Epigenetic regulation of TERRA transcription and metacyclogenesis by base J in Leishmania major.

The hyper-modified DNA base J helps control termination of Pol II transcription at polycistronic transcription units (PTUs) in T. brucei and L. major , allowing epigenetic control of gene expression. The Telomere Repeat-containing RNA (TERRA) is synthesized in T. brucei by Pol I readthrough transcription of a telomeric PTU. While little is understood regarding TERRA synthesis and function, the hyper-modified DNA base J is highly enriched at telomeres in L. major promastigotes. We now show that TERRA is synthesized by Pol II in L. major and loss of base J leads to increased TERRA. For at least one site, the increased TERRA is by Pol II readthrough transcription from an adjacent PTU. Furthermore, Pol II readthrough defects and increased TERRA correlate with increased differentiation of promastigotes to the infectious metacyclic life stage and decreased cell viability. These results help explain the essential nature of base J in Leishmania and provide insight regarding epigenetic control of coding and non-coding RNA expression and parasite development during the life cycle of L. major .

Live Organoid Cyclic Imaging.

Organoids are becoming increasingly relevant in biology and medicine for their physiological complexity and accuracy in modeling human disease. To fully assess their biological profile while preserving their spatial information, spatiotemporal imaging tools are warranted. While previously developed imaging techniques, such as four-dimensional (4D) live imaging and light-sheet imaging have yielded important clinical insights, these technologies lack the combination of cyclic and multiplexed analysis. To address these challenges, bioorthogonal click chemistry is applied to display the first demonstration of multiplexed cyclic imaging of live and fixed patient-derived glioblastoma tumor organoids. This technology exploits bioorthogonal click chemistry to quench fluorescent signals from the surface and intracellular of labeled cells across multiple cycles, allowing for more accurate and efficient molecular profiling of their complex phenotypes. Herein, the versatility of this technology is demonstrated for the screening of glioblastoma markers in patient-derived human glioblastoma organoids while conserving their viability. It is anticipated that the findings and applications of this work can be broadly translated into investigating physiological developments in other organoid systems.

Double Digital Assay for Single Extracellular Vesicle and Single Molecule Detection.

Extracellular vesicles (EVs) have emerged as a promising source of biomarkers for disease diagnosis. However, current diagnostic methods for EVs present formidable challenges, given the low expression levels of biomarkers carried by EV samples, as well as their complex physical and biological properties. Herein, a highly sensitive double digital assay is developed that allows for the absolute quantification of individual molecules from a single EV. Because the relative abundance of proteins is low for a single EV, tyramide signal amplification (TSA) is integrated to increase the fluorescent signal readout for evaluation. With the integrative microfluidic technology, the technology's ability to compartmentalize single EVs is successfully demonstrated, proving the technology's digital partitioning capacity. Then the device is applied to detect single PD-L1 proteins from single EVs derived from a melanoma cell line and it is discovered that there are ≈2.7 molecules expressed per EV, demonstrating the applicability of the system for profiling important prognostic and diagnostic cancer biomarkers for therapy response, metastatic status, and tumor progression. The ability to accurately quantify protein molecules of rare abundance from individual EVs will shed light on the understanding of EV heterogeneity and discovery of EV subtypes as new biomarkers.

Integrative analyses highlight functional regulatory variants associated with neuropsychiatric diseases.

Noncoding variants of presumed regulatory function contribute to the heritability of neuropsychiatric disease. A total of 2,221 noncoding variants connected to risk for ten neuropsychiatric disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, bipolar disorder, borderline personality disorder, major depression, generalized anxiety disorder, panic disorder, post-traumatic stress disorder, obsessive-compulsive disorder and schizophrenia, were studied in developing human neural cells. Integrating epigenomic and transcriptomic data with massively parallel reporter assays identified differentially-active single-nucleotide variants (daSNVs) in specific neural cell types. Expression-gene mapping, network analyses and chromatin looping nominated candidate disease-relevant target genes modulated by these daSNVs. Follow-up integration of daSNV gene editing with clinical cohort analyses suggested that magnesium transport dysfunction may increase neuropsychiatric disease risk and indicated that common genetic pathomechanisms may mediate specific symptoms that are shared across multiple neuropsychiatric diseases.

A multifault earthquake threat for the Seattle metropolitan region revealed by mass tree mortality.

