Adipocyte maturation impacts daunorubicin disposition and metabolism.

INTRODUCTION: Acute lymphoblastic leukaemia (ALL) is the most common type of childhood leukaemia with effective chemotherapeutic treatment. However, obesity has been associated with higher ALL chemoresistance rates and lower event-free survival rates. The molecular mechanism of how obesity promotes chemotherapy resistance is not well delineated. OBJECTIVES: This study evaluated the effect of adipocyte maturation on sequestration and metabolism of chemotherapeutic drug daunorubicin (DNR). METHODS: Using targeted LC-MS/MS multi-analyte assay, DNR sequestration and metabolism were studied in human preadipocyte and adipocyte cell lines, where expressions of DNR-metabolizing enzymes aldo-keto reductases (AKR) and carbonyl reductases (CBR) were also evaluated. In addition, to identify the most DNR-metabolizing AKR/CBR isoforms, recombinant human AKR and CBR enzymes were subject to DNR metabolism. The results were further validated by AKR-, CBR-specific inhibitors. RESULTS: This report shows that adipocyte maturation upregulates expressions of AKR and CBR enzymes (by 4- to 60- folds, p < .05), which is positively associated with enhanced sequestration and metabolism of DNR in adipocytes compared to preadipocytes (by ~30%, p < .05). In particular, adipocyte maturation upregulates AKR1C3 and CBR1, which are the predominate metabolic enzyme isoforms responsible for DNR biotransformation to its metabolites. CONCLUSION: Fat is an expandable tissue that can sequester and detoxify DNR when stimulated by obesity, likely through the upregulation of DNR-metabolizing enzymes AKR1C3 and CBR1. Our data partially explains why obese ALL patients may be more likely to become chemoresistant towards DNR, and provides evidence for potential clinical investigation targeting obesity to reduce DNR chemoresistance.

Immunonutrition with Omega-3 Fatty Acid Supplementation in Severe TBI: Retrospective Analysis of Patient Characteristics and Outcomes.

Early evidence-based medical interventions to improve patient outcomes after traumatic brain injury (TBI) are lacking. In patients admitted to the ICU after TBI, optimization of nutrition is an emerging field of interest. Specialized enteral nutrition (EN) formulas that include immunonutrition containing omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been developed and are used for their proposed anti-inflammatory and proimmune properties; however, their use has not been rigorously studied in human TBI populations. A single-center, retrospective, descriptive observational study was conducted at the LAC + USC Medical Center. Patients with severe TBI (sTBI, Glasgow Coma Scale score ≤ 8) who remained in the ICU for ≥2 weeks and received EN were identified between 2017 and 2022 using the institutional trauma registry. Those who received immunonutrition formulas containing n-3 PUFAs were compared with those who received standard, polymeric EN with regard to baseline characteristics, clinical markers of inflammation and immune function, and short-term clinical outcomes. A total of 151 patients with sTBI were analyzed. Those who received immunonutrition with n-3 PUFA supplementation were more likely to be male, younger, Hispanic/Latinx, and have polytrauma needing non-central nervous system surgery. No differences in clinical markers of inflammation or infection rate were found. In multivariate regression analysis, immunonutrition was associated with reduced hospital length of stay (LOS). ICU LOS was also reduced in the subgroup of patients with polytrauma and TBI. This study identifies important differences in patient characteristics and outcomes associated with the EN formula prescribed. Study results can directly inform a prospective pragmatic study of immunonutrition with n-3 PUFA supplementation aimed to confirm the biomechanistic and clinical benefits of the intervention.

Characterization of Induced Current Density During Transcorneal Electrical Stimulation to Promote Neuroprotection in the Degenerating Retina.

OBJECTIVE: Transcorneal electrical stimulation (TES) is a promising approach to delay retinal degeneration by inducing extracellular electric field-driven neuroprotective effects within photoreceptors. Although achieving precise electric field control is feasible in vitro, characterizing these fields becomes intricate and largely unexplored in vivo due to uneven distribution in the heterogeneous body. In this paper, we investigate and characterize electric fields within the retina during TES to assess the potential for therapeutic approaches Methods: We developed a computational model of a rat's head, enabling us to generate predictive simulations of the voltage and current density induced in the retina. Subsequently, an in vivo experimental setup involving Royal College of Surgeon (RCS) rats was implemented to measure the voltage across the retina using identical electrode configurations as employed in the simulations. RESULTS: A stimulation amplitude of 0.2-0.3 mA may be necessary during TES in rats to induce a current density of at least 20 A/m2 in the retina, which is the lower limit for triggering neuroprotective effects according to culture studies on neural cells. Measurement taken from cadaveric pigs' eyes revealed that a stimulation amplitude of 1 mA is necessary for achieving the same current density. CONCLUSION: The computational modeling approach presented in this study was validated with experimental data and can be leveraged for predictive simulations to optimize the electrode design and stimulation parameters of TES. SIGNIFICANCE: Once validated, the flexibility and low research cost of computational models are valuable in optimization studies where testing on live subjects is not feasible.

