objective measures of inflammation response in patients with ankylosing spondylitis (AS). Methods The COAST-V 52-week, double-blind, placebo-controlled, randomized phase III trial examined the efficacy of ixekizumab in patients with active AS; adalimumab was used as an active reference arm. Treatment effects on reduction in pain were assessed by objective measures of controlled and persisting inflammation (defined by magnetic resonance imaging [MRI], C-reactive protein [CRP], or MRI + CRP status). Pathway analysis was used to analyze treatment effect that was not attributable to reduction in inflammation biomarkers. Results In patients with AS, when inflammation was controlled as assessed by MRI, patients treated with ixekizumab experienced a reduction in spinal pain at night (SP-N, numeric rating scale, ixekizumab mean = − 3.9, p

Abstract Winter cover crop performance metrics (i.e., vegetative biomass quantity and quality) affect ecosystem services provisions, but they vary widely due to differences in agronomic practices, soil properties, and climate. Cereal rye (Secale cereale) is the most common winter cover crop in the United States due to its winter hardiness, low seed cost, and high biomass production. We compiled data on cereal rye winter cover crop performance metrics, agronomic practices, and soil properties across the eastern half of the United States. The dataset includes a total of 5,695 cereal rye biomass observations across 208 site-years between 2001–2022 and encompasses a wide range of agronomic, soils, and climate conditions. Cereal rye biomass values had a mean of 3,428 kg ha−1, a median of 2,458 kg ha−1, and a standard deviation of 3,163 kg ha−1. The data can be used for empirical analyses, to calibrate, validate, and evaluate process-based models, and to develop decision support tools for management and policy decisions.

Abstract Purpose The present study aimed at assessing the prevalences of post-traumatic stress disorder (PTSD) (main objective), anxiety, depression, and burnout syndrome (BOS) and their associated factors in intensive care unit (ICU) staff workers in the second year of the COVID-19 pandemic. Materials and methods An international cross-sectional multicenter ICU-based online survey was carried out among the ICU staff workers in 20 ICUs across 3 continents. ICUs staff workers (both caregivers and non-caregivers) were invited to complete PCL-5, HADS, and MBI questionnaires for assessing PTSD, anxiety, depression, and the different components of BOS, respectively. A personal questionnaire was used to isolate independent associated factors with these disorders. Results PCL-5, HADS, and MBI questionnaires were completed by 585, 570, and 539 responders, respectively (525 completed all questionnaires). PTSD was diagnosed in 98/585 responders (16.8%). Changing familial environment, being a non-caregiver staff worker, having not being involved in a COVID-19 patient admission, having not been provided with COVID-19-related information were associated with PTSD. Anxiety was reported in 130/570 responders (22.8%). Working in a public hospital, being a woman, being financially impacted, being a non-clinical healthcare staff member, having no theoretical or practical training on individual preventive measures, and fear of managing COVID-19 patients were associated with anxiety. Depression was reported in 50/570 responders (8.8%). Comorbidity at risk of severe COVID-19, working in a public hospital, looking after a child, being a non-caregiver staff member, having no information, and a request for moving from the unit were associated with depression. Having received no information and no adequate training for COVID-19 patient management were associated with all 3 dimensions of BOS. Conclusion The present study confirmed that ICU staff workers, whether they treated COVID-19 patients or not, have a substantial prevalence of psychological disorders.

Background: The AbSeS-classification defines specific phenotypes of patients with intra-abdominal infection based on the (1) setting of infection onset (community-acquired, early onset, or late-onset hospital-acquired), (2) presence or absence of either localized or diffuse peritonitis, and (3) severity of disease expression (infection, sepsis, or septic shock). This classification system demonstrated reliable risk stratification in intensive care unit (ICU) patients with intra-abdominal infection. This study aimed to describe the epidemiology of ICU patients with pancreatic infection and assess the relationship between the components of the AbSeS-classification and mortality. Methods: This was a secondary analysis of an international observational study (“AbSeS”) investigating ICU patients with intra-abdominal infection. Only patients with pancreatic infection were included in this analysis (n=165). Mortality was defined as ICU mortality within 28 days of observation for patients discharged earlier from the ICU. Relationships with mortality were assessed using logistic regression analysis and reported as odds ratio (OR) and 95% confidence interval (CI). Results: The overall mortality was 35.2% (n=58). The independent risk factors for mortality included older age (OR=1.03, 95% CI: 1.0 to 1.1 P=0.023), localized peritonitis (OR=4.4, 95% CI: 1.4 to 13.9 P=0.011), and persistent signs of inflammation at day 7 (OR=9.5, 95% CI: 3.8 to 23.9, P

