FEBS Open Bio

Rationale: Neuroblastoma is a devastating pediatric malignancy, for which surgical resection is a key factor in long-term survival. However, there are significant challenges in its resection, particularly in high-risk disease, as neuroblastoma encases surrounding critical structures, is often difficult to distinguish from desmoplastic or scar tissue, and can carry occult deposits of disease not readily identified on preoperative imaging or intraoperative visualization. Building on the principles of fluorescent and radio-guided surgery, in combination with the known overexpression of GD2 in neuroblastoma, we sought to develop and optimize 111In-Dinutuximab-IRDye800, a dual-modality GD2-targeted intraoperative molecular imaging agent, for use in pediatric neuroblastoma to help enhance patient safety while facilitating a more complete resection. Methods: Dinutuximab was conjugated to IRDye800 and DTPA, then radiolabeled with Indium-111 to yield 111In-Dinutuximab-IRDye800. Optimization occurred through ELISA assay to assess binding affinity, fluorescence intensity analysis to determine the optimal fluorescent degree of labeling, and phototoxicity testing through flow cytometry. Rodent models of neuroblastoma were then generated through injection of SK-N-BE(2) human neuroblastoma cells into the left adrenal glands of nude mice or RNU rats. A series of fluorescent and gamma biodistributions was performed, varying the dose, timing, and specific activity of the tracer. Tumor and organ uptake of the tracer was compared with one- or two-way ANOVA as appropriate, with Sidaks multiple comparison test to compare tumor uptake to individual organs. Once optimization was complete, a clinically significant events study modeled after human clinical trials was performed to evaluate the in vivo capabilities of 111In-Dinutuximab-IRDye800. Results: Increased ratios of IRDye800 per antibody led to decreased binding affinity for GD2 and was associated with formulation instability without significant return on fluorescence intensity. Specific activity of the tracer was not found to impact overall biodistribution of the tracer. A 45-50 microgram dose of 111In-Dinutuximab-IRDye800 with ratios around 1 DTPA and 1-1.5 IRDye800 per antibody imaged 4 days after tracer administration was found to be the optimal combination that maximized detectable tumor-specific signal. In the clinically significant events study mirroring human IMI clinical trials, fluorescent guidance identified additional malignant lesions not originally detected under white light in 64% of rodents. Conclusions: 111In-Dinutuximab-IRDye800 is a dual-modality GD2-targeted intraoperative imaging agent that is well-poised for clinical translation. As it preserves tumor specificity, yields clinically meaningful radiofluorescent signal, and is well-tolerated without adverse events after optimization was completed, it carries the potential to positively impact the safety and completeness of neuroblastoma resection.

Ulcerative colitis (UC) and radiation enteropathy involve intestinal epithelial injury, barrier dysfunction, and inflammation, but effective treatments remain limited. This study evaluated MXC-017, a novel vimentin-targeting urea compound, in mouse models of dextran sulfate sodium (DSS)-induced colitis and radiation-induced enteropathy. Acute colitis was induced in C57BL/6 mice using 3.5% DSS for seven days, followed by regular water for seven days. Radiation enteropathy was induced by 13 Gy total abdominal irradiation. Mice received MXC-017 (150 mg/kg) or vehicle. Disease activity, intestinal permeability, inflammatory and epithelial markers, and histopathology were assessed. MXC-017's effects on cancer stem cell frequency, sphere formation, and migration were also examined in PC-3 and DU-145 prostate cancer cells. MXC-017 reduced DSS-induced colitis severity, accelerated weight recovery, lowered disease activity, partially preserved colon length, and restored barrier function. It also reduced proinflammatory cytokines, macrophage infiltration, epithelial injury, and goblet cell loss while preserving epithelial proliferation and markers of intestinal stem cell function and tight-junction integrity. Following irradiation, MXC-017 improved weight recovery, reduced intestinal permeability, preserved epithelial architecture, and partially mitigated villus shortening. Importantly, MXC-017 did not protect prostate cancer stem cells from radiation. Instead, it reduced stem cell frequency, sphere-forming capacity, and cancer cell migration. These findings support vimentin targeting with MXC-017 as a potential treatment for UC and radiation-induced intestinal toxicity and as an adjunct to radiotherapy for pelvic and abdominal malignancies.

