Modulation of the pre-metastatic bone niche: molecular changes mediated by bone-homing prostate cancer extracellular vesicles.

Prostate cancer (PCa) is a leading male malignancy worldwide, often progressing to bone metastasis, with limited curative options. Extracellular vesicles (EVs) have emerged as key players in cancer communication and metastasis, promoting the formation of supportive microenvironments in distant sites. Our previous studies have highlighted the role of PCa EVs in modulating osteoblasts and facilitating tumor progression. However, the early pre-metastatic changes induced by PCa EVs within the bone microenvironment remain poorly understood. To investigate the early effects of repeated exposure to PCa EVs in vivo, mimicking EVs being shed from the primary tumor, PCa EVs isolated from cell line PC3MLuc2a were fluorescently labelled and repeatedly administered via tail vein injection to adult CD1 NuNu male mice for a period of 4 weeks. In vivo imagining, histological analysis and gene expression profiling were performed to assess the impact of PCa EVs on the bone microenvironment. We demonstrate for the first time that PCa EVs home to both bone and lymph nodes following repeated exposures. Furthermore, the accumulation of EVs within the bone leads to distinct molecular changes indicative of disrupted bone homeostasis (e.g., changes to signaling pathways such as Paxillin p = 0.0163, Estrogen Receptor p = 0.0271, RHOA p = 0.0287, Ribonucleotide reductase p = 0.0307 and ERK/MAPK p = 0.0299). Changes in key regulators of these pathways were confirmed in vitro on human osteoblasts. In addition, our data compares the known gene signature of osteocytes and demonstrates a high proportion of overlap (52.2%), suggesting a potential role for this cell type in response to PCa EV exposure. No changes in bone histology or immunohistochemistry were detected, indicating that PCa EV mediated changes were induced at the molecular level. This study provides novel insights into the alterations induced by PCa EVs on the bone microenvironment. The observed molecular changes indicate changes in key pathways and suggest a role for osteocytes in these EV mediated early changes to bone. Further research to understand these early events may aid in the development of targeted interventions to disrupt the metastatic cascade in PCa.

A Systematic Review and Meta-Analysis of the Pathology Underlying Aneurysm Enhancement on Vessel Wall Imaging.

Intracranial aneurysms are common, but only a minority rupture and cause subarachnoid haemorrhage, presenting a dilemma regarding which to treat. Vessel wall imaging (VWI) is a contrast-enhanced magnetic resonance imaging (MRI) technique used to identify unstable aneurysms. The pathological basis of MR enhancement of aneurysms is the subject of debate. This review synthesises the literature to determine the pathological basis of VWI enhancement. PubMed and Embase searches were performed for studies reporting VWI of intracranial aneurysms and their correlated histological analysis. The risk of bias was assessed. Calculations of interdependence, univariate and multivariate analysis were performed. Of 228 publications identified, 7 met the eligibility criteria. Individual aneurysm data were extracted for 72 out of a total of 81 aneurysms. Univariate analysis showed macrophage markers (CD68 and MPO, p = 0.001 and p = 0.002), endothelial cell markers (CD34 and CD31, p = 0.007 and p = 0.003), glycans (Alcian blue, p = 0.003) and wall thickness (p = 0.030) were positively associated with enhancement. Aneurysm enhancement therefore appears to be associated with inflammatory infiltrate and neovascularisation. However, all these markers are correlated with each other, and the literature is limited in terms of the numbers of aneurysms analysed and the parameters considered. The data are therefore insufficient to determine if these associations are independent of each other or of aneurysm size, wall thickness and rupture status. Thus, the cause of aneurysm-wall enhancement currently remains unknown.

Heparanase inhibition as a systemic approach to protect the endothelial glycocalyx and prevent microvascular complications in diabetes.

