A Biomimetic Leaflet Scaffold for Aortic Valve Remodeling.

Heart valve disease poses a significant clinical challenge, especially in pediatric populations, due to the inability of existing valve replacements to grow or respond biologically to their microenvironment. Tissue-engineered heart valves (TEHVs) provide a solution by facilitating patient-specific models for self-repair and remodeling. In this study, a 3D-bioprinted TEHV is designed to emulate the trilayer leaflet structure of an aortic valve. A cell-laden hydrogel scaffold made from gelatin methacrylate and polyethylene glycol diacrylate (GelMA/PEGDA) incorporates valvular interstitial-like (VIC-like) cells, being reinforced with a layer of polycaprolactone (PCL). The composition of the hydrogel scaffold remains stable over 7 days, having increased mechanical strength compared to pure GelMA. The scaffold maintains VIC-like cell function and promotes extracellular matrix (ECM) protein expression up to 14 days under two dynamic culture conditions: shear stress and stretching; replicating heart valve behavior within a more physiological-like setting and suggesting remodeling potential via ECM synthesis. This TEHV offers a promising avenue for valve replacements, closely replicating the structural and functional attributes of a native aortic valve, leading to mechanical and biological integration through biomaterial-cellular interactions.

The aconitate decarboxylase 1/itaconate pathway modulates immune dysregulation and associates with cardiovascular disease markers in SLE.

Objective: The Krebs cycle enzyme Aconitate Decarboxylase 1 (ACOD1) mediates itaconate synthesis in myeloid cells.. Previously, we reported that administration of 4-octyl itaconate abrogated lupus phenotype in mice. Here, we explore the role of the endogenous ACOD1/itaconate pathway in the development of murine lupus as well as their relevance in premature cardiovascular damage in SLE. Methods: We characterized Acod1 protein expression in bone marrow-derived macrophages and human monocyte-derived macrophages, following a TLR7 agonist (imiquimod, IMQ). Wild type and Acod1-/- mice were exposed to topical IMQ for 5 weeks to induce an SLE phenotype and immune dysregulation was quantified. Itaconate serum levels were quantified in SLE patients and associated to cardiometabolic parameters and disease activity. Results: ACOD1 was induced in mouse bone marrow-derived macrophages (BMDM) and human monocyte-derived macrophages following in vitro TLR7 stimulation. This induction was partially dependent on type I Interferon receptor signaling and specific intracellular pathways. In the IMQ-induced mouse model of lupus, ACOD1 knockout (Acod1-/-) displayed disruptions of the splenic architecture, increased serum anti-dsDNA and proinflammatory cytokine levels, enhanced kidney immune complex deposition and proteinuria, when compared to the IMQ-treated WT mice. Consistent with these results, Acod1-/- BMDM exposed to IMQ showed higher proinflammatory features in vitro. Itaconate levels were decreased in SLE serum compared to healthy control sera, in association with specific perturbed cardiometabolic parameters and subclinical vascular disease. Conclusion: These findings suggest that the ACOD1/itaconate pathway plays important immunomodulatory and vasculoprotective roles in SLE, supporting the potential therapeutic role of itaconate analogs in autoimmune diseases.

Determination of Ploidy Levels and Nuclear DNA Content in Cryptococcus neoformans by Flow Cytometry: Drawbacks with Variability.

Flow cytometry is commonly employed for ploidy determination and cell cycle analysis in cryptococci. The cells are subjected to fixation and staining with DNA-binding fluorescent dyes, most commonly with propidium iodide (PI), before undergoing flow cytometric analysis. In ploidy determination, cell populations are classified according to variations in DNA content, as evidenced by the fluorescence intensity of stained cells. As reported in Saccharomyces cerevisiae, we found drawbacks with PI staining that confounded the accurate analysis of ploidy by flow cytometry when the size of the cryptococci changed significantly. However, the shift in the fluorescence intensity, unrelated to ploidy changes in cells with increased size, could be accurately interpreted by applying the ImageStream system. SYTOX Green or SYBR Green I, reported to enable DNA analysis with a higher accuracy than PI in S. cerevisiae, were nonspecific for nuclear DNA staining in cryptococci. Until dyes or methods capable of reducing the variability inherent in the drastic changes in cell size or shape become available, PI appears to remain the most reliable method for cell cycle or ploidy analysis in Cryptococcus.

