Increased CX3CL1 in cerebrospinal fluid and ictal serum t-tau elevations in migraine: results from a cross-sectional exploratory case-control study.

BACKGROUND: To date, migraine is diagnosed exclusively based on clinical criteria, but fluid biomarkers are desirable to gain insight into pathophysiological processes and inform clinical management. We investigated the state-dependent profile of fluid biomarkers for neuroaxonal damage and microglial activation as two potentially relevant aspects in human migraine pathophysiology. METHODS: This exploratory study included serum and cerebrospinal fluid (CSF) samples of patients with migraine during the headache phase (ictally) (n = 23), between attacks (interictally) (n = 16), and age/sex-matched controls (n = 19). Total Tau (t-Tau) protein, glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and neurofilament light chain (NfL) were measured with the Neurology 4-plex kit on a Single Molecule Array SR-X Analyzer (Simoa® SR-X, Quanterix Corp., Lexington, MA). Markers of microglial activation, C-X3-C motif chemokine ligand 1 (CX3CL1) and soluble triggering receptor expressed on myeloid cells 2 (sTREM2), were assessed using an immunoassay. RESULTS: Concentrations of CX3CL1 but not sTREM2 were significantly increased both ictally and interictally in CSF but not in serum in comparison to the control cohort (p = 0.039). ROC curve analysis provided an AUC of 0.699 (95% CI 0.563 to 0.813, p = 0.007). T-Tau in serum but not in CSF was significantly increased in samples from patients taken during the headache phase, but not interictally (effect size: η2 = 0.121, p = 0.038). ROC analysis of t-Tau protein in serum between ictal and interictal collected samples provided an AUC of 0.729 (95% CI 0.558 to 0.861, p = 0.006). The other determined biomarkers for axonal damage were not significantly different between the cohorts in either serum or CSF. DISCUSSION: CX3CL1 in CSF is a novel potential fluid biomarker of migraine that is unrelated to the headache status. Serum t-Tau is linked to the headache phase but not interictal migraine. These data need to be confirmed in a larger hypothesis-driven prospective study.

Quantification of absolute labeling efficiency at the single-protein level.

State-of-the-art super-resolution microscopy allows researchers to spatially resolve single proteins in dense clusters. However, accurate quantification of protein organization and stoichiometries requires a general method to evaluate absolute binder labeling efficiency, which is currently unavailable. Here we introduce a universally applicable approach that uses a reference tag fused to a target protein of interest. By attaching high-affinity binders, such as antibodies or nanobodies, to both the reference tag and the target protein, and then employing DNA-barcoded sequential super-resolution imaging, we can correlate the location of the reference tag with the target molecule binder. This approach facilitates the precise quantification of labeling efficiency at the single-protein level.

Spatial proteomics in neurons at single-protein resolution.

To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution.

Normative body mass-adjusted reference ranges of magnetic resonance imaging signs commonly used in diagnosing idiopathic intracranial hypertension in a healthy standard population.

Patients with chronic daily headaches (CDH) are often a diagnostic challenge and frequently undergo neuroimaging. One common underlying cause of CDH is idiopathic intracranial hypertension (IIH). However, certain neuroimaging abnormalities that suggest IIH, such as optic nerve sheath diameters (ONSD), pituitary gland height, and venous sinus diameter, require interpretation due to the absence of established normative values. Notably, intracranial pressure is known to varies with age, sex and weight, further complicating the determination of objectively abnormal findings within a specific patient group. This study aims to assist clinical neuroradiologists in differentiating neuroimaging results in CDH by providing weight-adjusted normative values for imaging characteristics of IIH. In addition to age and BMI we here assessed 1924 population-based T1-weighted MRI datasets of healthy participants for relevant MRI aspects of IIH. Association to BMI was analyzed using linear/logistic regression controlled for age and stratified for sex. ONSD was 4.3 mm [2.8; 5.9]/4.6 mm [3.6; 5.7] and diameter of transverse sinus was 4.67 mm [1.6; 6.5]/4.45 mm [3.0; 7.9]. Height of pituitary gland was 5.1 mm [2.2;8.1]/4.6 mm [1.9;7.1] for female and male respectively. Values generally varied with BMI with regression slopes spanning 0.0001 to 0.05 and were therefor presented as normative values stratified by BMI. Protrusion of ocular papilla, empty sella and transverse sinus occlusion were rare in total. Our data show an association between BMI and commonly used MRI features for diagnosing IIH. We provide categorized normative BMI values for ONSD, pituitary gland height, and transverse sinus diameter. This distinction helps objectively identify potential IIH indicators compared to regular population norms, enhancing diagnostic accuracy for suspected IIH patients. Notably, optic nerve head protrusion, empty sella, and transverse sinus occlusion are rare in healthy individuals, solidifying their importance as imaging markers regardless of BMI.

