Shared decision making for perioperative antibiotic use during Mohs micrographic surgery on the lower extremities.

Background: While there is a higher risk of surgical site infection (SSI) on the lower extremities following Mohs micrographic surgery (MMS), antibiotic prophylaxis (AP) is debated. Objective: To determine the role of shared decision making (SDM) in guiding AP usage during MMS on the lower extremities. Materials and methods: A prospective observational study was conducted whereby patients received a standardized SDM discussion or routine counseling. Patient satisfaction quantified by the shared decision-making questionnaire (SDMQ9) survey, rate of SSI, and rate of AP prescription were recorded. Results: In total, 51 patients were included. While there were less antibiotics prescribed in the treatment group (20% versus 50%, P = .025), this did not affect incidence of SSI (8% in treatment group versus 7.7% in control group, P = .668). Patient satisfaction was statistically greater in SDM group (4.73 versus 2.18 in control (P < .001). Conclusion: Patient satisfaction scores were higher among the patients who received SDM. While the usage of AP was lower in the SDM group, this did not affect incidence of SSI. This study allows the opportunity to apply SDM in the setting of MMS, which to our knowledge has not yet been attempted in the field of dermatologic surgery.

A Multiomic Analysis to Identify Drivers of Subclinical Vascular Disease in Systemic Lupus Erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) increases cardiovascular disease (CVD) risk, and this is not explained by traditional risk factors. Characterization of blood immunologic signatures that associate with subclinical CVD and predict its progression has been challenging and may help identify subgroups at risk. METHODS: Patients with SLE (n = 77) and healthy controls (HCs) (n = 27) underwent assessments of arterial stiffness, vascular wall inflammation, and coronary atherosclerosis burden with cardio-ankle vascular index (CAVI); fluorodeoxyglucose-positron emission tomography/computed tomography (CT) (target-to-background ratio [TBR]); and coronary CT angiography. Whole blood bulk RNA sequencing was performed in a subset of study participants (HC n = 10, SLE n = 20). In a partially overlapping subset (HC n = 24, SLE n = 64), serum inflammatory protein biomarkers were quantified with an Olink platform. RESULTS: CAVI, TBR, and noncalcified coronary plaque burden (NCB) were increased in patients with SLE compared to HCs. When comparing patients with SLE with high CAVI scores to those with low CAVI scores or to HCs, there was a down-regulation of genes in pathways involved in the cell cycle and differentially regulated pathways related to metabolism. Distinct serum proteins associated with increased CAVI (CCL23, colony-stimulating factor 1, latency-activating peptide transforming growth factor ß1, interleukin 33 [IL-33], CD8A, and IL-12B), NCB (monocyte chemotactic protein 4 and FMS-like tyrosine kinase 3 ligand [Flt3L]), and TBR (CD5, IL-1α, AXIN1, cystatin D [CST5], and tumor necrosis factor receptor superfamily 9; P < 0.05). CONCLUSION: Blood gene expression patterns and serum proteins that associate with worse vascular phenotypes suggest dysregulated immune and metabolic pathways linked to premature CVD. Cytokines and chemokines identified in associations with arterial stiffness, inflammation, and NCB in SLE may allow for characterization of new CVD biomarkers in lupus.

Analysis of PM2.5, black carbon, and trace metals measurements from the Kansas City Transportation and Local-Scale Air Quality Study (KC-TRAQS).

