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Sáenz, Amets, Margarita Azpitarte, Rubén Armañanzas, France Leturcq, Ainhoa Alzualde, Iñaki Inza, Federico García-Bragado, et al. "Gene Expression Profiling in Limb-Girdle Muscular Dystrophy 2A." PLoS ONE 3, no. 11 (November 18, 2008): e3750. http://dx.doi.org/10.1371/journal.pone.0003750.
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Haikarainen, Teemu, Wei-Qiang Chen, Gert Lubec, and Petri Kursula. "Structure, modifications and ligand-binding properties of rat profilin 2a." Acta Crystallographica Section D Biological Crystallography 65, no. 4 (March 19, 2009): 303–11. http://dx.doi.org/10.1107/s0907444909000699.
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Lam, Angela M., Christine Espiritu, Shalini Bansal, Holly M. Micolochick Steuer, Congrong Niu, Veronique Zennou, Meg Keilman, et al. "Genotype and Subtype Profiling of PSI-7977 as a Nucleotide Inhibitor of Hepatitis C Virus." Antimicrobial Agents and Chemotherapy 56, no. 6 (March 19, 2012): 3359–68. http://dx.doi.org/10.1128/aac.00054-12.
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ABSTRACTPSI-7977, a prodrug of 2′-F-2′-C-methyluridine monophosphate, is the purified diastereoisomer of PSI-7851 and is currently being investigated in phase 3 clinical trials for the treatment of hepatitis C. In this study, we profiled the activity of PSI-7977 and its ability to select for resistance using a number of different replicon cells. Results showed that PSI-7977 was active against genotype (GT) 1a, 1b, and 2a (strain JFH-1) replicons and chimeric replicons containing GT 2a (strain J6), 2b, and 3a NS5B polymerase. Cross-resistance studies using GT 1b replicons confirmed that the S282T change conferred resistance to PSI-7977. Subsequently, we evaluated the ability of PSI-7977 to select for resistance using GT 1a, 1b, and 2a (JFH-1) replicon cells. S282T was the common mutation selected among all three genotypes, but while it conferred resistance to PSI-7977 in GT 1a and 1b, JFH-1 GT 2a S282T showed only a very modest shift in 50% effective concentration (EC50) for PSI-7977. Sequence analysis of the JFH-1 NS5B region indicated that additional amino acid changes were selected both prior to and after the emergence of S282T. These include T179A, M289L, I293L, M434T, and H479P. Residues 179, 289, and 293 are located within the finger and palm domains, while 434 and 479 are located on the surface of the thumb domain. Data from the JFH-1 replicon variants showed that amino acid changes within the finger and palm domains together with S282T were required to confer resistance to PSI-7977, while the mutations on the thumb domain serve to enhance the replication capacity of the S282T replicons.
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Fan, Xu-Xu, Yuan Gao, Long Shu, Yan-Quan Wei, Xue-Ping Yao, Sui-Zhong Cao, Guang-Neng Peng, Xiang-Tao Liu, and Shi-Qi Sun. "Transcriptome profiling indicating canine parvovirus type 2a as a potential immune activator." Virus Genes 52, no. 6 (June 23, 2016): 768–79. http://dx.doi.org/10.1007/s11262-016-1363-5.
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Kiviniemi, Anu, Petri Heinonen, and Harri Lönnberg. "Oligonucleotides bearing pentaerythritol-derived mass tags." Open Chemistry 5, no. 1 (March 1, 2007): 191–200. http://dx.doi.org/10.2478/s11532-006-0046-9.
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AbstractFive different di-O-alkylated pentaerythritol phosphoramidite building blocks (2a–e) were synthesized and introduced into oligonucleotides to obtain mass-tagged probes (6a–f) useful in RNA transcription profiling. These non-nucleosidic mass tags allow categorization of oligonucleotide probes having identical nucleoside content and, hence, identification of the probe hybridized to RNA by mass spectrometry analysis. Hybridization properties of the oligonucleotide conjugates were elucidated by melting temperature measurements.
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Murk, K., S. Buchmeier, B. M. Jockusch, and M. Rothkegel. "In birds, profilin-2a is ubiquitously expressed and contributes to actin-based motility." Journal of Cell Science 122, no. 7 (March 3, 2009): 957–64. http://dx.doi.org/10.1242/jcs.041715.
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Keira, Yoko, Satoru Noguchi, Rumi Kurokawa, Masako Fujita, Narihiro Minami, Yukiko K. Hayashi, Takashi Kato, and Ichizo Nishino. "Characterization of lobulated fibers in limb girdle muscular dystrophy type 2A by gene expression profiling." Neuroscience Research 57, no. 4 (April 2007): 513–21. http://dx.doi.org/10.1016/j.neures.2006.12.010.
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Severino, Patricia, Olivier Dussurget, Ricardo Z. N. Vêncio, Emilie Dumas, Patricia Garrido, Gabriel Padilla, Pascal Piveteau, et al. "Comparative Transcriptome Analysis of Listeria monocytogenes Strains of the Two Major Lineages Reveals Differences in Virulence, Cell Wall, and Stress Response." Applied and Environmental Microbiology 73, no. 19 (August 17, 2007): 6078–88. http://dx.doi.org/10.1128/aem.02730-06.
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ABSTRACT Listeria monocytogenes is a food-borne, opportunistic, bacterial pathogen causing a wide spectrum of diseases, including meningitis, septicemia, abortion, and gastroenteritis, in humans and animals. Among the 13 L. monocytogenes serovars described, human listeriosis is mostly associated with strains of serovars 4b, 1/2b, and 1/2a. Within the species L. monocytogenes, three phylogenetic lineages are described. Serovar 1/2a belongs to phylogenetic lineage I, while serovars 4b and 1/2b group in phylogenetic lineage II. To explore the role of gene expression in the adaptation of L. monocytogenes strains of these two major lineages to different environments, as well as in virulence, we performed whole-genome expression profiling of six L. monocytogenes isolates of serovars 4b, 1/2b, and 1/2a of distinct origins, using a newly constructed Listeria multigenome DNA array. Comparison of the global gene expression profiles revealed differences among strains. The expression profiles of two strains having distinct 50% lethal doses, as assessed in the mouse model, were further analyzed. Gene ontology term enrichment analysis of the differentially expressed genes identified differences in protein-, nucleic acid-, carbon metabolism-, and virulence-related gene expression. Comparison of the expression profiles of the core genomes of all strains revealed differences between the two lineages with respect to cell wall synthesis, the stress-related sigma B regulon and virulence-related genes. These findings suggest different patterns of interaction with host cells and the environment, key factors for host colonization and survival in the environment.
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Chuck, Chi-Pang, Hak-Fun Chow, David Chi-Cheong Wan, and Kam-Bo Wong. "Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses." PLoS ONE 6, no. 11 (November 2, 2011): e27228. http://dx.doi.org/10.1371/journal.pone.0027228.
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Pauwels, Frederik, Wendy Mostmans, Ludo M. M. Quirynen, Liesbet van der Helm, Carlo W. Boutton, Anne-Stéphanie Rueff, Erna Cleiren, et al. "Binding-Site Identification and Genotypic Profiling of Hepatitis C Virus Polymerase Inhibitors." Journal of Virology 81, no. 13 (April 25, 2007): 6909–19. http://dx.doi.org/10.1128/jvi.01543-06.
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ABSTRACT The search for hepatitis C virus polymerase inhibitors has resulted in the identification of several nonnucleoside binding pockets. The shape and nature of these binding sites differ across and even within diverse hepatitis C virus genotypes. These differences confront antiviral drug discovery with the challenge of finding compounds that are capable of inhibition in variable binding pockets. To address this, we have established a hepatitis C virus mutant and genotypic recombinant polymerase panel as a means of guiding medicinal chemistry through the elucidation of the site of action of novel inhibitors and profiling against genotypes. Using a genotype 1b backbone, we demonstrate that the recombinant P495L, M423T, M414T, and S282T mutant enzymes can be used to identify the binding site of an acyl pyrrolidine analog. We assess the inhibitory activity of this analog and other nonnucleoside inhibitors with our panel of enzyme isolates generated from clinical sera representing genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a.
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Anuja, A., H. Singh, D. Misra, V. Agarwal, and L. Gupta. "AB0149 PERIPHERAL T HELPER SUBSET PROFILING DIFFERS IN VARIOUS SUBSETS OF IDIOPATHIC INFLAMMATORY MYOSITIS." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1375.2–1375. http://dx.doi.org/10.1136/annrheumdis-2020-eular.5877.
