We developed a scalable pipeline for extracellular miRNA (ex-miRNA) profiling that integrates automated exRNA extraction, small RNA sequencing, and bioinformatic analysis including data processing and normalization. Automated extraction protocols, including doubling input volume or lyophilization to increase RNA yield, were benchmarked against leading manual methods, with donor pregnancy status serving as the primary biological variable. Small RNA sequencing was performed across conditions, enabling systematic evaluation of data quality and comparison of normalization strategies. Across methods and biofluids, DESeq2 most effectively reduced technical variability while preserving biological signal. Comparing specimen types, plasma exhibited the highest reproducibility and retention of biological signal, followed by serum, while urine exhibited greater variability and less differentially expressed miRNAs. Pregnancy-associated ex-miRNA signatures, including C19MC miRNAs, were consistently detected in both plasma and serum. Together, this study establishes a robust framework for scalable exRNA extraction and profiling, supporting standardized assay development for biomarker discovery and clinical applications.

Urine contains extracellular RNA (exRNA) carried by extracellular vesicles (EVs) and other biomolecular complexes. There is currently a need for studies focused on female cohorts to develop new methods for non-invasive analysis of biofluids to create reference profiles and for identification of biomarkers of reproductive and pregnancy disorders. The objective of this study was therefore to identify optimal methods for transcriptomic profiling of urine by testing different exRNA isolation and scalable library preparation methods that enable detection of biomarkers that reflect pregnancy-associated changes in the placenta and maternal tissues. RNA was extracted from pooled and individual urine samples obtained from normal non-pregnant and pregnant females, as well as males, using input volumes of either 0.6 mL, 1 mL, or 4 mL. Samples were extracted using methods that focused either on isolating vesicular (EV-associated) or total (EV-associated and non EV-associated) exRNA. Small RNA libraries (n=208) were prepared using the NEBNext Small RNA Library Prep kit and long RNA libraries (n=97) were prepared using the SMART-Seq v4 Ultra Low Input RNA or the SMARTer Stranded Total RNA-Seq Kit v2 Pico Input kits (Takara). Principal component analysis showed that the greatest source of variance amongst technical replicates of small RNA libraries (n=176 which passed quality control) was exRNA isolation method, and amongst long RNA libraries (n=97 which passed quality control) was library preparation method. Long RNA libraries prepared from exRNA extracted using miRCURY showed that the SMART-Seq v4 method yielded significantly more uniquely mapped reads compared to the Pico v2 method (p<0.05). We have established a scalable pipeline for small and long RNA-Seq profiling of exRNA in urine in a reproducible manner, which we used to identify differentially expressed urinary exRNAs in pregnancy, and will enable transcriptomic profiling of urinary exRNA in disorders of pregnancy, including preeclampsia.

Timely pathogen surveillance and reporting is essential for effective public health guidance. Web dashboards have become a key tool for communicating public health information to stakeholders, health care workers, and the broader community. Over the SARS-CoV-2 pandemic, wastewater surveillance has increasingly been incorporated into public health workflows for outbreak monitoring and response, enabling community-representative and low-cost monitoring to supplement clinical surveillance. However, the methods used for visualization and dissemination of clinical and wastewater surveillance data differ across programs, and best practices are yet to be defined. In this work, we demonstrate data workflows and dashboards used to perform wastewater-based public health surveillance in tandem with clinical data across local and national scales, leveraging custom-built, reproducible, and open-source software. Using a centralized data aggregation and analysis hub approach, we establish multiple data pipelines for data storage, wrangling, and standardized analyses, and deploy custom-built web dashboards that allow for immediate public release. We find that our approach is effective across scales, computing architectures, and dissemination strategies, and provides an adaptable model to incorporate additional pathogens and epidemiological data.