Effects of age on lacrimal gland bioactive lipids.

PURPOSE: Polyunsaturated fatty acids (PUFA) are a source of bioactive lipids regulating inflammation and its resolution. METHODS: Changes in PUFA metabolism were compared between lacrimal glands (LGs) from young and aged C57BL/6 J mice using a targeted lipidomics assay, as was the gene expression of enzymes involved in the metabolism of these lipids. RESULTS: Global reduction in PUFAs and their metabolites was observed in aged LGs compared to young controls, averaging between 25 and 66 % across all analytes. ꞷ-6 arachidonic acid (AA) metabolites were all reduced in aged LGs, where the changes in prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) were statistically significant. Several other 5-lipoxygenase (5-LOX) mediated metabolites were significantly reduced in the aged LGs, including D-series resolvins (e.g., RvD4, RvD5, and RvD6). Along with the RvDs, several ꞷ-3 docosahexaenoic acid (DHA) metabolites such as 14-HDHA, neuroprotectin D1 (NPD1), Maresin 2 (MaR2), and MaR 1 metabolite (22-COOH-MaR1) were significantly reduced in aged LGs. Similarly, ꞷ-3 eicosapentaenoic acid (EPA) and its metabolites were significantly reduced in aged LGs, where the most significantly reduced was 18-HEPE. Using metabolite ratios (product:precursor) for specific metabolic conversions as surrogate enzymatic measures, reduced 12-LOX activity was identified in aged LGs. CONCLUSION: In this study, global reduction of PUFAs and their metabolites was found in the LGs of aged female C57BL/6 J compared to young controls. A consistent reduction was observed across all detected lipid analytes except for ꞷ-3 docosapentaenoic acid (DPA) and its special pro-resolving mediator (SPM) metabolites in aged mice, suggesting an increased risk for LG inflammation.

Effects of APOE4 on omega-3 brain metabolism across the lifespan.

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), have important roles in human nutrition and brain health by promoting neuronal functions, maintaining inflammatory homeostasis, and providing structural integrity. As Alzheimer's disease (AD) pathology progresses, DHA metabolism in the brain becomes dysregulated, the timing and extent of which may be influenced by the apolipoprotein E ε4 (APOE4) allele. Here, we discuss how maintaining adequate DHA intake early in life may slow the progression to AD dementia in cognitively normal individuals with APOE4, how recent advances in DHA brain imaging could offer insights leading to more personalized preventive strategies, and how alternative strategies targeting PUFA metabolism pathways may be more effective in mitigating disease progression in patients with existing AD dementia.

Unveiling Drivers of Retinal Degeneration in RCS Rats: Functional, Morphological, and Molecular Insights.

Retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, significantly contribute to adult blindness. The Royal College of Surgeons (RCS) rat is a well-established disease model for studying these dystrophies; however, molecular investigations remain limited. We conducted a comprehensive analysis of retinal degeneration in RCS rats, including an immunodeficient RCS (iRCS) sub-strain, using ocular coherence tomography, electroretinography, histology, and molecular dissection using transcriptomics and immunofluorescence. No significant differences in retinal degeneration progression were observed between the iRCS and immunocompetent RCS rats, suggesting a minimal role of adaptive immune responses in disease. Transcriptomic alterations were primarily in inflammatory signaling pathways, characterized by the strong upregulation of Tnfa, an inflammatory signaling molecule, and Nox1, a contributor to reactive oxygen species (ROS) generation. Additionally, a notable decrease in Alox15 expression was observed, pointing to a possible reduction in anti-inflammatory and pro-resolving lipid mediators. These findings were corroborated by immunostaining, which demonstrated increased photoreceptor lipid peroxidation (4HNE) and photoreceptor citrullination (CitH3) during retinal degeneration. Our work enhances the understanding of molecular changes associated with retinal degeneration in RCS rats and offers potential therapeutic targets within inflammatory and oxidative stress pathways for confirmatory research and development.

Synthesis of site-specific Fab-drug conjugates using ADP-ribosyl cyclases.

Targeted delivery of small-molecule drugs via covalent attachments to monoclonal antibodies has proved successful in clinic. For this purpose, full-length antibodies are mainly used as drug-carrying vehicles. Despite their flexible conjugation sites and versatile biological activities, intact immunoglobulins with conjugated drugs, which feature relatively large molecular weights, tend to have restricted tissue distribution and penetration and low fractions of payloads. Linking small-molecule therapeutics to other formats of antibody may lead to conjugates with optimal properties. Here, we designed and synthesized ADP-ribosyl cyclase-enabled fragment antigen-binding (Fab) drug conjugates (ARC-FDCs) by utilizing CD38 catalytic activity. Through rapidly forming a stable covalent bond with a nicotinamide adenine dinucleotide (NAD+ )-based drug linker at its active site, CD38 genetically fused with Fab mediates robust site-specific drug conjugations via enzymatic reactions. Generated ARC-FDCs with defined drug-to-Fab ratios display potent and antigen-dependent cytotoxicity against breast cancer cells. This work demonstrates a new strategy for developing site-specific FDCs. It may be applicable to different antibody scaffolds for therapeutic conjugations, leading to novel targeted agents.