Communities near transportation sources can be impacted by higher concentrations of particulate matter (PM) and other air pollutants. Few studies have reported on air quality in complex urban environments with multiple transportation sources. To better understand these environments, the Kansas City Transportation and Local-Scale Air Quality Study (KC-TRAQS) was conducted in three neighborhoods in Southeast Kansas City, Kansas. This area has several emissions sources including transportation (railyards, vehicles, diesel trucks), light industry, commercial facilities, and residential areas. Stationary samples were collected for 1-year (October 24, 2017, to October 31, 2018) at six sites using traditional sampling methods and lower-cost air sensor packages. This work examines PM less than 2.5 µm in diameter (PM2.5), black carbon (BC), and trace metals data collected during KC-TRAQS. PM2.5 filter samples showed the highest 24-h mean concentrations (9.34 µg/m3) at the sites located within 20-50 m of the railyard. Mean 24-h PM2.5 concentrations, ranging from 7.96 to 9.34 µg/m3, at all sites were lower than that of the nearby regulatory site (9.83 µg/m3). Daily maximum PM2.5 concentrations were higher at the KC-TRAQS sites (ranging from 25.31 to 43.76 µg/m3) compared to the regulatory site (20.50 µg/m3), suggesting short-duration impacts of localized emissions sources. Across the KC-TRAQS sites, 24-h averaged PM2.5 concentrations from the sensor package (P-POD) ranged from 3.24 to 5.69 µg/m3 showing that, out-of-the-box, the PM sensor underestimated the reference concentrations. KC-TRAQS was supplemented by elemental and organic carbon (EC/OC) and trace metal analysis of filter samples. The EC/OC data suggested the presence of secondary organic aerosol formation, with the highest mean concentrations observed at the site within 20 m of the railyard. Trace metals data showed daily, monthly, and seasonal variations for iron, copper, zinc, chromium, and nickel, with elevated concentrations occurring during the summer at most of the sites.Implications: This work reports on findings from a year-long air quality study in Southeast Kansas City, Kansas to understand micro-scale air quality in neighborhoods impacted by multiple emissions sources such as transportation sources (including a large railyard operation), light industry, commercial facilities, and residential areas. While dozens of studies have reported on air quality near roadways, this work will provide more information on PM2.5, black carbon, and trace metals concentrations near other transportation sources in particular railyards. This work can also inform additional field studies near railyards.

Pinch Grafting: A Systematic Review of Modern Perspectives and Applications in Dermatologic Surgery and Wound Healing.

BACKGROUND: Pinch grafting has experienced a resurgence in interest in recent years, stemming from its simplicity, safety, and potential in restoring tissue integrity. While historically employed for chronic nonhealing wounds, pinch grafts have shown promise following surgical procedures, particularly those involving the lower extremities. OBJECTIVE: To systematically review the literature and present an updated overview of the current applications of pinch grafting. METHODS: A systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. In collaboration with a medical reference librarian, the PubMed, Embase, Scopus, and Web of Science databases were searched for studies reporting on the use of pinch grafting from 2000 onward. The references of each included article were also screened. RESULTS: Ten articles met final inclusion criteria. In total, 300 patients underwent pinch grafting for treatment of skin ulceration, while an additional 35 cases were performed as an alternative to primary closure following skin cancer resection. Overall, pinch grafting was safe and well tolerated, with minimal adverse outcomes reported. CONCLUSION: Pinch grafting is a safe, straightforward, and effective technique to promote the healing of chronic wounds. While the procedure shows early promise in emerging applications within dermatologic surgery, only about 10% of the reported cases involved this indication, reflecting a need for further research in this area.

Miniature Optical Fiber Fabry-Perot Interferometer Based on a Single-Crystal Metal-Organic Framework for the Detection and Quantification of Benzene and Ethanol at Low Concentrations in Nitrogen Gas.