Compound earthquakes involving simultaneous ruptures along multiple faults often define a region's upper threshold of maximum magnitude. Yet, the potential for linked faulting remains poorly understood given the infrequency of these events in the historic era. Geological records provide longer perspectives, although temporal uncertainties are too broad to clearly pinpoint single multifault events. Here, we use dendrochronological dating and a cosmogenic radiation pulse to constrain the death dates of earthquake-killed trees along two adjacent fault zones near Seattle, Washington to within a 6-month period between the 923 and 924 CE growing seasons. Our narrow constraints conclusively show linked rupturing that occurred either as a single composite earthquake of estimated magnitude 7.8 or as a closely spaced double earthquake sequence with estimated magnitudes of 7.5 and 7.3. These scenarios, which are not recognized in current hazard models, increase the maximum earthquake size needed for seismic preparedness and engineering design within the Puget Sound region of >4 million residents.

Prenatal vitamin D deficiency alters immune cell proportions of young adult offspring through alteration of long-term stem cell fates.

Vitamin D deficiency is a common deficiency worldwide, particularly among women of reproductive age. During pregnancy, it increases the risk of immune-related diseases in offspring later in life. However, exactly how the body remembers exposure to an adverse environment during development is poorly understood. Herein, we explore the effects of prenatal vitamin D deficiency on immune cell proportions in offspring using vitamin D deficient mice established by dietary manipulation. We show that prenatal vitamin D deficiency alters immune cell proportions in offspring by changing the transcriptional properties of genes downstream of vitamin D receptor signaling in hematopoietic stem and progenitor cells of both the fetus and adults. Further investigations of the associations between maternal vitamin D levels and cord blood immune cell profiles from 75 healthy pregnant women and their term babies also confirm that maternal vitamin D levels significantly affect immune cell proportions in the babies. Thus, lack of prenatal vitamin D, particularly at the time of hematopoietic stem cell migration from the liver to the bone marrow, has long-lasting effects on immune cell proportions. This highlights the importance of providing vitamin D supplementation at specific stages of pregnancy.

Single Extracellular Vesicle Analysis Using Droplet Microfluidics.

Extracellular vesicles (EVs) are lipid-bound nanometer-sized vesicles released by all cell types that contain molecular payload such as proteins and/or nucleic acids. EVs are a key facet of cell-to-cell communication and have the potential to be used in the diagnosis of numerous diseases, chief among them being cancer. However, most methods of EV analysis struggle to identify the rare, malformed proteins indicative of tumor cells as tumor EVs represent only a tiny fraction of the bulk EVs present in the bloodstream. Here, we present a method of single EV analysis, utilizing droplet microfluidics to encapsulate EVs, which are labeled with DNA barcodes linked to antibodies, in droplets with the DNA extension used to amplify the signals associated with each EV. The amplified DNA can then be sequenced to assess the protein content of individual EVs, enabling the detection of rare proteins and EV subpopulations within a bulk EV sample.

Highly parallel, wash-free, and ultrasensitive centrifugal droplet digital protein detection in sub-microliter blood.

The ability to efficiently detect low-abundance protein biomarkers in tiny blood samples is a significant challenge in clinical and laboratory settings. Currently, high-sensitivity approaches require specialized instrumentation, involve multiple washing steps, and lack the ability to parallelize, preventing their widespread implementation. Herein, we developed a parallelized, wash-free, and ultrasensitive centrifugal droplet digital protein detection (CDPro) technology that achieves a femtomolar limit of detection (LoD) of target proteins with sub-microliters of plasma. The CDPro combines two techniques, namely a centrifugal microdroplet generation device and a digital immuno-PCR assay. Miniaturized centrifugal devices enable emulsification of hundreds of samples within 3 minutes using a common centrifuge. The bead-free digital immuno-PCR assay not only eliminates the need for multistep washing, but also possesses ultra-high detection sensitivity and accuracy. We characterized the performance of CDPro using recombinant interleukins (IL-3 and IL-6) as example targets and reported a LoD of 0.0128 pg mL-1. We also quantified IL-6 from 7 human clinical blood samples using the CDPro with only 0.5 µL plasma, which showed excellent agreement with an existing clinical protein diagnostic system with 25 µL plasma from those samples (R2 = 0.98).

Single-Cell and Spatial Transcriptomic Analysis of Human Skin Delineates Intercellular Communication and Pathogenic Cells.