Loss of 15-Lipoxygenase in Retinodegenerative RCS Rats.

Retinitis pigmentosa (RP) is a retinal degenerative disease associated with a diversity of genetic mutations. In a natural progression study (NPS) evaluating the molecular changes in Royal College of Surgeons (RCS) rats using lipidomic profiling, RNA sequencing, and gene expression analyses, changes associated with retinal degeneration from p21 to p60 were evaluated, where reductions in retinal ALOX15 expression corresponded with disease progression. This important enzyme catalyzes the formation of specialized pro-resolving mediators (SPMs) such as lipoxins (LXs), resolvins (RvDs), and docosapentaenoic acid resolvins (DPA RvDs), where reduced ALOX15 corresponded with reduced SPMs. Retinal DPA RvD2 levels were found to correlate with retinal structural and functional decline. Retinal RNA sequencing comparing p21 with p60 showed an upregulation of microglial inflammatory pathways accompanied by impaired damage-associated molecular pattern (DAMP) clearance pathways. This analysis suggests that ALXR/FPR2 activation can ameliorate disease progression, which was supported by treatment with an LXA4 analog, NAP1051, which was able to promote the upregulation of ALOX12 and ALOX15. This study showed that retinal inflammation from activated microglia and dysregulation of lipid metabolism were central to the pathogenesis of retinal degeneration in RP, where ALXR/FPR2 activation was able to preserve retinal structure and function.

Hydroxypropyl-Beta Cyclodextrin Barrier Prevents Respiratory Viral Infections: A Preclinical Study.

The emergence and mutation of pathogenic viruses have been occurring at an unprecedented rate in recent decades. The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed into a global public health crisis due to extensive viral transmission. In situ RNA mapping has revealed angiotensin-converting enzyme 2 (ACE2) expression to be highest in the nose and lower in the lung, pointing to nasal susceptibility as a predominant route for infection and the cause of subsequent pulmonary effects. By blocking viral attachment and entry at the nasal airway using a cyclodextrin-based formulation, a preventative therapy can be developed to reduce viral infection at the site of entry. Here, we assess the safety and antiviral efficacy of cyclodextrin-based formulations. From these studies, hydroxypropyl beta-cyclodextrin (HPBCD) and hydroxypropyl gamma-cyclodextrin (HPGCD) were then further evaluated for antiviral effects using SARS-CoV-2 pseudotypes. Efficacy findings were confirmed with SARS-CoV-2 Delta variant infection of Calu-3 cells and using a K18-hACE2 murine model. Intranasal pre-treatment with HPBCD-based formulations reduced viral load and inflammatory signaling in the lung. In vitro efficacy studies were further conducted using lentiviruses, murine hepatitis virus (MHV), and influenza A virus subtype H1N1. These findings suggest HPBCD may be used as an agnostic barrier against transmissible pathogens, including but not limited to SARS-CoV-2.

Scaling up polarized RPE cell supernatant production on parylene membrane.

Age-related macular degeneration (AMD), a leading cause of vision loss, primarily arises from the degeneration of retinal pigment epithelium (RPE) and photoreceptors. Current therapeutic options for dry AMD are limited. Encouragingly, cultured RPE cells on parylene-based biomimetic Bruch's membrane demonstrate characteristics akin to the native RPE layer. In this study, we cultivated human embryonic stem cell-derived polarized RPE (hESC-PRPE) cells on parylene membranes at both small- and large-scale settings, collecting conditioned supernatant, denoted as PRPE-SF. We conducted a comprehensive analysis of the morphology of the cultured hESC-RPE cells and the secreted growth factors in PRPE-SF. To evaluate the in vivo efficacy of these products, the product was administered via intravitreal injections of PRPE-SF in immunodeficient Royal College of Surgeons (iRCS) rats, a model for retinal degeneration. Our study not only demonstrated the scalability of PRPE-SF production while maintaining RPE cell phenotype but also showed consistent protein concentrations between small- and large-scale batches. We consistently identified 10 key factors in PRPE-SF, including BMP-7, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, MANF, PEDF, PDGF-AA, TGFß1, and VEGF. Following intravitreal administration of PRPE-SF, we observed a significant increase in the thickness of the outer nuclear layer (ONL) and photoreceptor preservation in iRCS rats. Furthermore, correlation analysis revealed that IGFBP-3, IGFBP-4, MANF, PEDF, and TGFß1 displayed positive associations with in vivo bioactivity, while GDF-15 exhibited a negative correlation. Overall, this study highlights the feasibility of scaling up PRPE-SF production on parylene membranes without compromising its essential constituents. The outcomes of PRPE-SF administration in an animal model of retinal degeneration present substantial potential for photoreceptor preservation. Moreover, the identification of candidate surrogate potency markers, showing strong positive associations with in vivo bioactivity, lays a solid foundation for the development of a promising therapeutic intervention for retinal degenerative diseases.