Nuclei Isolation from Ocular Tissues of the Embryonic Chicken for Single-Nucleus Profiling.

The generation of quality data from a single-nucleus profiling experiment requires nuclei to be isolated from tissues in a gentle and efficient manner. Nuclei isolation must be carefully optimized across tissue types to preserve nuclear architecture, prevent nucleic acid degradation, and remove unwanted contaminants. Here, we present an optimized workflow for generating a single-nucleus suspension from ocular tissues of the embryonic chicken that is compatible with various downstream workflows. The described protocol enables the rapid isolation of a high yield of aggregate-free nuclei from the embryonic chicken eye without compromising nucleic acid integrity, and the nuclei suspension is compatible with single-nucleus RNA and ATAC sequencing. We detail several stopping points, either via cryopreservation or fixation, to enhance workflow adaptability. Further, we provide a guide through multiple QC points and demonstrate proof-of-principle using two commercially available kits. Finally, we demonstrate that existing in silico genotyping methods can be adopted to computationally derive biological replicates from a single pool of chicken nuclei, greatly reducing the cost of biological replication and allowing researchers to consider sex as a variable during analysis. Together, this tutorial represents a cost-effective, simple, and effective approach to single-nucleus profiling of embryonic chicken eye tissues and is likely to be easily modified to be compatible with similar tissue types.

Axon guidance during mouse central nervous system regeneration is required for specific brain innervation.

Reconstructing functional neuronal circuits is one major challenge of central nervous system repair. Through activation of pro-growth signaling pathways, some neurons achieve long-distance axon regrowth. Yet, functional reconnection has hardly been obtained, as these regenerating axons fail to resume their initial trajectory and reinnervate their proper target. Axon guidance is considered to be active only during development. Here, using the mouse visual system, we show that axon guidance is still active in the adult brain in regenerative conditions. We highlight that regenerating retinal ganglion cell axons avoid one of their primary targets, the suprachiasmatic nucleus (SCN), due to Slit/Robo repulsive signaling. Together with promoting regeneration, silencing Slit/Robo in vivo enables regenerating axons to enter the SCN and form active synapses. The newly formed circuit is associated with neuronal activation and functional recovery. Our results provide evidence that axon guidance mechanisms are required to reconnect regenerating axons to specific brain nuclei.

Spindle oscillations emerge at the critical state of electrically coupled networks in the thalamic reticular nucleus.

Spindle oscillation is a waxing-and-waning neural oscillation observed in the brain, initiated at the thalamic reticular nucleus (TRN) and typically occurring at 7-15 Hz. Experiments have shown that in the adult brain, electrical synapses, rather than chemical synapses, dominate between TRN neurons, suggesting that the traditional view of spindle generation via chemical synapses may need reconsideration. Based on known experimental data, we develop a computational model of the TRN network, where heterogeneous neurons are connected by electrical synapses. The model shows that the interplay between synchronizing electrical synapses and desynchronizing heterogeneity leads to multiple synchronized clusters with slightly different oscillation frequencies whose summed-up activity produces spindle oscillation as seen in local field potentials. Our results suggest that during spindle oscillation, the network operates at the critical state, which is known for facilitating efficient information processing. This study provides insights into the underlying mechanism of spindle oscillation and its functional significance.

Cognitive phenotyping of GBA1-Parkinson's disease: A study on deep brain stimulation outcomes.