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Background:There is dearth of biomarkers in Idiopathic Inflammatory Myositis(IIM) to identify ongoing inflammation in the muscle and distinguish it from inactivity or damage.Objectives:Since myositis is autoantibody mediated and tertiary lymphoid organogenesis (TLO) reported in the diseased muscles, we investigated peripheral blood T helper subset profiling as a reflection of ongoing muscle inflammation.Methods:Twenty-six patients of IIM (ACR EULAR criteria) were compared with 15 healthy controls (HC) and 21 patients with sarcoidosis (Table 1). Peripheral blood mononuclear cells were stained with combinations of antibodies to identify Th1, Th17, Th17.1 and Treg cells after stimulation assays (BD Biosciences). Myositis Specific and Associated autoantibodies were tested by the line immunoassay (Euroimmune, Germany).Table 1.Baseline characteristics of patients with inflammatory myositisCharacteristicsDemographic details (n, % or median, IQR)Healthy Control (median, IQR)Age37±25.2526.0±32Gender(M:F)5 vs. 2112 vs. 3Diagnosis PM3 DM15 OM4 ASS4Disease course Monocyclic5 Polycyclic7 Chronic continuous1 Undefined13Clinical Profile Myositis4 (15.3%) ILD5 (19.23%) Rash3 (11.53%) Arthritis6 (23.07%) Other16 (23.69)Disease duration (years)1.3 ± 6.91Disease activity Active12 [PM(1), OM(1), ASS(4), DM(5)] Inactive14 [PM(2),OM(2),ASS(0),DM(8)]Antinuclear AntibodiesPositive Nuclear Speckled9 (34.61%) Homogenous4 (15.38%) Nucleolar1 (3.8%) Other5 (19.23%)Cytoplasmic3 (11.53%)Negative4 (15.38%)Myositis Specific AntibodiesPositive ARS2(7.6%) Mi-23(11.53%) SAE-12(7.6%) NXP22(7.6%) MDA50MAA Ku1(3.8%) dsDNA0 U1RNP0 Ro524(15.38%)Negative12(46.15%)Results:All T helper subsets were higher in myositis as compared with healthy controls (figure 1A a-d). Between various IIM subsets, polymyositis had higher Th1 and Treg cells (Figure 1B b, c) while Th17 and Th17.1 cells(c) were higher in Overlap Myositis (Figure 1B a, d) as compared with healthy controls. Patients with sarcoidosis had similar subset profiling as myositis.(Figure 5a-f)Figure 1A. Representative plot depicting all T helper subsets quantified were higher in myositis as compared with healthy controls1B: Representative plot comparing %T cell subsets in various subsets of myositis with healthy controls showing that % Th1 cells (a) and Tregs (d) are highest in Polymyositis than controls while % Th17 (b) and % Th17.1 cells (c) are higher in Overlap MyositisPatients who were had either arthritis or were positive for myositis specific autoantibodies had higher Th17.1 cells (Figure 3 a(iii) & b(iii)) than those negative for MSA. There was no difference in T cell profile between the various autoantibody subsets (Figure 6a-d).There was no difference in subsets between active and inactive disease although active disease had lower Th1/Treg, Th17/Treg and Th17.1/Treg ratios.Conclusion:T Helper cell subsets are distinct from HC but similar to sarcoidosis patients. However, they differ in various subsets of myositis, suggesting different pathogenic mechanisms are operative. Autoantibody positivity is associated with elevated Th17.1 population suggesting plasticity in TLO which needs to be explored further. However, T cell profiling cannot distinguish active from inactive disease limited predictive potential as a biomarker.Figure 2A. Comparisons between various phenotypic subsets suggest patients positive for MSA had higher Th17.1 cells (Figure 2A a(iii)) than those negative for MSA. Similarly, patients with arthritis had higher Th17.1 cells(Figure 2A b (iii)). 2B Representative dot plot of T cell subsets ratio (Th1, Th17 & Th17.1) with Treg subsets (a) Th17/Treg ratio observed higher in lower cells in active as compared with inactive disease. 2C Representative dot plot T cell subsets in Sacodosis and myositis2D Representative dot plot comparing percentage of T cell subsets in various antibody subsets of myositisAcknowledgments:The authors thank APLAR for funding Myositis antibody testing.Disclosure of Interests:None declared
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Siegrist, Fredy, Thomas Singer, and Ulrich Certa. "MicroRNA Expression Profiling by Bead Array Technology in Human Tumor Cell Lines Treated with Interferon-Alpha-2a." Biological Procedures Online 11, no. 1 (July 23, 2009): 113–29. http://dx.doi.org/10.1007/s12575-009-9012-1.
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Mphahlele, Malose J., Emmanuel N. Agbo, Garland K. More, and Samantha Gildenhuys. "In Vitro Enzymatic and Kinetic Studies, and In Silico Drug-Receptor Interactions, and Drug-Like Profiling of the 5-Styrylbenzamide Derivatives as Potential Cholinesterase and β-Secretase Inhibitors with Antioxidant Properties." Antioxidants 10, no. 5 (April 22, 2021): 647. http://dx.doi.org/10.3390/antiox10050647.
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The 5-(styryl)anthranilamides were transformed into the corresponding 5-styryl-2-(p-tolylsulfonamido)benzamide derivatives. These 5-styrylbenzamide derivatives were evaluated through enzymatic assays in vitro for their capability to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-secretase (BACE-1) activities as well as for antioxidant potential. An in vitro cell-based antioxidant activity assay involving lipopolysaccharides (LPS)-induced reactive oxygen species (ROS) production revealed that compounds 2a and 3b have the capability of scavenging free radicals. The potential of the most active compound, 5-styrylbenzamide (2a), to bind copper (II) or zinc (II) ions has also been evaluated spectrophotometrically. Kinetic studies of the most active derivatives from each series against the AChE, BChE, and β-secretase activities have been performed. The experimental results are complemented with molecular docking studies into the active sites of these enzymes to predict the hypothetical protein–ligand binding modes. Their drug likeness properties have also been predicted.
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Murata, Yui, Miki Bundo, Fumiko Sunaga, Kiyoto Kasai, and Kazuya Iwamoto. "DNA Methylation Profiling in a Neuroblastoma Cell Line Exposed to the Antipsychotic Perospirone." Pharmacopsychiatry 52, no. 02 (February 27, 2018): 63–69. http://dx.doi.org/10.1055/s-0044-101467.
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Abstract Introduction Accumulating evidence suggests the importance of epigenetic changes in the brain induced by antipsychotic drugs. However, due to the lack of systematic investigation, their effects on epigenetic status remain largely unclear. During the course of examining the epigenetic effects of antipsychotics, we here focused on perospirone, an atypical antipsychotic drug mainly used in Japan. Methods Genomic DNA was obtained from human neuroblastoma cells exposed to 2 different doses of perospirone. Comprehensive DNA methylation analysis was performed using the Infinium HumanMethylation450 BeadChip. Results Of about 470,000 probes, perospirone exposure changed DNA methylation at 4098 probes. These probes were enriched to genes for neural development. Probes showing hypermethylation were mainly found at gene body and intergenic regions, whereas those that showed hypomethylation were located near promoter regions. Additionally, DNA methylation changes were found in the probes for dopamine receptor 2 and serotonin receptor (HTR) 2A and HTR1A, which are the pharmacological targets of atypical antipsychotics. Discussion Our comprehensive DNA methylation analyses will contribute to a better understanding of detailed pharmacological actions of perospirone.
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Do, Minh-Ha T., Sharon J. Santos, and Mark A. Lawson. "GNRH Induces the Unfolded Protein Response in the LβT2 Pituitary Gonadotrope Cell Line." Molecular Endocrinology 23, no. 1 (January 1, 2009): 100–112. http://dx.doi.org/10.1210/me.2008-0071.
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The neuropeptide GNRH 1 stimulates the secretion of the reproductive hormone LH in pituitary gonadotropes. Other secretory cell types depend on the unfolded protein response (UPR) pathway to regulate protein synthesis and protect against endoplasmic reticulum (ER) stress in response to differentiation or secretory stimuli. This study investigated the role of the UPR in GNRH action within the LβT2 gonadotrope model. Cells were treated with GNRH, and the activation of UPR signaling components and general translational status was examined. The ER-resident stress sensors, Atf6, Eif2ak3, and Ern1, are all present, and GNRH stimulation results in the phosphorylation of eukaryotic translation initiation factor 2A kinase 3 and its downstream effector, eukaryotic translation initiation factor 2A. Additionally, activation of the UPR was confirmed both in LβT2 as well as mouse primary pituitary cells through identifying GNRH-induced splicing of Xbp1 mRNA, a transcription factor activated by splicing by the ER stress sensor, ER to nucleus signaling 1. Ribosome profiling revealed that GNRH stimulation caused a transient attenuation in translation, a hallmark of the UPR, remodeling ribosomes from actively translating polysomes to translationally inefficient ribonucleoprotein complexes and monosomes. The transient attenuation of specific mRNAs was also observed. Overall, the results show that GNRH activates components of the UPR pathway, and this pathway may play an important physiological role in adapting the ER of gonadotropes to the burden of their secretory demand.
