Neuromuscular junction visualization in paraffin‐embedded thyroarytenoid muscle sections: Expanding options beyond frozen section analysis

Objectives: The current gold standard for immunofluorescent (IF) visualization of neuromuscular junctions (NMJs) in muscle utilizes frozen tissue sections with fluorescent conjugated antibodies to demarcate neurons and IF alpha-bungarotoxin (α-BTX) to demarcate motor endplates. Frozen tissue sectioning comes with inherent inescapable limitations, including cryosectioning artifact and limited sample shelf-life. However, a parallel approach to identify NMJs in paraffin-embedded tissue sections has not been previously described. Methods: Yucatan minipig thyroarytenoid (TA) muscle was harvested and prepared as 5-μm thick paraffin-embedded tissue sections. A variety of antibodies at various concentrations were selected to target nicotinic acetylcholine receptors, synaptic vesicles, and neurons. Results: Neurofilament (NEFL, Invitrogen, 1:500) and synaptic vesicle glycoprotein (SV2, DSHB, 1:230) bound and demarcated the neurons and synaptic vesicles, respectively. Following consistent visualization of nerve tissue, rabbit anti-nicotinic acetylcholine receptor alpha-1 subunit (CHRNα1, Abcam, 1:500) was used to identify the acetylcholine receptors within motor endplates. Complete NMJ visualization was then achieved with an optimized protocol using primary antibodies to the neurofilament light chain, nerve synaptic vesicle glycoprotein 2, and the alpha 1 subunit of the nicotinic acetylcholine receptor. Slide imaging was performed with the Echo Revolve Microscope (40×). Conclusions: Herein, we describe a new methodology to visualize NMJs within paraffin-embedded TA muscle sections. Our protocol avoids the known limitations associated with cryosectioned samples and introduces a new neurolaryngologic research tool that utilizes the advantageous ability of paraffin-embedded sectioning to preserve tissue morphology. In conjunction with standard cryosectioned methods, the described paraffin-embedded protocol serves to enhance histological analysis of NMJs

LRRK2-associated parkinsonism with and without in vivo evidence of alpha-synuclein aggregates: longitudinal clinical and biomarker characterization

Among LRRK2-associated parkinsonism cases with nigral degeneration, over two-thirds demonstrate evidence of pathologic alpha-synuclein, but many do not. Understanding the clinical phenotype and underlying biology in such individuals is critical for therapeutic development. Our objective was to compare clinical and biomarker features, and rate of progression over 4 years of follow-up, among LRRK2-associated parkinsonism cases with and without in vivo evidence of alpha-synuclein aggregates. Data were from the Parkinson's Progression Markers Initiative, a multicentre prospective cohort study. The sample included individuals diagnosed with Parkinson disease with pathogenic variants in LRRK2. Presence of CSF alpha-synuclein aggregation was assessed with seed amplification assay. A range of clinician- and patient-reported outcome assessments were administered. Biomarkers included dopamine transporter scan, CSF amyloid-beta1-42, total tau, phospho-tau181, urine bis(monoacylglycerol)phosphate levels and serum neurofilament light chain. Linear mixed-effects (LMMs) models examined differences in trajectory in CSF-negative and CSF-positive groups. A total of 148 LRRK2 parkinsonism cases (86% with G2019S variant), 46 negative and 102 positive for CSF alpha-synuclein seed amplification assay, were included. At baseline, the negative group was older than the positive group [median (inter-quartile range) 69.1 (65.2-72.3) versus 61.5 (55.6-66.9) years, P < 0.001] and a greater proportion were female [28 (61%) versus 43 (42%), P = 0.035]. Despite being older, the negative group had similar duration since diagnosis and similar motor rating scale [16 (11-23) versus 16 (10-22), P = 0.480] though lower levodopa equivalents. Only 13 (29%) of the negative group were hyposmic, compared with 75 (77%) of the positive group. The negative group, compared with the positive group, had higher per cent-expected putamenal dopamine transporter binding for their age and sex [0.36 (0.29-0.45) versus 0.26 (0.22-0.37), P < 0.001]. Serum neurofilament light chain was higher in the negative group compared with the positive group [17.10 (13.60-22.10) versus 10.50 (8.43-14.70) pg/mL; age-adjusted P-value = 0.013]. In terms of longitudinal change, the negative group remained stable in functional rating scale score in contrast to the positive group who had a significant increase (worsening) of 0.729 per year (P = 0.037), but no other differences in trajectory were found. Among individuals diagnosed with Parkinson disease with pathogenic variants in the LRRK2 gene, we found clinical and biomarker differences in cases without versus with in vivo evidence of CSF alpha-synuclein aggregates. LRRK2 parkinsonism cases without evidence of alpha-synuclein aggregates as a group exhibit less severe motor manifestations and decline. The underlying biology in LRRK2 parkinsonism cases without evidence of alpha-synuclein aggregates requires further investigation