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown great promise in treating bladder cancer and are now part of the standard treatment for advanced disease. However, many patients still fail to respond to treatment and at present many biomarkers are assessed but have yet shown only limited results. Therefore, with the advent of combination treatments and the increase of immune related adverse event, the search for reliable predictive biomarkers is paramount. Using a multiplexed enhanced FRET-FLIM based technique (QF-Pro) we quantified the interaction of PD-1/PD-L1, CTLA-4/CD80 and TIGIT/CD155 immune checkpoints in a pre-treatment TMA of 46 patients treated with atezolizumab. The association between higher PD-1/PD-L1 ICP interaction state and treatment efficacy was demonstrated in the male sample cohort, where it identified patients with better PFS. Conversely, patients exhibiting higher CTLA-4/CD80 engagement had a worse response to atezolizumab. Remarkably, the dual assessment of patients with high PD-1/PD-L1 and low CTLA-4/CD80 allowed to identify the best responders. These results indicate that the monitoring of patients immune profile in urothelial carcinoma might be critical in identifying patients who may benefit from combination therapy.

Introduction: Gliomas integrate into neural circuits and heighten neuronal excitability, engaging in bidirectional communication whereby neuronal activity promotes tumor growth and proliferation. Aging reshapes the brain microenvironment through extracellular matrix changes, altered secretory factors, and immune dysfunction, creating conditions permissive to tumorigenesis and limiting immunotherapy efficacy in glioblastoma. However, its effect on neuronal excitability and signaling in glioblastoma remains poorly understood. Methods: We developed a novel classification system for glioblastoma by leveraging three classes of DNA methylation-based aging biomarkers: chronological, biological, and mitotic clocks. This approach stratified tumors into accelerated and decelerated epigenetic aging subtypes, which we then characterized at the molecular, functional, and clinical levels using multimodal analyses. Guided by these profiles, we evaluated the in vitro effects of the FDA-approved agents levetiracetam and riluzole, alone and in combination with temozolomide, on U87MG and A172 cell lines. Specifically, we assessed changes in cell viability, apoptosis, and the expression of marker genes related to stemness, neuronal hyperexcitability, and immunosuppression. Results: Tumors with decelerated epigenetic aging showed expression modules and CpG hypomethylation associated with neuronal activity and stemness, and carried significantly worse prognosis. Single-cell and spatial multi-omics analyses revealed enrichment for neurons and malignant neural stem-like cells in these tumors. They also displayed enhanced intercellular communication, driven predominantly by glutamate signaling across the malignant, neuronal, and immune compartments of the tumor microenvironment. In vitro pharmacological inhibition of glutamatergic signaling with levetiracetam and riluzole reduced cell viability, induced apoptosis, and suppressed expression of stemness, neuronal hyperexcitability, and immunosuppression markers. Both agents potentiated the cytotoxic and apoptotic effects of temozolomide, supporting glutamatergic inhibition as a strategy for improving chemosensitivity. Conclusion: By establishing a framework for decoding glioblastoma heterogeneity through epigenetic aging, we identified the glutamatergic pathway as a clinically actionable vulnerability. Our findings suggest that combining anti-glutamatergic therapies with temozolomide exerts synergistic antitumor effects while mitigating adverse chemotherapy-induced phenotypes, such as increased stemness, neuronal hyperexcitability, and immunosuppression, thereby laying the groundwork for novel therapeutic strategies.

