Stop-ball small-sided games (SSGs-SB) have been proposed as an effective scoring method to influence physical and physiological responses in young soccer player. The aim of present study is to examine the differences in external and internal load variables across U-13, U-14 and U-15 players during SSGs-SB. Ninety-six male soccer players (32 players in each group) from teams competing in Italian regional championship were involved. The experimental design consisted of a 4 vs. 4 played on a 25×20 m pitch, including 4x3' of work separated by 2 min of passive recovery. During SSG-SB, players were monitored using 18.18 Hz global positioning system devices (GPEXE® SYSTEM, EXELIO srl, Udine, Italy). Total distance, peak velocity, distance covered at high-speed running 13-18 km∙h-1, sprint running >18 km∙h-1, number of sprint, acceleration >2.5 m∙s-2, deceleration <-2.5 m∙s-2, maximal acceleration and deceleration, high metabolic load distance and rating of perceived exertion were measured. U-15 team exhibited greater physical demands compared with U-13 team (P<0.001 for all comparisons), whereas younger counterparts significantly (P<0.001) reported higher RPE scores than U-15 players. The present study suggests that SSGs-SB designs should be tailored according to chronological age to support an appropriate long-term development process in youth soccer players.
Mineralocorticoid receptor (MR) antagonists (MRAs) are first-line therapy for primary aldosteronism and guideline-recommended treatment for heart failure (HF). The cardiovascular benefits of MRAs exceed blood pressure reduction, reflecting inhibition of inappropriate MR activation in cardiac and vascular tissues, and immune cells. Early preclinical studies demonstrated that aldosterone excess induces myocardial inflammation and fibrosis through MR-dependent pathways, and clinical trials established the efficacy of MRAs in reducing morbidity and mortality in HF. Nonsteroidal MRAs are now available, and aldosterone synthase inhibitors have commenced clinical testing; it is thus timely to revisit the mechanisms of MR activation in the heart and related cell types. This review will discuss key findings from preclinical and clinical studies of MRA use in HF and mineralocorticoid infusion, and update our understanding of MR actions in the normal myocardium and in the pathophysiology of primary aldosteronism and HF. Emerging data on aldosterone-mediated cardiovascular injury in patients with primary aldosteronism, including structural and functional cardiac changes and links to adverse outcomes, will be included, along with recent advances in MR-targeted pharmacology that offer improved selectivity and safety profiles. This review provides an updated understanding of MR-dependent pathways in the cardiovascular system and potential strategies for optimizing cardiovascular protection in both primary aldosteronism and HF.
Most genetic variants associated with complex heritability phenotypes lie in non-coding regions and are thought to influence disease risk by regulating gene expression. However, most transcriptome-wide association approaches primarily model local (cis) genetic effects, leaving much of gene regulation unexplained. Here, we show that incorporating distal (trans) regulatory effects improves the prediction of gene expression and the identification of disease-associated genes. Using RNA sequencing data from six human post-mortem brain regions, we developed INGENE and MODULE, two models capturing the combined influence of candidate trans-acting variants within gene coexpression networks. Integrating these models with conventional cis-based predictors improved gene expression imputation (maximum likelihood estimation, α = 0.05) for 18,744 genes across regions. Applying this framework to Psychiatric Genomics Consortium wave 3 genotypes identified 766 genes associated with schizophrenia (PFDR < 0.01), including 641 not previously reported by transcriptome-wide analyses. These findings highlight the contribution of distal regulatory mechanisms and gene network interactions to schizophrenia risk.
