Bone marrow niches orchestrate stem-cell hierarchy and immune tolerance.

Stem cells reside in specialized microenvironments, termed niches, at several different locations in tissues. The differential functions of heterogeneous stem cells and niches are important given the increasing clinical applications of stem-cell transplantation and immunotherapy. Whether hierarchical structures among stem cells at distinct niches exist and further control aspects of immune tolerance is unknown.

Human red blood cells release microvesicles with distinct sizes and protein composition that alter neutrophil phagocytosis.

Extracellular vesicles (EVs) are membrane-bound structures released by cells and tissues into biofluids, involved in cell-cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell-type in the body, generating daily large numbers of microvesicles. RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187.

Circulating extracellular vesicles in human cardiorenal syndrome promote renal injury in a kidney-on-chip system.

BACKGROUNDCardiorenal syndrome (CRS) - renal injury during heart failure (HF) - is linked to high morbidity. Whether circulating extracellular vesicles (EVs) and their RNA cargo directly impact its pathogenesis remains unclear.METHODSWe investigated the role of circulating EVs from patients with CRS on renal epithelial/endothelial cells using a microfluidic kidney-on-chip (KOC) model.

Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases.

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases.

Electrophoretic mobility shift as a molecular beacon-based readout for miRNA detection.

MicroRNAs are short, non-coding RNA sequences involved in gene expression regulation. Quantification of miRNAs in biological fluids involves time consuming and laborious methods such as Northern blotting or PCR-based techniques. Molecular beacons (MB) are an attractive means for rapid detection of miRNAs, although the need for sophisticated readout methods limits their use in research and clinical settings.

Measurement and standardization challenges for extracellular vesicle therapeutic delivery vectors.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are nonreplicating lipid bilayer particles shed by most cell types which have the potential to revolutionize the development and efficient delivery of clinical therapeutics. This article provides an introduction to the landscape of EV-based vectors under development for the delivery of protein- and nucleic acid-based therapeutics. We highlight some of the most pressing measurement and standardization challenges that limit the translation of EVs to the clinic.

VWF maturation and release are controlled by 2 regulators of Weibel-Palade body biogenesis: exocyst and BLOC-2.

von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized in endothelial cells and stored in Weibel-Palade bodies (WPBs). Understanding the mechanisms underlying WPB biogenesis and exocytosis could enable therapeutic modulation of endogenous VWF, yet optimal targets for modulating VWF release have not been established. Because biogenesis of lysosomal related organelle-2 (BLOC-2) functions in the biogenesis of platelet dense granules and melanosomes, which like WPBs are lysosome-related organelles, we hypothesized that BLOC-2-dependent endolysosomal trafficking is essential for WPB biogenesis and sought to identify BLOC-2-interacting proteins.

Rational Design of a Bifunctional AND-Gate Ligand To Modulate Cell-Cell Interactions.

Protein "AND-gate" systems, in which a ligand acts only on cells with two different receptors, direct signaling activity to a particular cell type and avoid action on other cells. In a bifunctional AND-gate protein, the molecular geometry of the protein domains is crucial. Here we constructed a tissue-targeted erythropoietin (EPO) that stimulates red blood cell (RBC) production without triggering thrombosis.

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy.

A Novel Implementation of Magnetic Levitation to Quantify Leukocyte Size, Morphology, and Magnetic Properties to Identify Patients With Sepsis.

Background: We have developed a novel, easily implementable methodology using magnetic levitation to quantify circulating leukocyte size, morphology, and magnetic properties, which may help in rapid, bedside screening for sepsis.

Objective: Our objectives were to describe our methodological approach to leukocyte assessment, and to perform a pilot investigation to test the ability of magnetic levitation to identify and quantify changes in leukocyte size, shape, density, and/or paramagnetic properties in healthy controls and septic patients.

Methods: This prospective, observational cohort study was performed in a 56,000/y visit emergency department (ED) and affiliated outpatient phlebotomy laboratory.

Detection and quantification of subtle changes in red blood cell density using a cell phone.