Background: Acute phase response is an early immunometabolic response to brain injuries, primarily coordinated by the liver via the activation of acute phase proteins. These immune responses can be both beneficial, promoting tissue repair, and detrimental, exacerbating neurological deficits, if not properly controlled. Despite the central role of the liver in immunometabolism, how hepatic metabolism dynamically adapts to traumatic brain injury remains under explored, primarily due to limited liver-specific modalities that can assess metabolic pathways in vivo. 13C MRI utilizing hyperpolarized 13C-pyruvate can assess key regulatory enzyme activities in hepatic metabolism. Methods: Rats with controlled cortical impact were studied in vivo using hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate under fed and fasted conditions 3-4 days after injury. Hyperpolarized 13C products, including [13C]bicarbonate from [1-13C]pyruvate and [5-13C]glutamate, [1-13C]acetyl-L-carnitine, and [2-13C]phosphoenolpyruvate from [2-13C]pyruvate, were evaluated to assess mitochondrial and gluconeogenic metabolism. In parallel, liver tissues were collected following [U-13C3]pyruvate injection for NMR isotopomer analysis of phosphoenolpyruvate, glucose, and glutamate. Results: While no metabolic differences were detected under fed condition, [13C]bicarbonate and [2-13C]phosphoenolpyruvate increased after brain injury under fasted condition, indicating an upregulation of the hepatic gluconeogenic pathway after injury. 13C NMR of liver tissue extracts from injured rats showed an elevated [2,3-13C2]glutamate-to-[4,5-13C2]glutamate ratio and increased 13C-labeling in phosphoenolpyruvate than controls, confirming enhanced hepatic gluconeogenic pathway. Conclusion: This study demonstrates that hepatic acute phase response to brain injuries can be monitored in vivo by hyperpolarized pyruvate, which may be further utilized for longitudinal immunometabolic evaluation of the liver during pathogenesis and therapeutic interventions.

BCL2L13 is a mitochondrial BCL2 family protein linked to mitophagy and ceramide metabolism, but its role in NSCLC metastatic plasticity remains unclear. Human lung cancer Tissue Microarray and matched patient specimens showed subtype and site dependent BCL2L13 expression, with higher cytoplasmic granular staining in primary NSCLC and reduced, heterogeneous staining in lymph node metastases, most evident in adenocarcinoma and squamous cell carcinoma. Because Epithelial mesenchymal transition and anoikis resistance are central requirements for metastatic dissemination, this primary to node attenuation provided the rationale to test BCL2L13 knockdown and overexpression in metastasis relevant NSCLC models. In A549 and LLC cell lines. TGF beta 1 induced coordinated mitophagy and EMT with mitochondrial enrichment of BCL2L13. BCL2L13 knockdown impaired TGF beta 1 and carbonyl cyanide m chlorophenyl hydrazone associated mitophagy, reducing LC3 beta mitochondria colocalization, TOMM20, LAMP1 overlap and mitochondrial LC3 II, p62, TOMM20 turnover; BNIP3 and NIX redistribution did not compensate. BCL2L13 loss enhanced EMT marker switching and migration, whereas overexpression partially opposed these changes. During detachment, BCL2L13 knockdown reduced anoikis associated apoptosis despite preserved mitochondrial recruitment of BAX, BAK, BNIP3,NIX, altered BID processing, non parallel caspase activity and shifted FAK phosphorylation. Pharmacological autophagy modulation did not reverse this anoikis phenotype. Lipidomics identified adhesion state dependent ceramide synthases CerS2, CerS6 linked sphingolipid remodeling: BCL2L13 knockdown increased C24 linked sphingolipid species in attached cells but reduced C16, C24 ceramide related profiles during anoikis. These findings identify BCL2L13 downregulation as a metastasis associated mitochondrial-lipid state that limits mitophagic quality control while favoring EMT and detachment survival in NSCLC adenocarcinoma.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by progressive motor neuron degeneration. Mutations in the SOD1 gene represent the second most common genetic cause of ALS (ALS), and distinct SOD1 missense variants present with markedly different clinical profiles. A4V leads to an aggressive form of the disease (median survival [~]1y), H46R confers a mild, slowly progressive course and I113T exhibits an intermediate phenotype. The molecular basis by which these mutations produce divergent clinical outcomes remains poorly understood. We performed extensive classical molecular dynamics simulations of wild-type SOD1 and the three ALS-associated variants in the apo monomeric state to attempt to investigate the mechanisms behind such phenotypic differences. Structural stability, global compactness, and conformational flexibility, as well as analysis of collective motions between residues and estimation of free energy, were assessed. The H46R, A4V, and I113T variants exhibited distinct dynamic behaviours, highlighting differences in structural stability, local flexibility, and intramolecular interactions. These findings suggest that specific structural regions may contribute differently to protein dysfunction and could represent key elements for understanding the relationship between molecular dynamic properties and the differing clinical severity associated with these variants. Most strikingly, H46R exhibited exceptional structural stability across every analytical level, the lowest global deviation, most attenuated local flexibility, strongest internal dynamic coordination, and the deepest, most confined free energy basins of any system examined. This convergent multi-layered evidence of structural restraint provides a compelling mechanistic basis for the mild and slowly progressive clinical course of H46R ALS, suggesting that enhanced conformational rigidity, rather than bulk destabilisation, is the defining biophysical feature of this variant, and that its pathogenic mechanism operates through a route fundamentally decoupled from the aggregation-driven toxicity that characterises the more aggressive SOD1-ALS mutations.

