Creatine Deficiency Syndromes: A Clinical, Molecular and Functional Approach

Salomons, G.S. [Promotor]Jakobs, C.A.J.M. [Promotor]Martinez Munoz, C. [Copromotor

Profiling Non-Coding RNA Changes Associated with 16 Different Engineered Nanomaterials in a Mouse Airway Exposure Model

Perturbations in cellular molecular events and their associated biological processes provide opportunities for hazard assessment based on toxicogenomic profiling. Long non-coding RNAs (lncRNAs) are transcribed from DNA but are typically not translated into full-length proteins. Via epigenetic regulation, they play important roles in organismal response to environmental stress. The effects of nanoparticles on this important part of the epigenome are understudied. In this study, we investigated changes in lncRNA associated with hazardous inhalatory exposure of mice to 16 engineered nanomaterials (ENM)-4 ENM (copper oxide, multi-walled carbon nanotubes, spherical titanium dioxide, and rod-like titanium dioxide particles) with 4 different surface chemistries (pristine, COOH, NH2, and PEG). Mice were exposed to 10 mu g of ENM by oropharyngeal aspiration for 4 consecutive days, followed by cytological analyses and transcriptomic characterization of whole lung tissues. The number of significantly altered non-coding RNA transcripts, suggestive of their degrees of toxicity, was different for each ENM type. Particle surface chemistry and shape also had varying effects on lncRNA expression. NH2 and PEG caused the strongest and weakest responses, respectively. Via correlational analyses to mRNA expression from the same samples, we could deduce that significantly altered lncRNAs are potential regulators of genes involved in mitotic cell division and DNA damage response. This study sheds more light on epigenetic mechanisms of ENM toxicity and also emphasizes the importance of the lncRNA superfamily as toxicogenomic markers of adverse ENM exposure.Peer reviewe

Elucidating differential nano-bio interactions of multi-walled andsingle-walled carbon nanotubes using subcellular proteomics

Understanding the relationship between adverse exposure events and specific material properties will facilitate predictive classification of carbon nanotubes (CNTs) according to their mechanisms of action, and a safe-by-design approach for the next generation of CNTs. Mass-spectrometry-based proteomics is a reliable tool to uncover the molecular dynamics of hazardous exposures, yet challenges persist with regards to its limited dynamic range when sampling whole organisms, tissues or cell lysates. Here, the simplicity of the sub-cellular proteome was harnessed to unravel distinctive adverse exposure outcomes at the molecular level, between two CNT subtypes. A549, MRC9 and human macrophage cells, were exposed for 24h to non-cytotoxic doses of single-walled or multi-walled CNTs (swCNTs or mwCNTs). Label-free proteomics on enriched cytoplasmic fractions was complemented with analyses of reactive oxygen species (ROS) production and mitochondrial integrity. The extent/number of modulated proteoforms indicated the single-walled variant was more bioactive. Greater enrichment of pathways corresponding to oxido-reductive activity was consistent with greater intracellular ROS induction and mitochondrial dysfunction capacities of swCNTs. Other compromised cellular functions, as revealed by pathway analysis were; ribosome, spliceosome and DNA repair. Highly upregulated proteins (fold change in abundance >6) such as, APOC3, RBP4 and INS are also highlighted as potential markers of hazardous CNT exposure. We conclude that, changes in cytosolic proteome abundance resulting from nano-bio interactions, elucidate adverse response pathways and their distinctive molecular components. Our results indicate that CNT-protein interactions might have a thus far unappreciated significance for protein trafficking, and this warrants further investigation.Peer reviewe

Profiling Non-Coding RNA Changes Associated with 16 Different Engineered Nanomaterials in a Mouse Airway Exposure Model

