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Sökning: WFRF:(Cristobal Susana)

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1.
  • Garcia-Sanchez, Susana, et al. (författare)
  • Early response to nanoparticles in the Arabidopsis transcriptome compromises plant defence and root-hair development through salicylic acid signalling
  • 2015
  • Ingår i: BMC Genomics. - : BioMed Central. - 1471-2164. ; 16:341
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: The impact of nano-scaled materials on photosynthetic organisms needs to be evaluated. Plants represent the largest interface between the environment and biosphere, so understanding how nanoparticles affect them is especially relevant for environmental assessments. Nanotoxicology studies in plants allude to quantum size effects and other properties specific of the nano-stage to explain increased toxicity respect to bulk compounds. However, gene expression profiles after exposure to nanoparticles and other sources of environmental stress have not been compared and the impact on plant defence has not been analysed. Results: Arabidopsis plants were exposed to TiO2-nanoparticles, Ag-nanoparticles, and multi-walled carbon nanotubes as well as different sources of biotic (microbial pathogens) or abiotic (saline, drought, or wounding) stresses. Changes in gene expression profiles and plant phenotypic responses were evaluated. Transcriptome analysis shows similarity of expression patterns for all plants exposed to nanoparticles and a low impact on gene expression compared to other stress inducers. Nanoparticle exposure repressed transcriptional responses to microbial pathogens, resulting in increased bacterial colonization during an experimental infection. Inhibition of root hair development and transcriptional patterns characteristic of phosphate starvation response were also observed. The exogenous addition of salicylic acid prevented some nano-specific transcriptional and phenotypic effects, including the reduction in root hair formation and the colonization of distal leaves by bacteria. Conclusions: This study integrates the effect of nanoparticles on gene expression with plant responses to major sources of environmental stress and paves the way to remediate the impact of these potentially damaging compounds through hormonal priming.
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2.
  • Almeida, A. M., et al. (författare)
  • Animal board invited review: advances in proteomics for animal and food sciences
  • 2015
  • Ingår i: Animal. - : Cambridge University Press (CUP): STM Journals. - 1751-7311 .- 1751-732X. ; 9:1
  • Forskningsöversikt (refereegranskat)abstract
    • Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid - i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002 - Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East-West and North-South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.
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4.
  • Amelina, Hanna, et al. (författare)
  • Proteomic study on gender differences in aging kidney of mice
  • 2009
  • Ingår i: Proteome Science. - : Springer Science and Business Media LLC. - 1477-5956. ; 7:16
  • Tidskriftsartikel (refereegranskat)abstract
    • BACKGROUND: This study aims to analyze sex differences in mice aging kidney. We applied a proteomic technique based on subfractionation, and liquid chromatography coupled with 2-DE. Samples from male and female CD1-Swiss outbred mice from 28 weeks, 52 weeks, and 76 weeks were analysed by 2-DE, and selected proteins were identified by matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS).RESULTS: This proteomic analysis detected age-related changes in protein expression in 55 protein-spots, corresponding to 22 spots in males and 33 spots in females. We found a protein expression signature (PES) of aging composed by 8 spots, common for both genders. The identified proteins indicated increases in oxidative and proteolytic proteins and decreases in glycolytic proteins, and antioxidant enzymes.CONCLUSION: Our results provide insights into the gender differences associated to the decline of kidney function in aging. Thus, we show that proteomics can provide valuable information on age-related changes in expression levels of proteins and related modifications. This pilot study is still far from providing candidates for aging-biomarkers. However, we suggest that the analysis of these proteins could suggest mechanisms of cellular aging in kidney, and improve the kidney selection for transplantation.
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5.
  • Amelina, Hanna, et al. (författare)
  • Proteomics-based method for the assessment of marine pollution using liquid chromatography coupled with two-dimensional electrophoresis
  • 2007
  • Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:6, s. 2094-2104
  • Tidskriftsartikel (refereegranskat)abstract
    • Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography (LC) coupled with two-dimensional electrophoresis (2-DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel “blue mussel” (Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern (PES) composed of 13 proteins. Principal component analysis (PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry (ESI−MS/MS), and they are involved in β-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.
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6.
  • Amelina, Hanna, 1983- (författare)
  • Proteomics in biomarker research : Insights into the effects of aging and environment on biological systems
  • 2011
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Proteomics is the global analysis of proteins that covers a broad range of technologies aimed at determining the identity and quantity of proteins expressed in the cell, their three-dimensional structure and interaction partners. In contrast to genome, proteome reflects more accurately on the dynamic state of the cell, tissue, or an organism. Therefore much is expected from proteomics to yield better disease markers for early diagnosis and therapy monitoring, as well as biomarkers that would indicate environmental exposure or provide prediction of biological age. In this thesis, I have developed and applied robust and sensitive subproteomic approaches to study the effect of aging as well as and environmental pollution using different animal models. In the first part, a high-throughput proteomic method based on liquid chromatography coupled to 2-dimensional gel electrophoresis (LC/2-DE) was developed. The usefulness of this method has been demonstrated by applying it to the assessment of marine pollution in a field experiment. Next, I have utilized this subproteomic approach to study the effect of aging in mouse kidney of both genders. As a result, a protein expression signature of aging kidney was obtained, revealing gender-dependent alterations in proteome profiles of aging mouse kidney. In order to further reduce the dynamic range of protein expression and increase the sensitivity of proteomic analysis, I have applied a shotgun mass spectrometry-based proteomic approach using isobaric tags for relative and absolute quantification (iTRAQ) coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to study age-related differences in peroxisome-enriched fractions from mouse liver. Only eight proteins showed statistically significant difference in expression (p<0.05) with moderate folds. This study indicates that age-depended changes in the liver proteome are minimal, suggesting that its proteome is efficiently maintained until certain age. Finally, in the context of aging studies and the role of peroxisomes in aging, I have tested the utility of cell-penetrating peptides (CPPs) as agents for protein delivery into acatalasemic peroxisomes using yeast as a model. The results obtained suggest that CPPs may be suitable for the delivery of antioxidants to peroxisomes and in future could provide a tool for the protein therapy of age-related diseases.
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7.
  • Amelina, Hanna, et al. (författare)
  • Quantitative subproteomic analysis of age-related changes in mouse liver peroxisomes by iTRAQ LC-MS/MS
  • 2011
  • Ingår i: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 879:30, s. 3393-3400
  • Tidskriftsartikel (refereegranskat)abstract
    • Aging is a complex multifactorial phenomenon, which is believed to result from the accumulation of cellular damage to biological macromolecules. Peroxisomes recently emerged as another important source of reactive oxygen species (ROS) production in addition to mitochondria. However, the role of these organelles in the process of aging is still not clear. The aim of this study was to characterize the changes in protein expression profiles of young (10 weeks old) versus old (18 months old) mouse liver peroxisome-enriched fractions. We have applied shotgun proteomic approach based on liquid chromatography and tandem mass spectrometry (LC-MS/MS) combined with iTRAQ (isobaric tags for relative and absolute quantitation) labeling that allows comparative quantitative multiplex analysis. Our analysis led to identification and quantification of 150 proteins, 8 out of which were differentially expressed between two age groups at a statistically significant level (p < 0.05), with folds ranging from 1.2 to 4.1. These proteins involved in peroxisornal beta-oxidation, detoxification of xenobiotics and production of ROS. Noteworthy, differences in liver proteome have been observed between as well as within different age groups. In conclusion, our subproteomic quantitative study suggests that mouse liver proteome is sufficiently maintained until certain age.
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11.
  • Apraiz, Itxaso, et al. (författare)
  • Peroxisomal proteomics, biomonitoring in mussles after the Prestige's oil spill
  • 2009
  • Ingår i: Marine Pollution Bulletin. - : Elsevier BV. - 0025-326X .- 1879-3363. ; 58:12, s. 1815-1826
  • Tidskriftsartikel (refereegranskat)abstract
    • Peroxisomal proteomics was applied to assess possible biological effects after the Prestige's oil spill. Mussels were sampled in July 2004 and 2005 in four stations in the NW (closest to the spill) and NE coasts of the Iberian Peninsula. Principal components analysis (PCA) suggested differences in protein expression among stations and sampling years. Several proteins were putatively identified by mass spectrometry and immunolocalization. PC1 separated the NW stations in 2004 from the rest of the stations and sampling years mainly due to up-regulation of peroxisomal β-oxidation proteins and PMP70. PC3 separated the NE-stations, based on up-regulation of the antioxidant enzyme catalase in 2004 compared to 2005. PC4 separated the stations in the NE and the NW. This work shows that environmental proteomics, together with multivariate data analysis, could provide information to interpret the effects of oil spills at cellular level in mussels also in the absence of historical data.