Evidence for converging pathophysiology in complex regional pain-syndrome and primary headache disorders: results from a case-control study.

BACKGROUND: Neuroinflammation and maladaptive neuroplasticity play pivotal roles in migraine (MIG), trigeminal autonomic cephalalgias (TAC), and complex regional pain syndrome (CRPS). Notably, CRPS shares connections with calcitonin gene-related peptide (CGRP) in its pathophysiology. This study aims to assess if the documented links between CRPS and MIG/TAC in literature align with clinical phenotypes and disease progressions. This assessment may bolster the hypothesis of shared pathophysiological mechanisms. METHODS: Patients with CRPS (n = 184) and an age-/gender-matched control group with trauma but without CRPS (n = 148) participated in this case-control study. Participant answered well-established questionnaires for the definition of CRPS symptoms, any headache complaints, headache entity, and clinical management. RESULTS: Patients with CRPS were significantly more likely to suffer from migraine (OR: 3.23, 95% CI 1.82-5.85), TAC (OR: 8.07, 95% CI 1.33-154.79), or non-classified headaches (OR: 3.68, 95% CI 1.88-7.49) compared to the control group. Patients with MIG/TAC developed CRPS earlier in life (37.2 ± 11.1 vs 46.8 ± 13.5 years), had more often a central CRPS phenotype (60.6% vs. 37.0% overall) and were three times more likely to report allodynia compared to CRPS patients with other types of headaches. Additionally, these patients experienced higher pain levels and more severe CRPS, which intensified with an increasing number of headache days. Patients receiving monoclonal antibody treatment targeting the CGRP pathway for headaches reported positive effects on CRPS symptoms. CONCLUSION: This study identified clinically relevant associations of MIG/TAC and CRPS not explained by chance. Further longitudinal investigations exploring potentially mutual pathomechanisms may improve the clinical management of both CRPS and primary headache disorders. TRIAL REGISTRATION: German Clinical Trials Register (DRKS00022961).

Nanoscale organization of the endogenous ASC speck.

The NLRP3 inflammasome is a central component of the innate immune system. Its activation leads to formation of the ASC speck, a supramolecular assembly of the inflammasome adaptor protein ASC. Different models, based on ASC overexpression, have been proposed for the structure of the ASC speck. Using dual-color 3D super-resolution imaging (dSTORM and DNA-PAINT), we visualized the ASC speck structure following NLRP3 inflammasome activation using endogenous ASC expression. A complete structure was only obtainable by labeling with both anti-ASC antibodies and nanobodies. The complex varies in diameter between ∼800 and 1000 nm, and is composed of a dense core with emerging filaments. Dual-color confocal fluorescence microscopy indicated that the ASC speck does not colocalize with the microtubule-organizing center at late time points after Nigericin stimulation. From super-resolution images of whole cells, the ASC specks were sorted into a pseudo-time sequence indicating that they become denser but not larger during formation.

Reliability of a telephone interview for the classification of headache disorders.

Objective: The study aimed to test the reliability of a semi-structured telephone interview for the classification of headache disorders according to the ICHD-3. Background: Questionnaire-based screening tools are often optimized for single primary headache diagnoses [e.g., migraine (MIG) and tension headache (TTH)] and therefore insufficiently represent the diagnostic precision of the ICHD-3, which limits epidemiological research of rare headache disorders. Brief semi-structured telephone interviews could be an effective alternative to improve classification. Methods: A patient population representative of different primary and secondary headache disorders (n = 60) was recruited from the outpatient clinic (HSA) of a tertiary care headache center. These patients completed an established population-based questionnaire for the classification of MIG, TTH, or trigeminal autonomic cephalalgia (TAC). In addition, they received a semi-structured telephone interview call from three blinded headache specialists individually. The agreement of diagnoses made either using the questionnaires or interviews with the HSA diagnoses was evaluated. Results: Of the 59 patients (n = 1 dropout), 24% had a second-order and 5% had a third-order headache disorder. The main diagnoses were as follows: frequent primary headaches with 61% MIG, 10% TAC, 9% TTH, and 5% rare primary and 16% secondary headaches. Second-order diagnosis was chronic migraine throughout, and third-order diagnoses were medication overuse headache and TTH. Agreement between main headaches from the HSA was significantly better for the telephone interview than for the questionnaire (questionnaire: κ = 0.330; interview: κ = 0.822; p < 0.001). Second-order diagnoses were not adequately captured by questionnaires, while there was a trend for good agreement with the telephone interview (κ = 0.433; p = 0.074). Headache frequency and psychiatric comorbidities were independent predictors of HSA and telephone interview agreement. Male sex, headache frequency, severity, and depressive disorders were independently predictive for agreement between the questionnaire and HSA. The telephone interview showed high sensitivity (≥71%) and specificity (≥92%) for all primary headache disorders, whereas the questionnaire was below 50% in either sensitivity or specificity. Conclusion: The semi-structured telephone interview appears to be a more reliable tool for accurate diagnosis of headache disorders than self-report questionnaires. This offers the potential to improve epidemiological headache research and care even in underserved areas.

