Development of a new method for the detection of vanadium complexes bound to DNA, using Agarose Gel Electrophoresis

Dissertação de mest., Qualidade em Análises, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2012Existem apenas alguns métodos disponíveis para o estudo da ligação de metais ao ADN. Estes são baseados em técnicas espectroscópicas, que podem apenas ser utilizadas quando determinados cromóforos quer da molécula de ADN ou dos complexos metálicos estão directamente envolvidos na ligação de metais ao ADN. O objectivo deste projecto foi desenvolver um novo método que pode ser utilizado para detectar a ligação de um metal de transição ao ADN, utilizando Electroforese em gel de agarose (EGA). O método tem sido estudado para complexos de vanádio, mas pode ser adaptado para quaisquer metais de transição susceptíveis de sofrer reacções de tipo Fenton. O novo método envolve a adição ao gel de agarose de ácido tereftálico (TPA), um indicador bem conhecido para os radicais hidroxilo. Embebendo o gel desenvolvido em peróxido de hidrogénio, a presença de vanádio será revelada por uma reacção do tipo Fenton que irá gerar radicais hidroxilo e irá hidroxilar TPA em 2-hidroxi-TPA, uma molécula altamente fluorescente. Bandas electroforéticas contendo vanádio podem então ser fotografadas e analisadas utilizando um sistema de imagem. A metodologia envolveu quatro passos: 1. Um estudo preliminar do sistema utilizando a EGA. 2. O desenvolvimento do método para a quantificação dos complexos de vanádio baseado no mesmo sistema de reacção utilizando amostras aquosas. 3. Desenvolvimento do método em soluções aquosas com uma matriz semelhante à de agarose. 4. Aplicação em géis de agarose. Os resultados preliminares foram mal sucedidos para detectar a presença de vanádio em géis de agarose. Concluiu-se que para ser usado um sistema de imagem, o método requer a utilização de um filtro (464 ± 40) nm. Foram efectuados mais testes utilizando um espectrofluorímetro. Quando se utilizou um concentrado peroxide (~ 9M), a intensidade máxima de fluorescência de 2-OH-TPA formado diminuiu ao longo do tempo. Com um peróxido menos concentrado (1,8 mM) foi obtida uma boa sensibilidade. O LOD (limite de detecção) determinado para o vanádio foi de 4,1 μM. O sistema foi depois testado num sistema aquoso com galactose. O sistema tem uma boa gama de trabalho, até 300 μM na presença de 0,50% de galactose. A sensibilidade diminuiu significativamente na presença de galactose. O LOD determinado para o vanádio foi de 5,4 μM. O sistema foi finalmente testado em géis de agarose. Houve alguma dificuldade em assegurar a homogeneidade da preparação, necessária para um estudo espectrofluorimétrico. Num gel onde a homogeneidade foi conseguida, foi observada uma banda de emissão muito fraca na região do espectro onde o complexo 2-hidroxi-TPA emite. A sensibilidade obtida nesta preparação foi muito baixa. Não foi possível completar os testes em EGA devido à falta de tempo disponível para este trabalho. No entanto, os resultados em solução aquosa mostram que o método é viável e pode proporcionar uma boa sensibilidade quando aplicado em matrizes EGA, desde que o método para a preparação de gel seja optimizado

Micro-RNA and Proteomic Profiles of Plasma-Derived Exosomes from Irradiated Mice Reveal Molecular Changes Preventing Apoptosis in Neonatal Cerebellum

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury

Lysine 53 Acetylation of Cytochrome c in Prostate Cancer: Warburg Metabolism and Evasion of Apoptosis

Prostate cancer is the second leading cause of cancer-related death in men. Two classic cancer hallmarks are a metabolic switch from oxidative phosphorylation (OxPhos) to glycolysis, known as the Warburg effect, and resistance to cell death. Cytochrome c (Cytc) is at the intersection of both pathways, as it is essential for electron transport in mitochondrial respiration and a trigger of intrinsic apoptosis when released from the mitochondria. However, its functional role in cancer has never been studied. Our data show that Cytc is acetylated on lysine 53 in both androgen hormone-resistant and -sensitive human prostate cancer xenografts. To characterize the functional effects of K53 modification in vitro, K53 was mutated to acetylmimetic glutamine (K53Q), and to arginine (K53R) and isoleucine (K53I) as controls. Cytochrome c oxidase (COX) activity analyzed with purified Cytc variants showed reduced oxygen consumption with acetylmimetic Cytc compared to the non-acetylated Cytc (WT), supporting the Warburg effect. In contrast to WT, K53Q Cytc had significantly lower caspase-3 activity, suggesting that modification of Cytc K53 helps cancer cells evade apoptosis. Cardiolipin peroxidase activity, which is another proapoptotic function of the protein, was lower in acetylmimetic Cytc. Acetylmimetic Cytc also had a higher capacity to scavenge reactive oxygen species (ROS), another pro-survival feature. We discuss our experimental results in light of structural features of K53Q Cytc, which we crystallized at a resolution of 1.31 Å, together with molecular dynamics simulations. In conclusion, we propose that K53 acetylation of Cytc affects two hallmarks of cancer by regulating respiration and apoptosis in prostate cancer xenografts