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12.
  • Apraiz, Itxaso, 1980-, et al. (författare)
  • Proteomic analysis of mussels exposed to fresh and weathered Prestige's oil
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Biomonitoring
programs
that
use
mussels to assess the
water
quality
around
the
world, could
benefit
from
the
use
 of
proteomics
techniques. These
could
be
applied to obtain
protein expression
signatures
of
exposure to
pollution
that
could
be further
used
for
prediction purposes. This
would
require
that
a
combination
of
univariate
and
multivariate
 statistical
analyses
of
proteomics data
were
utilized
to
obtain
robust
models.
We
show an
application
of
this approach
on
mussels
exposed
to
fresh
fuel, and weathered
fuel
in
a
laboratory experiment
that
tried
to
mimic
the
effects of the
Prestige's
oil
spill.
A
set
of
protein
spots
were
selected
that
could
be used
to
classify
mussels exposed
to
the two
types
of
fuel
oil.
As
an
example
of the possibilities
this
approach
could offer to biomonitoring
programs, mussels were collected from ten sampling stations
along
the
NW
and
NE of the Iberian Peninsula, and their protein expression patterns monitored.    
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13.
  • Apraiz Larrucea, Itxaso, 1980- (författare)
  • Development and application of a proteomic approach to the assessment of pollution in the marine environment
  • 2009
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Today, assessment of the health of coastal waters is recognized as being important for both the conservation of nature and well-being of humans. Anthropogenic pollution has been the focus of extensive research for some time and a variety of programs for the monitoring and assessment of environmental pollution have been developed. Determination of the levels of pollution in sensitive ‘sentinels’ such as mussels, allows monitoring of these levels in a given area over a prolonged period of time. Furthermore, the biological effects of pollution are reflected in a series of biomarkers, none of which provides a general picture of the sentinel’s state of health and all of which are individually specific for certain pollutants and influenced by both biotic and abiotic factors. In an attempt to improve biomonitoring of marine pollution, we have developed two proteomic approaches here. In the first portion of the thesis, a proteomic analysis was performed on peroxisomes isolated from mussels exposed either to one of three model anthropogenic pollutants, or two different types of crude oil, or from mussels exposed to the Prestige oil spill. Application of two-dimensional electrophoresis (2-DE) provided protein expression signatures (PES) for exposure to these different pollutants.Furthermore, several individual protein components of these PES could be putatively identified. In the second portion of this work, such analysis of subproteomes was developed further in order to improve the applicability of this approach to biomonitoring. A simple fractionation procedure in combination with liquid chromatography and 2-DE provided samples from mussels residing in different regions of a pollution gradient around the harbor of Gothenburg, as well as from mussels exposed to two types of fuel oil similar to that of the Prestige that were suitable for environmental proteomics. In addition, we constructed a model for this approach that can be cross-validated in the future and applied to assess sources of fuel oil pollution in connection with biomonitoring programs.
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14.
  • Bayat, Narges, et al. (författare)
  • Assessment of functionalized iron oxide nanoparticles in vitro : introduction to integrated nanoimpact index
  • 2015
  • Ingår i: Environmental Science: Nano. - : Royal Society of Chemistry (RSC). - 2051-8153 .- 2051-8161. ; 2:4, s. 380-394
  • Tidskriftsartikel (refereegranskat)abstract
    • Functionalization of super paramagnetic iron oxide NPs (SPIONs) with different coatings renders them with unique physicochemical properties that allow them to be used in a broad range of applications such as drug targeting and water purification. However, it is required to fill the gap between the promises of any new functionalized SPIONs and the effects of these coatings on the NPs safety. Nanotoxicology is offering diverse strategies to assess the effect of exposure to SPIONs in a case-by-case manner but an integrated nanoimpact scale has not been developed yet. We have implemented the classical integrated biological response (IBR) into an integrated nanoimpact index (INI) as an early warning scale of nano-impact based on a combination of toxicological end points such as cell proliferation, oxidative stress, apoptosis and genotoxicity. Here, the effect of SPIONs functionalized with tri-sodium citrate (TSC), polyethylenimine (PEI), aminopropyl-triethoxysilane (APTES) and Chitosan (chitosan) were assessed on human keratinocytes and endothelial cells. Our results show that endothelial cells were more sensitive to exposure than keratinocytes and the initial cell culture density modulated the toxicity. PEI-SPIONs had the strongest effects in both cell types while TSC-SPIONS were the most biocompatible. This study emphasizes not only the importance of surface coatings but also the cell type and the initial cell density on the selection of toxicity assays. The INI developed here could offer an initial rationale to choose either modifying SPIONs properties to reduce its nanoimpact or performing a complete risk assessment to define the risk boundaries.
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15.
  • Bayat, Narges, 1982-, et al. (författare)
  • Assessment of the safety of functionalized iron oxide nanoparticles in vitro : introduction to integrated nanoimpact index
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Functionalization of super paramagnetic iron oxide NPs (SPIONs) with different coatings renders them with unique physicochemicalproperties that allow them to be used in a broad range of applications such as drug targeting and water purification. However, it is required tofill the gap between the promises of any new functionalized SPIONs and the effects of these coatings on the NPs safety. Nanotoxicology isoffering diverse strategies to assess the effect of exposure to SPIONs in a case-by-case manner but an integrated nanoimpact scale has notbeen developed yet. We have implemented the classical integrated biological response (IBR) into an integrated nanoimpact index (INI) as anearly warning scale of nano-impact based on a combination of toxicological end points such as cell proliferation, oxidative stress, apoptosisand genotoxicity. Here, the effect of SPIONs functionalized with tri-sodium citrate (TSC), polyethylenimine (PEI), aminopropyltriethoxysilane(APTES) and Chitosan (chitosan) were assessed on human keratinocytes and endothelial cells. Our results show thatendothelial cells were more sensitive to exposure than keratinocytes and the initial cell culture density modulated the toxicity. PEI-SPIONshad the strongest effects in both cell types while TSC-SPIONS were the most biocompatible. This study emphasizes not only the importanceof surface coatings but also the cell type and the initial cell density on the selection of toxicity assays. The INI developed here could offer aninitial rationale to choose either modifying SPIONs properties to reduce its nanoimpact or performing a complete risk assessment to definethe risk boundaries.
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16.
  • Bayat, Narges, et al. (författare)
  • The effects of engineered nanoparticles on the cellular structure and growth of Saccharomyces cerevisiae
  • 2014
  • Ingår i: Nanotoxicology. - : Informa UK Limited. - 1743-5390 .- 1743-5404. ; 8:4, s. 363-373
  • Tidskriftsartikel (refereegranskat)abstract
    • In order to study the effects of nanoparticles (NPs) with different physicochemical properties on cellular viability and structure, Saccharomyces cerevisiae were exposed to different concentrations of TiO2-NPs (1-3 nm), ZnO-NPs (<100 nm), CuO-NPs (<50 nm), their bulk forms, Ag-NPs (10 nm) and single-walled carbon nanotubes (SWCNTs). The GreenScreen assay was used to measure cyto- and genotoxicity, and transmission electron microscopy (TEM) used to assess ultrastructure. Cu-ONPs were highly cytotoxic, reducing the cell density by 80% at 9 cm(2)/ml, and inducing lipid droplet formation. Cells exposed to Ag-NPs (19 cm(2)/ml) and TiO2-NPs (147 cm(2)/ml) contained dark deposits in intracellular vacuoles, the cell wall and vesicles, and reduced cell density (40 and 30%, respectively). ZnO-NPs (8 cm(2)/ml) caused an increase in the size of intracellular vacuoles, despite not being cytotoxic. SWCNTs did not cause cytotoxicity or significant alterations in ultrastructure, despite high oxidative potential. Two genotoxicity assays, GreenScreen and the comet assay, produced different results and the authors discuss the reasons for this discrepancy. Classical assays of toxicity may not be the most suitable for studying the effects of NPs in cellular systems, and the simultaneous assessment of other measures of the state of cells, such as TEM are highly recommended.
