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1
Ernkvist, Mira, Nathalie Luna Persson, Stéphane Audebert, Patrick Lecine, Indranil Sinha, Miaoliang Liu, Marc Schlueter, et al. "The Amot/Patj/Syx signaling complex spatially controls RhoA GTPase activity in migrating endothelial cells." Blood 113, no. 1 (January 1, 2009): 244–53. http://dx.doi.org/10.1182/blood-2008-04-153874.
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Abstract Controlled regulation of Rho GTPase activity is an essential component mediating growth factor–stimulated migration. We have previously shown that angiomotin (Amot), a membrane-associated scaffold protein, plays a critical role during vascular patterning and endothelial migration during embryogenesis. However, the signaling pathways by which Amot controls directional migration are not known. Here we have used peptide pull-down and yeast 2-hybrid (Y2H) screening to identify proteins that interact with the C-terminal PDZ-binding motifs of Amot and its related proteins AmotL1 and 2. We report that Amot and its related proteins bind to the RhoA GTPase exchange factor (RhoGEF) protein Syx. We show that Amot forms a ternary complex together with Patj (or its paralogue Mupp1) and Syx. Using FRET analysis, we provide evidence that Amot controls targeting of RhoA activity to lamellipodia in vitro. We also report that, similar to Amot, morpholino knockdown of Syx in zebrafish results in inhibition of migration of intersegmental arteries. Taken together, our results indicate that the directional migration of capillaries in the embryo is governed by the Amot:Patj/Mupp1:Syx signaling that controls local GTPase activity.
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Cox, Christopher M., Edward K. Mandell, Lorraine Stewart, Ruifeng Lu, Debra L. Johnson, Sarah D. McCarter, Andre Tavares, Ray Runyan, Sourav Ghosh, and Jean M. Wilson. "Endosomal regulation of contact inhibition through the AMOT:YAP pathway." Molecular Biology of the Cell 26, no. 14 (July 5, 2015): 2673–84. http://dx.doi.org/10.1091/mbc.e15-04-0224.
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Contact-mediated inhibition of cell proliferation is an essential part of organ growth control; the transcription coactivator Yes-associated protein (YAP) plays a pivotal role in this process. In addition to phosphorylation-dependent regulation of YAP, the integral membrane protein angiomotin (AMOT) and AMOT family members control YAP through direct binding. Here we report that regulation of YAP activity occurs at the endosomal membrane through a dynamic interaction of AMOT with an endosomal integral membrane protein, endotubin (EDTB). EDTB interacts with both AMOT and occludin and preferentially associates with occludin in confluent cells but with AMOT family members in subconfluent cells. EDTB competes with YAP for binding to AMOT proteins in subconfluent cells. Overexpression of the cytoplasmic domain or full-length EDTB induces translocation of YAP to the nucleus, an overgrowth phenotype, and growth in soft agar. This increase in proliferation is dependent upon YAP activity and is complemented by overexpression of p130-AMOT. Furthermore, overexpression of EDTB inhibits the AMOT:YAP interaction. EDTB and AMOT have a greater association in subconfluent cells compared with confluent cells, and this association is regulated at the endosomal membrane. These data provide a link between the trafficking of tight junction proteins through endosomes and contact-inhibition-regulated cell growth.
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Wang, Chenji, Jian An, Pingzhao Zhang, Chen Xu, Kun Gao, Di Wu, Dejie Wang, Hongxiu Yu, Jun O. Liu, and Long Yu. "The Nedd4-like ubiquitin E3 ligases target angiomotin/p130 to ubiquitin-dependent degradation." Biochemical Journal 444, no. 2 (May 11, 2012): 279–89. http://dx.doi.org/10.1042/bj20111983.
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AMOT (angiomotin) is a membrane-associated protein that is expressed in ECs (endothelial cells) and controls migration, TJ (tight junction) formation, cell polarity and angiogenesis. Recent studies have revealed that AMOT and two AMOT-like proteins, AMOTL1 and AMOTL2, play critical roles in the Hippo pathway by regulating the subcellular localization of the co-activators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif). However, it has been unclear how AMOT is regulated. In the present study, we report that AMOT undergoes proteasomal degradation. We identify three members of Nedd4 (neural-precursor-cell-expressed developmentally down-regulated)-like ubiquitin E3 ligases, Nedd4, Nedd4-2 and Itch, as the ubiquitin E3 ligases for the long isoform of AMOT, AMOT/p130. We demonstrate that Nedd4, Nedd4-2 and Itch mediate poly-ubiquitination of AMOT/p130 in vivo. Overexpression of Nedd4, Nedd4-2 or Itch leads to AMOT/p130 proteasomal degradation. Knockdown of Nedd4, Nedd4-2 and Itch causes an accumulation of steady-state level of AMOT/p130. We also show that three L/P-PXY motifs of AMOT/p130 and the WW domains of Nedd4 mediate their interaction. Furthermore, Nedd4-like ubiquitin E3 ligases might compete with YAP for the binding to AMOT/p130, and subsequently targeting AMOT/p130 for ubiquitin-dependent degradation. Together, these observations reveal a novel post-translational regulatory mechanism of AMOT/p130.
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Han, Ziying, Gordon Ruthel, Shantoshini Dash, Corbett T. Berry, Bruce D. Freedman, Ronald N. Harty, and Olena Shtanko. "Angiomotin regulates budding and spread of Ebola virus." Journal of Biological Chemistry 295, no. 25 (May 7, 2020): 8596–601. http://dx.doi.org/10.1074/jbc.ac120.013171.
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The Ebola virus (EBOV) VP40 matrix protein (eVP40) orchestrates assembly and budding of virions in part by hijacking select WW-domain–bearing host proteins via its PPxY late (L)-domain motif. Angiomotin (Amot) is a multifunctional PPxY-containing adaptor protein that regulates angiogenesis, actin dynamics, and cell migration/motility. Amot also regulates the Hippo signaling pathway via interactions with the WW-domain–containing Hippo effector protein Yes-associated protein (YAP). In this report, we demonstrate that endogenous Amot is crucial for positively regulating egress of eVP40 virus-like particles (VLPs) and for egress and spread of authentic EBOV. Mechanistically, we show that ectopic YAP expression inhibits eVP40 VLP egress and that Amot co-expression rescues budding of eVP40 VLPs in a dose-dependent and PPxY-dependent manner. Moreover, results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot's role in actin organization and dynamics also contributes to promoting eVP40-mediated egress. In summary, these findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. We propose that our results have wide-ranging implications for understanding the biology and pathology of EBOV infections.
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Shen, Pengfei, Hao Zeng, Angelica Ortiz, Chien-Jui Cheng, Yu-Chen Lee, Guoyu Yu, Song-Chang Lin, Li-Yuan Yu-Lee, and Sue-Hwa Lin. "Angiomotin to regulate prostate cancer cell proliferation by signaling through Hippo-YAP pathway." Journal of Clinical Oncology 35, no. 6_suppl (February 20, 2017): e559-e559. http://dx.doi.org/10.1200/jco.2017.35.6_suppl.e559.
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e559 Background: Angiomotin (AMOT) is a family of proteins found to be a component of the apical junctional complex of vertebrate epithelial cells and is recently found to play important roles in neurofibromatosis type 2 (NF-2). Whether AMOT plays a role in prostate cancer (PCa) is unknown. Methods: Purified GST-AMOTp80 was used as immunogen for antibody generation. Real-time PCR, western blot and immunohistochemistry were used to identify the expression of AMOT. To study the function of AMOT, retroviral vector were constructed, also shRNA was used to knockdown AMOT in cells. Cell migration and invasion assays were performed by using transwell chambers. Nuclear and cytoplasmic protein fractions were prepared by using NE-PER reagents (Pierce). The SPSS 19.0 software was used for statistical analysis. Chi-square test and t test were used for the comparisons between groups. Results: AMOT is expressed as two isoforms, AMOTp80 and AMOTp130, which has a 409 aa N-terminal domain that is absent in AMOTp80. Both AMOTp80 and AMOTp130 are expressed in LNCaP and C4-2B4, but at a low to undetectable level in PC3 cells. Further study showed that AMOTp130 and AMOTp80 have distinct functions in PCa cells. We found that AMOTp80 functioned as a tumor promoter by enhancing PCa cell proliferation while AMOTp130 did not. Mechanistic studies showed that AMOTp80 signaled through the Hippo pathway by promoting the nuclear translocation of YAP, resulting in an increased expression of YAP target protein BMP4. Moreover, inhibition of BMP receptor activity by LDN-193189 abrogates AMOTp80-mediated cell proliferation. Conclusions: Together, this study reveals a novel mechanism whereby the AMOTp80-Merlin-MST1-LATS-YAP-BMP4 pathway leads to AMOTp80-induced tumor cell proliferation.
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Wigerius, Michael, Dylan Quinn, and James P. Fawcett. "Emerging roles for angiomotin in the nervous system." Science Signaling 13, no. 655 (October 27, 2020): eabc0635. http://dx.doi.org/10.1126/scisignal.abc0635.
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Angiomotins are a family of molecular scaffolding proteins that function to organize contact points (called tight junctions in vertebrates) between adjacent cells. Some angiomotin isoforms bind to the actin cytoskeleton and are part of signaling pathways that influence cell morphology and migration. Others cooperate with components of the Hippo signaling pathway and the associated networks to control organ growth. The 130-kDa isoform, AMOT-p130, has critical roles in neural stem cell differentiation, dendritic patterning, and synaptic maturation—attributes that are essential for normal brain development and are consistent with its association with autism. Here, we review and discuss the evidence that supports a role for AMOT-p130 in neuronal development in the central nervous system.
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Peck, Cameron James, Piia Virtanen, Derrick Johnson, and Ann Kimble-Hill. "Using the Predicted Structure of the Amot Coiled Coil Homology Domain to Understand Lipid Binding." IU Journal of Undergraduate Research 4, no. 1 (December 16, 2018): 27–46. http://dx.doi.org/10.14434/iujur.v4i1.24528.
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Angiomotins (Amots) are a family of adapter proteins that modulate cellular polarity, differentiation, proliferation, and migration. Amot family members also have a characteristic lipid-binding domain, the coiled coil homology (ACCH) domain that selectively targets the protein to membranes, which has been directly linked to its regulatory role in the cell. Several spot blot assays were used to validate the regions of the domain that participate in its membrane association, deformation, and vesicle fusion activity, which suggested the need for a structure to define the mechanism. Therefore, we endeavored to understand the structure-function relationship of this domain with the desire to find ways to modulate these signaling pathways. After many failed attempts to crystallize the ACCH domain of each of the Amot family members for structural analysis, we decided to pursue homologous models that could be refined using small angle x-ray scattering data. Theoretical models were produced using the homology software SWISS-MODEL and threading software I-TASSER and LOMETS, followed by comparison to SAXS data for model selection and refinement. As a result, we present a theoretical model of the domain that is driven by alpha helices and short random coil regions. These alpha helical regions form a classic dimer interface followed by two wide spread legs that we predict to be the lipid binding interface.
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de Oliveira, Nathalia Barth, Ana Carolina Irioda, Priscila Elias Ferreira Stricker, Bassam Felipe Mogharbel, Nádia Nascimento da Rosa, Dilcele Silva Moreira Dziedzic, and Katherine Athayde Teixeira de Carvalho. "Natural Membrane Differentiates Human Adipose-Derived Mesenchymal Stem Cells to Neurospheres by Mechanotransduction Related to YAP and AMOT Proteins." Membranes 11, no. 9 (September 5, 2021): 687. http://dx.doi.org/10.3390/membranes11090687.