Odorants in the inhaled air bind to odorant receptors embedded in cilia of olfactory receptor neurons, triggering the opening of the two ciliary olfactory transduction channels; first, a cationic heterotetrameric cAMP-gated channel, followed by an excitatory Ca2+-activated Cl- channel TMEM16B (also called anoctamin 2), which gives rise to a transduction current. While cholesterol is known to modulate ion channels, its effect on the olfactory channels, specifically TMEM16B, is unclear. We addressed how membrane cholesterol regulates these transduction channels. We heterologously expressed the main subunit of the olfactory cyclic nucleotide-gated (CNG) channel, CNGA2, and the Cl- channel TMEM16B and recorded their function in control, cholesterol-depleted, and cholesterol-enriched membranes. Maximal cAMP- or Ca2+-evoked currents were not altered by varying cholesterol levels. TMEM16B showed a progressive reduction in current upon repeated exposure to Ca2+ ("rundown"), which was accelerated in cholesterol-depleted membranes but decelerated in cholesterol-enriched patches when compared with control patches. Maximal cAMP-gated currents were stable over time, but their sensitivity was altered, with lower and higher cholesterol levels increasing and decreasing the channel's sensitivity, respectively. Cholesterol depletion of patches excised from mouse olfactory cilia containing native channels entirely abolished the TMEM16B current, while the maximal current of the native CNG channel remained unaltered. Reduced cholesterol did not change the sensitivity of the native CNG channel. Thus, while both olfactory transduction channels are sensitive to changes in membrane cholesterol in differing ways, the precise channel stoichiometry and the specific membrane environment also contribute to their function.
Constitutively active mutations (CAMs) in the thyrotropin receptor (TSHR) are the major cause of nonautoimmune hyperthyroidism. TSHR is a key regulator of thyroid hormone synthesis, which is essential for skeletal formation, bone turnover, and craniofacial development. In addition to its role in thyroid hormone regulation, thyrotropin and its receptor have been proposed to independently influence bone formation and resorption. However, the specific effects of constitutively active TSHR mutations on cranial and skeletal development have not been investigated in mouse models. Cranial morphometry, micro-computed tomography, and three-point bending tests were performed in established TSHR knock-in mouse models carrying patient-derived TSHR D633H or M453T CAMs. Homozygous TSHR D633H mice exhibit mild, transient hyperthyroidism at 2 months of age, more pronounced in females, whereas homozygous TSHR M453T mice develop a more severe, iodine-dependent hyperthyroid phenotype. Both TSHR CAM lines showed altered craniofacial morphology, particularly in nasal bone dimensions, resulting in a shorter snout compared with wild-type (WT) controls. The incidence of malocclusion was significantly increased in both homozygous and heterozygous mice, regardless of sex. TSHR D633H mice displayed no significant changes in femoral or tibial bone structure or biomechanical properties. In contrast, TSHR M453T mice exhibited hyperthyroidism-dependent alterations in trabecular bone mineral density (BMD) and architecture, while cortical bone was unaffected. Body and tail lengths were unchanged in TSHR D633H mice. M453T homozygous mice had reduced tail length at weaning in an iodine-dependent manner, which normalized with age. This in vivo study demonstrates that TSHR CAM-induced hyperthyroidism alters craniofacial morphology and increases the incidence of malocclusion in mice. Structural changes and altered BMD in M453T mice were dependent on the severity of hyperthyroidism. These findings highlight the role of TSHR signaling and thyroid status in craniofacial and skeletal development, warranting further mechanistic investigation.