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17.
  • Bayat, Narges, 1982-, et al. (författare)
  • The effects of ultra-small TiO2 nanoparticle and single walled carbon nanotubes on endothelial cells : next generation sequencing and transcriptome sequencing (RNA-seq) analysis
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • The cardiovascular system is a key route of exposure to nanoparticles (NPs). The exposure could costendothelial cell dysfunction and impairment in blood circulation that could lead to cardiovascular diseasessuch as atherosclerosis. Currently, ultra-small nanoparticles (USNPs) at 1-3 nm, are receiving growingattention due to their unique properties. Emerging application for rutile TiO2-USNPs in medicine areexploring due to their insoluble nature, lack of oxidative activity and strong luminescence not observed inlarger NPs. On organic nanoparticle side, single walled carbon nanotubes (SWCNTs) are candidatemolecules for drug delivery from the chemical perspective. However their potential applications arehindered by their high oxidative activity and potential toxicity. Here we used transcriptome sequencing(RNA-seq) to evaluate the effects of exposure to sub-lethal concentration of TiO2-USNPs, TiO2-NPs andSWCNTs on human dermal microvascular endothelial cells. Specific toxicological effects were inferredfrom the functions of genes whose transcripts either increased or decreased. Our results show that TiO2-USNPs mostly induced the up-regulation of transcripts involved in lipid and cholesterol metabolism.TiO2-NPs induced the highest number of differentially expressed transcripts involved in cellularsenescence, endoplasmic reticulum (ER) stress, and heat shock responses as well lipid metabolism.Finally, SWCNTs affected to those genes involved in early stress and inflammatory responses.
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18.
  • Bayat, Narges, 1982- (författare)
  • Toxicity and biological impact of metal and metal oxide nanoparticles : Focus on the vascular toxicity of ultra-small titanium dioxide nanoparticles
  • 2015
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The application of nanoparticles (NPs) in different technologies has led to tremendous advancement in those fields.  Moreover, there is growing interest in application of ultra-small NPs (USNPs) at 1-3 nm due to their distinct molecule like features. Parallel to these promises, there is a growing concern regarding their safety. The main goal of this thesis was to investigate the toxicity and underlying mechanisms following exposure to different metal and metal oxide NPs as well as USNPs. Their effects were studied on Saccharomyces cerevisiae, on hepatocytes and endothelial cells and finally in vivo on zebrafish embryos (Danio rerio). By selecting the rutile form of titanium dioxide (TiO2-USNPs) without intrinsic or intracellular reactive oxygen species (ROS) production, we could study biological impacts solely due to size and direct interaction with the cells. We showed that TiO2-USNPs were not cytotoxic but induced DNA damage. They had anti-angiogenic effects both in vitro and in vivo. Also, at high concentrations they caused complete mortality in zebrafish embryos exposed in water, while at lower concentrations induced delay in hatching. When injected they caused malformations. They specifically induced the differential overexpression of transcripts involved in lipid and cholesterol metabolism in endothelial cells. In hepatocytes they induced the overexpression of proteins in the electron transport chain and decreased lipids in lipid rafts. At 30 nm, TiO2-NPs, were also not cytotoxic but were genotoxic. They had no effects in vivo or on angiogenesis. However, they induced differential expression of transcripts involved in endoplasmic reticulum (ER) stress and heat shock response as well as cholesterol metabolism. This suggests a more toxic response in the cells compared to TiO2-USNPs.  Single walled carbon nanotubes (SWCNTs) despite having the highest oxidative activity among the NPs studied, were not severely cyto- or genotoxic but induced expression of transcripts involved in early ER stress response. Copper oxide (CuO-NPs) was the most toxic NPs studied due to both ion release and ROS production, affecting lipid metabolism of the cells. Silver (Ag-NPs) were also cytotoxic and caused the disruption of cellular components and lipids. ZnO-NPs were not cytotoxic, did not affect cellular lipids but they increased the size of vacuoles in yeast cells. Finally by using superparamagnetic iron oxide NPs (SPIONs) with different coatings, and using a mathematical model, a nano impact index (INI) was developed as a tool to enable the comparison of nanotoxicology data.
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19.
  • Bayat, Narges, 1982-, et al. (författare)
  • Vascular toxicity of ultra-small TiO2 nanoparticles and single walled carbon nanotubes in vitro and in vivo
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Ultra-small nanoparticles (USNPs) at 1-3 nm are a subset of nanoparticles (NPs) that exhibit intermediate physicochemical properties between molecular dispersions and larger NPs. Despite interest in their utilization in applications such as theranostics, limited data about their toxicity exist. Here the effect of TiO2-USNPs on endothelial cells in vitro, and zebrafish embryos in vivo, was studied. The findings were compared to TiO2-NPs (30 nm) and single walled carbon nanotubes (SWCNTs). TiO2-USNPs were not cytotoxic, had no oxidative ability yet were genotoxic in vitro. They caused mortality at high concentrations in water possibly by acidifying the water and caused malformations in the form of pericardial edema when injected in early developing zebrafish embryos. Myo1C involved in glomerular development of zebrafish embryos was upregulated in embryos exposed to TiO2-USNPs. They also exhibited anti-angiogenic effects both in vitro and in vivo plus decreased nitric oxide concentration. TiO2-NPs were genotoxic but not cytotoxic. SWCNTs were cytotoxic in vitro and had the highest oxidative ability. Neither of these NPs had significant effects in vivo. To our knowledge this is the first study evaluating the effects of TiO2-USNPs on vascular toxicity in vitro and in vivo, demonstrating their potency and necessity for more focus in nanotoxicology.
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20.
  • Bayat, Narges, et al. (författare)
  • Vascular toxicity of ultra-small TiO2 nanoparticles and single walled carbon nanotubes in vitro and in vivo
  • 2015
  • Ingår i: Biomaterials. - : Elsevier BV. - 0142-9612 .- 1878-5905. ; 63, s. 1-13
  • Tidskriftsartikel (refereegranskat)abstract
    • Ultra-small nanoparticles (USNPs) at 1-3 nm are a subset of nanoparticles (NPs) that exhibit intermediate physicochemical properties between molecular dispersions and larger NPs. Despite interest in their utilization in applications such as theranostics, limited data about their toxicity exist. Here the effect of TiO2-USNPs on endothelial cells in vitro, and zebrafish embryos in vivo, was studied and compared to larger TiO2-NPs (30 nm) and to single walled carbon nanotubes (SWCNTs). In vitro exposure showed that TiO2-USNPs were neither cytotoxic, nor had oxidative ability, nevertheless were genotoxic. In vivo experiment in early developing zebrafish embryos in water at high concentrations of TiO2-USNPs caused mortality possibly by acidifying the water and caused malformations in the form of pericardial edema when injected. Myo1C involved in glomerular development of zebrafish embryos was upregulated in embryos exposed to TiO2-USNPs. They also exhibited anti-angiogenic effects both in vitro and in vivo plus decreased nitric oxide concentration. The larger TiO2-NPs were genotoxic but not cytotoxic. SWCNTs were cytotoxic in vitro and had the highest oxidative ability. Neither of these NPs had significant effects in vivo. To our knowledge this is the first study evaluating the effects of TiO2-USNPs on vascular toxicity in vitro and in vivo and this strategy could unravel USNPs potential applications.
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21.
  • Bendz, Maria, et al. (författare)
  • Membrane protein shaving with thermolysin can be used to evaluate topology predictors
  • 2013
  • Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 13:9, s. 1467-1480
  • Tidskriftsartikel (refereegranskat)abstract
    • Topology analysis of membrane proteins can be obtained by enzymatic shaving in combination with MS identification of peptides. Ideally, such analysis could provide quite detailed information about the membrane spanning regions. Here, we examine the ability of some shaving enzymes to provide large-scale analysis of membrane proteome topologies. To compare different shaving enzymes, we first analyzed the detected peptides from two over-expressed proteins. Second, we analyzed the peptides from non-over-expressed Escherichia coli membrane proteins with known structure to evaluate the shaving methods. Finally, the identified peptides were used to test the accuracy of a number of topology predictors. At the end we suggest that the usage of thermolysin, an enzyme working at the natural pH of the cell for membrane shaving, is superior because: (i) we detect a similar number of peptides and proteins using thermolysin and trypsin; (ii) thermolysin shaving can be run at a natural pH and (iii) the incubation time is quite short. (iv) Fewer detected peptides from thermolysin shaving originate from the transmembrane regions. Using thermolysin shaving we can also provide a clear separation between the best and the less accurate topology predictors, indicating that using data from shaving can provide valuable information when developing new topology predictors.