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Klein, Sharon, Antonin R. de Fougerolles, Pamela Blaikie, Leila Khan, Angela Pepe, Cynthia D. Green, Victor Koteliansky, and Filippo G. Giancotti. "α5β1 Integrin Activates an NF-κB-Dependent Program of Gene Expression Important for Angiogenesis and Inflammation." Molecular and Cellular Biology 22, no. 16 (August 15, 2002): 5912–22. http://dx.doi.org/10.1128/mcb.22.16.5912-5922.2002.
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ABSTRACT GeneCalling, a genome-wide method of mRNA profiling, reveals that endothelial cells adhering to fibronectin through the α5β1 integrin, but not to laminin through the α2β1 integrin, undergo a complex program of gene expression. Several of the genes identified are regulated by the NF-κB transcription factor, and many are implicated in the regulation of inflammation and angiogenesis. Adhesion of endothelial cells to fibronectin activates NF-κB through a signaling pathway requiring Ras, phosphatidylinositol 3-kinase, and Rho family proteins, whereas adhesion to laminin has a limited effect. Retroviral transfer of the superrepressor of NF-κB, IκB-2A, blocks basic fibroblast growth factor-induced angiogenesis in vivo. These results suggest that engagement of the α5β1 integrin promotes an NF-κB-dependent program of gene expression that coordinately regulates angiogenesis and inflammation.
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Abdelhameed, Reda F. A., Eman S. Habib, Nermeen A. Eltahawy, Hashim A. Hassanean, Amany K. Ibrahim, Anber F. Mohammed, Shaimaa Fayez, et al. "New Cytotoxic Natural Products from the Red Sea Sponge Stylissa carteri." Marine Drugs 18, no. 5 (May 3, 2020): 241. http://dx.doi.org/10.3390/md18050241.
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Bioactivity-guided isolation supported by LC-HRESIMS metabolic profiling led to the isolation of two new compounds, a ceramide, stylissamide A (1), and a cerebroside, stylissoside A (2), from the methanol extract of the Red Sea sponge Stylissa carteri. Structure elucidation was achieved using spectroscopic techniques, including 1D and 2D NMR and HRMS. The bioactive extract’s metabolomic profiling showed the existence of various secondary metabolites, mainly oleanane-type saponins, phenolic diterpenes, and lupane triterpenes. The in vitro cytotoxic activity of the isolated compounds was tested against two human cancer cell lines, MCF-7 and HepG2. Both compounds, 1 and 2, displayed strong cytotoxicity against the MCF-7 cell line, with IC50 values at 21.1 ± 0.17 µM and 27.5 ± 0.18 µM, respectively. They likewise showed a promising activity against HepG2 with IC50 at 36.8 ± 0.16 µM for 1 and IC50 30.5 ± 0.23 µM for 2 compared to the standard drug cisplatin. Molecular docking experiments showed that 1 and 2 displayed high affinity to the SET protein and to inhibitor 2 of protein phosphatase 2A (I2PP2A), which could be a possible mechanism for their cytotoxic activity. This paper spreads light on the role of these metabolites in holding fouling organisms away from the outer surface of the sponge, and the potential use of these defensive molecules in the production of novel anticancer agents.
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Burzynski, Stanislaw R., Tomasz J. Janicki, and Gregory S. Burzynski. "Comprehensive Genomic Profiling of Recurrent Classic Glioblastoma in a Patient Surviving Eleven Years Following Antineoplaston Therapy." Cancer and Clinical Oncology 4, no. 2 (October 22, 2015): 41. http://dx.doi.org/10.5539/cco.v4n2p41.
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Most patients with recurrent glioblastoma (RGBM) die within 6 months regardless of treatment. In phase II studies of Antineoplaston A10 and AS2-1 injections (ANP), our investigators have reported objective responses and long-term survival in RGBM. Using a next-generation sequencing (NGS) based assay of 343 cancer-related genes and introns, comprehensive genomic profiling of tumor tissue obtained from a RGBM patient (who remains alive and well) was performed 11 years after diagnosis and permitted assignment of the patient’s RGBM to the classical subgroup. The most important genomic alterations included amplification of epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitor 2A and 2B (CDKN2A/B), loss of phosphatase and tensin homolog (PTEN) and telomerase reverse transcriptase (TERT) mutation. An analysis of the signaling networks and other targets of ANP therapy was recently performed and presented in this publication. Based on our findings, patients with classical RGBM have a reasonable possibility of responding to ANP therapy and experiencing long-term survival. It is proposed that this subgroup of RGBM patients be enrolled in a genomics-driven clinical trial of ANP therapy.
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Hong, Jung Yong, Hee Jin Cho, Seung Tae Kim, Young Suk Park, Sang Hyun Shin, In Woong Han, Jeeyun Lee, Jin Seok Heo, and Joon Oh Park. "Comprehensive molecular profiling to predict clinical outcomes in pancreatic cancer." Therapeutic Advances in Medical Oncology 13 (January 2021): 175883592110384. http://dx.doi.org/10.1177/17588359211038478.
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Background: Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among common cancers. The genomic landscape of PDAC is defined by four mutational pathways: kirsten rat sarcoma virus ( KRAS), cellular tumor antigen p53 ( TP53), cyclin dependent kinase inhibitor 2A ( CDKN2A), and SMAD family member 4 ( SMAD4). However, there is a paucity of data on the molecular features associated with clinical outcomes after surgery or chemotherapy. Methods: We performed comprehensive molecular characterization of tumor specimens from 83 patients with PDAC who received surgery, using whole-exome sequencing and ribonucleic acid sequencing on tumor and matched normal tissues derived from patients. We also systematically performed integrative analysis, combining genomic, transcriptomic, and clinical features to identify biomarkers and possible therapeutic targets. Results: KRAS (75%), TP53 (67%), CDKN2A (12%), SMAD4 (20%), and ring finger protein 43 ( RNF43) (13%) were identified as significantly mutated genes. The tumor-specific transcriptome was classified into two clusters (tumor S1 and tumor S2), which resembled the Moffitt tumor classification. Tumor S1 displayed two distinct subclusters (S1-1 and S1-2). The transcriptome of tumor S1-1 overlapped with the exocrine-like (Collisson)/ADEX (Bailey) subtype, while tumor S1-2 mostly consisted of the classical (Collisson)/progenitor (Bailey) subtype. In the analysis of combinatorial gene alterations, concomitant mutations of KRAS with low-density lipoprotein receptor related protein 1B (LRP1B) were associated with significantly worse disease-free survival after surgery ( p = 0.034). One patient (1.2%) was an ultrahypermutant with microsatellite instability. We also identified high protein kinase C lota ( PRKCI) expression as an overlapping, poor prognostic marker between our dataset and the TCGA dataset. Conclusion: We identified potential prognostic biomarkers and therapeutic targets of patients with PDAC. Understanding these molecular aberrations that determine patient outcomes after surgery and chemotherapy has the potential to improve the treatment outcomes of PDAC patients.
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Kuzmanov, Uros, Hongbo Guo, Diana Buchsbaum, Jake Cosme, Cynthia Abbasi, Ruth Isserlin, Parveen Sharma, Anthony O. Gramolini, and Andrew Emili. "Global phosphoproteomic profiling reveals perturbed signaling in a mouse model of dilated cardiomyopathy." Proceedings of the National Academy of Sciences 113, no. 44 (October 14, 2016): 12592–97. http://dx.doi.org/10.1073/pnas.1606444113.
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Phospholamban (PLN) plays a central role in Ca2+ homeostasis in cardiac myocytes through regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase 2A (SERCA2A) Ca2+ pump. An inherited mutation converting arginine residue 9 in PLN to cysteine (R9C) results in dilated cardiomyopathy (DCM) in humans and transgenic mice, but the downstream signaling defects leading to decompensation and heart failure are poorly understood. Here we used precision mass spectrometry to study the global phosphorylation dynamics of 1,887 cardiac phosphoproteins in early affected heart tissue in a transgenic R9C mouse model of DCM compared with wild-type littermates. Dysregulated phosphorylation sites were quantified after affinity capture and identification of 3,908 phosphopeptides from fractionated whole-heart homogenates. Global statistical enrichment analysis of the differential phosphoprotein patterns revealed selective perturbation of signaling pathways regulating cardiovascular activity in early stages of DCM. Strikingly, dysregulated signaling through the Notch-1 receptor, recently linked to cardiomyogenesis and embryonic cardiac stem cell development and differentiation but never directly implicated in DCM before, was a prominently perturbed pathway. We verified alterations in Notch-1 downstream components in early symptomatic R9C transgenic mouse cardiomyocytes compared with wild type by immunoblot analysis and confocal immunofluorescence microscopy. These data reveal unexpected connections between stress-regulated cell signaling networks, specific protein kinases, and downstream effectors essential for proper cardiac function.
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Liu, Dongcheng, Shixuan Li, Jiaqing Wang, Xuefeng Sun, Guofeng Li, Bin Peng, Peikun Ding, Zhiqiang Cheng, Chang Zou, and Guangsuo Wang. "Characterization of heterogeneity in three lung cancer patients with multiple nodules by whole exome sequencing." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e21530-e21530. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e21530.