Hippocampal sclerosis of aging (HS-A), a common cause of dementia, can currently only be diagnosed at autopsy. We aimed to identify and evaluate MRI metrics to distinguish HS-A from Alzheimer's Disease neuropathologic change (ADNC) and cases with limited/no pathology. HS-A (N = 5), ADNC (N = 10), and limited/no pathology (N = 12) cases were compared on postmortem MRI signatures: manually measured cornu ammonis (CA)1/subiculum thickness, grey matter signal intensity, and automated hippocampal subfield thickness metrics. Similar metrics were obtained in T1-weighted antemortem MRI in an initial dataset (HS-A = 4, ADNC = 7, limited/no pathology = 25) for group differences and discrimination (HS-A vs ADNC). T1-weighted metrics were then evaluated in a second dataset (HS-A = 6, ADNC = 18) and in a pooled post-hoc analysis combining HS-A and ADNC cases from both datasets (NHS-A = 10, NADNC = 25). Postmortem MRI showed hippocampal thinning and grey matter hypointensity in HS-A at the CA1-subiculum junction more severe than in ADNC and limited/no pathology cases. In antemortem MRI, differentiating HS-A and ADNC based on anterior/posterior manual measures of CA1/subiculum thickness and hippocampal volumes displayed good discrimination in dataset 1, but lower discriminative performance in dataset 2. In complementary analyses pooling both datasets and adjusting for age, manual thickness achieved good performance (area under the curve (AUC) = 0.80-0.87), while anterior, posterior, and whole hippocampal volumes showed excellent discrimination (AUC = 0.94-0.98). The study included a relatively small and neuropathologically heterogeneous sample. The final antemortem analyses were exploratory, reflecting challenges in replicating findings across independent datasets. Classification was limited to HS-A and ADNC, leaving it uncertain how the metrics perform when comparing HS-A with other diagnostic groups. HS-A diagnoses were determined postmortem, often several years after MRI, and most measures relied on standard imaging sequences with limited resolution to assess fine-grained hippocampal subfields. HS-A displays distinct changes in the hippocampus that are detectable through structural MRI. Associated quantifiable MRI metrics may serve as promising tools in aiding antemortem HS-A diagnosis but require further validation in larger cohorts and against other dementia-related diseases.
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Conventional data visualization techniques in single-cell analysis (such as two-dimensional dot plots, SPADE, PCA, t-SNE, or UMAP) often fall short in enabling an intuitive understanding of high-parameter flow cytometry data. These methods tend to oversimplify complex biological relationships, lack biologically meaningful interpretations, and offer no principled framework for downstream quantitative analysis. To address these limitations, we present a graph-based (network-based) visualization framework grounded in optimal transport theory. In this framework, cell populations are defined by their marker-expression profiles, and inter-population similarity is quantified using an efficiently computable optimal transport formulation known as the Sinkhorn distance. Our approach produces biologically consistent two-dimensional graph layouts using a phenotype-aware Hamming distance. Structural differences between sample graphs are characterized through a customized graph-edit distance that captures changes in population size, marker expression, and relationships between populations. We demonstrate our methods on two flow cytometry datasets: one from a clinical trial of dendritic cell-based immunotherapy in malignant peritoneal mesothelioma, involving 14 patients sampled at three time points with 14-color panels, and another from FlowCAP-II, which involved 43 acute myeloid leukemia patient samples analyzed with 7-color panels. Our framework produces robust, quantitative visual summaries of cell populations and supports statistical analysis based on graph edit distances, thereby offering new insights into disease progression and treatment response. Ultimately, our method bridges the gap between flow cytometry data visualization and biological interpretation.
Sample pretreatment plays a central role in analytical workflows, particularly for complex matrices where efficiency, reproducibility, and sustainability are essential. In recent years, increasing levels of automation have driven a gradual transition from labor-intensive manual procedures toward integrated and standardized pretreatment strategies. This review examines two representative automation paradigms that have significantly influenced recent developments in sample pretreatment: surface-based liquid microjunction sampling and flow-based automated platforms. Liquid microjunction techniques, including liquid extraction surface analysis, liquid microjunction surface sampling probe, and the MasSpec Pen, enable localized and minimally invasive extraction directly from solid or semi-solid substrates. These approaches have extended the applicability of mass spectrometry to in situ biological and clinical analysis, while also presenting challenges related to spatial resolution, extraction selectivity, and quantitative consistency. In contrast, flow-based platforms such as lab-in-syringe and online solid-phase extraction emphasize controlled fluid handling, process integration, and operational reproducibility, and have become important tools for high-throughput and trace-level analysis in biomedicine, food safety, and environmental monitoring. This review focuses on the methodological characteristics, design considerations, and practical limitations of these automation strategies, highlighting how platform architecture influences analytical performance and application scope. Current trends toward greater integration, intelligent control, and improved standardization are also discussed, with reference to future directions in automated analytical workflows.