Background: A dose finding trial evaluated safety and adherence for pre-cardiac surgery administration of sodium valproate. Integrated multi-omics analyses of myocardium were used to characterise mechanisms underlying the treatment effects. Methods: Adults undergoing cardiac surgery were randomised 1:1:1:1 with concealed allocation to no treatment (Controls), sodium valproate 15mg/kg/day for 1-2 weeks, 15mg/kg/day for 4-6 weeks, or 25mg/kg/day for 4-6 weeks pre-surgery. The primary analysis evaluated adherence and toxicity. Myocardial injury was defined by high sensitivity serum troponin at 24 hours post-surgery. Single-nucleus Assay for Transposase-Accessible Chromatin with sequencing (snATACseq) and single nuclei RNA sequencing (snRNAseq) of myocardial biopsies collected at surgery assessed treatment effects on chromatin accessibility and gene expression. Candidate mechanisms were validated in in vitro. Results: The analysis cohort included 42 participants enrolled between January 2020 and August 2024. Non-compliance (38%) was highest with longer and higher dosing. Sodium valproate 15mg/kg/day for 1-2 weeks had the highest levels of complete treatment adherence (70%), with 20% experiencing moderate/severe drug related adverse effects. An as-treated analyses demonstrated reductions in troponin release in participants receiving Valproate[≤]14 days. Myocardial biopsies from trial participants demonstrated activation of hormetic p53 and Akt-GSK-3{beta} ferroptosis protection pathways. Treatment effects were not attributable to chromatin accessibility. Treatment >14 days resulted in a heart failure phenotype with suppression of ferroptosis protection pathways, endothelial mesenchymal transition, and increased myocardial injury. Conclusions: Sodium valproate 15mg/kg/day for [≤]14 days pre-surgery is well tolerated in adults awaiting cardiac surgery. This treatment was associated with upregulation of ferroptosis protection pathways and reductions in myocardial injury.

Hybridoma screening in semi-solid medium typically employs antigens labeled with visible fluorophores (e.g., FITC, AF488) to enable single-step identification of antibody-secreting clones. However, conventional chemical conjugation via NHS-esters or isothiocyanate groups frequently modifies lysine residues located within epitopes, potentially abrogating antibody recognition of these critical regions. Here, we describe a SpyTag SpyCatcher-based site-specific labeling strategy that circumvents epitope damage during semi-solid medium screening. A 16-amino-acid SpyTag was genetically fused to the C-terminus of the target antigen, enabling covalent conjugation to an sfGFP SpyCatcher fluorescent probe. In semi-solid medium supplemented with SpyTag-antigen and sfGFPSpyCatcher, positive hybridoma clones were readily identified by distinct fluorescent halos, whereas negative clones showed no detectable signal. Notably, the site-specific method yielded a significantly higher frequency of fluorescence-positive clones compared to the conventional AF488-labeled antigen method, suggesting that epitope preservation enhances screening recovery. Furthermore, this approach did not impair hybridoma growth or final clone positivity, offering a simple, rapid, and epitope-compatible method for monoclonal antibody screening.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