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22.
  • Benede, Sara, et al. (författare)
  • New applications of advanced instrumental techniques for the characterization of food allergenic proteins
  • 2022
  • Ingår i: Critical reviews in food science and nutrition. - : TAYLOR & FRANCIS INC. - 1040-8398 .- 1549-7852. ; 62:31, s. 8686-8702
  • Forskningsöversikt (refereegranskat)abstract
    • Current approaches based on electrophoretic, chromatographic or immunochemical principles have allowed characterizing multiple allergens, mapping their epitopes, studying their mechanisms of action, developing detection and diagnostic methods and therapeutic strategies for the food and pharmaceutical industry. However, some of the common structural features related to the allergenic potential of food proteins remain unknown, or the pathological mechanism of food allergy is not yet fully understood. In addition, it is also necessary to evaluate new allergens from novel protein sources that may pose a new risk for consumers. Technological development has allowed the expansion of advanced technologies for which their whole potential has not been entirely exploited and could provide novel contributions to still unexplored molecular traits underlying both the structure of food allergens and the mechanisms through which they sensitize or elicit adverse responses in human subjects, as well as improving analytical techniques for their detection. This review presents cutting-edge instrumental techniques recently applied when studying structural and functional aspects of proteins, mechanism of action and interaction between biomolecules. We also exemplify their role in the food allergy research and discuss their new possible applications in several areas of the food allergy field.
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23.
  • Berlin, Emmanuel, et al. (författare)
  • Nonionic Surfactants can Modify the Thermal Stability of Globular and Membrane Proteins Interfering with the Thermal Proteome Profiling Principles to Identify Protein Targets
  • 2023
  • Ingår i: Analytical Chemistry. - : AMER CHEMICAL SOC. - 0003-2700 .- 1520-6882. ; 95:8, s. 4033-4042
  • Tidskriftsartikel (refereegranskat)abstract
    • The membrane proteins are essential targets for understanding cellular function. The unbiased identification of membrane protein targets is still the bottleneck for a system-level understanding of cellular response to stimuli or perturbations. It has been suggested to enrich the soluble proteome with membrane proteins by introducing nonionic surfactants in the solubilization solution. This strategy aimed to simultaneously identify the globular and membrane protein targets by thermal proteome profiling principles. However, the thermal shift assay would surpass the cloud point temperature from the nonionic surfactants frequently utilized for membrane protein solubilization. It is expected that around the cloud point temperature, the surfactant micelles would suffer structural modifications altering protein solubility. Here, we show that the presence of nonionic surfactants can alter protein thermal stability from a mixed, globular, and membrane proteome. In the presence of surfactant micelles, the changes in protein solubility analyzed after the thermal shift assay was affected by the thermally dependent modification of the micellar size and its interaction with proteins. We demonstrate that the introduction of nonionic surfactants for the solubilization of membrane proteins is not compatible with the principles of target identification by thermal proteome profiling methodologies. Our results lead to exploring thermally independent strategies for membrane protein solubilization to assure confident membrane protein target identification. The proteome-wide thermal shift methods have already shown their capability to elucidate mechanisms of action from pharma, biomedicine, analytical chemistry, or toxicology, and finding strategies, free from surfactants, to identify membrane protein targets would be the next challenge.
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24.
  • Bertile, Fabrice, et al. (författare)
  • Diversifying the concept of model organisms in the age of -omics
  • 2023
  • Ingår i: Communications Biology. - : NATURE PORTFOLIO. - 2399-3642. ; 6:1
  • Tidskriftsartikel (refereegranskat)abstract
    • In today's post-genomic era, it is crucial to rethink the concept of model organisms. While a few historically well-established organisms, e.g. laboratory rodents, have enabled significant scientific breakthroughs, there is now a pressing need for broader inclusion. Indeed, new organisms and models, from complex microbial communities to holobionts, are essential to fully grasp the complexity of biological principles across the breadth of biodiversity. By fostering collaboration between biology, advanced molecular science and omics communities, we can collectively adopt new models, unraveling their molecular functioning, and uncovering fundamental mechanisms. This concerted effort will undoubtedly enhance human health, environmental quality, and biodiversity conservation. The concept of model organisms in biological studies needs to be re-evaluated to reflect novel technological advances and help further scientific discovery.
  •  
25.
  • Campos, Alexandre, et al. (författare)
  • Proteomic research in bivalves Towards the identification of molecular markers of aquatic pollution
  • 2012
  • Ingår i: Journal of Proteomics. - : Elsevier. - 1874-3919 .- 1876-7737. ; 75:14, s. 4346-4359
  • Forskningsöversikt (refereegranskat)abstract
    • Biomonitoring of aquatic environment and assessment of ecosystem health play essential roles in the development of effective strategies for the protection of the environment, human health and sustainable development. Biomarkers of pollution exposure have been extensively utilized in the last few decades to monitor the health of organisms and hence assess environmental status. However, the use of single biomarkers against biotic or abiotic stressors may be limited by the lack of sensitivity and specificity. Therefore, more recently, the search for novel biomarkers has been focused on the application of OMICS methodologies. Environmental proteomics focuses on the analysis of an organisms proteome and the detection of changes in the level of individual proteins/peptides in response to environmental stressors. Proteomics can provide a more robust approach for the assessment of environmental stress and therefore exposure to pollutants. This review aims to summarize the proteomic research in bivalves, a group of sessile and filter feeding organisms that play an important function as "sentinels" of the aquatic environment. A description of the main proteomic methodologies is provided. The current knowledge in bivalves toxicology, achieved with proteomics, is reported describing the main biochemical markers identified. A brief discussion regarding future challenges in this area of research emphasizing the development of more descriptive gene/protein databases that could support the OMICs approaches is presented. less thanbrgreater than less thanbrgreater thanThis article is part of a Special Issue entitled: Farm animal proteomics.
  •  
26.
  • Campos, Alexandre, et al. (författare)
  • Shotgun analysis of the marine mussel Mytilus edulis hemolymph proteome and mapping the innate immunity elements
  • 2015
  • Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 15:23-24, s. 4021-4029
  • Tidskriftsartikel (refereegranskat)abstract
    • The marine mussel innate immunity provides protection to pathogen invasion and inflammation. In this regard, the mussel hemolymph takes a main role in the animal innate response. Despite the importance of this body fluid in determining the physiological condition of the animal, little is known about the molecular mechanisms underlying the cellular and humoral responses. In this work, we have applied a MS (nano-LC-MS/MS) strategy integrating genomic and transcriptomic data with the aim to: (i) identify the main protein functional groups that characterize hemolymph and (ii) to map the elements of innate immunity in the marine mussel Mytilus edulis hemolymph proteome. After sample analysis and first protein identification based on MS/MS data comparison, proteins with unknown functions were annotated with blast using public database (nrNCBI) information. Overall 595 hemolymph proteins were identified with high confidence and annotated. These proteins encompass primary cellular metabolic processes: energy production and metabolism of biomolecules, as well as processes related to oxidative stress defence, xenobiotic detoxification, drug metabolism, and immune response. A group of proteins was identified with putative immune effector, receptor, and signaling functions in M. edulis. Data are available via ProteomeXchange with identifier PXD001951 (http://proteomecentral.proteomexchange.org/dataset/PXD001951).
  •  
27.
