18 results on '"Kanashova, Tamara"'
Search Results
2. Patient-derived xenograft (PDX) models of colorectal carcinoma (CRC) as a platform for chemosensitivity and biomarker analysis in personalized medicine
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Rivera, Maria, Fichtner, Iduna, Wulf-Goldenberg, Annika, Sers, Christine, Merk, Johannes, Patone, Giannino, Alp, Keziban M., Kanashova, Tamara, Mertins, Philipp, Hoffmann, Jens, Stein, Ulrike, and Walther, Wolfgang
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- 2021
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3. Effect of Atmospheric Aging on Soot Particle Toxicity in Lung Cell Models at the Air-Liquid Interface: Differential Toxicological Impacts of Biogenic and Anthropogenic Secondary Organic Aerosols (SOAs)
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Offer, Svenja, Hartner, Elena, Bucchianico, Sebastiano Di, Bisig, Christoph, Bauer, Stefanie, Pantzke, Jana, Zimmermann, Elias J., Cao, Xin, Binder, Stefanie, Kuhn, Evelyn, Huber, Anja, Jeong, Seongho, Kafer, Uwe, Martens, Patrick, Mesceriakovas, Arunas, Bendl, Jan, Brejcha, Ramona, Buchholz, Angela, Gat, Daniella, Hohaus, Thorsten, Rastak, Narges, Jakobi, Gert, Kalberer, Markus, Kanashova, Tamara, Hu, Yue, Ogris, Christoph, Marsico, Annalisa, Theis, Fabian, Pardo, Michal, Groger, Thomas, Oeder, Sebastian, Orasche, Jurgen, Paul, Andreas, Ziehm, Till, Zhang, Zhi-Hui, Adam, Thomas, Sippula, Olli, Sklorz, Martin, Schnelle-Kreis, Jurgen, Czech, Hendryk, Kiendler-Scharr, Astrid, Rudich, Yinon, and Zimmermann, Ralf
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Lung diseases -- Risk factors -- Development and progression ,Aerosols -- Environmental aspects -- Health aspects ,Air pollution -- Health aspects ,Soot -- Environmental aspects -- Health aspects ,Environmental issues ,Health - Abstract
Background: Secondary organic aerosols (SOAs) formed from anthropogenic or biogenic gaseous precursors in the atmosphere substantially contribute to the ambient fine particulate matter [PM [less than or equal to] 2.5 [micro]m in aerodynamic diameter (P[M.sub.2.5])] burden, which has been associated with adverse human health effects. However, there is only limited evidence on their differential toxicological impact. OBJECTIVES: We aimed to discriminate toxicological effects of aerosols generated by atmospheric aging on combustion soot particles (SPs) of gaseous biogenic ([beta]-pinene) or anthropogenic (naphthalene) precursors in two different lung cell models exposed at the air-liquid interface (ALI). Methods: Mono- or cocultures of lung epithelial cells (A549) and endothelial cells (EA.hy926) were exposed at the ALI for 4 h to different aerosol concentrations of a photochemically aged mixture of primary combustion SP and [beta]-pinene ([SOA.sub.[beta]PIN]-SP) or naphthalene ([SOA.sub.NAP]-SP). The internally mixed soot/SOA particles were comprehensively characterized in terms of their physical and chemical properties. We conducted toxicity tests to determine cytotoxicity, intracellular oxidative stress, primary and secondary genotoxicity, as well as inflammatory and angiogenic effects. Results: We observed considerable toxicity-related outcomes in cells treated with either SOA type. Greater adverse effects were measured for [SOA.sub.NAP]-SP compared with [SOA.sub.[beta]PIN]-SP in both cell models, whereas the nano-sized soot cores alone showed only minor effects. At the functional level, we found that [SOA.sub.NAP]-SP augmented the secretion of malondialdehyde and interleukin-8 and may have induced the activation of endothelial cells in the coculture system. This activation was confirmed by comet assay, suggesting secondary genotoxicity and greater angiogenic potential. Chemical characterization of PM revealed distinct qualitative differences in the composition of the two secondary aerosol types. Discussion: In this study using A549 and EA.hy926 cells exposed at ALI, SOA compounds had greater toxicity than primary SPs. Photochemical aging of naphthalene was associated with the formation of more oxidized, more aromatic SOAs with a higher oxidative potential and toxicity compared with [beta]-pinene. Thus, we conclude that the influence of atmospheric chemistry on the chemical PM composition plays a crucial role for the adverse health outcome of emissions. https://doi.org/10.1289/EHP9413, Introduction Over the past decades, the impact of ambient fine particulate matter [PM [less than or equal to] 2.5 [micro]m (P[M.sub.2.5])] on the global burden of disease has gained substantial [...]