BACKGROUND: Heterozygous variants in the glucocerebrosidase (GBA1) gene are the most common genetic risk factor for Parkinson's Disease (PD). GBA1-PD patients exhibit earlier disease onset, severe motor impairment, and heightened cognitive decline. Deep Brain Stimulation (DBS) offers motor improvement for PD patients, but its cognitive effects, particularly in GBA1-PD, are debated. METHODS: This study involved 96 PD patients who underwent subthalamic nucleus DBS at Hospital de la Santa Creu i Sant Pau between 2004 and 2023. Clinical and neuropsychological assessments were conducted pre- and post-surgery, focusing on Mattis Dementia Rating Scale (MDRS) and Frontal Systems Behavior Scale (FrSBe). Patients were categorized into GBA1-PD and non-GBA1-PD groups, with non-GBA1-PD further divided into cognitive fast-progressors and slow-progressors. RESULTS: GBA1 variants were present in 13.5 % of patients. GBA1-PD patients showed greater cognitive decline over time, particularly in attention, conceptualization, and memory, compared to non-GBA1-PD. Non-GBA1-PD fast-progressors exhibited significant cognitive deterioration in initiation and conceptualization within the first year post-DBS. Motor outcomes improved similarly across all groups, but slow-progressors showed a greater reduction in Levodopa Equivalent Daily Dose (LEDD). CONCLUSIONS: GBA1-PD patients experience more rapid cognitive decline, particularly in posterior-cortical and fronto-striatal functions. Additionally, a subset of non-GBA1-PD patients shows significant early cognitive decline post-DBS, especially in executive functions. Baseline MDRS scores do not predict cognitive outcomes, highlighting the need for further research to refine prognostic tools. Despite cognitive challenges, GBA1-PD patients benefit from DBS in terms of motor outcomes, underscoring the importance of individualized assessments for DBS suitability, regardless of genetic status.

Optimized protocol for the multi-omics processing of cryopreserved human kidney tissue.

Biobanking of tissue from clinically obtained kidney biopsies for later use with multi-omic and imaging techniques is an inevitable step to overcome the need of disease model systems and towards translational medicine. Hence, collection protocols ensuring integration into daily clinical routines using preservation media not requiring liquid nitrogen but instantly preserving kidney tissue for clinical and scientific analyses are of paramount importance. Thus, we modified a robust single nucleus dissociation protocol for kidney tissue stored snap frozen or in the preservation media RNAlaterand CellCover. Using porcine kidney tissue as surrogate for human kidney tissue, we conducted single nucleus RNA sequencing with the Chromium 10X Genomics platform. The resulting data sets from each storage condition were analyzed to identify any potential variations in transcriptomic profiles. Furthermore, we assessed the suitability of the preservation media for additional analysis techniques (proteomics, metabolomics) and the preservation of tissue architecture for histopathological examination including immunofluorescence staining. In this study, we show that in daily clinical routines the RNAlater facilitates the collection of highly preserved human kidney biopsies and enables further analysis with cutting-edge techniques like single nucleus RNA sequencing, proteomics, and histopathological evaluation. Only metabolome analysis is currently restricted to snap frozen tissue. This work will contribute to build tissue biobanks with well-defined cohorts of the respective kidney disease that can be deeply molecularly characterized, opening new horizons for the identification of unique cells, pathways and biomarkers for the prevention, early identification, and targeted therapy of kidney diseases.

Adaptations in glutathione-based redox protein signaling pathways and alcohol drinking across species.

Alcohol use disorder (AUD) is the most prevalent substance use disorder but there is incomplete knowledge of the underlying molecular etiology. Here, we examined the cytosolic proteome from the nucleus accumbens core (NAcC) of ethanol drinking rhesus macaques to identify ethanol-sensitive signaling proteins. The targets were subsequently investigated using bioinformatics, genetic, and pharmacological manipulations in mouse models of ethanol drinking. Of the 1000+ cytosolic proteins identified in our screen, 50 proteins differed significantly between control and ethanol drinking macaques. Gene Ontology analysis of the differentially expressed proteins identified enrichment in pathways regulating metabolic processes and proteasome activity. Because the family of Glutathione S-transferases (GSTs) was enriched in these pathways, validation studies targeted GSTs using bioinformatics and genetically diverse mouse models. Gstp1 and Gstm2 were identified in Quantitative Trait Loci and published gene sets for ethanol-related phenotypes (e.g., ethanol preference, conditioned taste aversion, differential expression), and recombinant inbred strains that inherited the C57BL/6J allele at the Gstp2 interval consumed higher amounts of ethanol than those that inherited the DBA/2J allele. Genetic deletion of Gstp1/2 led to increased ethanol consumption without altering ethanol metabolism or sucrose preference. Administration of the pharmacologic activator of Gstp1/2, carnosic acid, decreased voluntary ethanol drinking. Proteomic analysis of the NAcC cytosolic of heavy drinking macaques that were validated in mouse models indicate a role for glutathione-mediated redox regulation in ethanol-related neurobiology and the potential of pharmacological interventions targeting this system to modify excessive ethanol drinking.

