Lipid availability influences ferroptosis sensitivity in cancer cells by regulating polyunsaturated fatty acid trafficking.

Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic acid and adrenic acid. These PUFAs then accumulate in phospholipids, particularly ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools dictates their sensitivity to ferroptosis.

Stealth transgenes enable CAR-T cells to evade host immune responses.

BACKGROUND: Adoptive cell therapy, such as chimeric antigen receptor (CAR)-T cell therapy, has improved patient outcomes for hematological malignancies. Currently, four of the six FDA-approved CAR-T cell products use the FMC63-based αCD19 single-chain variable fragment, derived from a murine monoclonal antibody, as the extracellular binding domain. Clinical studies demonstrate that patients develop humoral and cellular immune responses to the non-self CAR components of autologous CAR-T cells or donor-specific antigens of allogeneic CAR-T cells, which is thought to potentially limit CAR-T cell persistence and the success of repeated dosing. METHODS: In this study, we implemented a one-shot approach to prevent rejection of engineered T cells by simultaneously reducing antigen presentation and the surface expression of both Classes of the major histocompatibility complex (MHC) via expression of the viral inhibitors of transporter associated with antigen processing (TAPi) in combination with a transgene coding for shRNA targeting class II MHC transactivator (CIITA). The optimal combination was screened in vitro by flow cytometric analysis and mixed lymphocyte reaction assays and was validated in vivo in mouse models of leukemia and lymphoma. Functionality was assessed in an autologous setting using patient samples and in an allogeneic setting using an allogeneic mouse model. RESULTS: The combination of the Epstein-Barr virus TAPi and an shRNA targeting CIITA was efficient and effective at reducing cell surface MHC classes I and II in αCD19 'stealth' CAR-T cells while retaining in vitro and in vivo antitumor functionality. Mixed lymphocyte reaction assays and IFNγ ELISpot assays performed with T cells from patients previously treated with autologous αCD19 CAR-T cells confirm that CAR T cells expressing the stealth transgenes evade allogeneic and autologous anti-CAR responses, which was further validated in vivo. Importantly, we noted anti-CAR-T cell responses in patients who had received multiple CAR-T cell infusions, and this response was reduced on in vitro restimulation with autologous CARs containing the stealth transgenes. CONCLUSIONS: Together, these data suggest that the proposed stealth transgenes may reduce the immunogenicity of autologous and allogeneic cellular therapeutics. Moreover, patient data indicate that repeated doses of autologous FMC63-based αCD19 CAR-T cells significantly increased the anti-CAR T cell responses in these patients.

Heterogeneity in breeding productivity is driven largely by factors affecting nestlings and young fledglings in an imperiled migratory passerine.

Identifying factors that drive variation in vital rates among populations is a prerequisite to understanding a species' population biology and, ultimately, to developing effective conservation strategies. This is especially true for imperiled species like the golden-winged warbler (Vermivora chrysoptera) that exhibit strong spatial heterogeneity in demography and responds variably to conservation interventions. Habitat management actions recommended for breeding grounds conservation include timber harvest, shrub shearing, and prescribed fire that maintain or create early successional woody communities. Herein, we assessed variation in the survival of nests [n = 145] and fledglings [n = 134] at 17 regenerating timber harvest sites within two isolated populations in Pennsylvania that differed in productivity and response to habitat management. Although the overall survival of nests and fledglings was higher in the eastern population than the central population, this was only true when the nest phases and fledgling phases were considered wholly. Indeed, survival rates of nestlings and recently fledged young (1-5 days post-fledging) were lower in the central population, whereas eggs and older fledglings (6-30 days post-fledging) survived at comparable rates in both populations. Fledglings in the central population were smaller (10% lower weight) and begged twice as much as those in the eastern population, suggesting food limitation may contribute to lower survival rates. Fledgling survival in the central population, but not the eastern, also was a function of habitat features (understory vegetation density [positive] and distance to mature forest [negative]) and individual factors (begging effort [negative]). Our findings illustrate how identifying how survival varies across specific life stages can elucidate potential underlying demographic drivers, such as food resources in this case. In this way, our work underscores the importance of studying and decomposing stage-specific demography in species of conservation concern.