BACKGROUND:The health effects of smoking vary substantially between individuals. Novel strategies are needed to identify individuals with increased risk of smoking-related morbidity. Extracellular vesicle-encapsulated microRNAs (EV-miRNAs) may be viable biomarkers of smoking-related harm. Here, we measured associations of smoking status with plasma EV-miRNA expression levels. METHODS:Participants who were free of smoking-related diseases were sampled from the prospective Normative Aging Study (NAS). Smoking histories were evaluated using standardized smoking questionnaires. EV-miRNAs were measured in plasma. Vital status was monitored using death records. Associations of smoking status with EV-miRNA expression levels were modeled using linear regression. Results were validated in the Strong Heart Study (SHS). Associations of candidate EV-miRNAs with all-cause mortality were modeled using Cox proportional hazards models. RESULTS:Among 88 NAS participants (mean age 70.9 ± 8.0 years), recent smoking was associated with differential expression of 16 plasma EV-miRNAs, including decreased expression of miR-30b-5p (fold change in EV-miRNA expression: 0.26, 95 % CI 0.11-0.58). The association between smoking and low miR-30b-5p expression was replicated in the SHS cohort (fold change: 0.71, 95 % CI 0.51-0.97). A pathway analysis showed miR-30b-5p targeted genes that modulate oncogenic and pro-inflammatory signaling pathways (q-value<0.05). In the NAS cohort, low miR-30b-5p expression was associated with higher all-cause mortality (adjusted Hazard Ratio 3.36, 95 % CI 1.35-8.35). CONCLUSIONS:Recent cigarette smoking was associated with low miR-30b-5p expression in two distinct populations. Circulating miR-30b-5p is a robust biomarker of smoking-related harm and may represent a novel target for the prevention and treatment of smoking-related diseases.

The trophectoderm (TE), the first lineage specified during mammalian development, initiates implantation and gives rise to placental trophoblasts. While animal models have elucidated key conserved signaling pathways involved in early TE specification, including bone morphogenetic protein (BMP), WNT, and HIPPO, species-specific differences during early development emphasize the need for human-specific models. We previously identified VGLL1, a coactivator of TEAD transcription factors, as a human-specific placental marker. In this study, we employed a pluripotent stem cell (PSC)-based model of TE induction by BMP4 to investigate chromatin remodeling and transcriptional dynamics during TE formation. BMP4-induced chromatin accessibility changes promoted a trophoblast gene expression program, while mesoderm lineage markers were only transiently expressed upon canonical WNT activation. We found that VGLL1 was expressed downstream of key TE transcription factors (GATA2/3, TFAP2A/C) but was essential for establishment of full trophoblast identity by up-regulating the epidermal growth factor receptor (EGFR) and reinforcing GATA3 expression through positive feedback. Notably, VGLL1 enhanced canonical WNT signaling via direct regulation of WNT receptors and effectors. We also identified KDM6B, a histone demethylase that removes H3K27me3 repressive marks, as a direct VGLL1 target. KDM6B facilitated activation of bivalent promoters associated with TE markers, linking epigenetic regulation to lineage identity. Our findings establish a mechanistic framework positioning VGLL1 as a central regulator that integrates HIPPO, BMP, and WNT signaling pathways to drive establishment of human TE.