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- 2022
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4. Influence of wood species on toxicity of log-wood stove combustion aerosols: a parallel animal and air-liquid interface cell exposure study on spruce and pine smoke
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Ihantola, Tuukka, Di Bucchianico, Sebastiano, Happo, Mikko, Ihalainen, Mika, Uski, Oskari, Bauer, Stefanie, Kuuspalo, Kari, Sippula, Olli, Tissari, Jarkko, Oeder, Sebastian, Hartikainen, Anni, Rönkkö, Teemu J., Martikainen, Maria-Viola, Huttunen, Kati, Vartiainen, Petra, Suhonen, Heikki, Kortelainen, Miika, Lamberg, Heikki, Leskinen, Ari, Sklorz, Martin, Michalke, Bernhard, Dilger, Marco, Weiss, Carsten, Dittmar, Gunnar, Beckers, Johannes, Irmler, Martin, Buters, Jeroen, Candeias, Joana, Czech, Hendryk, Yli-Pirilä, Pasi, Abbaszade, Gülcin, Jakobi, Gert, Orasche, Jürgen, Schnelle-Kreis, Jürgen, Kanashova, Tamara, Karg, Erwin, Streibel, Thorsten, Passig, Johannes, Hakkarainen, Henri, Jokiniemi, Jorma, Zimmermann, Ralf, Hirvonen, Maija-Riitta, and Jalava, Pasi I.
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- 2020
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5. Senescence-associated reprogramming promotes cancer stemness
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Milanovic, Maja, Fan, Dorothy N. Y., Belenki, Dimitri, Dbritz, J. Henry M., Zhao, Zhen, Yu, Yong, Drr, Jan R., Dimitrova, Lora, Lenze, Dido, Monteiro Barbosa, Ines A., Mendoza-Parra, Marco A., Kanashova, Tamara, Metzner, Marlen, Pardon, Katharina, Reimann, Maurice, Trumpp, Andreas, Drken, Bernd, Zuber, Johannes, Gronemeyer, Hinrich, Hummel, Michael, Dittmar, Gunnar, Lee, Soyoung, and Schmitt, Clemens A.
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Cancer -- Physiological aspects ,Aging (Biology) -- Health aspects ,Cancer cells -- Physiological aspects ,Stem cells -- Health aspects ,Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
Author(s): Maja Milanovic [1]; Dorothy N. Y. Fan [1, 2, 3, 4]; Dimitri Belenki [1]; J. Henry M. Dbritz [1]; Zhen Zhao [5]; Yong Yu [6]; Jan R. Drr [1]; [...]
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- 2018
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6. Stable Isotope-Assisted Untargeted Metabolomics Identifies ALDH1A1-Driven Erythronate Accumulation in Lung Cancer Cells.