The differential effect of optogenetic serotonergic manipulation on sustained motor actions and waiting for future rewards in mice.

Serotonin is an essential neuromodulator that affects behavioral and cognitive functions. Previous studies have shown that activation of serotonergic neurons in the dorsal raphe nucleus (DRN) promotes patience to wait for future rewards. However, it is still unclear whether serotonergic neurons also regulate persistence to act for future rewards. Here we used optogenetic activation and inhibition of DRN serotonergic neurons to examine their effects on sustained motor actions for future rewards. We trained mice to perform waiting and repeated lever-pressing tasks with variable reward delays and tested effects of optogenetic activation and inhibition of DRN serotonergic neurons on task performance. Interestingly, in the lever-pressing task, mice tolerated longer delays as they repeatedly pressed a lever than in the waiting task, suggesting that lever-pressing actions may not simply be costly, but may also be subjectively rewarding. Optogenetic activation of DRN serotonergic neurons prolonged waiting duration in the waiting task, consistent with previous studies. However, its effect on lever presses was nuanced, and was detected only by focusing on the period before premature reward check and by subtracting the trends within and across sessions using generalized linear model. While optogenetic inhibition decreased waiting, it did not affect lever pressing time or numbers. These results revealed that the necessity of motor actions may increase motivation for delayed rewards and that DRN serotonergic neurons more significantly promote waiting rather than persistent motor actions for future rewards.

Machine learning for the localization of Subthalamic Nucleus during deep brain stimulation surgery: a systematic review and Meta-analysis.

INTRODUCTION: Delineating subthalamic nucleus (STN) boundaries using microelectrode recordings (MER) and trajectory history is a valuable resource for neurosurgeons, aiding in the accurate and efficient positioning of deep brain stimulation (DBS) electrodes within the STN. Here, we aimed to assess the application of artificial intelligence, specifically Hidden Markov Models (HMM), in the context of STN localization. METHODS: A comprehensive search strategy was employed, encompassing electronic databases, including PubMed, EuroPMC, and MEDLINE. This search strategy entailed a combination of controlled vocabulary (e.g., MeSH terms) and free-text keywords pertaining to "artificial intelligence," "machine learning," "deep learning," and "deep brain stimulation." Inclusion criteria were applied to studies reporting the utilization of HMM for predicting outcomes in DBS, based on structured patient-level health data, and published in the English language. RESULTS: This systematic review incorporated a total of 14 studies. Various machine learning compared wavelet feature to proposed features in diagnosing the STN, with the HMM yielding a diagnostic odds ratio (DOR) of 838.677 (95% CI: 203.309-3459.645). Similarly, the K-Nearest Neighbors (KNN) model produced parameter estimates, including a diagnostic odds ratio of 25.151 (95% CI: 12.270-51.555). Meanwhile, the support vector machine (SVM) model exhibited parameter estimates, with a DOR of 13.959 (95% CI: 10.436-18.671). CONCLUSIONS: MER data demonstrates significant variability in neural activity, with studies employing a wide range of methodologies. Machine learning plays a crucial role in aiding STN diagnosis, though its accuracy varies across different approaches.

Replication and extension of the subregion selectivity of glutamate-related changes within the nucleus accumbens associated with the incubation of cocaine-craving.