Use of molecularly imprinted polymers for enrichment of phosphopeptides and alpha-Synuclein, and the mass spectrometric analyses of alpha-Synuclein in post−mortem\it post-mortem human brains

Alpha-Synuclein ($\alpha$Syn) ist ein putativer Biomarker der Parkinson-Krankheit (PK). Aggregierte $\alpha$Syn werden als Lewy-Körperchen (LK) bezeichnet und sind in der Gehirnregion $\textit {substancia nigra}$ ein Kennzeichen für die PK. Phosphoryliertes $\alpha$Syn ist die häufigste Form in LK. Die Konzentration von $\alpha$Syn in Körperflüssigkeiten wie z.B. in der Cerebrospinalflüssigkeit ist sehr gering und liegt unter der Nachweisgrenze für die Massenspektrometrie (MS)-analyse. In dieser Arbeit wurden molekular geprägte Polymere (MIPs) für die Anreicherung von $\alpha$Syn und anderen Phosphopeptiden verwendet. Mit Hilfe der MIPs konnten sowohl $\alpha$Syn aus Human Embryonic Kidney (HEK)- als auch Phosphopeptide aus dem HeLa-Zelllysat angereichert werden. Außerdem wurde die MS-basierte Methode $\textit {parallel reaction monitoring}$ zur Quantifizierung der $\alpha$Syn-Konzentration in humanen Gehirn erfolgreich etabliert. Mit Hilfe der neu etablierten Methode konnte der Massenanteil von $\alpha$Syn im humanen Gehirn auf 0,3% bestimmt werden

Omics in Radiation Biology: Surprised but Not Disappointed

High-throughput omics platforms have pioneered our approach to understanding biological and cellular processes. Omics technologies provide powerful tools for studying various molecules, such as genes, proteins, and metabolites, in a particular state and at a particular time. Although omics has had a presence in the radiation community for more than 3 decades, the use of it is still in its infancy. Omics studies enable radiation researchers to understand the molecular mechanism underlying the biological effects of radiation exposure on normal and cancerous tissues, and to answer critical questions such as individual sensitivity, risk assessment, and biomarker discovery. In this commentary, we take a look back at the omics studies that have been conducted in radiation research in the last 20 years and discuss whether omics has fulfilled expectations by examining the knowledge and research gaps in radiation omics

Ionizing Radiation Protein Biomarkers in Normal Tissue and Their Correlation to Radiosensitivity: A Systematic Review

Background and objectives: Exposure to ionizing radiation (IR) has increased immensely over the past years, owing to diagnostic and therapeutic reasons. However, certain radiosensitive individuals show toxic enhanced reaction to IR, and it is necessary to specifically protect them from unwanted exposure. Although predicting radiosensitivity is the way forward in the field of personalised medicine, there is limited information on the potential biomarkers. The aim of this systematic review is to identify evidence from a range of literature in order to present the status quo of our knowledge of IR-induced changes in protein expression in normal tissues, which can be correlated to radiosensitivity. Methods: Studies were searched in NCBI Pubmed and in ISI Web of Science databases and field experts were consulted for relevant studies. Primary peer-reviewed studies in English language within the time-frame of 2011 to 2020 were considered. Human non-tumour tissues and human-derived non-tumour model systems that have been exposed to IR were considered if they reported changes in protein levels, which could be correlated to radiosensitivity. At least two reviewers screened the titles, keywords, and abstracts of the studies against the eligibility criteria at the first phase and full texts of potential studies at the second phase. Similarly, at least two reviewers manually extracted the data and accessed the risk of bias (National Toxicology Program/Office for Health Assessment and Translation—NTP/OHAT) for the included studies. Finally, the data were synthesised narratively in accordance to synthesis without meta analyses (SWiM) method. Results: In total, 28 studies were included in this review. Most of the records (16) demonstrated increased residual DNA damage in radiosensitive individuals compared to normo-sensitive individuals based on γH2AX and TP53BP1. Overall, 15 studies included proteins other than DNA repair foci, of which five proteins were selected, Vascular endothelial growth factor (VEGF), Caspase 3, p16INK4A (Cyclin-dependent kinase inhibitor 2A, CDKN2A), Interleukin-6, and Interleukin-1β, that were connected to radiosensitivity in normal tissue and were reported at least in two independent studies. Conclusions and implication of key findings: A majority of studies used repair foci as a tool to predict radiosensitivity. However, its correlation to outcome parameters such as repair deficient cell lines and patients, as well as an association to moderate and severe clinical radiation reactions, still remain contradictory. When IR-induced proteins reported in at least two studies were considered, a protein network was discovered, which provides a direction for further studies to elucidate the mechanisms of radiosensitivity. Although the identification of only a few of the commonly reported proteins might raise a concern, this could be because (i) our eligibility criteria were strict and (ii) radiosensitivity is influenced by multiple factors. Registration: PROSPERO (CRD42020220064)