Telemedicine Use During the COVID-19 Pandemic in 8 Countries From the International Sexual Health and Reproductive Health Consortium: Web-Based Cross-Sectional Survey Study

Background: Telemedicine is an important way to fill in the access gap to in-person health care services during challenging times like pandemics. Objective: This study aimed to investigate the role that telemedicine played during the COVID-19 pandemic by multicountry comparison of the use of telemedicine prior to and during the pandemic. Methods: This study analyzes data from the second wave of the International Sexual Health and Reproductive Health study. This included data collected between April 2021 and July 2022 in 8 countries, including Armenia (n=296), Egypt (n=889), Germany (n=138), Moldova (n=311), Nigeria (n=205), Portugal (n=951), Singapore (n=13), and Spain (n=54). This study covered sociodemographics, sexual and reproductive health (SRH), and telemedicine use. Descriptive statistics and multilevel modeling were used to assess the factors influencing the use of telemedicine. Results: Overall, 2857 participants were recruited. Approximately 57.6% (n=1646) of participants had never used telemedicine prior to COVID-19 measures, while 45.9% (n=1311) of participants required health care but reported not using telemedicine services following the introduction of COVID-19 measures. In high-income countries, the most common mode reported was audio-based telemedicine services, with 283 (71.8%) and 417 (73.5%) participants doing so before and during COVID-19, respectively. This was followed by text-based telemedicine services, with 152 (38.6%) and 173 (30.5%) participants doing so before and during COVID-19, respectively. In low- to middle-income countries, many participants also reported using audio-based telemedicine services, with 288 (35.3%) and 237 (40.8%) participants doing so before and during COVID-19, respectively. This was followed by chat-based telemedicine services, with 265 (32.4%) and 217 (37.3%) participants doing so before and during COVID-19, respectively. Multilevel modeling revealed that those who were older (adjusted odds ratio [aOR] 0.99, 95% CI 0.99-1.00) and were in countries with a higher gross domestic product per capita (aOR 0.99, 95% CI 0.98-1.00) were less likely to have ever used telemedicine. Participants who were of male sex assigned at birth (aOR 0.79, 95% CI 0.65-0.96) were less likely to use telemedicine during the pandemic. Participants who perceived that they were worse off financially were more likely to have switched to telemedicine during COVID-19 (aOR 1.39, 95% CI 1.02-1.89) and were more likely to report having a poor or fair experience of telemedicine services (aOR 1.75, 95% CI 1.34-2.29). When sexual orientation was included in the model, nonheterosexual individuals were more likely to ever use telemedicine prior to COVID-19 (aOR 1.35, 95% CI 1.08-1.69), more likely to have used telemedicine during COVID-19 (aOR 1.58, 95% CI 1.24-2.02), and more likely to have switched to telemedicine during COVID-19 (aOR 1.55, 95% CI 1.09-2.21). Conclusions: Telemedicine played a key role in addressing health care needs during the COVID-19 pandemic. Age, sex, economic status, and sexual orientation influenced its use

