An experimental approach to probe conformational changes in proteinstructure using a biotin derivative followed by mass spectrometry

The biotinylation patterns of two soluble model proteins (BSA and CA II) were analyzed under different conditions. In a first step these proteins were biotinylated with an increasing molar excess of a biotin derivative. In this case increases in biotin concentration led to an increase in the number of biotinylated lysine residues until a point of saturation was reached. A further increase in biotin concentration did not result in additional lysine residues being biotinylated. The biotinylation pattern was reproducible and under any given conditions, only specific lysine residues are biotinylated. After showing that the biotinylation of proteins is specific and reproducible, the same approach was used to map artificially induced conformation changes in BSA and CA II. These proteins were subjected to elevated temperatures that induce conformation changes. The biotinylation pattern of these proteins at room temperature and after exposure to 80°C was compared. For BSA, heating results in the biotinylation of two additional lysine residues. This is most likely due to conformation changes in the protein structure induced by the high temperature. In the case of CA II there was no difference in the biotinylation pattern of the protein at room temperature and at 80°C.Having established that biotinylation patterns can be used to reliably detect conformational changes in protein structure, the biotinylation pattern of Band III (a transmembrane protein of the human erythrocyte plasma membrane) in infected and noninfected erythrocyte was analyzed. These experiments confirmed previous observations that this protein undergoes a conformational change upon infection. The biotinylation pattern was different between RBC and IRBC in respect to a single lysine residue which is biotinylated only in RBC. These results suggest that, upon invasion by P. falciparum, the Band III protein undergoes conformation changes which are probably important for the survival of the parasite within the host cell

Editorial: Cardiotoxicity induced by radiotherapy and/or chemotherapy after cancer treatment

International audienceIt is our privilege to present 11 articles in this Frontiers Oncology Research Topic on "cardiotoxicity induced by radiotherapy and/or chemotherapy after cancer treatment". The therapeutic management of cancer has progressed over the last 10 years and has led to a significant increase in patient survival rates. Radiotherapy (RT) is classically applied in multiple fractions administered over several weeks to kill cancer while sparing normal tissue as much as possible. However, the corollary is the development of delayed toxicities that alter the quality of life of patients. Chemo- and/or RT-induced cardiovascular disease (CVD) is recognized as a worrisome side effect in patients with thoracic cancers

An eye-tracking study on how the popularity and gender of the endorsers affected the audience’s attention on the advertisement

Funding Information: This work was funded by the Ferdowsi University of Mashhad. Publisher Copyright: © 2023, The Author(s).Nowadays, advertising is regarded as one of the vital elements of marketing tools’ promotional strategies. Therefore, advertising is very important to businesses’ marketing strategies and policies. In light of this, the purpose of the current study was to ascertain the influence of endorsers’ gender and the level of attention given to various aspects of advertising. A quasi-experimental study was used for the current investigation. All students at Mashhad’s Ferdowsi University made up the study’s statistical population. 80 students were chosen as the research sample out of the entire student body. Eye motions were captured using an eye tracking gadget. According to research findings, the number and length of fixes on advertisement items were significantly influenced by the popularity of their endorsers. However, there was no statistically significant difference between the genders in terms of the quantity and length of fixations. These conclusions suggest that the popularity of the endorser is a key factor in commercials, but that the endorser’s gender has little effect.publishersversionpublishe

Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole

<p>Abstract</p> <p>Background</p> <p>The <it>Plasmodium falciparum </it>PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s) of the three different forms of the enzyme (p120, p96 and p68) are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed.</p> <p>Methods</p> <p>By using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before.</p> <p>Results</p> <p>The high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus.</p> <p>Conclusions</p> <p>The PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole <it>via </it>the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96 form. This p96 form is eventually redirected into the parasite to be converted into the processed p68 form that is only marginally delivered to the parasite food vacuole. These results provide insights on PfA-M1 topology regarding key compartments of the infected red blood cells that have important implications for the development of inhibitors targeting this plasmodial enzyme.</p

