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Eric Lancaster - One of the best experts on this subject based on the ideXlab platform.
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antibodies to delta notch like epidermal growth factor related receptor in patients with anti tr paraneoplastic cerebellar degeneration and hodgkin lymphoma
JAMA Neurology, 2014Co-Authors: Maxwell Greene, Yongjie Lai, Nicolle Baella, Josep Dalmau, Eric LancasterAbstract:Importance The anti-Tr immune response is associated with paraneoplastic cerebellar degeneration and Hodgkin lymphoma (HL). One case series has reported that the Delta/notch-like epidermal growth factor–related receptor (DNER) is the actual target for anti-Tr antibodies, but this result has not been replicated. Objective To describe a patient with anti-Tr and confirm that DNER is the autoantigen for a series of patients with anti-Tr. Design, Setting, and Participants Observational study and analysis of biological samples for antibodies to DNER at the hospital of the University of Pennsylvania. We examined a cerebrospinal fluid sample from 1 patient with anti-Tr and serum and/or cerebrospinal fluid samples from 5 other patients with anti-Tr. Exposure Transfection of HEK293T and Hela cells to express DNER coupled to an enhanced green fluorescent Protein Tag using a plasmid previously used to detect human DNER antibodies. Results A man in his 30s with paraneoplastic cerebellar degeneration and anti-Tr underwent treatment with corticosteroids and intravenous immunoglobulin, resulting in clinical improvement before chemotherapy. Despite close oncologic follow-up, a biopsy, positron emission tomography, and computed tomography, he was not diagnosed as having HL until 6 months after symptom onset. The cerebrospinal fluid sample from this patient reacted with cells transfected to express DNER, as did cerebrospinal fluid and/or serum samples from 5 other patients with paraneoplastic cerebellar degeneration, HL, and anti-Tr. Only 4 of the 5 serum samples reacted to permeabilized cells enough to be distinguished from background, but all 5 serum samples convincingly labeled live cells, which had considerably less background. All 6 control serum samples and 1 serum sample from a patient previously diagnosed as having anti-Tr (but without HL or cerebellitis) did not recognize DNER. Conclusions and Relevance This case demonstrates the importance of testing for the anti-Tr immune response in patients with cerebellar degeneration. The strong association of anti-Tr with HL requires careful surveillance for this tumor. We also confirm that DNER is the target antigen of the anti-Tr immune response. Screening for DNER antibodies against living transfected cells may offer an improved signal-to-noise characteristic compared with immunostaining of fixed, permeabilized cells.
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antibodies to delta notch like epidermal growth factor related receptor in patients with anti tr paraneoplastic cerebellar degeneration and hodgkin lymphoma
JAMA Neurology, 2014Co-Authors: Maxwell Greene, Nicolle Baella, Josep Dalmau, Eric LancasterAbstract:Importance The anti-Tr immune response is associated with paraneoplastic cerebellar degeneration and Hodgkin lymphoma (HL). One case series has reported that the Delta/notch-like epidermal growth factor–related receptor (DNER) is the actual target for anti-Tr antibodies, but this result has not been replicated. Objective To describe a patient with anti-Tr and confirm that DNER is the autoantigen for a series of patients with anti-Tr. Design, Setting, and Participants Observational study and analysis of biological samples for antibodies to DNER at the hospital of the University of Pennsylvania. We examined a cerebrospinal fluid sample from 1 patient with anti-Tr and serum and/or cerebrospinal fluid samples from 5 other patients with anti-Tr. Exposure Transfection of HEK293T and Hela cells to express DNER coupled to an enhanced green fluorescent Protein Tag using a plasmid previously used to detect human DNER antibodies. Results A man in his 30s with paraneoplastic cerebellar degeneration and anti-Tr underwent treatment with corticosteroids and intravenous immunoglobulin, resulting in clinical improvement before chemotherapy. Despite close oncologic follow-up, a biopsy, positron emission tomography, and computed tomography, he was not diagnosed as having HL until 6 months after symptom onset. The cerebrospinal fluid sample from this patient reacted with cells transfected to express DNER, as did cerebrospinal fluid and/or serum samples from 5 other patients with paraneoplastic cerebellar degeneration, HL, and anti-Tr. Only 4 of the 5 serum samples reacted to permeabilized cells enough to be distinguished from background, but all 5 serum samples convincingly labeled live cells, which had considerably less background. All 6 control serum samples and 1 serum sample from a patient previously diagnosed as having anti-Tr (but without HL or cerebellitis) did not recognize DNER. Conclusions and Relevance This case demonstrates the importance of testing for the anti-Tr immune response in patients with cerebellar degeneration. The strong association of anti-Tr with HL requires careful surveillance for this tumor. We also confirm that DNER is the target antigen of the anti-Tr immune response. Screening for DNER antibodies against living transfected cells may offer an improved signal-to-noise characteristic compared with immunostaining of fixed, permeabilized cells.
