Behavioral immune landscapes of inflammation.

Transcriptional or proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues. These approaches, however, fail to describe dynamic scenarios in which cells can change their biochemical properties and downstream “behavioral” outputs every few seconds or minutes. Here, we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamism of individual leukocytes at sites of active inflammation. By analyzing over 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioral descriptors of individual cells and used these high-dimensional datasets to build behavioral landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and inside blood vessels uncovered a continuum of neutrophil states, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioral in vivo screening of thousands of cells from 24 different mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and genetic or pharmacological interference of Fgr protected from inflammatory injury. Thus, behavioral landscapes report unique biological properties of dynamic environments at high cellular, spatial and temporal resolution.pre-print4302 K

Isolation and Characterization of Extracellular Vesicles from Various Biological Matrices using Capillary-Channeled Polymer (C-CP) Fiber Solid-Phase Extraction Spin-Down Tips

A number of recent works have emphasized the need to isolate nanometer-scale analytes, like extracellular vesicles (EVs), from various biologically-relevant fluids. Exosomes are a subset of small EVs that range from 30-200 nm in diameter that serve as biomolecular snapshots of their cell of origin containing mother cell-specific DNA, miRNA, mRNA, and proteins. As critical components of intercellular communication, exosomes and other EVs play significant roles in many physiological and pathological processes. Diverse populations of these vesicles can be collected from biofluids, including blood, saliva, and urine, from cell culture conditioned media and primary cells, and even from plant fluid stocks. With their characteristic vector-like activities and accessible collection from renewable sources, the large-scale processing of EVs from patient biofluids for clinical diagnostics and from plant fluids or high-yield bioreactors for use as therapeutic vectors has been previously proposed. However, these applications are limited by extremely impure, low-yield exosome recoveries, despite the large availability of exosome sources. Hence, an isolation method that provides high concentrations of pure, bioactive EVs from diverse sources on reasonable scales of time and cost is of much interest. Employed in this work is a rapid EV isolation method using a hydrophobic interaction chromatography (HIC) workflow on a capillary-channeled polymer (C-CP) fiber spin-down tip. Here, EVs are isolated from several biofluid sources, including mock biofluid matrices, clinical patient biofluid samples, cellular milieu from mammalian and amoeba cell lines, and over 20 fruit and vegetable sample stocks. Representative populations of EVs are obtained using the C-CP tip method, where up to 12 samples are simultaneously processed in a standard tabletop centrifuge in less than 15 minutes. This batch solid-phase extraction technique allows up to 1 x 1012 EVs to be obtained from each μL-scale aliquot of the original biofluid. The tip-isolated EVs were characterized using transmission electron microscopy (TEM), multi-angle light scattering (MALS), nanoparticle tracking analysis (NTA), absorbance quantification, protein purity assay, and immunoassays to EV and source-specific proteins. The efficient HIC C-CP tip isolation method produces the required integrity and purity of recovered EVs to enable fundamental research to be performed and their therapeutic vector and clinical diagnostic potentials to be better explored

Micro- and Nanostructured Microfluidic Devices for Localized Protein Immobilization and Other Biomedical Applications

A new immobilization method for the localized adsorption of proteins on thermoplastic surfaces is introduced. Artificial three-phase interfaces were realized by surface structuring to control the wetting behavior which lead to a preferred adsorption in these modified areas. Additionally, different fabrication methods were analyzed to determine mass fabrication capabilities. These fabrication methods also allowed the production of fully structured microchannels to tune the fluids behavior within

From Animaculum to single-molecules : 300 years of the light microscope

Although not laying claim to being the inventor of the light microscope, Antonj van Leeuwenhoek (1632-1723) was arguably the first person to bring this new technological wonder of the age properly to the attention of natural scientists interested in the study of living things (people we might now term 'biologists'). He was a Dutch draper with no formal scientific training. From using magnifying glasses to observe threads in cloth, he went on to develop over 500 simple single lens microscopes (Baker & Leeuwenhoek 1739 Phil. Trans. 41, 503-519. (doi:10.1098/rstl.1739.0085)) which he used to observe many different biological samples. He communicated his finding to the Royal Society in a series of letters (Leeuwenhoek 1800 The select works of Antony Van Leeuwenhoek, containing his microscopical discoveries in many of the works of nature, vol. 1) including the one republished in this edition of Open Biology. Our review here begins with the work of van Leeuwenhoek before summarizing the key developments over the last ca 300 years, which has seen the light microscope evolve from a simple single lens device of van Leeuwenhoek's day into an instrument capable of observing the dynamics of single biological molecules inside living cells, and to tracking every cell nucleus in the development of whole embryos and plants

Monitoring of Immune Cell Response to B Cell Depletion Therapy and Nerve Root Injury Using Spio Enhanced MRI