Rationale: Cigarette smoking is the leading cause of preventable death and disease in the world. However, only a fraction of people who smoke die from smoking-related diseases. Thus, there is an urgent need to develop robust markers of smoking-related damage. Extracellular vesicles and their microRNA cargo (EV-miRNAs) may represent viable biomarkers of smoking-related harm. Methods: Participants who were free of known medical conditions including hypertension, diabetes, and coronary artery disease were sampled from the prospective Normative Aging Study (NAS). Smoking histories were evaluated using standardized smoking questionnaires. Plasma EV-miRNAs were measured in the baseline visit. Vital status was monitored using periodic mailings. Associations of smoking status with plasma EV-miRNA expression levels were modeled using linear regression models adjusted for age, body mass index (BMI), education, and alcohol consumption. False discovery rate correction was used to account for multiple comparisons. Results were replicated in the Strong Heart Study (SHS). The gene targets of smoking-associated EV-miRNAs were identified using biological pathway analyses. Associations of candidate EV-miRNAs with all-cause mortality were modeled using Kaplan-Meier curves. Results: Among 91 participants contributing 603 person-years of follow-up in the NAS cohort, the mean age was 71.3±7.9 years. In total, 11 (12.1%) participants reported current or recent cigarette use, which was defined as cigarette smoking within 10 years of the baseline visit. Compared to 80 (87.9%) participants who never smoked cigarettes or had more remote smoking exposures, participants reporting current or recent cigarette use had differential expression of 14 plasma EV-miRNAs, including decreased expression of miR-30b-5p (Fold change in EV-miRNA expression: 0.27, 95% CI 0.13-0.58). The replication in the SHS cohort confirmed the association of recent smoking with decreased miR-30b-5p expression (Fold change: 0.65, 95% CI 0.48-0.88). A biological pathway analysis showed miR-30b-5p targeted pro-inflammatory pathways including the p53 signaling pathway and the Hippo signaling pathway as well as oncogenic pathways related to small cell lung cancer and colorectal cancer. In the NAS cohort, participants with lower miR-30b-5p expression had higher risk of all-cause mortality (log-rank p=0.05) (Figure 1). Conclusions: Current and recent cigarette smoking was associated with decreased expression of miR-30b-5p in two distinct populations. A pathway analysis showed miR-30b-50 targeted genes related to pro-inflammatory signaling and oncogenic pathways. Lower miR-30b-50 expression was associated with higher risk of all-cause mortality. MiR-30b-5p may be a viable mechanistic biomarker of smoking-related harm and may represent a novel target for the prevention and treatment of smoking-related diseases.

BACKGROUND:SARS-CoV-2 antibody levels have been proposed as a correlate of protection from infection. Yet, large-scale prospective studies of cost-effective scalable antibody measures as predictors of infection under real-world conditions are limited. We examined whether antibody levels measured by high-throughput variant-specific SARS-CoV-2 anti-spike immunoglobulin G (IgG) and angiotensin-converting enzyme 2 (ACE2) neutralization assays correlate with cell-based neutralizing antibody measurements and whether they can serve as a reasonable correlate of protection from SARS-CoV-2 infection. METHODS:We conducted a large institutional cohort study between January 2022 and March 2023. Participants (N = 2513) provided dried blood spot (DBS) samples for assessment of anti-spike IgG and ACE2 inhibition levels through high-throughput assays. Comparison with authentic cell-based SARS-CoV-2 neutralizing antibody assays was conducted with serum samples (n = 105). Associations between antibody levels and risk of infection were evaluated. RESULTS:Correlation between serum and DBS sampling and between cell-based neutralizing and high-throughput antibody binding assays was high for anti-spike IgG and ACE2 neutralization, though the degree of correlation varied by variant. Longitudinal evaluation suggested that DBS-based IgG and ACE2 inhibition levels were anticorrelated with infection risk, with higher sensitivity noted for ACE2 inhibition and variant-matched measures. IgG and ACE2 inhibition levels decreased over time, with more durable responses observed in participants whose most recent priming event was infection vs vaccination. CONCLUSIONS:Findings suggest that variant-specific SARS-CoV-2 antibody levels may be a useful correlate of protection for infection, which has important implications for vaccination recommendations and evaluating infection risk. High-throughput assays measured via DBS may have utility in timing of boosters at the population or individual level.

BACKGROUND:Infection prevention (IP) measures are designed to mitigate the transmission of pathogens in healthcare. Using large-scale viral genomic and social network analyses, we determined if IP measures used during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic were adequate in protecting healthcare workers (HCWs) and patients from acquiring SARS-CoV-2. METHODS:We performed retrospective cross-sectional analyses of viral genomics from all available SARS-CoV-2 viral samples collected at UC San Diego Health and social network analysis using the electronic medical record to derive temporospatial overlap of infections among related viromes and supplemented with contact tracing data. The outcome measure was any instance of healthcare transmission, defined as cases with closely related viral genomes and epidemiological connection within the healthcare setting during the infection window. Between November 2020 through January 2022, 12 933 viral genomes were obtained from 35 666 patients and HCWs. RESULTS:Among 5112 SARS-CoV-2 viral samples sequenced from the second and third waves of SARS-CoV-2 (pre-Omicron), 291 pairs were derived from persons with a plausible healthcare overlap. Of these, 34 pairs (12%) were phylogenetically linked: 19 attributable to household and 14 to healthcare transmission. During the Omicron wave, 2106 contact pairs among 7821 sequences resulted in 120 (6%) related pairs among 32 clusters, of which 10 were consistent with healthcare transmission. Transmission was more likely to occur in shared spaces in the older hospital compared with the newer hospital (2.54 vs 0.63 transmission events per 1000 admissions, P < .001). CONCLUSIONS:IP strategies were effective at identifying and preventing healthcare SARS-CoV-2 transmission.