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.

Myocardial Matrix Hydrogels Mitigate Negative Remodeling and Improve Function in Right Heart Failure Model.

This study evaluates the effectiveness of myocardial matrix (MM) hydrogels in mitigating negative right ventricular (RV) remodeling in a rat model of RV heart failure. The goal was to assess whether a hydrogel derived from either the right or left ventricle could promote cardiac repair. Injured rat right ventricles were injected with either RV-or left ventricular-derived MM hydrogels. Both hydrogels improved RV function and morphology and reduced negative remodeling. This study supports the potential of injectable biomaterial therapies for treating RV heart failure.

Pacemaking in the lymphatic system.

Lymphatic collecting vessels exhibit spontaneous phasic contractions that are critical for lymph propulsion and tissue fluid homeostasis. This rhythmic activity is driven by action potentials conducted across the lymphatic muscle cell (LMC) layer to produce entrained contractions. The contraction frequency of a lymphatic collecting vessel displays exquisite mechanosensitivity, with a dynamic range from <1 to >20 contractions per minute. A myogenic pacemaker mechanism intrinsic to the LMCs was initially postulated to account for pressure-dependent chronotropy. Further interrogation into the cellular constituents of the lymphatic vessel wall identified non-muscle cell populations that shared some characteristics with interstitial cells of Cajal, which have pacemaker functions in the gastrointestinal and lower urinary tracts, thus raising the possibility of a non-muscle cell pacemaker. However, recent genetic knockout studies in mice support LMCs and a myogenic origin of the pacemaker activity. LMCs exhibit stochastic, but pressure-sensitive, sarcoplasmic reticulum calcium release (puffs and waves) from IP3R1 receptors, which couple to the calcium-activated chloride channel Anoctamin 1, causing depolarisation. The resulting electrical activity integrates across the highly coupled lymphatic muscle electrical syncytia through connexin 45 to modulate diastolic depolarisation. However, multiple other cation channels may also contribute to the ionic pacemaking cycle. Upon reaching threshold, a voltage-gated calcium channel-dependent action potential fires, resulting in a nearly synchronous calcium global calcium flash within the LMC layer to drive an entrained contraction. This review summarizes the key ion channels potentially responsible for the pressure-dependent chronotropy of lymphatic collecting vessels and various mechanisms of IP3R1 regulation that could contribute to frequency tuning.

ChatGPT Yields a Passing Score on a Pediatric Board Preparatory Exam but Raises Red Flags.

Objectives: We aimed to evaluate the performance of a publicly-available online artificial intelligence program (OpenAI's ChatGPT-3.5 and -4.0, August 3 versions) on a pediatric board preparatory examination, 2021 and 2022 PREP® Self-Assessment, American Academy of Pediatrics (AAP). Methods: We entered 245 questions and answer choices from the Pediatrics 2021 PREP® Self-Assessment and 247 questions and answer choices from the Pediatrics 2022 PREP® Self-Assessment into OpenAI's ChatGPT-3.5 and ChatGPT-4.0, August 3 versions, in September 2023. The ChatGPT-3.5 and 4.0 scores were compared with the advertised passing scores (70%+) for the PREP® exams and the average scores (74.09%) and (75.71%) for all 10 715 and 6825 first-time human test takers. Results: For the AAP 2021 and 2022 PREP® Self-Assessments, ChatGPT-3.5 answered 143 of 243 (58.85%) and 137 of 247 (55.46%) questions correctly on a single attempt. ChatGPT-4.0 answered 193 of 243 (79.84%) and 208 of 247 (84.21%) questions correctly. Conclusion: Using a publicly-available online chatbot to answer pediatric board preparatory examination questions yielded a passing score but demonstrated significant limitations in the chatbot's ability to assess some complex medical situations in children, posing a potential risk to this vulnerable population.

Current and Emerging Technologies to Address the Placebo Response Challenge in CNS Clinical Trials: Promise, Pitfalls, and Pathways Forward.