Hepatocellular carcinoma (HCC) is particularly aggressive and difficult to treat. Due to the lack of early clinical diagnosis and the unsatisfactory clinical treatment effect, it is particularly important to identify novel markers that can predict tumor behavior in HCC. biogenesis of ribosomes BRX1 (BRIX1) is abundant in various tissues of the human body. However, the regulatory mechanisms and its role in various tissues are not fully understood. Here, we analyzed the expression pattern of BRIX1 in HCC from public gene expression databases and tissue samples from clinical HCC. We confirmed that BRIX1 was upregulated in both HCC cell lines and HCC paraffin section samples. BRIX1 depletion significantly dicreased the capacity of cells to grow and migrate in vitro, and knockdown BRIX1 suppressed tumor growth in xenograft tumor model. Mechanistically, BRIX1 depletion suppressed the MAPK/ERK pathway, as reflected by reduced phosphorylated ERK (p-ERK) levels. In summary, we provide a rational clue for the further investigation of BRIX1 as an invaluable biological marker for diagnosing and predicting prognosis of patients with HCC.

Background: Prostate cancer enzalutamide treatment is approved at a standard dose of 160 mg daily. Concerns for real-world patients -- older and more fragile than those enrolled in clinical trials -- have prompted consideration of initiating treatment with lower doses, but the long-term efficacy of this approach remains unknown. We evaluate the long-term survival and longevity in patients treated with standard versus upfront low-dose enzalutamide. Methods: Retrospective analysis of 151 patients treated with enzalutamide (102 receiving 160 mg; 49 receiving [≤]80 mg) between 2014--2021 at the Centre Hospitalier Universitaire de Martinique, with complete follow-up through end of life (98.7% completeness of follow-up). Primary outcomes were overall survival (OS), progression-free survival (PFS), and longevity (attained age). Results: Doses [≤]80 mg were associated with longer median OS (36.3 vs. 20.7 months), improved restricted mean OS (difference of 0.7 years, p=0.05), and enhanced longevity (median 82.5 vs. 78.3 years, p=0.004). PSA response rate at 12 weeks was higher with lower-dose (71.4% vs. 48.8%, p=0.016). In multivariable models adjusted for prognostic factors, [≤]40 mg compared with 160 mg was non-inferior regarding OS (HR=0.61, 95% CI 0.36--1.06), superior regarding PFS (HR=0.59, 95% CI 0.35--0.99), and superior regarding longevity (HR=0.48, 95% CI 0.28--0.84). Bone metastasis, poor performance status, PSA response, time to PSA nadir, and disease duration were independent predictors of outcomes. A post-hoc analysis revealed a strong association between dose and physician-prescribing profiles, ranging from "endorse-lowest-dose" to "never-deviate-from-full-dose". Conclusions: Lower doses of enzalutamide were non-inferior to full-dose. Dose-adapted strategies warrant further investigation.

IMPORTANCE: A recent meta-analysis showed that chemoimmunotherapy was associated with improved overall survival (OS) compared with immune checkpoint inhibitor (ICI) monotherapy for programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) [&ge;] 50% advanced non-small-cell lung cancer (NSCLC). However, whether this benefit reflects chemotherapy effect or ICI heterogeneity remains unclear. OBJECTIVE: To reassess the survival benefit of adding chemotherapy to ICI monotherapy using agent-stratified comparisons anchored to chemotherapy. DATA SOURCES: The 24 phase 3 randomized clinical trials included in the original meta-analysis (search date, August 3, 2025). DATA EXTRACTION AND SYNTHESIS: Hazard ratios (HRs) for OS and progression-free survival (PFS) were extracted from each trial in the original meta-analysis. Two analytic frameworks were used: within-agent comparisons (same ICI in both chemoimmunotherapy and monotherapy) and across-agent comparisons (ICI in one treatment strategy only). For within-agent comparisons, a two-stage random-effects meta-analysis was conducted. In stage 1, ICI-specific HRs for chemoimmunotherapy and ICI monotherapy versus chemotherapy were pooled and their ratio was calculated (RHR = HRchemoimmuno/HRmono; RHR < 1 favors chemoimmunotherapy). The RHRs were pooled in stage 2. For across-agent comparisons, RHR was derived from pooled HRs by treatment strategy. MAIN OUTCOMES AND MEASURES: Endpoints were OS and PFS. RESULTS: In within-agent comparisons (4 ICIs; 13 trials; N = 3252), pooled RHR was 0.94 (95% CI, 0.78-1.13; P = .48; I2 = 0.0%) for OS and 0.85 (95% CI, 0.68-1.06; P = .14; I2 = 0.0%) for PFS. In across-agent comparisons (7 ICIs; 11 trials; N = 2231), RHR favored chemoimmunotherapy for OS (0.68; 95% CI, 0.50-0.92; P = .01) and PFS (0.46; 95% CI, 0.37-0.58; P < .001). In a sensitivity analysis restricted to trials of NCCN-recommended regimens, pooled RHR was 1.02 (95% CI, 0.81-1.28; P = .87) for OS. CONCLUSIONS AND RELEVANCE: In the within-agent comparisons, adding chemotherapy to ICI monotherapy did not improve OS or PFS in patients with PD-L1 TPS [&ge;] 50% advanced NSCLC. The benefit in the original meta-analysis appears driven by across-ICI heterogeneity. These findings are consistent with ICI monotherapy as a standard first-line option and underscore the need for agent-level stratification in across-trial comparisons.

