Homogeneous peptide-break assay for luminescent detection of enzymatic protein post-translational modification activity utilizing charged peptides

We have developed a rapid and sensitive universal peptide-based time-resolved luminescence assay for detection of enzymatic post-translational modifications (PTMs). PTMs play essential roles in intracellular signaling and cell regulation, thus providing functional protein diversity in cell. Due this, impaired PTM patterns have been linked to multiple disease states. Clear link between PTMs and pathological conditions have also driven assay development further, but still today most of the methodologies are based on single-specificity or group-specific PTM-recognition. We have previously introduced leuzine-zipper based peptide-break technology as a viable option for universal PTM detection. Here, we introduce peptide-break technology utilizing single-label homogeneous quenching resonance energy transfer (QRET) and charge-based peptide-peptide interaction. We demonstrate the functionality of the new assay concept in phosphorylation, deacetylation, and citrullination. In a comparable study between previously introduced leucine-zipper and the novel charge-based approach, we found equal PTM detection performance and sensitivity, but the peptide design for new targets is simplified with the charged peptides. The new concept allows the use of short </p

Direct transfection of clonal organoids in matrigel microbeads : a promising approach toward organoid-based genetic screens

Organoid cultures in 3D matrices are relevant models to mimic the complex in vivo environment that supports cell physiological and pathological behaviors. For instance, 3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen. Effective genetic engineering in organoids would bring new insights in organogenesis and carcinogenesis. However, direct 3D transfection on already formed organoids remains challenging. One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques. To address this issue, we developed an innovative approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads using a microfluidic device and electroporation. We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency. Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues. We believe that the combination of controlled organoid generation and efficient 3D transfection developed here opens new perspectives for flow-based high-throughput genetic screening and functional genomic applications

Nanomolar Protein-Protein Interaction Monitoring with a Label-Free Protein-Probe Technique

Protein-protein interactions (PPIs) are an essential part of correct cellular functionality, making them increasingly interesting drug targets. While Forster resonance energy transfer-based methods have traditionally been widely used for PPI studies, label-free techniques have recently drawn significant attention. These methods are ideal for studying PPIs, most importantly as there is no need for labeling of either interaction partner, reducing potential interferences and overall costs. Already, several different label-free methods are available, such as differential scanning calorimetry and surface plasmon resonance, but these biophysical methods suffer from low to medium throughput, which reduces suitability for high-throughput screening (HTS) of PPI inhibitors. Differential scanning fluorimetry, utilizing external fluorescent probes, is an HTS compatible technique, but high protein concentration is needed for experiments. To improve the current concepts, we have developed a method based on time-resolved luminescence, enabling PPI monitoring even at low nanomolar protein concentrations. This method, called the protein probe technique, is based on a peptide conjugated with Eu3+ chelate, and it has already been applied to monitor protein structural changes and small molecule interactions at elevated temperatures. Here, the applicability of the protein probe technique was demonstrated by monitoring single-protein pairing and multiprotein complexes at room and elevated temperatures. The concept functionality was proven by using both artificial and multiple natural protein pairs, such as KRAS and eIF4A together with their binding partners, and C-reactive protein in a complex with its antibody

Single-Peptide TR-FRET Detection Platform for Cysteine-Specific Post-Translational Modifications

Post-translational modifications (PTMs) are one of the most important regulatory mechanisms in cells, and they play key roles in cell signaling both in health and disease. PTM catalyzing enzymes have become significant drug targets, and therefore, tremendous interest has been focused on the development of broad-scale assays to monitor several different PTMs with a single detection platform. Most of the current methodologies suffer from low throughput or rely on antibody recognition, increasing the assay costs, and decreasing the multifunctionality of the assay. Thus, we have developed a sensitive time-resolved Forster resonance energy transfer (TR-FRET) detection method for PTMs of cysteine residues using a single-peptide approach performed in a 384-well format. In the developed assay, the enzyme-specific biotinylated substrate peptide is post-translationally modified at the cysteine residue, preventing the subsequent thiol coupling with a reactive AlexaFluor 680 acceptor dye. In the absence of enzymatic activity, increase in the TR-FRET signal between the biotin-bound Eu(III)-labeled streptavidin donor and the cysteine-coupled AlexaFluor 680 acceptor dye is observed. We demonstrate the detection concept with cysteine modifying S-nitrosylation and ADP-ribosylation reactions using a chemical nitric oxide donor S-nitrosoglutathione and enzymatic ADP-ribosyltransferase PtxS1-subunit of pertussis toxin, respectively. As a proof of concept, three peptide substrates derived from the small GTPase K-Ras and the inhibitory alpha-subunit of the heterotrimeric G-protein G alpha i showed expected functionality in both chemical and enzymatic assays. Measurements yielded signal-to-background ratios of 28.7, 33.0, and 8.7 between the modified and the nonmodified substrates for the three peptides in the S-nitrosylation assay, 5.8 in the NAD(+) hydrolysis assay, and 6.8 in the enzymatic ADP-ribosyltransferase inhibitor dose-response assay. The developed antibody-free assay for cysteine-modifying enzymes provides a detection platform with low nanomolar peptide substrate consumption, and the assay is potentially applicable to investigate various cysteine-modifying enzymes in a high throughput compatible format