Reynoutria japonica is an economically vital, resveratrol-producing medicinal plant whose growth and quality are increasingly threatened by climate-induced heat stress. To elucidate the post-translational mechanisms underlying thermal adaptation, we established the first comprehensive, proteome-wide lysine succinylation (Ksucc) landscape in R. japonica. A total of 1722 Ksucc sites were mapped across 650 proteins, including eight distinct sites on histones H3 and H4. Evolutionary analysis of these histone marks revealed a combination of conserved (H3K56, H3K79, H4K31, H4K77, and H4K91) and species-specific sites. Functional profiling demonstrated that succinylated proteins are heavily enriched in central metabolism, particularly photosynthesis and the tricarboxylic acid (TCA) cycle, with extensive modifications targeting key enzymes tied to succinic acid metabolism. Under acute heat stress (42 °C), R. japonica seedlings exhibited pronounced physiological chlorosis, accumulation of reactive oxygen species, and a robust global hyper-succinylation response. Beyond transcriptional screening, a combined strategy of pharmacological inhibition and in planta transient expression assays successfully identified candidate acetyltransferase RjapHAC2-like and deacetylase RjapHDA5-like as the functional writer and eraser, respectively driving Ksucc homeostasis. In conclusion, these findings provide a comprehensive lysine succinylation dataset for R. japonica and highlight its dynamic response to heat stress, offering a valuable reference for future succinylome investigations across medicinal plants.
Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) is an inflammatory demyelinating disorder that overlaps clinically with multiple sclerosis (MS) but immunopathologically distinct. Although often considered an acute inflammatory disease, recurrent attacks in MOGAD can lead to demyelination, axonal injury, and secondary neurodegeneration. Reliable biomarkers associated with relapse risk and disease subphenotypes, including optic neuritis, remain limited. Here, we aimed to define molecular and cellular signatures that distinguish MOGAD from MS as a prototypical neuroinflammatory disease and from Alzheimer disease (AD) as a proxy of neurodegeneration and to identify candidate immune-proteomic features associated with relapse frequency and clinical phenotype in MOGAD. CSF, serum, and whole-blood samples from patients with MOGAD (n = 67), MS (n = 49), and AD (n = 36) were profiled using NULISAseq™ CSF proteomics, Olink Explore 3072 CSF and serum proteomics, and high-dimensional mass cytometry for immune cell characterization. In MOGAD, longitudinal clinical data, including total attack counts from the earliest documented attack through follow-up, were integrated with immune and proteomic profiles to assess associations with disease course and clinical phenotype. CSF and blood proteomic profiling revealed distinct inflammatory and cardiometabolic proteomic profiles in MOGAD, differentiating it from both MS and AD. Compared with MS, MOGAD showed relative reductions in lymphocyte populations with regulatory phenotypes. Within MOGAD, relapsing disease was associated with reduced frequencies of CD8+CCR7+CD31+CTLA4+ T cells and concurrent expansion of double-negative γδ T-cell subsets. IL-13 correlated positively with relapse frequency and inversely with circulating regulatory T cells, whereas IL-32 and CASP4 showed opposite associations, correlating negatively with relapse count and positively with Treg frequency. IL-13 was also inversely associated with CD31-expressing CD8+ T cells. Phenotype-stratified analyses suggested that these immune-proteomic relationships differed according to clinical presentation, including optic neuritis vs nonoptic neuritis phenotypes. This integrative immune-proteomic analysis identifies cellular and molecular features associated with relapsing vs monophasic MOGAD, suggesting a model of impaired peripheral immune regulation in relapsing disease. While exploratory, these findings generate a concrete hypothesis for future longitudinal and functional studies aimed at refining biomarker-based monitoring and informing individualized therapeutic strategies in MOGAD.
