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.

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).

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.

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.

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.

Reduced Axial Scan Length Coronary Calcium Scoring Reduces Radiation Dose and Provides Adequate Clinical Decision-Making Before Coronary CT Angiography.

Extensive coronary artery calcium (CAC) diminishes the accuracy of coronary computed tomography angiography (CCTA). Many imagers adjust CCTA acquisition parameters depending on a preCCTA Agatston CAC score to optimize diagnostic accuracy. Typical preCCTA CAC imaging adds considerably to radiation exposure, partially attributable to imaging beyond the area known for highest CAC, the proximal coronary arteries. We aimed to determine whether a z-axis reduced scan length (RSL) would identify the majority of CAC and provide adequate information to computed tomography angiography providers relative to a standard full-scan length (FSL) preCCTA noncontrast CT. We retrospectively examined 200 subjects. The mean CAC scores detected in RSL and FSL were 77.4 (95% CI 50.6 to 104.3) and 93.9 (95% CI 57.3 to 130.5), respectively. RSL detected 81% of the FSL CAC. Among false negatives, with no CAC detected in RSL, FSL CAC severity was minimal (mean score 2.8). There was high concordance, averaging 88%, between CCTA imaging parameter adjustment decisions made by 2 experienced imagers based on either RSL or FSL. CAC detected and decision concordance decreased with increasing CAC burden. CAC detected was lower, and false negatives were more common in the right coronary artery owing to its anatomic course, placing larger segments outside RSL. Axial scan length and effective dose decreased 59% from FSL (∼14.5 cm/∼1.1 mSv) to RSL (∼5.9 cm/∼0.45 mSv). This retrospective study suggests that RSL identifies most CAC, results in similar CCTA acquisition parameter modifications, and reduces radiation exposure. Our colleagues corroborated these results in a recently published prospective study.

Multimodal Analysis of Composition and Spatial Architecture in Human Squamous Cell Carcinoma.

To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.

Manufacturing of poly(ethylene glycol diacrylate)-based hollow microvessels using microfluidics.

The microvasculature is a vital organ that distributes nutrients within tissues, and collects waste products from them, and which defines the environmental conditions in both normal and disease situations. Here, a microfluidic chip was developed for the fabrication of poly(ethylene glycol diacrylate) (PEGDA)-based hollow self-standing microvessels having inner dimensions ranging from 15 µm to 73 µm and displaying biocompatibility/cytocompatibility. Macromer solutions were hydrodynamically focused into a single microchannel to form a concentric flow regime, and were subsequently solidified through photopolymerization. This approach uniquely allowed the fabrication of hollow microvessels having a defined structure and integrity suitable for cell culturing.

Human Pluripotent Stem Cell-Derived Multipotent Vascular Progenitors of the Mesothelium Lineage Have Utility in Tissue Engineering and Repair.

In this report we describe a human pluripotent stem cell-derived vascular progenitor (MesoT) cell of the mesothelium lineage. MesoT cells are multipotent and generate smooth muscle cells, endothelial cells, and pericytes and self-assemble into vessel-like networks in vitro. MesoT cells transplanted into mechanically damaged neonatal mouse heart migrate into the injured tissue and contribute to nascent coronary vessels in the repair zone. When seeded onto decellularized vascular scaffolds, MesoT cells differentiate into the major vascular lineages and self-assemble into vasculature capable of supporting peripheral blood flow following transplantation. These findings demonstrate in vivo functionality and the potential utility of MesoT cells in vascular engineering applications.

A short perspective on the long road to effective treatments for Alzheimer's disease.

Globally, there are approximately 47 million people living with dementia, and about two thirds of those have Alzheimer's disease (AD). Age is the single biggest risk factor for the vast majority of sporadic AD cases, and because the world's population is aging, the number of people living with AD is set to rise dramatically over the coming decades. There are currently no disease-modifying treatments for AD, and the few symptomatic agents available have limited impact on the disease. Perhaps surprisingly, there is relatively little activity in the AD research and development field compared with other diseases with a high mortality burden, such as cancer. There is enormous economic incentive to discover and develop the first disease-modifying treatment, but previous failure has significantly reduced further industrial investment in this field. The short review looks at the historical path trodden to develop treatments and reflects on the journey down the road to truly effective treatments for people living with AD. LINKED ARTICLES: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc.

Publisher Correction: Myelodysplastic syndrome progression to acute myeloid leukemia at the stem cell level.

In the version of this article originally published, Ulrich Steidl's name was listed as "and Ulrich Steidl." His name has been updated to "Ulrich Steidl." The error has been fixed in the print, PDF and HTML versions of this article.

Myelodysplastic syndrome progression to acute myeloid leukemia at the stem cell level.