Perturbations in cellular molecular events and their associated biological processes provide opportunities for hazard assessment based on toxicogenomic profiling. Long non-coding RNAs (lncRNAs) are transcribed from DNA but are typically not translated into full-length proteins. Via epigenetic regulation, they play important roles in organismal response to environmental stress. The effects of nanoparticles on this important part of the epigenome are understudied. In this study, we investigated changes in lncRNA associated with hazardous inhalatory exposure of mice to 16 engineered nanomaterials (ENM)–4 ENM (copper oxide, multi-walled carbon nanotubes, spherical titanium dioxide, and rod-like titanium dioxide particles) with 4 different surface chemistries (pristine, COOH, NH2, and PEG). Mice were exposed to 10 µg of ENM by oropharyngeal aspiration for 4 consecutive days, followed by cytological analyses and transcriptomic characterization of whole lung tissues. The number of significantly altered non-coding RNA transcripts, suggestive of their degrees of toxicity, was different for each ENM type. Particle surface chemistry and shape also had varying effects on lncRNA expression. NH2 and PEG caused the strongest and weakest responses, respectively. Via correlational analyses to mRNA expression from the same samples, we could deduce that significantly altered lncRNAs are potential regulators of genes involved in mitotic cell division and DNA damage response. This study sheds more light on epigenetic mechanisms of ENM toxicity and also emphasizes the importance of the lncRNA superfamily as toxicogenomic markers of adverse ENM exposure

Profiling Non-Coding RNA Changes Associated with 16 Different Engineered Nanomaterials in a Mouse Airway Exposure Model

Perturbations in cellular molecular events and their associated biological processes provide opportunities for hazard assessment based on toxicogenomic profiling. Long non-coding RNAs (lncRNAs) are transcribed from DNA but are typically not translated into full-length proteins. Via epigenetic regulation, they play important roles in organismal response to environmental stress. The effects of nanoparticles on this important part of the epigenome are understudied. In this study, we investigated changes in lncRNA associated with hazardous inhalatory exposure of mice to 16 engineered nanomaterials (ENM)–4 ENM (copper oxide, multi-walled carbon nanotubes, spherical titanium dioxide, and rod-like titanium dioxide particles) with 4 different surface chemistries (pristine, COOH, NH2, and PEG). Mice were exposed to 10 µg of ENM by oropharyngeal aspiration for 4 consecutive days, followed by cytological analyses and transcriptomic characterization of whole lung tissues. The number of significantly altered non-coding RNA transcripts, suggestive of their degrees of toxicity, was different for each ENM type. Particle surface chemistry and shape also had varying effects on lncRNA expression. NH2 and PEG caused the strongest and weakest responses, respectively. Via correlational analyses to mRNA expression from the same samples, we could deduce that significantly altered lncRNAs are potential regulators of genes involved in mitotic cell division and DNA damage response. This study sheds more light on epigenetic mechanisms of ENM toxicity and also emphasizes the importance of the lncRNA superfamily as toxicogenomic markers of adverse ENM exposure

Molecular insights into a putative potyvirus RNA encapsidation pathway and potyvirus particles as enzyme nano-carriers