Estimating the size and scope of the academic emergency physician workforce.

BACKGROUND: Academic emergency medicine (EM) is foundational to the EM specialty through the development of new knowledge and clinical training of resident physicians. Despite recent increased attention to the future of the EM workforce, no evaluations have specifically characterized the U.S. academic EM workforce. We sought to estimate the national proportion of emergency physicians (EPs) identified as academic and the proportion of emergency department (ED) visits that take place at academic sites. METHODS: We performed a cross-sectional analysis of EPs and EDs using data from the American Hospital Association, the Centers for Medicare & Medicaid Services, and Doximity's Residency Navigator. EPs were identified as "academic" if they were affiliated with at least one facility determined to be academic, defined as EDs officially designated by the Accreditation Council for Graduate Medical Education (ACGME) as clinical training sites at accredited EM residency programs. Our primary outcomes were to estimate the national proportion of EPs identified as academic and the proportion of ED visits performed at academic sites. RESULTS: Our analytic sample included 26,937 EPs practicing clinically across 4920 EDs and providing care during 130,471,386 ED visits. Among EPs, 11,720 (43.5%) were identified as academic, and among EDs, 635 (12.9%) were identified as academic sites, including 585 adult/general sites, 45 pediatric-specific sites, and 10 sites affiliated with the Department of Veterans Affairs. In 2021, academic EDs provided care for 42,794,106 ED visits or 32.8% of all ED visits nationally. CONCLUSIONS: Approximately four in 10 EPs practice in at least one clinical training site affiliated with an ACGME-accredited EM residency program, and approximately one in three ED visits nationally occur in these academic EDs. We encourage further work using alternative definitions of an academic EPs and EDs, along with longitudinal research to identify trends in the workforce's composition.

Unveiling the proteome-wide autoreactome enables enhanced evaluation of emerging CAR-T therapies in autoimmunity.

Given the global surge in autoimmune diseases, it is critical to evaluate emerging therapeutic interventions. Despite numerous new targeted immunomodulatory therapies, comprehensive approaches to apply and evaluate the effects of these treatments longitudinally are lacking. Here, we leveraged advances in programmable-phage immunoprecipitation (PhIP-Seq) methodology to explore the modulation, or lack thereof, of autoantibody profiles, proteome-wide, in both health and disease. Using a custom set of over 730,000 human derived peptides, we demonstrated that each individual, regardless of disease state, possesses a distinct and complex constellation of autoreactive antibodies. For each individual, the set of resulting autoreactivites constituted a unique immunological fingerprint, or "autoreactome," that was remarkably stable over years. Using the autoreactome as a primary output, we evaluated the relative effectiveness of various immunomodulatory therapies in altering autoantibody repertoires. We found that therapies targeting B-Cell Maturation Antigen (BCMA) profoundly altered an individual's autoreactome, while anti-CD19 and CD20 therapies had minimal effects. These data both confirm that the autoreactome is comprised of autoantibodies secreted by plasma cells, and strongly suggest that BCMA or other plasma cell targeting therapies may be highly effective in treating currently refractory autoantibody mediated diseases.

Elevated sympathetic-mediated vasoconstriction at rest but intact functional sympatholysis during exercise in heart failure with reduced ejection fraction.