Magnetic resonance (MR) is a robust platform for non-invasive, high-resolution anatomical imaging. However, MR imaging lacks the requisite sensitivity and contrast for imaging at the cellular level. This represents a clinical impediment to greater diagnostic accuracy. Recent advances have allowed for the in vivo visualization of populations and even of individual cells using superparamagnetic iron oxide (SPIO) MR contrast agents. These nanoparticles, commonly manifested as a core of a single iron oxide crystal or cluster of crystals coated in a biocompatible shell, function to shorten proton relaxation times. In MR imaging these constructs locally dephase protons, resulting in a decrease in signal (hypointensity) localized to the region of accumulation of SPIO. In the context of immune cell imaging, SPIO can provide insight into the cellular migration patterns, trafficking, temporal dynamics and progression of diseases and their related pathological states. Furthermore, by visualizing the presence and activity of immune cells, SPIO-enabled cellular imaging can help evaluate the efficacy of therapy in immune disorders. This thesis examines the production, modification and application of SPIO in a range of in vitro and in vivo immune-response-relevant cellular systems. The role of different nanoparticle characteristics including diameter, surface charge and concentration are investigated in the labeling of T cells in culture. Following optimization of SPIO loading conditions for lymphocytes, the effect these particles have on the activation of primary B cells are elucidated. B cells are tracked using a variety of modalities, with and without the application of B cell depleting therapy. This is to evaluate the efficacy of SPIO as in vivo marker for B cell distribution. Unmodified SPIO were applied to monitor macrophage infiltration in a transient nerve root compression model, with implications for neck pain diagnosis and treatment. Nanoparticle accumulation and MR hypointensity was correlated to the presence of activated macrophage at the site of injury. Taken together, the application of SPIO to study nanoparticle uptake in vitro and visualization of immune cells in vivo provide a basis for advanced study and diagnosis of diverse pathologies

Clearance of damaged mitochondria via mitophagy is important to the protective effect of ischemic preconditioning in kidneys

<p>Ischemic preconditioning (IPC) affords tissue protection in organs including kidneys; however, the underlying mechanism remains unclear. Here we demonstrate an important role of macroautophagy/autophagy (especially mitophagy) in the protective effect of IPC in kidneys. IPC induced autophagy in renal tubular cells in mice and suppressed subsequent renal ischemia-reperfusion injury (IRI). The protective effect of IPC was abolished by pharmacological inhibitors of autophagy and by the ablation of <i>Atg7</i> from kidney proximal tubules. Pretreatment with BECN1/Beclin1 peptide induced autophagy and protected against IRI. These results suggest the dependence of IPC protection on renal autophagy. During IPC, the mitophagy regulator PINK1 (PTEN induced putative kinase 1) was activated. Both IPC and BECN1 peptide enhanced mitolysosome formation during renal IRI in mitophagy reporter mice, suggesting that IPC may protect kidneys by activating mitophagy. We further established an in vitro model of IPC by inducing ‘chemical ischemia’ in kidney proximal tubular cells with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Brief treatment with CCCP protected against subsequent injury in these cells and the protective effect was abrogated by autophagy inhibition. In vitro IPC increased mitophagosome formation, enhanced the delivery of mitophagosomes to lysosomes, and promoted the clearance of damaged mitochondria during subsequent CCCP treatment. IPC also suppressed mitochondrial depolarization, improved ATP production, and inhibited the generation of reactive oxygen species. Knockdown of <i>Pink1</i> suppressed mitophagy and reduced the cytoprotective effect of IPC. Together, these results suggest that autophagy, especially mitophagy, plays an important role in the protective effect of IPC.</p> <p><b>Abbreviations</b>: ACTB: actin, beta; ATG: autophagy related; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; BUN: blood urea nitrogen; CASP3: caspase 3; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; COX4I1: cytochrome c oxidase subunit 4I1; COX8: cytochrome c oxidase subunit 8; DAPI: 4ʹ,6-diamidino-2-phenylindole; DNM1L: dynamin 1 like; EGFP: enhanced green fluorescent protein; EM: electron microscopy; ER: endoplasmic reticulum; FC: floxed control; FIS1: fission, mitochondrial 1; FUNDC1: FUN14 domain containing 1; H-E: hematoxylin-eosin; HIF1A: hypoxia inducible factor 1 subunit alpha; HSPD1: heat shock protein family D (Hsp60) member 1; IMMT/MIC60: inner membrane mitochondrial protein; IPC: ischemic preconditioning; I-R: ischemia-reperfusion; IRI: ischemia-reperfusion injury; JC-1: 5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazolylcarbocyanine iodide; KO: knockout; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; mito-QC: mito-quality control; mRFP: monomeric red fluorescent protein; NAC: N-acetylcysteine; PINK1: PTEN induced putative kinase 1; PPIB: peptidylprolyl isomerase B; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; RPTC: rat proximal tubular cells; SD: standard deviation; sIPC: simulated IPC; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling</p

Development of Extracellular Vesicle Isolation and Model Systems Toward Early Ovarian Cancer Diagnostics

Ovarian cancer (OC) is characterized by late stage discovery and low survivability. However, when diagnosed early (Stages I or II) the 5-year survival rate is 92% up from 29%.5 The extreme dichotomy in survivability is what makes OC a prime candidate for early diagnosis techniques. Exosomes, a subtype of extracellular vesicles, may bridge the gap between early and late diagnosis, but are lacking consistent isolation and detection technologies. Here poly(ethylene terephthalate) (PET) capillary channeled polymer (C-CP) fibers employing an HIC protocol are investigated as a novel exosome isolation method and a quick, inexpensive, and easy-to-use platform for OC diagnosis. The cell model system, immunoaffinity protocols, and biomarker identification tools developed here will aid in the refinement of a selective PET C-CP exosome isolation. The exosome isolation and diagnostic technique developed as a result of these investigations will allow for earlier and routine diagnosis of OC and save many women from one of the deadliest cancers