Molecularly imprinted polymers synthesized via template immobilization on fumed silica nanoparticles for the enrichment of phosphopeptides

Phosphorylation is a protein post-translational modification (PTM) that plays an important role in cell signaling, cell differentiation, and metabolism. The hyper phosphorylated forms of certain proteins have been appointed as biomarkers for neurodegenerative diseases, and phosphorylation-related mutations are important for detecting cancer pathways. Due to the low abundance of phosphorylated proteins in biological fluids, sample enrichment is beneficial prior to detection. Thus, a need to find new strategies for enriching phosphopeptides has emerged. Molecularly imprinted polymers (MIPs) are synthetic polymeric materials manufactured to exhibit affinity for a target molecule. In this study, MIPs have been synthesized using a new approach based on the use of fumed silica as sacrificial support acting as solid porogen with the template (phosphotyrosine) immobilized on its surface. Phosphotyrosine MIPs were tested against a mixture of peptides and phosphopeptides by performing micro-solid phase extraction using MIPs (μMISPE) packed in a pipette tip. First, the capability of the materials to preferentially enrich phosphopeptides was evaluated. In a next step, the enrichment of phosphopeptides from a whole-cell lysate of human embryonic kidney (HEK) 293T cells was performed. The eluates were analyzed using MALDI-MS in the first case and with nano-HPLC-ESI-MS/MS in the second case. The results showed that the MIPs provided affinity for phosphopeptides, binding preferentially to multi-site phosphorylated peptides. The MIPs could enrich phosphopeptides in over 10-fold compared with the number of phosphopeptides found in a cell lysate without enrichment

Validation of Molecularly Imprinted Polymers for side chain selective phosphopeptide enrichment

Selective enrichment techniques are essential for mapping of protein posttranslational modifications (PTMs). Phosphorylation is one of the PTMs which continues to be associated with significant analytical challenges. Particularly problematic are tyrosine-phosphorylated peptides (pY-peptides) resulting from tryptic digestion which commonly escape current chemo- or immuno- affinity enrichments and hence remain undetected. We here report on significant improvements in this regard using pY selective molecularly imprinted polymers (pY-MIPs). The pY-MIP was compared with titanium dioxide (TiO2) affinity based enrichment and immunoprecipitation (IP) with respect to selective enrichment from a mixture of 13 standard peptides at different sample loads. At a low sample load (1 pmol of each peptide), IP resulted in enrichment of only a triply phosphorylated peptide whereas TiO2 enriched phosphopeptides irrespective of the amino acid side chain. However, with increased sample complexity, TiO2 failed to enrich the doubly phosphorylated peptides. This contrasted with the pY-MIP showing enrichment of all four tyrosine phosphorylated peptides at 1 pmol sample load of each peptide with a few other peptides binding unselectively. At an increased sample complexity consisting of the standard peptides spiked into mouse brain digest, the MIP showed clear enrichment of all four pYpeptides

Nanoplasmonic modification of the local morphology, shape, and wetting properties of nanoflake microparticles

Inducing a phase transition of a self-organized object may trigger its structural transformation. Here, we demonstrate local control of the morphology and shape of selforganized microparticles with a nanoflake outer surface by nanoplasmonic heating. To increase the photothermal efficiency of the microparticles, gold nanoparticles (AuNPs) or single-walled carbon nanotubes (SWCNTs) were incorporated. AuNPs and SWCNTs, which have excellent photothermal activity, acts as photoresponsive heat converters. Because they have distinct absorption characteristics, visible or nearinfrared lasers can be used to induce local heating. The photothermal effect was used to spatially confine the melting to the space within the particle and the aggregate; as a result, microparticles with various shapes and morphologies have been fabricated. Such morphological changes lead to a uperhydrophobic−hydrophobic wetting transition, which was confirmed by the films constituting the microparticles. The work presented is seen useful for anisotropic particle synthesis, local wetting control, lithography, and morphological control of functional materials

Anisotropic multicompartment micro- and nano-capsules produced via embedding into biocompatible PLL/HA films

We present a novel strategy to fabricate anisotropic multi-compartment Janus capsules by embedding larger containers into a soft poly-L-lysine/hyaluronic acid (PLL/HA) polymeric film, followed by adsorption of smaller containers on top of their unmasked surface. This research is also attractive for developing substrates for cell cultures