BACKGROUND:American Indian populations have experienced marked disparities in respiratory disease burden. Extracellular vesicle-encapsulated microRNAs (EV-miRNAs) are a novel class of biomarkers that may improve recognition of lung damage in indigenous populations in the United States. RESEARCH QUESTION:Are plasma EV-miRNAs viable biomarkers of respiratory health in American Indian populations? STUDY DESIGN AND METHODS:The Strong Heart Study is a prospective cohort study that enrolled American Indian patients aged 45 to 74 years. EV-miRNA expression was measured in plasma (1993-1995). Respiratory health outcomes, including prebronchodilator FEV1, FVC, and respiratory symptom burden, were ascertained in the same study visit. Club cell secretory protein (CC-16), an antiinflammatory pneumoprotein implicated in COPD pathogenesis, was measured in serum. Linear and logistic regression were used for statistical analyses. Biological pathway analyses were used to elucidate gene targets of significant EV-miRNAs. RESULTS:Among 853 American Indian adults, three EV-miRNAs were associated with FEV1, four EV-miRNAs were associated with FVC, and one EV-miRNA was associated with FEV1/FVC (P < .05). Increased miR-1294 expression was associated with higher odds of airflow limitation (OR, 1.29; 95% CI, 1.07-1.55), whereas increased expression of miR-1294 (OR, 1.32; 95% CI, 1.07-1.63) and miR-532-5p (OR, 1.57; 95% CI, 1.02-2.40) was associated with higher odds of restriction. Increased miR-451a expression was associated with lower odds of exertional dyspnea (OR, 0.71; 95% CI, 0.59-0.85). Twenty-two EV-miRNAs were associated with serum CC-16 levels (q < 0.05), suggesting that EV-miRNAs may play a role in the pathway linking CC-16 to COPD pathogenesis. A pathway analysis showed key EV-miRNAs targeted biological pathways that modulate inflammation, immunity, and structural integrity in the lungs. INTERPRETATION:Circulating EV-miRNAs are novel mechanistic biomarkers of respiratory health and may facilitate the early detection and treatment of lung damage in American Indian populations that have been disproportionately affected by chronic lung diseases.

Background Mechanistic studies of the effects of environmental risk factors have been exploring the potential role of microRNA(miRNAs) as a possible pathway to clinical disease. In this study we examine whether levels of toenail metals are associated with changes in extracellular miRNA(ex-miRNA) expression. Methods We used data derived from the Normative Aging Study from 1996 to 2014 to conduct our analyses. We looked at associations between measured toenail metals: arsenic, cadmium, lead, manganese, and mercury and 282 ex-miRNAs in this population using canonical correlation analyses (CCAs) and longitudinal median regression. We adjusted for covariates such as age, education, body mass index, drinking and smoking behaviors, diabetes, and where available, seafood consumption. The p-values obtained from regression analyses were corrected for multiple comparisons. Ex-miRNAs identified to be associated with toenail metal levels were further examined using pathway analyses. Results Our dataset included 937 observations from 589 men with an average age of 72.9 years at baseline. Both our correlation and regression analyses identified lead and cadmium as exposures most strongly associated with ex-miRNA expression. Numerous ex-miRNAs were identified as being associated with toenail metal levels. miR-27b-3p, in particular, was found to have high correlation with the first canonical dimension in the CCA and was significantly associated with cadmium in the regression analysis. Pathway analyses revealed messenger RNA (mRNA) targets for the ex-miRNAs that were associated with a number of clinical disorders including cancer, cardiovascular disease, and neurological disorders, etc. Conclusion Toenail metals were associated with changes in ex-miRNA levels in both correlational and regression analyses. The ex-miRNAs identified can be linked to a variety of clinical disorders. Further studies are required to validate these findings.