BACKGROUND: Diabetes mellitus is a chronic disease which is detrimental to cardiovascular health, often leading to secondary microvascular complications, with huge global health implications. Therapeutic interventions that can be applied to multiple vascular beds are urgently needed. Diabetic retinopathy (DR) and diabetic kidney disease (DKD) are characterised by early microvascular permeability changes which, if left untreated, lead to visual impairment and renal failure, respectively. The heparan sulphate cleaving enzyme, heparanase, has previously been shown to contribute to diabetic microvascular complications, but the common underlying mechanism which results in microvascular dysfunction in conditions such as DR and DKD has not been determined. METHODS: In this study, two mouse models of heparan sulphate depletion (enzymatic removal and genetic ablation by endothelial specific Exotosin-1 knock down) were utilized to investigate the impact of endothelial cell surface (i.e., endothelial glycocalyx) heparan sulphate loss on microvascular barrier function. Endothelial glycocalyx changes were measured using fluorescence microscopy or transmission electron microscopy. To measure the impact on barrier function, we used sodium fluorescein angiography in the eye and a glomerular albumin permeability assay in the kidney. A type 2 diabetic (T2D, db/db) mouse model was used to determine the therapeutic potential of preventing heparan sulphate damage using treatment with a novel heparanase inhibitor, OVZ/HS-1638. Endothelial glycocalyx changes were measured as above, and microvascular barrier function assessed by albumin extravasation in the eye and a glomerular permeability assay in the kidney. RESULTS: In both models of heparan sulphate depletion, endothelial glycocalyx depth was reduced and retinal solute flux and glomerular albumin permeability was increased. T2D mice treated with OVZ/HS-1638 had improved endothelial glycocalyx measurements compared to vehicle treated T2D mice and were simultaneously protected from microvascular permeability changes associated with DR and DKD. CONCLUSION: We demonstrate that endothelial glycocalyx heparan sulphate plays a common mechanistic role in microvascular barrier function in the eye and kidney. Protecting the endothelial glycocalyx damage in diabetes, using the novel heparanase inhibitor OVZ/HS-1638, effectively prevents microvascular permeability changes associated with DR and DKD, demonstrating a novel systemic approach to address diabetic microvascular complications.

Measurement of solute permeability in the mouse spinal cord.

BACKGROUND: Sensory perception and motor dexterity is coordinated by the spinal cord, which remains effective due to maintenance of neuronal homeostasis. This is stringently controlled by the blood spinal cord barrier. Therefore, the function of the spinal cord is susceptible to alterations in the microvessel integrity (e.g. vascular leakage) and/or perfusion (e.g. changes in blood flow). NEW METHOD: Spinal cord solute permeability was measured in anaesthetised mice. The lumbar spinal cord vertebra were stabilised and a coverslip secured to allow fluorescent tracers of vascular function and anatomy to be visualised in the vascular network. Fluorescence microscopy allowed real time measurements of vascular leakage and capillary perfusion within the spinal cord. RESULTS: Capillaries were identified through fluorescent labelling of the endothelial luminal glycocalyx (wheat germ agglutin 555). Real time estimation of vascular permeability through visualisation of sodium fluorescein transport was recorded from identified microvessels in the lumbar dorsal horn of the spinal cord. COMPARISON WITH EXISTING METHOD(S): Current approaches have used histological and/or tracer based in-vivo assays alongside cell culture to determine endothelium integrity and/or function. These only provide a snapshot of the developing vasculopathy, restricting the understanding of physiological function or disease progression over time. CONCLUSIONS: These techniques allow for direct visualisation of cellular and/or mechanistic influences upon vascular function and integrity, which can be applied to rodent models including disease, transgenic and/or viral approaches. This combination of attributes allows for real time understanding of the function of the vascular network within the spinal cord.

PBMC-derived extracellular vesicles in a smoking-related inflammatory disease model.