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Zhang, Jie, Keibler, Mark A., Dong, Wentao, Ghelfi, Jenny, Cordes, Thekla, Kanashova, Tamara, Pailot, Arnaud, Linster, Carole L., Dittmar, Gunnar, Metallo, Christian M., Lautenschlaeger, Tim, Hiller, Karsten, and Stephanopoulos, Gregory
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CANCER cells ,PENTOSE phosphate pathway ,LUNG cancer ,PROTEIN fractionation ,ALDEHYDE dehydrogenase ,DEHYDROGENASES - Abstract
Using an untargeted stable isotope-assisted metabolomics approach, we identify erythronate as a metabolite that accumulates in several human cancer cell lines. Erythronate has been reported to be a detoxification product derived from off-target glycolytic metabolism. We use chemical inhibitors and genetic silencing to define the pentose phosphate pathway intermediate erythrose 4-phosphate (E4P) as the starting substrate for erythronate production. However, following enzyme assay-coupled protein fractionation and subsequent proteomics analysis, we identify aldehyde dehydrogenase 1A1 (ALDH1A1) as the predominant contributor to erythrose oxidation to erythronate in cell extracts. Through modulating ALDH1A1 expression in cancer cell lines, we provide additional support. We hence describe a possible alternative route to erythronate production involving the dephosphorylation of E4P to form erythrose, followed by its oxidation by ALDH1A1. Finally, we measure increased erythronate concentrations in tumors relative to adjacent normal tissues from lung cancer patients. These findings suggest the accumulation of erythronate to be an example of metabolic reprogramming in cancer cells, raising the possibility that elevated levels of erythronate may serve as a biomarker of certain types of cancer. [ABSTRACT FROM AUTHOR]
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- 2023
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7. Simultaneous extraction of proteins and metabolites from cells in culture
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Sapcariu, Sean C., Kanashova, Tamara, Weindl, Daniel, Ghelfi, Jenny, Dittmar, Gunnar, and Hiller, Karsten
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- 2014
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8. Differential proteomic analysis of mouse macrophages exposed to adsorbate-loaded heavy fuel oil derived combustion particles using an automated sample-preparation workflow
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Kanashova, Tamara, Popp, Oliver, Orasche, Jürgen, Karg, Erwin, Harndorf, Horst, Stengel, Benjamin, Sklorz, Martin, Streibel, Thorsten, Zimmermann, Ralf, and Dittmar, Gunnar
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- 2015
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9. Isolation of native plasma membrane H+‐ATPase (Pma1p) in both the active and basal activation states.
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Pedersen, Jesper Torbøl, Kanashova, Tamara, Dittmar, Gunnar, and Palmgren, Michael
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CELL membranes ,ADENOSINE triphosphatase ,POLYMERSOMES ,PROTEIN hydrolysates ,SACCHAROMYCES cerevisiae - Abstract
The yeast plasma membrane H
+ ‐ATPase Pma1p is a P‐type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between ATP hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H+ ‐ATPases are autoinhibited. [ABSTRACT FROM AUTHOR]- Published
- 2018
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10. Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p.
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Laub, Katrine Rude, Marek, Magdalena, Stanchev, Lyubomir Dimitrov, Herrera, Sara Abad, Kanashova, Tamara, Bourmaud, Adèle, Dittmar, Gunnar, and Günther Pomorski, Thomas
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CELL membranes ,ATP-binding cassette transporters ,SACCHAROMYCES cerevisiae ,STEROLS analysis ,GREEN fluorescent protein - Abstract
The ATP binding cassette (ABC) transporters Pdr11p and its paralog Aus1p are expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and are required for sterol uptake. However, the precise mechanism by which these ABC transporters facilitate sterol movement is unknown. In this study, an overexpression and purification procedure was developed with the aim to characterise the Pdr11p transporter. Engineering of Pdr11p variants fused at the C terminus with green fluorescent protein (Pdr11p-GFP) and containing a FLAG tag at the N terminus facilitated expression analysis and one-step purification, respectively. The detergent-solubilised and purified protein displayed a stable ATPase activity with a broad pH optimum near 7.4. Mutagenesis of the conserved lysine to methionine (K788M) in the Walker A motif abolished ATP hydrolysis. Remarkably, and in contrast to Aus1p, ATPase activity of Pdr11p was insensitive to orthovanadate and not specifically stimulated by phosphatidylserine upon reconstitution into liposomes. Our results highlight distinct differences between Pdr11p and Aus1p and create an experimental basis for further biochemical studies of both ABC transporters to elucidate their function. [ABSTRACT FROM AUTHOR]
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- 2017
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11. Metabolic Profiling as Well as Stable Isotope Assisted Metabolic and Proteomic Analysis of RAW 264.7 Macrophages Exposed to Ship Engine Aerosol Emissions: Different Effects of Heavy Fuel Oil and Refined Diesel Fuel.