Myelodysplastic syndromes (MDS) frequently progress to acute myeloid leukemia (AML); however, the cells leading to malignant transformation have not been directly elucidated. As progression of MDS to AML in humans provides a biological system to determine the cellular origins and mechanisms of neoplastic transformation, we studied highly fractionated stem cell populations in longitudinal samples of patients with MDS who progressed to AML. Targeted deep sequencing combined with single-cell sequencing of sorted cell populations revealed that stem cells at the MDS stage, including immunophenotypically and functionally defined pre-MDS stem cells (pre-MDS-SC), had a significantly higher subclonal complexity compared to blast cells and contained a large number of aging-related variants. Single-cell targeted resequencing of highly fractionated stem cells revealed a pattern of nonlinear, parallel clonal evolution, with distinct subclones within pre-MDS-SC and MDS-SC contributing to generation of MDS blasts or progression to AML, respectively. Furthermore, phenotypically aberrant stem cell clones expanded during transformation and stem cell subclones that were not detectable in MDS blasts became dominant upon AML progression. These results reveal a crucial role of diverse stem cell compartments during MDS progression to AML and have implications for current bulk cell-focused precision oncology approaches, both in MDS and possibly other cancers that evolve from premalignant conditions, that may miss pre-existing rare aberrant stem cells that drive disease progression and leukemic transformation.

The Prevalence of Ultrarapid Metabolizers of Codeine in a Diverse Urban Population.

OBJECTIVE: To examine the prevalence of ultrarapid metabolizers of codeine among children in an ethnically diverse urban community. STUDY DESIGN: Cross-sectional study. SETTING: A tertiary care academic children's hospital in the Bronx, New York. SUBJECTS AND METHODS: In total, 256 children with nonsyndromic congenital sensorineural hearing loss were analyzed. DNA was assessed for 63 previously described single-nucleotide polymorphisms (SNPs) and copy number variants (CNVs) known to alter the function and expression of the CYP2D6 gene primarily responsible for codeine metabolism. The rate of CYP2D6 metabolism was predicted based on participants' haplotype. RESULTS: Ethnic distribution in the study subjects paralleled recent local census data, with the largest portion (115 children, 45.8%) identified as Hispanic or Latino. A total of 154 children (80.6%) had a haplotype that corresponds to extensive codeine metabolism, 18 children (9.42%) were identified as ultrarapid metabolizers (UMs), and 16 children (8.37%) were intermediate metabolizers. Only 3 children in our cohort (1.57%) were poor metabolizers. Patients identifying as Caucasian or Hispanic had an elevated incidence of UMs (11.3% and 11.2%, respectively) with extensive variability within subpopulations. CONCLUSIONS: The clinically significant rate of ultrarapid metabolizers reinforces safety concerns regarding the use of codeine and related opiates. A patient-targeted approach using pharmacogenomics may mitigate adverse effects by individualizing the selection and dosing of these analgesics.

Metastasis to the Heart: A Radiologic Approach to Diagnosis With Pathologic Correlation.

OBJECTIVE: Evaluating metastatic disease to the heart and pericardium, from detection to diagnosis, often requires a multimodality imaging approach. A radiologist's ability to evaluate cardiac metastases hinges on an understanding of the epidemiology, anatomy, and imaging features of this disease process. CONCLUSION: On surveillance imaging of patients with cancer or when metastatic disease is suspected, detection of metastatic disease may be greatly enhanced by an understanding of which primary tumors metastasize to the heart and the most common routes of spread.

Polo kinase Cdc5 associates with centromeres to facilitate the removal of centromeric cohesin during mitosis.

Sister chromatid cohesion is essential for tension-sensing mechanisms that monitor bipolar attachment of replicated chromatids in metaphase. Cohesion is mediated by the association of cohesins along the length of sister chromatid arms. In contrast, centromeric cohesin generates intrastrand cohesion and sister centromeres, while highly cohesin enriched, are separated by >800 nm at metaphase in yeast. Removal of cohesin is necessary for sister chromatid separation during anaphase, and this is regulated by evolutionarily conserved polo-like kinase (Cdc5 in yeast, Plk1 in humans). Here we address how high levels of cohesins at centromeric chromatin are removed. Cdc5 associates with centromeric chromatin and cohesin-associated regions. Maximum enrichment of Cdc5 in centromeric chromatin occurs during the metaphase-to-anaphase transition and coincides with the removal of chromosome-associated cohesin. Cdc5 interacts with cohesin in vivo, and cohesin is required for association of Cdc5 at centromeric chromatin. Cohesin removal from centromeric chromatin requires Cdc5 but removal at distal chromosomal arm sites does not. Our results define a novel role for Cdc5 in regulating removal of centromeric cohesins and faithful chromosome segregation.

Bilateral gluteal ischemic necrosis mistaken for stage IV pressure wound: case report and discussion.

BACKGROUND: Sacral and gluteal pressure wounds are a common problem in elderly and critically ill patients. Prompt and accurate diagnosis is important to determine the best plan of treatment. CASE: A 48-year-old female was hospitalized with severe trunk pain and bilateral necrotic gluteal wounds present for more than 1 year initially diagnosed as stage IV pressure ulcers; she reported pain radiating to her lower extremities. She had multiple comorbid conditions, including peripheral vascular disease, smoking, and hypertension. CONCLUSION: Wound care providers should be aware of this differential diagnosis especially in patients with history of vascular disease.