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e21530 Background: Tumor heterogeneity is an important characteristic of malignant tumors that reflects the high complexity and diversity in the process of evolution. It can lead to differences in tumor growth rate, invasion, metastasis, drug sensitivity and prognosis. At present, there exist few genome-wide studies on heterogeneous cases of multiple nodules for patients with non-small-cell lung cancer (NSCLC). Methods: In this study, we performed genetic profiling using a combination of targeted panel and whole exon genome sequencing (WES) on all lesions of three NSCLC patients with multiple nodules. Results: Driver gene alterations were detected in all lesion samples of the three patients. One patient presented with three nodules, which harbored EGFR L858R, ERBB2 exon 20 insertion and a EML4-ALK fusion. Evolutionary analysis showed strong heterogeneity among the three lesions. The three lesions of patient 2 showed EGFR L858R in two lesions (2A and 2C), and a ERBB2 exon 20 insertions in the other (2B). Mutational signatures and cluster analysis of somatic mutations also showed commonality between sample 2A and 2C, suggesting that they might arise from the same clone. Patient 3 had seven lesions. Analysis of somatic mutations and copy number variations revealed possible route of metastasis. Conclusions: Detailed analysis of genetic mutation characteristics and biological evolution trees showed that there were substantial heterogeneity and distinct evolutionary relationships among the different lesions of the same individual. Consequently, such information could aid in the determination of primary and metastasis of tumor lesions and guide the selection of treatment options.
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Schiff, David, Martin Van den Bent, Michael A. Vogelbaum, Wolfgang Wick, C. Ryan Miller, Martin Taphoorn, Whitney Pope, et al. "Recent developments and future directions in adult lower-grade gliomas: Society for Neuro-Oncology (SNO) and European Association of Neuro-Oncology (EANO) consensus." Neuro-Oncology 21, no. 7 (February 8, 2019): 837–53. http://dx.doi.org/10.1093/neuonc/noz033.
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Abstract The finding that most grades II and III gliomas harbor isocitrate dehydrogenase (IDH) mutations conveying a relatively favorable and fairly similar prognosis in both tumor grades highlights that these tumors represent a fundamentally different entity from IDH wild-type gliomas exemplified in most glioblastoma. Herein we review the most recent developments in molecular neuropathology leading to reclassification of these tumors based upon IDH and 1p/19q status, as well as the potential roles of methylation profiling and deletional analysis of cyclin-dependent kinase inhibitor 2A and 2B. We discuss the epidemiology, clinical manifestations, benefit of surgical resection, and neuroimaging features of lower-grade gliomas as they relate to molecular subtype, including advanced imaging techniques such as 2-hydroxyglutarate magnetic resonance spectroscopy and amino acid PET scanning. Recent, ongoing, and planned studies of radiation therapy and both cytotoxic and targeted chemotherapies are summarized, including both small molecule and immunotherapy approaches specifically targeting the mutant IDH protein.
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Wan, Chunhua, Sylvia Mahara, Claire Sun, Anh Doan, Hui Kheng Chua, Dakang Xu, Jia Bian, et al. "Genome-scale CRISPR-Cas9 screen of Wnt/β-catenin signaling identifies therapeutic targets for colorectal cancer." Science Advances 7, no. 21 (May 2021): eabf2567. http://dx.doi.org/10.1126/sciadv.abf2567.
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Aberrant activation of Wnt/β-catenin pathway is a key driver of colorectal cancer (CRC) growth and of great therapeutic importance. In this study, we performed comprehensive CRISPR screens to interrogate the regulatory network of Wnt/β-catenin signaling in CRC cells. We found marked discrepancies between the artificial TOP reporter activity and β-catenin–mediated endogenous transcription and redundant roles of T cell factor/lymphoid enhancer factor transcription factors in transducing β-catenin signaling. Compiled functional genomic screens and network analysis revealed unique epigenetic regulators of β-catenin transcriptional output, including the histone lysine methyltransferase 2A oncoprotein (KMT2A/Mll1). Using an integrative epigenomic and transcriptional profiling approach, we show that KMT2A loss diminishes the binding of β-catenin to consensus DNA motifs and the transcription of β-catenin targets in CRC. These results suggest that KMT2A may be a promising target for CRCs and highlight the broader potential for exploiting epigenetic modulation as a therapeutic strategy for β-catenin–driven malignancies.
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Gamblin, T. Clark, Sydney D. Finkelstein, Neil Upsal, Jonathan D. Kaye, and David Blumberg. "Microdissection-Based Allelotyping: A Novel Technique to Determine the Temporal Sequence and Biological Aggressiveness of Colorectal Cancer." American Surgeon 72, no. 5 (May 2006): 445–53. http://dx.doi.org/10.1177/000313480607200516.
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Pathologic staging in colorectal adenocarcinoma (CA) is based on the concept that the timing of metastatic tumor spread is directly related to the depth of the primary tumor invasion. To evaluate the temporal sequence of CA metastasis, we performed microdissection mutational profiling at multiple microscopic sites of primary and metastatic CA specimens. Twenty-one cases of CA were selected from fixed-tissue archives. Primary tumors were microdissected at the deepest point of invasion. Comparative mutational profiling for different genomic loci [1p36(CCM = cutaneous malignant melanoma], 3p26(OGGI = 8 oxoguanine DNA glycosylase), 5q23 (APC, MCC = mutated in colorectal cancer), 9p21(p16/CDKN2A = cyclin-dependent kinase 2A), 10q23(PTEN = phosphatase and tensin homolog [mutated in multiple advanced cancers 1]), 12p12(K-ras-2 point mutation), 17p13(TP53), 18q25(DCC= deleted in colorectal cancer) was carried out on each microdissected tissue target using microsatellite loss of heterozygosity determination or DNA sequencing. All primary and metastatic sites of CA manifested acquired mutational change in 18 to 91 per cent of the genomic markers. In 15/21 (71%) cases, metastatic sites lacked a specific allelic loss seen in the corresponding primary tumor, indicating that the metastasis occurred before maximal depth of primary invasion. This was further supported by discordant mutational profiles between primary and secondary tumors, requiring divergent clonal evolution. This is the first report describing the temporal sequence and significance of sequential mutational acquisition in clinical tissue specimens with potential implications for a new molecular pathology approach to classify human cancer.
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Alsina, Katherina M., Mohit Hulsurkar, Sören Brandenburg, Daniel Kownatzki-Danger, Christof Lenz, Henning Urlaub, Issam Abu-Taha, et al. "Loss of Protein Phosphatase 1 Regulatory Subunit PPP1R3A Promotes Atrial Fibrillation." Circulation 140, no. 8 (August 20, 2019): 681–93. http://dx.doi.org/10.1161/circulationaha.119.039642.
Full textAbstract:
Background: Abnormal calcium (Ca 2+ ) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca 2+ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN. Methods: Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln -knockout mice. Ppp1r3a -knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a -knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca 2+ imaging. Results: Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca 2+ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF. Conclusions: PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca 2+ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.
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Engelmann, D., D. Koczan, P. Ricken, U. Rimpler, J. Pahnke, Z. Li, and B. M. Pützer. "Transcriptome analysis in mouse tumors induced by Ret-MEN2/FMTC mutations reveals subtype-specific role in survival and interference with immune surveillance." Endocrine-Related Cancer 16, no. 1 (March 2009): 211–24. http://dx.doi.org/10.1677/erc-08-0158.
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Activating mutations in the Ret proto-oncogene are responsible for occurrence of multiple endocrine neoplasia (MEN) type 2A and 2B, and familial medullary thyroid carcinoma (FMTC). A striking genotype–phenotype correlation between the mutated RET codon and clinical manifestation implies that tumorigenesis is conditioned by the type of mutation. We investigated gene expression profiles between and within distinct MEN2 subtypes through whole-genome microarray analysis in tumors induced by NIH-3T3 cells transformed with defined RET-MEN2A (C609Y, C634R), MEN2B, (A883F, M918T), and FMTC (Y791F) mutations. Expression profiling identified a statistically significant modification of 1494 genes, 628 down- and 866 upregulated in MEN2B compared with MEN2A/FMTC tumors. By contrast, no obvious alterations were observed among individual MEN2B and MEN2A type mutations, or between MEN2A and FMTC. Functional clustering of differential genes revealed RET-MEN2B specific upregulation of genes associated with novel growth and survival pathways. Intriguingly, RET-MEN2A/FMTC-specific tumors were characterized by a considerable number of genes involved in the host antitumor immune response via stimulation of natural killer/T-cell proliferation, migration, and cytotoxicity, which were completely absent in RET-MEN2B related cancers. QPCR on tumors versus cultured NIH-RET cell lines demonstrated that they are largely attributed to the host innate immune system, whereas expression of CX3CL1 involved in leukocyte recruitment is exclusively RET-MEN2A/FMTC tumor cell dependent. In correlation, massive inflammatory infiltrates were apparent only in tumors carrying MEN type 2A/FMTC mutations, suggesting that RET-MEN2B receptors specifically counteract immune infiltration by preventing chemokine expression, which may contribute to the different clinical outcome of both subtypes.