Extracellular vesicles (EVs) function as mediators of intercellular communication and as such influence the recipient cell function. EVs derived from immune cells can carry out many of the same functions as their parental cells, as they carry costimulatory molecules, antigens, and antigen-MHC complexes. As a result, there is a strong interest in understanding the composition and origin of immune cell-derived EVs in order to understand their role in the pathogenesis of diseases. This study aimed to optimize methodologies to study immune cell-derived EVs. Peripheral blood mononuclear cell-derived small EVs were isolated and observed using conventional transmission electron microscopy and sized by nanoparticle tracking analysis. They were then enumerated and profiled using imaging flow cytometry and were further characterized using a flow cytometric multiplex bead assay. These techniques were then applied to our current research, namely smoking-related inflammatory disease. We present here a comprehensive approach to analyze PBMC-derived small EVs in smoking-related inflammatory disease following the Minimal Information for Studies of Extracellular Vesicle 2018 guidelines.

Hydrogel-Based Pre-Clinical Evaluation of Repurposed FDA-Approved Drugs for AML.

In vivo models of acute myeloid leukemia (AML) are low throughput, and standard liquid culture models fail to recapitulate the mechanical and biochemical properties of the extracellular matrix-rich protective bone marrow niche that contributes to drug resistance. Candidate drug discovery in AML requires advanced synthetic platforms to improve our understanding of the impact of mechanical cues on drug sensitivity in AML. By use of a synthetic, self-assembling peptide hydrogel (SAPH) of modifiable stiffness and composition, a 3D model of the bone marrow niche to screen repurposed FDA-approved drugs has been developed and utilized. AML cell proliferation was dependent on SAPH stiffness, which was optimized to facilitate colony growth. Three candidate FDA-approved drugs were initially screened against the THP-1 cell line and mAF9 primary cells in liquid culture, and EC50 values were used to inform drug sensitivity assays in the peptide hydrogel models. Salinomycin demonstrated efficacy in both an 'early-stage' model in which treatment was added shortly after initiation of AML cell encapsulation, and an 'established' model in which time-encapsulated cells had started to form colonies. Sensitivity to Vidofludimus treatment was not observed in the hydrogel models, and Atorvastatin demonstrated increased sensitivity in the 'established' compared to the 'early-stage' model. AML patient samples were equally sensitive to Salinomycin in the 3D hydrogels and partially sensitive to Atorvastatin. Together, this confirms that AML cell sensitivity is drug- and context-specific and that advanced synthetic platforms for higher throughput are valuable tools for pre-clinical evaluation of candidate anti-AML drugs.

John squire and endothelial glycocalyx structure: an unfinished story.

John Squire did not only produce leading works in the muscle field, he also significantly contributed to the vascular permeability field by ultrastructural analysis of the endothelial glycocalyx. Presented here is a review of his involvement in the field by his main collaborator C.C. Michel and his last postdoctoral researcher KP Arkill. We end on a reinterpretation of his work that arguably links to our current understanding of endothelial glycocalyx structure and composition predicting 6 glycosaminoglycans fibres per syndecan core protein, only achieved in the endothelium by dimerization.

The role of extracellular vesicle miRNAs and tRNAs in synovial fibroblast senescence.

Extracellular vesicles are mediators of intercellular communication with critical roles in cellular senescence and ageing. In arthritis, senescence is linked to the activation of a pro-inflammatory phenotype contributing to chronic arthritis pathogenesis. We hypothesised that senescent osteoarthritic synovial fibroblasts induce senescence and a pro-inflammatory phenotype in non-senescent osteoarthritic fibroblasts, mediated through extracellular vesicle cargo. Small RNA-sequencing and mass spectrometry proteomics were performed on extracellular vesicles isolated from the secretome of non-senescent and irradiation-induced senescent synovial fibroblasts. ß-galactosidase staining confirmed senescence in SFs. RNA sequencing identified 17 differentially expressed miRNAs, 11 lncRNAs, 14 tRNAs and one snoRNA and, 21 differentially abundant proteins were identified by mass spectrometry. Bioinformatics analysis of miRNAs identified fibrosis, cell proliferation, autophagy, and cell cycle as significant pathways, tRNA analysis was enriched for signaling pathways including FGF, PI3K/AKT and MAPK, whilst protein analysis identified PAX3-FOXO1, MYC and TFGB1 as enriched upstream regulators involved in senescence and cell cycle arrest. Finally, treatment of non-senescent synovial fibroblasts with senescent extracellular vesicles confirmed the bystander effect, inducing senescence in non-senescent cells potentially through down regulation of NF-κß and cAMP response element signaling pathways thus supporting our hypothesis. Understanding the exact composition of EV-derived small RNAs of senescent cells in this way will inform our understanding of their roles in inflammation, intercellular communication, and as active molecules in the senescence bystander effect.