Do the genetic and clinical outcomes of monopronuclear blastocysts (MPBs) differ between standard insemination and intracytoplasmic sperm injection cycles, and what do these differences imply for risk stratification and individualized clinical decision-making? IVF-derived MPBs demonstrate significantly lower rates of uniparental inheritance than ICSI-derived MPBs (96.9% vs 65.9%) biparental inheritance (P < 0.001), with comparable clinical and neonatal outcomes to 2PN blastocysts following transfer of euploid biparental embryos. A proportion of monopronuclear (1PN) zygotes can develop into euploid blastocysts and, following transfer, result in healthy live births, yet these embryos are widely discarded following fertilization check due to atypical pronucleation. The mechanisms underlying 1PN formation are varied and include asynchronous pronuclear formation, early pronuclear fusion, and premature pronuclear breakdown, meaning a subset may represent normally fertilized diploid zygotes missed at static assessment. Retrospective cohort study of 1PN embryos (N = 13 203) derived from IVF (n = 5266) or ICSI (n = 5464) inseminations across 10 730 cycles performed at multiple Australian clinics between January 2010 and December 2023. Embryos were defined as 1PN by the appearance of a single pronucleus at fertilization check 16-18 h post-insemination. Time-lapse footage was reviewed on Day 3 to identify late appearing 1PNs and exclude late second pronucleus appearance. Suitable blastocysts underwent trophectoderm biopsy for pre-implantation genetic testing for aneuploidy (PGT-A) and short tandem repeat (STR)-based biparental inheritance testing; only euploid embryos with confirmed biparental inheritance were available for frozen embryo transfer. Outcomes assessed included ploidy, biparental inheritance, blastocyst development, utilization, morphokinetics, pregnancy, live birth, and maternal and neonatal outcomes. IVF-derived MPBs had similar aneuploidy rates to two pronuclei (2PN) embryos (37.3% vs 33.9%) and 440/454 (96.9%) demonstrated biparental inheritance. ICSI-derived MPBs had higher aneuploidy rates (45.5% vs 31.5%, P < 0.05) and only 108/164 (65.9%) had biparental inheritance. Uniparental inheritance was predominantly maternal (IVF 92.8%; ICSI 94.6%). Both IVF and ICSI MPBs were less likely to reach blastocyst stage by Day 5 than 2PN embryos (IVF 19.3% vs 63.3%; ICSI 9.7% vs 60.6%, P < 0.05), and biparental IVF-1PN zygotes were more likely to have more nucleoli compared with uniparental IVF-1PN zygotes (P = 0.008). For embryos with confirmed biparental inheritance, there was no significant difference in clinical pregnancy, ongoing pregnancy, live birth rates, or neonatal outcomes compared with 2PN blastocysts. In approximately one in six cycles containing a 1PN embryo, no utilizable 2PN embryo were available (IVF 15.6%; ICSI 16.7%), with the 1PN embryo representing the sole option for embryo utilization. Retrospective single-entity design introduces potential selection bias and limits generalizability. Uniform protocols across sites preclude the level of evidence required for formal guideline revision. Differential use of time-lapse imaging for ICSI versus static assessment for IVF embryos may contribute to differences in 1PN identification rates between fertilization methods. The STR-based biparental classification platform has not been validated against an orthogonal technology for parental origin calling in 1PN embryos, and the possibility of triploid misclassification or absorption into unreported inconclusive outcomes cannot be excluded. These findings support a risk-stratified approach to MPB management based on fertilization method. IVF-derived MPBs meeting specific morphological and developmental criteria demonstrate a low-risk profile that warrants reconsideration of genetic testing requirements and may inform individualized consent discussions, particularly where 2PN embryos are unavailable. ICSI-derived MPBs carry a substantially higher risk of uniparental inheritance and comprehensive genetic testing remains indicated. No funding was attached to this study. The authors declare no conflict of interest. N/A.