The role of ipsilateral motor network in upper limb movement.

The execution of voluntary movements is primarily governed by the cerebral hemisphere contralateral to the moving limb. Previous research indicates that the ipsilateral motor network, comprising the primary motor cortex (M1), supplementary motor area (SMA), and premotor cortex (PM), plays a crucial role in the planning and execution of limb movements. However, the precise functions of this network and its interplay in different task contexts have yet to be fully understood. Twenty healthy right-handed participants (10 females, mean age 26.1 ± 4.6 years) underwent functional MRI scans while performing biceps brachii representations such as bilateral, unilateral flexion, and bilateral flexion-extension. Ipsilateral motor evoked potentials (iMEPs) were obtained from the identical set of participants in a prior study using transcranial magnetic stimulation (TMS) targeting M1 while employing the same motor tasks. The voxel time series was extracted based on the region of interest (M1, SMA, ventral PM and dorsal PM). Directed functinal connectivity was derived from the extracted time series using time-resolved partial directed coherence. We found increased connectivity from left-PMv to both sides M1, as well as right-PMv to both sides SMA, in unilateral flexion compared to bilateral flexion. Connectivity from left M1 to left-PMv, and left-SMA to right-PMd, also increased in both unilateral flexion and bilateral flexion-extension compared to bilateral flexion. However, connectivity between PMv and right-M1 to left-PMd decreased during bilateral flexion-extension compared to unilateral flexion. Additionally, during bilateral flexion-extension, the connectivity from right-M1 to right-SMA had a negative relationship with the area ratio of iMEP in the dominant side. Our results provide corroborating evidence for prior research suggesting that the ipsilateral motor network is implicated in the voluntary movements and underscores its involvement in cognitive processes such as movement planning and coordination. Moreover, ipsilateral connectivity from M1 to SMA on the dominant side can modulate the degree of ipsilateral M1 activation during bilateral antagonistic contraction.

Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions.

DNA-PAINT based single-particle tracking (DNA-PAINT-SPT) has recently significantly enhanced observation times in in vitro SPT experiments by overcoming the constraints of fluorophore photobleaching. However, with the reported implementation, only a single target can be imaged and the technique cannot be applied straight to live cell imaging. Here we report on leveraging this technique from a proof-of-principle implementation to a useful tool for the SPT community by introducing simultaneous live cell dual-color DNA-PAINT-SPT for quantifying protein dimerization and tracking proteins in living cell membranes, demonstrating its improved performance over single-dye SPT.

Participatory design of teacher dashboards: navigating the tension between teacher input and theories on teacher professional vision.

In the field of AI in education, there is a movement toward human-centered design in which the primary stakeholders are collaborators in establishing the design and functionality of the AI system (participatory design). Several authors have noted that there is a potential tension in participatory design between involving stakeholders and, thus, increasing uptake of the system on the one hand, and the use of educational theory on the other hand. The goal of the present perspective article is to unpack this tension in more detail, focusing on the example of teacher dashboards. Our contribution to theory is to show that insights from the research field of teacher professional vision can help explain why stakeholder involvement may lead to tension. In particular, we discuss that the sources of information that teachers use in their professional vision, and which data sources could be included on dashboards, might differ with respect to whether they actually relate to student learning or not. Using this difference as a starting point for participatory design could help navigate the aforementioned tension. Subsequently, we describe several implications for practice and research that could help move the field of human centered design further.

Ångström-resolution fluorescence microscopy.

Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches1-6 can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations7-14 have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with Ångström resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems.

Experimental and Simulative Analysis of the Pressure Development in a Closed Injection Pultrusion Process with Multiple Chamber Geometries.