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Lin, Jiunn-Chang, Tsang-Pai Liu, and Pei-Ming Yang. "CDKN2A-Inactivated Pancreatic Ductal Adenocarcinoma Exhibits Therapeutic Sensitivity to Paclitaxel: A Bioinformatics Study." Journal of Clinical Medicine 9, no. 12 (December 12, 2020): 4019. http://dx.doi.org/10.3390/jcm9124019.
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The mutation of cyclin dependent kinase inhibitor 2A (CDKN2A) is frequently found in pancreatic ductal adenocarcinoma (PDAC). However, its prognostic and therapeutic roles in PDAC have not been extensively investigated yet. In this study, we mined and integrated the cancer genomics and chemogenomics data to investigate the roles of CDKN2A genetic alterations in PDAC patients’ prognosis and treatment. We found that functional CDKN2A inactivation caused by mutations and deep deletions predicted poor prognosis in PDAC patients. CDKN2A inactivation was associated with the upregulation of genes related to estrogen response, which can be overcome by CDKN2A restoration. Chemosensitivity profiling of PDAC cell lines and patient-derived organoids found that CDKN2A inactivation was associated with the increased sensitivity to paclitaxel and SN-38 (the active metabolite of irinotecan). However, only paclitaxel can mimic the effect of CDKN2A restoration, and its drug sensitivity was correlated with genes related to estrogen response. Therefore, our study suggested that CDKN2A-inactivated PDAC patients could benefit from the precision treatment with paclitaxel, whose albumin-stabilized nanoparticle formulation (nab-paclitaxel) has been approved for treating PDAC.
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Mafficini, Andrea, Rita T. Lawlor, Claudio Ghimenton, Davide Antonello, Cinzia Cantù, Gaetano Paolino, Alessia Nottegar, et al. "Solid Pseudopapillary Neoplasm of the Pancreas and Abdominal Desmoid Tumor in a Patient Carrying Two Different BRCA2 Germline Mutations: New Horizons from Tumor Molecular Profiling." Genes 12, no. 4 (March 26, 2021): 481. http://dx.doi.org/10.3390/genes12040481.
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This case report describes the history of a 41 year-old woman with a solid pseudopapillary neoplasm (SPN) of the pancreas and a metachronous abdominal desmoid tumor (DT) that occurred two years after the SPN surgical resection. At next-generation sequencing of 174 cancer-related genes, both neoplasms harbored a CTNNB1 somatic mutation which was different in each tumor. Moreover, two BRCA2 pathogenic mutations were found in both tumors, confirmed as germline by the sequencing of normal tissue. The BRCA2 mutations were c.631G>A, resulting in the amino-acid change p.V211I, and c.7008-2A>T, causing a splice acceptor site loss. However, as the two neoplasms showed neither loss of heterozygosity nor somatic mutation in the second BRCA2 allele, they cannot be considered as BRCA-dependent tumors. Nevertheless, this study highlights the important opportunities opened by extensive tumor molecular profiling. In this particular case, it permitted the detection of BRCA2-germline mutations, essential for addressing the necessary BRCA-related genetic counseling, surveillance, and screening for the patient and her family.
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Hu, Xiao, Xikui Liu, Hongxiu Li, Elizabeth Zawidzka, Garry Alan Weems, Yilin Gao, Brian Fox, H. Jeffrey Wilkins, and Laura Carter. "Novel small-molecule RORγ agonist immuno-oncology agent LYC-55716: Tumor selection and evaluation of renal cell and bladder cancer for inclusion in phase 2a expansion." Journal of Clinical Oncology 36, no. 6_suppl (February 20, 2018): 424. http://dx.doi.org/10.1200/jco.2018.36.6_suppl.424.
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424 Background: The master transcription factor retinoic acid receptor–related orphan receptor γt (RORγ) controls Type 17 effector T cell differentiation and function. Synthetic RORγ agonists modulate gene expression to enhance immune effector functions and decrease regulatory T cell (Treg) development and expression of checkpoint pathways. RORγ agonists have shown promise as mono- and combination therapy in syngeneic tumor models. Translational and bioinformatics analyses were performed to evaluate renal cell carcinoma (RCC) and bladder cancer (BC) for inclusion in a Phase 2a expansion trial of the investigational RORγ agonist LYC-55716 (NCT02929862). Methods: A gene signature was identified through transcriptional profiling of murine and human T cells treated ± RORγ agonists. Bioinformatics analyses were then conducted using data on RCC and BC patients from The Cancer Genome Atlas (TCGA) to determine (a) RORγ and RORγ-inducing cytokine expression; (b) signature genes associated with RORγ biology, biomarkers for endogenous RORγ ligands, and correlations with patient survival rates; and (c) tumor microenvironment (TME) immune profiles. Results: Expression: RNA sequencing analysis identified RORγ+ cells in a significant fraction of RCC and BC samples. RORγ-inducing cytokines IL6, IL23a, and IL1b as well as other genes that support Type 17 differentiation were highly expressed in RCC and BC. Biology: Low expression of sterol efflux genes in RCC and BC tumors suggested low levels of endogenous RORγ ligands in the TME. Importantly, analysis revealed a positive correlation between patient survival and expression of RORγ (or the RORγ signature gene IL17A) for RCC. Immune profile: Analysis of RCC and BC tumors indicated high infiltration of CD3+, CD4+, and CD8+ T cells and high mutation burden, which are associated with immunotherapy efficacy. Conclusions: Translational and bioinformatics studies of RORγ expression, biology, and tumor immune profiles support the inclusion of RCC and BC patients in an ongoing Phase 2a expansion trial of LYC-55716.
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Kobow, Katja, Samir Jabari, Tom Pieper, Manfred Kudernatsch, Tilman Polster, Friedrich G. Woermann, Thilo Kalbhenn, et al. "Mosaic trisomy of chromosome 1q in human brain tissue associates with unilateral polymicrogyria, very early-onset focal epilepsy, and severe developmental delay." Acta Neuropathologica 140, no. 6 (September 26, 2020): 881–91. http://dx.doi.org/10.1007/s00401-020-02228-5.
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AbstractPolymicrogyria (PMG) is a developmental cortical malformation characterized by an excess of small and frustrane gyration and abnormal cortical lamination. PMG frequently associates with seizures. The molecular pathomechanisms underlying PMG development are not yet understood. About 40 genes have been associated with PMG, and small copy number variations have also been described in selected patients. We recently provided evidence that epilepsy-associated structural brain lesions can be classified based on genomic DNA methylation patterns. Here, we analyzed 26 PMG patients employing array-based DNA methylation profiling on formalin-fixed paraffin-embedded material. A series of 62 well-characterized non-PMG cortical malformations (focal cortical dysplasia type 2a/b and hemimegalencephaly), temporal lobe epilepsy, and non-epilepsy autopsy controls was used as reference cohort. Unsupervised dimensionality reduction and hierarchical cluster analysis of DNA methylation profiles showed that PMG formed a distinct DNA methylation class. Copy number profiling from DNA methylation data identified a uniform duplication spanning the entire long arm of chromosome 1 in 7 out of 26 PMG patients, which was verified by additional fluorescence in situ hybridization analysis. In respective cases, about 50% of nuclei in the center of the PMG lesion were 1q triploid. No chromosomal imbalance was seen in adjacent, architecturally normal-appearing tissue indicating mosaicism. Clinically, PMG 1q patients presented with a unilateral frontal or hemispheric PMG without hemimegalencephaly, a severe form of intractable epilepsy with seizure onset in the first months of life, and severe developmental delay. Our results show that PMG can be classified among other structural brain lesions according to their DNA methylation profile. One subset of PMG with distinct clinical features exhibits a duplication of chromosomal arm 1q.
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Li, Huaqin, Lianjie Hou, Yu Zhang, Fangyi Jiang, Yifan Zhu, Qing X. Li, Ching Yuan Hu, and Chong Wang. "PFN2a Suppresses C2C12 Myogenic Development by Inhibiting Proliferation and Promoting Apoptosis via the p53 Pathway." Cells 8, no. 9 (August 23, 2019): 959. http://dx.doi.org/10.3390/cells8090959.