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Adipose tissue-derived mesenchymal stem cells (ADMSCs) are promising candidates for regenerative medicine, as they have good cell yield and can differentiate into several cell lines. When induced to the neuronal differentiation, they form neurospheres composed of neural precursors (NPs) that can be an alternative in treating neurodegenerative diseases. This study aimed to characterize NPs from neurospheres obtained after seeding ADMSCs on a natural polyisoprene-based membrane. The ADMSCs were isolated from adipose tissue by enzymatic dissociation, were subjected to trilineage differentiation, and were characterized by flow cytometry for specific ADMSC surface markers. For neuronal differentiation, the cells were seeded on polystyrene flasks coated with the membrane and were characterized by immunocytochemistry and RT-PCR. The results demonstrated that the isolated cells showed characteristics of ADMSCs. At 15 to 25 days, ADMSCs seeded on the natural membrane developed neurospheres. Then, after dissociation, the cells demonstrated characteristic neuronal markers expressed on NPs: nestin, ß-III tubulin, GFAP, NeuN, and the YAP1/AMOT in the cytoplasm. In conclusion, it was demonstrated that this membrane differentiates the ADMSCs to NPs without any induction factors, and suggests that their differentiation mechanisms are related to mechanotransduction regulated by the YAP and AMOT proteins.
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Wells, Clark D., James P. Fawcett, Andreas Traweger, Yojiro Yamanaka, Marilyn Goudreault, Kelly Elder, Sarang Kulkarni, et al. "A Rich1/Amot Complex Regulates the Cdc42 GTPase and Apical-Polarity Proteins in Epithelial Cells." Cell 125, no. 3 (May 2006): 535–48. http://dx.doi.org/10.1016/j.cell.2006.02.045.
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Paramasivam, Murugan, Ali Sarkeshik, John R. Yates, Maria J. G. Fernandes, and Dannel McCollum. "Angiomotin family proteins are novel activators of the LATS2 kinase tumor suppressor." Molecular Biology of the Cell 22, no. 19 (October 2011): 3725–33. http://dx.doi.org/10.1091/mbc.e11-04-0300.
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LATS2 kinase functions as part of the Hippo pathway to promote contact inhibition of growth and tumor suppression by phosphorylating and inhibiting the transcriptional coactivator YAP. LATS2 is activated by the MST2 kinase. How LATS2 is activated by MST2 in response to changes in cell density is unknown. Here we identify the angiomotin-family tight junction protein AMOTL2 as a novel activator of LATS2. Like AMOTL2, the other angiomotin-family proteins AMOT and AMOTL1 also activate LATS2 through a novel conserved domain that binds and activates LATS2. AMOTL2 binds MST2, LATS2, and YAP, suggesting that AMOTL2 might serve as a scaffold protein. We show that LATS2, AMOTL2, and YAP all localize to tight junctions, raising the possibility that clustering of Hippo pathway components at tight junctions might function to trigger LATS2 activation and growth inhibition in response to increased cell density.
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Ray, Greeshma, Phuong Schmitt, and Anthony Schmitt. "Angiomotin-Like 1 Links Paramyxovirus M Proteins to NEDD4 Family Ubiquitin Ligases." Viruses 11, no. 2 (January 31, 2019): 128. http://dx.doi.org/10.3390/v11020128.
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To define the links between paramyxovirus budding and cellular ESCRT machinery, we previously identified angiomotin-like 1 (AMOTL1) in a screen for host factors that bind to the matrix (M) protein of parainfluenza virus 5 (PIV5). This protein harbors three L/PPXY sequences, allowing it to interact with WW domain containing proteins including NEDD4 family members. We hypothesize that paramyxoviruses use AMOTL1 as a linker to indirectly recruit the same NEDD4 ubiquitin ligases for budding that other enveloped viruses recruit directly through their PPXY late domains. In support of this hypothesis, we found that AMOTL1 could link together M proteins and NEDD4 family proteins in three-way co-IP experiments. Both PIV5 and mumps virus M proteins could be linked to the NEDD4 family proteins NEDD4-1, NEDD4L, and NEDL1, provided that AMOTL1 was co-expressed as a bridging protein. AMOT and AMOTL2 could not substitute for AMOTL1, as they lacked the ability to bind with paramyxovirus M proteins. Attachment of a PPXY late domain sequence to PIV5 M protein obviated the need for AMOTL1 as a linker between M and NEDD4 proteins. Together, these results suggest a novel host factor recruitment strategy for paramyxoviruses to achieve particle release.
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Nguyen, Hung Thanh, Jan-Michael Kugler, and Stephen M. Cohen. "DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins." PLOS ONE 12, no. 1 (January 6, 2017): e0169587. http://dx.doi.org/10.1371/journal.pone.0169587.
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Mana-Capelli, Sebastian, and Dannel McCollum. "Angiomotins stimulate LATS kinase autophosphorylation and act as scaffolds that promote Hippo signaling." Journal of Biological Chemistry 293, no. 47 (September 28, 2018): 18230–41. http://dx.doi.org/10.1074/jbc.ra118.004187.
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The Hippo pathway controls cell proliferation, differentiation, and survival by regulating the Yes-associated protein (YAP) transcriptional coactivator in response to various stimuli, including the mechanical environment. The major YAP regulators are the LATS1/2 kinases, which phosphorylate and inhibit YAP. LATS1/2 are activated by phosphorylation on a hydrophobic motif (HM) outside of the kinase domain by MST1/2 and other kinases. Phosphorylation of the HM motif then triggers autophosphorylation of the kinase in the activation loop to fully activate the kinase, a process facilitated by MOB1. The angiomotin family of proteins (AMOT, AMOTL1, and AMOTL2) bind LATS1/2 and promote its kinase activity and YAP phosphorylation through an unknown mechanism. Here we show that angiomotins increase Hippo signaling through multiple mechanisms. We found that, by binding LATS1/2, SAV1, and YAP, angiomotins function as a scaffold that connects LATS1/2 to both its activator SAV1–MST1 and its target YAP. Deletion of all three angiomotins reduced the association of LATS1 with SAV1–MST1 and decreased MST1/2-mediated LATS1/2-HM phosphorylation. Angiomotin deletion also reduced LATS1/2's ability to associate with and phosphorylate YAP. In addition, we found that angiomotins have an unexpected function along with MOB1 to promote autophosphorylation of LATS1/2 on the activation loop motif independent of HM phosphorylation. These results indicate that angiomotins enhance Hippo signaling by stimulating LATS1/2 autophosphorylation and by connecting LATS1/2 with both its activator SAV1–MST1/2 and its substrate YAP.
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El Sheikh, Amal F., Amisha T. Poret-Peterson, and Martin G. Klotz. "Characterization of Two New Genes, amoR and amoD, in the amo Operon of the Marine Ammonia Oxidizer Nitrosococcus oceani ATCC 19707." Applied and Environmental Microbiology 74, no. 1 (November 9, 2007): 312–18. http://dx.doi.org/10.1128/aem.01654-07.
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ABSTRACT Molecular analysis of the amo gene cluster in Nitrosococcus oceani revealed that it consists of five genes, instead of the three known genes, amoCAB. The two additional genes, orf1 and orf5, were introduced as amoR and amoD, respectively. Putative functions of the AmoR and AmoD proteins are discussed.
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Sharma, Jyoti, Monica Antenos, and Pavneesh Madan. "A Comparative Analysis of Hippo Signaling Pathway Components during Murine and Bovine Early Mammalian Embryogenesis." Genes 12, no. 2 (February 16, 2021): 281. http://dx.doi.org/10.3390/genes12020281.
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The time required for successful blastocyst formation varies among multiple species. The formation of a blastocyst is governed by numerous molecular cell signaling pathways, such as the Hippo signaling pathway. The Hippo signaling pathway is initiated by increased cell–cell contact and via apical polarity proteins (AMOT, PARD6, and NF2) during the period of preimplantation embryogenesis. Cell–cell contact and cell polarity activate (phosphorylates) the core cascade components of the pathway (mammalian sterile twenty like 1 and 2 (MST1/2) and large tumor suppressor 1 and 2 (LATS1/2)), which in turn phosphorylate the downstream effectors of the pathway (YAP1/TAZ). The Hippo pathway remains inactive with YAP1 (Yes Associated protein 1) present inside the nucleus in the trophectoderm (TE) cells (polar blastomeres) of the mouse blastocyst. In the inner cell mass (ICM) cells (apolar blastomeres), the pathway is activated with p-YAP1 present in the cytoplasm. On the contrary, during bovine embryogenesis, p-YAP1 is exclusively present in the nucleus in both TE and ICM cells. Contrary to mouse embryos, transcription co activator with PDZ-binding motif (TAZ) (also known as WWTR1) is also predominantly present in the cytoplasm in all the blastomeres during bovine embryogenesis. This review outlines the major differences in the localization and function of Hippo signaling pathway components of murine and bovine preimplantation embryos, suggesting significant differences in the regulation of this pathway in between the two species. The variance observed in the Hippo signaling pathway between murine and bovine embryos confirms that both of these early embryonic models are quite distinct. Moreover, based on the similarity of the Hippo signaling pathway between bovine and human early embryo development, bovine embryos could be an alternate model for understanding the regulation of the Hippo signaling pathway in human embryos.
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Musiani, Francesco, Valquiria Broll, Elisa Evangelisti, and Stefano Ciurli. "The model structure of the copper-dependent ammonia monooxygenase." JBIC Journal of Biological Inorganic Chemistry 25, no. 7 (September 14, 2020): 995–1007. http://dx.doi.org/10.1007/s00775-020-01820-0.
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Abstract Ammonia monooxygenase is a copper-dependent membrane-bound enzyme that catalyzes the first step of nitrification in ammonia-oxidizing bacteria to convert ammonia to hydroxylamine, through the reductive insertion of a dioxygen-derived O atom in an N–H bond. This reaction is analogous to that carried out by particulate methane monooxygenase, which catalyzes the conversion of methane to methanol. The enzymatic activity of ammonia monooxygenase must be modulated to reduce the release of nitrogen-based soil nutrients for crop production into the atmosphere or underground waters, a phenomenon known to significantly decrease the efficiency of primary production as well as increase air and water pollution. The structure of ammonia monooxygenase is not available, rendering the rational design of enzyme inhibitors impossible. This study describes a successful attempt to build a structural model of ammonia monooxygenase, and its accessory proteins AmoD and AmoE, from Nitrosomonas europaea, taking advantage of the high sequence similarity with particulate methane monooxygenase and the homologous PmoD protein, for which crystal structures are instead available. The results obtained not only provide the structural details of the proteins ternary and quaternary structures, but also suggest a location for the copper-containing active site for both ammonia and methane monooxygenases, as well as support a proposed structure of a CuA-analogue dinuclear copper site in AmoD and PmoD. Graphic abstract
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Ben-Ning, Cao, Wang Jun-Heng, and Wang Bao-An. "Differences in the Membrane Skeleton Proteins of RBC of AMoL Patients." Membrane Biochemistry 8, no. 4 (January 1989): 241–46. http://dx.doi.org/10.3109/09687688909026818.
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Tabe, Yoko, Linhua Jin, Saiko Kazuno, Tsutomu Fujimura, Hiromichi Matsushita, Takashi Ueno, Takashi Miida, Michael Andreeff, and Marina Konopleva. "Molecular Mechanisms Of Adipocyte-Leukemia Interactions Revealed By The Quantitative Proteomics Technology: Pro-Survival Function Of Adipocyte-Derived Free Fatty Acids On Acute Monoblastic Leukemia Mitochondrial Biogenesis." Blood 122, no. 21 (November 15, 2013): 3881. http://dx.doi.org/10.1182/blood.v122.21.3881.3881.