Excessive placebo response rates have long been a major challenge for central nervous system (CNS) drug discovery. As CNS trials progressively shift toward digitalization, decentralization, and novel remote assessment approaches, questions are emerging about whether innovative technologies can help mitigate the placebo response. This article begins with a conceptual framework for understanding placebo response. We then critically evaluate the potential of a range of innovative technologies and associated research designs that might help mitigate the placebo response and enhance detection of treatment signals. These include technologies developed to directly address placebo response; technology-based approaches focused on recruitment, retention, and data collection with potential relevance to placebo response; and novel remote digital phenotyping technologies. Finally, we describe key scientific and regulatory considerations when evaluating and selecting innovative strategies to mitigate placebo response. While a range of technological innovations shows potential for helping to address the placebo response in CNS trials, much work remains to carefully evaluate their risks and benefits.

Evaluating injuries and illnesses that occurred during the Yukon Quest International sled dog race, 2018-2020.

Introduction: The purpose of this study was to evaluate medical record data from the 2018-2020 Yukon Quest International Sled Dog race to examine injury patterns and risk factors for dogs competing in multi-day ultra-endurance events. Specifically, we summarized injuries and illnesses that resulted in canine athletes being removed ("dropped") from competition, and in orthopedic injuries diagnosed in both dropped and finished dogs. Methods: The records of 989 dogs that started the race were examined, but only records from dogs in teams that went on to finish the race were included, for a total of 711 records. Results and discussion: Three hundred and sixty five dogs (51.3%) were noted to have at least one abnormal finding in their veterinary medical record during the race. Orthopedic injuries were most common, and 291 injuries were ultimately diagnosed in 234 dogs (32.9%). Ultimately, 206 dogs (29%) were dropped from competition, for any reason. The most common reasons for dropping dogs were orthopedic injuries (156 dogs; 188 injuries), gastrointestinal illness (22 dogs), and cardiorespiratory disease (7 dogs). Most orthopedic injuries in dropped dogs occurred in the thoracic limb (n = 121 dogs; 151 injuries). Of those, injuries to the shoulder were most common (n = 77), followed by injuries to the carpus (n = 59), and injury to the pelvic limb (n = 32). Carpal injuries were the most prevalent injury diagnosed in dogs that went on to finish the race (71 of 85 injuries). Carpal injuries were the most prevalent injuries overall in 2018 (51%) and 2019 (52%). In 2020, shoulder injuries were most prevalent (27%), suggesting that trail conditions may have differed between years. The majority of dogs with an orthopedic injury ultimately were removed from competition (156 of 234, or 66.6%), but the likelihood of finishing the race with an injury depended on the type of injury sustained; 71 of 130 dogs (54.6%) with a carpal injury went on to finish the race, whereas only 9 of 86 dogs with a shoulder injury (10.5%) went on to finish. The results of this study can assist mushers and veterinarians in preparing for races, and in decision making during endurance sled dog races.

Developmental progression of lymphatic valve morphology and function.

Introduction: The bileaflet valves found in collecting lymphatic vessels and some veins are essential for maintaining a unidirectional flow, which is important for lymphatic and venous function. Under an adverse pressure gradient, the two leaflets tightly overlap to prevent backflow. Valves are proposed to share four main stages of development, based on images obtained from randomly oriented valves in fixed mouse embryos, with the best structural views obtained from larger venous valves. It is not known at what stage lymphatic valves (LVs) become functional (e.g., able to oppose backflow), although a requirement for stage 4 is presumed. Methods: To gain an insight into this sequence of events for LVs, we used Prox1CreER T2 :Foxo1 fl/fl mice and Foxc2CreER T2 :Foxo1 fl/fl mouse models, in which deletion of the valve repressor factor Foxo1 promotes the development of new LVs in adult lymphatic vessels. Both strains also contained a Prox1eGFP reporter to image the lymphatic endothelium. Mesenteric collecting lymphatic vessels were dissected, cannulated, and pressurized for ex vivo tests of valve function. LVs at various stages (1-4 and intermediate) were identified in multi-valve segments, which were subsequently shortened to perform the backleak test on single valves. The GFP signal was then imaged at high magnification using a confocal microscope. Z-stack reconstructions enabled 1:1 comparisons of LV morphology with a quantitative measurement of back leak. Results: As expected, LVs of stages 1-3 were completely leaky in response to outflow pressure elevation. Stage 4 valves were generally not leaky, but valve integrity depended on the Cre line used to induce new valve formation. A high percentage of valves at leaflet an intermediate stage (3.5), in which there was an insertion of a second commissure, but without proper luminal alignment, effectively resisted back leak when the outflow pressure was increased. Discussion: Our findings represent the first 3D images of developing lymphatic valves and indicate that valves become competent between stages 3 and 4 of development.