The present study intended to identify new strategies for the selective presentation of biocatalyst on the surface of viral nanoparticles with potential application in biosensor technology or protein chips. Potyviruses were chosen as model nano-scaffolds for biocatalysts. Potyviruses are the largest genus in the family Potyviridae and cause significant plant damage. They form flexible rod-shaped capsids surrounding a single stranded positive sense RNA molecule. The molecular events leading to the specific selection and encapsidation of potyviral RNA are unknown. To better exploit the potential of these viruses as nano-carriers, the first step in this study was to look into their in vivo RNA encapsidation process. Earlier studies showed that Potato virus A (PVA) coat protein (CP) interferes with viral RNA translation when provided in excess in trans and it was suggested this could occur to initiate viral RNA encapsidation. In this follow up study, we used the ago-infiltration approach for the transient expression of full length, truncated or mutated viral RNAs with wild type CP (CPwt) and showed that this inhibition is mediated by co-translational CP-CP interactions occurring between two CP populations, produced in trans and in cis. Because CP inhibited translation of the entire viral genome and virus particles were formed (which were less abundant and appeared latter than during normal infection), it was assumed that the CP acted during this inhibition process to specifically recruit viral RNA for encapsidation. In line with previously published in vitro assembly studies, we propose a mechanism through which viral RNA encapsidation is initiated through co-translational CP-CP interactions. The second part of this work entailed the investigation of novel approaches for organizing biocatalyst on virus platforms. The aim was to be able to control the display of enzymes on virus surfaces while maximizing channelling of reaction intermediates. Three strategies were investigated: the first involved the use of specific PVA CP binding peptides selected by phage display, the second involved the use of heterodimeric leucine zippers and the last involved the use of an antibody binding peptide, the z33 peptide, from Staphylococcus aureus. The first two strategies were unsuccessful. On the other hand, the z33 strategy enabled an 87 % occupancy of accessible sites on the potyvirus particles by the enzyme. To further test the potential of potyviruses as multi-enzyme nano-carriers, two enzymes, 4-coumarate:coenzyme A ligase (4CL2) and stilbene synthase (STS), catalyzing consecutive steps in resveratrol synthetic pathway were employed as model biocatalysts. The z33-peptide was fused to the N-terminus of these enzymes, z4CL2His and zSTSHis and a chimera-protein, z4CL2::STSHis was also generated by the genetic fusion of both enzymes. All enzymes were active and resveratrol was synthesized from both mono-enzymes and the protein chimera either in solution or adsorbed on potyvirus particles. The latter was illustrated by trapping the mono-enzymes or the protein chimera from clarified soluble E. coli cell lysates on to the surface of potyvirus particles immobilized on the surface of a polypropylene tube. This strategy brings together a bottom-up and top down approach for designing virus based nano-materials and offers a cost effective and efficient way to co-immobilize and purify enzymes.Työn tavoitteena oli löytää uusia strategioita entsyymien järjestämiseksi viruspartikkeleiden pinnoille halutulla tavalla. Potentiaalisia sovellutusalueita entsyymi-pinnoitetuille nanopartikkeleille ovat esimerkiksi biosensori ja biosiruteknologiat. Työssä tutkittiin, voidaanko potyviruksia käyttää entsyymien nanokokoisina tarttumapintoina ja reaktioalustoina. Potyvirukset ovat Potyviridae heimon suurin suku. Ne aiheuttavat merkittäviä satotappioita. Potyvirusten RNA genomia suojaa joustava, nauhamainen virus partikkeli. Rakenneproteiini, jota kutsutaan kuoriproteiiniksi, muodostaa 95 %:a ja viruksen RNA genomi 5 %:a partikkelin painosta. Potyviruspartikkelin muodostumisen infektion aikaista mekanismia ei täysin tunneta, mutta tämän prosessin tutkiminen saattaa avata uusia mahdollisuuksia hyödyntää potyviruksia entsyymien kantajina. Koska partikkelinmuodostus on tärkeä virusinfektion vaihe, saatu informaatio voi samalla palvella etsittäessä virusten lisääntymistä estäviä strategioita. Tässä työssä osoitimme, että potyviruksilla partikkelinmuodostusta mahdollisesti ohjaa tuotettujen kuoriproteiinimolekyylien ja parhaillaan tuotettavana olevan kuoriproteiinimolekyylin väliset vuorovaikutukset proteiinisynteesin aikana. Potyviruspartikkelit tarjoavat suuren yhtenäisen tarttumapinnan entsyymeille. Työn seuraavassa vaiheessa tutkimme erilaisia entsyymien kiinnittämismenetelmiä ja havaitsimme, että Staphylococcus aureus bakteerista peräisin olevaa vasta-aineisiin sitoutuvaa z33 peptidiä voidaan hyödyntää monipuolisesti tähän tarkoitukseen. Z33 peptidi sitoutui partikkeleihin potyviruksen kuoriproteiinia tunnistavan vasta-aineen kautta, siten että kiinnittyneet entsyymit kattoivat 87 %:a viruspartikkeleista. Käytimme tätä strategiaa kahta peräkkäistä resveratrolin biosynteesireitin reaktiota katalysoivan entsyymin, 4-kumaraatti:koentsyymi A ligaasin (4CL2) ja stilbeeni syntaasin (STS), kiinnittämiseen. Tuotimme 4CL2 ja STS entsyymit Escherichia coli bakteereissa ja keräsimme liukoiset entsyymit vasta-aine-partikkelikompleksien pinnalle suoraan rikottujen solujen vapauttamasta nesteestä. Aktiivinen resveratrolin synteesi osoitettiin polypropyleeniputkeen kiinnitetyn viruskompleksin pinnalla. Työssä yhdistettiin bottom-up lähestymistapa, joka hyödyntää potyvirusten partikkelinmuodostumiskykyä ja top down lähestymistapa, jossa entsyymejä kantavat partikkelit kiinnitetään alustoille, tuotettaessa potyvirus-pohjaisia entsyymikantajia pyrkimyksenä kehittää taloudellinen ja tehokas entsyymien puhdistus- ja kiinnitysstrategia