Brain Radiation Information Data Exchange (BRIDE): Integration of experimental data from low-dose ionising radiation research for pathway discovery

Background: The underlying molecular processes representing stress responses to low-dose ionising radiation (LDIR) in mammals are just beginning to be understood. In particular, LDIR effects on the brain and their possible association with neurodegenerative disease are currently being explored using omics technologies. Results: We describe a light-weight approach for the storage, analysis and distribution of relevant LDIR omics datasets. The data integration platform, called BRIDE, contains information from the literature as well as experimental information from transcriptomics and proteomics studies. It deploys a hybrid, distributed solution using both local storage and cloud technology. Conclusions: BRIDE can act as a knowledge broker for LDIR researchers, to facilitate molecular research on the systems biology of LDIR response in mammals. Its flexible design can capture a range of experimental information for genomics, epigenomics, transcriptomics, and proteomics. The data collection is available at:

Nuclear Fragility in Radiation-Induced Senescence: Blebs and Tubes Visualized by 3D Electron Microscopy

Irreparable DNA damage following ionizing radiation (IR) triggers prolonged DNA dam age response and induces premature senescence. Cellular senescence is a permanent state of cell-cycle arrest characterized by chromatin restructuring, altered nuclear morphology and acquisition of secretory phenotype, which contributes to senescence-related inflammation. However, the mech anistic connections for radiation-induced DNA damage that trigger these senescence-associated hallmarks are poorly understood. In our in vitro model of radiation-induced senescence, mass spectrometry-based proteomics was combined with high-resolution imaging techniques to investi gate the interrelations between altered chromatin compaction, nuclear envelope destabilization and nucleo-cytoplasmic chromatin blebbing. Our findings confirm the general pathophysiology of the senescence-response, with disruption of nuclear lamin organization leading to extensive chromatin restructuring and destabilization of the nuclear membrane with release of chromatin fragments into the cytosol, thereby activating cGAS-STING-dependent interferon signaling. By serial block-face scanning electron microscopy (SBF-SEM) whole-cell datasets were acquired to investigate the mor phological organization of senescent fibroblasts. High-resolution 3-dimensional (3D) reconstruction of the complex nuclear shape allows us to precisely visualize the segregation of nuclear blebs from the main nucleus and their fusion with lysosomes. By multi-view 3D electron microscopy, we identified nanotubular channels formed in lamin-perturbed nuclei of senescent fibroblasts; the potential role of these nucleo-cytoplasmic nanotubes for expulsion of damaged chromatin has to be examined

Radiation–Induced Signaling Results in Mitochondrial Impairment in Mouse Heart at 4 Weeks after Exposure to X-Rays

BACKGROUND: Radiation therapy treatment of breast cancer, Hodgkin's disease or childhood cancers expose the heart to high local radiation doses, causing an increased risk of cardiovascular disease in the survivors decades after the treatment. The mechanisms that underlie the radiation damage remain poorly understood so far. Previous data show that impairment of mitochondrial oxidative metabolism is directly linked to the development of cardiovascular disease. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the radiation-induced in vivo effects on cardiac mitochondrial proteome and function were investigated. C57BL/6N mice were exposed to local irradiation of the heart with doses of 0.2 Gy or 2 Gy (X-ray, 200 kV) at the age of eight weeks, the control mice were sham-irradiated. After four weeks the cardiac mitochondria were isolated and tested for proteomic and functional alterations. Two complementary proteomics approaches using both peptide and protein quantification strategies showed radiation-induced deregulation of 25 proteins in total. Three main biological categories were affected: the oxidative phophorylation, the pyruvate metabolism, and the cytoskeletal structure. The mitochondria exposed to high-dose irradiation showed functional impairment reflected as partial deactivation of Complex I (32%) and Complex III (11%), decreased succinate-driven respiratory capacity (13%), increased level of reactive oxygen species and enhanced oxidation of mitochondrial proteins. The changes in the pyruvate metabolism and structural proteins were seen with both low and high radiation doses. CONCLUSION/SIGNIFICANCE: This is the first study showing the biological alterations in the murine heart mitochondria several weeks after the exposure to low- and high-dose of ionizing radiation. Our results show that doses, equivalent to a single dose in radiotherapy, cause long-lasting changes in mitochondrial oxidative metabolism and mitochondria-associated cytoskeleton. This prompts us to propose that these first pathological changes lead to an increased risk of cardiovascular disease after radiation exposure