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Abstract Acute monocytic leukemia (AMoL) is a distinct subtype of AML with an average 3-year overall survival of 31% (Tallman, J Clin Oncol 2004), and the majority of patients die from disease progression after relapse. Adipocytes represent an essential component of the adults bone marrow (BM) microenvironment (Battula, Blood 2013, Tabe, Blood 2004, Konopleva, Blood 1999), and promote survival of monoblastic leukemia cells (Tabe, ASH, 2012). In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to examine the molecular mechanism of pro-survival properties of BM adipocytes in co-culture with AMoL. The specific pathway alterations were identified by Metacore (GeneGo, St. Joseph, MI). We first confirmed the more prominent protective role of BM-derived adipocytes as compared to BM stromal cells (MSC) on AMoL cells. Co-culture with adipocytes derived from differentiated MSCs significantly protected U937 and primary AMoL cells (n = 5) from serum starvation-induced apoptosis compared to MSCs (U937 p = 0.029, primary samples p = 0.034). Gene expression analysis demonstrated striking upregulation of mRNA expression levels of CD36 (4.1+0.4-fold),FABP4 (569.3+40.1-fold), and PPARG (2.1+0.2-fold) in U937 cells co-cultured with adipocytes compared to the MSC co-cultured cells. Notably, Bcl-2 mRNA expression in U937 cells was significantly upregulated after adipocyte co-culture compared to MSCs (5.4+0.3-fold). We previously reported that fatty acids (FAs) promote leukemic cell survival via leukemia cells metabolic shifting from pyruvate oxidation to fatty acid oxidation for glycolysis, which links to the Bcl-2 anti-apoptotic machinery (Samudio, J Clin Invest. 2010). Mature adipocytes are capable of releasing abundant free fatty acids (Fas), the essential ligands of a nuclear receptor PPARgamma (PPARG), which are internalized by leukemic cells via scavenger receptor CD36 and ligate PPARG through fatty acid binding protein 4 (FABP4). Enhanced transcriptional activity of FA-ligated PPARG may in turn stimulate glucose metabolism, regulate fatty acids storage, maintain mitochondrial membrane potential, and prevents apoptosis by upregulating anti-apoptotic Bcl-2 resulting in further increase of its downstream target genes including CD36 and FABP4. To gain further insights into metabolic alterations induced in adipocyte-leukemia co-cultures, we performed iTRAQ proteomic analysis of U937 cells cultured in the presence of MSCs or adipocytes. A total of 1,610 proteins were detected by this technology, with 106 proteins differentially expressed between co-culture conditions; 50 genes were up-regulated and 56 genes were down-regulated by adipocytes co-culture compared to MSCs. Among the upregulated proteins, FA synthase (p = 0.007) ATP-citrate synthase responsible for cytosolic acetyl-CoA synthesis (p = 0.019); and glyceraldehyde-3-phosphate dehydrogenase (p = 0.028) involved in the activation of glycolysis and gluconeogenesis pathways (p< 0.0001) were identified. Of note, 20% of the upregulated proteins (10/50) were ribosomal proteins, accompanied by an increase in six DNA elongation or replication related proteins. Anti-apoptotic chaperone proteins known to assist ribosome biogenesis, HSP70, HSP90alpha and HSP90beta were also increased by adipocyte co-culture (p<0.01). The mass spectrometry further revealed more prominent repression of Cytochrome c (p=0.01) as well as the proline metabolism pathway (p=0.0011) under adipocyte co-culture conditions, consistent with inhibition of mitochondrial apoptosis of U937 cells. Summary our results indicate that abundant FAs produced by BM adipocytes support AMoL cell survival via stimulation of PPARG transcription which directly increases the FA metabolism through uptake and translocation of FAs by CD36 and FABP4 in AMoL cells. FA metabolism in turn facilitates supply of Acetyl-CoA via oxidative phosphorylation, which drives the TCA cycle; and enhances ribosomal biogenesis of AMoL cells, resulting in pro-survival signaling in AMoL cells. It is conceivable that increased adipocyte content of the BM in adult AML patients may promote leukemogenesis and negatively affect the responsiveness to chemotherapy. Disclosures: No relevant conflicts of interest to declare.
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Sheehan, David. "Introduction to Proteins: Structure, Function and Motion. By Amit Kessel and Nir Ben-Tal." ChemBioChem 12, no. 10 (May 12, 2011): 1603–4. http://dx.doi.org/10.1002/cbic.201100254.
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Pagani, Lisa, Clizia Chinello, Allia Mahajneh, Francesca Clerici, Lucrezia Criscuolo, Andrea Favalli, Paola Gruarin, et al. "Untargeted Mass Spectrometry Approach to Study SARS-CoV-2 Proteins in Human Plasma and Saliva Proteome." BioChem 2, no. 1 (February 8, 2022): 64–83. http://dx.doi.org/10.3390/biochem2010005.
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Since the start of the COVID-19 outbreak, more than four million people have died of this disease. Given its ability to provide a precise response, mass spectrometry-based proteomics could represent a useful tool to study this pathology. To this end, an untargeted nLC-ESI-MS/MS-based method to characterise SARS-CoV-2 proteins, including possible variants, and investigate human saliva and plasma proteome in a single analysis was developed for further application in patients. Four SARS-CoV-2 recombinant proteins, three (S1–S2–RBD) belonging to the spike glycoprotein (S) and one corresponding to the nucleoprotein (N), were prepared and analysed with nLC-UHRTOF by injecting decreasing amounts to establish the limit of detection (LOD) of the method. This was determined as 10 pg for all the components of the S protein and for N (71 amol and 213 amol, respectively). Various viral inactivation strategies plus deglycosylation and digestion approaches were then tested in saliva and plasma spiked with different quantities of SARS-CoV-2 recombinant proteins. The limit of characterisation (LOC) in saliva for the N and S proteins was observed at 100 pg (coverage of 20% and 3%, respectively); instead, in plasma, it was 33 pg for N and 330 pg for the S protein, with a coverage of 4% for both. About 300 and 800 human proteins were identified in plasma and saliva, respectively, including several key effectors and pathways that are known to be altered in COVID-19 patients. In conclusion, this approach allows SARS-CoV-2 proteins and the human proteome to be simultaneously explored, both for plasma and saliva, showing a high relevant potential for retrospective studies aimed at investigating possible virus variants and for patient stratification.
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Lacasse, Vincent, Rene Zahedi, Vincent Richard, Hangjun Wang, Georgia Mitsa, Olivier Poetz, Margaret Redpath, et al. "Liquid chromatography coupled to multiple reaction monitoring (LC-MRM) for quantification of PD-L1 and PD1-signaling proteins in non-small cell lung carcinoma (NSCLC)." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e21040-e21040. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e21040.
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e21040 Background: Improving the predictive biomarkers arena of checkpoint inhibitors (CPIs) beyond PD-L1 immunohistochemistry (IHC) is one of the most important unmet need in NSCLC. Up to 50% of patients that show positive PD-L1 expression by IHC do not respond to anti-PD-L1 treatments, and patients with low/undetectable PD-L1 have significantly improved survival with CPI. Moreover, PD-L1 IHC is heterogeneous, can be affected by tissue fixation time and post-translational modifications like glycosylation. Also, multiple studies indicated that PD-L1 expression alone does not reliably reflect the immune status of the tumor, thus requiring the measurement of other members of the PD1 signalling pathway. Methods: To address these issues, we developed a multiplexed targeted mass spectrometry-based (MS) assay for the quantitation of protein members of the PD-1/PD-L1 axis in formalin fixed paraffin-embedded (FFPE) tissue.The effect of fixation time on protein recovery was determined using differentially fixed H1915 cells. Liquid chromatography (LC) coupled to MRM was used to develop a targeted assay for PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70. Results: After 30 minutes fixation 33.6 µg of protein were extracted per mg of FFPE H1915 cells, while protein recovery after 7 days was 55.8 µg/mg, with greater variability (18% and 28% CV, p-value = ns). The optimized LC-MRM method allows the quantitation of PD-L1 and PD-1 down to 23 amol on-column. We evaluated the utility of our MRM assays using the H1915 FFPE cells (PD-L1 3+ by IHC) and determined an endogenous concentration of PD-L1 and NT5E as being 33.2±0.1 amol/µg of total protein and 4.8±0.5 fmol/µg respectively. Noteworthy, a known glycosylation site of PD-L1 was quantified at 10.9±0.3 amol/µg of total protein (30% of total). As increases sensitivity was required, anti-peptide antibodies were generated against the 15 best peptides. We then evaluated this LC-MRM method using MDA-MB-436 cells with lower levels of PD-L1 protein assessed by IHC. Determination of endogenous PD-L1 concentration (3.0 amol/µg of total protein) was achieved using anti-peptide immuno-enrichment followed by MRM. Conclusions: We developed a fixation time independent extraction technique for FFPE and optimized a highly sensitive LC-MRM method that allows the absolute quantification of our targets. This proteomic workflow allows absolute quantification of the PD-1/PD-L1 axis from FFPE tissue using immuno-enrichment and a multiplexed LC-MRM method.
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Lai, Eric C. "Drosophila Tufted Is a Gain-of-Function Allele of the Proneural Gene amos." Genetics 163, no. 4 (April 1, 2003): 1413–25. http://dx.doi.org/10.1093/genetics/163.4.1413.
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Abstract Tufted is a classical Drosophila mutant characterized by a large number of ectopic mechanosensory bristles on the dorsal mesothorax. Unlike other ectopic bristle mutants, Tufted is epistatic to achaete and scute, the proneural genes that normally control the development of these sensory organs. In this report, I present genetic and molecular evidence that Tufted is a gain-of-function allele of the proneural gene amos that ectopically activates mechanosensory neurogenesis. I also systematically examine the ability of the various proneural bHLH proteins to cross-activate each other and find that their ability to do so is in general relatively limited, despite their common ability to induce the formation of mechanosensory bristles. This phenomenon seems instead to be related to their shared ability to activate Asense and Senseless.
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Askri, Cunin, Ouni, Béal, Rachidi, Sakly, Amara, Lehmann, and Sève. "Effects of Iron Oxide Nanoparticles (γ-Fe2O3) on Liver, Lung and Brain Proteomes following Sub-Acute Intranasal Exposure: A New Toxicological Assessment in Rat Model Using iTRAQ-Based Quantitative Proteomics." International Journal of Molecular Sciences 20, no. 20 (October 19, 2019): 5186. http://dx.doi.org/10.3390/ijms20205186.
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Iron Oxide Nanoparticles (IONPs) present unique properties making them one of the most used NPs in the biomedical field. Nevertheless, for many years, growing production and use of IONPs are associated with risks that can affect human and the environment. Thus, it is essential to study the effects of these nanoparticles to better understand their mechanism of action and the molecular perturbations induced in the organism. In the present study, we investigated the toxicological effects of IONPs (γ-Fe2O3) on liver, lung and brain proteomes in Wistar rats. Exposed rats received IONP solution during 7 consecutive days by intranasal instillation at a dose of 10 mg/kg body weight. An iTRAQ-based quantitative proteomics was used to study proteomic variations at the level of the three organs. Using this proteomic approach, we identified 1565; 1135 and 1161 proteins respectively in the brain, liver and lung. Amon them, we quantified 1541; 1125 and 1128 proteins respectively in the brain, liver and lung. Several proteins were dysregulated comparing treated samples to controls, particularly, proteins involved in cytoskeleton remodeling, cellular metabolism, immune system stimulation, inflammation process, response to oxidative stress, angiogenesis, and neurodegenerative diseases.