Endotyping asthma related to 3 different work exposures

Background: Work exposures play a significant role in adult-onset asthma, but the mechanisms of work-related asthma are not fully elucidated. Objective: We aimed to reveal the molecular mechanisms of work-related asthma associated with exposure to flour (flour asthma), isocyanate (isocyanate asthma), or welding fumes (welding asthma) and identify potential biomarkers that distinguish these groups from each other. Methods: We used a combination of clinical tests, transcriptomic analysis, and associated pathway analyses to investigate the underlying disease mechanisms of the blood immune cells and the airway epithelium of 61 men. Results: Compared with the healthy controls, the welding asthma patients had more differentially expressed genes than the flour asthma and isocyanate asthma patients, both in the airway epithelia and in the blood immune cells. In the airway epithelia, active inflammation was detected only in welding asthma patients. In contrast, many differentially expressed genes were detected in blood cells in all 3 asthma groups. Disease-related immune functions in blood cells, including leukocyte migration and inflammatory responses, and decreased expression of upstream cytokines such as TNF and IFN-gamma were suppressed in all the asthma groups. In transcriptomephenotype correlations, hyperresponsiveness (R similar to vertical bar 0.6 vertical bar) had the highest clinical relevance and was associated with a set of exposure group-specific genes. Finally, biomarker subsets of only 5 genes specifically distinguished each of the asthma exposure groups. Conclusions: This study provides novel data on the molecular mechanisms underlying work-related asthma. We identified a set of 5 promising biomarkers in asthma related to flour, isocyanate, and welding fume exposure to be tested and clinically validated in future studies.Peer reviewe

Silver, titanium dioxide, and zinc oxide nanoparticles trigger miRNA/isomiR expression changes in THP-1 cells that are proportional to their health hazard potential

After over a decade of nanosafety research, it is indisputable that the vast majority of nano-sized particles induce a plethora of adverse cellular responses - the severity of which is linked to the material's physicochemical properties. Differentiated THP-1 cells were previously exposed for 6 h and 24 h to silver, titanium dioxide, and zinc oxide nanoparticles at the maximum molar concentration at which no more than 15% cellular cytotoxicity was observed. All three nanoparticles differed in extent of induction of biological pathways corresponding to immune response signaling and metal ion homeostasis. In this study, we integrated gene and miRNA expression profiles from the same cells to propose miRNA biomarkers of adverse exposure to metal-based nanoparticles. We employed RNA sequencing together with a quantitative strategy that also enables analysis of the overlooked repertoire of length and sequence miRNA variants called isomiRs. Whilst only modest changes in expression were observed within the first 6 h of exposure, the miRNA/isomiR (miR) profiles of each nanoparticle were unique. Via canonical correlation and pathway enrichment analyses, we identified a co-regulated miR-mRNA cluster, predicted to be highly relevant for cellular response to metal ion homeostasis. These miRs were annotated to be canonical or variant isoforms of hsa-miR-142-5p, -342-3p, -5100, -6087, -6894-3p, and -7704. Hsa-miR-5100 was differentially expressed in response to each nanoparticle in both the 6 h and 24 h exposures. Taken together, this co-regulated miR-mRNA cluster could represent potential biomarkers of sub-toxic metal-based nanoparticle exposure.Peer reviewe

Bet v 1 triggers antiviral-type immune signalling in birch-pollen-allergic individuals

Background In allergic patients, clinical symptoms caused by pollen remind of symptoms triggered by viral respiratory infections, which are also the main cause of asthmatic exacerbations. In patients sensitized to birch pollen, Bet v 1 is the major symptom-causing allergen. Immune mechanisms driving Bet v 1-related responses of human blood cells have not been fully characterized. Objective To characterize the immune response to Bet v 1 in peripheral blood in patients allergic to birch pollen. Methods The peripheral blood mononuclear cells of birch-allergic (n = 24) and non-allergic (n = 47) adolescents were stimulated ex-vivo followed by transcriptomic profiling. Systems-biology approaches were employed to decipher disease-relevant gene networks and deconvolution of associated cell populations. Results Solely in birch-allergic patients, co-expression analysis revealed activation of networks of innate immunity and antiviral signalling as the immediate response to Bet v 1 stimulation. Toll-like receptors and signal transducer transcription were the main drivers of gene expression patterns. Macrophages and dendritic cells were the main cell subsets responding to Bet v 1. Conclusions and clinical relevance In birch-pollen-allergic patients, the activated innate immune networks seem to be, in part, the same as those activated during viral infections. This tendency of the immune system to read pollens as viruses may provide new insight to allergy prevention and treatment.Peer reviewe