Maxwell Greene - One of the best experts on this subject based on the ideXlab platform.
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antibodies to delta notch like epidermal growth factor related receptor in patients with anti tr paraneoplastic cerebellar degeneration and hodgkin lymphoma
JAMA Neurology, 2014Co-Authors: Maxwell Greene, Yongjie Lai, Nicolle Baella, Josep Dalmau, Eric LancasterAbstract:Importance The anti-Tr immune response is associated with paraneoplastic cerebellar degeneration and Hodgkin lymphoma (HL). One case series has reported that the Delta/notch-like epidermal growth factor–related receptor (DNER) is the actual target for anti-Tr antibodies, but this result has not been replicated. Objective To describe a patient with anti-Tr and confirm that DNER is the autoantigen for a series of patients with anti-Tr. Design, Setting, and Participants Observational study and analysis of biological samples for antibodies to DNER at the hospital of the University of Pennsylvania. We examined a cerebrospinal fluid sample from 1 patient with anti-Tr and serum and/or cerebrospinal fluid samples from 5 other patients with anti-Tr. Exposure Transfection of HEK293T and Hela cells to express DNER coupled to an enhanced green fluorescent Protein Tag using a plasmid previously used to detect human DNER antibodies. Results A man in his 30s with paraneoplastic cerebellar degeneration and anti-Tr underwent treatment with corticosteroids and intravenous immunoglobulin, resulting in clinical improvement before chemotherapy. Despite close oncologic follow-up, a biopsy, positron emission tomography, and computed tomography, he was not diagnosed as having HL until 6 months after symptom onset. The cerebrospinal fluid sample from this patient reacted with cells transfected to express DNER, as did cerebrospinal fluid and/or serum samples from 5 other patients with paraneoplastic cerebellar degeneration, HL, and anti-Tr. Only 4 of the 5 serum samples reacted to permeabilized cells enough to be distinguished from background, but all 5 serum samples convincingly labeled live cells, which had considerably less background. All 6 control serum samples and 1 serum sample from a patient previously diagnosed as having anti-Tr (but without HL or cerebellitis) did not recognize DNER. Conclusions and Relevance This case demonstrates the importance of testing for the anti-Tr immune response in patients with cerebellar degeneration. The strong association of anti-Tr with HL requires careful surveillance for this tumor. We also confirm that DNER is the target antigen of the anti-Tr immune response. Screening for DNER antibodies against living transfected cells may offer an improved signal-to-noise characteristic compared with immunostaining of fixed, permeabilized cells.