Earlier studies have revealed microRNAs (miRNAs) as potential biomarkers for neurological conditions, however, such evidence on psychiatric outcomes is limited. We utilized the Normative Aging Study (NAS) cohort to investigate the associations between extracellular miRNAs (ex-miRNA) and psychiatric symptoms among a group of older male adults, along with the targeted genes and biological pathways. We studied 569 participants with miRNA profile primarily measured in extracellular vesicles isolated from plasma, and psychiatric symptoms reported over 1996-2014 with repeated measures. Global and dimension scales of psychiatric symptoms were measured via the administration of Brief Symptom Inventory (BSI) per visit covering nine aspects of psychiatric health, such as anxiety, depression, hostility, psychoticism, etc. Ex-miRNAs were profiled using small RNA sequencing. Associations of expression of 395 ex-miRNAs (present in >70% samples) with current mental status were assessed using single-miRNA as well as Least Absolute Shrinkage and Selection Operator (LASSO)-based multi-miRNAs linear mixed effects models adjusting for key demographic and behavioral factors. Biological functions were explored using pathway analyses. We identified ex-miRNAs associated with each BSI scale. In particular, hsa-miR-320d was consistently identified for two global scales. Similar overlapping miRNAs across global and dimension scores included hsa-miR-379-3p, hsa-miR-1976, hsa-miR-151a-5p, hsa-miR-151b, hsa-miR-144-3p, etc. Top KEGG pathways for identified miRNAs included p53 signaling, Hippo signaling, FoxO signaling, protein processing in endoplasmic reticulum and several pathways related with cancer and neurological diseases. This study provided early evidence supporting the associations between extracellular miRNAs and psychiatric conditions. MiRNAs may serve as biomarkers of subclinical psychiatric illness in older adults.

Antibodies are critical tools for research into extracellular vesicles (EVs) and other extracellular nanoparticles (ENPs), where they can be used for their identification, characterization, and isolation. However, the lack of a centralized antibody platform where researchers can share validation results thus minimizing wasted personnel time and reagents, has been a significant obstacle. Moreover, because the performance of antibodies varies among assay types and conditions, detailed information on assay variables and protocols is also of value. To facilitate sharing of results on antibodies that are relevant to EV/ENP research, the EV Antibody Database has been developed by the investigators of the Extracellular RNA Communication Consortium (ERCC). Hosted by the ExRNA Portal (https://exrna.org/resources/evabdb/), this interactive database aggregates and shares results from antibodies that have been tested by research groups in the EV/ENP field. Currently, the EV Antibody Database includes modules for antibodies tested for western Blot, EV Flow Cytometry, and EV Sandwich Assays, and holds 110 records contributed by 6 laboratories from the ERCC. Detailed information on antibody sources, assay conditions, and results is provided, including negative results. We encourage ongoing expert input and community feedback to enhance the database's utility, making it a valuable resource for comprehensive validation data on antibodies and protocols in EV biology.