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Vervoort, Michel, and Valerie Ledent. "The Evolution of the Neural Basic Helix-Loop-Helix Proteins." Scientific World JOURNAL 1 (2001): 396–426. http://dx.doi.org/10.1100/tsw.2001.68.
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Basic Helix-Loop-Helix (bHLH) transcription factors control various aspects of the formation of the nervous system in the metazoans. In Drosophila some bHLH (such as the achaete-scuteatonal, and amos genes) act as proneural genes, directing ectodermal cells toward a neural fate. Their vertebrate orthologs, however, probably do not assume such a neural determination function, but rather control the decision made by neural precursors to generate neurons and not glial cells, as well as the progression of neuronal precursors toward differentiation into mature neurons. The proneural function of Drosophila bHLH genes may be an innovation that occurs in the evolutive lineage that leads to arthropods. In addition, although neural bHLH appear to be involved in the specification of neuronal identities, they probably do not confer by themselves neuronal type-specific properties to the cells. Rather, neural bHLH allow neural cells to correctly interpret specification and positional cues provided by other factors. Although bHLH genes are often expressed in complementary subsets of neural cells and/or expressed sequentially in those cells, the coding regions of the various neural bHLH appear largely interchangeable. We propose that the specific expression patterns have been acquired, following gene duplications, by subfunctional-ization, i.e., the partitioning of ancestral expression patterns among the duplicates and, by extension, we propose that subfunctionalization is a key process to understand the evolution of neural bHLH genes.
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Ueda, Kenji, Hideaki Takano, Madoka Nishimoto, Hiromi Inaba, and Teruhiko Beppu. "Dual Transcriptional Control of amfTSBA, Which Regulates the Onset of Cellular Differentiation in Streptomyces griseus." Journal of Bacteriology 187, no. 1 (January 1, 2005): 135–42. http://dx.doi.org/10.1128/jb.187.1.135-142.2005.
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ABSTRACT The amf gene cluster encodes a probable secretion system for a peptidic morphogen, AmfS, which induces aerial mycelium formation in Streptomyces griseus. Here we examined the transcriptional control mechanism for the promoter preceding amfT (PamfT) directing the transcription of the amfTSBA operon. High-resolution S1 analysis mapped a transcriptional start point at 31 nucleotides upstream of the translational start codon of amfT. Low-resolution analysis showed that PamfT is developmentally regulated in the wild type and completely abolished in an amfR mutant. The −35 region of PamfT contained the consensus sequence for the binding of BldD, a pleiotropic negative regulator for morphological and physiological development in Streptomyces coelicolor A3(2). The cloned bldD locus of S. griseus showed high sequence similarity to the S. coelicolor counterpart. Transcription of bldD occurred constitutively in both the wild type and an A-factor-deficient mutant of S. griseus, which suggests that the regulatory role of BldD is independent of A-factor. The gel retardation assay revealed that purified BldD and AmfR recombinant proteins specifically bind PamfT. Overproduction of BldD in the wild-type cell conferred a bald phenotype (defective in aerial growth and streptomycin production) and caused marked repression of PamfT activity. An amfT-depleted mutant also showed a bald phenotype but PamfT activity was not affected. Both the bldD-overproducing wild-type strain and the amfT mutant were unable to induce aerial growth of an amfS mutant in a cross-feeding assay, which indicates that these strains are defective in the production of an active AmfS peptide. The results overall suggests that two independent regulators, AmfR and BldD, control PamfT activity via direct binding to determine the transcriptional level of the amf operon responsible for the production and secretion of AmfS peptide, which induces the erection of aerial hyphae in S. griseus.
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Thyparambil, Sheeno P., Wei-Li Liao, Eunkyung An, Anuja Bhalkikar, Robert Heaton, Karl G. Sylvester, and Xuefeng B. Ling. "Proteomic profiling to identify therapeutics targets in glioblastoma (GBM)." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 2555. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.2555.
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2555 Background: Glioblastoma (GBM) is an aggressive primary brain tumor with poor prognosis. Treatment at diagnosis is largely confined to surgery, radiation and temozolomide (TMZ) with median progression-free survival (PFS) of 7 months and median overall survival (mOS) of 15 months. GBM tumors recur in most cases and in patients with recurrent GBM, the mOS is 6.2 months. The lack of effective therapies underscores the importance of exploring other agents. We propose that quantitating therapy-associated protein biomarkers can improve treatment personalization for GBM. Methods: 97 FFPE GBM tissues were microdissected and solubilized for mass spectrometry-based proteomic analysis of therapy-associated protein biomarkers in our CLIA certified lab. We quantified protein levels of MGMT, hENT1, RRM1, TOPO1 and EGFR/TUBB3 (antibody target and payload resistance markers, respectively, for anti-EGFR ADCs) simultaneously. The multiplexed assay also quantified additional 24 clinically relevant proteins. Results: 43/57 patients were predicted to respond to TMZ based on undetectable levels of MGMT, confirming wide utility of this agent. 42/97(43%) patients were predicted to have gemcitabine sensitivity based on high expression of the response marker (hENT1 > 100 amol/ug) and low expression of the resistance marker (RRM1 < 700 amol/ug). 11/97(11%) patients expressed TOPO1 > 1350 amol/ug (75th percentile of all indications tested by author’s laboratory), suggesting likely response to irinotecan and topotecan. EGFR expression ranged from < 100 amol/ug to > 25000 amol/ug, including overexpression (> 1500 amol/ug) in 22%(21/97) of cases. While expression of EGFR(81/97, 84%) suggested likely response to anti-EGFR ADC, concurrent expression of TUBB3(78/81) may indicate resistance to several known payloads, such as taxanes and MMAE. Conjugation with another payload that targets sensitivity marker TOPO1 (68% expression) is a likely option. Proteomic analysis also revealed detectable levels of multiple RTKs (FGFR(4), AXL(20), IGF1R(10), MET overexpression(1), and HER2 overexpression(2)), indicating potential response to RTK inhibitors. Exploratory investigation in tumor vs TME using proteomics and metabolomics is ongoing. Conclusions: In this population of GBM patients, proteomic analysis identified protein targets of multiple approved and investigational therapies. Gemcitabine, which crosses the blood-brain barrier, may be considered as a salvage option after TMZ failure. Proteomic quantitation of EGFR and TUBB3 may improve patient selection for EGFR-targeting ADCs.
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Cicero, Marco P., Meghan M. Sharp, Carol A. Gross, and Kenneth N. Kreuzer. "Substitutions in Bacteriophage T4 AsiA andEscherichia coli ς70 That Suppress T4motA Activation Mutations." Journal of Bacteriology 183, no. 7 (April 1, 2001): 2289–97. http://dx.doi.org/10.1128/jb.183.7.2289-2297.2001.
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ABSTRACT Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the ς70 subunit. AmotA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five ς70 mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant ς70 proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The ς70 substitutions affected the 4.2 region, which binds the −35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. colipromoters. This defect could not be explained solely by a disruption in −35 recognition since similar results were obtained with extended −10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth.
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Freitas, M. R., B. N. Araujo, R. A. N. Soares, J. J. S. Gouveia, M. M. Costa, and G. V. Gouveia. "Detection of Fur, AmoA and pvcAB genes in Aeromonas hydrophila isolated from aquatic organisms and impact on bacterial growth under different iron concentrations." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 74, no. 4 (August 2022): 671–76. http://dx.doi.org/10.1590/1678-4162-12491.
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ABSTRACT Infection caused by Aeromonas brings great harm to fish farming. Among the factors associated with bacterial pathogenesis, iron uptake can contribute to the survival and virulence of bacteria within hosts. The aim of this study was to check the presence of genes related to iron uptake in Aeromonas hydrophila deriving from aquatic organisms in the São Francisco Valley and associate the presence of these genes with the ability to grow in media containing different concentrations of iron. The DNAs of 41 isolates were extracted and used in PCRs to verify the presence of the Fur, AmoA and pvcAB genes related to iron uptake. The growth of the isolates belonging to different genetic profiles was verified in culture media containing different iron concentrations. Two isolates were positive for the presence of the Fur gene, seven for the AmoA gene and two for the pvcAB gene. The growth test showed that the low availability of iron did not interfere in the growth of the isolates, nor in the isolate that did not contain any of the genes evaluated in this study, suggesting that the iron uptake’s mechanisms of the tested isolates may be related to other genes and proteins.
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Lacasse, Vincent, Vincent Richard, Hangjun Wang, Georgia Mitsa, Olivier Poetz, Andreas I. Papadakis, Mounib Elchebly, et al. "Immuno-multiple reaction monitoring (iMRM) for quantitation of PD-L1 and PD-1-signaling proteins in non-small cell lung carcinoma (NSCLC)." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): 2627. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.2627.
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2627 Background: More accurate predictive biomarkers of response to checkpoint inhibitors (CPIs) still is a major unmet need in oncology. PD-L1 immunohistochemistry (IHC) limitations include its analytical variability and the post-translational modifications of PD-1 signaling-associated proteins like glycosylation. Moreover, PD-L1 IHC is an imperfect surrogate of the tumor immune microenvironment, and immunoscoring is important but difficult to assess in a clinical setting. Proteomic based technologies can overcome these challenges, but the low concentration of these proteins and the presence of high background noise in formalin-fixed paraffin embedded (FFPE) tumors were limiting obstacles. In this study, we evaluate the benefit of a new approach we used with anti-peptide antibodies to purify surrogate peptides, while liquid chromatography (LC) was coupled to multiple reaction monitoring mass spectrometry (iMRM) to improve specificity and precision of protein quantitation. Methods: To determine the concentration of PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70, we used unique and well detectable proteolytic peptides as surrogates. In a refined protocol, we optimized protein extraction and digestion, peptide immuno-enrichment, LC and MRM parameters to maximize recovery, increase target-specific signal and reduce noise. Plus, we assessed the glycosylation status of PD-L1, PD-L2, and PD-1. The entire workflow was fully validated using 31 NSCLC FFPE tumors. PD-L1 quantitation by iMRM was compared to PD-L1 IHC clone 22C3. Results: On average, 71±29 µg (n = 52) of protein could be extracted from each 1–3 mm3 NSCLC tumor FFPE core. The optimized iMRM method allowed the quantitation of PD-L1 and PD-1 down to 21 amol on-column. Inter- and intra-day repeatability were well below FDA guidelines (coefficients of variation [CV] < 20%) with average CVs of 5.2±4.0% (intra-day) and 4.5±2.6% (inter-day). Sample storage had no significant effect on peptide quantitation. The final multiplexed iMRM assay enables quantitation all targets and glycosylation states for > 40 samples in only 3 days (including external calibration and quality controls) and was used to quantify the PD-1/PD-L1 axis proteins successfully in all 31 NSCLC FFPE tumors. PD-L1 expression ranged from 2 amol/μg to 61 amol/μg of total protein. As expected, iMRM results correlated moderately (R = 0.56, ρ < 0.001) with PD-L1 IHC. PD-L1 glycosylation status ranged from 99.9±0.2%, and therefore did not explain the discrepancies between IHC and iMRM for these samples. Conclusions: Herein a robust iMRM workflow was developed for the quantitation of the PD-1/PD-L1 axis in FFPE. This proof-of-concept supports that MS-based assay can provide otherwise unavailable data (e.g., PD-L1 concentration, glycosylation status). CPI treated patient tumors are being currently processed to validate the predictive value of the assay.