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antibodies to delta notch like epidermal growth factor related receptor in patients with anti tr paraneoplastic cerebellar degeneration and hodgkin lymphoma
JAMA Neurology, 2014Co-Authors: Maxwell Greene, Nicolle Baella, Josep Dalmau, Eric LancasterAbstract:Importance The anti-Tr immune response is associated with paraneoplastic cerebellar degeneration and Hodgkin lymphoma (HL). One case series has reported that the Delta/notch-like epidermal growth factor–related receptor (DNER) is the actual target for anti-Tr antibodies, but this result has not been replicated. Objective To describe a patient with anti-Tr and confirm that DNER is the autoantigen for a series of patients with anti-Tr. Design, Setting, and Participants Observational study and analysis of biological samples for antibodies to DNER at the hospital of the University of Pennsylvania. We examined a cerebrospinal fluid sample from 1 patient with anti-Tr and serum and/or cerebrospinal fluid samples from 5 other patients with anti-Tr. Exposure Transfection of HEK293T and Hela cells to express DNER coupled to an enhanced green fluorescent Protein Tag using a plasmid previously used to detect human DNER antibodies. Results A man in his 30s with paraneoplastic cerebellar degeneration and anti-Tr underwent treatment with corticosteroids and intravenous immunoglobulin, resulting in clinical improvement before chemotherapy. Despite close oncologic follow-up, a biopsy, positron emission tomography, and computed tomography, he was not diagnosed as having HL until 6 months after symptom onset. The cerebrospinal fluid sample from this patient reacted with cells transfected to express DNER, as did cerebrospinal fluid and/or serum samples from 5 other patients with paraneoplastic cerebellar degeneration, HL, and anti-Tr. Only 4 of the 5 serum samples reacted to permeabilized cells enough to be distinguished from background, but all 5 serum samples convincingly labeled live cells, which had considerably less background. All 6 control serum samples and 1 serum sample from a patient previously diagnosed as having anti-Tr (but without HL or cerebellitis) did not recognize DNER. Conclusions and Relevance This case demonstrates the importance of testing for the anti-Tr immune response in patients with cerebellar degeneration. The strong association of anti-Tr with HL requires careful surveillance for this tumor. We also confirm that DNER is the target antigen of the anti-Tr immune response. Screening for DNER antibodies against living transfected cells may offer an improved signal-to-noise characteristic compared with immunostaining of fixed, permeabilized cells.
Giuseppe Perugino - One of the best experts on this subject based on the ideXlab platform.
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An AGT-based Protein-Tag system for the labelling and surface immobilization of enzymes on E. coli outer membrane
Taylor & Francis Group, 2019Co-Authors: Rosa Merlo, Anna Valenti, Sonia Del Prete, Rosanna Mattossovich, Vincenzo Carginale, Claudiu T. Supuran, Clemente Capasso, Giuseppe PeruginoAbstract:The use of natural systems, such as outer membrane Protein A (OmpA), phosphoporin E (PhoE), ice nucleation Protein (INP), etc., has been proved very useful for the surface exposure of Proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advanTage of unifying in a one-step the production, the purification and the in vivo immobilisation of Proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-Protein-Tag (ASLTag), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised Proteins with the engineered alkylguanine-DNA-alkyl-transferase (H5) from Sulfolobus solfataricus. Our results demonstrated that this Tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the β-glycoside hydrolase (SsβGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest
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crystal structure of a thermophilic o6 alkylguanine dna alkyltransferase derived self labeling Protein Tag in covalent complex with a fluorescent probe
Biochemical and Biophysical Research Communications, 2018Co-Authors: Franca Rossi, Anna Valenti, Giuseppe Perugino, Castrese Morrone, Alberto Massarotti, Davide M Ferraris, Riccardo MiggianoAbstract:Abstract The self-labeling Protein Tags are robust and versatile tools for studying different molecular aspects of cell biology. In order to be suitable for a wide spectrum of experimental conditions, it is mandatory that these systems are stable after the fluorescent labeling reaction and do not alter the properties of the fusion partner. SsOGT-H5 is an engineered variant alkylguanine-DNA-alkyl-transferase (OGT) of the hyperthermophilic archaeon Sulfolobus solfataricus, and it represents an alternative solution to the SNAP-Tag® technology under harsh reaction conditions. Here we present the crystal structure of SsOGT-H5 in complex with the fluorescent probe SNAP-Vista Green® (SsOGT-H5-SVG) that reveals the conformation adopted by the Protein upon the trans-alkylation reaction with the substrate, which is observed covalently bound to the catalytic cysteine residue. Moreover, we identify the amino acids that contribute to both the overall Protein stability in the post-reaction state and the coordination of the fluorescent moiety stretching-out from the Protein active site. We gained new insights in the conformational changes possibly occurring to the OGT Proteins upon reaction with modified guanine base bearing bulky adducts; indeed, our structural analysis reveals an unprecedented conformation of the active site loop that is likely to trigger Protein destabilization and consequent degradation. Interestingly, the SVG moiety plays a key role in restoring the interaction between the N- and C-terminal domains of the Protein that is lost following the new conformation adopted by the active site loop in the SsOGT-H5-SVG structure. Molecular dynamics simulations provide further information into the dynamics of SsOGT-H5-SVG structure, highlighting the role of the fluorescent ligand in keeping the Protein stable after the trans-alkylation reaction.
