Reduced Monocyte and Neutrophil Infiltration and Activation by P-Selectin/CD62P Inhibition Enhances Thrombus Resolution in Mice.

Background: Venous thromboembolism is a major health problem. After thrombus formation, its resolution is essential to re-establish blood flow, which is crucially mediated by infiltrating neutrophils and monocytes in concert with activated platelets and endothelial cells. Thus, we aimed to modulate leukocyte function during thrombus resolution post-thrombus formation by blocking P-selectin/CD62P-mediated cell interactions.

Estimating the Interaction Strength Between PTS1-Peptides and Their Receptor PEX5 in Living Cells Using Flow-Cytometer-Based FRET (flowFRET) Measurements.

The import of many peroxisomal matrix proteins is initiated by the interaction of type-1 peroxisomal targeting signals (PTS1) residing at the extreme C-terminus of cargo proteins and their receptor protein PEX5. This interaction has been amply investigated by biophysical methods using isolated proteins and peptides or heterologous systems such as two-hybrid assays. However, a recently developed novel application of Fluorescence resonance energy transfer (FRET) allows a quantifying measurement of this interaction in living cells.

Studying the interaction between PEX5 and its full-length cargo proteins in living cells by a novel Försteŕs resonance energy transfer-based competition assay.

The import of the majority of soluble peroxisomal proteins is initiated by the interaction between type-1 peroxisomal targeting signals (PTS1) and their receptor PEX5. PTS1 motifs reside at the extreme C-terminus of proteins and consist of a characteristic tripeptide and a modulatory upstream region. Various PTS1-PEX5 interactions have been studied by biophysical methods using isolated proteins or in heterologous systems such as two-hybrid assays, but a recently established approach based on Försters resonance energy transfer (FRET) allows a quantifying investigation in living cells.

PI3K Signaling in Dendritic Cells Aggravates DSS-Induced Colitis.

Aberrant innate immune responses to the gut microbiota are causally involved in the pathogenesis of inflammatory bowel diseases (IBD). The exact triggers and main signaling pathways activating innate immune cells and how they modulate adaptive immunity in IBD is still not completely understood. Here, we report that the PI3K/PTEN signaling pathway in dendritic cells enhances IL-6 production in a model of DSS-induced colitis.

The Tyrosine Kinase Tec Regulates Effector Th17 Differentiation, Pathogenicity, and Plasticity in T-Cell-Driven Intestinal Inflammation.

T helper (Th) 17 cells are not only key in controlling infections mediated by extracellular bacteria and fungi but are also triggering autoimmune responses. Th17 cells comprise heterogeneous subsets, some with pathogenic functions. They can cease to secrete their hallmark cytokine IL-17A and even convert to other T helper lineages, a process known as transdifferentiation relying on plasticity.

Potential of unglycosylated horseradish peroxidase variants for enzyme prodrug cancer therapy.

Fighting cancer still relies on chemo- and radiation therapy, which is a trade-off between effective clearance of malignant cells and severe side effects on healthy tissue. Targeted cancer treatment on the other hand is a promising and refined strategy with less systemic interference. The enzyme horseradish peroxidase (HRP) exhibits cytotoxic effects on cancer cells in combination with indole-3-acetic acid (IAA).

MicroRNA Signatures in Plasma of Patients With Venous Thrombosis: A Preliminary Report.

Background: Venous thromboembolism (VTE) is a frequent and potentially fatal disease, but its pathophysiology is incompletely understood. microRNAs (miR) dysregulate hemostatic proteins and influence thrombotic pathology by posttranscriptional regulation of gene expression. Consensus in defining VTE-related miR clusters and functionally relevant miR has not been reached.

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

Host and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles.

Rare Human Missense Variants can affect the Function of Disease-Relevant Proteins by Loss and Gain of Peroxisomal Targeting Motifs.

Single nucleotide variants (SNVs) resulting in amino acid substitutions (i.e., missense variants) can affect protein localization by changing or creating new targeting signals.

MicroRNA-155 Controls T Helper Cell Activation During Viral Infection.

MicroRNA (miR) 155 has been implicated in the regulation of innate and adaptive immunity as well as autoimmune processes. Importantly, it has been shown to regulate several antiviral responses, but its contribution to the immune response against cytopathic viruses such as vesicular stomatitis virus (VSV) infections is not known. Using transgenic/recombinant VSV expressing ovalbumin, we show that miR-155 is crucially involved in regulating the T helper cell response against this virus.

Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells.

FRET (Fluorescence Resonance Energy Transfer) measurements are commonly applied to proof protein-protein interactions. However, standard methods of live cell FRET microscopy and signal normalization only allow a principle assessment of mutual binding and are unable to deduce quantitative information of the interaction. We present an evaluation and normalization procedure for 3-filter FRET measurements, which reflects the process of complex formation by plotting FRET-saturation curves.

PI3K activity in dendritic cells exerts paradoxical effects during autoimmune inflammation.