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Lacasse, Vincent, Vincent Richard, Hangjun Wang, Georgia Mitsa, Olivier Poetz, Andreas I. Papadakis, Mounib Elchebly, et al. "Immuno-multiple reaction monitoring (iMRM) for quantitation of PD-L1 and PD-1-signaling proteins in non-small cell lung carcinoma (NSCLC)." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): 2627. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.2627.
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2627 Background: More accurate predictive biomarkers of response to checkpoint inhibitors (CPIs) still is a major unmet need in oncology. PD-L1 immunohistochemistry (IHC) limitations include its analytical variability and the post-translational modifications of PD-1 signaling-associated proteins like glycosylation. Moreover, PD-L1 IHC is an imperfect surrogate of the tumor immune microenvironment, and immunoscoring is important but difficult to assess in a clinical setting. Proteomic based technologies can overcome these challenges, but the low concentration of these proteins and the presence of high background noise in formalin-fixed paraffin embedded (FFPE) tumors were limiting obstacles. In this study, we evaluate the benefit of a new approach we used with anti-peptide antibodies to purify surrogate peptides, while liquid chromatography (LC) was coupled to multiple reaction monitoring mass spectrometry (iMRM) to improve specificity and precision of protein quantitation. Methods: To determine the concentration of PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70, we used unique and well detectable proteolytic peptides as surrogates. In a refined protocol, we optimized protein extraction and digestion, peptide immuno-enrichment, LC and MRM parameters to maximize recovery, increase target-specific signal and reduce noise. Plus, we assessed the glycosylation status of PD-L1, PD-L2, and PD-1. The entire workflow was fully validated using 31 NSCLC FFPE tumors. PD-L1 quantitation by iMRM was compared to PD-L1 IHC clone 22C3. Results: On average, 71±29 µg (n = 52) of protein could be extracted from each 1–3 mm3 NSCLC tumor FFPE core. The optimized iMRM method allowed the quantitation of PD-L1 and PD-1 down to 21 amol on-column. Inter- and intra-day repeatability were well below FDA guidelines (coefficients of variation [CV] < 20%) with average CVs of 5.2±4.0% (intra-day) and 4.5±2.6% (inter-day). Sample storage had no significant effect on peptide quantitation. The final multiplexed iMRM assay enables quantitation all targets and glycosylation states for > 40 samples in only 3 days (including external calibration and quality controls) and was used to quantify the PD-1/PD-L1 axis proteins successfully in all 31 NSCLC FFPE tumors. PD-L1 expression ranged from 2 amol/μg to 61 amol/μg of total protein. As expected, iMRM results correlated moderately (R = 0.56, ρ < 0.001) with PD-L1 IHC. PD-L1 glycosylation status ranged from 99.9±0.2%, and therefore did not explain the discrepancies between IHC and iMRM for these samples. Conclusions: Herein a robust iMRM workflow was developed for the quantitation of the PD-1/PD-L1 axis in FFPE. This proof-of-concept supports that MS-based assay can provide otherwise unavailable data (e.g., PD-L1 concentration, glycosylation status). CPI treated patient tumors are being currently processed to validate the predictive value of the assay.
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Platonova, I. L., M. I. Sakhelashvili, G. D. Shtybel, and O. I. Sakhelashvili-Bil. "Laboratory evaluation of the efficacy of Liastene application in complex therapy of multiresistant pulmonary tuberculosis." Infusion & Chemotherapy, no. 4 (December 27, 2021): 25–31. http://dx.doi.org/10.32902/2663-0338-2021-4-25-31.
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OBJECTIVE. Evaluating according to laboratory tests the effectiveness of Liasten in the treatment of patients with multidrug-resistant pulmonary tuberculosis (MDR-TB).
MATERIALS AND METHODS. Evaluation of the effectiveness of etiotropic and etiopathogenetic therapy in 57 patients with MDR-TB was performed. According to the treatment schemes, patients were divided into groups. The control group (n=22) received individualized antimycobacterial therapy (AMBT) regimens. The experimental group (n=35) received AMBT in combination with Liasten. Evaluation of the effectiveness of treatment regimens was performed on the basis of indicators of general clinical blood tests, immunological and bacteriological studies.
RESULTS AND DISCUSSION. In patients of the experimental group, compared with the control in 1.5 times more often found positive changes in the hemogram of blood and ESR (p<0.05-0.001), the establishment of a dynamic balance between the pools of lymphocyte cells CD4+ and СD8+ (immunoregulatory index, p<0.05), an increase in the number of phagocytosis active cells (phagocytic index, p<0.05), the content of cationic lysosomal proteins of granulocyte leukocytes (p<0.05), a 1.4-fold decrease in the cytochemical coefficient of neutrophils (p<0.05), the number of proliferated under the action of PPD-L lymphocytes (p<0.05), normalization of phagocytic counts and total redox activity of neutrophils (p<0.05), increase in frequency and reduction of anesthesia was stated.
CONCLUSIONS. Restoration of the body’s immune status, blood hemogram, increase in frequency and reduction of the time of decontamination were more active and occurred 1.5 times more often in patients receiving a complex combination of AMBT with Liasten.
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Ansari, Sahar, Marcelo O. Freire, Eun-Kyoung Pang, Alaa I. Abdelhamid, Mohammad Almohaimeed, and Homayoun H. Zadeh. "Immobilization of Murine Anti-BMP-2 Monoclonal Antibody on Various Biomaterials for Bone Tissue Engineering." BioMed Research International 2014 (2014): 1–10. http://dx.doi.org/10.1155/2014/940860.
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Biomaterials are widely used as scaffolds for tissue engineering. We have developed a strategy for bone tissue engineering that entails application of immobilized anti-BMP-2 monoclonal antibodies (mAbs) to capture endogenous BMPs in vivo and promote antibody-mediated osseous regeneration (AMOR). The purpose of the current study was to compare the efficacy of immobilization of a specific murine anti-BMP-2 mAb on three different types of biomaterials and to evaluate their suitability as scaffolds for AMOR. Anti-BMP-2 mAb or isotype control mAb was immobilized on titanium (Ti) microbeads, alginate hydrogel, and ACS. The treated biomaterials were surgically implanted in rat critical-sized calvarial defects. After 8 weeks,de novobone formation was assessed using micro-CT and histomorphometric analyses. Results showedde novobone regeneration with all three scaffolds with immobilized anti-BMP-2 mAb, but not isotype control mAb. Ti microbeads showed the highest volume of bone regeneration, followed by ACS. Alginate showed the lowest volume of bone. Localization of BMP-2, -4, and -7 antigens was detected on all 3 scaffolds with immobilized anti-BMP-2 mAb implanted in calvarial defects. Altogether, these data suggested a potential mechanism for bone regeneration through entrapment of endogenous BMP-2, -4, and -7 proteins leading to bone formation using different types of scaffoldsviaAMOR.
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Besse, Andrej, Christopher R. Cogle, Leylah M. Drusbosky, Shireen Vali, Taher Abbasi, Neeraj Kumar Singh, Neha G. Gupta, et al. "Predicting Carfilzomib Resistance Mechanisms and Therapeutics Using Computational Modelling of Genomics and Proteomics." Blood 132, Supplement 1 (November 29, 2018): 3193. http://dx.doi.org/10.1182/blood-2018-99-116939.
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Abstract Background: Multiple myeloma (MM) is a malignancy of plasma cells accounting for around 10% of all hematologic cancers. MM is an incurable heterogeneous malignancy which impacts the response rate due to complex nature of the disease. Although with standard of care treatment, including proteasome inhibitors (PI), a significant response and remission is achieved, the majority of patients still develops resistance. There is no precise method for determining the pathways which govern the acquired resistance to PI. Computational biology modelling (CBM), which uses genomics for creating the MM specific disease characteristics, can be used for deciphering the dominance of signaling changes in acquired resistance and accordingly select right treatment combination. Predicting a priori treatment response based on disease characteristics would enable optimal treatment selection and potentially reduce the trial and error approach that impacts outcome and health care costs. Aim: The objective of the study was to predict response to proteasome inhibitor Carfilzomib (CFZ) in AMO1 MM disease model and its resistant counterparts using CBM, to determine mechanism of resistance to CFZ and identify customized therapy options for CFZ resistant AMO1 (AMO-CFZ) cells. Methods: AMO-CFZ resistant cells were derived by long-term exposure of the cells to increasing doses of CFZ. AMO1 PI-sensitive and AMO-CFZ were analyzed using cytogenetics and mutations (profiled by next-generation sequencing; NGS). At the same time quantitative proteomic analysis was done to find proteins which were upregulated as well as downregulated in the resistant cells. Genomic and proteomic data was input to generate disease-specific protein network maps, biomarkers and pathway characteristics unique to AMO1 and AMO-CFZ cells respectively. Digital drug models of CFZ and other targeted-FDA approved drugs are simulated on the disease models individually and in combination at varying doses. Drug impact was assessed by quantitatively measuring the effect on a cell growth score, which is a composite of cell proliferation, viability and apoptosis. Unique therapy combinations identified in AMO-CFZ cells will be prospectively experimentally validated; at the same time key signaling changes were deciphered in AMO-CFZ resistance with respect to AMO1 PI-sensitive MM model. Results: Genomic analysis using CBM showed that AMO1 MM model harbored amplification of MYC, POU2AF1, POU2F1, TXNIP and knock down (KD) of proteasome subunit PSMA6 which lead to Carfilzomib sensitivity. The predictions in AMO-CFZ identified loss of PSMA6 and upregulation of proteasomal subunits PSMB5/7/2/1 which reduced ER stress and ATF4. Predictions in AMO-CFZ cells identified higher glycolysis and pentose phosphate pathway resulting in higher glutathione levels compared to AMO1 PI-sensitive cells. The anti-oxidant proteins NQO1, TXN, PRDX1/4/5 were higher in AMO-CFZ-resistant cells, in addition to PRDX6 overexpression (OE), which was common with PI-sensitive cells that reduced oxidative stress. Carfilzomib drug exporter ABCB1 was overexpressed in CFZ-resistant cells and Nelfinavir increased sensitivity of the cells towards Carfilzomib. CBM identified novel therapeutic targets CDK5 and NFE2L2 that can be used to overcome CFZ-resistance. Moreover, Venetoclax was more cytotoxic in AMO-CFZ cells than in AMO-1 PI-sensitive, because BCL2 was higher in CFZ-resistant version than the sensitive one due to its overexpression and TP53 knockdown. CBM predicted novel therapeutics for investigation which include: GEMCITABINE plus MELPHALAN; GEMCITABINE plus OLAPARIB; MELPHALAN plus OLAPARIB. DNA damage is high in AMO-CFZ due to knockdown of CHEK1, ERCC3, FANCB/G, LIG4, XRCC6 and loss of function of FANCD2, ATM. Conclusions: Using genomics and proteomics, CBM identified the resistance mechanisms of AMO-CFZ and new treatment strategies. These were validated showcasing a methodology for a clinically relevant workflow using NGS information. Disclosures Cogle: Celgene: Other: Steering Committee Member of Connect MDS/AML Registry. Vali:Cell Works Group Inc.: Employment. Abbasi:Cell Works Group Inc.: Employment. Singh:Cellworks Research India Private Limited: Employment. Gupta:Cellworks Research India Private Limited: Employment. Kushwaha:Cellworks Research India Private Limited: Employment. Kumari:Cellworks Research India Private Limited: Employment. Raju:Cellworks Research India Private Limited: Employment. Naga:Cellworks Research India Private Limited: Employment. Basu:Cellworks Research India Private Limited: Employment.