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Sapcariu, Sean C., Kanashova, Tamara, Dilger, Marco, Diabaté, Silvia, Oeder, Sebastian, Passig, Johannes, Radischat, Christian, Buters, Jeroen, Sippula, Olli, Streibel, Thorsten, Paur, Hanns-Rudolf, Schlager, Christoph, Mülhopt, Sonja, Stengel, Benjamin, Rabe, Rom, Harndorf, Horst, Krebs, Tobias, Karg, Erwin, Gröger, Thomas, and Weiss, Carsten
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AIR pollution , *HEALTH , *FOSSIL fuels , *COMBUSTION , *STABLE isotopes , *PROTEOMICS , *MACROPHAGES , *ATMOSPHERIC aerosols , *METABOLISM - Abstract
Exposure to air pollution resulting from fossil fuel combustion has been linked to multiple short-term and long term health effects. In a previous study, exposure of lung epithelial cells to engine exhaust from heavy fuel oil (HFO) and diesel fuel (DF), two of the main fuels used in marine engines, led to an increased regulation of several pathways associated with adverse cellular effects, including pro-inflammatory pathways. In addition, DF exhaust exposure was shown to have a wider response on multiple cellular regulatory levels compared to HFO emissions, suggesting a potentially higher toxicity of DF emissions over HFO. In order to further understand these effects, as well as to validate these findings in another cell line, we investigated macrophages under the same conditions as a more inflammation-relevant model. An air-liquid interface aerosol exposure system was used to provide a more biologically relevant exposure system compared to submerged experiments, with cells exposed to either the complete aerosol (particle and gas phase), or the gas phase only (with particles filtered out). Data from cytotoxicity assays were integrated with metabolomics and proteomics analyses, including stable isotope-assisted metabolomics, in order to uncover pathways affected by combustion aerosol exposure in macrophages. Through this approach, we determined differing phenotypic effects associated with the different components of aerosol. The particle phase of diluted combustion aerosols was found to induce increased cell death in macrophages, while the gas phase was found more to affect the metabolic profile. In particular, a higher cytotoxicity of DF aerosol emission was observed in relation to the HFO aerosol. Furthermore, macrophage exposure to the gas phase of HFO leads to an induction of a pro-inflammatory metabolic and proteomic phenotype. These results validate the effects found in lung epithelial cells, confirming the role of inflammation and cellular stress in the response to combustion aerosols. [ABSTRACT FROM AUTHOR]
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- 2016
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12. Muscle RING-finger 2 and 3 maintain striated-muscle structure and function.