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Skeletal muscle plays a crucial role in physical activity and in regulating body energy and protein balance. Myoblast proliferation, differentiation, and apoptosis are indispensable processes for myoblast myogenesis. Profilin 2a (PFN2a) is a ubiquitous actin monomer-binding protein and promotes lung cancer growth and metastasis through suppressing the nuclear localization of histone deacetylase 1 (HDAC1). However, how PFN2a regulates myoblast myogenic development is still not clear. We constructed a C2C12 mouse myoblast cell line overexpressing PFN2a. The CRISPR/Cas9 system was used to study the function of PFN2a in C2C12 myogenic development. We find that PFN2a suppresses proliferation and promotes apoptosis and consequentially downregulates C2C12 myogenic development. The suppression of PFN2a also decreases the amount of HDAC1 in the nucleus and increases the protein level of p53 during C2C12 myogenic development. Therefore, we propose that PFN2a suppresses C2C12 myogenic development via the p53 pathway. Si-p53 (siRNA-p53) reverses the PFN2a inhibitory effect on C2C12 proliferation and the PFN2a promotion effect on C2C12 apoptosis, and then attenuates the suppression of PFN2a on myogenic differentiation. Our results expand understanding of PFN2a regulatory mechanisms in myogenic development and suggest potential therapeutic targets for muscle atrophy-related diseases.
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Gong, Jun, Vincent M. Chung, Joel Picus, Scott T. Tagawa, Sumati Gupta, Julie Poore, Christine Peterson, Ely Benaim, and Sumanta K. Pal. "Activity of RX-3117, an oral antimetabolite nucleoside, in subjects with metastatic bladder cancer resistant to gemcitabine: Preliminary results of a phase Ia/IIb study." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 4544. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.4544.
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4544 Background: RX-3117 is an oral small molecule antimetabolite, cyclopentyl pyrimidyl nucleoside that is activated by uridine cytidine kinase 2. RX-3117 has shown efficacy in xenograft models of gemcitabine resistant pancreatic, bladder and colorectal cancer. Preliminary data from an analysis of a phase 1b/2a clinical study of RX3117 in metastatic bladder cancer is described. Methods: This phase 1b/2a study (NCT02030067) was designed to evaluate safety, tolerability and efficacy following treatment with 700 mg administered orally once-daily for 5 consecutive days with 2 days off per week for 3 weeks with 1 week off in each 4 week cycle in a 2-stage design. Eligible subjects (aged ≥ 18 years) were those with relapsed/refractory metastatic bladder cancer with any number of prior therapies. Prior therapy with platinum-based chemotherapy was required. The primary endpoint was to assess the efficacy and safety of RX-3117 in metastatic bladder cancer, with secondary aims of evaluating PFS and CBR. Results: With 9 subjects enrolled, median age was 66 years, ECOG PS was 0-1. All subjects had received gemcitabine/cisplatin in the perioperative or metastatic setting, and 4 subjects had received 3 or more prior therapies. The most frequent related adverse events were anemia, mild-moderate fatigue, vomiting and diarrhea. No dose limiting toxicities were observed. PFS and CBR will be presented at the meeting, as 5 subjects continue to receive therapy at the time of this submission. One subject continues on treatment at 139 days with persistent stable disease. Molecular profiling of his bladder tumor showed alterations in ARID1A, FBXW7, FGFR3, NF1, and TERT. The patient previously responded to an FGFR3 inhibitor but progressed after 9 months, with ctDNA assessments showing incurrence of TP53 alteration. Clinical benefit with RX-3117 was achieved in spite of incurrence of this alteration. Conclusions: RX-3117 demonstrated an excellent safety profile, and prolonged stable disease was seen in 1 subject who failed prior cisplatin/gemcitabine and FGFR3 inhibition. Activity persisted despite development a putative resistance alteration detected by ctDNA. Clinical trial information: NCT02030067.
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Feldman, Rebecca, Sandeep K. Reddy, Zoran Gatalica, Joanne R. Mahanes, and Charles E. Myers. "Biomarker patterns of localized and metastatic prostate cancer." Journal of Clinical Oncology 33, no. 7_suppl (March 1, 2015): 192. http://dx.doi.org/10.1200/jco.2015.33.7_suppl.192.
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192 Background: Presence or absence of metastases is a determining factor for prostate cancer prognosis. Common sites of metastasis include bone (B), lymph nodes (LN) or visceral organs (V). We sought to determine theranostic biomarker differences between primary (P) and metastatic (M) specimens, with subset analysis for differences between metastatic sites. Methods: 497 prostate cancer cases referred to Caris Life Sciences between 2009 thru 2014 were evaluated. Specific testing was performed and included a multiplatform approach: sequencing (Sanger, NGS), protein expression (IHC) and gene amplification (CISH/FISH). Results: 58% (287/497) were M specimens, of which 23% (66), 24% (68) and 52% (149) were from B, LN and V, respectively. Significant differences for EGFR amplification/overexpression, low MGMT expression, TOPO1/2A overexpression and higher PTEN mutation rate were found in the M subgroup. These alterations indicate a role for EGFR/PTEN in progression of prostate cancer, and potential role of MAPK/PAM-targeted therapies, alkylating agents and topoisomerase inhibitors in M disease. Mutational profiles of the M subgroup are more genetically unstable exhibiting mutations in 60% of genes tested (notable events include APC & β-Catenin, PTEN, TP53 and BRCA1/2) vs. 30% observed in the P subgroup (p=0.0067). In the subset analysis, EGFR amplification was higher in B (29%) and V (24%) compared to LN (13%); (not significant). Low TS expression was more frequent in B vs. LN (p=0.004), TOPO2A overexpression was higher in V vs. B (p=0.0001) and there was a trend for PD1 positive infiltrating lymphocytes being more abundant in LN vs B (p=0.09). Interestingly, cMET overexpression (7/230) and amplification (1/162) were rarely observed across the cohort, providing support for the surprising failure of cabozantinib in castration-resistant prostate cancer. Conclusions: Our data indicate a role for multiplatform profiling to identify therapeutic options that are appropriate for subsets of patients. Molecular changes associated with M disease including EGFR, PTEN, MGMT and TOPO1/2A, present opportunities for developing therapeutic strategies which may include a combination of targeted therapies, as well as traditional cytotoxic chemotherapies.
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Reddy, Kalpana S., David K. Crockett, Marshall E. Kadin, Kojo S. J. Elenitoba-Johnson, and Megan S. Lim. "Quantitative Proteomic Profiling of Primary Cutaneous CD30+ T-Cell Lymphoma Progression." Blood 104, no. 11 (November 16, 2004): 2270. http://dx.doi.org/10.1182/blood.v104.11.2270.2270.
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Abstract Primary cutaneous CD30+ T-cell lymphoproliferative disorders (LPD) represent a complex spectrum of related conditions that are not very well understood. Many may progress to more aggressive or systemic lymphomas. The factors involved in the progression of these LPDs are largely unknown. Advances in robust high throughput proteomics techniques permit elucidation of the molecular mechanisms underlying disease development and progression. In this study we applied a quantitative proteomics methodology involving isotope coded affinity tag reagents (ICAT™), 3-dimensional liquid chromatography (3-D LC), and tandem mass spectrometry (MS/MS) to evaluate the differential protein expression patterns of two unique T-cell lymphoma derived cell lines. The Mac-1 and Mac-2A are clonally related cell lines derived from the same cutaneous T-cell lymphoma (CTCL) at relatively indolent versus aggressive stages of tumor progression, respectively. Equivalent quantities of total cell lysates obtained from the two cell lines were labeled with cleavable ICAT™ and dialyzed prior to digestion with proteinase K. The labeled peptides were then subjected to strong cation-exchange (SCX) chromatography followed by further purification of each offline fraction using avidin affinity chromatography. These cysteine enriched ICAT™ labeled peptides were analyzed by reverse phase nanospray LC-MS/MS. Differential expression of 490 unique proteins was determined. Of these, a total of 59 proteins showed a 1.5 fold or greater increase in abundance while 86 proteins exhibited a similar decrease in the clinically aggressive lymphoma cells as compared to the more indolent form. Functional classification of the differentially expressed proteins revealed increased expression of those involved in cell signaling (neurotrophic tyrosine kinase receptor, ATP binding cassette transporter 1, SH3 protein interacting with NCK), transcription (HFK1, general transcription factor IIIC), and cell adhesion (ICAM, protocadherin) in the advanced lymphoma. There was also decreased expression in the advanced lymphoma of proteins which were involved in apoptosis (caspase 8), cell signaling (STAT4, LIM kinase), cell cycle regulation (CDC10 homolog, ankyrin 3), cell adhesion (laminin, periostin), and ion transport (voltage-gated potassium channel). Overexpression of MALT1/paracaspase that leads to activation of the NF-kappaB pathway was observed in the advanced lymphoma. Proteins such as Smad4 which is involved in regulation of the TGF-beta signaling pathway and ubiquitin specific protease of the ubiquitin-proteasomal degradation pathway that in turn regulates Smads were differentially expressed. Some of the proteins identified by MS were selected for confirmation by Western blot analysis. This study implicates deregulation of multiple signal transduction pathways during progression of CTCL and provides novel insights into the underlying molecular pathogenetic mechanisms involved.