This study reports for the first time, to the best of our knowledge, a real-time detection of ultralow-concentration chemical gases using fiber-optic technology, combining a miniaturized Fabry-Perot interferometer (FPI) with metal-organic frameworks (MOFs). The sensor consists of a short and thick-walled silica capillary segment spliced to a lead-in single-mode fiber (SMF), housing a tiny single crystal of HKUST-1 MOF, imparting chemoselectivity features. Ethanol and benzene gases were tested, resulting in a shift in the FPI interference signal. The sensor demonstrated high sensitivity, detecting ethanol gas concentrations (EGCs) with a sensitivity of 0.428 nm/ppm between 24.9 and 40.11 ppm and benzene gas concentrations (BGCs) with a sensitivity of 0.15 nm/ppm between 99 and 124 ppm. The selectivity study involved a combination of three ultralow concentrations of ethanol, benzene, and toluene gases, revealing an enhancement factor of 436% for benzene and 140% for toluene, attributed to the improved miscibility of these conjugated ring molecules with the alkane chains of the ethanol-modified HKUST-1. Experimental tests confirmed the sensor's viability, demonstrating significantly improved response time and spectral characteristics through crystal polishing, indicating its potential for quantifying and detecting chemical gases at ultralow concentrations. This technology may prevent energy resource losses, and the sensor's small size and robust construction make it applicable in confined and hazardous locations.

Circulating T cell specific extracellular vesicle profiles in cardiac allograft acute cellular rejection.

There is a critical need for biomarkers of acute cellular rejection (ACR) in organ transplantation. We hypothesized that ACR leads to changes in donor-reactive T cell small extracellular vesicle (sEV) profiles in transplant recipient circulation that match the kinetics of alloreactive T cell activation. In rodent heart transplantation, circulating T cell sEV quantities (P < .0001) and their protein and mRNA cargoes showed time-specific expression of alloreactive and regulatory markers heralding early ACR in allogeneic transplant recipients but not in syngeneic transplant recipients. Next generation sequencing of their microRNA cargoes identified novel candidate biomarkers of ACR, which were validated by stem loop quantitative reverse transcription polymerase chain reaction (n = 10). Circulating T cell sEVs enriched from allogeneic transplant recipients mediated targeted cytotoxicity of donor cardiomyocytes by apoptosis assay (P < .0001). Translation of the concept and EV methodologies to clinical heart transplantation demonstrated similar upregulation of circulating T cell sEV profiles at time points of grade 2 ACR (n = 3 patients). Furthermore, T cell receptor sequencing of T cell sEV mRNA cargo demonstrated expression of T cell clones with intact complementarity determining region 3 signals. These data support the diagnostic potential of T cell sEVs as noninvasive biomarker of ACR and suggest their potential functional roles.

Intraoperative margin assessment for basal cell carcinoma with deep learning and histologic tumor mapping to surgical site.

Successful treatment of solid cancers relies on complete surgical excision of the tumor either for definitive treatment or before adjuvant therapy. Intraoperative and postoperative radial sectioning, the most common form of margin assessment, can lead to incomplete excision and increase the risk of recurrence and repeat procedures. Mohs Micrographic Surgery is associated with complete removal of basal cell and squamous cell carcinoma through real-time margin assessment of 100% of the peripheral and deep margins. Real-time assessment in many tumor types is constrained by tissue size, complexity, and specimen processing / assessment time during general anesthesia. We developed an artificial intelligence platform to reduce the tissue preprocessing and histological assessment time through automated grossing recommendations, mapping and orientation of tumor to the surgical specimen. Using basal cell carcinoma as a model system, results demonstrate that this approach can address surgical laboratory efficiency bottlenecks for rapid and complete intraoperative margin assessment.

Validation of a robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of VK-2019, a selective EBNA1 inhibitor.

EBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV-associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK-2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV-associated carcinomas. A sensitive and reliable method was developed to quantify VK-2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK-2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK-2019, two purported metabolites, and the internal standard, VK-2019-d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK-2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5-500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK-2019 was stable in plasma at -70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK-2019 in a patient who received a single and multiple oral daily doses of 120 mg.

Associations Between Oral Health Fatalism and Demographic Factors, Dental Practices, Fatalism, and Oral Health Self-Efficacy.