The use of innovative higher-performance highly reactive resin systems requires an enhancement of the established method of fiber impregnation (open bath) towards closed resin-injection pultrusion (CIP), due to the short pot life of the resin systems. The result is that the open bath is developed into a closed injection and impregnation chamber ("ii-chamber"). In this study, three parameters-resin viscosity, opening angle and opening factor at the injection point on the ii-chamber-are varied, each in three stages. For each set of parameters, a pultrusion trial is conducted and the process pressures in the ii-chamber and pultrusion die measured. This enables direct feedback via the process conditions of the as yet uncured composite. The data obtained are used to validate a newly developed simulation model. The model is based on Darcy's law, which has been extended to take fiber movement into account and thus represent the resulting pressure increase in the die. The flexible ii-chamber and die concept enhance our understanding of the processes taking place in the die system. The sensitivity of the process pressures can be shown for the three influencing variables. The experiment shows that of the three influencing variables investigated, viscosity has the greatest sensitivity to pressure development. In general, it can be said that over the length of the pultrusion die system, the pressure level increases across the three measuring points.

Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus.

Rotaviruses transcribe 11 distinct RNAs that must be co-packaged prior to their replication to make an infectious virion. During infection, nontranslating rotavirus transcripts accumulate in cytoplasmic protein-RNA granules known as viroplasms that support segmented genome assembly and replication via a poorly understood mechanism. Here, we analysed the RV transcriptome by combining DNA-barcoded smFISH of rotavirus-infected cells. Rotavirus RNA stoichiometry in viroplasms appears to be distinct from the cytoplasmic transcript distribution, with the largest transcript being the most enriched in viroplasms, suggesting a selective RNA enrichment mechanism. While all 11 types of transcripts accumulate in viroplasms, their stoichiometry significantly varied between individual viroplasms. Accumulation of transcripts requires the presence of 3' untranslated terminal regions and viroplasmic localisation of the viral polymerase VP1, consistent with the observed lack of polyadenylated transcripts in viroplasms. Our observations reveal similarities between viroplasms and other cytoplasmic RNP granules and identify viroplasmic proteins as drivers of viral RNA assembly during viroplasm formation.

Slow-Off-Rate-Modified Aptamer Labeling for Fluorescence Microscopy and DNA-PAINT.

Super-resolution microscopy methods enable the visualization of biological processes on the level of a few nanometers. However, the application of these techniques in biological systems is limited by the availability of small affinity reagents. Slow off-rate-modified aptamers as nucleic acid analogues to antibodies have been successfully applied to improve the resolution and quantification of DNA-PAINT. In this chapter, we describe a protocol for using SOMAmers as labeling reagents for super-resolution microscopy.

Polypharmacy in patients with multiple sclerosis and the impact on levels of care and therapy units.

Background: The aim of this study was to examine the societal costs of polypharmacy in patients with multiple sclerosis (MS). We therefore focused on the association between the number of medications on the level of care (LOC), the German classification of the need for care, and the number of therapy sessions (TTU). Methods: In addition to demographic information and medication, 101 MS patients performed the Multiple Sclerosis Health Resource Utilization Survey (MS-HRS). Medications were subdivided into a total number of medications (TD), MS-related medication [MSD, i.e., disease-modifying drugs (DMDs) and symptomatic treatment (SD)], and medication for comorbidities (CDs). Multivariate linear regression models were performed to estimate if the amount of each medication type affects LOC or TTU. Results: Polypharmacy appeared in 54 patients at the time of the survey. The relative risk (RR) of LOC 1 increased significantly by 2.46 (p = 0.001) per TD and by 2.55 (p = 0.004) per MSD, but not per CD (RR 1.44; p = 0.092). The effect of RR on MSD was driven by SD (RR 2.2; p = 0.013) but not DMD (RR 2.6; p = 0.4). RR of MSD remained significant for LOC 2 (1.77; p = 0.009) and LOC 3/4 (1.91; p = 0.015), with a strong trend in RR of SD, but not DMD. TTU increased significantly per MSD (p = 0.012), but not per TD (p = 0.081) and CD (p = 0.724). Conclusion: The number of MSDs is related to the likelihood of a higher level of care and the number of therapy sessions and is therefore a good indication of the extent of the societal costs.

Effect of non-invasive transcutaneous auricular vagus nerve stimulation on cerebral motor excitability-Study protocol for a randomized, sham controlled trial.

