NanoLuc Binary Technology as a methodological approach: an important new tool for studying the localization of androgen receptor and androgen receptor splice variant V7 homo and heterodimers.

Background: The androgen/androgen receptor (AR)-signaling axis plays a central role in prostate cancer (PCa). Upon androgen-binding the AR dimerizes with another AR, and translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. Consequently, treatments for PCa are commonly based on androgen deprivation therapy (ADT).

A partial epithelial-mesenchymal transition signature for highly aggressive colorectal cancer cells that survive under nutrient restriction.

Partial epithelial-mesenchymal transition (p-EMT) has recently been identified as a hybrid state consisting of cells with both epithelial and mesenchymal characteristics and is associated with the migration, metastasis, and chemoresistance of cancer cells. Here, we describe the induction of p-EMT in starved colorectal cancer (CRC) cells and identify a p-EMT gene signature that can predict prognosis. Functional characterisation of starvation-induced p-EMT in HCT116, DLD1, and HT29 cells showed changes in proliferation, morphology, and drug sensitivity, supported by in vivo studies using the chorioallantoic membrane model.

Functional In Vivo Imaging of Macrophages.

Resident tissue macrophages (RTMs) are specialized phagocytes that are widely distributed throughout the body and are responsible for maintaining homeostasis. Recent advances in experimental techniques have enabled us to gain a greater insight into the actual in vivo biology of RTMs by observing their spatiotemporal dynamics directly in their native environment. Here, we detail a method for live tracking macrophages in a prototypical stromal tissue with high spatial and temporal resolution and great experimental versatility.

How enhancers regulate wavelike gene expression patterns.

A key problem in development is to understand how genes turn on or off at the right place and right time during embryogenesis. Such decisions are made by non-coding sequences called 'enhancers.' Much of our models of how enhancers work rely on the assumption that genes are activated de novo as stable domains across embryonic tissues.

A monoclonal Trd chain supports the development of the complete set of functional γδ T cell lineages.

The clonal selection theory describes key features of adaptive immune responses of B and T cells. For αβ T cells and B cells, antigen recognition and selection principles are known at a detailed molecular level. The precise role of the antigen receptor in γδ T cells remains less well understood.

3Dscript.server: true server-side 3D animation of microscopy images using a natural language-based syntax.

Summary: Creating 3D animations from microscopy data is computationally expensive and requires high-end hardware. We therefore developed 3Dscript.server, a 3D animation software that runs as a service on dedicated, shared workstations.

3D Spheroids Versus 3D Tumor-Like Microcapsules: Confinement and Mechanical Stress May Lead to the Expression of Malignant Responses in Cancer Cells.

As 2D surfaces fail to resemble the tumoral milieu, current discussions are focused on which 3D cell culture strategy may better lead the cells to express in vitro most of the malignant hints described in vivo. In this study, this question is assessed by analyzing the full genetic profile of MCF7 cells cultured either as 3D spheroids-considered as "gold standard" for in vitro cancer research- or immobilized in 3D tumor-like microcapsules, by RNA-Seq and transcriptomic methods, allowing to discriminate at big-data scale, which in vitro strategy can better resemble most of the malignant features described in neoplastic diseases. The results clearly show that mechanical stress, rather than 3D morphology only, stimulates most of the biological processes involved in cancer pathogenicity, such as cytoskeletal organization, migration, and stemness.

Natural human knockouts and Mendelian disorders: deep phenotyping in Italian isolates.

Whole genome sequencing (WGS) allows the identification of human knockouts (HKOs), individuals in whom loss of function (LoF) variants disrupt both alleles of a given gene. HKOs are a valuable model for understanding the consequences of genes function loss. Naturally occurring biallelic LoF variants tend to be significantly enriched in "genetic isolates," making these populations specifically suited for HKO studies.

Investigating Intestinal Barrier Breakdown in Living Organoids.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques.

Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases.

Papillon-Lefèvre syndrome (PLS) is characterized by nonfunctional neutrophil serine proteases (NSPs) and fulminant periodontal inflammation of unknown cause. Here we investigated neutrophil extracellular trap (NET)-associated aggregation and cytokine/chemokine-release/degradation by normal and NSP-deficient human and mouse granulocytes. Stimulated with solid or soluble NET inducers, normal neutrophils formed aggregates and both released and degraded cytokines/chemokines.

ROS-Responsive N-Alkylaminoferrocenes for Cancer-Cell-Specific Targeting of Mitochondria.

Mitochondrial membrane potential is more negative in cancer cells than in normal cells, allowing cancer targeting by delocalized lipophilic cations (DLCs). However, as the difference is rather small, these drugs affect also normal cells. Now a concept of pro-DLCs is proposed based on an N-alkylaminoferrocene structure.

A New Fluorogenic Small-Molecule Labeling Tool for Surface Diffusion Analysis and Advanced Fluorescence Imaging of β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Based on Silicone Rhodamine: SiR-BACE1.

β-site APP-cleaving enzyme 1 (BACE1) is a major player in the pathogenesis of Alzheimer's disease. Structural and functional fluorescence microscopy offers a powerful approach to learn about the physiology and pathophysiology of this protease. Up to now, however, common labeling techniques require genetic manipulation, use large antibodies, or are not compatible with live cell imaging.

A miRNA181a/NFAT5 axis links impaired T cell tolerance induction with autoimmune type 1 diabetes.