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Lodka, Dörte, Pahuja, Aanchal, Geers ‐ Knörr, Cornelia, Scheibe, Renate J., Nowak, Marcel, Hamati, Jida, Köhncke, Clemens, Purfürst, Bettina, Kanashova, Tamara, Schmidt, Sibylle, Glass, David J., Morano, Ingo, Heuser, Arnd, Kraft, Theresia, Bassel ‐ Duby, Rhonda, Olson, Eric N., Dittmar, Gunnar, Sommer, Thomas, and Fielitz, Jens
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FINGERS ,STRIATED muscle ,UBIQUITIN ligases ,BIOLOGICAL adaptation ,CELL culture - Abstract
Background The Muscle-specific RING-finger (MuRF) protein family of E3 ubiquitin ligases is important for maintenance of muscular structure and function. MuRF proteins mediate adaptation of striated muscles to stress. MuRF2 and MuRF3 bind to microtubules and are implicated in sarcomere formation with noticeable functional redundancy. However, if this redundancy is important for muscle function in vivo is unknown. Our objective was to investigate cooperative function of MuRF2 and MuRF3 in the skeletal muscle and the heart in vivo. Methods MuRF2 and MuRF3 double knockout mice (DKO) were generated and phenotypically characterized. Skeletal muscle and the heart were investigated by morphological measurements, histological analyses, electron microscopy, immunoblotting, and real-time PCR. Isolated muscles were subjected to in vitro force measurements. Cardiac function was determined by echocardiography and working heart preparations. Function of cardiomyocytes was measured in vitro. Cell culture experiments and mass-spectrometry were used for mechanistic analyses. Results DKO mice showed a protein aggregate myopathy in skeletal muscle. Maximal force development was reduced in DKO soleus and extensor digitorum longus. Additionally, a fibre type shift towards slow/type I fibres occurred in DKO soleus and extensor digitorum longus. MuRF2 and MuRF3-deficient hearts showed decreased systolic and diastolic function. Further analyses revealed an increased expression of the myosin heavy chain isoform beta/slow and disturbed calcium handling as potential causes for the phenotype in DKO hearts. Conclusions The redundant function of MuRF2 and MuRF3 is important for maintenance of skeletal muscle and cardiac structure and function in vivo. [ABSTRACT FROM AUTHOR]
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- 2016
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13. Particulate Matter from Both Heavy Fuel Oil and Diesel Fuel Shipping Emissions Show Strong Biological Effects on Human Lung Cells at Realistic and Comparable In Vitro Exposure Conditions.
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Oeder, Sebastian, Kanashova, Tamara, Sippula, Olli, Sapcariu, Sean C., Streibel, Thorsten, Arteaga-Salas, Jose Manuel, Passig, Johannes, Dilger, Marco, Paur, Hanns-Rudolf, Schlager, Christoph, Mülhopt, Sonja, Diabaté, Silvia, Weiss, Carsten, Stengel, Benjamin, Rabe, Rom, Harndorf, Horst, Torvela, Tiina, Jokiniemi, Jorma K., Hirvonen, Maija-Riitta, and Schmidt-Weber, Carsten
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LUNG diseases , *PARTICULATE matter , *DIESEL fuels , *OXIDATIVE stress , *CELLULAR signal transduction , *PROTEOMICS - Abstract
Background: Ship engine emissions are important with regard to lung and cardiovascular diseases especially in coastal regions worldwide. Known cellular responses to combustion particles include oxidative stress and inflammatory signalling. Objectives: To provide a molecular link between the chemical and physical characteristics of ship emission particles and the cellular responses they elicit and to identify potentially harmful fractions in shipping emission aerosols. Methods: Through an air-liquid interface exposure system, we exposed human lung cells under realistic in vitro conditions to exhaust fumes from a ship engine running on either common heavy fuel oil (HFO) or cleaner-burning diesel fuel (DF). Advanced chemical analyses of the exhaust aerosols were combined with transcriptional, proteomic and metabolomic profiling including isotope labelling methods to characterise the lung cell responses. Results: The HFO emissions contained high concentrations of toxic compounds such as metals and polycyclic aromatic hydrocarbon, and were higher in particle mass. These compounds were lower in DF emissions, which in turn had higher concentrations of elemental carbon (“soot”). Common cellular reactions included cellular stress responses and endocytosis. Reactions to HFO emissions were dominated by oxidative stress and inflammatory responses, whereas DF emissions induced generally a broader biological response than HFO emissions and affected essential cellular pathways such as energy metabolism, protein synthesis, and chromatin modification. Conclusions: Despite a lower content of known toxic compounds, combustion particles from the clean shipping fuel DF influenced several essential pathways of lung cell metabolism more strongly than particles from the unrefined fuel HFO. This might be attributable to a higher soot content in DF. Thus the role of diesel soot, which is a known carcinogen in acute air pollution-induced health effects should be further investigated. For the use of HFO and DF we recommend a reduction of carbonaceous soot in the ship emissions by implementation of filtration devices. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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14. Exposure to naphthalene and β-pinene-derived secondary organic aerosol induced divergent changes in transcript levels of BEAS-2B cells.