Progressive multifocal leukoencephalopathy in a lung transplant recipient.

Progressive multifocal leukoencephalopathy (PML) is a rare and fatal demyelinating disease of the central nervous system (CNS). The case we describe highlights the importance of considering a diagnosis of PML early (<1 year) after lung transplant.

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.

Antigen stasis and airway nitrosative stress in human primary ciliary dyskinesia.

Nasal nitric oxide (nNO) is low in most patients with primary ciliary dyskinesia (PCD). Decreased ciliary motion could lead to antigen stasis, increasing oxidant production and NO oxidation in the airways. This could both decrease gas phase NO and increase nitrosative stress. We studied primary airway epithelial cells from healthy controls (HCs) and patients with PCD with several different genotypes. We measured antigen clearance in fenestrated membranes exposed apically to the fluorescently labeled antigen Dermatophagoides pteronyssinus (Derp1-f). We immunoblotted for 3-nitrotyrosine (3-NT) and for oxidative response enzymes. We measured headspace NO above primary airway cells without and with a PCD-causing genotype. We measured nNO and exhaled breath condensate (EBC) H2O2 in vivo. Apical Derp1-f was cleared from HC better than from PCD cells. DUOX1 expression was lower in HC than in PCD cells at baseline and after 24-h Derp1-f exposure. HC cells had less 3-NT and NO3- than PCD cells. However, NO consumption by HC cells was less than that by PCD cells; NO loss was prevented by superoxide dismutase (SOD) and by apocynin. nNO was higher in HCs than in patients with PCD. EBC H2O2 was lower in HC than in patients with PCD. The PCD airway epithelium does not optimally clear antigens and is subject to oxidative and nitrosative stress. Oxidation associated with antigen stasis could represent a therapeutic target in PCD, one with convenient monitoring biomarkers.NEW & NOTEWORTHY The PCD airway epithelium does not optimally clear antigens, and antigen exposure can lead to NO oxidation and nitrosative stress. Oxidation caused by antigen stasis could represent a therapeutic target in PCD, and there are convenient monitoring biomarkers.

A Concise Review of Exhaled Breath Testing for Respiratory Clinicians and Researchers.

Exhaled breath contains an extensive reservoir of biomolecules. The collection of exhaled breath is noninvasive and low risk. Therefore, its testing is an appealing strategy for the discovery of biomarkers of respiratory diseases. In this concise review, we summarize the evidence of exhaled breath tests for airways diseases and respiratory infections. An overview of breath collection methods in both individuals who are spontaneously breathing and those receiving mechanical ventilation is outlined. We also highlight the challenges in exhaled breath testing and areas for future research.

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.

Nasopharyngeal lymphatic plexus is a hub for cerebrospinal fluid drainage.

Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long been known to drain through the lymphatics to cervical lymph nodes1-17, but the connections and regulation have been challenging to identify. Here, using fluorescent CSF tracers in Prox1-GFP lymphatic reporter mice18, we found that the nasopharyngeal lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This plexus had unusual valves and short lymphangions but no smooth-muscle coverage, whereas downstream deep cervical lymphatics had typical semilunar valves, long lymphangions and smooth muscle coverage that transported CSF to the deep cervical lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells regulated CSF drainage through the transport properties of deep cervical lymphatics. During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical lymphatics were not similarly altered, and CSF outflow could still be increased by adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed increased type I interferon signalling and other inflammatory cytokines. The importance of evidence for the nasopharyngeal lymphatic plexus functioning as a CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant pharmacological activation of deep cervical lymphatic transport towards lymph nodes can still increase CSF outflow, offering an approach for augmenting CSF clearance in age-related neurological conditions in which greater efflux would be beneficial.

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.