Novel perspectives of sodium handling in type 2 diabetes mellitus.

INTRODUCTION: As a key regulator of body water, sodium homeostasis forms an essential component of human physiology. Type 2 Diabetes Mellitus (T2D)-associated sodium overload stems from chronic renal retention of sodium, contributing toward the development of adverse cardiovascular sequelae. AREAS COVERED: Our traditional model of sodium regulation invokes two compartments: extracellular fluid (ECF [plasma and interstitial fluid]) and intracellular fluid (ICF). Data from the Mars program reveal inconsistencies with this two-space model, including mismatches between net body sodium and water. Recent data utilizing 23Na magnetic resonance imaging (MRI) show a preponderance of bound sodium within human dermis, consistent with a third space repository and providing compelling evidence to support a three-space model in which dermal sodium binding facilitates sodium homeostasis within the ECF and ICF. This buffer is impaired in T2D, with diminishment of dermal bound sodium that may promote deleterious sequelae of sodium overload within the ECF and ICF. EXPERT OPINION: Future studies should focus on novel therapeutic opportunities for sodium regulation in T2D and other conditions of sodium dysregulation. The ratio of free:bound dermal sodium (reflecting sodium storage capacity) could be utilized as a clinical biomarker for salt and water balance, to improve diagnostic accuracy and facilitate clinical decision-making.

Serine-arginine-rich protein kinase-1 inhibition for the treatment of diabetic retinopathy.

Angiogenic VEGF isoforms are upregulated in diabetic retinopathy (DR), driving pathological growth and fluid leakage. Serine-arginine-rich protein kinase-1 (SRPK1) regulates VEGF splicing, and its inhibition blocks angiogenesis. We tested the hypothesis that SRPK1 is activated in diabetes, and an SRPK1 inhibitor (SPHINX31) switches VEGF splicing in DR and prevents increased vascular permeability into the retina. SRPK1 was activated by high glucose (HG), in a PKC-dependent manner, and was blocked by SPHINX31. HG induced release of SRSF1 from the nuclear speckles, which was also SRPK1 dependent, and increased retinal pigment epithelial (RPE) monolayer admittance, which was reversed by SRPK1 inhibition (P < 0.05). Diabetes increased retinal permeability and thickness after 14 days which was blocked by treatment with SPHINX31 eye drops (P < 0.0001). These results show that SRPK1 inhibition, administered as an eye drop, protected the retinal barrier from hyperglycemia-associated loss of integrity in RPE cells in vitro and in diabetic rats in vivo. A clinical trial of another SRPK1 inhibitor has now been initiated in patients with diabetic macular edema.NEW & NOTEWORTHY VEGF-A165b splicing is induced by hyperglycemia through PKC-mediated activation of SRPK1 in RPE cells, increasing their permeability and angiogenic capability. SRPK1 inhibitors can be given as eye drops to reduce retinal permeability and edema in diabetic retinopathy.

Endothelial glycocalyx is damaged in diabetic cardiomyopathy: angiopoietin 1 restores glycocalyx and improves diastolic function in mice.