Neutron scattering is a powerful technique to investigate atomic structures and molecular dynamics of proteins at the nano-scale. When it comes to dynamics, incoherent and coherent scattering respectively provide information on the single and collective dynamics of nuclei. In proteins, hydrogen has the highest incoherent cross-section, and it is common practice to overlook the contribution of coherent terms stemming from all nuclei. However, the fast collective dynamics of heavier nuclei could also be studied if coherent scattering and incoherent scattering were experimentally separated. The recent advent of polarized neutron spectroscopy with sufficient flux and energy resolution has made it possible, and opens new perspectives to investigate the relative importance of coherent scattering and the information it provides on biological samples. The present study reports on the use of polarized quasi-elastic neutron scattering (QENS) and the application of a minimalistic model adapted to both individual and collective dynamics. Using a perdeuterated green fluorescent protein as a model globular protein, the study provides an interpretation of the dynamical parameters obtained with QENS, and a comparative study of the Elastic Coherent and Incoherent Scattering Factor. Based on both experiments and calculations, we discuss the relative importance of distinct and self components of coherent scattering, which is often wrongly assumed to be representative of collective dynamics only. The results highlight the current impediments rendering complicated a straightforward analysis of fast collective dynamics in hydrated protein samples.

Liver metastases (LM) remain a major cause of death from different cancer types, in particular malignancies of the gastrointestinal tract. Liver metastases predict a poor response to immunotherapy due, among others, to the immunotolerant microenvironment (ME) of the liver and loss of local and systemic cytotoxic T cells. Thus, strategies that can reprogram the immune ME of the liver and restore cytotoxic T cell reactivity are being sought. We previously reported that estrogen signaling blockade impedes the growth of LM by reducing MDSC accumulation and monocyte/macrophage polarization. The aim of this study was to elucidate the underlying mechanism(s) and assess whether estrogen signaling in the myeloid lineage was driving the immunotolerant ME of LM. To this end, we generated mice with conditional myeloid cell-specific deletions of estrogen receptors (ER) or ER{beta} and analyzed in these mice the effect of ER loss on the liver immune ME and the outgrowth of LM. In mice with ER, but not with ER{beta} deletion, we observed a marked reduction in the growth of murine colon carcinoma MC-38 liver metastases as compared to their respective controls. Flow cytometry and immunohistochemistry revealed a decrease in macrophages that were polarized to the pro-tumorigenic M2-like phenotype and a concomitant increase in activated CD8+ T and NK cells relative to controls. Bulk RNAseq analysis performed on hepatic immune cells infiltrating the liver revealed changes in the expression of key cytokines/chemokines mediating immune cell recruitment, activation and polarization, including Ccl5 (upregulated) and Csf1 (downregulated). Taken together, the data suggest that ER signaling in myeloid-derived cells programs the immune landscape and contributes to an immunosuppressive and metastases-growth permissive ME in the liver.