Cue-elicited drug-seeking behavior intensifies with the passage of time during withdrawal from drug taking and this "incubation of cocaine-craving" involves alterations in nucleus accumbens (NA) glutamate transmission. Here, we employed a combination of in vivo microdialysis and immunoblotting approaches to further examine changes in biochemical indices of glutamate transmission within NA subregions that accompany the incubation of cocaine-craving exhibited by male rats with a 10-day history of 6-h access to intravenous cocaine (0.25 mg/infusion). Immunoblotting on whole cell lysates from the core subregion (NAc core) revealed interactions between cocaine self-administration history, withdrawal and drug cue re-exposure for Homer2a/b, mGlu1, and GluN2b expression, as well as indices of Akt and ERK activity. With the exception of PKCε phosphorylation, most protein changes within the shell subregion (NAc shell) depended on drug cue re-exposure and cocaine history rather than varying in a consistent time-dependent manner. Reduced basal extracellular glutamate content was apparent only in the NAc core of cocaine-experienced rats during protracted (30 days) withdrawal and this was accompanied by a markedly blunted capacity of the mGlu1/5 agonist DHPG to elevate glutamate levels within this subregion. Finally, over-expressing neither Homer1c nor Homer2b within the NAc core during protracted cocaine withdrawal altered the magnitude of cue-elicited responding, its extinction or cocaine-primed reinstatement of drug-seeking behavior. The present findings are consistent with the extant literature implicating changes in Group 1 mGlu receptor function within the NAc core subregion as central to incubated cocaine-craving and provide further evidence against a major role for Homer proteins in gating incubated cocaine-craving. Further, our results provide novel correlational evidence implicating elevated Akt and blunted ERK activity within the NAc core as potential contributors to the expression of incubated cocaine-craving, worthy of future investigation.

Nucleus reuniens: Modulating emotional overgeneralization in peri-adolescents with anxiety.

BACKGROUND: Anxiety affects 4.4-million children in the USA with an onset between childhood and adolescence, a period marked by neural changes that impact emotions and memory. Negative overgeneralization - or responding similarly to innocuous events that share features with past aversive experiences - is common in anxiety but remains mechanistically underspecified. The nucleus reuniens (RE) has been considered a crucial candidate in the modulation of memory specificity. Our study investigated its activation and functional connectivity with the medial prefrontal cortex (mPFC) and hippocampus (HPC) as neurobiological mechanisms of negative overgeneralization in anxious youth. METHODS: As part of a secondary data analysis, we examined data from 34 participants between 9 and 14 years of age (mean age ± SD, 11.4 ± 2.0 years; 16 females) with varying degrees of anxiety severity. During the Study session participants rated images as negative, neutral, and positive. After 12 h, participants returned for a Test session, where they performed a memory recognition test with repeated (targets) and similar (lures) images. Labeling negative relative to neutral lures as "old" (false alarms) was our operational definition of negative overgeneralization. RESULTS: Negative relative to neutral false alarmed stimuli displayed elevated RE activation (at Study and Test) and increased functional connectivity with the Cornu Ammonis (CA) 1 (at Test). Elevated anxiety severity was associated with reductions in the RE-mPFC functional coupling for neutral relative to negative stimuli. Exploratory analyses revealed similar patterns in activation and functional connectivity with positive stimuli. CONCLUSIONS: Our findings demonstrate the importance of the RE in negative overgeneralization and anxiety.

Dopamine transients encode reward prediction errors independent of learning rates.

Biological accounts of reinforcement learning posit that dopamine encodes reward prediction errors (RPEs), which are multiplied by a learning rate to update state or action values. These values are thought to be represented by corticostriatal synaptic weights, which are updated by dopamine-dependent plasticity. This suggests that dopamine release reflects the product of the learning rate and RPE. Here, we characterize dopamine encoding of learning rates in the nucleus accumbens core (NAcc) in a volatile environment. Using a task with semi-observable states offering different rewards, we find that rats adjust how quickly they initiate trials across states using RPEs. Computational modeling and behavioral analyses show that learning rates are higher following state transitions and scale with trial-by-trial changes in beliefs about hidden states, approximating normative Bayesian strategies. Notably, dopamine release in the NAcc encodes RPEs independent of learning rates, suggesting that dopamine-independent mechanisms instantiate dynamic learning rates.

Magnetic resonance-guided focused ultrasound thalamotomy for tremor.