Epidermal homeostasis is governed by a balance between keratinocyte proliferation and differentiation with contributions from cell-cell interactions, but conserved or divergent mechanisms governing this equilibrium across species and how an imbalance contributes to skin disease are largely undefined. To address these questions, human skin single-cell RNA sequencing and spatial transcriptomics data were integrated and compared with mouse skin data. Human skin cell-type annotation was improved using matched spatial transcriptomics data, highlighting the importance of spatial context in cell-type identity, and spatial transcriptomics refined cellular communication inference. In cross-species analyses, we identified a human spinous keratinocyte subpopulation that exhibited proliferative capacity and a heavy metal processing signature, which was absent in mouse and may account for species differences in epidermal thickness. This human subpopulation was expanded in psoriasis and zinc-deficiency dermatitis, attesting to disease relevance and suggesting a paradigm of subpopulation dysfunction as a hallmark of the disease. To assess additional potential subpopulation drivers of skin diseases, we performed cell-of-origin enrichment analysis within genodermatoses, nominating pathogenic cell subpopulations and their communication pathways, which highlighted multiple potential therapeutic targets. This integrated dataset is encompassed in a publicly available web resource to aid mechanistic and translational studies of normal and diseased skin.

An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies.

BACKGROUND: Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies. On the other hand, while several methods have been published, multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide-conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. RESULTS: We performed multiplexing snATAC-seq analyses on a mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The analyses on nuclei with at least 10,000 read counts showed that the demultiplexing accuracy of NuHash was high, and only ten out of 9144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. The differential open chromatin region (OCR) analysis between female and male samples revealed that male-specific OCRs were enriched in chromosome Y (four out of nine). We also found that five female-specific OCRs (20 OCRs) were on chromosome X. A comparative analysis between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples revealed that the bulk ATAC-seq signal intensity was positively correlated with the number of cell clusters detected in snATAC-seq. Moreover, when we categorized snATAC-seq peaks based on the number of cell clusters in which the peak was present, we observed different distributions over different genomic features between the groups. This result suggests that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. CONCLUSIONS: Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high-accuracy demultiplexing of samples. The NuHash protocol is straightforward, works on frozen samples, and requires no modifications for snATAC-seq library preparation.

JAK-STAT inhibition reduces endothelial prothrombotic activation and leukocyte-endothelial proadhesive interactions.

BACKGROUND: Vascular activation is characterized by increased proinflammatory, pro thrombotic, and proadhesive signaling. Several chronic and acute conditions, including Bcr-abl-negative myeloproliferative neoplasms (MPNs), graft-vs-host disease, and COVID-19 have been noted to have increased activation of the janus kinase (JAK)-signal transducer and downstream activator of transcription (STAT) pathways. Two notable inhibitors of the JAK-STAT pathway are ruxolitinib (JAK1/2 inhibitor) and fedratinib (JAK2 inhibitor), which are currently used to treat MPN patients. However, in some conditions, it has been noted that JAK inhibitors can increase the risk of thromboembolic complications. OBJECTIVES: We sought to define the anti-inflammatory and antithrombotic effects of JAK-STAT inhibitors in vascular endothelial cells. METHODS: We assessed endothelial activation in the presence or absence of ruxolitinib or fedratinib by using immunoblots, immunofluorescence, qRT-PCR, and function coagulation assays. Finally, we used endothelialized microfluidics perfused with blood from normal and JAK2V617F+ individuals to evaluate whether ruxolitinib and fedratinib changed cell adhesion. RESULTS: We found that both ruxolitinib and fedratinib reduced endothelial cell phospho-STAT1 and STAT3 signaling and attenuated nuclear phospho-NK-κB and phospho-c-Jun localization. JAK-STAT inhibition also limited secretion of proadhesive and procoagulant P-selectin and von Willebrand factor and proinflammatory IL-6. Likewise, we found that JAK-STAT inhibition reduced endothelial tissue factor and urokinase plasminogen activator expression and activity. CONCLUSIONS: By using endothelialized microfluidics perfused with whole blood samples, we demonstrated that endothelial treatment with JAK-STAT inhibitors prevented rolling of both healthy control and JAK2V617F MPN leukocytes. Together, these findings demonstrate that JAK-STAT inhibitors reduce the upregulation of critical prothrombotic pathways and prevent increased leukocyte-endothelial adhesion.

Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

Comparative Diagnostic Performance of Estimated Fetal Weight and Isolated Abdominal Circumference for the Detection of Fetal Growth Restriction.