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Tabe, Yoko, Masako Harada, Yuka Miyamae, Kaoru Mogushi, Saiko Kazuno, Tsutomu Fujimura, Hiromichi Matsushita, et al. "Bone Marrow Adipocyte-Derived Free Fatty Acids Induce Gene Signature Linking Transcription with Metabolic Changes That Contribute to Survival of Acute Monocytic Leukemia Cells." Blood 124, no. 21 (December 6, 2014): 1013. http://dx.doi.org/10.1182/blood.v124.21.1013.1013.
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Abstract Adipocytes are the prevalent stromal cell type in adult bone marrows (BM). With increasing age, BM stroma-resident mesenchymal stem cells (MSCs), increase their capacity to differentiate into adipocytes, which leads to the progressive accumulation of fat in the BM space. It is conceivable that the increased BM adipocyte content promotes leukemogenesis and negatively affects responsiveness to chemotherapy. We previously reported that free fatty acids (FFAs) promote the metabolic shift from pyruvate oxidation to fatty acid oxidation (FAO), which causes uncoupling of mitochondrial oxidative phosphorylation and promotes leukemia cell survival (Samudio, J Clin Invest. 2010). We further demonstrated the prominent antiapoptotic effects of BM-derived adipocytes co-cultured with cells from acute monocytic leukemia (AMoL), a poor-prognosis subtype of AML (Tabe ASH. 2013). Proteomic analysis with isobaric tags for relative and absolute quantification (iTRAQ) showed upregulation of protein folding pathways which increases the expression of antiapoptotic chaperone proteins HSP70 and HSP90, of integrin-mediated cell adhesion and migration pathways and downregulation of oxidative phosphorylation along with repression of cytochrome c.Metacore gene ontology (GO) analysis identified NF-kB, c-Jun, SP1, AP-1, and HMG as the potent relevant transcription factors that closely interact with and activate chaperone proteins, chromatin, and gene transcription. In this study, we characterized a gene signature linking transcription with metabolic changes that contribute to AMoL cell survival under conditions mimicking aging BM with prevalent adipocytes. We confirmed the antiapoptotic role of FFAs produced by the primary BM MSC-derived adipocytes via pharmacologic inhibition of FAO by etomoxir (EX), which inhibits fatty acid entry into the mitochondria. EX (50mM) treatment reversed the prosurvival effects of adipocytes on serum-starved U937 monoblast cells (% Annexin V, -/+ EX: mono-culture, 30.1±9.0 / 31.5±4.6; co-culture with adipocytes, 8.9±2.1 / 29.6±9.2; P=0.02). To assess the molecular links between metabolic pathways and gene expression triggered by BM adipocytes, we performed RNA-seq transcriptome analysis with a next generation sequencer system HiSeq1500 (Illumina) using TopHat software for alignment and Cufflinks software for identifying differential gene expression. RNA-Seq detected upregulation of 21 genes in U937 cells after co-culture with BM-derived adipocytes (false discovery rate, <0.05). Specifically, 10 of these upregulated genes were significantly decreased by EX treatment, including transcription factor–activating PPARg2 promoter KLF9, co-chaperone immunophilin protein FKBP5, chemokine CXCL12 receptor CXCR4, receptor tyrosine kinase FLT3, PI3K negative regulator PIK3IP1, and immunosuppressive transcriptional regulator TSC22D3. GO pathway analysis further revealed that co-culture with adipocytes induced upregulation of antioxidant thioredoxin peroxidase activity, which was reversed by EX. Together with the iTRAQ GO results, the antioxidative chaperone proteins might play critical roles in regulation of FAO with repression of oxidative phosphorylation in AMoL cells in adipocytes abundant BM. DNA array (GeneSQUARE) analysis and quantitative RT-PCR detected upregulation of fatty acid binding protein 4 (FABP4), scavenger receptor CD36, nuclear receptor PPARG, and antiapoptotic Bcl-2 in U937 cells co-cultured with adipocytes. It is known that FFAs, the ligand of nuclear receptor PPARγ, activate PPARγ and promote FFA uptake through transcriptional induction of CD36 and FABP4 in monocytic cells. EX treatment, which blocks the entry of fatty acids into the mitochondria, induced prominent elevation of FABP4 in U937 cells co-cultured with adipocytes. These results indicate that the FABP4-mediated internalization and ligation of fatty acids to PPARγ facilitates transcriptional activation. From the transcriptome analysis and the mitochondrial uncoupling metabolic changes, we conclude that the survival of AMoL cells depends on the cooperative interactions between lipid metabolism and transcriptional activation of factors associated with chaperones, chemokines, and integrins. Strategies targeting FAO warrant further exploration in patients with monocytic leukemia, which is highly dependent on altered lipid metabolism. Disclosures No relevant conflicts of interest to declare.
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Yan, Dongyao, Ji Hyung Hong, Hee Yeon Lee, Jae Ho Byun, Fabiola Cecchi, Yuan Tian, Sarit Schwartz, Eunkyung An, and Todd A. Hembrough. "Selecting patients with stage II/III colorectal cancer for 5-fluorouracil-based adjuvant chemotherapy using proteomic analysis." Journal of Clinical Oncology 36, no. 4_suppl (February 1, 2018): 708. http://dx.doi.org/10.1200/jco.2018.36.4_suppl.708.
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708 Background: 5-fluorouracil (5-FU) is a common adjuvant treatment for stage III and high-risk stage II colorectal cancer (CRC). However, about 20% of patients relapse within 48 months of treatment with 5-FU, even when combined with oxaliplatin. To improve patient selection, tumor biomarkers that predict sensitivity to 5-FU have been proposed. These include proteins involved in 5-FU activation or metabolism such as uridine-cytidine kinase 2 (UCK2) and thymidylate synthase (TYMS). We used multiplexed mass spectrometry to evaluate the utility these biomarkers in the archived tumor samples of patients with stage II/III CRC. Methods: Tumor samples were from 143 patients with stage II/III CRC who received adjuvant 5-FU, folinic acid, and oxaliplatin during 2000-2014; 83% of patients received 12 cycles and the others received ≤ 11 cycles. Tumor cells were microdissected and solubilized, and 67 candidate biomarkers were quantitated using mass spectrometry. Overall survival (OS) and relapse-free survival (RFS) were assessed using the Kaplan-Meier method and log-rank test. Protein expression by tumor stage, lymph node (LN) status, tumor sidedness was compared using the Student’s t-test. Results: Of 143 patients, 45 had recurrence and 98 patients did not. UCK2 was detected in all samples, ranging from 187 to 1606 attomoles per microgram of total protein (amol/µg). Patients with UCK2 expression above 335 amol/μg (n = 109) had significantly longer OS than patients with lower expression (n = 34; HR: 0.42; p= 0.009). There was no significant difference in RFS (HR: 0.6; p= 0.088). UCK2 expression did not differ by disease stage, LN metastasis status, or tumor sidedness. TYMS expression was not associated with survival in this cohort. Analysis of other biomarkers associated with response to 5-FU and platinum is in progress. Conclusions: In stage II/III CRC, UCK2 expression above 335 amol/μg identifies a subgroup of 5-FU-treated CRC patients with longer survival, suggesting that quantitated UCK2 has potential for use in selecting patients for treatment. These findings warrant validation in larger cohorts.
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Steven Tebbets, J., and Patrick V. Vail. "Dose Response of Omnivorous Leafroller to Commercial Formulations of Bacillus Thuringiensis Var. Kurstaki in the Laboratory, 1992." Arthropod Management Tests 19, no. 1 (January 1, 1994): 370. http://dx.doi.org/10.1093/amt/19.1.370.
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Abstract We compared three formulations of B. thuringiensis var. kurstaki (Btk) for their insecticidal activity against first-instar OLR. Products tested included: (1) NOVO Laboratories Biobit, a flowable concentrate containing live bacteria with spores and parasporal crystal protein bodies; (2) Abbott Laboratories Dipel 2X, a wettable powder also containing active bacteria with spores and parasporal crystal protein bodies; and (3) Mycogen Corporations MVP Bioinsecticide, a flowable concentrate containing dead bacterial cells which bioencapsulate the Btk proteins. The AI in each of these products is the insecticidal protein, or delta-endotoxin, of Btk. Dose-response was determined by feeding trials exposing first-instar OLR to each product. Serial dilutions of each formulation were made and 10 μ1 of each dilution was applied to the surface of an agar-based diet (Bioserv No. 9370) and then allowed to air dry. One larva was placed in each vial containing ca. 1 ml of surface-contaminated diet (surface area = 1 cm2). Thirty to 60 larvae were observed for each dilution and formulation tested. Doses were expressed in mg AI per sq. cm. Mortality evaluations were made after 3, 5, and 7 days. Response data were analyzed by the POLO-PC PROBIT procedure (LeOra Software, 1987).
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Shahpoury, Pourya, Tom Harner, Gerhard Lammel, Steven Lelieveld, Haijie Tong, and Jake Wilson. "Development of an antioxidant assay to study oxidative potential of airborne particulate matter." Atmospheric Measurement Techniques 12, no. 12 (December 9, 2019): 6529–39. http://dx.doi.org/10.5194/amt-12-6529-2019.
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Abstract. Oxidative potential is a measure of redox activity of airborne particulate matter (PM) and is often used as a surrogate to estimate one form of PM toxicity. The evaluation of oxidative potential in a physiologically relevant environment is always challenging. In this work, we developed a chromatographic method, employing an ultra-high-performance liquid chromatograph coupled to a triple–quadruple mass spectrometer, to determine the oxidative potential of PM from different sources. To this purpose, we measured the PM-induced oxidation of glutathione, cysteine, and ascorbic acid, and formation of glutathione disulfide and cystine, following PM addition to simulated epithelial lining fluids, which, in addition to the antioxidants, contained inorganic salts, a phospholipid, and proteins. The new method showed high precision and, when applied to standard reference PM, the oxidative potential was found to increase with the reaction time and PM concentration in the lung fluid. The antioxidant depletion rates were considerably higher than the rates found with the conventional dithiothreitol assay, indicating the higher sensitivity of the new method. The presence of the lung fluid inorganic species increased the oxidative potential determined through glutathione and cysteine, but showed an opposite effect with ascorbic acid, whereas the presence of proteins resulted in a moderate decrease in the oxidative potential. In the presence of PM2.5, glutathione and cysteine demonstrated similar depletion patterns, which were noticeably different from that of ascorbic acid, suggesting that cysteine could be used as an alternative to glutathione for probing oxidative potential.
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More, E., T. Fellner, H. Doppelmayr, C. Hauser-Kronberger, N. Dandachi, P. Obrist, F. Sandhofer, and B. Paulweber. "Activation of the MAP kinase pathway induces chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) expression in human breast cancer cell lines." Journal of Endocrinology 176, no. 1 (January 1, 2003): 83–94. http://dx.doi.org/10.1677/joe.0.1760083.
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Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function. COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/ micro g total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3.5 amol/ micro g total RNA). While treatment of EGF, TGFalpha and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression. In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.
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Ostlie, K. R., and K. M. Helgeson. "BT Corn Performance Against First- and Second-Generation Infestations of European Corn Borer, 1997." Arthropod Management Tests 23, no. 1 (January 1, 1998): 374–75. http://dx.doi.org/10.1093/amt/23.1.374.