Cholesterol efflux is an important determinant of cellular lipid homeostasis. However, how microglial excessive cholesterol accumulation affects neuronal synaptic integrity remains poorly understood, particularly in the context of Alzheimer's disease. Here, we utilized a conditional knockout mouse model targeting the cholesterol transporters ABCA1 and ABCG1 in microglia. The microglia-specific ABCA1/ABCG1 deficiency triggered marked cholesterol accumulation, microglial hypertrophy, downregulation of the homeostatic marker P2ry12, and upregulation of the reactivity-associated marker CD11b, indicating shift toward a reactive phenotype. This phenotype was accompanied by increased reactive oxygen species, consistent with enhanced oxidative stress in ABCA1/ABCG1-deficient microglia compared with control. Using organotypic hippocampal slice cultures, we investigated the downstream neuronal outcomes of microglial ABCA1/ABCG1 deficiency. Under basal conditions, microglial ABCA1/ABCG1 knockdown did not significantly alter dendritic spine density in CA1 pyramidal neurons. However, upon exposure to amyloid-beta (A{beta}) stress, microglial ABCA1/ABCG1 deficiency markedly exacerbated dendritic spine loss in CA1 pyramidal neurons. Taken together, our findings highlight an important role for ABCA1/ABCG1-dependent cholesterol efflux in maintaining microglial homeostasis and limiting neuronal synaptic vulnerability to A{beta}-associated stress. These results support further investigation of microglial cholesterol transport as a potential target for preserving synaptic resilience in Alzheimer's disease.

Carbapenems are the most potent {beta}-lactams, key antibiotics for healthcare-associated infections by Gram-negative bacteria and evade hydrolysis by most {beta}-lactamases, but are increasingly threatened by emergence of enzymes exhibiting hydrolytic activity towards them. Of the four recognised {beta}-lactamase subclasses, class A (active-site serine enzymes that hydrolyse {beta}-lactams via a covalent acylenzyme intermediate) is the most widely disseminated and, while the majority of such enzymes react with carbapenems to form long-lasting acylenzyme complexes, several possess carbapenem-hydrolyzing activity (carbapenemases). Here, we investigate the basis for these differences in a panel of class A {beta}-lactamases using molecular dynamics (MD) simulations of the respective acylenzyme complexes and tetrahedral intermediates (TI). The simulations reveal multiple features associated with catalytic activity across the spectrum of enzymes tested, including more extensive interactions of the carbapenem acylenzyme carbonyl and generally increased lifetimes of active site water molecules positioned for deacylation. Analysis of the dynamic trajectories shows carbapenemases to have reduced root mean-squared fluctuation (RMSF) differences between the acylenzyme and TI, that are not limited to the active site, indicating that the acylenzyme complex is pre-organised for reaction in carbapenemases but not in carbapenem-inhibited enzymes. Similarly, Principal Component Analysis (PCA) of acylenzyme and TI dynamics shows greater overlap between the two states in carbapenemases, providing further evidence for acylenzyme pre-organisation. Such simulations may represent an effective computational assay able to identify enzymes with carbapenemase activity at relatively modest computational cost.

Patients with history of neck radiation for Human Papilloma Virus (HPV)-associated head and neck cancer experience rapid progression of carotid artery stenosis. The present study sought to determine whether screening and treating asymptomatic carotid artery stenosis in patients with a history of neck radiation is cost-effective. This study is a cost-utility analysis using a probabilistic Markov model over a thirty-year time horizon assessing carotid screening and treatment to avoid neurologic consequences of neck radiation for HPV-associated head and neck cancer. A strategy of no carotid surveillance was associated with a 14.8% cumulative risk of stroke and a strategy of ultrasound surveillance and treatment with TCAR was associated with a 3.0% cumulative risk of stroke. The latter had a median incremental cost of $1.04 million USD and provided a median 39.1 additional QALYs, resulting in a median incremental cost-effectiveness ratio of $26,556 per QALY. In conclusion, this study suggests that ultrasound surveillance and treatment with TCAR for asymptomatic carotid artery stenosis is likely to be cost-effective for patients who have been successfully treated with radiation therapy for HPV-associated head and neck cancer.