Background Schools are high-risk settings for SARS-CoV-2 transmission, but necessary for children's educational and social-emotional wellbeing. Previous research suggests that wastewater monitoring can detect SARS-CoV-2 infections in controlled residential settings with high levels of accuracy. However, its effective accuracy, cost, and feasibility in non-residential community settings is unknown. Methods The objective of this study was to determine the effectiveness and accuracy of community-based passive wastewater and surface (environmental) surveillance to detect SARS-CoV-2 infection in neighborhood schools compared to weekly diagnostic (PCR) testing. We implemented an environmental surveillance system in nine elementary schools with 1700 regularly present staff and students in southern California. The system was validated from November 2020 to March 2021. Findings In 447 data collection days across the nine sites 89 individuals tested positive for COVID-19, and SARS-CoV-2 was detected in 374 surface samples and 133 wastewater samples. Ninety-three percent of identified cases were associated with an environmental sample (95% CI: 88%-98%); 67% were associated with a positive wastewater sample (95% CI: 57%-77%), and 40% were associated with a positive surface sample (95% CI: 29%-52%). The techniques we utilized allowed for near-complete genomic sequencing of wastewater and surface samples. Interpretation Passive environmental surveillance can detect the presence of COVID-19 cases in non-residential community school settings with a high degree of accuracy. Copyright (c) 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The maturation of genomic surveillance in the past decade has enabled tracking of the emergence and spread of epidemics at an unprecedented level. During the COVID-19 pandemic, for example, genomic data revealed that local epidemics varied considerably in the frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage importation and persistence, likely due to a combination of COVID-19 restrictions and changing connectivity. Here, we show that local COVID-19 epidemics are driven by regional transmission, including across international boundaries, but can become increasingly connected to distant locations following the relaxation of public health interventions. By integrating genomic, mobility, and epidemiological data, we find abundant transmission occurring between both adjacent and distant locations, supported by dynamic mobility patterns. We find that changing connectivity significantly influences local COVID-19 incidence. Our findings demonstrate a complex meaning of "local" when investigating connected epidemics and emphasize the importance of collaborative interventions for pandemic prevention and mitigation.

We report on the identification of extracellular miRNA (ex-miRNA) biomarkers for early diagnosis and prognosis of preeclampsia (PE). Small RNA sequencing of maternal serum prospectively collected from participants undergoing evaluation for suspected PE revealed distinct patterns of ex-miRNA expression among different categories of hypertensive disorders in pregnancy. Applying an iterative machine learning method identified three bivariate miRNA biomarkers ( miR-522-3p/miR-4732-5p , miR-516a-5p/miR-144-3p , and miR-27b-3p/let-7b-5p ) that, when applied serially, distinguished between PE cases of different severity and differentiated cases from controls with a sensitivity of 93%, specificity of 79%, positive predictive value (PPV) of 55%, and negative predictive value (NPV) of 89%. In a small independent validation cohort, these ex-miRNA biomarkers had a sensitivity of 91% and specificity of 57%. Combining these ex-miRNA biomarkers with the established sFlt1:PlGF protein biomarker ratio performed better than either set of biomarkers alone (sensitivity of 89.4%, specificity of 91.3%, PPV of 95.5%, and NPV of 80.8%).

BACKGROUND:While the health effects of air pollution and temperature are widely studied, the molecular effects are poorly understood. Extracellular microRNAs (ex-miRNAs) have the potential to serve as diagnostic or prognostic biomarkers and/or to act as intercellular signaling molecules that mediate the effects of environmental exposures on health outcomes.METHODS:We examined the relationship between short-term exposure to air pollution and ambient temperature and the ex-miRNA profiles of participants in the Normative Aging Study (NAS) from 1999 to 2015. Our exposures were defined as same-day, two-day, three-day, one-week, two-week, and three-week moving averages of PM2.5, NO2, O3, and temperature which were derived from high-resolution spatio-temporal models. The ex-miRNA profiles of the subjects were obtained during follow-up visits. We analyzed the data using a longitudinal quantile regression model adjusted for individual covariates, batch effects, and time trends. We adjusted for multiple comparisons using a false discovery rate (FDR) correction. Ex-miRNAs that were significantly associated with exposures were further investigated using pathway analyses.RESULTS:We found that all the examined exposures were associated with changes in ex-miRNA profiles in our study, particularly PM2.5 which was responsible for most of the statistically significant results. We found 110 statistically significant exposure-outcome relationships that revealed associations with the levels of 52 unique ex-miRNAs. Pathway analyses showed these ex-miRNAs have been linked to target mRNAs, genes, and biological mechanisms that could affect virtually every organ system, and as such may be linked to multiple clinical disease presentations such as cardiovascular disease, respiratory disease, and neurological disease.CONCLUSIONS:Air pollution and temperature exposures were significantly associated with alterations in the ex-miRNA profiles of NAS subjects with possible biological consequences.