Purpose: To evaluate the relationship between demographics, dental beliefs and practices, fatalism, oral health self-efficacy, and oral health fatalism (OHF) among parent (guardian, caregivers). Methods: English-speaking parents of children presenting for dental care at a hospital dental clinic, a dental surgery center, and two private practices answered a 33-item questionnaire regarding demographics, general fatalistic views, and dental beliefs, practices, and history. Participants rated their agreement with the OHF statement: "Most children eventually develop dental cavities." Results: More than half (58.4 percent) of parent respondents (n equals 332) were Caucasian, and 44.6 percent had education beyond high school. Most were female (81.3 percent), with public (Medicaid) insurance (67.5 percent), and were raising three (average) children. Less than 30 percent endorsed the OHF statement, and 42.5 percent were neutral. Higher OHF was found in parents of children with Medicaid insurance (P=0.02), fair (P=0.01) or poor (P=0.03) dental health, previous caries history (P=0.02), and those attending their first dental visit (P=0.03). Higher OHF was found in parents whose children do not brush their teeth when asked (P=0.02) or who do not behave when a parent helps (P=0.02), as well as those who subscribe to general fatalism beliefs (P=0.002). Conclusions: Higher oral health fatalism was associated with general fatalism, low oral health self-efficacy, parents of children with Medicaid insurance, suboptimal dental health, and first dental visits. Future studies investigating whether OHF can change over time and the role providers play in OHF can help dental professionals understand parent health behaviors and plan for health promotion interventions.

Angioinvasive Trichophyton rubrum associated necrotizing fasciitis in an immunocompromised patient.

Angioinvasive dermatophytosis with necrotizing fasciitis can be a rare complication in immunocompromised patients with early surgical debridement, 12 weeks of oral terbinafine, and reduction in immunosuppression being a viable management strategy.

ERG K+ channels mediate a major component of action potential repolarization in lymphatic muscle.

Smooth muscle cells in the walls of collecting lymphatic vessels fire spontaneous action potentials (APs), which conduct rapidly over the muscle layer to initiate contractions that propel lymph. Several ion channels have been implicated in the currents underlying the AP spike and the preceding diastolic depolarization, but the molecular identities of K+ channels involved in AP repolarization are unknown. Based on previous studies of other rhythmically active smooth muscles, we hypothesized that ether-a-go-go related gene (ERG) K+ channels (Kv11) play an important role in repolarization of the AP in lymphatic muscle. Message for one or more ERG channel isoforms was detected by RT-PCR analysis of lymphatic vessels from mice, rats and humans. Membrane potential recordings in smooth muscle cells of rat and human lymphatics revealed that nanomolar concentrations of ERG-1 inhibitors (E-4031 and BeKm-1) prolonged the duration of the AP plateau (normally ~ 1 s in duration) and induced multiple spikes, whereas ERG-1 activators (ICA-105574 and RPR-260243) shortened the plateau and could completely inhibit spontaneous APs. At relatively high inhibitor concentrations, the AP plateau duration lasted as long as 24 s. ERG activators reversed the effects of ERG inhibitors and vice-versa. In pressure myograph studies, ERG channel inhibition prolonged the diastolic repolarization phase of the contraction cycle and reduced the frequency of spontaneous contractions. This is the first evidence for a specific K+ channel contributing to the AP in lymphatic muscle. Our results imply that lymphatic contractile dysfunction may occur in long QT type II patients with mutations that result in ERG channel loss-of-function or impaired trafficking of the channel to the cell membrane.

Metformin is associated with improved clinical outcomes in patients with melanoma: a retrospective, multi-institutional study.