OBJECTIVES: To describe the comparative incidence, detection of small-for-gestational age (SGA), and composite perinatal morbidity (CPM) associated with diagnostic criteria of fetal growth restriction (FGR) by estimated fetal weight (EFW) <10% with those with isolated abdominal circumference (AC) measurements <10%. METHODS: We performed a retrospective cohort study of 1587 patients receiving prenatal care and delivery at our institution. We included all patients with ultrasounds and delivery outcomes available, and excluded terminations, second trimester losses, and pregnancies without ultrasounds. EFW was calculated from Hadlock and use of the Duryea centiles, and AC from Hadlock's reference curves. We determined SGA at birth and defined CPM as birthweight less than 3% or birthweight less than 10% with neonatal morbidity. RESULTS: Of 1587 patients, 28 (1.8%) were classified as FGR by EFW <10%. Three of 12 patients with isolated AC <10% developed EFW <10% later in pregnancy (25%). The performance of each diagnostic criteria were comparable for the outcomes of SGA and CPM, with similar sensitivities, but with decreased specificity for SGA outcome, and an increased false positive rate for patients classified as FGR by isolated AC <10, with a tradeoff of decreased false negatives. CONCLUSIONS: Broadening the diagnosis of FGR to include patients with isolated AC <10 did not significantly increase the detection of pregnancies at risk for SGA or CPM. Our conclusions may be limited by a lack of statistical power given a low frequency of SGA and CPM.

Equine mesenchymal stem cell derived extracellular vesicle immunopathology biomarker discovery.

The use of mesenchymal stem cells (MSCs) in human and veterinary clinical applications has become a subject of increasing importance due to their roles in immunomodulation and regenerative processes. MSCs are especially relevant in equine medicine because they may have the ability to treat prevalent musculoskeletal disorders, among other conditions. However, recent evidence suggests that the components secreted by MSCs, particularly extracellular vesicles (EVs), are responsible for these properties. EVs contain proteins and nucleic acids, which possess an active role in intercellular communication and can be used as therapeutics. However, because the intersection of equine veterinary medicine with EVs remains a relatively new field, there is a demand to identify biomarkers that can discern and enrich for therapeutic EVs, progressing their clinical efficacy. In this study, we identified and characterized 84 miRNAs, between three equine donors involved in immunomodulation in cell and EV subjects. We discovered distinct groups of shared miRNAs, like miR-21-5p and miR-451a, that are abundant and enriched between the donors' EVs, respectively. By mapping and comparing the MSC-EV miRNA expression, we discovered many pathways that are involved in immunomodulation and tissue regenerative processes related to equine clinical applications. Therefore, the miRNAs highlighted in this article can be used as valuable biomarkers for screening MSC-derived EVs for potential equine therapy.

A Customizable and Low-Cost Ultraviolet Exposure System for Photolithography.

For microfluidic device fabrication in the research, industry, and commercial areas, the curing and transfer of patterns on photoresist relies on ultraviolet (UV) light. Often, this step is performed by commercial mask aligner or UV lamp exposure systems; however, these machines are often expensive, large, and inaccessible. To find an alternative solution, we present an inexpensive, customizable, and lightweight UV exposure system that is user-friendly and readily available for a homemade cleanroom. We fabricated a portable UV exposure system that costs under $200. The wafer holder's adjustable height enabled for the selection of the appropriate curing distance, demonstrating our system's ability to be easily tailored for different applications. The high light uniformity across a 4" diameter wafer holder (light intensity error ~2.9%) was achieved by adding a light diffusing film to the apparatus. These values are comparable to the light uniformity across a 5" diameter wafer holder from a commercial mask aligner (ABM 3000HR Mask Aligner), that has a light intensity error of ~4.0%. We demonstrated the ability to perform photolithography with high quality by fabricating microfluidic devices and generating uniform microdroplets. We achieved comparable quality to the wafer patterns, microfluidic devices, and droplets made from the ABM 3000HR Mask Aligner.

PROBER identifies proteins associated with programmable sequence-specific DNA in living cells.

DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.

Destabilisation of the Subpolar North Atlantic prior to the Little Ice Age.

The cooling transition into the Little Ice Age was the last notable shift in the climate system prior to anthropogenic global warming. It is hypothesised that sea-ice to ocean feedbacks sustained an initial cooling into the Little Ice Age by weakening the subpolar gyre circulation; a system that has been proposed to exhibit bistability. Empirical evidence for bistability within this transition has however been lacking. Using statistical indicators of resilience in three annually-resolved bivalve proxy records from the North Icelandic shelf, we show that the subpolar North Atlantic climate system destabilised during two episodes prior to the Little Ice Age. This loss of resilience indicates reduced attraction to one stable state, and a system vulnerable to an abrupt transition. The two episodes preceded wider subpolar North Atlantic change, consistent with subpolar gyre destabilisation and the approach of a tipping point, potentially heralding the transition to Little Ice Age conditions.