BACKGROUND: Patients with heart failure with reduced ejection fraction (HFrEF) have exaggerated sympathoexcitation and impaired peripheral vascular conductance. Evidence demonstrating consequent impaired functional sympatholysis is limited in HFrEF. This study aimed to determine the magnitude of reduced limb vascular conductance during sympathoexcitation and whether functional sympatholysis would abolish such reductions in HFrEF. METHODS: Twenty patients with HFrEF and 22 age-matched controls performed the cold pressor test (left foot 2-min in -0.5[1] °C water) alone (CPT) and with right handgrip exercise (EX+CPT). Right forearm vascular conductance (FVC), forearm blood flow (FBF), and mean arterial pressure (MAP) were measured. RESULTS: Patients with HFrEF had greater decreases in %ΔFVC and %ΔFBF during CPT (both P<0.0001) but not EX+CPT (P=0.449, P=0.199) compared to controls, respectively. %ΔFVC and %ΔFBF decreased from CPT to EX+CPT in patients with HFrEF (both P<0.0001) and controls (P=0.018, P=0.015), respectively. MAP increased during CPT and EX+CPT in both groups (all P<0.0001). MAP was greater in controls compared to patients with HFrEF during EX+CPT (P=0.025) but not CPT (P=0.209). CONCLUSIONS: Acute sympathoexcitation caused exaggerated peripheral vasoconstriction and reduced peripheral blood flow in patients with HFrEF. Handgrip exercise abolished sympathoexcitatory-mediated peripheral vasoconstriction and normalized peripheral blood flow in patients with HFrEF. These novel data reveal intact functional sympatholysis in the upper limb and suggest exercise-mediated, local control of blood flow is preserved when cardiac limitations that are cardinal to HFrEF are evaded with dynamic handgrip exercise.

Detection of Podosphaera macularis in Air Samples by Quantitative PCR.

Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis, for detection of the organism from air samples. The assay utilizes primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit (LSU) of the nuclear ribosomal rDNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development.

Low-index mesoscopic surface reconstructions of Au surfaces using Bayesian force fields.

Metal surfaces have long been known to reconstruct, significantly influencing their structural and catalytic properties. Many key mechanistic aspects of these subtle transformations remain poorly understood due to limitations of previous simulation approaches. Using active learning of Bayesian machine-learned force fields trained from ab initio calculations, we enable large-scale molecular dynamics simulations to describe the thermodynamics and time evolution of the low-index mesoscopic surface reconstructions of Au (e.g., the Au(111)-'Herringbone,' Au(110)-(1 × 2)-'Missing-Row,' and Au(100)-'Quasi-Hexagonal' reconstructions). This capability yields direct atomistic understanding of the dynamic emergence of these surface states from their initial facets, providing previously inaccessible information such as nucleation kinetics and a complete mechanistic interpretation of reconstruction under the effects of strain and local deviations from the original stoichiometry. We successfully reproduce previous experimental observations of reconstructions on pristine surfaces and provide quantitative predictions of the emergence of spinodal decomposition and localized reconstruction in response to strain at non-ideal stoichiometries. A unified mechanistic explanation is presented of the kinetic and thermodynamic factors driving surface reconstruction. Furthermore, we study surface reconstructions on Au nanoparticles, where characteristic (111) and (100) reconstructions spontaneously appear on a variety of high-symmetry particle morphologies.

Calculation of Overall Hospital Quality Star Ratings With and Without Inclusion of the Peer Grouping Step.