Background: Pre-clinical studies have shown that metformin reduces intratumoral hypoxia, improves T-cell function, and increases sensitivity to PD-1 blockade, and metformin exposure has been associated with improved clinical outcomes in various types of cancer. However, the impact of this drug in diabetic melanoma patients has not yet been fully elucidated. Methods: We reviewed 4,790 diabetic patients with stage I-IV cutaneous melanoma treated at the UPMC-Hillman Cancer Center and Memorial Sloan Kettering Cancer Center between 1996-2020. The primary endpoints included recurrence rates, progression free survival (PFS), and overall survival (OS) with and without metformin exposure. Tabulated variables included BRAF mutational status, immunotherapy (IMT) by type, and incidence of brain metastases. Results: The five-year incidence of recurrence in stage I/II patients was significantly reduced with metformin exposure (32.3% vs 47.7%, p=0.012). The five-year recurrence rate for stage III patients was also significantly reduced (58.3% vs 77.3%, p=0.013) in the metformin cohort. OS was numerically increased in nearly all stages exposed to metformin, though this did not reach statistical significance. The incidence of brain metastases was significantly lower in the metformin cohort (8.9% vs 14.6%, p=0.039). Conclusion: This is the first study to demonstrate significantly improved clinical outcomes in diabetic melanoma patients exposed to metformin. Overall, these results provide further rationale for ongoing clinical trials studying the potential augmentation of checkpoint blockade with metformin in advanced melanoma.

Methods to Detect Volatile Organic Compounds for Breath Biopsy Using Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry.

Volatile organic compounds (VOCs) are byproducts from metabolic pathways that can be detected in exhaled breath and have been reported as biomarkers for different diseases. The gold standard for analysis is gas chromatography-mass spectrometry (GC-MS), which can be coupled with various sampling methods. The current study aims to develop and compare different methods for sampling and preconcentrating VOCs using solid-phase microextraction (SPME). An in-house sampling method, direct-breath SPME (DB-SPME), was developed to directly extract VOCs from breath using a SPME fiber. The method was optimized by exploring different SPME types, the overall exhalation volume, and breath fractionation. DB-SPME was quantitatively compared to two alternative methods involving the collection of breath in a Tedlar bag. In one method, VOCs were directly extracted from the Tedlar bag (Tedlar-SPME) and in the other, the VOCs were cryothermally transferred from the Tedlar bag to a headspace vial (cryotransfer). The methods were verified and quantitatively compared using breath samples (n = 15 for each method respectively) analyzed by GC-MS quadrupole time-of-flight (QTOF) for compounds including but not limited to acetone, isoprene, toluene, limonene, and pinene. The cryotransfer method was the most sensitive, demonstrating the strongest signal for the majority of the VOCs detected in the exhaled breath samples. However, VOCs with low molecular weights, including acetone and isoprene, were detected with the highest sensitivity using the Tedlar-SPME. On the other hand, the DB-SPME was less sensitive, although it was rapid and had the lowest background GC-MS signal. Overall, the three breath-sampling methods can detect a wide variety of VOCs in breath. The cryotransfer method may be optimal when collecting a large number of samples using Tedlar bags, as it allows the long-term storage of VOCs at low temperatures (-80 °C), while Tedlar-SPME may be more effective when targeting relatively small VOCs. The DB-SPME method may be the most efficient when more immediate analyses and results are required.

Nocturnal High-Flow Nasal Cannula Therapy and Sinonasal Symptoms During Cystic Fibrosis Exacerbations.

BACKGROUND: Both nasal obstruction and sleep disturbance are common in patients with cystic fibrosis (CF). In patients with obstructive sleep apnea (OSA), studies suggest that these conditions are related and that nasal congestion improves with CPAP therapy. We hypothesized that subjects admitted to hospital for therapy of an exacerbation of CF would have both nasal symptoms and sleep disturbance and that these would improve with the initiation of nocturnal high-flow nasal cannula therapy (HFNC). METHODS: Twenty-five subjects with an exacerbation of CF were enrolled to randomly receive either 5 d of nocturnal HFNC at 20 L/min in the treatment group or 5 L/min of nocturnal nasal cannula air at ambient temperature and humidity in the low-flow group. On the first and last day of the study, the Sino-Nasal Outcome Test (SNOT-20) was administered to evaluate nasal symptoms, and sleep quality was measured using the Actiwatch 2. RESULTS: Fifteen subjects completed the study (6 HFNC, 9 low flow). We confirmed that subjects had significant sleep disturbance that did not improve over the 5 d of the study. Subjects also had disturbing nasal symptoms that significantly improved only in those receiving HFNC (pre 14 [20] vs post 6 [13], P = .027). CONCLUSIONS: Similar to what has been reported in older subjects with OSA, nocturnal HFNC improves sinonasal symptoms in subjects with an exacerbation of CF. There was no measurable effect on sleep quality, which may be due to the short duration of the study, or to subjects being evaluated while being treated in a hospital setting.