Early Detection of Treatment's Side Effects: A Sequential Approach

IUIWith the emergence and spread of infectious diseases with pandemic potential, such as COVID-19, the urgency for vaccine development has led to unprecedented compressed and accelerated schedules that shortened the standard development timeline. To monitor the potential side effect(s) of the vaccine during the (initial) vaccination campaign, we developed an optimal sequential test that allows for the early detection of potential side effect(s). This test employs a rule to stop the vaccination process once the observed number of side effect incidents exceeds a certain (pre-determined) threshold. The optimality of the proposed sequential test is justified when compared with the (α,β) optimality of the non-randomized fixed-sample Uniformly Most Powerful (UMP) test. Firstly, we construct an optimal sequential procedure for the case of a single side effect. We study the properties of the sequential test and derive the exact expressions of the Average Sample Number (ASN) curve of the stopping time (and its variance) via the regularized incomplete beta function. Additionally, we derive the asymptotic distribution of the relative ’savings’ in ASN as compared to fixed sample size. Moreover, we construct the post-test parameter estimate and studied its sampling properties, including its asymptotic behavior under local-type alternatives. These limiting behavior results are the consistency and asymptotic normality of the post-test parameter estimator. We conclude this part with a small simulation study illustrating the asymptotic performance of the point and interval estimation and provide a detailed example, based on COVID-19 side effect data (see Beatty et al. (2021)) of our suggested testing procedure. Next, we propose an optimal sequential procedure for the case of two (or more) side effects. While the sequential procedure we employ, simultaneously monitors several of the treatment’s side effects, the (α,β)-optimal test we propose does not require any information about the inter-correlation between these potential side effects to obtain the optimal sample size and critical values. However, in all of the subsequent analyses, including the derivations of the exact expressions of the ASN, the power function, and the properties of the post-test (or post-detection) estimators, we accounted specifically, for the correlation between the potential side effects. In the real-life application (such as post-marketing surveillance), the number of available observations is large enough to justify asymptotic analyses of the sequential procedure (testing and post-detection estimation) properties. Accordingly, we also derive the consistency and asymptotic normality of our post-test estimators. Moreover, to compare two specific side effects, their relative risk plays an important role. We derive the distribution of the estimated relative risk in the asymptotic framework to provide appropriate inference. To illustrate the theoretical results presented, we provide two detailed examples based on the data of side effects on COVID-19 vaccine collected in Nigeria (see Ilori et al. (2022)). Finally, we consider the Bayesian framework and construct a Bayesian Sequential testing procedure to test the Relative Risk between two specific treatments based on the binary data obtained from the two-arm clinical trial. We noticed that the optimal sequential test mentioned in the first part can be utilized here to test Relative Risk as a one-sided hypothesis testing procedure. To apply the optimal sequential test more straightforward, we introduce the Bayesian framework into our analysis. Since by the Stopping Rule Principle (SRP), the posterior probabilities remain unaffected by the stopping rule used to reach that point with accumulated data. Accordingly, using the Bayesian test, we obtain the corresponding decision on each data point without affecting by the optimal stopping rule as in classical sequential test. Moreover, with the modified Bayesian test, we obtain the corresponding conditional error probabilities on each data point. Specifically, we utilized the data from Silva, Kulldorff, and Katherine Yih (2020) to analyze the results obtained from two tests under several different priors and make the conclusion

Health and Financial Literacy and the Acquisition of COVID-19 Knowledge in Older Adults

Knowledge about COVID-19 enters into many aspects of decision making, especially for older people who are at increased risk of severe disease or death. Yet little is known about the resources that supported older people's uptake of COVID-19 knowledge. Here, we hypothesized that higher pre-pandemic health and financial literacy was associated with higher COVID-19 knowledge. Participants were 434 community-based older people without dementia. COVID-19 knowledge was assessed via a 5-item measure, and health and financial literacy was assessed via a 32-item measure. In an ordinal regression model adjusted for age, gender, and education, higher literacy was associated with higher COVID-19 knowledge (p < .0001), and this association persisted after further adjusting for robust measures of global cognition or one of five specific cognitive domains (all p's ≤ .0001). These findings suggest that literacy plays a key role in supporting older people's acquisition of impactful knowledge in the real world

Sabirnetug (ACU193) Lowers CSF Levels of Synaptic Biomarkers in INTERCEPT‐AD Phase 1 Study in Early AD

Background: Sabirnetug (ACU193) is a humanized IgG2 antibody targeting soluble, synaptotoxic amyloid β oligomers (AβOs). AβOs accumulate in Alzheimer’s disease (AD) and induce pre‐ and post‐synaptic changes, resulting in dendritic spine loss, neuronal degeneration, and release of synaptic proteins into the CSF. Recently, we reported that three administrations of sabirnetug in an early AD population (INTERCEPT‐AD Phase 1 study, NCT04931459) significantly lowered CSF levels of the post‐synaptic protein neurogranin as well as pTau181. Here, we present data on additional CSF synaptic biomarkers and AD plasma biomarkers measured in INTERCEPT‐AD. Method: INTERCEPT‐AD was a randomized, placebo‐controlled study with two parts: single ascending dose (SAD) randomized in a 6:2 ratio to sabirnetug (2, 10, 25, 60 mg/kg) or placebo and multiple ascending dose (MAD) randomized 8:2 to sabirnetug (three administrations at 10 or 60 mg/kg every 4 weeks [Q4W] or 25 mg/kg Q2W) or placebo. Biomarkers were measured blinded in CSF and EDTA‐plasma, before and after drug administration, by Amsterdam UMC. Result: CSF levels of vesicle‐associated membrane protein 2 (VAMP2), a protein involved in pre‐ and post‐synaptic vesicle trafficking, were significantly normalized (lowered) in sabirnetug treated participants relative to placebo in all three MAD cohorts. Levels of the pre‐synaptic protein neuronal pentraxin 2 (NPTX2), which acts on post‐synaptic excitatory synapses, regulates complement activity, and lowers with cognitive decline, trended lower with sabirnetug than placebo. Plasma levels of GFAP, pTau181, and pTau217 trended towards normalization (lowering) of AD‐dependent changes in the 60 mg/kg Q4W cohort. Conclusion: In INTERCEPT‐AD, three administrations of sabirnetug lowered CSF levels of both pre‐ and post‐synaptic proteins, consistent with sabirnetug’s proposed mechanism of action to inhibit AβO synaptic binding. VAMP2 appeared most sensitive to sabirnetug in this study, lowering significantly in all three MAD cohorts; other markers previously reported to have a statistically significant response to sabirnetug ‐ neurogranin and pTau181 ‐ did so at the highest dose. No statistically significant changes in plasma biomarkers were observed in this short study. Long‐term changes in biomarker levels and their relationship to clinical efficacy will be evaluated in the 18‐month ALTITUDE‐AD phase 2 study of sabirnetug beginning in the first half of 2024