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Knight, Pamela A., Alan D. Pemberton, Kevin A. Robertson, Douglas J. Roy, Steven H. Wright, and Hugh R. P. Miller. "Expression Profiling Reveals Novel Innate and Inflammatory Responses in the Jejunal Epithelial Compartment during Infection with Trichinella spiralis." Infection and Immunity 72, no. 10 (October 2004): 6076–86. http://dx.doi.org/10.1128/iai.72.10.6076-6086.2004.
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ABSTRACT Infection with intestinal nematodes induces profound pathological changes to the gut that are associated with eventual parasite expulsion. We have applied expression profiling as an initial screening process with oligonucleotide microarrays (Affymetrix MG-U74AV2 gene chips) and time course kinetics to investigate gene transcription triggered by the intraepithelial nematode Trichinella spiralis in jejunal epithelium from BALB/c mice. Of the 4,114 genes detected, 2,617 were present in all uninfected and T. spiralis-infected replicates, 8% of which were notably upregulated, whereas 12% were downregulated at the time of worm expulsion (day 14 postinfection). Upregulation of goblet cell mucin gene transcripts intestinal mucin gene 3 (MUC3), calcium chloride channel 5 (CLCA5), and goblet cell gene 4 (GOB4) is consistent with enhanced production and alteration of mucus, whereas a 60- to 70-fold upregulation of transcripts for mast cell proteases 1 and 2 (MCPT-1 and -2) is consistent with intraepithelial mucosal mast cell recruitment. Importantly, there was novel expression of sialyltransferase 4C (SIAT4C), small proline-rich protein 2A (SPRR2A), and resistin-like molecule β (RELMβ) on day 14 postinfection. In contrast, DNase I and regenerating protein 3 (REG3) transcripts were substantially downregulated. Time course analyses revealed early (within 48 h of infection) induction of Siat4c, Sprr2A, and Relmβ and later (within 120 h) induction of Mcpt-1 and -2. The findings demonstrate early innate responses and later inflammatory changes within the epithelium. The early epithelial responses may be associated both with repair (Sprr2A) and with the development of innate immunity (Siat4c and Relmβ).
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Wei, Kevin, Aaron C. Logan, Heather Wakelee, M. Celeste Simon, and Calvin J. Kuo. "Systemic VEGF Inhibition Induces Hepatic EPO Production and Erythrocytosis Via HIF-2a-Dependent and -Independent Mechanisms." Blood 112, no. 11 (November 16, 2008): 482. http://dx.doi.org/10.1182/blood.v112.11.482.482.
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Abstract Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We have previously reported increased hematocrit (60–75%) and erythrocytosis after high-grade VEGF inhibition by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Erythrocytosis occurred in both mice and primate models in an erythropoietin (Epo)-dependent manner, with unexpected >40-fold induction of hepatic erythropoietin through a hypoxia- and HIF-1α-independent mechanism involving disruption of endothelial-hepatocyte cross-talk. To identify candidate transcription factors required for VEGF inhibition-dependent hepatic Epo induction, we performed hepatic gene expression profiling, revealing up-regulation of HIF-2α target genes (Loxl2 and Cited2). Systemic VEGF inhibition resulted in hepatic stabilization of the HIF-2α protein without concomitant liver hypoxia. Conversely, hepatocyte-specific deletion of the Hif2a gene in Hif2aflox/flox mice strongly diminished the hematocrit elevation and hepatic Epo synthesis after systemic VEGF inhibition, indicating an essential role for HIF-2α. These results are consistent with other reports indicating potent regulation of hepatic Epo production by HIF-2α. In addition to the previously characterized Epo-dependent erythrocytosis, RBC mass monitoring in VEGF inhibitor-treated mice reveals that an erythrocytosis-independent mechanism also contributes to elevated hematocrit. VEGF inhibition initially moderately elevates hematocrit from a baseline of 47% to approximately 50–55% without a parallel increase in RBC mass or reticulocyte index. This initial hematocrit elevation (phase 1) occurs over the first 5 days of VEGF inhibition and is associated with decreased plasma volume (PV) as measured by Evans blue dye injection. Subsequently, a persistent erythrocytosis ensues, as reflected by increased RBC mass and reticulocytosis (phase 2) and is responsible for the vast majority of the erythrocytosis observed with stringent in vivo VEGF inhibition. Together, decreased PV (phase 1) with elevated erythrocytosis (phase 2) combine to maximally elevate hematocrit (near 75%) under stringent VEGF inhibition. Notably, phase 2 but not phase 1 hematocrit elevation and Epo induction were ablated by hepatocyte Hif2a gene deletion, again suggesting that phase 1 elevation arises through distinct mechanisms. Clinical implications of our work include the use of VEGF inhibitors for the simultaneous treatment of malignancy and anemia as well as the use of hematocrit and/or Epo as surrogate markers for the efficacy of in vivo VEGF inhibition. Accordingly, we will summarize our initial attempts at prospective monitoring of these parameters in cancer patient populations treated with VEGF inhibitors. Overall, these data highlight unexpected physiologic consequences of VEGF inhibition and indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythrocytosis via HIF-2α-dependent and -independent mechanisms.
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Mphahlele, Malose J., Emmanuel Ndubuisi Agbo, and Yee Siew Choong. "Synthesis, Structure, Carbohydrate Enzyme Inhibition, Antioxidant Activity, In Silico Drug-Receptor Interactions and Drug-Like Profiling of the 5-Styryl-2-Aminochalcone Hybrids." Molecules 26, no. 9 (May 4, 2021): 2692. http://dx.doi.org/10.3390/molecules26092692.
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The 2-amino-5-(3/4-fluorostyryl)acetophenones were prepared and reacted with benzaldehyde derivatives to afford the corresponding 5-styryl-2-aminochalcone hybrids. The trans geometry of the styryl and α,β-unsaturated carbonyl arms, and the presence of NH…O intramolecular hydrogen bond were validated using 1H-NMR and X-ray data. The 2-amino-5-styrylacetophenones and their 5-styryl-2-aminochalcone derivatives were screened in vitro for their capability to inhibit α-glucosidase and/or α-amylase activities. Their antioxidant properties were evaluated in vitro through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) free radical scavenging assays. Kinetic studies of the most active derivatives from each series against α-glucosidase and/or α-amylase activities have been performed supported by molecular docking studies to determine plausible protein–ligand interactions on a molecular level. The key aspects of the pharmacokinetics of these compounds, i.e., absorption, distribution, metabolism, and excretion have also been simulated at theoretical level. The most active compounds from each series, namely, 2a and 3e, were evaluated for cytotoxicity against the normal monkey kidney cells (Vero cells) and the adenocarcinomic human epithelial (A549) cell line to establish their safety profile at least in vitro.
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Abdelhameed, Reda F. A., Enas E. Eltamany, Dina M. Hal, Amany K. Ibrahim, Asmaa M. AboulMagd, Tarfah Al-Warhi, Khayrya A. Youssif, et al. "New Cytotoxic Cerebrosides from the Red Sea Cucumber Holothuria spinifera Supported by In-Silico Studies." Marine Drugs 18, no. 8 (August 1, 2020): 405. http://dx.doi.org/10.3390/md18080405.
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Bioactivity-guided fractionation of a methanolic extract of the Red Sea cucumber Holothuria spinifera and LC-HRESIMS-assisted dereplication resulted in the isolation of four compounds, three new cerebrosides, spiniferosides A (1), B (2), and C (3), and cholesterol sulfate (4). The chemical structures of the isolated compounds were established on the basis of their 1D NMR and HRMS spectral data. Metabolic profiling of the H. spinifera extract indicated the presence of diverse secondary metabolites, mostly hydroxy fatty acids, diterpenes, triterpenes, and cerebrosides. The isolated compounds were tested for their in vitro cytotoxicities against the breast adenocarcinoma MCF-7 cell line. Compounds 1, 2, 3, and 4 displayed promising cytotoxic activities against MCF-7 cells, with IC50 values of 13.83, 8.13, 8.27, and 35.56 µM, respectively, compared to that of the standard drug doxorubicin (IC50 8.64 µM). Additionally, docking studies were performed for compounds 1, 2, 3, and 4 to elucidate their binding interactions with the active site of the SET protein, an inhibitor of protein phosphatase 2A (PP2A), which could explain their cytotoxic activity. This study highlights the important role of these metabolites in the defense mechanism of the sea cucumber against fouling organisms and the potential uses of these active molecules in the design of new anticancer agents.
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Minamitani, Takeharu, Teruhito Yasui, Yijie Ma, Hufeng Zhou, Daisuke Okuzaki, Chiau-Yuang Tsai, Shuhei Sakakibara, Benjamin E. Gewurz, Elliott Kieff, and Hitoshi Kikutani. "Evasion of affinity-based selection in germinal centers by Epstein–Barr virus LMP2A." Proceedings of the National Academy of Sciences 112, no. 37 (August 24, 2015): 11612–17. http://dx.doi.org/10.1073/pnas.1514484112.