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Abstract Six transgenic corn hybrids expressing insecticidal proteins produced by genes from Bacillus thuringiensis (Bt) were evaluated at the Rosemount Experiment Station in separate experiments that targeted first and second generations. The hybrids representing 5 different insertion events, and their non-Bt isolines were evaluated in a RCB design with tour replications. Test hybrids were planted in the center 2 rows of a 4-row plot (22 ft long, 30-inch row spacing). Border rows were planted to NK 4640Bt to preclude inter-plot movement of larvae. Plots were planted on 21 May using a cone planter and thinned to 30,000 plants/A. In view of extensive natural first generation infestation, the second-generation experiment was treated with Warrier 1 E (zeta cypermethrin) @ 3.8 fl oz/acre on 15 Jul. In each plot, 15 consecutive plants were tested for Bt expression and non-expressers were rogued on 29 Jul. The plants intended for first generation evaluation were infested at the V6 with neonate larvae in grits deposited in the whorl on 14 Jul. Heavy natural egg laying from second generation eliminated the need for manually infesting second-generation plots. First generation evaluations included: number and length of tunnels on 21 Aug and % of plants with leaf injury and a leaf injury rating on 24 Sep. On 9 Oct the second generation measurements were recorded: number of overwintering larvae, tunnel number and length, and shank and ear damage.
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Cai, Mei, Joseph Amor, and Maiyon Park. "Abstract 5834: Examining the potential oncogenic function of septins and their interaction with Chmp1A tumor suppressor in pancreatic cancer." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5834. http://dx.doi.org/10.1158/1538-7445.am2022-5834.
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Abstract Introduction: Chmp1A functions as a tumor suppressor by the activation of ATM and p53 in pancreatic cancer. The nuclear localization signal (NLS) of Chmp1A is required for cell growth inhibition and activation of ATM and p53. Proteomic analysis has identified Septins (a group of GTP-binding proteins) to be associated with the NLS-deleted Chmp1A. Since NLS-deleted Chmp1A promotes cancer cell growth, we hypothesized that Septins may function as oncogenes and that Chmp1A functions as a tumor suppressor partly by inhibiting the oncogenic action of Septin proteins. Objective: To examine whether Septin proteins exhibit oncogenic activity and to investigate whether Chmp1A inhibits the oncogenic function of Septins. Methodology: Using normal pancreatic and cancer cells, we compared Septin transcripts using PCR assays, Septin protein expression using Western blot, and Septin cellular expression using immunocytochemistry. We performed immunohistochemical analysis to examine whether Septin expression is increased in pancreatic cancer tissues compared to corresponding normal tissues. Silencing technology was used to test whether Septin expression is regulated by Chmp1A. Results: Our preliminary data indicates that the transcript and protein of Septins are increased in various pancreatic cancer cell lines compared to normal. Septin protein expression was increased or altered in human pancreatic ductal adenocarcinoma tissues compared to corresponding normal tissues. Additionally, our data implies that Chmp1A negatively regulates Septin expression, since Septin transcripts and proteins are increased when Chmp1A protein is silenced. Summary and future direction: Our data suggests that Septin functions as an oncogene in pancreatic cancer cells and that Chmp1A functions as a tumor suppressor partly by inhibiting the oncogenic action of Septin proteins. For the completion of the project in the future, we plan to confirm the function of Septins by investigating the effect of Septin overexpression on pancreatic cancer cell proliferation. In addition, we will further investigate the interaction between Septins and Chmp1A by examining Septin expression and localization in pancreatic cancer cells that overexpress Chmp1A. Citation Format: Mei Cai, Joseph Amor, Maiyon Park. Examining the potential oncogenic function of septins and their interaction with Chmp1A tumor suppressor in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5834.
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Pöhlker, C., J. A. Huffman, J. D. Förster, and U. Pöschl. "Autofluorescence of atmospheric bioaerosols: spectral fingerprints and taxonomic trends of pollen." Atmospheric Measurement Techniques 6, no. 12 (December 9, 2013): 3369–92. http://dx.doi.org/10.5194/amt-6-3369-2013.
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Abstract. Primary biological aerosol particles (PBAP) are important factors in atmospheric cycling, climate, and public health. Pollen is a major fraction of PBAP and is receiving increasing attention due to its high allergenic potential and the associated impacts on personal life quality and economy. Recently, autofluorescence-based techniques have proven to be valuable tools for real time, in situ quantification and classification of PBAP. First studies suggest that the autofluorescence of pollen may be sufficiently selective to be utilized for an automated and real-time monitoring of pollen in ambient air. However, the degree of selectivity autofluorescence can provide is still in question and actively debated. This study addresses the origin, properties, and selectivity of autofluorescence from natural pollen by fluorescence microscopy and spectroscopy measurements along with a systematic synthesis of related literature. We show that dry pollen reveals characteristic and reproducible autofluorescence signatures which are shaped by cell wall associated fluorophores, such as phenolic compounds and carotenoid pigments. In addition, fluorescence signals from proteins and chlorophyll a were observed in some species. The abundance and intensity of the individual fluorescence signals show certain taxonomic trends and allow systematic differentiation from bacteria and fungal spores due to the lack of proteins on the grain surface. Principal component analysis was used to explore the discrimination potential of pollen autofluorescence, in combination with size and shape, revealing a differentiation of pollen on family level. Our results help explore the levels of selectivity that autofluorescence-based techniques can provide to PBAP analysis and will support the development and application of autofluorescence-based detectors for monitoring of allergenic pollen in the atmosphere.
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Hinman, Veronica F., Albert T. Nguyen, and Eric H. Davidson. "Expression and function of a starfish Otx ortholog, AmOtx: a conserved role for Otx proteins in endoderm development that predates divergence of the eleutherozoa." Mechanisms of Development 120, no. 10 (October 2003): 1165–76. http://dx.doi.org/10.1016/j.mod.2003.08.002.
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El-Mohammady, Hanan, Hind Ibrahim Shaheen, John David Klena, Isabelle Antoun Nakhla, Mattew A. Weiner, and Adam Wilson Armstrong. "Specific IgA antibodies in the diagnosis of acute brucellosis." Journal of Infection in Developing Countries 6, no. 02 (September 30, 2011): 192–200. http://dx.doi.org/10.3855/jidc.1411.
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An Egyptian female with night sweats, headache, and back pain was diagnosed with acute brucellosis one week after returning from a North African country. Humoral immune responses to specific immunogenic proteins were investigated before and after treatment. ELISA was performed to detect levels of specific antibody (Ab) titers. Immunoblot analysis of Ab recognizing specific Brucella antigenic bands was also performed. IgA was detected on the day of disease onset. Specific agglutination titer was 1:160; it doubled three days later and treatment was implemented. Blood culture yielded Gram-negative coccobacilli after one month, confirmed as B. melitensis by AMOS-PCR. Immunoblotting revealed IgM Abs against two protein bands of 112 and 130-kDa observed only during the acute stage. On the other hand, the intensity of IgG Abs against 21 and 21.5-kDa protein bands positively correlated with the time of convalescence. Based on our observations we conclude that specific IgA levels may be used as an early diagnostic marker for Brucella and high molecular weight protein bands may be useful in the differentiation between acute and chronic brucellosis.
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Kazuma, Yasuhiro, Kotaro Shirakawa, Anamaria Daniela Sarca, Yoshihito Horisawa, Hirofumi Fukuda, Hiroyuki Matsui, Hiroyuki Yamazaki, et al. "Interactome Analysis of APOBEC3B in Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 1259. http://dx.doi.org/10.1182/blood-2019-126856.
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Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family proteins restrict retroviruses and retrotransposons by inducing hypermutation or degradation of the replication intermediates through their DNA cytidine deaminase activity. APOBECs can also act as endogenous sources of DNA damage that mutate many human cancers. Accumulation of APOBEC signature mutations is associated with disease progression and poor overall survival in multiple myeloma (Walker et al. Nat Commun, 2015). Among APOBEC3 enzymes, APOBEC3B (A3B) is the only family member that is predominantly located in the nucleus throughout the cell cycle. We previously reported that A3B knockdown decreased cytidine deaminase activity in myeloma cells, suggesting that among APOBECs, A3B plays a major role in cytidine deamination-related mutagenesis in myeloma cells (Yamazaki et al., Sci Rep, 2019). Recent studies showed that cofactors of A3B could affect the functions of A3B: heterogeneous nuclear ribonucleoproteins (hnRNPs) interact with surface hydrophobic residues of the N-terminal domain in order to bind to A3B (Xiao et al., Nuc. Acids Res, 2017; Zhang al., Cell Microbiol, 2008); BORF2, an Epstein-Barr viral protein, interacts with the A3B catalytic domain and inhibits A3B DNA cytidine deaminase activity (Cheng et al., Nat Microbiol, 2018). However, the biological mechanisms of how endogenous A3B induces mutations in genomic DNA are still unclear. In this study, we aim to ascertain the cofactors for nucleic acid binding and elucidate the regulatory mechanisms that prevent APOBEC-mediated genomic mutagenesis. Because of the high homology between APOBEC3 proteins, a specific antibody against A3B is not available, and it is difficult to analyze A3B-interaction at the endogenous expression level. To overcome this technical impediment, we used a lentiCRISPR system to insert a FLAG-tag sequence at the C-terminus of the A3B gene in A3B highly expressing myeloma cell lines (AMO1 and RPMI8226). We then conducted A3B-immunoprecipitation with the anti-FLAG M2 antibody, followed by mass spectrometry (MS) analysis to identify potential A3B interacting proteins. MS analysis identified 40 putative interacting proteins and these proteins were clustered largely into two interaction networks: ribonucleoprotein complex and ribosomal-associated proteins. We also performed Gene Ontology (GO) enrichment analysis and revealed that spliceosome, ribosome, and RNA transport were significantly enriched terms. We confirmed the binding between A3B and selected A3B interacting proteins: hnRNPs, interleukin enhancer-binding factor 2 and 3 (ILF2, ILF3) in myeloma cell lines by co-immunoprecipitation assays. Next, we tested the intracellular colocalization of overexpressed A3B and interacting proteins in Hela cells by immunofluorescence microscopy. We found that ILF2 presents strong colocalization with A3B in the nuclei of cells. We also employed density-gradient sedimentation analysis to test if these proteins form high molecular mass (HMM) complexes with A3B in the nucleus using HEK293T cells expressing FLAG-tagged A3B. We detected that ILF2 is one of the components of HMM A3B complexes. To check whether these putative interacting proteins affect A3B cytidine deaminase activity, we next performed an in vitro luminescence-based screening assay (AlphaScreen)using a FLAG-GST protein library which was produced by the wheat cell-free protein production system. We found that hnRNP A1 and ILF2 decreased A3B cytidine deaminase activity. This study provides for the first time a proteomic characterization of A3B interactome in a myeloma cell context. Our findings reveal putative A3B cofactors in myeloma cell lines which may regulate the catalytic activity of A3B. We discuss how these proteins bind A3B and affect its activity in myeloma cells. Disclosures Takaori-Kondo: Pfizer: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ono: Research Funding; Takeda: Research Funding; Chugai: Research Funding; Kyowa Kirin: Research Funding.
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Wei, Yi, Thomas W. Southworth, Hilde Kloster, Masahiro Ito, Arthur A. Guffanti, Anne Moir, and Terry A. Krulwich. "Mutational Loss of a K+ and NH4+ Transporter Affects the Growth and Endospore Formation of Alkaliphilic Bacillus pseudofirmus OF4." Journal of Bacteriology 185, no. 17 (September 1, 2003): 5133–47. http://dx.doi.org/10.1128/jb.185.17.5133-5147.2003.