The peripheral activation of autoreactive T cells and subsequent central nervous system (CNS) immune cell infiltration are key events relevant for experimental autoimmune encephalomyelitis (EAE), a commonly employed multiple sclerosis (MS) model, influenced by T1 and T17 mediated immunity. The phosphoinositide-3-kinase (PI3K)-AKT kinase pathway modulates outcome during EAE, with direct actions of PI3K on adaptive immunity implicated in deleterious and effects on antigen presenting cells involved in beneficial responses during EAE. Here, by genetically deleting the regulatory subunit of Class Ia PI3K, p85α, in selective myeloid cells, we aimed to resolve the impact of PI3K in EAE.

Constituents of Mediterranean Spices Counteracting Vascular Smooth Muscle Cell Proliferation: Identification and Characterization of Rosmarinic Acid Methyl Ester as a Novel Inhibitor.

Scope: Aberrant vascular smooth muscle cell (VSMC) proliferation is involved in atherosclerotic plaque formation and restenosis. Mediterranean spices have been reported to confer cardioprotection, but their direct influence on VSMCs has largely not been investigated. This study aims at examining rosmarinic acid (RA) and 11 related constituents for inhibition of VSMC proliferation in vitro, and at characterizing the most promising compound for their mode of action and influence on neointima formation in vivo.

Targeting ncRNAs by plant secondary metabolites: The ncRNAs game in the balance towards malignancy inhibition.

The current trend of combining state of the art technologies with quondam treatments in order to overcome existing gaps in the clinical area determined an increased interest into polyphenols, common dietary phytochemicals, for the prevention and treatment of chronic diseases, especially cancer. The reemergence of polyphenols in the cancer field is sustained by advanced-omics technologies able to identify coding and non-coding genes and their related signaling pathways modulated by natural compounds. Identification of the structural correspondence between interacting molecules will allow the development of more targeted and informed therapeutic strategies for cancer management.

Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells.

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface.

Xanthohumol Blocks Proliferation and Migration of Vascular Smooth Muscle Cells in Vitro and Reduces Neointima Formation in Vivo.

Xanthohumol (1) is a principal prenylated chalcone found in hops. The aim of this study was to examine its influence on platelet-derived growth factor (PDGF)-BB-triggered vascular smooth muscle cell (VSMC) proliferation and migration in vitro and on experimentally induced neointima formation in vivo. Quantification of resazurin conversion indicated that 1 can inhibit PDGF-BB-induced VSMC proliferation concentration-dependently (IC = 3.

Myeloid PTEN deficiency impairs tumor-immune surveillance via immune-checkpoint inhibition.

Tumor-host interaction is determined by constant immune surveillance, characterized by tumor infiltration of myeloid and lymphoid cells. A malfunctioning or diverted immune response promotes tumor growth and metastasis. Recent advances had been made, by treating of certain tumor types, such as melanoma, with T-cell checkpoint inhibitors.

Plumericin inhibits proliferation of vascular smooth muscle cells by blocking STAT3 signaling via S-glutathionylation.

The etiology of atherosclerosis and restenosis involves aberrant inflammation and proliferation, rendering compounds with both anti-inflammatory and anti-mitogenic properties as promising candidates for combatting vascular diseases. A recent study identified the iridoid plumericin as a new scaffold inhibitor of the pro-inflammatory NF-κB pathway in endothelial cells. We here examined the impact of plumericin on the proliferation of primary vascular smooth muscle cells (VSMC).

Fluorescent proteins as genetically encoded FRET biosensors in life sciences.

Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET.

Recent advances in the genetic analysis of PTEN and PI3K innate immune properties.

The role of the PI3-kinase pathway and its antagonist PTEN in the regulation of innate immune responses has only recently attracted the attention of the scientific community. The PI3K/PTEN signaling axis is most renowned for its critical involvement in the malignant transformation of cells leading to tumorigenesis. PI3K function in the regulation of innate immunity, either pro-inflammatory or anti-inflammatory, is still a controversial issue.

A membrane-anchored Theileria parva cyclophilin with a non-cleaved amino-terminal signal peptide for entry into the endoplasmic reticulum.

Recent studies suggest that peptidyl-prolyl isomerases of the cyclophilin family, that access the secretory pathway, can be involved in the interaction of parasitic protozoa with mammalian host cells. The amino acid sequence of a cDNA encoding a cyclophilin family member of the intracellular protozoan parasite of cattle Theileria parva contains a conserved C-terminal domain that exhibits 70% amino acid identity to cyclophilin proteins from other organisms, and a unique 60 amino acid novel N-terminal extension. Cell-free expression of the cDNA revealed a 26kDa amino translation product, indicating expression of the N-terminal domain.

Serum albumin is a specific inhibitor of apoptosis in human endothelial cells.

Excess blood vessels are removed by apoptosis of endothelial cells, however, the signals responsible for this have not been defined. Apoptosis of cultured human umbilical vein endothelial cells is induced by deprivation of serum or adhesion. In this paper, apoptosis in human umbilical vein and microvascular endothelium was induced by deprivation of serum and or adhesion.