  • Campos, Alexandre, et al. (författare)
  • Shotgun proteomics to unravel marine mussel (Mytilus edulis) response to long-term exposure to low salinity and propranolol in a Baltic Sea microcosm
  • 2016
  • Ingår i: Journal of Proteomics. - : Elsevier. - 1874-3919 .- 1876-7737. ; 137, s. 97-106
  • Tidskriftsartikel (refereegranskat)abstract
    • Pharmaceuticals, among them the β-adrenoreceptor blocker propranolol, are an important group of environmental contaminants reported in European waters. Laboratory exposure to pharmaceuticals on marine species has been performed without considering the input of the ecosystem flow. To unravel the ecosystem response to long-term exposure to propranolol we have performed long-term exposure to propranolol and low salinity in microcosms. We applied shotgun proteomic analysis to gills of Mytilus edulis from those Baltic Sea microcosms and identified 2071 proteins with a proteogenomic strategy. The proteome profiling patterns from the 587 highly reproductive proteins among groups define salinity as a key factor in the mussel´s response to propranolol. Exposure at low salinity drives molecular mechanisms of adaptation based on a decrease in the abundance of several cytoskeletal proteins, signalling and intracellular membrane trafficking pathway combined with a response towards the maintenance of transcription and translation. The exposure to propranolol combined with low salinity modulates the expression of structural proteins including cilia functions and decrease the expression membrane protein transporters. This study reinforces the environment concerns of the impact of low salinity in combination with anthropogenic pollutants and anticipate critical physiological conditions for the survival of the blue mussel in the northern areas.
  •  
28.
  • Carrasco Del Amor, Ana Maria, et al. (författare)
  • Application of Bioactive Thermal Proteome Profiling to Decipher the Mechanism of Action of the Lipid Lowering 13(2)-Hydroxy-pheophytin Isolated from a Marine Cyanobacteria
  • 2019
  • Ingår i: Marine Drugs. - : MDPI. - 1660-3397. ; 17:6
  • Tidskriftsartikel (refereegranskat)abstract
    • The acceleration of the process of understanding the pharmacological application of new marine bioactive compounds requires identifying the compound protein targets leading the molecular mechanisms in a living cell. The thermal proteome profiling (TPP) methodology does not fulfill the requirements for its application to any bioactive compound lacking chemical and functional characterization. Here, we present a modified method that we called bTPP for bioactive thermal proteome profiling that guarantees target specificity from a soluble subproteome. We showed that the precipitation of the microsomal fraction before the thermal shift assay is crucial to accurately calculate the melting points of the protein targets. As a probe of concept, the protein targets of 13(2)-hydroxy-pheophytin, a compound previously isolated from a marine cyanobacteria for its lipid reducing activity, were analyzed on the hepatic cell line HepG2. Our improved method identified 9 protein targets out of 2500 proteins, including 3 targets (isocitrate dehydrogenase, aldehyde dehydrogenase, phosphoserine aminotransferase) that could be related to obesity and diabetes, as they are involved in the regulation of insulin sensitivity and energy metabolism. This study demonstrated that the bTPP method can accelerate the field of biodiscovery, revealing protein targets involved in mechanisms of action (MOA) connected with future applications of bioactive compounds.
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29.
  • Carrasco Del Amor, Ana Maria, et al. (författare)
  • Insights into the mechanism of action of the chlorophyll derivative 13-2-hydroxypheophytine a on reducing neutral lipid reserves in zebrafish larvae and mice adipocytes
  • 2023
  • Ingår i: European Journal of Pharmacology. - : ELSEVIER. - 0014-2999 .- 1879-0712. ; 960
  • Tidskriftsartikel (refereegranskat)abstract
    • Obesity is a worldwide epidemic and natural products may hold promise in its treatment. The chlorophyll derivative 13-2-hydroxypheophytine (hpa) was isolated in a screen with zebrafish larvae to identify lipid reducing molecules from cyanobacteria. However, the mechanisms underlying the lipid-reducing effects of hpa in zebrafish larvae remain poorly understood. Thus, investigating the mechanism of action of hpa and validation in other model organisms such as mice represents important initial steps.In this study, we identified 14 protein targets of hpa in zebrafish larvae by thermal proteome profiling, and selected two targets (malate dehydrogenase and pyruvate kinase) involved in cellular metabolism for further validation by enzymatic measurements. Our findings revealed a dose-dependent inhibition of pyruvate kinase by hpa exposure using protein extracts of zebrafish larvae in vitro, and in exposure experiments from 3 to 5 days post fertilization in vivo. Analysis of untargeted metabolomics of zebrafish larvae detected 940 mass peaks (66 increased, 129 decreased) and revealed that hpa induced the formation of various phospholipid species (phosphoinositol, phosphoethanolamine, phosphatidic acid). Inter-species validation showed that brown adipocytes exposed to hpa significantly reduced the size of lipid droplets, increased maximal mitochondrial respiratory capacity, and the expression of PPARy during adipocyte differentiation.In line with our data, previous work described that reduced pyruvate kinase activity lowered hepatic lipid content via reduced pyruvate and citrate, and improved mitochondrial function via phospholipids. Thus, our data provide new insights into the molecular mechanism underlying the lipid reducing activities of hpa in zebrafish larvae, and species overlapping functions in reduction of lipids.
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30.
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31.
  • Emanuelsson, Olof, et al. (författare)
  • In silico prediction of the peroxisomal proteome in fungi, plants and animals.
  • 2003
  • Ingår i: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 330:2, s. 443-456
  • Tidskriftsartikel (refereegranskat)abstract
    • In an attempt to improve our abilities to predict peroxisomal proteins, we have combined machine-learning techniques for analyzing peroxisomal targeting signals (PTS1) with domain-based cross-species comparisons between eight eukaryotic genomes. Our results indicate that this combined approach has a significantly higher specificity than earlier attempts to predict peroxisomal localization, without a loss in sensitivity. This allowed us to predict 430 peroxisomal proteins that almost completely lack a localization annotation. These proteins can be grouped into 29 families covering most of the known steps in all known peroxisomal pathways. In general, plants have the highest number of predicted peroxisomal proteins, and fungi the smallest number.
  •  
32.
  • Fleenor, Courtney J., et al. (författare)
  • Zinc Finger Protein 521 Regulates Early Hematopoiesis through Cell-Extrinsic Mechanisms in the Bone Marrow Microenvironment
  • 2018
  • Ingår i: Molecular and Cellular Biology. - : AMER SOC MICROBIOLOGY. - 0270-7306 .- 1098-5549. ; 38:17
  • Tidskriftsartikel (refereegranskat)abstract
    • Zinc finger protein 521 (ZFP521), a DNA-binding protein containing 30 Kruppel-like zinc fingers, has been implicated in the differentiation of multiple cell types, including hematopoietic stem and progenitor cells (HSPC) and B lymphocytes. Here, we report a novel role for ZFP521 in regulating the earliest stages of hematopoiesis and lymphoid cell development via a cell-extrinsic mechanism. Mice with inactivated Zfp521 genes (Zfp521(-/-)) possess reduced frequencies and numbers of hematopoietic stem and progenitor cells, common lymphoid progenitors, and B and T cell precursors. Notably, ZFP521 deficiency changes bone marrow microenvironment cytokine levels and gene expression within resident HSPC, consistent with a skewing of hematopoiesis away from lymphopoiesis. These results advance our understanding of ZFP521s role in normal hematopoiesis, justifying further research to assess its potential as a target for cancer therapies.
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33.
  • Ge, Yue, et al. (författare)
  • Environmental OMICS: Current Status and Future Directions
  • 2013
  • Ingår i: JOURNAL OF INTEGRATED OMICS. - : Proteomass Scientific Society. - 2182-0287. ; 3:2, s. 75-87
  • Tidskriftsartikel (refereegranskat)abstract
    • Applications of OMICS to high throughput studies of changes of genes, RNAs, proteins, metabolites, and their associated functionsin cells or organisms exposed to environmental chemicals has led to the emergence of a very active research field: environmental OMICS.This developing field holds an important key for improving the scientific basis for understanding the potential impacts of environmentalchemicals on both health and the environment. Here we describe the state of environmental OMICS with an emphasis on its recent accomplishmentsand its problems and potential solutions to facilitate the incorporation of OMICS into mainstream environmental and healthresearch.Data sources: We reviewed relevant and recently published studies on the applicability and usefulness of OMICS technologies to the identificationof toxicity pathways, mechanisms, and biomarkers of environmental chemicals for environmental and health risk monitoring andassessment, including recent presentations and discussions on these issues at The First International Conference on Environmental OMICS(ICEO), held in Guangzhou, China during November 8-12, 2011. This paper summarizes our review.Synthesis: Environmental OMICS aims to take advantage of powerful genomics, transcriptomics, proteomics, and metabolomics tools toidentify novel toxicity pathways/signatures/biomarkers so as to better understand toxicity mechanisms/modes of action, to identify/categorize/prioritize/screen environmental chemicals, and to monitor and predict the risks associated with exposure to environmental chemicalson human health and the environment. To improve the field, some lessons learned from previous studies need to be summarized, aresearch agenda and guidelines for future studies need to be established, and a focus for the field needs to be developed.Conclusions: OMICS technologies for identification of RNA, protein, and metabolic profiles and endpoints have already significantly improvedour understanding of how environmental chemicals affect our ecosystem and human health. OMICS breakthroughs are empoweringthe fields of environmental toxicology, chemical toxicity characterization, and health risk assessment. However, environmental OMICS is stillin the data generation and collection stage. Important data gaps in linking and/or integrating toxicity data with OMICS endpoints/profilesneed to be filled to enable understanding of the potential impacts of chemicals on human health and the environment. It is expected thatfuture environmental OMICS will focus more on real environmental issues and challenges such as the characterization of chemical mixturetoxicity, the identification of environmental and health biomarkers, and the development of innovative environmental OMICS approachesand assays. These innovative approaches and assays will inform chemical toxicity testing and prediction, ecological and health risk monitoringand assessment, and natural resource utilization in ways that maintain human health and protects the environment in a sustainable manner.