Importance: The Centers for Medicare & Medicaid Services (CMS) Overall Star Rating is widely used by patients and consumers, and there is continued stakeholder curiosity surrounding the inclusion of a peer grouping step, implemented to the 2021 Overall Star Rating methods. Objective: To calculate hospital star rating scores with and without the peer grouping step, with the former approach stratifying hospitals into 3-, 4-, and 5-measure group peer groups based on the number of measure groups with at least 3 reported measures. Design, Setting, and Participants: This cross-sectional study used Care Compare website data from January 2023 for 3076 hospitals that received a star rating in 2023. Data were analyzed from April 2023 to December 2023. Exposure: Peer grouping vs no peer grouping. Main Outcomes and Measures: The primary outcome was the distribution of star ratings, with 1 star being the lowest-performing hospitals and 5 stars, the highest. Analyses additionally identified the number of hospitals with a higher, lower, or identical star rating with the use of the peer grouping step compared with its nonuse, stratified by certain hospital characteristics. Results: Among 3076 hospitals that received a star rating in 2023, most were nonspecialty (1994 hospitals [64.8%]), nonteaching (1807 hospitals [58.7%]), non-safety net (2326 hospitals [75.6%]), non-critical access (2826 hospitals [91.9%]) hospitals with fewer than 200 beds (1822 hospitals [59.2%]) and located in an urban geographic designations (1935 hospitals [62.9%]). The presence of the peer grouping step resulted in 585 hospitals (19.0%) being assigned a different star rating than if the peer grouping step was absent, including considerably more hospitals receiving a higher star rating (517 hospitals) rather than a lower (68 hospitals) star rating. Hospital characteristics associated with a higher star rating included urbanicity (351 hospitals [67.9%]), non-safety net status (414 hospitals [80.1%]), and fewer than 200 beds (287 hospitals [55.6%]). Collectively, the presence of the peer grouping step supports a like-to-like comparison among hospitals and supports the ability of patients to assess overall hospital quality. Conclusions and Relevance: In this cross-sectional study, inclusion of the peer grouping in the CMS star rating method resulted in modest changes in hospital star ratings compared with application of the method without peer grouping. Given improvement in face validity and the close association between the current peer grouping approach and stakeholder needs for peer-comparison, the current CMS Overall Star Rating method allows for durable comparisons in hospital performance.

Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1ß protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

Two-year retest reliability and predictive validity of the Self- and Informant-Personality Inventory for ICD-11 in older adults.

The International Classification of Diseases, 11th edition (ICD-11) adopted a fully dimensional model of personality disorder. The Personality Inventory for ICD-11 (PiCD) and Informant-Personality Inventory for ICD-11 (IPiC) were developed to assess the ICD-11 trait model, and the PiCD has since received significant validation support. However, there has only been one prior study of longitudinal predictive validity of the PiCD, two relatively short test-retest reliability studies of the PiCD, and no prior longitudinal tests of the IPiC. Longitudinal psychometric support for psychological assessment measures is essential. The present study provides a longer, larger, 2-year psychometric validation test of the PiCD and IPiC. Participants (N = 711) and their informants (N = 569) were recruited in the St. Louis Personality and Aging Network. The results demonstrated strong 2-year retest reliability for the PiCD and IPiC, as well as mean-level stability. Additionally, we explored the relationships between the PiCD and IPiC and important life outcome measures (depressive symptoms, satisfaction with life, and health status). The analysis revealed several significant associations between PiCD and IPiC scales and the outcome variables across time. Further, the PiCD Negative Affectivity and IPiC Detachment scales demonstrated incremental validity over each other and the outcome variables at Wave 1 in the prediction of depressive symptoms and satisfaction with life, respectively. The findings provide essential longitudinal test-retest reliability and predictive validity support for the PiCD and IPiC. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Classifying Future Healthcare Utilization in COPD Using Quantitative CT Lung Imaging and Two-Step Feature Selection via Sparse Subspace Learning with the CanCOLD Study.

RATIONALE: Although numerous candidate features exist for predicting risk of higher risk of healthcare utilization in patients with chronic obstructive pulmonary disease (COPD), the process for selecting the most discriminative features remains unclear. OBJECTIVE: The objective of this study was to develop a robust feature selection method to identify the most discriminative candidate features for predicting healthcare utilization in COPD, and compare the model performance with other common feature selection methods. MATERIALS AND METHODS: In this retrospective study, demographic, lung function measurements and CT images were collected from 454 COPD participants from the Canadian Cohort Obstructive Lung Disease study from 2010-2017. A follow-up visit was completed approximately 1.5 years later and participants reported healthcare utilization. CT analysis was performed for feature extraction. A two-step hybrid feature selection method was proposed that utilized: (1) sparse subspace learning with nonnegative matrix factorization, and, (2) genetic algorithm. Seven commonly used feature selection methods were also implemented that reported the top 10 or 20 features for comparison. Performance was evaluated using accuracy. RESULTS: Of the 454 COPD participants evaluated, 161 (35%) utilized healthcare services at follow-up. The accuracy for predicting subsequent healthcare utilization for the seven commonly used feature selection methods ranged from 72%-76% with the top 10 features, and 77%-80% with the top 20 features. Relative to these methods, hybrid feature selection obtained significantly higher accuracy for predicting subsequent healthcare utilization at 82% ± 3% (p < 0.05). Selected features with the proposed method included: DLCO, FEV1, RV, FVC, TAC, LAA950, Pi-10, LAA856, LAC total hole count, outer area RB1, wall area RB1, wall area and Jacobian. CONCLUSION: The hybrid feature selection method identified the most discriminative features for classifying individuals with and without future healthcare utilization, and increased the accuracy compared to other state-of-the-art approaches.