AIMS/HYPOTHESIS: Diabetic cardiomyopathy (DCM) is a serious and under-recognised complication of diabetes. The first sign is diastolic dysfunction, which progresses to heart failure. The pathophysiology of DCM is incompletely understood but microcirculatory changes are important. Endothelial glycocalyx (eGlx) plays multiple vital roles in the microcirculation, including in the regulation of vascular permeability, and is compromised in diabetes but has not previously been studied in the coronary microcirculation in diabetes. We hypothesised that eGlx damage in the coronary microcirculation contributes to increased microvascular permeability and hence to cardiac dysfunction. METHODS: We investigated eGlx damage and cardiomyopathy in mouse models of type 1 (streptozotocin-induced) and type 2 (db/db) diabetes. Cardiac dysfunction was determined by echocardiography. We obtained eGlx depth and coverage by transmission electron microscopy (TEM) on mouse hearts perfusion-fixed with glutaraldehyde and Alcian Blue. Perivascular oedema was assessed from TEM images by measuring the perivascular space area. Lectin-based fluorescence was developed to study eGlx in paraformaldehyde-fixed mouse and human tissues. The eGlx of human conditionally immortalised coronary microvascular endothelial cells (CMVECs) in culture was removed with eGlx-degrading enzymes before measurement of protein passage across the cell monolayer. The mechanism of eGlx damage in the diabetic heart was investigated by quantitative reverse transcription-PCR array and matrix metalloproteinase (MMP) activity assay. To directly demonstrate that eGlx damage disturbs cardiac function, isolated rat hearts were treated with enzymes in a Langendorff preparation. Angiopoietin 1 (Ang1) is known to restore eGlx and so was used to investigate whether eGlx restoration reverses diastolic dysfunction in mice with type 1 diabetes. RESULTS: In a mouse model of type 1 diabetes, diastolic dysfunction (confirmed by echocardiography) was associated with loss of eGlx from CMVECs and the development of perivascular oedema, suggesting increased microvascular permeability. We confirmed in vitro that eGlx removal increases CMVEC monolayer permeability. We identified increased MMP activity as a potential mechanism of eGlx damage and we observed loss of syndecan 4 consistent with MMP activity. In a mouse model of type 2 diabetes we found a similar loss of eGlx preceding the development of diastolic dysfunction. We used isolated rat hearts to demonstrate that eGlx damage (induced by enzymes) is sufficient to disturb cardiac function. Ang1 restored eGlx and this was associated with reduced perivascular oedema and amelioration of the diastolic dysfunction seen in mice with type 1 diabetes. CONCLUSIONS/INTERPRETATION: The association of CMVEC glycocalyx damage with diastolic dysfunction in two diabetes models suggests that it may play a pathophysiological role and the enzyme studies confirm that eGlx damage is sufficient to impair cardiac function. Ang1 rapidly restores the CMVEC glycocalyx and improves diastolic function. Our work identifies CMVEC glycocalyx damage as a potential contributor to the development of DCM and therefore as a therapeutic target.

Transmission Electron Microscopy of Endothelium.

Transmission electron microscopy using resin sections still remains an exceptionally useful tool in evaluating cellular ultrastucture within tissue. For the endothelium the best method for maintaining such structure is perfusion fixation: fixing the tissue under physiological pressure. Here the focus is on a method of maintaining the vascular wall structure including the endothelial glycocalyx and extending this with tilt series tomography. Shown are typical histological sections from multiple capillary beds including brain, heart and retina using a lanthanide staining technique (LaDy GAGa) to highlight that the differences in normo-physiology are substantial.It is hoped that users will find the notes useful in deciding which specific staining and imaging method would suit their needs so this technically challenging, and low throughput methodology, is used to its best effect.

Noninvasive Measurement of Retinal Microvascular Permeability During Loss of Endothelial Quiescence.