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erratum to a novel thermostable Protein Tag optimization of the sulfolobus solfataricus dna alkyl transferase by Protein engineering
Extremophiles, 2016Co-Authors: Antonella Vettone, Mario Serpe, Aurelio Hidalgo, Jose Berenguer, Giovanni Del Monaco, Anna Valenti, Mose Rossi, Maria Ciaramella, Giuseppe PeruginoAbstract:In the last decade, a powerful biotechnological tool for the in vivo and in vitro specific labeling of Proteins (SNAP-Tag™ technology) was proposed as a valid alternative to classical Protein-Tags (green fluorescent Proteins, GFPs). This was made possible by the discovery of the irreversible reaction of the human alkylguanine-DNA-alkyl-transferase (hAGT) in the presence of benzyl-guanine derivatives. However, the mild reaction conditions and the general instability of the mesophilic SNAP-Tag™ make this new approach not fully applicable to (hyper-)thermophilic and, in general, extremophilic organisms. Here, we introduce an engineered variant of the thermostable alkylguanine-DNA-alkyl-transferase from the Archaea Sulfolobus solfataricus (SsOGT-H5), which displays a catalytic efficiency comparable to the SNAP-Tag™ Protein, but showing high intrinsic stability typical of Proteins from this organism. The successful heterologous expression obtained in a thermophilic model organism makes SsOGT-H5 a valid candidate as Protein-Tag for organisms living in extreme environments.
Neal J. Zondlo - One of the best experts on this subject based on the ideXlab platform.
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design of a Protein motif responsive to tyrosine nitration and an encoded turn off sensor of tyrosine nitration
Biochemistry, 2019Co-Authors: Andrew R Urmey, Neal J. ZondloAbstract:Tyrosine nitration is a Protein post-translational modification that is predominantly non-enzymatic and is observed to be increased under conditions of nitrosative stress and in numerous disease states. A small Protein motif (14-18 amino acids) responsive to tyrosine nitration has been developed. In this design, nitrotyrosine replaced the conserved Glu12 of an EF-hand metal-binding motif. Thus, the non-nitrated peptide bound terbium weakly. In contrast, tyrosine nitration resulted in a 45-fold increase in terbium affinity. Nuclear magnetic resonance spectroscopy indicated direct binding of nitrotyrosine to the metal and EF-hand-like metal contacts in this designed peptide. Nitrotyrosine is an efficient quencher of fluorescence. To develop a sensor of tyrosine nitration, the initial design was modified to incorporate Glu residues at EF-hand positions 9 and 16 as additional metal-binding residues, to increase the terbium affinity of the peptide with unmodified tyrosine. This peptide with a tyrosine at residue 12 bound terbium and effectively sensitized terbium luminescence. Tyrosine nitration resulted in a 180-fold increase in terbium affinity ( Kd = 1.6 μM) and quenching of terbium luminescence. This sequence was incorporated as an encoded Protein Tag and applied as a turn-off fluorescent Protein sensor of tyrosine nitration. The sensor was responsive to nitration by peroxynitrite, with fluorescence quenched upon nitration. The greater terbium affinity upon tyrosine nitration resulted in a large dynamic range and sensitivity to substoichiometric nitration. An improved approach for the synthesis of peptides containing nitrotyrosine was also developed, via the in situ silyl protection of nitrotyrosine. This work represents the first designed, encodable Protein motif that is responsive to tyrosine nitration.
Nicolle Baella - One of the best experts on this subject based on the ideXlab platform.