Barriers and Potential Solutions to Implementing Evidence-Based PTSD Treatment in Military Treatment Facilities.

INTRODUCTION: Prolonged exposure therapy is an effective treatment for posttraumatic stress disorder that is underutilized in health systems, including the military health system. Organizational barriers to prolonged exposure implementation have been hypothesized but not systematically examined. This multisite project sought to identify barriers to increasing the use of prolonged exposure across eight military treatment facilities and describe potential solutions to addressing these barriers. MATERIALS AND METHODS: As part of a larger project to increase the use of prolonged exposure therapy in the military health system, we conducted a needs assessment at eight military treatment facilities. The needs assessment included analysis of clinic administrative data and a series of stakeholder interviews with behavioral health clinic providers, leadership, and support staff. Key barriers were matched with potential solutions using a rubric developed for this project. Identified facilitators, barriers, and potential solutions were summarized in a collaboratively developed implementation plan for increasing prolonged exposure therapy tailored to each site. RESULTS: There was a greater than anticipated consistency in the barriers reported by the sites, despite variation in the size and type of facility. The identified barriers were grouped into four categories: time-related barriers, provider-related barriers, barriers related to patient education and matching patients to providers, and scheduling-related barriers. Potential solutions to each barrier are described. CONCLUSIONS: The findings highlight the numerous organizational-level barriers to implementing evidence-based psychotherapy in the military health system and offer potential solutions that may be helpful in addressing the barriers.

In-home environmental exposures predicted from geospatial characteristics of the built environment and electronic health records of children with asthma.

PURPOSE: Children may be exposed to numerous in-home environmental exposures (IHEE) that trigger asthma exacerbations. Spatially linking social and environmental exposures to electronic health records (EHR) can aid exposure assessment, epidemiology, and clinical treatment, but EHR data on exposures are missing for many children with asthma. To address the issue, we predicted presence of indoor asthma trigger allergens, and estimated effects of their key geospatial predictors. METHODS: Our study samples were comprised of children with asthma who provided self-reported IHEE data in EHR at a safety-net hospital in New England during 2004-2015. We used an ensemble machine learning algorithm and 86 multilevel features (e.g., individual, housing, neighborhood) to predict presence of cockroaches, rodents (mice or rats), mold, and bedroom carpeting/rugs in homes. We reduced dimensionality via elastic net regression and estimated effects by the G-computation causal inference method. RESULTS: Our models reasonably predicted presence of cockroaches (area under receiver operating curves [AUC] = 0.65), rodents (AUC = 0.64), and bedroom carpeting/rugs (AUC = 0.64), but not mold (AUC = 0.54). In models adjusted for confounders, higher average household sizes in census tracts were associated with more reports of pests (cockroaches and rodents). Tax-exempt parcels were associated with more reports of cockroaches in homes. Living in a White-segregated neighborhood was linked with lower reported rodent presence, and mixed residential/commercial housing and newer buildings were associated with more reports of bedroom carpeting/rugs in bedrooms. CONCLUSIONS: We innovatively applied a machine learning and causal inference mixture methodology to detail IHEE among children with asthma using EHR and geospatial data, which could have wide applicability and utility.

Transport of Maternally Administered Pharmaceutical Agents Across the Placental Barrier In Vitro.

To understand the transport of pharmaceutical agents and their effects on developing fetus, we have created a placental microsystem that mimics structural phenotypes and physiological characteristic of a placental barrier. We have shown the formation of a continuous network of epithelial adherens junctions and endothelial cell-cell junctions confirming the integrity of the placental barrier. More importantly, the formation of elongated microvilli under dynamic flow condition is demonstrated. Fluid shear stress acts as a mechanical cue triggering the microvilli formation. Pharmaceutical agents were administered to the maternal channel, and the concentration of pharmaceutical agents in fetal channel for coculture and control models were evaluated. In fetal channel, the coculture model exhibited about 2.5 and 2.2% of the maternal initial concentration for naltrexone and 6ß-naltrexol, respectively. In acellular model, fetal channel showed about 10.5 and 10.3% of the maternal initial concentration for naltrexone and 6ß-naltrexol, respectively. Gene expressions of epithelial cells after direct administration of naltrexone and 6ß-naltrexol to the maternal channel and endothelial cells after exposure due to transport through placental barrier are also reported.