Medical Ethics integrates scientific approaches from ethics, philosophy, religious studies, history, and sociology into all relevant fields and subdisciplines of medicine, biomedicine, and healthcare. However, there is a lack of detailed analysis of research activity and networking of peer-reviewed research in Medical Ethics. Consequently, this study employs established bibliometric methods to examine the chronological, geographical, and thematic patterns of global research, as well as network structures, by analyzing metadata retrieved from the Web of Science. The analysis identified a total of 11,663 articles published in journals in the field of Medical Ethics. The number of articles peaked slightly in 2015 but remained more or less constant otherwise. From a global perspective, the USA was the dominant country in absolute numbers, followed by China and Japan. By contrast, the European countries Sweden, Austria, and Norway were positioned first when the research activity was related to the population size. Large parts of Africa, South Asia, and South America/Caribbean are virtually not present in the global landscape of Medical Ethics research, although these areas offer many open questions. Although a far-reaching, global network has been established, networking primarily takes place among English-speaking countries such as the USA, the UK, Canada, and Australia, while developing countries in particular are underrepresented. The growth in publication numbers is not as steep as in other fields and is imbalanced from a global viewpoint. Therefore, countries with weaker economies should be systematically encouraged to participate in international research collaborations.
Insulin secretion can be stimulated by immune and neuronal processes prior to a rise in blood glucose, exemplified by the cephalic phase of insulin response in the anticipation of food. Pancreatic α-cells prevent hypoglycemia by releasing glucagon. Here, we identified α-cells as critical mediators of IL-1β- and cholinergic agonist-driven insulin secretion. Cholinergic blockade prevented glucagon-stimulated insulin secretion in mice. Selective ablation of α-cells abolished cephalic phase insulin release. Islets from α-cell-deficient mice also failed to secrete insulin in response to IL-1β or muscarinic receptor activation. However, glucagon, acting on glucagon and GLP-1 receptors, rescued this insulin-stimulatory response. Mechanistically, intracellular Ca2+ mobilization at fasting glucose mediated cholinergic and IL-1β-stimulated insulin release. Short-term high-fat diet impaired glucagon-induced insulin secretion in vivo, while isolated islets showed increased insulin secretion after cholinergic stimulation versus chow-fed controls. These findings reveal α-cell-derived glucagon as a gatekeeper of immune and neuronal control of insulin secretion at fasting glucose.
Autism spectrum disorder (ASD) is a pervasive neurodevelopmental condition characterized by social communication deficits, exhibiting a male bias in prevalence. Emerging evidence suggests that prenatal exposure to bisphenol A (BPA) may perturb neurodevelopmental trajectories relevant to ASD. While the cerebellum is increasingly recognized as a brain region implicated in ASD pathophysiology, the impact of gestational BPA exposure on its post-transcriptional alternative splicing machinery remains fundamentally undefined. Here, we investigated sex-dependent effects of prenatal BPA exposure on the alternative splicing landscape of the neonatal rat cerebellum. We utilized RNA-seq to profile differential alternative splicing (DAS) events. Ingenuity Pathway Analysis (IPA) was used to predict biological functions and canonical pathways, and to construct the interactome network of DAS genes. To explore candidate upstream regulatory mechanisms, we performed in silico molecular docking and used high-resolution melting (HRM) qRT-PCR to validate selected splicing events. Furthermore, we assessed in vitro cellular phenotypes in primary cerebellar neurons by measuring MTS-based viability and Syn1/Psd95 puncta colocalization. Prenatal BPA exposure was associated with widespread DAS in genes enriched for ASD-relevant pathways in the neonatal rat cerebellum. To our knowledge, this study is the first to report molecular docking analyses predicting favorable interactions between BPA and several candidate RNA-binding proteins (RBPs), including CPEB1, RALYL, HNRNPDL, and ACO1. Our findings support a model in which BPA may perturb RBP-associated splicing regulation, including altered splicing of chromatin regulators such as Ccar1 in males. These molecular and cellular findings were accompanied by sex-stratified differences in neuronal viability and synaptic puncta measurements. BPA exposure was associated with an increased MTS viability signal in male primary cerebellar neurons, together with significant reductions in Psd95 and Syn1 puncta density, whereas female neurons showed significantly increased synaptic puncta colocalization together with reduced viability. In this study, we propose that prenatal BPA may be relevant to ASD-related neurodevelopmental pathways through sex-dependent changes in RBP-associated alternative splicing, including altered splicing of Ccar1 in males, together with distinct cellular outcomes. Together, these findings identify the developing cerebellum as a sensitive target of prenatal BPA exposure and highlight alternative splicing as a candidate pathway relevant to ASD biology.