Deep enteroscopy in children: techniques, applications, and future directions

Deep enteroscopy, encompassing push enteroscopy (PE) and balloon-assisted enteroscopy (BAE), has revolutionized the diagnosis and treatment of pediatric small bowel disorders. This review examines the evolving role of these techniques in managing conditions such as obscure gastrointestinal bleeding, Crohn's disease, polyposis syndromes, strictures, and small bowel tumors. While PE is effective for both diagnostic and therapeutic interventions in the proximal small bowel, its limited insertion depth has driven the adoption of BAE techniques. These include single-balloon enteroscopy (SBE) and double-balloon enteroscopy (DBE), which provide deeper and more comprehensive access. Both BAE modalities offer greater insertion depth and stability, enabling advanced therapeutic interventions such as polypectomy, stricture dilation, and hemostasis. Pediatric-specific data demonstrate high diagnostic yields for BAE, with comparable outcomes between SBE and DBE. These techniques have proven safe across diverse indications, though younger children may experience slightly higher complication rates due to anatomical considerations. Despite these advancements, challenges persist, including a limited evidence base in pediatrics, barriers to training, and the need for standardized protocols. Additionally, emerging innovations such as artificial intelligence offer opportunities to enhance diagnostic accuracy and procedural efficiency. Comparative analyses of PE, BAE, and capsule endoscopy are necessary to refine procedural selection and optimize outcomes in pediatric patients. Furthermore, structured pediatric training programs and simulation-based learning could address competency gaps, ensuring safe and effective application of these techniques. By addressing current research gaps, embracing technological advancements, and tailoring approaches to pediatric populations, deep enteroscopy can continue to transform the management of small bowel disorders in children

Over‐Representation of Extremely Wealthy Neighborhood Social Exposomes for Brain Donors within Alzheimer’s Disease Research Center Brain Banks assessed by the Neighborhoods Study

Background: Adverse social exposome (indexed by national Area Deprivation Index [ADI] 80‐100 or ‘high ADI’) is linked to structural inequities and increased risk of Alzheimer’s disease neuropathology. Twenty percent of the US population resides within high ADI areas, predominantly in inner cities, tribal reservations and rural areas. The percentage of brain donors from high ADI areas within the Alzheimer’s Disease Research Center (ADRC) brain bank system is unknown. Objective: Determine ADI for brain donors from 21 ADRC sites as part of the on‐going Neighborhoods Study. Methods: All brain donors in participating ADRC sites with NACC neuropathology data and personal identifiers for ADI linkage (N = 8,637) were included (Figure 1). Geocoded donor addresses were linked to time‐concordant ADI percentiles for year of death. Results: Overall, only 5.6% of ADRC brain donors (N = 488) resided in a high ADI (disadvantaged) neighborhood at death. The remaining donors resided in more advantaged neighborhoods, with nearly 40% of donors living in the wealthiest quintile of neighborhoods, and over 300 brain donors originating from the wealthiest 1% of US neighborhoods (Figure 2). Donors from high ADI (disadvantaged) neighborhoods identified as 87% White (n = 424), 11% Black (55), 1% Multiracial (6) and <1% other/unknown race (3), with 1% Hispanic (5). None identified as American Indian/Alaska Native or Native Hawaiian/Pacific Islander/Asian. In comparison, donors from low ADI neighborhoods were 94% White (n = 7680), 3% Black (273), 1% Multiracial (75), <1% American Indian/Alaska Native (11), <1% Native Hawaiian/Pacific Islander/Asian (60), and <1% other/unknown race (50), with 3% Hispanic (230). Sex distribution was similar (54%, 51% female, respectively). Inclusion of high ADI donors varied dramatically across the 21 ADRC brain banks from a low of 0.6% to high of 20% of all a site’s donors (Figure 3). Conclusions: ADI was determined for over 8,600 brain donors in the ADRC system, demonstrating a marked over‐representation of donors from very low ADI (extremely wealthy) neighborhoods, in addition to site‐to‐site variability. This is the first time a comprehensive cross‐sectional social exposome assessment of this nature has been performed, opening windows for additional mechanistic study of the social exposome on brain pathology. Life course ADI assessments are on‐going