Transcutaneous auricular vagus nerve stimulation (taVNS) is becoming increasingly established in the treatment of various neurological and psychiatric diseases. However, only a few studies have focused on the overall influence of taVNS on cortical excitability in general. The planned study will investigate the effect of taVNS on the excitability of the motor cortex in young healthy subjects. The aim of the study is to gain better understand of the physiological mechanism of taVNS to contribute to new fields of application of taVNS in new areas such as the treatment of stroke or multiple sclerosis. This protocol describes a single-center, prospective, double blind, sham-controlled trial that evaluates the effect of taVNS on motor cortex excitability with a planned sample size of 30 participants. The effect of taVNS is investigated by neuronavigation and electromyography (EMG) coupled transcranial magnetic stimulation (TMS) applied before and after taVNS stimulation. The following parameters are assessed: resting motor threshold (RMT), active motor threshold (AMT), recruitment curve (RC), short intracortical inhibition (SICI), intracortical facilitation (ICF). All parameters will be assessed for taVNS on the basis of perception threshold and tolerance threshold. All investigations performed in the study were reviewed and approved by the local ethics committee of the University Medical Center Greifswald (study reference number: BB048/22). Clinical trial registration: www.drks.de, number: DRKS00029937.

The Cortical Silent Period and Its Association with Fatigue in Multiple Sclerosis: The Need for Standardized Data Collection.

The cortical silent period (CSP), assessed with transcranial magnetic stimulation (TMS), provides insights into motor cortex excitability. Alterations in the CSP have been observed in multiple sclerosis (MS), although a comparison of the sometimes contradictory results is difficult due to methodological differences. The aim of this study is to provide a more profound neurophysiological understanding of fatigue's pathophysiology and its relationship to the CSP. Twenty-three patients with MS, along with a matched control group, underwent comprehensive CSP measurements at four intensities (125, 150, 175, and 200% resting motor threshold), while their fatigue levels were assessed using the Fatigue Scale for Motor and Cognitive Functions (FSMC) and its motor and cognitive subscore. MS patients exhibited a significantly increased CSP duration compared to controls (p = 0.02), but CSP duration was not associated with the total FSMC, or the motor or cognitive subscore. Our data suggest a systematic difference in MS patients compared to healthy controls in the CSP but no association with fatigue when measured with the FSMC. Based on these results, and considering the heterogeneous literature in the field, our study highlights the need for a more standardized approach to neurophysiological data collection and validation. This standardization is crucial for exploring the link between TMS and clinical impairments in diseases like MS.

Impaired Pain Processing at a Brainstem Level Is Involved in Maladaptive Neuroplasticity in Patients with Chronic Complex Regional Pain Syndrome.

Neuroinflammatory mechanisms and maladaptive neuroplasticity underlie the progression of complex regional pain syndrome (CRPS), which is prototypical of central neuropathic pain conditions. While cortical maladaptive alterations are well described, little is known about the contribution of the brainstem to the pathophysiology. This study investigates the role of pain-modulatory brainstem pathways in CRPS using the nociceptive blink reflex (nBR), which not only provides a direct read-out of brainstem excitability and habituation to painful stimuli but may also be suitable for use as a diagnostic biomarker for CRPS. Thirteen patients with CRPS and thirteen healthy controls (HCs) participated in this prospective case-control study investigating the polysynaptic trigemino-cervical (R2) nBR response. The R2 area and its habituation were assessed following repeated supraorbital electrical stimulation. Between-group comparisons included evaluations of diagnostic characteristics as a potential biomarker for the disease. Patients with CRPS showed a substantial decrease in habituation on the stimulated (Cohen's d: 1.3; p = 0.012) and the non-stimulated side (Cohen's d: 1.1; p = 0.04). This is the first study to reveal altered nBR habituation as a pathophysiological mechanism and potential diagnostic biomarker in CRPS. We confirmed previous findings of altered nBR excitability, but the diagnostic accuracy was inferior. Future studies should investigate the nBR as a marker of progression to central mechanisms in CRPS and as a biomarker to predict treatment response or prognosis.

High-precision estimation of emitter positions using Bayesian grouping of localizations.

Single-molecule localization microscopy super-resolution methods rely on stochastic blinking/binding events, which often occur multiple times from each emitter over the course of data acquisition. Typically, the blinking/binding events from each emitter are treated as independent events, without an attempt to assign them to a particular emitter. Here, we describe a Bayesian method of inferring the positions of the tagged molecules by exploring the possible grouping and combination of localizations from multiple blinking/binding events. The results are position estimates of the tagged molecules that have improved localization precision and facilitate nanoscale structural insights. The Bayesian framework uses the localization precisions to learn the statistical distribution of the number of blinking/binding events per emitter and infer the number and position of emitters. We demonstrate the method on a range of synthetic data with various emitter densities, DNA origami constructs and biological structures using DNA-PAINT and dSTORM data. We show that under some experimental conditions it is possible to achieve sub-nanometer precision.