Lonidamine Induced Selective Acidification and De-Energization of Prostate Cancer Xenografts: Enhanced Tumor Response to Radiation Therapy.

Prostate cancer is a multi-focal disease that can be treated using surgery, radiation, androgen deprivation, and chemotherapy, depending on its presentation. Standard dose-escalated radiation therapy (RT) in the range of 70-80 Gray (GY) is a standard treatment option for prostate cancer. It could be used at different phases of the disease (e.g., as the only primary treatment when the cancer is confined to the prostate gland, combined with other therapies, or as an adjuvant treatment after surgery). Unfortunately, RT for prostate cancer is associated with gastro-intestinal and genitourinary toxicity. We have previously reported that the metabolic modulator lonidamine (LND) produces cancer sensitization through tumor acidification and de-energization in diverse neoplasms. We hypothesized that LND could allow lower RT doses by producing the same effect in prostate cancer, thus reducing the detrimental side effects associated with RT. Using the Seahorse XFe96 and YSI 2300 Stat Plus analyzers, we corroborated the expected LND-induced intracellular acidification and de-energization of isolated human prostate cancer cells using the PC3 cell line. These results were substantiated by non-invasive 31P magnetic resonance spectroscopy (MRS), studying PC3 prostate cancer xenografts treated with LND (100 mg/kg, i.p.). In addition, we found that LND significantly increased tumor lactate levels in the xenografts using 1H MRS non-invasively. Subsequently, LND was combined with radiation therapy in a growth delay experiment, where we found that 150 µM LND followed by 4 GY RT produced a significant growth delay in PC3 prostate cancer xenografts, compared to either control, LND, or RT alone. We conclude that the metabolic modulator LND radio-sensitizes experimental prostate cancer models, allowing the use of lower radiation doses and diminishing the potential side effects of RT. These results suggest the possible clinical translation of LND as a radio-sensitizer in patients with prostate cancer.

Chest Tube Size Selection: Evaluating Provider Practices, Treatment Efficacy, and Complications in Management of Thoracic Trauma.

BACKGROUND: The standard for managing traumatic pneumothorax (PTX), hemothorax (HTX), and hemopneumothorax (HPTX) has historically been large-bore (LB) chest tubes (>20-Fr). Previous studies have shown equal efficacy of small-bore (SB) chest tubes (≤19-Fr) in draining PTX and HTX/HPTX. This study aimed to evaluate provider practice patterns, treatment efficacy, and complications related to the selection of chest tube sizes for patients with thoracic trauma. METHODS: A retrospective chart review was performed on adult patients who underwent tube thoracostomy for traumatic PTX, HTX, or HPTX at a Level 1 Trauma Center from January 2016 to December 2021. Comparison was made between SB and LB thoracostomy tubes. The primary outcome was indication for chest tube placement based on injury pattern. Secondary outcomes included retained hemothorax, insertion-related complications, and duration of chest tube placement. Univariate and multivariate analyses were performed. RESULTS: Three hundred and forty-one patients were included and 297 (87.1%) received LB tubes. No significant differences were found between the groups concerning tube failure and insertion-related complications. LB tubes were more frequently placed in patients with penetrating MOI, higher average ISS, and higher average thoracic AIS. Patients who received LB chest tubes experienced a higher incidence of retained HTX. DISCUSSION: In patients with thoracic trauma, both SB and LB chest tubes may be used for treatment. SB tubes are typically placed in nonemergent situations, and there is apparent provider bias for LB tubes. A future randomized clinical trial is needed to provide additional data on the usage of SB tubes in emergent situations.