- Author
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Pardo M, Offer S, Hartner E, Di Bucchianico S, Bisig C, Bauer S, Pantzke J, Zimmermann EJ, Cao X, Binder S, Kuhn E, Huber A, Jeong S, Käfer U, Schneider E, Mesceriakovas A, Bendl J, Brejcha R, Buchholz A, Gat D, Hohaus T, Rastak N, Karg E, Jakobi G, Kalberer M, Kanashova T, Hu Y, Ogris C, Marsico A, Theis F, Shalit T, Gröger T, Rüger CP, Oeder S, Orasche J, Paul A, Ziehm T, Zhang ZH, Adam T, Sippula O, Sklorz M, Schnelle-Kreis J, Czech H, Kiendler-Scharr A, Zimmermann R, and Rudich Y
- Abstract
The health effects of exposure to secondary organic aerosols (SOAs) are still limited. Here, we investigated and compared the toxicities of soot particles (SP) coated with β-pinene SOA (SOA
βPin -SP) and SP coated with naphthalene SOA (SOANap -SP) in a human bronchial epithelial cell line (BEAS-2B) residing at the air-liquid interface. SOAβPin -SP mostly contained oxygenated aliphatic compounds from β-pinene photooxidation, whereas SOANap -SP contained a significant fraction of oxygenated aromatic products under similar conditions. Following exposure, genome-wide transcriptome responses showed an Nrf2 oxidative stress response, particularly for SOANap -SP. Other signaling pathways, such as redox signaling, inflammatory signaling, and the involvement of matrix metalloproteinase, were identified to have a stronger impact following exposure to SOANap -SP. SOANap -SP also induced a stronger genotoxicity response than that of SOAβPin -SP. This study elucidated the mechanisms that govern SOA toxicity and showed that, compared to SOAs derived from a typical biogenic precursor, SOAs from a typical anthropogenic precursor have higher toxicological potency, which was accompanied with the activation of varied cellular mechanisms, such as aryl hydrocarbon receptor. This can be attributed to the difference in chemical composition; specifically, the aromatic compounds in the naphthalene-derived SOA had higher cytotoxic potential than that of the β-pinene-derived SOA., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)- Published
- 2022
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15. Pairwise Stimulations of Pathogen-Sensing Pathways Predict Immune Responses to Multi-adjuvant Combinations.
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Pandey S, Gruenbaum A, Kanashova T, Mertins P, Cluzel P, and Chevrier N
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- Adjuvants, Immunologic pharmacology, Animals, Female, Immunity immunology, Immunity, Innate immunology, Immunotherapy methods, Male, Mice, Mice, Inbred C57BL, Receptors, Pattern Recognition immunology, Immunity physiology, Immunity, Innate physiology, Receptors, Pattern Recognition physiology
- Abstract
The immune system makes decisions in response to combinations of multiple microbial inputs. We do not understand the combinatorial logic governing how higher-order combinations of microbial signals shape immune responses. Here, using coculture experiments and statistical analyses, we discover a general property for the combinatorial sensing of microbial signals, whereby the effects of triplet combinations of microbial signals on immune responses can be predicted by combining the effects of single and pairs. Mechanistically, we find that singles and pairs dictate the information signaled by triplets in mouse and human DCs at the levels of transcription, chromatin, and protein secretion. We exploit this simplifying property to develop cell-based immunotherapies prepared with adjuvant combinations that trigger protective responses in mouse models of cancer. We conclude that the processing of multiple input signals by innate immune cells is governed by pairwise effects, which will inform the rationale combination of adjuvants to manipulate immunity., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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16. TMT Labeling for the Masses: A Robust and Cost-efficient, In-solution Labeling Approach.