Radiation Adverse Outcome pathways (AOPs): examining priority questions from an international horizon-style exercise

Purpose: The Organisation for Economic Co-operation and Development (OECD) Adverse Outcome Pathway (AOP) Development Programme is being explored in the radiation field, as an overarching framework to identify and prioritize research needs that best support strengthening of radiation risk assessment and risk management strategies. To advance the use of AOPs, an international horizon-style exercise (HSE) was initiated through the Radiation/Chemical AOP Joint Topical Group (JTG) formed by the OECD Nuclear Energy Agency (NEA) High-Level Group on Low Dose Research (HLG-LDR) under the auspices of the Committee on Radiological Protection and Public Health (CRPPH). The intent of the HSE was to identify key research questions for consideration in AOP development that would help to reduce uncertainties in estimating the health risks following exposures to low dose and low dose-rate ionizing radiation. The HSE was conducted in several phases involving the solicitation of relevant questions, a collaborative review of open-ended candidate questions and an elimination exercise that led to the selection of 25 highest priority questions for the stated purpose. These questions were further ranked by over 100 respondents through an international survey. This final set of questions was judged to provide insights into how the OECD’s AOP approach can be put into practice to meet the needs of hazard and risk assessors, regulators, and researchers. This paper examines the 25 priority questions in the context of hazard/risk assessment framework for ionizing radiation. Conclusion: By addressing the 25 priority questions, it is anticipated that constructed AOPs will have a high level of specificity, making them valuable tools for simplifying and prioritizing complex biological processes for use in developing revised radiation hazard and risk assessment strategies.publishedVersio

Experimenteller Ansatz zur Untersuchung von Konformationsänderungen in Proteinen mit Hilfe von Biotinderivaten und Massenspektrometrie