Integrated Filtration and Washing Modeling: Optimization of Impurity Rejection for Filtration and Washing of Active Pharmaceutical Ingredients.

A digital design tool that can transfer material property information between unit operations to predict the product attributes in integrated purification processes has been developed to facilitate end-to-end integrated pharmaceutical manufacturing. This work aims to combine filtration and washing operations frequently using active pharmaceutical ingredient (API) isolation. This is achieved by coupling predicted and experimental data produced during the upstream crystallization process. To reduce impurities in the isolated cake, a mechanistic model-based workflow was used to optimize an integrated filtration and washing process model. The Carman-Kozeny filtration model has been combined with a custom washing model that incorporates diffusion and axial dispersion mechanisms. The developed model and approach were applied to two systems, namely, mefenamic acid and paracetamol, which are representative compounds, and various crystallization and wash solvents and related impurities were used. The custom washing model provides a detailed evolution of species concentration during washing, simulating the washing curve with the three stages of the wash curve: constant rate, intermediate stage, and diffusion stage. A model validation approach was used to estimate cake properties (e.g., specific cake resistance, cake volume, cake composition after washing, and washing curve). A global systems analysis was conducted by using the calibrated model to explore the design space and aid in the setup of the optimization decision variables. Qualitative optimization was performed in order to reduce the concentration of impurities in the final cake after washing. The findings of this work were translated into a final model to simulate the optimal isolation conditions.

Targeting Recycling Endosomes to Potentiate mRNA Lipid Nanoparticles.

mRNA lipid nanoparticles (LNPs) have emerged as powerful modalities for gene therapies to control cancer and infectious and immune diseases. Despite the escalating interest in mRNA-LNPs over the past few decades, endosomal entrapment of delivered mRNAs vastly impedes therapeutic developments. In addition, the molecular mechanism of LNP-mediated mRNA delivery is poorly understood to guide further improvement through rational design. To tackle these challenges, we characterized LNP-mediated mRNA delivery using a library of small molecules targeting endosomal trafficking. We found that the expression of delivered mRNAs is greatly enhanced via inhibition of endocytic recycling in cells and in live mice. One of the most potent small molecules, endosidine 5 (ES5), interferes with recycling endosomes through Annexin A6, thereby promoting the release and expression of mRNA into the cytoplasm. Together, these findings suggest that targeting endosomal trafficking with small molecules is a viable strategy to potentiate the efficacy of mRNA-LNPs.

Imbalance in Glucose Metabolism Regulates the Transition of Microglia from Homeostasis to Disease-Associated Microglia Stage 1.

Microglia undergo two-stage activation in neurodegenerative diseases, known as disease-associated microglia (DAM). TREM2 mediates the DAM2 stage transition, but what regulates the first DAM1 stage transition is unknown. We report that glucose dyshomeostasis inhibits DAM1 activation and PKM2 plays a role. As in tumors, PKM2 was aberrantly elevated in both male and female human AD brains, but unlike in tumors, it is expressed as active tetramers, as well as among TREM2+ microglia surrounding plaques in 5XFAD male and female mice. snRNAseq analyses of microglia without Pkm2 in 5XFAD mice revealed significant increases in DAM1 markers in a distinct metabolic cluster, which is enriched in genes for glucose metabolism, DAM1, and AD risk. 5XFAD mice incidentally exhibited a significant reduction in amyloid pathology without microglial Pkm2 Surprisingly, microglia in 5XFAD without Pkm2 exhibited increases in glycolysis and spare respiratory capacity, which correlated with restoration of mitochondrial cristae alterations. In addition, in situ spatial metabolomics of plaque-bearing microglia revealed an increase in respiratory activity. These results together suggest that it is not only glycolytic but also respiratory inputs that are critical to the development of DAM signatures in 5XFAD mice.