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34.
  • Gouveia, Duarte, et al. (författare)
  • Ecotoxicoproteomics : A decade of progress in our understanding of anthropogenic impact on the environment
  • 2019
  • Ingår i: Journal of Proteomics. - : Elsevier. - 1874-3919 .- 1876-7737. ; 198, s. 66-77
  • Tidskriftsartikel (refereegranskat)abstract
    • Anthropogenic pollutants are found worldwide. Their fate and effects on human and ecosystem health must be appropriately monitored. Today, ecotoxicology is focused on the development of new methods to assess the impact of pollutant toxicity on living organisms and ecosystems. In situ biomonitoring often uses sentinel animals for which, ideally, molecular biomarkers have been defined thanks to which environmental quality can be assessed. In this context, high-throughput proteomics methods offer an attractive approach to study the early molecular responses of organisms to environmental stressors. This approach can be used to identify toxicity pathways, to quantify more precisely novel biomarkers, and to draw the possible adverse outcome pathways. In this review, we discuss the major advances in ecotoxicoproteomics made over the last decade and present the current state of knowledge, emphasizing the technological and conceptual advancements that allowed major breakthroughs in this field, which aims to “make our planet great again”.SignificanceEcotoxicoproteomics is a protein-centric methodology that is useful for ecotoxicology and could have future applications as part of chemical risk assessment and environmental monitoring. Ecotoxicology employing non-model sentinel organisms with highly divergent phylogenetic backgrounds aims to preserve the functioning of ecosystems and the overall range of biological species supporting them. The classical proteomics workflow involves protein identification, functional annotation, and extrapolation of toxicity across species. Thus, it is essential to develop multi-omics approaches in order to unravel molecular information and construct the most suitable databases for protein identification and pathway analysis in non-model species. Current instrumentation and available software allow relevant combined transcriptomic/proteomic studies to be performed for almost any species. This review summarizes these approaches and illustrates how they can be implemented in ecotoxicology for routine biomonitoring.
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35.
  • Hedin, Linnea E, 1981- (författare)
  • Intra- and intermolecular interactions in proteins : Studies of marginally hydrophobic transmembrane alpha-helices and protein-protein interactions.
  • 2010
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Most of the processes in a living cell are carried out by proteins. Depending on the needs of the cell, different proteins will interact and form the molecular machines demanded for the moment. A subset of proteins called integral membrane proteins are responsible for the interchange of matter and information across the biological membrane, the lipid bilayer enveloping and defining the cell. Most of these proteins are co-translationally integrated into the membrane by the Sec translocation machinery. This thesis addresses two questions that have emerged during the last decade. The first concerns membrane proteins: a number of α-helices have been observed to span the membrane in the obtained three-dimensional structures even though these helices are predicted not to be hydrophobic enough to be recognized by the translocon for integration. We show for a number of these marginally hydrophobic protein segments that they indeed do not insert well outside of their native context, but that their local sequence context can improve the level of integration mediated by the translocon. We also find that many of these helices are overlapped by more hydrophobic segments. We propose, supported by experimental results, that the latter are initially integrated into the membrane, followed by post-translational structural rearrangements. Finally, we investigate whether the integration of the marginally hydrophobic TMHs of the lactose permease of Escherichia coli is facilitated by the formation of hairpin structures. However our combined efforts of computational simulations and experimental investigations find no evidence for this. The second question addressed in this thesis is that of the interpretation of the large datasets on which proteins that interact with each other in a cell. We have analyzed the results from several large-scale investigations concerning protein interactions in yeast and draw conclusions regarding the biases, strengths and weaknesses of these datasets and the methods used to obtain them.
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36.
  • Hedin, Linnea E., et al. (författare)
  • Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context
  • 2010
  • Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 396:1, s. 221-229
  • Tidskriftsartikel (refereegranskat)abstract
    • In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.
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37.
  • Helander, Sara, et al. (författare)
  • Pre-Anchoring of Pin1 to Unphosphorylated c-Myc in a Fuzzy Complex Regulates c-Myc Activity
  • 2015
  • Ingår i: Structure. - : Cell Press. - 0969-2126 .- 1878-4186. ; 23:12, s. 2267-2279
  • Tidskriftsartikel (refereegranskat)abstract
    • Hierarchic phosphorylation and concomitant Pin1-mediated proline isomerization of the oncoprotein c-Myc controls its cellular stability and activity. However, the molecular basis for Pin1 recognition and catalysis of c-Myc and other multisite, disordered substrates in cell regulation and disease is unclear. By nuclear magnetic resonance, surface plasmon resonance, and molecular modeling, we show that Pin1 subdomains jointly pre-anchor unphosphorylated c-Myc1–88 in the Pin1 interdomain cleft in a disordered, or “fuzzy”, complex at the herein named Myc Box 0 (MB0) conserved region N-terminal to the highly conserved Myc Box I (MBI). Ser62 phosphorylation in MBI intensifies previously transient MBI-Pin1 interactions in c-Myc1–88 binding, and increasingly engages Pin1PPIase and its catalytic region with maintained MB0 interactions. In cellular assays, MB0 mutated c-Myc shows decreased Pin1 interaction, increased protein half-life, but lowered rates of Myc-driven transcription and cell proliferation. We propose that dynamic Pin1 recognition of MB0 contributes to the regulation of c-Myc activity in cells
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38.
  • Kauko, Anni, et al. (författare)
  • Repositioning of transmembrane alpha-helices during membrane protein folding
  • 2010
  • Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 397:1, s. 190-201
  • Tidskriftsartikel (refereegranskat)abstract
    • We have determined the optimal placement of individual transmembrane helices in the Pyrococcus horikoshii Glt(Ph) glutamate transporter homolog in the membrane. The results are in close agreement with theoretical predictions based on hydrophobicity, but do not, in general, match the known three-dimensional structure, suggesting that transmembrane helices can be repositioned relative to the membrane during folding and oligomerization. Theoretical analysis of a database of membrane protein structures provides additional support for this idea. These observations raise new challenges for the structure prediction of membrane proteins and suggest that the classical two-stage model often used to describe membrane protein folding needs to be modified.
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39.
  • Kuruvilla, Jacob, et al. (författare)
  • Proteomic Analysis of Endothelial Cells Exposed to Ultrasmall Nanoparticles Reveals Disruption in Paracellular and Transcellular Transport
  • 2019
  • Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 19:5
  • Tidskriftsartikel (refereegranskat)abstract
    • The large interactive surfaces of nanoparticles (NPs) increase the opportunities to develop NPs for vascular targeting. Proteomic analysis of endothelial cells exposed to NPs reveals the cellular response and turns the focus into the impairment of the endothelial permeability. Here, quantitative proteomics and transcriptome sequencing are combined to evaluate the effects of exposure to sub-lethal concentrations of TiO2-USNPs and TiO2-NPs on human dermal microvascular endothelial cells. Endothelial cells react to preserve the semi-permeable properties that are essential for vascular tissue fluid homeostasis, vascular development, and angiogenesis. The main impact of the exposure was alteration of functional complexes involved in cell adhesion, vesicular transport, and cytoskeletal structure. Those are the core cellular structures that are linked to the permeability and the integrity of the endothelial tissue. Moreover, the extracellular proteins uptake along wih the NPs into the endothelial cells escape the lysosomal degradation pathway. These findings improve the understanding of the interaction of NPs with endothelial cell. The effects of the studied NPs modulating cell-cell adhesion and vesicular transport can help to evaluate the distribution of NPs via intravenous administration.