Associations between Amyloid, Cardiovascular Risk, and Cognitive Function in Korean Older Adults: Insights from the KBASE Cohort

Background: Understanding the relationship between cardiovascular burden, amyloid, and cognition in Alzheimer’s disease (AD) is essential for targeted interventions, especially in ethnically diverse populations where research remains limited. This study aimed to investigate these relationships in a cohort of Korean older adults along the AD spectrum. Method: 526 participants from the Korean Brain Aging Study for the Early Diagnosis and Prediction of Alzheimer’s Disease (KBASE) cohort were included in this study. Vascular burden was quantified using Framingham Risk Score (FRS) and participants were categorized into four groups based on combinations of FRS (FRS High or FRS Low with a median split) and amyloid status (Aβ+ or Aβ‐ based on a cut‐off of 1.2373). Cognitive function was evaluated using standardized neuropsychological tests processed with structural equation models to produce domain scores for memory, executive functioning, language, and visuospatial. ANOVA was employed at baseline to analyze cognitive differences among these groups and within each clinical diagnosis. Longitudinal mixed effects models spanning a period of four years from the initial visit captured cognitive changes over time within these groups (Figure 1). Result: Significant group and pairwise differences were observed among the four groups in all cognitive domains (p < 0.0001). Stratified analysis within each clinical diagnoses group revealed that CN individuals in FRS high Aβ‐ demonstrated significantly lower memory scores compared to those with FRS low Aβ‐ (p < 0.0001), this trend was absent from MCI and AD groups (Figure 2). Longitudinally, FRS high Aβ+ and FRS low Aβ+ groups consistently demonstrated lower memory scores compared to the FRS low Aβ‐ group. Interestingly, no significant difference in cognition was observed between FRS high Aβ‐ and FRS low Aβ‐ groups over time. However, the most pronounced divergence in longitudinal cognition of the four FRS and Amyloid groups was observed within the MCI diagnosis group (Figure 3). Conclusion: This study highlights the differential impact of cardiovascular risk on cognition depending on amyloid status and clinical diagnosis group. This underscores the importance of considering both cardiovascular risk factors and amyloid pathology early‐on in understanding clinical manifestation and cognitive decline in the AD spectrum, particularly in ethnically diverse populations

Evaluating Meta‐Learners to Analyze Treatment Heterogeneity in Survival Data: Application to Electronic Health Records of Pediatric Asthma Care in COVID‐19 Pandemic

An important aspect of precision medicine focuses on characterizing diverse responses to treatment due to unique patient characteristics, also known as heterogeneous treatment effects (HTE) or individualized treatment effects (ITE), and identifying beneficial subgroups with enhanced treatment effects. Estimating HTE with right-censored data in observational studies remains challenging. In this paper, we propose a pseudo-ITE-based framework for analyzing HTE in survival data, which includes a group of meta-learners for estimating HTE, a variable importance metric for identifying predictive variables to HTE, and a data-adaptive procedure to select subgroups with enhanced treatment effects. We evaluate the finite sample performance of the framework under various observational study settings. Furthermore, we applied the proposed methods to analyze the treatment heterogeneity of a written asthma action plan (WAAP) on time-to-ED (Emergency Department) return due to asthma exacerbation using a large asthma electronic health records dataset with visit records expanded from pre- to post-COVID-19 pandemic. We identified vulnerable subgroups of patients with poorer asthma outcomes but enhanced benefits from WAAP and characterized patient profiles. Our research provides valuable insights for healthcare providers on the strategic distribution of WAAP, particularly during disruptive public health crises, ultimately improving the management and control of pediatric asthma