Variable gut pH as a potential mechanism of tolerance to Bacillus thuringiensis subsp. israelensis toxins in the biting midge Culicoides sonorensis.

BACKGROUND: Toxins of Bacillus thuringiensis subsp. israelensis (Bti) are safer alternatives for controlling dipteran pests such as black flies and mosquitoes. The biting midge Culicoides sonorensis (Diptera: Ceratopogonidae) is an important pest of livestock in much of the United States and larval midges utilize semi-aquatic habitats which are permissive for Bti product application. Reports suggest that Bti products are ineffective at killing biting midges despite their taxonomic relation to black flies and mosquitoes. Here, we investigate the toxicity of a Bti-based commercial insecticide and its active ingredient in larval Culicoides sonorensis. A suspected mechanism of Bti tolerance is an acidic larval gut, and we used a pH indicator dye to examine larval Culicoides sonorensis gut pH after exposure to Bti. RESULTS: The lethal concentration to kill 90% (LC90) of larvae of the commercial product (386 mg/L) was determined to be almost 10 000 times more than that of some mosquito species, and no concentration of active ingredient tested achieved 50% larval mortality. The larval gut was found to be more acidic after exposure to Bti which inhibits Bti toxin activity. By comparison, 100% mortality was achieved in larval Aedes aegypti at the product's label rate for this species and mosquito larvae had alkaline guts regardless of treatment. Altering the larval rearing water to alkaline conditions enhanced Bti efficacy when using the active ingredient. CONCLUSION: We conclude that Bti is not practical for larval Culicoides sonorensis control at the same rates as mosquitos but show that alterations or additives to the environment could make the products more effective. © 2024 Society of Chemical Industry.

Geriatric Emergency Medication Safety Recommendations (GEMS-Rx): Modified Delphi Development of a High-Risk Prescription List for Older Emergency Department Patients.

STUDY OBJECTIVE: Half of emergency department (ED) patients aged 65 years and older are discharged with new prescriptions. Potentially inappropriate prescriptions contribute to adverse drug events. Our objective was to develop an evidence- and consensus-based list of high-risk prescriptions to avoid among older ED patients. METHODS: We performed a modified, 3-round Delphi process that included 10 ED physician experts in geriatrics or quality measurement and 1 pharmacist. Consensus members reviewed all 35 medication categories from the 2019 American Geriatrics Society Beers Criteria and ranked each on a 5-point Likert scale (5=highest) for overall priority for avoidance (Round 1), risk of short-term adverse events and avoidability (Round 2), and reasonable medical indications for high-risk medication use (Round 3). RESULTS: For each round, questionnaire response rates were 91%, 82%, and 64%, respectively. After Round 1, benzodiazepines (mean, 4.60 [SD, 0.70]), skeletal muscle relaxants (4.60 [0.70]), barbiturates (4.30 [1.06]), first-generation antipsychotics (4.20 [0.63]) and first-generation antihistamines (3.70 [1.49]) were prioritized for avoidance. In Rounds 2 and 3, hypnotic "Z" drugs (4.29 [1.11]), metoclopramide (3.89 [0.93]), and sulfonylureas (4.14 [1.07]) were prioritized for avoidability, despite lower concern for short-term adverse events. All 8 medication classes were included in the final list. Reasonable indications for prescribing high-risk medications included seizure disorders, benzodiazepine/ethanol withdrawal, end of life, severe generalized anxiety, allergic reactions, gastroparesis, and prescription refill. CONCLUSION: We present the first expert consensus-based list of high-risk prescriptions for older ED patients (GEMS-Rx) to improve safety among older ED patients.