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40.
  • Kuruvilla, Jacob (författare)
  • Proteomics as a multifaceted tool in medicine and environmental assessment
  • 2017
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Proteomics is evolving as a multi-faceted tool for addressing various biochemical and biomedical queries in the field of scientific research. This involves various stages, ranging from sample preparation to data analysis and biological interpretation. Sample preparation involves isolating proteins from the sample source, purifying and digesting them to initiate shotgun proteomics. Shotgun proteomics identifies proteins by bottom-up proteomic approaches where proteins are identified from the fragmentation spectra of their own peptides.Paper I: deals with the simplification of functional characterization for nanoparticles intended for use in biomedicine. Proteomics was constructive in differentiating and semi-quantifying the surface of protein corona. This could be beneficial in predicting the interactions between nanoparticles and a biological entity like the cell or a receptor protein and provide initial valuable information related to targeting, uptake and safety.Paper II: deals with understanding effects of TiO2 nanoparticles on endothelial cells. A combinatorial approach, involving transcriptomics and proteomics was used to identify aberrations in the permeability and integrity of endothelial cells and tissues. Our study also investigated the correlation of size and how they motivated a differential cellular response. In case of intravenous entry for nanoparticles in targeted drug delivery systems, endothelial cells are the first barrier encountered by these drug carriers. This evaluation involving endothelial cell response could be very instrumental during the designing of NP based drug delivery systems.Paper III: Pharmaceuticals and its metabolites could be very hazardous, especially if its disposal is not managed properly. Since water bodies are the ultimate sink, these chemicals could end up there, culminating in toxicity and other ‘mixture effects’ in combination with other factors. To evaluate the effects of the pharmaceutical, propranolol and climatic factors like low salinity conditions, a microcosm exposure was designed and shotgun proteomics helped understand its impact on mussel gills. In this study too, a combination of transcriptomics and proteomics unveiled molecular mechanisms altered in response to stressors, both individually and in combination.Paper IV: An interplay of various factors like EBF1 and PAX5 determines B-cell lineage and commitment. This might have been materialized by direct and transient proteinprotein interactions. A unique method called BioID helped screen relevant interactions in living cells by the application of a promiscuous biotin ligase enzyme capable of tagging proteins through biotinylation based on a proximity radius. Biotinylation of endogenous proteins enabled their selective isolation by exploiting the high affinity of biotin and streptavidin on streptavidin coated agarose beads, leading to their identification by mass spectrometry. The biotinylated proteins were potential candidate interactors of EBF1 and PAX5, which were later confirmed by sequencing techniques like ChIP-Seq, ATAC seq, and visualization techniques like proximity ligation assay (PLA).
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41.
  • Kuruvilla, Jacob, et al. (författare)
  • Surface proteomics on nanoparticles, a step to simplify the rapid prototyping of nanoparticles
  • 2017
  • Ingår i: Nanoscale Horizons. - : Royal Society of Chemistry. - 2055-6764 .- 2055-6756. ; :1, s. 55-64
  • Tidskriftsartikel (refereegranskat)abstract
    • Engineered nanoparticles for biomedical applications requireincreasing effectiveness in targeting specific cells while preservingnon-target cell’s safety. We developed a surface proteomicsmethod for a rapid and systematic analysis of the interphasebetween the nanoparticle protein corona and the targeting cellsthat could implement the rapid prototyping of nanomedicines.Native nanoparticles entering in a protein-rich liquid mediaquickly form a macromolecular structure called protein corona.This protein structure defines the physical interaction betweennanoparticles and target cells. The surface proteins compose thefirst line of interaction between this macromolecular structureand the cell surface of a target cell. We demonstrated that SUSTU(SUrface proteomics, Safety, Targeting, Uptake) provides aqualitative and quantitative analysis from the protein coronasurface. With SUSTU, the spatial dynamics of the protein coronasurface can be studied. Data from SUSTU would ascertain thenanoparticle functionalized groups exposed at destiny that couldcircumvent preliminary in vitro experiments. Therefore thismethod could implement the analysis of nanoparticle targetingand uptake capability and could be integrated into a rapidprototyping strategy which is a major challenge in nanomaterialscience. Data are available via ProteomeXchange with identifierPXD004636.
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42.
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43.
  • Legler, Juliette, et al. (författare)
  • The GOLIATH Project : Towards an Internationally Harmonised Approach for Testing Metabolism Disrupting Compounds
  • 2020
  • Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 21:10
  • Tidskriftsartikel (refereegranskat)abstract
    • The purpose of this project report is to introduce the European "GOLIATH" project, a new research project which addresses one of the most urgent regulatory needs in the testing of endocrine-disrupting chemicals (EDCs), namely the lack of methods for testing EDCs that disrupt metabolism and metabolic functions. These chemicals collectively referred to as "metabolism disrupting compounds" (MDCs) are natural and anthropogenic chemicals that can promote metabolic changes that can ultimately result in obesity, diabetes, and/or fatty liver in humans. This project report introduces the main approaches of the project and provides a focused review of the evidence of metabolic disruption for selected EDCs. GOLIATH will generate the world's first integrated approach to testing and assessment (IATA) specifically tailored to MDCs. GOLIATH will focus on the main cellular targets of metabolic disruption-hepatocytes, pancreatic endocrine cells, myocytes and adipocytes-and using an adverse outcome pathway (AOP) framework will provide key information on MDC-related mode of action by incorporating multi-omic analyses and translating results from in silico, in vitro, and in vivo models and assays to adverse metabolic health outcomes in humans at real-life exposures. Given the importance of international acceptance of the developed test methods for regulatory use, GOLIATH will link with ongoing initiatives of the Organisation for Economic Development (OECD) for test method (pre-)validation, IATA, and AOP development.
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44.
  • Lizano Fallas, Veronica, et al. (författare)
  • Prediction of Molecular Initiating Events for Adverse Outcome Pathways Using High-Throughput Identification of Chemical Targets
  • 2023
  • Ingår i: Toxics. - : MDPI. - 2305-6304. ; 11:2
  • Tidskriftsartikel (refereegranskat)abstract
    • The impact of exposure to multiple chemicals raises concerns for human and environmental health. The adverse outcome pathway method offers a framework to support mechanism-based assessment in environmental health starting by describing which mechanisms are triggered upon interaction with different stressors. The identification of the molecular initiating event and the molecular interaction between a chemical and a protein target is still a challenge for the development of adverse outcome pathways. The cellular response to chemical exposure studied with omics could not directly identify the protein targets. However, recent mass spectrometry-based methods are offering a proteome-wide identification of protein targets interacting with s but unrevealing a molecular initiating event from a set of targets is still dependent on available knowledge. Here, we directly coupled the target identification findings from the proteome integral solubility alteration assay with an analytical hierarchy process for the prediction of a prioritized molecular initiating event. We demonstrate the applicability of this combination of methodologies with a test compound (TCDD), and it could be further studied and integrated into AOPs. From the eight protein targets identified by the proteome integral solubility alteration assay after analyzing 2824 human hepatic proteins, the analytical hierarchy process can select the most suitable protein for an AOP. Our combined method solves the missing links between high-throughput target identification and prediction of the molecular initiating event. We anticipate its utility to decipher new molecular initiating events and support more sustainable methodologies to gain time and resources in chemical assessment.
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45.