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ABSTRACT A putative transport protein (Orf9) of alkaliphilic Bacillus pseudofirmus OF4 belongs to a transporter family (CPA-2) of diverse K+ efflux proteins and cation antiporters. Orf9 greatly increased the concentration of K+ required for growth of a K+ uptake mutant of Escherichia coli. The cytoplasmic K+ content of the cells was reduced, consistent with an efflux mechanism. Orf9-dependent translocation of K+ in E. coli is apparently bidirectional, since ammonium-sensitive uptake of K+ could be shown in K+-depleted cells. The upstream gene product Orf8 has sequence similarity to a subdomain of KTN proteins that are associated with potassium-translocating channels and transporters; Orf8 modulated the transport capacities of Orf9. No Orf9-dependent K+(Na+)/H+ antiport activity was found in membrane vesicles. Nonpolar deletion mutants in the orf9 locus of the alkaliphile chromosome exhibited no K+-related phenotype but showed profound phenotypes in medium containing high levels of amine-nitrogen. Their patterns of growth and ammonium content suggested a physiological role for the orf9 locus in bidirectional ammonium transport. Orf9-dependent ammonium uptake was observed in right-side-out membrane vesicles of the alkaliphile wild type and the mutant with an orf8 deletion. Uptake was proton motive force dependent and was inhibited by K+. Orf9 is proposed to be designated AmhT (ammonium homeostasis). Ammonium homeostasis is important in high-amine-nitrogen settings and is particularly crucial at high pH since cytosolic ammonium accumulation interferes with cytoplasmic pH regulation. Endospore formation in amino-acid-rich medium was significantly defective and germination was modestly defective in the orf9 and orf7-orf10 deletion mutants.
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Tereba, Allan. "High Density Protein Kinase Assay With Subattomole Sensitivity." Journal of Biomolecular Screening 3, no. 1 (February 1998): 29–35. http://dx.doi.org/10.1177/108705719800300104.
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A rapid assay system based on incorporation of [γ-32P]ATP into biotinylated peptide substrates and their subsequent capture onto a high capacity streptavidin-coated membrane, SAM2™, has been developed for the detection of protein kinases. The system uses prenumbered and partially cut membrane squares for analyzing a limited number of samples or can be formatted to analyze up to 1,536 samples per microtiter plate footprint of 7.0 cm X 10.6 cm. The high biotin-binding capacity and low background of the membrane allows the use of nearly saturating amounts of most substrates, giving this system very high signal-to-noise ratios at low enzyme concentrations. Using cAMP-dependent Protein Kinase A (PKA) as a model system, as little as 0.3 amol of purified enzyme in 0.2 μl can be detected with a linear response range of over 3 orders of magnitude. cAMP-dependent kinase activity can be measured directly in tissue extracts by using a specific substrate and harsh washing procedures to reduce nonspecific backgrounds from proteins phosphorylated by other kinases. For increased assay flexibility, results can be analyzed either by PhosphorImager™ quantitation or by scintillation counting.
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Chen, Dafu, Huazhi Chen, Yu Du, Dingding Zhou, Sihai Geng, Haipeng Wang, Jieqi Wan, Cuiling Xiong, Yanzhen Zheng, and Rui Guo. "Genome-Wide Identification of Long Non-Coding RNAs and Their Regulatory Networks Involved in Apis mellifera ligustica Response to Nosema ceranae Infection." Insects 10, no. 8 (August 9, 2019): 245. http://dx.doi.org/10.3390/insects10080245.
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Long non-coding RNAs (lncRNAs) are a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins, and lncRNAs have been proven to play pivotal roles in a wide range of biological processes in animals and plants. However, knowledge of expression patterns and potential roles of honeybee lncRNA response to Nosema ceranae infection is completely unknown. Here, we performed whole transcriptome strand-specific RNA sequencing of normal midguts of Apis mellifera ligustica workers (Am7CK, Am10CK) and N. ceranae-inoculated midguts (Am7T, Am10T), followed by comprehensive analyses using bioinformatic and molecular approaches. A total of 6353 A. m. ligustica lncRNAs were identified, including 4749 conserved lncRNAs and 1604 novel lncRNAs. These lncRNAs had minimal sequence similarities with other known lncRNAs in other species; however, their structural features were similar to counterparts in mammals and plants, including shorter exon and intron length, lower exon number, and lower expression level, compared with protein-coding transcripts. Further, 111 and 146 N. ceranae-responsive lncRNAs were identified from midguts at 7-days post-inoculation (dpi) and 10 dpi compared with control midguts. Twelve differentially expressed lncRNAs (DElncRNAs) were shared by Am7CK vs. Am7T and Am10CK vs. Am10T comparison groups, while the numbers of unique DElncRNAs were 99 and 134, respectively. Functional annotation and pathway analysis showed that the DElncRNAs may regulate the expression of neighboring genes by acting in cis and trans fashion. Moreover, we discovered 27 lncRNAs harboring eight known miRNA precursors and 513 lncRNAs harboring 2257 novel miRNA precursors. Additionally, hundreds of DElncRNAs and their target miRNAs were found to form complex competitive endogenous RNA (ceRNA) networks, suggesting that these DElncRNAs may act as miRNA sponges. Furthermore, DElncRNA-miRNA-mRNA networks were constructed and investigated, the results demonstrated that a portion of the DElncRNAs were likely to participate in regulating the host material and energy metabolism as well as cellular and humoral immune host responses to N. ceranae invasion. Our findings revealed here offer not only a rich genetic resource for further investigation of the functional roles of lncRNAs involved in the A. m. ligustica response to N. ceranae infection, but also a novel insight into understanding the host-pathogen interaction during honeybee microsporidiosis.
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Xia, Jun, Mu Lin, Zhuoyi Song, Gail Fernandes, Susan Rosenberg, and Chris Amos. "Abstract A018: Lung cancer associated DNA Damageome reveals a genome destabilizing role of Aquaporin 3." Cancer Research 82, no. 10_Supplement (May 15, 2022): A018. http://dx.doi.org/10.1158/1538-7445.evodyn22-a018.
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Abstract Lung cancer is a multifactorial disease caused by both environmental exposures and genetic factors. Genome and transcriptome-wide association studies (GWAS and TWAS) successfully identified many genetic variants and candidate causal genes associated with lung cancer. However, the lack of functional studies of these genes remains a major bottleneck to clinical translation. Recently, we discovered that large networks of evolutionarily conserved DNA damageome proteins (DDPs) promote DNA damage and genome instability. Here, we discovered a lung cancer-associated DNA damageome network. We further discovered that one of the lung cancer DDPs, Aquaporin 3 (AQP3), can promote ROS dependent DNA damage upon overproduction. Furthermore, it potentiates DNA damage by a low dose of arsenic exposure, creating unique double strand break hotspots which help predict mutational signatures. We screened for high endogenous DNA damage levels for more than 40 TWAS and GWAS-nominated candidates using high-throughput single cell-based flow cytometric assays. We optimized and performed Mre11-Cut&Tag (Cleavage Under Targets and Tagmentation) to map double strand breaks, which are the most deleterious form of DNA damage. In addition, we used END-Seq to provide a much higher resolution of the DSB map and uncovered hotspots in arsenic-detoxifying genes. We are implementing duplex sequencing to map the mutational signatures caused by arsenic and the arsenic-Aquaporin 3 interaction. Our study brings function to endogenous DNA damage and DNA damageome proteins that interact with environmental toxicants. Mechanistic insights into how low-dose arsenic interacts with risk genes yield critical knowledge for the prevention, diagnosis, and treatment of arsenic-associated diseases. We also uncovered several early biomarkers to potentially predict the long-term health impacts of arsenic. Citation Format: Jun Xia, Mu Lin, Zhuoyi Song, Gail Fernandes, Susan Rosenberg, Chris Amos. Lung cancer associated DNA Damageome reveals a genome destabilizing role of Aquaporin 3 [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A018.
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Valle-Rios, Ricardo, Amanda Burkhardt, Peter Hevezi, and Albert Zlotnik. "Identification of a novel secreted molecule expressed by Th17 cells: potential association with inflammatory diseases. (59.3)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 59.3. http://dx.doi.org/10.4049/jimmunol.188.supp.59.3.
Full textAbstract:
Abstract CD4 T cells (Th cells) play an important role in adaptive immune responses. These processes are controlled in part by a pattern of soluble molecules (cytokines) secreted by different types of Th cells. Here we describe a novel secreted molecule which we have called barrier tissue and activated T cell-associated molecule (BATAM). Using a comprehensive database of human gene expression that comprises more than 115 tissues or organs of the body, we identified a secreted protein that is produced by activated CD4 T cells as well as skin and various mucosal tissues. BATAM encodes a ~60 KD protein that contains a thrombospondin type 1 repeat (TSR) and the adhesion associated domain AMOP, which are known to mediate cell adhesion and are also present in various proteins involved in inflammation. We confirmed the BATAM expression pattern by qPCR in mouse tissues. The production of BATAM by activated spleen CD4 T cells is significant but not strong. We therefore hypothesized that BATAM may be preferentially produced by a polarized subset of Th cells. To investigate this possibility, we polarized CD4 T cells into Th1, Th2, Treg and Th17 in vitro. The results indicate that BATAM expression is highly associated with Th17 cells. We have also confirmed that it is a secreted protein. Using recombinant BATAM, we are currently probing its biological activity. We conclude that BATAM is a novel pro-inflammatory molecule that may be associated with diseases mediated by Th17 cells.
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Pöhlker, C., J. A. Huffman, and U. Pöschl. "Autofluorescence of atmospheric bioaerosols – fluorescent biomolecules and potential interferences." Atmospheric Measurement Techniques 5, no. 1 (January 9, 2012): 37–71. http://dx.doi.org/10.5194/amt-5-37-2012.
Full textAbstract:
Abstract. Primary biological aerosol particles (PBAP) are an important subset of air particulate matter with a substantial contribution to the organic aerosol fraction and potentially strong effects on public health and climate. Recent progress has been made in PBAP quantification by utilizing real-time bioaerosol detectors based on the principle that specific organic molecules of biological origin such as proteins, coenzymes, cell wall compounds and pigments exhibit intrinsic fluorescence. The properties of many fluorophores have been well documented, but it is unclear which are most relevant for detection of atmospheric PBAP. The present study provides a systematic synthesis of literature data on potentially relevant biological fluorophores. We analyze and discuss their relative importance for the detection of fluorescent biological aerosol particles (FBAP) by online instrumentation for atmospheric measurements such as the ultraviolet aerodynamic particle sizer (UV-APS) or the wide issue bioaerosol sensor (WIBS). In addition, we provide new laboratory measurement data for selected compounds using bench-top fluorescence spectroscopy. Relevant biological materials were chosen for comparison with existing literature data and to fill in gaps of understanding. The excitation-emission matrices (EEM) exhibit pronounced peaks at excitation wavelengths of ~280 nm and ~360 nm, confirming the suitability of light sources used for online detection of FBAP. They also show, however, that valuable information is missed by instruments that do not record full emission spectra at multiple wavelengths of excitation, and co-occurrence of multiple fluorophores within a detected sample will likely confound detailed molecular analysis. Selected non-biological materials were also analyzed to assess their possible influence on FBAP detection and generally exhibit only low levels of background-corrected fluorescent emission. This study strengthens the hypothesis that ambient supermicron particle fluorescence in wavelength ranges used for most FBAP instruments is likely to be dominated by biological material and that such instrumentation is able to discriminate between FBAP and non-biological material in many situations. More detailed follow-up studies on single particle fluorescence are still required to reduce these uncertainties further, however.