Quantification of Dynamic Morphological Drug Responses in 3D Organotypic Cell Cultures by Automated Image Analysis

Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate large-scale, cell-based 3D assays in basic research, drug discovery, and target validation.</p

TRIB1 constitutes a molecular link between regulation of sleep and lipid metabolism in humans

Epidemiological studies show association between sleep duration and lipid metabolism. In addition, inactivation of circadian genes induces insulin resistance and hyperlipidemia. We hypothesized that sleep length and lipid metabolism are partially controlled by the same genes. We studied the association of total sleep time (TST) with 60 genetic variants that had previously been associated with lipids. The analyses were performed in a Finnish population-based sample (N = 6334) and replicated in 2189 twins. Finally, RNA expression from mononuclear leucocytes was measured in 10 healthy volunteers before and after sleep restriction. The genetic analysis identified two variants near TRIB1 gene that independently contributed to both blood lipid levels and to TST (rs17321515, P = 8.92(*)10(-5), Bonferroni corrected P = 0.0053, β = 0.081 h per allele; rs2954029, P = 0.00025, corrected P = 0.015, β = 0.076; P<0.001 for both variants after adjusting for blood lipid levels or body mass index). The finding was replicated in the twin sample (rs17321515, P = 0.022, β = 0.063; meta-analysis of both samples P = 8.1(*)10(-6), β = 0.073). After the experimentally induced sleep restriction period TRIB1 expression increased 1.6-fold and decreased in recovery phase (P = 0.006). In addition, a negative correlation between TRIB1 expression and slow wave sleep was observed in recovery from sleep restriction. These results show that allelic variants of TRIB1 are independently involved in regulation of lipid metabolism and sleep. The findings give evidence for the pleiotropic nature of TRIB1 and may reflect the shared roots of sleep and metabolism. The shared genetic background may at least partially explain the mechanism behind the well-established connection between diseases with disrupted metabolism and sleep.Peer reviewe

La estrategia Educativa 2020 o las limitaciones del Banco Mundial para promover el "aprendizaje para todos"

La nueva Estrategia Educativa 2020 del Banco Mundial establece las prioridades de reforma educativa en paises en vias de desarrollo para la decada siguiente. El titulo explicito de la estrategia, Aprendizaje para Todos, es un claro reconocimiento de que, mas alla de politicas centradas en el acceso, se debe hacer algo mas para asegurar que la educacion derive en experiencias positivas de aprendizaje. Sin embargo, como este articulo sostiene, las opciones de politicas explicitas y latentes en la Estrategia 2020 no son las mas adecuadas para lograr el Aprendizaje para Todos. El articulo desarrolla tres tipos de argumentos al respecto. El primero se refiere al fuerte apego del Banco a un conocimiento disciplinario y un enfoque metodológico que es insufi ciente para entender lo que aprenden los niños en la escuela y por que. El segundo argumento se refiere al sesgo pro-mercado de la Estrategia por lo que respecta a la reforma del sector publico y a nuevas formas de oferta educativa. En tercer lugar, el articulo senala las principales ausencias de la Estrategia, con especial atencion a las omisiones relacionadas con la compleja relación entre educación y pobreza.The World Bank's 2020 Education Strategy establishes the new education priorities in developing countries for the next decade. Its title, Learning for All, clearly recognizes that, beyond policies focusing on access, something else must be done to ensure that schooling involves positive learning experiences. However, as this paper argues, the 2020 Strategy explicit and latent policy options might not be adequate to achieve Learning for All. This paper develops three arguments on that matter. The fi rst one refers to the Bank's strong attachment to a disciplinary knowledge and a methodological approach that do not suffi ce to understand what children learn at school and why. The second one addresses its pro-market bias when it approaches the public sector reforms and the new forms of providing education. The last argument points out the main omissions of this Strategy, especially in what regards the complex relation between education and poverty