In the retina EC dysfunction and angiogenesis are driven by an altered microenvironment e.g., diabetes, leading to hypoxia and inflammation in the retinal layers, resulting in excessive vascular leakage and growth. The gold standard for measuring blood-retinal barrier permeability in response to disease and or therapy has been the gold standard Evans blue (EB) assay. However, this technique has limitations in vivo, including nonspecific tissue binding and toxicity. Here we describe a novel imaging methodology combining sodium fluorescein fundus angiography (FFA) with mathematical quantification allowing retinal permeability to be noninvasively and accurately measured at multiple time points in the same animal, minimizing animal use in line with the 3Rs framework. In addition, this technique is a nontoxic, high throughput, sensitive, and cost-effective alternative technique to the Evans blue assay. Moreover, this technique can be translated to other species.

Transcriptomic Analysis of Cardiomyocyte Extracellular Vesicles in Hypertrophic Cardiomyopathy Reveals Differential snoRNA Cargo.

Hypertrophic cardiomyopathy (HCM) is characterized by increased left ventricular wall thickness that can lead to devastating conditions such as heart failure and sudden cardiac death. Despite extensive study, the mechanisms mediating many of the associated clinical manifestations remain unknown and human models are required. To address this, human-induced pluripotent stem cell (hiPSC) lines were generated from patients with a HCM-associated mutation (c.ACTC1G301A) and isogenic controls created by correcting the mutation using CRISPR/Cas9 gene editing technology. Cardiomyocytes (hiPSC-CMs) were differentiated from these hiPSCs and analyzed at baseline, and at increased contractile workload (2 Hz electrical stimulation). Released extracellular vesicles (EVs) were isolated and characterized after a 24-h culture period and transcriptomic analysis performed on both hiPSC-CMs and released EVs. Transcriptomic analysis of cellular mRNA showed the HCM mutation caused differential splicing within known HCM pathways, and disrupted metabolic pathways. Analysis at increasing contraction frequency showed further disruption of metabolic gene expression, with an additive effect in the HCM background. Intriguingly, we observed differences in snoRNA cargo within HCM released EVs that specifically altered when HCM hiPSC-CMs were subjected to increased workload. These snoRNAs were predicted to have roles in post-translational modifications and alternative splicing, processes differentially regulated in HCM. As such, the snoRNAs identified in this study may unveil mechanistic insight into unexplained HCM phenotypes and offer potential future use as HCM biomarkers or as targets in future RNA-targeting therapies.

A Reinterpretation of Evidence for the Endothelial Glycocalyx Filtration Structure.

The endothelial glycocalyx (eGlx) is thought to be the primary macromolecular filter for fluid flux out of the vasculature. This filter maintains the higher protein concentration within the vessel lumen relative to the tissue. Whilst the arguments for the eGlx being the size filter are convincing the structural evidence has been limited to specialized stains of perfusion fixed tissue, which are further processed for resin embedding for transmission electron microscopy. The staining and processing of the delicate pore structure has left many researchers struggling to interpret the observed surface coat. Previous work has alluded to a 19.5 nm spacing between fibers; however, whilst repeatable it does not give an eGlx pore size consistent with known glycosaminoglycan (GAG) molecular structure due to the required fiber thickness of >10 nm. Here a new interpretation is proposed based on the likelihood that the electron micrographs of are often of collapsed eGlx. The 19.5 nm spacing measured may therefore be the core protein of the proteoglycans (PGs) with the GAGs wrapped up around them rather than in an expanded in vivo state. The concept is explored to determine that this is indeed consistent with experimental measurements of permeability if the syndecans are predominately dimerized. Further an alteration of core protein lattice from hexagonal packing to square packing dramatically changes the permeability which could be facilitated via known mechanisms such as transient actin binding.

A new view of macula densa cell microanatomy.