Background: Stress research in nursing education has largely focused on distress, stressors, and negative outcomes, although challenging experiences may also support motivation, confidence, learning, and growth when appraised positively. Objective: To develop and evaluate the psychometric properties of the Nursing Student Positive Stress Scale (NSPSS). Design: A methodological instrument development and psychometric evaluation study. Methods: The NSPSS was developed using a deductive, theory-driven approach informed by the transactional theory of stress and coping and positive psychology perspectives. Content validity was assessed by an international nursing expert panel. Psychometric evaluation used national survey data from nursing students in New Zealand. Of 539 responses, 507 were analysed. Exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) were conducted using separate subsamples. Internal consistency was assessed using Cronbach's alpha and McDonald's omega, and convergent validity through correlation with Perceived Stress Scale-10 scores. Results: Content validity was strong (I-CVI = .88-1.00; S-CVI/Ave = .975; S-CVI/UA = .800). EFA identified a dominant factor explaining 41.38% of variance (loadings = .528-.735). CFA supported a two-context Academic and Clinical Positive Stress model with correlated residuals between five parallel item pairs, chi-square(29) = 60.49, CFI = .970, TLI = .954, RMSEA = .063, SRMR = .065. Internal consistency was good (alpha = .839; omega = .843). NSPSS scores correlated negatively with PSS-10 scores (r = -.298, p < .001). Conclusion: The NSPSS demonstrated strong content validity, preliminary evidence of structural and convergent validity, and good internal consistency reliability for assessing positive stress appraisal among nursing students. Further validation in independent samples is warranted.

Ethnopharmacological relevance: Australian Indigenous medicinal plants represent a valuable yet underexplored source of bioactive compounds with potential therapeutic relevance. The Iningai community of Central Queensland has traditionally used native plants to manage conditions associated with inflammation, pain, infection, and general illness. Scientific evaluation of these plants may provide evidence for their customary applications and identify bioactivities relevant to anticancer biodiscovery. Aim of the study: This study evaluated leaf and stem extracts of seven medicinal plants-Pittosporum angustifolium, Alphitonia excelsa, Calytrix microcoma, Geijera parviflora, Melaleuca uncinata, Gossypium australe, and Eucalyptus similis-traditionally used by the Iningai community, focusing on three biological processes relevant to cancer: oxidative stress, inflammation, and cellular proliferation. Materials and methods: Antioxidant activity was assessed using DPPH radical-scavenging and ferric reducing antioxidant power (FRAP) assays. Anti-inflammatory activity was evaluated in lipopolysaccharide (LPS)-stimulated THP-1 macrophage-like cells by profiling IFN-, TNF-, IL-6, IL-12, IL-18, and IL-23. Antiproliferative activity was assessed using MTT-based viability assays in human and murine liver cancer cell lines (Huh7, Hep3B, Hep-55.1c, and A52). Results: The extracts exhibited distinct biological activity profiles. G. parviflora stem and C. microcoma leaf extracts showed the strongest antioxidant activities, whereas P. angustifolium stem exhibited the weakest radical-scavenging capacity. Cytokine responses were extract-specific, with E. similis leaf extract demonstrating broad and pronounced suppression of multiple LPS-induced pro-inflammatory cytokines. Several extracts produced concentration-dependent reductions in liver cancer cell viability, with P. angustifolium stem exhibiting the most consistent and potent antiproliferative activity across the cell lines tested. Notably, strong antioxidant or anti-inflammatory activity did not necessarily correspond with antiproliferative activity. Conclusion: Australian Indigenous medicinal plant extracts demonstrated distinct antioxidant, immunomodulatory, and antiproliferative activities rather than uniform bioactivity across experimental systems. The divergent activities of G. parviflora, C. microcoma, E. similis, and P. angustifolium highlight the importance of integrated biological screening and support the value of Indigenous knowledge-guided biodiscovery. These plants represent promising sources for further investigation of selective bioactive compounds with potential relevance to anticancer drug discovery.