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antibodies to delta notch like epidermal growth factor related receptor in patients with anti tr paraneoplastic cerebellar degeneration and hodgkin lymphoma
JAMA Neurology, 2014Co-Authors: Maxwell Greene, Yongjie Lai, Nicolle Baella, Josep Dalmau, Eric LancasterAbstract:Importance The anti-Tr immune response is associated with paraneoplastic cerebellar degeneration and Hodgkin lymphoma (HL). One case series has reported that the Delta/notch-like epidermal growth factor–related receptor (DNER) is the actual target for anti-Tr antibodies, but this result has not been replicated. Objective To describe a patient with anti-Tr and confirm that DNER is the autoantigen for a series of patients with anti-Tr. Design, Setting, and Participants Observational study and analysis of biological samples for antibodies to DNER at the hospital of the University of Pennsylvania. We examined a cerebrospinal fluid sample from 1 patient with anti-Tr and serum and/or cerebrospinal fluid samples from 5 other patients with anti-Tr. Exposure Transfection of HEK293T and Hela cells to express DNER coupled to an enhanced green fluorescent Protein Tag using a plasmid previously used to detect human DNER antibodies. Results A man in his 30s with paraneoplastic cerebellar degeneration and anti-Tr underwent treatment with corticosteroids and intravenous immunoglobulin, resulting in clinical improvement before chemotherapy. Despite close oncologic follow-up, a biopsy, positron emission tomography, and computed tomography, he was not diagnosed as having HL until 6 months after symptom onset. The cerebrospinal fluid sample from this patient reacted with cells transfected to express DNER, as did cerebrospinal fluid and/or serum samples from 5 other patients with paraneoplastic cerebellar degeneration, HL, and anti-Tr. Only 4 of the 5 serum samples reacted to permeabilized cells enough to be distinguished from background, but all 5 serum samples convincingly labeled live cells, which had considerably less background. All 6 control serum samples and 1 serum sample from a patient previously diagnosed as having anti-Tr (but without HL or cerebellitis) did not recognize DNER. Conclusions and Relevance This case demonstrates the importance of testing for the anti-Tr immune response in patients with cerebellar degeneration. The strong association of anti-Tr with HL requires careful surveillance for this tumor. We also confirm that DNER is the target antigen of the anti-Tr immune response. Screening for DNER antibodies against living transfected cells may offer an improved signal-to-noise characteristic compared with immunostaining of fixed, permeabilized cells.
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antibodies to delta notch like epidermal growth factor related receptor in patients with anti tr paraneoplastic cerebellar degeneration and hodgkin lymphoma
JAMA Neurology, 2014Co-Authors: Maxwell Greene, Nicolle Baella, Josep Dalmau, Eric LancasterAbstract:Importance The anti-Tr immune response is associated with paraneoplastic cerebellar degeneration and Hodgkin lymphoma (HL). One case series has reported that the Delta/notch-like epidermal growth factor–related receptor (DNER) is the actual target for anti-Tr antibodies, but this result has not been replicated. Objective To describe a patient with anti-Tr and confirm that DNER is the autoantigen for a series of patients with anti-Tr. Design, Setting, and Participants Observational study and analysis of biological samples for antibodies to DNER at the hospital of the University of Pennsylvania. We examined a cerebrospinal fluid sample from 1 patient with anti-Tr and serum and/or cerebrospinal fluid samples from 5 other patients with anti-Tr. Exposure Transfection of HEK293T and Hela cells to express DNER coupled to an enhanced green fluorescent Protein Tag using a plasmid previously used to detect human DNER antibodies. Results A man in his 30s with paraneoplastic cerebellar degeneration and anti-Tr underwent treatment with corticosteroids and intravenous immunoglobulin, resulting in clinical improvement before chemotherapy. Despite close oncologic follow-up, a biopsy, positron emission tomography, and computed tomography, he was not diagnosed as having HL until 6 months after symptom onset. The cerebrospinal fluid sample from this patient reacted with cells transfected to express DNER, as did cerebrospinal fluid and/or serum samples from 5 other patients with paraneoplastic cerebellar degeneration, HL, and anti-Tr. Only 4 of the 5 serum samples reacted to permeabilized cells enough to be distinguished from background, but all 5 serum samples convincingly labeled live cells, which had considerably less background. All 6 control serum samples and 1 serum sample from a patient previously diagnosed as having anti-Tr (but without HL or cerebellitis) did not recognize DNER. Conclusions and Relevance This case demonstrates the importance of testing for the anti-Tr immune response in patients with cerebellar degeneration. The strong association of anti-Tr with HL requires careful surveillance for this tumor. We also confirm that DNER is the target antigen of the anti-Tr immune response. Screening for DNER antibodies against living transfected cells may offer an improved signal-to-noise characteristic compared with immunostaining of fixed, permeabilized cells.