Although macula densa (MD) cells are chief regulatory cells in the nephron with unique microanatomical features, they have been difficult to study in full detail due to their inaccessibility and limitations in earlier microscopy techniques. The present study used a new mouse model with a comprehensive imaging approach to visualize so far unexplored microanatomical features of MD cells, their regulation, and functional relevance. MD-GFP mice with conditional and partial induction of green fluorescent protein (GFP) expression, which specifically and intensely illuminated only single MD cells, were used with fluorescence microscopy of fixed tissue and live MD cells in vitro and in vivo with complementary electron microscopy of the rat, rabbit, and human kidney. An elaborate network of major and minor cell processes, here named maculapodia, were found at the cell base, projecting toward other MD cells and the glomerular vascular pole. The extent of maculapodia showed upregulation by low dietary salt intake and the female sex. Time-lapse imaging of maculapodia revealed highly dynamic features including rapid outgrowth and an extensive vesicular transport system. Electron microscopy of rat, rabbit, and human kidneys and three-dimensional volume reconstruction in optically cleared whole-mount MD-GFP mouse kidneys further confirmed the presence and projections of maculapodia into the extraglomerular mesangium and afferent and efferent arterioles. The newly identified dynamic and secretory features of MD cells suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD cells and between MD and other target cells.NEW & NOTEWORTHY This study illuminated a physiologically regulated dense network of basal cell major and minor processes (maculapodia) in macula densa (MD) cells. The newly identified dynamic and secretory features of these microanatomical structures suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus between MD and other target cells. Detailed characterization of the function and molecular details of MD cell intercellular communications and their role in physiology and disease warrant further studies.

Structural assessment of SARS-CoV2 accessory protein ORF7a predicts LFA-1 and Mac-1 binding potential.

ORF7a is an accessory protein common to SARS-CoV1 and the recently discovered SARS-CoV2, which is causing the COVID-19 pandemic. The ORF7a protein has a structural homology with ICAM-1 which binds to the T lymphocyte integrin receptor LFA-1. As COVID-19 has a strong immune component as part of the disease, we sought to determine whether SARS-CoV2 would have a similar structural interaction with LFA-1. Using molecular docking simulations, we found that SARS-CoV2 ORF7a has the key structural determinants required to bind LFA-1 but also the related leukocyte integrin Mac-1, which is also known to be expressed by macrophages. Our study shows that SARS-CoV2 ORF7a protein has a conserved Ig immunoglobulin-like fold containing an integrin binding site that provides a mechanistic hypothesis for SARS-CoV2's interaction with the human immune system. This suggests that experimental investigation of ORF7a-mediated effects on immune cells such as T lymphocytes and macrophages (leukocytes) could help understand the disease further and develop effective treatments.

Non-invasive measurement of retinal permeability in a diabetic rat model.

OBJECTIVE: The gold standard for measuring blood-retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non-specific tissue binding and toxicity. This study describes a non-toxic, high-throughput, and cost-effective alternative technique that minimizes animal usage. METHODS: Sodium fluorescein fundus angiography was performed in non-diabetic and diabetic Brown Norway rats on days 0, 7, 14, 21, and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post-study eyes were fixed, dissected, and stained (isolectin B4) to measure required parameters for permeability quantification including total vessel length per retinal volume, radius, and thickness. RESULTS: In the non-diabetic cohort retinal permeability remained constant over the 28-day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from days 14-28 (P < .01, P < .001, P < .0001). CONCLUSIONS: This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non-invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non-toxic, rapid, sensitive, and cost-effective alternative to the Evans blue assay.

Local microvascular leakage promotes trafficking of activated neutrophils to remote organs.

Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Although neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we report that vascular permeability-enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of neutrophil rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the bloodstream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labeling method we provide direct evidence for the systemic dissemination of rTEM neutrophils, and showed them to exhibit an activated phenotype and be capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrate that increased microvascular leakage reverses the localization of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to reenter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.

Dye Aggregate-Mediated Self-Assembly of Bacteriophage Bioconjugates.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.