The biotinylation patterns of two soluble model proteins (BSA and CA II) were analyzed under different conditions. In a first step these proteins were biotinylated with an increasing molar excess of a biotin derivative. In this case increases in biotin concentration led to an increase in the number of biotinylated lysine residues until a point of saturation was reached. A further increase in biotin concentration did not result in additional lysine residues being biotinylated. The biotinylation pattern was reproducible and under any given conditions, only specific lysine residues are biotinylated. After showing that the biotinylation of proteins is specific and reproducible, the same approach was used to map artificially induced conformation changes in BSA and CA II. These proteins were subjected to elevated temperatures that induce conformation changes. The biotinylation pattern of these proteins at room temperature and after exposure to 80°C was compared. For BSA, heating results in the biotinylation of two additional lysine residues. This is most likely due to conformation changes in the protein structure induced by the high temperature. In the case of CA II there was no difference in the biotinylation pattern of the protein at room temperature and at 80°C.Having established that biotinylation patterns can be used to reliably detect conformational changes in protein structure, the biotinylation pattern of Band III (a transmembrane protein of the human erythrocyte plasma membrane) in infected and noninfected erythrocyte was analyzed. These experiments confirmed previous observations that this protein undergoes a conformational change upon infection. The biotinylation pattern was different between RBC and IRBC in respect to a single lysine residue which is biotinylated only in RBC. These results suggest that, upon invasion by P. falciparum, the Band III protein undergoes conformation changes which are probably important for the survival of the parasite within the host cell.Das Biotinylierungsmuster zweier löslicher Proteine (BSA und CA II), sowie das des Erythrozytenmembranproteins Bande III wurde unter unterschiedlichen Bedingungen analysiert. Im ersten Schritt wurden diese Proteine mit einem Überschuss an Biotin in einem molaren Verhältnis biotinyliert. Für die löslichen Proteine führt eine Erhöhung der Biotinkonzentration zu einer Zunahme der Anzahl an biotinylierten Lysinresten bis zu einem Punkt der Sättigung. Eine weitere Erhöhung der Biotinkonzentration führt nicht zu zusätzlich mit Biotin markierten Lysinresten. Das Biotinylierungsmuster dieser beiden löslichen Proteine ist reproduzierbar und zeigt unter allen gewählten Bedingungen eine Biotinylierung spezifischer Lysinreste. Nachdem gezeigt war, dass die Biotinylierung der Proteine spezifisch und reproduzierbar ist, wurde die gleiche Methode gewählt, um künstlich verursachte Konformationsänderungen in BSA und CA II anhand eines veränderten Biotinylierungsmusters zu zeigen. Diese Proteine wurden erhöhten Temperaturen ausgesetzt, um eine Konformationsänderung hervorzurufen, und anschliessend wurden die Biotinylierungsmuster bei Raumtemperatur und 80 °C miteinander verglichen. Bei BSA führt die Erhitzung auf 80 °C zu einer Biotinylierung von zwei zusätzlichen Lysinresten. Dabei ist es wahrscheinlich, dass die durch die Erwärmung verursachte Konformationsänderung die beobachtete Änderung des Biotinylierungsmusters zur Folge hat. Im Fall von CA II gibt es keinen Unterschied das Biotinylierungsmusters zwischen Raumtemperatur und 80 °C. Da es Hinweise dafür gibt, dass sich die Konformation des Bande III Proteins in der Plasmamembran von Erythrozyten nach einer Infektion mit P. falciparum verändert, wurden intakte infizierte und nichtinfizierte Erythrozyten biotinyliert, um etwaige Unterschiede in den Biotinylierungsmustern festzustellen. Das Biotinylierungsmuster des Proteins unterschied sich zwischen RBC und IRBC nur in einem einzelnen Lysinrest, der nur in RBC biotinyliert ist. Diese Beobachtung bestätigt die Annahme, dass Bande III nach der Infektion durch P. falciparum eine Konformationsänderung erfährt und erlaubt es, die Position dieser Veränderung einzugrenzen

Integrative proteomics and targeted transcriptomics analyses in cardiac endothelial cells unravel mechanisms of long-term radiation-induced vascular dysfunction.

Epidemiological data from radiotherapy patients show the damaging effect of ionizing radiation on heart and vasculature. The endothelium is the main target of radiation damage and contributes essentially to the development of cardiac injury. However, the molecular mechanisms behind the radiation-induced endothelial dysfunction are not fully understood. In the present study, 10-week-old C57Bl/6 mice received local X-ray heart doses of 8 or 16 Gy and were sacrificed after 16 weeks; the controls were sham-irradiated. The cardiac microvascular endothelial cells were isolated from the heart tissue using streptavidin-CD31-coated microbeads. The cells were lysed and proteins were labeled with duplex isotope-coded protein label methodology for quantification. All samples were analyzed by LC-ESI-MS/MS and Proteome Discoverer software. The proteomics data were further studied by bioinformatics tools and validated by targeted transcriptomics, immunoblotting, immunohistochemistry, and serum profiling. Radiation-induced endothelial dysfunction was characterized by impaired energy metabolism and perturbation of the insulin/IGF-PI3K-Akt signaling pathway. The data also strongly suggested premature endothelial senescence, increased oxidative stress, decreased NO availability, and enhanced inflammation as main causes of radiation-induced long-term vascular dysfunction. Detailed data on molecular mechanisms of radiation-induced vascular injury as compiled here are essential in developing radiotherapy strategies that minimize cardiovascular complications