Auditory information plays a critical role in human posture and locomotion. Beyond its obvious contribution to spatial awareness, sound provides continuous cues about body orientation, movement dynamics and environmental context. This review summarizes current knowledge on how auditory inputs modulate postural stability, locomotor kinematics and muscle coordination through multisensory integration with visual, somatosensory and vestibular systems. We highlight evidence from behavioural, neurophysiological and clinical studies showing that auditory features, for example coming from spatialised sounds, rhythmic stimulation and auditory feedback, can influence gait timing, trajectory control and balance recovery. A dedicated section explores the interaction between the auditory and vestibular systems, focusing on shared neural pathways in brainstem and cerebellum that contribute to motion perception and equilibrium. We also discuss alterations of auditory-motor coupling in neurological and sensory disorders, including vestibular deficits, Parkinson's Disease and developmental coordination disorders, and we consider the implications for auditory-based rehabilitation. Understanding how sound informs and stabilizes human movement may open new perspectives for multisensory training, neuroprosthetic design and fall prevention strategies.
As an anatomical extension of the central nervous system (CNS), the eye harbors rich neural and immune interfaces with the brain. However, the integrated immunological and neurological nexus between the eye and CNS remains unexplored. Here, we identify the existence of an eye-brain neuroimmune axis by analyzing immune and neuronal responses to eye-brain electrical stimulation (ES). ES-assisted intravitreal immunization (IVT) prolonged survival in a murine glioblastoma model, with 33.3% of mice surviving to day 50. Mechanistically, the eye-brain neuroimmune axis serves two pivotal roles: (1) Immune activation: ES accelerates the rapid drainage of intravitreally delivered antigens to deep cervical lymph nodes (dCLNs), bypassing the BBB and triggering robust CNS-specific immune responses; (2) Disruption of pathological neuron connectivity: the expression of synaptogenic factors and neuronal excitability can also be mediated under ES treatments. These results uncover an unexplored brain neuroimmune connection, offer new insights into the pathophysiology of eye-brain diseases and suggest promising avenues for precise diagnostic and therapeutic strategies targeting both ocular and CNS disorders.
Liquid chromatographic (LC) and capillary electrokinetic chromatography (EKC) methods with ultraviolet/diode array detection were developed for the enantioseparation of five azoles: econazole (ECO), miconazole (MICO), imazalil (IMA), ketoconazole (KET), and penconazole (PEN). The chiral selectors investigated were human serum albumin (HSA) and eight cyclodextrins (CDs), exploited in LC and EKC, respectively. HSA LC offered partial enantioseparation of IMA and, less effectively, of PEN. Interestingly, these azoles share a high degree of molecular similarity, as confirmed by in silico calculations. This may suggest that a single enantiomer achieves preferential access to enantioselective sites of the protein. To address the limited enantioselectivity of HSA for these azoles, CDs were explored as chiral selectors where EKC was preferable to LC due to its superior separation efficiency. The chiral discrimination capabilities of eight CDs were evaluated for these azole derivatives, aiming for fast separations with minimal amounts of CDs. Effective enantioseparation was achieved for ECO, MICO, IMA, and KET, using two of the tested CDs: 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD). Satisfactory resolutions were obtained using a 40 mM phosphate buffer (pH 3.2) with 1 mM HP-β-CD for ECO and MICO and 8 mM TM-β-CD for KET. For IMA, the optimal conditions consisted of a 20 mM phosphate buffer with 3 mM HP-β-CD. None of the tested CDs were effective for the enantiomeric separation of PEN; therefore, a dual CD system was developed for the first time for this compound, employing succinyl-β-CD (Succ-β-CD) in combination with HP-β-CD in a borax buffer under basic conditions (pH 9.4), enabling complete enantioseparation of PEN. The low concentration of CDs required under optimal conditions renders the developed methods highly cost-effective.