Lanthanide-based time-resolved luminescence immunoassays

The sensitive and specific detection of analytes such as proteins in biological samples is critical for a variety of applications, for example disease diagnosis. In immunoassays a signal in response to the concentration of analyte present is generated by use of antibodies labeled with radioisotopes, luminophores, or enzymes. All immunoassays suffer to some extent from the problem of the background signal observed in the absence of analyte, which limits the sensitivity and dynamic range that can be achieved. This is especially the case for homogeneous immunoassays and surface measurements on tissue sections and membranes, which typically have a high background because of sample autofluorescence. One way of minimizing background in immunoassays involves the use of lanthanide chelate labels. Luminescent lanthanide complexes have exceedingly long-lived luminescence in comparison with conventional fluorophores, enabling the short-lived background interferences to be removed via time-gated acquisition and delivering greater assay sensitivity and a broader dynamic range. This review highlights the potential of using lanthanide luminescence to design sensitive and specific immunoassays. Techniques for labeling biomolecules with lanthanide chelate tags are discussed, with aspects of chelate design. Microtitre plate-based heterogeneous and homogeneous assays are reviewed and compared in terms of sensitivity, dynamic range, and convenience. The great potential of surface-based time-resolved imaging techniques for biomolecules on gels, membranes, and tissue sections using lanthanide tracers in proteomics applications is also emphasized

Lysophosphatidic acid and sphingosine-1-phosphate promote morphogenesis and block invasion of prostate cancer cells in three-dimensional organotypic models

Normal prostate and some malignant prostate cancer (PrCa) cell lines undergo acinar differentiation and form spheroids in three-dimensional (3-D) organotypic culture. Acini formed by PC-3 and PC-3M, less pronounced also in other PrCa cell lines, spontaneously undergo an invasive switch, leading to the disintegration of epithelial structures and the basal lamina, and formation of invadopodia. This demonstrates the highly dynamic nature of epithelial plasticity, balancing epithelial-to-mesenchymal transition against metastable acinar differentiation. This study assessed the role of lipid metabolites on epithelial maturation. PC-3 cells completely failed to form acinar structures in delipidated serum. Adding back lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) rescued acinar morphogenesis and repressed invasion effectively. Blocking LPA receptor 1 (LPAR1) functions by siRNA (small interference RNA) or the specific LPAR1 inhibitor Ki16425 promoted invasion, while silencing of other G-protein-coupled receptors responsive to LPA or S1P mainly caused growth arrest or had no effects. The G-proteins Gα12/13 and Gαi were identified as key mediators of LPA signalling via stimulation of RhoA and Rho kinases ROCK1 and 2, activating Rac1, while inhibition of adenylate cyclase and accumulation of cAMP may be secondary. Interfering with these pathways specifically impeded epithelial polarization in transformed cells. In contrast, blocking the same pathways in non-transformed, normal cells promoted differentiation. We conclude that LPA and LPAR1 effectively promote epithelial maturation and block invasion of PrCa cells in 3-D culture. The analysis of clinical transcriptome data confirmed reduced expression of LPAR1 in a subset of PrCa's. Our study demonstrates a metastasis-suppressor function for LPAR1 and Gα12/13 signalling, regulating cell motility and invasion versus epithelial maturation

Physiological and autonomic stress responses after prolonged sleep restriction and subsequent recovery sleep in healthy young men

Purpose Sleep restriction is increasingly common and associated with the development of health problems. We investigated how the neuroendocrine stress systems respond to prolonged sleep restriction and subsequent recovery sleep in healthy young men. Methods After two baseline (BL) nights of 8 h time in bed (TIB), TIB was restricted to 4 h per night for five nights (sleep restriction, SR, n = 15), followed by three recovery nights (REC) of 8 h TIB, representing a busy workweek and a recovery weekend. The control group (n = 8) had 8 h TIB throughout the experiment. A variety of autonomic cardiovascular parameters, together with salivary neuropeptide Y (NPY) and cortisol levels, were assessed. Results In the control group, none of the parameters changed. In the experimental group, heart rate increased from 60 +/- 1.8 beats per minute (bpm) at BL, to 63 +/- 1.1 bpm after SR and further to 65 +/- 1.8 bpm after REC. In addition, whole day low-frequency to-high frequency (LF/HF) power ratio of heart rate variability increased from 4.6 +/- 0.4 at BL to 6.0 +/- 0.6 after SR. Other parameters, including salivary NPY and cortisol levels, remained unaffected. Conclusions Increased heart rate and LF/HF power ratio are early signs of an increased sympathetic activity after prolonged sleep restriction. To reliably interpret the clinical significance of these early signs of physiological stress, a follow-up study would be needed to evaluate if the stress responses escalate and lead to more unfavourable reactions, such as elevated blood pressure and a subsequent elevated risk for cardiovascular health problems.Peer reviewe