Molecular checkpoints that trigger the onset of islet autoimmunity or progression to human type 1 diabetes (T1D) are incompletely understood. Using T cells from children at an early stage of islet autoimmunity without clinical T1D, we find that a microRNA181a (miRNA181a)-mediated increase in signal strength of stimulation and costimulation links nuclear factor of activated T cells 5 (NFAT5) with impaired tolerance induction and autoimmune activation. We show that enhancing miRNA181a activity increases NFAT5 expression while inhibiting FOXP3 regulatory T cell (T) induction in vitro.

Helicobacter pylori Employs a Unique Basolateral Type IV Secretion Mechanism for CagA Delivery.

The Helicobacter pylori (Hp) type IV secretion system (T4SS) forms needle-like pili, whose binding to the integrin-β receptor results in injection of the CagA oncoprotein. However, the apical surface of epithelial cells is exposed to Hp, whereas integrins are basolateral receptors. Hence, the mechanism of CagA delivery into polarized gastric epithelial cells remains enigmatic.

A Stat6/Pten Axis Links Regulatory T Cells with Adipose Tissue Function.

Obesity and type 2 diabetes are associated with metabolic defects and adipose tissue inflammation. Foxp3 regulatory T cells (Tregs) control tissue homeostasis by counteracting local inflammation. However, if and how T cells interlink environmental influences with adipocyte function remains unknown.

A re-investigation of the phytochemical composition of the edible herb Amaranthus retroflexus L.

In this paper the presence of selected prenylated and unprenylated phenylpropanoids of nutraceutical value, namely umbelliferone, apigenin, 4'-geranyloxyferulic acid, 7-isopentenyloxycoumarin, auraptene, and umbelliprenin have been determined in all parts of the edible herb Amaranthus retroflexus extracted with different methodologies. Roots were seen to contain the widest variety of unprenylated and prenylated phenylpropanoids both in terms of number of secondary metabolites and their quantitites. Findings described in the present study underline how A.

Using simulated fluorescence cell micrographs for the evaluation of cell image segmentation algorithms.

Background: Manual assessment and evaluation of fluorescent micrograph cell experiments is time-consuming and tedious. Automated segmentation pipelines can ensure efficient and reproducible evaluation and analysis with constant high quality for all images of an experiment. Such cell segmentation approaches are usually validated and rated in comparison to manually annotated micrographs.

Quantification of biologically active O-prenylated and unprenylated phenylpropanoids in dill (Anethum graveolens), anise (Pimpinella anisum), and wild celery (Angelica archangelica).

An analytical strategy based on different extraction methodologies and HPLC with spectrophotometric (UV-vis) detection has been developed to investigate the presence of and to quantitate biologically active selected unprenylated and O-prenylated phenylpropanoids, namely umbelliferone, 4'-geranyloxyferulic acid, 7-isopentenyloxycoumarin, auraptene, and umbelliprenin in dill (Anethum graveolens L.), anise (Pimpinella anisum L.), and wild celery (Angelica archangelica L.

LAP proteins are localized at the post-synaptic membrane of neuromuscular junctions and appear to modulate synaptic morphology and transmission.

Erbin, Lano, Scribble, and Densin-180 belong to LAP (leucine-rich repeats and PDZ domain) adaptor proteins involved in cell signaling pathways. Previously, we identified Erbin, Lano, and Scribble, but not Densin-180, in muscle cells, where they are involved in regulating the aggregation of nicotinic acetylcholine receptors in vitro. Here, we analyzed their cellular localization at the neuromuscular junction (NMJ) in skeletal muscles of mice.

In Liddle Syndrome, Epithelial Sodium Channel Is Hyperactive Mainly in the Early Part of the Aldosterone-Sensitive Distal Nephron.

The epithelial sodium channel (ENaC) is rate limiting for Na(+) absorption in the aldosterone-sensitive distal nephron comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT), and the entire collecting duct. Liddle syndrome (pseudohyperaldosteronism), a severe form of salt-sensitive hypertension, is caused by gain-of-function mutations of ENaC, but the precise tubular site of increased ENaC function is unknown. In the cortical collecting duct (CCD), ENaC is known to be regulated by aldosterone.

Discovery and Characterization of Biased Allosteric Agonists of the Chemokine Receptor CXCR3.

In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound 11 (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces weak chemotaxis and leads to receptor internalization and the β-arrestin 2 recruitment with potency comparable to that of the chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, 14 (FAUC1104)) led to a contrasting biased allosteric partial agonist that activated solely G proteins, induced chemotaxis, but failed to induce receptor internalization or β-arrestin 2 recruitment.

Review of free software tools for image analysis of fluorescence cell micrographs.

An increasing number of free software tools have been made available for the evaluation of fluorescence cell micrographs. The main users are biologists and related life scientists with no or little knowledge of image processing. In this review, we give an overview of available tools and guidelines about which tools the users should use to segment fluorescence micrographs.

Using multi-channel level sets to measure the cytoplasmic localization of HCMV pUL97 in GFP-B-gal fusion constructs.

Human cytomegalovirus UL97-encoded protein kinase (pUL97) phosphorylates cellular and viral proteins and is critical for viral replication. To quantify the efficiency of nuclear translocation and to elucidate the role of putative nuclear localization signal (NLS) elements, immunofluorescence analysis of different pUL97 expression constructs was performed. Since manual quantitation of respective expression levels lacks objectivity and reproducibility, and is time-consuming as well, a computer-based model is established.