Lymphatic contractile dysfunction in mouse models of Cantú Syndrome with KATP channel gain-of-function.

Cantú Syndrome (CS) is an autosomal dominant disorder caused by gain-of-function (GoF) mutations in the Kir6.1 and SUR2 subunits of KATP channels. KATP overactivity results in a chronic reduction in arterial tone and hypotension, leading to other systemic cardiovascular complications. However, the underlying mechanism of lymphedema, developed by >50% of CS patients, is unknown. We investigated whether lymphatic contractile dysfunction occurs in mice expressing CS mutations in Kir6.1 (Kir6.1[V65M]) or SUR2 (SUR2[A478V], SUR2[R1154Q]). Pressure myograph tests of contractile function of popliteal lymphatic vessels over the physiological pressure range revealed significantly impaired contractile strength and reduced frequency of spontaneous contractions at all pressures in heterozygous Kir6.1[V65M] vessels, compared to control littermates. Contractile dysfunction of intact popliteal lymphatics in vivo was confirmed using near-infrared fluorescence microscopy. Homozygous SUR2[A478V] vessels exhibited profound contractile dysfunction ex vivo, but heterozygous SUR2[A478V] vessels showed essentially normal contractile function. However, further investigation of vessels from all three GoF mouse strains revealed significant disruption in contraction wave entrainment, decreased conduction speed and distance, multiple pacemaker sites, and reversing wave direction. Tests of 2-valve lymphatic vessels forced to pump against an adverse pressure gradient revealed that all CS-associated genotypes were essentially incapable of pumping under an imposed outflow load. Our results show that varying degrees of lymphatic contractile dysfunction occur in proportion to the degree of molecular GoF in Kir6.1 or SUR2. This is the first example of lymphatic contractile dysfunction caused by a smooth muscle ion channel mutation and potentially explains the susceptibility of CS patients to lymphedema.

Fifteen-Year Outcomes after Monitoring, Surgery, or Radiotherapy for Prostate Cancer.

BACKGROUND: Between 1999 and 2009 in the United Kingdom, 82,429 men between 50 and 69 years of age received a prostate-specific antigen (PSA) test. Localized prostate cancer was diagnosed in 2664 men. Of these men, 1643 were enrolled in a trial to evaluate the effectiveness of treatments, with 545 randomly assigned to receive active monitoring, 553 to undergo prostatectomy, and 545 to undergo radiotherapy. METHODS: At a median follow-up of 15 years (range, 11 to 21), we compared the results in this population with respect to death from prostate cancer (the primary outcome) and death from any cause, metastases, disease progression, and initiation of long-term androgen-deprivation therapy (secondary outcomes). RESULTS: Follow-up was complete for 1610 patients (98%). A risk-stratification analysis showed that more than one third of the men had intermediate or high-risk disease at diagnosis. Death from prostate cancer occurred in 45 men (2.7%): 17 (3.1%) in the active-monitoring group, 12 (2.2%) in the prostatectomy group, and 16 (2.9%) in the radiotherapy group (P = 0.53 for the overall comparison). Death from any cause occurred in 356 men (21.7%), with similar numbers in all three groups. Metastases developed in 51 men (9.4%) in the active-monitoring group, in 26 (4.7%) in the prostatectomy group, and in 27 (5.0%) in the radiotherapy group. Long-term androgen-deprivation therapy was initiated in 69 men (12.7%), 40 (7.2%), and 42 (7.7%), respectively; clinical progression occurred in 141 men (25.9%), 58 (10.5%), and 60 (11.0%), respectively. In the active-monitoring group, 133 men (24.4%) were alive without any prostate cancer treatment at the end of follow-up. No differential effects on cancer-specific mortality were noted in relation to the baseline PSA level, tumor stage or grade, or risk-stratification score. No treatment complications were reported after the 10-year analysis. CONCLUSIONS: After 15 years of follow-up, prostate cancer-specific mortality was low regardless of the treatment assigned. Thus, the choice of therapy involves weighing trade-offs between benefits and harms associated with treatments for localized prostate cancer. (Funded by the National Institute for Health and Care Research; ProtecT Current Controlled Trials number, ISRCTN20141297; ClinicalTrials.gov number, NCT02044172.).