  • Lizano-Fallas, Veronica, et al. (författare)
  • Systematic analysis of chemical-protein interactions from zebrafish embryo by proteome-wide thermal shift assay, bridging the gap between molecular interactions and toxicity pathways
  • 2021
  • Ingår i: Journal of Proteomics. - : Elsevier. - 1874-3919 .- 1876-7737. ; 249
  • Tidskriftsartikel (refereegranskat)abstract
    • The molecular interaction between chemicals and proteins often promotes alteration of cellular function. One of the challenges of the toxicology is to predict the impact of exposure to chemicals. Assessing the impact of exposure implies to understand their mechanism of actions starting from identification of specific protein targets of the interaction. Current methods can mainly predict effects of characterized chemicals with knowledge of its targets, and mechanism of actions. Here, we show that proteome-wide thermal shift methods can identify chemical-protein interactions and the protein targets from bioactive chemicals. We analyzed the identified targets from a soluble proteome extracted from zebrafish embryo, that is a model system for toxicology. To evaluate the utility to predict mechanism of actions, we discussed the applicability in four cases: single chemicals, chemical mixtures, novel chemicals, and novel drugs. Our results showed that this methodology could identify the protein targets, discriminate between protein increasing and decreasing in solubility, and offering additional data to complement the map of intertwined mechanism of actions. We anticipate that the proteome integral solubility alteration (PISA) assay, as it is defined here for the unbiased identification of protein targets of chemicals could bridge the gap between molecular interactions and toxicity pathways. Significance: One of the challenges of the environmental toxicology is to predict the impact of exposure to chemicals on environment and human health. Our phenotype should be explained by our genotype and the environmental exposure. Genomic methodologies can offer a deep analysis of human genome that alone cannot explain our risks of disease. We are starting to understand the key role of exposure to chemicals on our health and risks of disease. Here, we present a proteomic-based method for the identification of soluble proteins interacting with chemicals in zebrafish embryo and discuss the opportunities to complement the map of toxicity pathway perturbations. We anticipate that this PISA assay could bridge the gap between molecular interactions and toxicity pathways.
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46.
  • Lizano Fallas, Verónica, 1985- (författare)
  • Toxicoproteomics, from finding molecular targets to evaluating the impact on human health
  • 2023
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The exposome refers to all exposures, including exposures to chemicals, that an individual may encounter over the whole life, from conception to death, that influence the individual’s health. To date, over 200,000 chemicals have been registered under the legislative framework of the European Union. Exposomics studies have revealed that individuals are exposed to chemical mixtures consisting of hundreds of compounds simultaneously. The risks to human health posed by many of these chemicals and chemical mixtures are still unknown and require evaluation. Traditional methods for assessing chemicals and chemical mixtures have been inadequate in addressing the increasing number of potentially toxic compounds in the environment. Current high-throughput toxicology methods, which involve the application of batteries of in vitro bioassays, can reduce the time and costs of analysis. However, these methods evaluate the impact on well-established pathways that have already been identified as being affected by exposure, making it difficult to discover new modes of action. The goal of this thesis is to provide a method to unravel the targets of chemicals for a better understanding of the mechanisms of action of chemicals and chemical mixtures under the scenario of the exposome. The proteome integral solubility alteration (PISA) assay is a proteome-wide approach for drug-target identification. However, implementing the PISA assay to address toxicological challenges requires different experimental considerations from chemical properties and toxicology principles. Moreover, it is necessary to translate the data from target identification to an understanding of the potential impact on human health. Therefore, three steps were followed to implement the PISA method in the field of toxicology: i) experimental considerations of the method for toxicology and chemical assessment purposes, ii) analysis of the method capability in the field of toxicology, and iii) development of pipelines from the target identification to the understanding of potential impact on human health. The results showed the capability of the PISA assay to identify the protein targets of single chemicals and chemical mixtures, extending, in an unbiased manner, the list of evaluated biological pathways in current available methodologies. The approach presented here reduces the time and cost associated with experimental and data analysis work, which could aid in the chemical risk assessment process in the context of the exposome.
  •  
47.
  • Lopes, Viviana R, et al. (författare)
  • Dose-dependent autophagic effect of titanium dioxide nanoparticles in human HaCaT cells at non-cytotoxic levels
  • 2016
  • Ingår i: Journal of Nanobiotechnology. - : Springer Science and Business Media LLC. - 1477-3155. ; 14
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Interactions between nanoparticles and cells are now the focus of a fast-growing area of research. Though many nanoparticles interact with cells without any acute toxic responses, metal oxide nanoparticles including those composed of titanium dioxide (TiO2-NPs) may disrupt the intracellular process of macroautophagy. Autophagy plays a key role in human health and disease, particularly in cancer and neurodegenerative diseases. We herein investigated the in vitro biological effects of TiO2-NPs (18 nm) on autophagy in human keratinocytes (HaCaT) cells at non-cytotoxic levels. Results: TiO2-NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering techniques. Cellular uptake, as evaluated by TEM and NanoSIMS revealed that NPs internalization led to the formation of autophagosomes. TiO2-NPs treatment did not reduce cell viability of HaCaT cells nor increased oxidative stress. Cellular autophagy was additionally evaluated by confocal microscopy using eGFP-LC3 keratinocytes, western blotting of autophagy marker LC3I/II, immunodetection of p62 and NBR1 proteins, and gene expression of LC3II, p62, NBR1, beclin1 and ATG5 by RT-qPCR. We also confirmed the formation and accumulation of autophagosomes in NPs treated cells with LC3-II upregulation. Based on the lack of degradation of p62 and NBR1 proteins, autophagosomes accumulation at a high dose (25.0 mu g/ml) is due to blockage while a low dose (0.16 mu g/ml) promoted autophagy. Cellular viability was not affected in either case. Conclusions: The uptake of TiO2-NPs led to a dose-dependent increase in autophagic effect under non-cytotoxic conditions. Our results suggest dose-dependent autophagic effect over time as a cellular response to TiO2-NPs. Most importantly, these findings suggest that simple toxicity data are not enough to understand the full impact of TiO2-NPs and their effects on cellular pathways or function.
  •  
48.
  • Marco-Ramell, Anna, et al. (författare)
  • Proteomics and the search for welfare and stress biomarkers in animal production in the one health context
  • 2016
  • Ingår i: Molecular Biosystems. - : Royal Society of Chemistry. - 1742-206X .- 1742-2051. ; 12:7, s. 2024-2035
  • Forskningsöversikt (refereegranskat)abstract
    • Stress and welfare are important factors to animal production in a context of growing production optimization and scrutiny by the general public. In a context in which animal and human health are intertwined aspects of the one-health concept it is of utmost importance to define markers for stress and welfare. These are important tools for producers, retailers, regulatory agents and ultimately consumers to effectively monitor and assess the welfare state of production animals. Proteomics is the science that studies the proteins existing in a given tissue or fluid. In this review we address this topic by showing clear examples where proteomics has been used to study stress-induced changes at various levels. We adopt a multi-species (cattle, swine, small ruminants, poultry, fish and shellfish) approach under the effect of varied stress inducers (handling, transport, management, nutritional, thermal and exposure to pollutants) clearly demonstrating how Proteomics and Systems Biology are key elements to the study of stress and welfare on farm animals and a powerful tool to animal welfare, health and productivity.
  •  
49.
  •  
50.
  • Mi, Jia, et al. (författare)
  • Age-related subproteomic analysis of mouse liver and kidney peroxisomes
  • 2007
  • Ingår i: Proteome Science. - : Springer Science and Business Media LLC. - 1477-5956. ; 5, s. 19-
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Despite major recent advances in the understanding of peroxisomal functions and how peroxisomes arise, only scant information is available regarding this organelle in cellular aging. The aim of this study was to characterize the changes in the protein expression profile of aged versus young liver and kidney peroxisome-enriched fractions from mouse and to suggest possible mechanisms underlying peroxisomal aging. Peroxisome-enriched fractions from 10 weeks, 18 months and 24 months C57bl/6J mice were analyzed by quantitative proteomics. Results: Peroxisomal proteins were enriched by differential and density gradient centrifugation and proteins were separated by two-dimensional electrophoresis (2-DE), quantified and identified by mass spectrometry (MS). In total, sixty-five proteins were identified in both tissues. Among them, 14 proteins were differentially expressed in liver and 21 proteins in kidney. The eight proteins differentially expressed in both tissues were involved in beta-oxidation, alpha-oxidation, isoprenoid biosynthesis, amino acid metabolism, and stress response. Quantitative proteomics, clustering methods, and prediction of transcription factors, all indicated that there is a decline in protein expression at 18 months and a recovery at 24 months. Conclusion: These results indicate that some peroxisomal proteins show a tissue-specific functional response to aging. This response is probably dependent on their differential regeneration capacity. The differentially expressed proteins could lead several cellular effects: such as alteration of fatty acid metabolism that could alert membrane protein functions, increase of the oxidative stress and contribute to decline in bile salt synthesis. The ability to detect age-related variations in the peroxisomal proteome can help in the search for reliable and valid aging biomarkers.
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