Magnetic resonance-guided focused ultrasound (MRgFUS) thalamotomy has emerged as an effective treatment for tremor, particularly in those patients who are excluded from deep brain stimulation. The authors illustrate an example of MRgFUS thalamotomy, targeting the ventralis intermedius nucleus, in a 78-year-old patient with tremor who had features of essential tremor and tremor-predominant Parkinson's disease. Significant tremor improvement was seen during the procedure. The authors review step-by-step the preoperative considerations, Vim targeting, treatment, and outcomes for this evolving treatment modality. The video can be found here: https://stream.cadmore.media/r10.3171/2024.7.FOCVID249.

The concept of the Schwann cell by Louis Ranvier and his school: The 'interannular segment' as a cell unit.

The hundredth anniversary of the death of French histologist Louis Ranvier (1835‒1922) is an opportunity to reexamine his elaboration of the first concept of the Schwann cell. A loyal supporter of Theodor Schwann and his discoveries, and an attentive reader of the work of Albert von Kölliker, Ranvier studied the anatomic details of the myelinated nerve fiber with picrocarminate staining. The diffusion of the dye into the nerve fiber at the cut ends and at the sites of the annular constrictions (Ranvier's nodes) set him on the path to defining a new cellular entity surrounding the axon, the "interannular segment," comprising a Schwann nucleus, myelin, and cytoplasm. Ramón y Cajal recognized in 1913 that this concept of the Schwann cell according to Ranvier and his pupil William Vignal had been a brilliant intuition, but it was widely rejected until it was rediscovered using electron microscopy in the 1950s. The article reconstructs the steps of Ranvier and Vignal in building this Schwann cell concept, as well as establishing bridges with the discoveries of the 1950s.

Nucleus-Spike 3D Hierarchical Superstructures via a Lecithin-Mediated Biomineralization Approach.

3D hierarchical superstructures (3DHSs) are key products of nature's evolution and have raised wide interest. However, the preparation of 3DHSs composed of building blocks with different structures is rarely reported, and regulating their structural parameters is challenging. Herein, a simple lecithin-mediated biomineralization approach is reported for the first time to prepare gold 3DHSs composed of 0D nucleus and 1D protruding dendritic spikes. It is demonstrated that a hydrophobic complex by coordination of disulfiram (DSF) with a share of chloroauric acid is the key to forming the 3DHSs. Under the lecithin mediation, chloroauric acid is first reduced to form the 0D nucleus, followed by the spike growth through the reduction of the hydrophobic complex. The prepared 3DHSs possess well-defined morphology with a spike length of ≈95 nm. Notably, the hierarchical spike density is systematically manipulated from 38.9% to 74.3% by controlling DSF concentrations. Moreover, the spike diameter is regulated from 9.2 to 12.9 nm by selecting different lecithin concentrations to tune the biomineralization process. Finite-difference time-domain (FDTD) simulations reveal that the spikes form "hot spots". The dense spike structure endows the 3DHSs with sound performance in surface-enhanced Raman scattering (SERS) applications.

Tanshinone IIA Exerts Cardioprotective Effects Through Improving Gut-Brain Axis Post-Myocardial Infarction.

Myocardial infarction (MI) is a lethal cardiovascular disease worldwide. Emerging evidence has revealed the critical role of gut dysbiosis and impaired gut-brain axis in the pathological progression of MI. Tanshinone IIA (Tan IIA), a traditional Chinese medicine, has been demonstrated to exert therapeutic effects for MI. However, the effects of Tan IIA on gut-brain communication and its potential mechanisms post-MI are still unclear. In this study, we initially found that Tan IIA significantly reduced myocardial inflammation, apoptosis and fibrosis, therefore alleviating hypertrophy and improving cardiac function following MI, suggesting the cardioprotective effect of Tan IIA against MI. Additionally, we observed that Tan IIA improved the gut microbiota as evidenced by changing the α-diversity and ß-diversity, and reduced histopathological impairments by decreasing inflammation and permeability in the intestinal tissues, indicating the substantial improvement of Tan IIA in gut function post-MI. Lastly, Tan IIA notably reduced lipopolysaccharides (LPS) level in serum, inflammation responses in paraventricular nucleus (PVN) and sympathetic hyperexcitability following MI, suggesting that restoration of Tan IIA on MI-induced brain alterations. Collectively, these results indicated that the cardioprotective effects of Tan IIA against MI might be associated with improvement in gut-brain axis, and LPS might be the critical factor linking gut and brain. Mechanically, Tan IIA-induced decreased intestinal damage reduced LPS release into serum, and reduced serum LPS contributes to decreased neuroinflammation with PVN and sympathetic inactivation, therefore protecting the myocardium against MI-induced injury.