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Zecha J, Satpathy S, Kanashova T, Avanessian SC, Kane MH, Clauser KR, Mertins P, Carr SA, and Kuster B
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- HeLa Cells, Humans, Jurkat Cells, Peptides metabolism, Proteome metabolism, Proteomics, Reference Standards, Reproducibility of Results, Cost-Benefit Analysis, Isotope Labeling economics, Mass Spectrometry
- Abstract
Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an experiment. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800 μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concentrations. When reaction volumes were reduced to maintain TMT and peptide concentrations of at least 10 mm and 2 g/l, respectively, TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlaboratory reproducibility. The utility of the optimized protocol was further demonstrated in a deep-scale proteome and phosphoproteome analysis of patient-derived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chemistry and provide guidance on adopting efficient labeling protocols for different peptide quantities., (© 2019 Zecha et al.)
- Published
- 2019
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17. Isolation of native plasma membrane H + -ATPase (Pma1p) in both the active and basal activation states.
- Author
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Pedersen JT, Kanashova T, Dittmar G, and Palmgren M
- Abstract
The yeast plasma membrane H
+ -ATPase Pma1p is a P-type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between ATP hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H+ -ATPases are autoinhibited.- Published
- 2018
- Full Text
- View/download PDF
18. Protein kinase Ymr291w/Tda1 is essential for glucose signaling in saccharomyces cerevisiae on the level of hexokinase isoenzyme ScHxk2 phosphorylation*.
- Author
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Kaps S, Kettner K, Migotti R, Kanashova T, Krause U, Rödel G, Dittmar G, and Kriegel TM
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- Amino Acid Sequence, Gene Expression Regulation, Fungal, Hexokinase biosynthesis, Hexokinase metabolism, Isoenzymes genetics, Kluyveromyces, Phosphorylation, Protein Serine-Threonine Kinases biosynthesis, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins biosynthesis, Saccharomyces cerevisiae Proteins metabolism, Serine genetics, Signal Transduction genetics, Substrate Specificity, Glucose metabolism, Glycolysis, Hexokinase genetics, Protein Serine-Threonine Kinases genetics, Saccharomyces cerevisiae Proteins genetics
- Abstract
The enzyme ScHxk2 of Saccharomyces cerevisiae is a dual-function hexokinase that besides its catalytic role in glycolysis is involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by the phosphorylation of the nuclear fraction of ScHxk2 at serine 15 and the translocation of the phosphoenzyme into the cytosol. Different studies suggest different serine/threonine protein kinases, Ymr291w/Tda1 or Snf1, to accomplish ScHxk2-S15 phosphorylation. The current paper provides evidence that Ymr291w/Tda1 is essential for that modification, whereas protein kinases Ydr477w/Snf1, Ynl307c/Mck1, Yfr014c/Cmk1, and Ykl126w/Ypk1, which are co-purified during Ymr291w/Tda1 tandem affinity purification, as well as protein kinase PKA and PKB homolog Sch9 are dispensable. Taking into account the detection of a significantly higher amount of the Ymr291w/Tda1 protein in cells grown in low-glucose media as compared with a high-glucose environment, Ymr291w/Tda1 is likely to contribute to glucose signaling in S. cerevisiae on the level of ScHxk2-S15 phosphorylation in a situation of limited external glucose availability. The evolutionary conservation of amino acid residue serine 15 in yeast hexokinases and its phosphorylation is illustrated by the finding that YMR291W/TDA1 of S. cerevisiae and the homologous KLLA0A09713 gene of Kluyveromyces lactis allow for cross-complementation of the respective protein kinase single-gene deletion strains., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
- Full Text
- View/download PDF
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