Intramyocardial cell-based therapy with Lomecel-B during bidirectional cavopulmonary anastomosis for hypoplastic left heart syndrome: the ELPIS phase I trial.

Aims: Hypoplastic left heart syndrome (HLHS) survival relies on surgical reconstruction of the right ventricle (RV) to provide systemic circulation. This substantially increases the RV load, wall stress, maladaptive remodelling, and dysfunction, which in turn increases the risk of death or transplantation. Methods and results: We conducted a phase 1 open-label multicentre trial to assess the safety and feasibility of Lomecel-B as an adjunct to second-stage HLHS surgical palliation. Lomecel-B, an investigational cell therapy consisting of allogeneic medicinal signalling cells (MSCs), was delivered via intramyocardial injections. The primary endpoint was safety, and measures of RV function for potential efficacy were obtained. Ten patients were treated. None experienced major adverse cardiac events. All were alive and transplant-free at 1-year post-treatment, and experienced growth comparable to healthy historical data. Cardiac magnetic resonance imaging (CMR) suggested improved tricuspid regurgitant fraction (TR RF) via qualitative rater assessment, and via significant quantitative improvements from baseline at 6 and 12 months post-treatment (P < 0.05). Global longitudinal strain (GLS) and RV ejection fraction (EF) showed no declines. To understand potential mechanisms of action, circulating exosomes from intramyocardially transplanted MSCs were examined. Computational modelling identified 54 MSC-specific exosome ribonucleic acids (RNAs) corresponding to changes in TR RF, including miR-215-3p, miR-374b-3p, and RNAs related to cell metabolism and MAPK signalling. Conclusion: Intramyocardially delivered Lomecel-B appears safe in HLHS patients and may favourably affect RV performance. Circulating exosomes of transplanted MSC-specific provide novel insight into bioactivity. Conduct of a controlled phase trial is warranted and is underway.Trial registration number NCT03525418.

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle.

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Multiomics in primary and metastatic breast tumors from the AURORA US network finds microenvironment and epigenetic drivers of metastasis.

The AURORA US Metastasis Project was established with the goal to identify molecular features associated with metastasis. We assayed 55 females with metastatic breast cancer (51 primary cancers and 102 metastases) by RNA sequencing, tumor/germline DNA exome and low-pass whole-genome sequencing and global DNA methylation microarrays. Expression subtype changes were observed in ~30% of samples and were coincident with DNA clonality shifts, especially involving HER2. Downregulation of estrogen receptor (ER)-mediated cell-cell adhesion genes through DNA methylation mechanisms was observed in metastases. Microenvironment differences varied according to tumor subtype; the ER+/luminal subtype had lower fibroblast and endothelial content, while triple-negative breast cancer/basal metastases showed a decrease in B and T cells. In 17% of metastases, DNA hypermethylation and/or focal deletions were identified near HLA-A and were associated with reduced expression and lower immune cell infiltrates, especially in brain and liver metastases. These findings could have implications for treating individuals with metastatic breast cancer with immune- and HER2-targeting therapies.