A Novel Superparamagnetic-Responsive Hydrogel Facilitates Disc Regeneration by Orchestrating Cell Recruitment, Proliferation, and Differentiation within Hostile Inflammatory Niche.

In situ disc regeneration is a meticulously orchestrated process, which involves cell recruitment, proliferation and differentiation within a local inflammatory niche. Thus far, it remains a challenge to establish a multi-staged regulatory framework for coordinating these cellular events, therefore leading to unsatisfactory outcome. This study constructs a super paramagnetically-responsive cellular gel, incorporating superparamagnetic iron oxide nanoparticles (SPIONs) and aptamer-modified palladium-hydrogen nanozymes (PdH-Apt) into a double-network polyacrylamide/hyaluronic acid (PAAm/HA) hydrogel. The Aptamer DB67 within magnetic hydrogel (Mag-gel) showed a high affinity for disialoganglioside (GD2), a specific membrane ligand of nucleus pulposus stem cells (NPSCs), to precisely recruit them to the injury site. The Mag-gel exhibits remarkable sensitivity to a magnetic field (MF), which exerts tunable micro/nano-scale forces on recruited NPSCs and triggers cytoskeletal remodeling, consequently boosting cell expansion in the early stage. By altering the parameters of MF, the mechanical cues within the hydrogel facilitates differentiation of NPSCs into nucleus pulposus cells to restore disc structure in the later stage. Furthermore, the PdH nanozymes within the Mag-gel mitigate the harsh inflammatory microenvironment, favoring cell survival and disc regeneration. This study presents a remote and multi-staged strategy for chronologically regulating endogenous stem cell fate, supporting disc regeneration without invasive procedures.

Evaluating a motor progression connectivity model across Parkinson's disease stages.

BACKGROUND: Stimulation of a specific site in the dorsolateral subthalamic nucleus (STN) was recently associated with slower motor progression in Parkinson's Disease (PD), based on the deep brain stimulation (DBS) in early-stage PD pilot clinical trial. Here, subject-level visualizations are presented of this early-stage PD dataset to further describe the relationship between active contacts and motor progression. This study also evaluates whether stimulation of the sweet spot and connectivity model associated with slower motor progression is also associated with improvements in long-term motor outcomes in patients with advanced-stage PD. METHODS: Active contacts of the early-stage PD cohort (N = 14) were analyzed alongside the degree of two-year motor progression. Sweet spot and connectivity models derived from the early-stage PD cohort were then used to determine how well they can estimate the variance in long-term motor outcomes in an independent STN-DBS cohort of advanced-stage PD patients (N = 29). RESULTS: In early-stage PD, proximity of stimulation to the dorsolateral STN was associated with slower motor progression. In advanced-stage PD, stimulation proximity to the early PD connectivity model and sweet spot were associated with better long-term motor outcomes (R = 0.60, P < 0.001; R = 0.37, P = 0.046, respectively). CONCLUSIONS: Results suggest stimulation of a specific site in the dorsolateral STN is associated with both slower motor progression and long-term motor improvements in PD.

Seizure freedom using a regional approach to responsive neurostimulation for multifocal drug-resistant epilepsy: illustrative case.

