EVALUATION OF THE ALAMARBLUE ASSAY FOR ADHERENT CELL IRRADIATION EXPERIMENTS

The AlamarBlue assay is based on fluorometric detection of metabolic mitochondrial activity of cells. In this study, we determined the methodology for application of the assay to radiation response experiments in 96-well plates. AlamarBlue was added and its reduction measured 7 hours later. Selection of the initial number of plated cells was important so that the number of proliferating cells remains lower than the critical number that produced full AlamarBlue reduction (plateau phase) at the time points of measurements. Culture medium was replaced twice a week to avoid suppression of viability due to nutrient competition and metabolic waste accumulation. There was no need to replace culture medium before adding AlamarBlue. Cell proliferation continued after irradiation and the suppression effect on cell viability was most evident on day 8. At this time point, by comparing measurements from irradiated vs. non-irradiated cells, for various dose levels, a viability dose response curve was plotted. Immediately after the 8th day (nadir), cells started to re-grow at a rate inversely related to the radiation dose. By comparing measurements at the time point of nadir vs. a convenient subsequent time point, re-growth dose response abilities were plotted, simulating clonogenic assays

Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform

The dynamics and coordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required the earliest, followed by auto-phosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps, whereas ULK1 and ULK2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way, suggesting multiple initiation events. Targeted ubiquitinated mitochondria are cradled by endoplasmic reticulum (ER) strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands, providing platforms for formation of the mitophagosomes

Cholera- and Anthrax-Like Toxins Are among Several New ADP-Ribosyltransferases

Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins we identified and characterized using in silico and cell-based techniques. We also uncovered medically relevant toxins from Mycobacterium avium and Enterococcus faecalis. We found agriculturally relevant toxins in Photorhabdus luminescens and Vibrio splendidus. These toxins belong to the ADP-ribosyltransferase family that has conserved structure despite low sequence identity. Therefore, our search for new toxins combined fold recognition with rules for filtering sequences – including a primary sequence pattern – to reduce reliance on sequence identity and identify toxins using structure. We used computers to build models and analyzed each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. We confirmed activity using a yeast growth test. In this era where an expanding protein structure library complements abundant protein sequence data – and we need high-throughput validation – our approach provides insight into the newest toxin ADP-ribosyltransferases

The EU territorial cohesion discourse and the spatial planning system in Greece

This paper argues the changing nature of spatial planning in the recent decades, under the light of the relevant progressions that have been taking place at the EU level. It is suggested that the EU territorial cohesion discourse and its relevant -although less developed- policy tools are in line with and promote a new orientation of spatial planning that is characterized of an integrated approach which is spatially aware and aims to guide development in order to avoid or minimize spatial imbalances and to increase the effectiveness of policies according to the territorial characteristics. This is an attempt to bring into light the new dimensions of spatial planning through the analysis of the relevant European concepts. In the first part, the developments at the EU level and their contribution to this new approach of planning are analysed. Following that, what is examined is how these developments and the EU territorial cohesion perspective influenced the Greek planning system and, more specifically, how the new traits of planning have been integrated and the degree to which the new approach is reflected. © 2019, © 2019 Informa UK Limited, trading as Taylor & Francis Group

Oxidized LDL, serum oxidizability and serum lipid levels in patients with breast or ovarian cancer

Objectives: The aim of this study was to evaluate the extent of oxidative stress in patients with breast or ovarian cancer by analyzing the magnitude of serum oxidizability and the involvement of oxidized low-density lipoproteins (oxLDL) in the disease. Design and methods: The study was conducted on 32 patients diagnosed with breast or ovarian cancer but who had not undergone any kind of treatment and 30 healthy individuals of similar age. The evaluation of oxidative stress was assessed by: (a) the ex-vivo susceptibility of serum lipids to oxidation and (b) the detection of oxLDL and anti-oxLDL autoantibodies. Total cholesterol, LDL-cholesterol and HDL-cholesterol were co-estimated. Results: The results indicated that the levels of oxLDL were increased among both breast and ovarian cancer patients as compared to the control subjects. Additionally in patients with breast cancer, serum total cholesterol, LDL-cholesterol, anti-oxLDL antibodies and the maximal rate of diene formation (RA), the index of oxidizable components load, were increased in comparison to controls. There is statistically significant evidence that serum oxLDL levels are associated with increased risk of breast and ovarian cancer. Conclusions: The findings exhibit a correlation between oxLDL and malignancy, supporting the contribution of oxidative stress to carcinogenesis and the possible involvement of oxLDL in the process of malignancy. The clinical evaluation of the oxLDL measurement is under investigation. © 2007 The Canadian Society of Clinical Chemists

Rancang Bangun Battery Charger Cc-cv Dengan Forward Converter

Perkembangan pada bidang transportasi darat saat ini mengalami peningkatan yang drastis. Salah satu perkembangan yang diutamakan saat ini adalah mobil listrik. Pada mobil listrik sumber energi yang dibutuhkan yaitu baterai. Baterai merupakan salah satu komponen yang sangat penting. Baterai mampu menyimpan energi sebagai sumber daya suatu alat dan energi tersebut dapat diisi kembali apabila telah habis, sehingga diperlukan perawatan untuk menjaga kondisi baterai agar tetap baik. Kinerja baterai dengan kondisi yang baik merupakan sumber daya utama pada mobil listrik. Semakin baik kinerja baterai, maka kinerja mobil listrik akan semakin baik juga. Metode pengisian ulang daya pada baterai menjadi hal utama dalam membangun sebuah sistem electrical vehicle, sehingga perlu adanya sebuah sistem pengisian yang dapat mengisi baterai yang aman bagi baterai untuk mempertahankan masa kinerja baterai