Abstract Background Direct assessment of microcirculatory function remains a critical care research tool but approaches for analysis of microcirculatory videomicroscopy clips are shifting from manual to automated algorithms, with a view to clinical application in the intensive care unit. Automated analysis software associated with current sidestream darkfield videomicroscopy systems is demonstrably unreliable; therefore, semi-automated analysis of captured clips should be undertaken in older generations of software. We present a method for capture of microcirculatory clips using current version videomicroscope hardware and resizing of clips to allow compatibility with legacy analysis software. The interobserver reliability of this novel approach is examined, in addition to a comparison of this approach with the current generation of automated analysis software. Results Resizing microcirculatory clips did not significantly change image quality. Assessment of bias between observers for manual analysis of resized clips; and between manually analysed clips and automated software analysis was undertaken by Bland–Altman analysis. Bias was demonstrated for all parameters for manual analysis of resized clips (total vessel density = 6.8, perfused vessel density = 6.3, proportion of perfused vessels = − 8.79, microvascular flow index = − 0.08). Marked bias between manual analysis and automated analysis was also evident (total vessel density = 16.6, perfused vessel density = 16.0, proportion of perfused vessels = 1.8). The difference between manual and automated analysis was linearly related to the magnitude of the measured parameter. Conclusions Poor reliability of automated analysis is a significant hurdle for clinical translation of microcirculatory monitoring. The method presented here allows capture of microcirculatory clips using current hardware that are backwards compatible with older versions of manual analysis software. We conclude that this approach is appropriate for research applications in the intensive care unit, however the time delay to results limits utility for clinical translation.

Peatland plays an important role not just as a carbon store but also in facilitating the flux of greenhouse gasses into the atmosphere. Apart from that, peatland is also home to a diverse population of microorganisms such as bacteria, fungi, and actinomycetes. Actinomycetes were known to be one of the most ubiquitous microbes that can be found in most of the soil types including peat soil. In this study, seventy isolates of actinomycetes were isolated from the peat soil using the soil dilution method. The 70 isolates of actinomycetes were later screened for their ability to produce secondary metabolites and antimicrobial activities using the agar diffusion method before the selected potential isolates were identified by targeting their 16S rRNA region. The results obtained showed 34.3% produce cellulase followed by, 12.8, 31.7, 80.0, and 51.4% for mannanase, xylanase, lipase, and protease respectively. The percentage of actinomycetes producing antimicrobial activity was 27.1 and 21.4% for Ralstonia solanacearum and Colletotrichum gleosporioides respectively. All the selected isolates of actinomycetes were identified as belonging to the genus of Streptomycetes spp. The potential actinomycetes were stored in freeze-dried form for future usage. This study showed that more diverse population of actinomycetes was obtained from the undisturbed forested peat soil area ecosystem compared to the agricultural peat soil area.

Waterborne transmission of the bacterium Legionella pneumophila has emerged as a major cause of severe nosocomial infections of major public health impact. The major route of transmission involves the uptake of aerosolized bacteria, often from the contaminated hot water systems of large buildings. Public health regulations aimed at controlling the mesophilic pathogen are generally concerned with acute pasteurization and maintaining high temperatures at the heating systems and throughout the plumbing of hot water systems, but L. pneumophila is often able to survive these treatments due to both bacterium-intrinsic and environmental factors. Previous work has established an experimental evolution system to model the observations of increased heat resistance in repeatedly but unsuccessfully pasteurized L. pneumophila populations. Here, we show rapid fixation of novel alleles in lineages selected for resistance to heat shock and shifts in mutational profile related to increases in the temperature of selection. Gene-level and nucleotide-level parallelisms between independently-evolving lineages show the centrality of the DnaJ/DnaK chaperone system in the heat resistance of L. pneumophila. Inference of epistatic interactions through reverse genetics shows an unexpected interaction between DnaJ/DnaK and the polyhydroxybutyrate-accumulation energy storage mechanism used by the species to survive long-term starvation in low-nutrient environments.

Ossicular pathology is a recognized etiology of conductive hearing loss. Ossicular pathology includes 2 main categories, that is, ossicular chain fixation and ossicular discontinuity. Ossicular discontinuity can be congenital or acquired. Auto-incudotomy is an uncommon form of acquired ossicular discontinuity that usually occurs as a sequel of spontaneous expulsion of cholesteatoma. Typically, it manifests with conductive hearing loss without evidence of cholesteatoma. In this report, we presented CT imaging finding of a 34-year-old male with tympanic membrane perforation and defective long process of the incus (auto-incudotomy) with minimal middle ear granulation tissue and adhesions, sequela of cholesteatoma. Radiologists should pay attention for evaluation of ossicles especially in patients presented with conductive hearing loss.

This paper synthesizes insights from a workshop on fleet electrification at the 2023 Transportation Research Board Annual Meeting, which included participants from transit agencies, national labs, industry, government and academia. Participants identified barriers to fleet electrification including lack of knowledge on fleet electrification, high utility demand charges, lack of charging infrastructure, delays in grid infrastructure upgrades and high up-front costs. To overcome these challenges, panelists emphasized partnering with OEMs and utilities, and having flexible charging infrastructure with software support tools. Finally, participants identified opportunities for integrating with electricity markets on peak demand management, time-of-use charging, participation in wholesale markets, and various vehicle-to-grid solutions. Based on these findings, we propose specific steps that various stakeholders like fleet operators, utilities and regulatory authorities could take.