Magnetic levitation has emerged as a technique that offers the ability to differentiate between cells with different densities. We have developed a magnetic levitation system for this purpose that distinguishes not only different cell types but also density differences in cells of the same type. This small-scale system suspends cells in a paramagnetic medium in a capillary placed between two rare earth magnets, and cells levitate to an equilibrium position determined solely by their density.

Ligation of Glycophorin A Generates Reactive Oxygen Species Leading to Decreased Red Blood Cell Function.

Acute, inflammatory conditions associated with dysregulated complement activation are characterized by significant increases in blood concentration of reactive oxygen species (ROS) and ATP. The mechanisms by which these molecules arise are not fully understood. In this study, using luminometric- and fluorescence-based methods, we show that ligation of glycophorin A (GPA) on human red blood cells (RBCs) results in a 2.

Smart-Phone Based Magnetic Levitation for Measuring Densities.

Magnetic levitation, which uses a magnetic field to suspend objects in a fluid, is a powerful and versatile technology. We develop a compact magnetic levitation platform compatible with a smart-phone to separate micro-objects and estimate the density of the sample based on its levitation height. A 3D printed attachment is mechanically installed over the existing camera unit of a smart-phone.

Levitational Image Cytometry with Temporal Resolution.

A simple, yet powerful magnetic-levitation-based device is reported for real-time, label-free separation, as well as high-resolution monitoring of cell populations based on their unique magnetic and density signatures. This method allows a wide variety of cellular processes to be studied, accompanied by transient or permanent changes in cells' fundamental characteristics as a biological material.

Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane.

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e.

C4d deposits on the surface of RBCs in trauma patients and interferes with their function.

Objective: Complement system is activated in patients with trauma. Although complement activation is presumed to contribute to organ damage and constitutional symptoms, little is known about the involved mechanisms. Because complement components may deposit on RBCs, we asked whether complement deposits on the surface of RBC in trauma and whether such deposition alters RBC function.

CR1-mediated ATP release by human red blood cells promotes CR1 clustering and modulates the immune transfer process.

Humans and other higher primates are unique among mammals in using complement receptor 1 (CR1, CD35) on red blood cells (RBC) to ligate complement-tagged inflammatory particles (immune complexes, apoptotic/necrotic debris, and microbes) in the circulation for quiet transport to the sinusoids of spleen and liver where resident macrophages remove the particles, but allow the RBC to return unharmed to the circulation. This process is called immune-adherence clearance. In this study we found using luminometric- and fluorescence-based methods that ligation of CR1 on human RBC promotes ATP release.

Systemic lupus erythematosus serum deposits C4d on red blood cells, decreases red blood cell membrane deformability, and promotes nitric oxide production.

Objective: Systemic lupus erythematosus (SLE) is characterized by intravascular activation of the complement system and deposition of complement fragments (C3 and C4) on plasma membranes of circulating cells, including red blood cells (RBCs). The aim of this study was to address whether this process affects the biophysical properties of RBCs.

Methods: Serum and RBCs were isolated from patients with SLE and healthy controls.

Introduction to fluorescence microscopy.

This chapter is an overview of basic principles of fluorescence microscopy, including a brief history on the invention of this type of microscopy. The chapter highlights important points related to properties of fluorochromes, resolution in fluorescence microscopy, phase contrast and fluorescence, fluorescence filters, construction of a fluorescence microscope, and tips on the correct use of this equipment.

Ligation of complement receptor 1 increases erythrocyte membrane deformability.

Microbes as well as immune complexes and other continuously generated inflammatory particles are efficiently removed from the human circulation by red blood cells (RBCs) through a process called immune-adherence clearance. During this process, RBCs use complement receptor 1 (CR1, CD35) to bind circulating complement-opsonized particles and transfer them to resident macrophages in the liver and spleen for removal. We here show that ligation of RBC CR1 by antibody and complement-opsonized particles induces a transient Ca(++) influx that is proportional to the RBC CR1 levels and is inhibited by T1E3 pAb, a specific inhibitor of TRPC1 channels.