BACKGROUND: Responsive neurostimulation (RNS) has emerged as an effective neuromodulatory intervention for patients with medically refractory epilepsy who are not candidates for resective or ablative surgery. However, in patients with multifocal seizures arising from a widely distributed network, optimizing lead placement can be challenging. OBSERVATIONS: Here, the authors present the case of a patient with drug-resistant multifocal, nonlateralizing seizures and multiple developmental brain lesions who underwent phase II monitoring with stereoelectroencephalography electrodes targeting the lesion and surrounding cortex as well as the centromedian thalamus. Neurophysiological signals observed during recorded events implicated a seizure network within the left perisylvian polymicrogyria, involving the left parietal operculum, insula, and centromedian thalamic regions rather than a single focus. LESSONS: Using a regional RNS approach to modulate this network, the patient improved from 5 seizures a day to freedom from disabling seizures shortly after lead implantation despite low stimulation parameters. This has implications for understanding the timescale of adaptive mechanisms that occur in response to stimulation and supports the use of RNS as a surgical treatment for drug-resistant epilepsy. https://thejns.org/doi/10.3171/CASE24369.

Task-evoked pupillary responses as potential biomarkers of mild cognitive impairment.

INTRODUCTION: Eye movement alterations are effective biomarkers for Alzheimer's disease (AD). This study examines task-evoked pupillary responses (TEPRs) as potential biomarkers of the mild cognitive impairment (MCI), the symptomatic stage preceding AD. METHODS: The prospective cohort study included 213 MCI patients and 514 cognitively normal controls (CNs). Participants performed a prosaccade (PS) or antisaccade (AS) task while their eye movements were tracked using a Tobii Pro Spectrum system. RESULTS: The CNs showed unique TEPRs linked to better performance, characterized by larger baselines, greater PS target-onset variability, and smaller AS target-onset variability. Conversely, for MCI patients, better performance was linked to larger AS target-onset sizes. Furthermore, MCI patients displayed reduced dilation during the cue and target-onset periods compared to CNs. DISCUSSION: MCI patients showed altered pupillary response patterns associated with cognitive task performance, highlighting the potential of oculomotor changes as a biomarker for early cognitive decline. Highlights: MCI patients displayed markedly smaller pupil dilation than CNs in response to cue and target stimuli.For MCI patients, larger pupil size upon target appearance during antisaccades correlated with better performance.Faster and more consistent prosaccades were linked to better performance in both groups.For MCI patients, the association between longer AS latencies and better performance was more pronounced than in CNs.Combined analysis of TEPRs and saccade performances in a sizeable cohort strengthens the generalizability of our findings to the broader MCI population.

Single nucleus DNA sequencing of flow sorted archived frozen and formalin fixed paraffin embedded solid tumors.

BACKGROUND: Archived samples, including frozen and formalin fixed paraffin embedded (FFPE) tissues, are a vast resource of clinically annotated materials for the application of high-definition genomics to improve patient management and provide a molecular basis for the delivery of personalized cancer therapeutics. Notably, FFPE tissues are stable, provide repeat sampling of tissues of interest, and can be stored indefinitely at ambient temperature. The development of single cell DNA sequencing (scDNA-seq) technologies provides an unparalleled opportunity for the study of tumor heterogeneity and the identification of often rare subclonal cell populations that drive tumor evolution and progression to advanced therapy resistant disease. However, major limitations to the use of archived tissues for scDNA-seq include the low yields of intact cells in the presence of high levels of subcellular debris in biopsies, and the highly variable quantity and quality of the DNA extracted from samples of interest. The latter is of high significance for the use of FFPE tissues due to the presence of DNA-protein crosslinks. In addition, many samples, notably tumors arising in solid tissues, contain admixtures of reactive stroma, inflammatory cells, and necrosis in immediate contact with tumor cells. RESULTS: To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived fresh frozen (FF) and FFPE tumor tissues. Our methods, which include isolation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching FF and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest. CONCLUSIONS: Our flow sorting based protocol provides intact nuclei suitable for snDNA-seq from archival FF and FFPE tissues. Furthermore, we have developed QC steps that optimize the preparation and selection of samples for deep single cell clonal profiling. Our data processing pipeline captures rare subclones in tumors with highly variable genomes based on variants in genes of interest.