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Epstein–Barr virus (EBV) infects germinal center (GC) B cells and establishes persistent infection in memory B cells. EBV-infected B cells can cause B-cell malignancies in humans with T- or natural killer-cell deficiency. We now find that EBV-encoded latent membrane protein 2A (LMP2A) mimics B-cell antigen receptor (BCR) signaling in murine GC B cells, causing altered humoral immune responses and autoimmune diseases. Investigation of the impact of LMP2A on B-cell differentiation in mice that conditionally express LMP2A in GC B cells or all B-lineage cells found LMP2A expression enhanced not only BCR signals but also plasma cell differentiation in vitro and in vivo. Conditional LMP2A expression in GC B cells resulted in preferential selection of low-affinity antibody-producing B cells despite apparently normal GC formation. GC B-cell–specific LMP2A expression led to systemic lupus erythematosus-like autoimmune phenotypes in an age-dependent manner. Epigenetic profiling of LMP2A B cells found increased H3K27ac and H3K4me1 signals at the zinc finger and bric-a-brac, tramtrack domain-containing protein 20 locus. We conclude that LMP2A reduces the stringency of GC B-cell selection and may contribute to persistent EBV infection and pathogenesis by providing GC B cells with excessive prosurvival effects.
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Hassan-Zahraee, M., Z. Ye, L. Xi, M. L. Baniecki, X. Li, W. Farin, L. Tibaldi, et al. "P446 Transcriptional and microbial biomarkers of response to anti-TL1A therapy in ulcerative colitis: the Phase 2a TUSCANY study." Journal of Crohn's and Colitis 14, Supplement_1 (January 2020): S401—S402. http://dx.doi.org/10.1093/ecco-jcc/jjz203.575.
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Abstract Background Tumour necrosis factor α-like ligand 1A (TL1A) acts as a synergistic cytokine that amplifies pro-inflammatory signals to increase T-cell, natural killer (NK) cell, and innate lymphoid cell production of cytokines, which are thought to be key drivers of inflammatory bowel disease (IBD) and intestinal fibrosis. The recent Phase 2a TUSCANY study demonstrated the efficacy of PF-06480605, a fully human immunoglobulin G1 monoclonal antibody targeting TL1A, in the treatment of participants with moderate to severe active Ulcerative Colitis (UC). Here, we present transcriptomic analysis of intestinal tissue biopsies and metagenomic analysis of the faecal microbiome to further evaluate the treatment effect of PF-06480605. Methods The Phase 2a, open-label, multicentre, single-arm TUSCANY study (NCT02840721) evaluated the safety, tolerability and efficacy of PF-06480605 in participants with moderate to severe UC. Participants received 500 mg intravenous (IV) PF-06480605 every 2 weeks (Q2W) for a total of 7 doses, with a 14-week follow-up period. We performed RNA sequencing of colonic biopsies and metagenomic sequencing (>40 million reads per sample) of faecal samples taken prior to and after 12 weeks of therapy. Differential expression and cell subset deconvolution by CytoReason were used to identify genes and immune cells involved in response to therapy. Differential abundance analyses based on taxonomic profiling was performed using the GOTTCHA metagenome mapping algorithm to discover whether microbial taxa changed in response to treatment. Results Tissue transcriptomic analysis identified a robust inhibition of adaptive (Th1, Th2 and Th17) and innate (macrophage) inflammatory pathways known to be associated with UC pathogenesis and a significant reduction in fibrosis-associated pathways (MMP3, MMP7 and MMP10). Transcriptomic responses were most pronounced in participants achieving endoscopic improvement (Mayo endoscopic subscore of ≤1). A candidate baseline gene signature was predictive of endoscopic improvement (area under the curve [AUC] 0.75; p-value = 0.03). Metagenomic analysis revealed a significant reduction in inflammatory pathobiont species (S. salivarius, S. parasanguinis, and H. parainfluenzae) following treatment. Conclusion These results provide insight into the molecular and microbial mechanisms underlying the efficacy of anti-TL1A therapy for UC. We defined a baseline transcriptomic signature to predict response and a pathobiont treatment signature. These findings may enable the development of precision medicine strategies for anti-TL1A therapy in the treatment of intestinal inflammation.
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Khatlani, Tanvir, Subhashree Pradhan, Qi Da, Tanner Shaw, Vladimir L. Buchman, Miguel A. Cruz, and K. Vinod Vijayan. "A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in αIIbβ3 Integrin Signaling." Journal of Biological Chemistry 291, no. 33 (June 22, 2016): 17360–68. http://dx.doi.org/10.1074/jbc.m115.704296.
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The transduction of signals generated by protein kinases and phosphatases are critical for the ability of integrin αIIbβ3 to support stable platelet adhesion and thrombus formation. Unlike kinases, it remains unclear how serine/threonine phosphatases engage the signaling networks that are initiated following integrin ligation. Because protein-protein interactions form the backbone of signal transduction, we searched for proteins that interact with the catalytic subunit of protein phosphatase 2A (PP2Ac). In a yeast two-hybrid study, we identified a novel interaction between PP2Ac and an adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). Truncation and alanine mutagenesis studies revealed that PP2Ac binds to the P3 block (396PAIPPKKPRP405) of the proline-rich region in CIN85. The interaction of purified PP2Ac with CIN85 suppressed phosphatase activity. Human embryonal kidney 293 αIIbβ3 cells overexpressing a CIN85 P3 mutant, which cannot support PP2Ac binding, displayed decreased adhesion to immobilized fibrinogen. Platelets contain the ∼85 kDa CIN85 protein along with the PP2Ac-CIN85 complex. A myristylated cell-permeable peptide derived from residues 395–407 of CIN85 protein (P3 peptide) disrupted the platelet PP2Ac-CIN85 complex and decreased αIIbβ3 signaling dependent functions such as platelet spreading on fibrinogen and thrombin-mediated fibrin clot retraction. In a phospho-profiling study P3 peptide treated platelets also displayed decreased phosphorylation of several signaling proteins including Src and GSK3β. Taken together, these data support a role for the novel PP2Ac-CIN85 complex in supporting integrin-dependent platelet function by dampening the phosphatase activity.
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Mulaw, Medhanie Assmelash, Alexandre Krause, Alexander Riedel, Belay Tizazu, Hendrik Reuter, and Stefan K. Bohlander. "Early Target Genes of CALM/AF10 as Revealed by Gene Expression Profiling." Blood 112, no. 11 (November 16, 2008): 2260. http://dx.doi.org/10.1182/blood.v112.11.2260.2260.
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Abstract The t(10;11)(p12;q14) is a recurring chromosomal translocation that is found in acute myeloid and acute lymphoblastic leukemia as well as in malignant lymphoma. This translocation results in the fusion of AF10, a putative zinc finger transcription factor containing an N-terminal LAP/PHD zinc finger motif, a nuclear localization signal, an AT-hook domain, and a leucine zipper and with the CALM gene (Clathrin assembly protein lymphoid myeloid leukemia gene) that encodes a clathrin assembly protein. In the monocytic cell line U937 both the CALM/AF10 and the AF10/CALM fusion mRNAs can be identified. The CALM/AF10 fusion mRNA codes for the CALM/AF10 fusion protein which contains almost the complete CALM protein fused in frame to about 90% of the AF10 protein without the N terminal two PHD zinc fingers. CALM/AF10 is highly leukemogenic with its expression in primary bone marrow cells leading to the development of an aggressive acute leukemia with a short latency of 10 weeks in a murine bone marrow transplant model. We set out to identify immediate target genes of CALM/AF10. The fusion gene was cloned into pRTS-1, an episomally replicating tetracycline/doxycycline inducible (tet-on) expression vector. The construct was stably transfected into the Burkitt’s lymphoma B cell line DG75. The expression of CALM/AF10 after induction was confirmed by RT-PCR. Gene expression profiling experiments were conducted using the Affymetrix® Human Genome U133 Plus 2.0 Array analyzing non-induced and induced (24 and 72 hours after induction) samples. As controls, DG75 cells transfected with the pRTS-1 vector without the CALM/AF10 fusion gene were used. The results were analyzed using the R statistical package and dChip (DNA Chip Analyzer) software. The expression of 1237 genes was found to be changed at least 2 fold 24 hours after the induction of CALM/AF10. 594 (48%) genes were downregulated, while 643 (52%) were upregulated. Downregulated genes included genes involved in DNA repair (DDB2, TOP2A, BRCA1), cell cycle check point control (CCNE2, CHEK1, CDC2), and chromosome maintenance (MCM3, MCM7, MCM10). Upregulated genes included signal transduction molecules like RAB8B (a member of the RAS oncogene family) and STAT family members (STAT1 and STAT2), chromatin remodeling factors like BAZ2A (bromodomain adjacent to zinc finger domain, 2A), and MAML3 (master mind like 3), a positive regulator of Notch signaling. Pathway analysis using KegArray showed an enrichment of differentially regulated genes in processes like cell cycle regulation and DNA replication and repair. Interestingly, no significant upregulation of Hox genes was observed, as was previously reported in a study of CALM/AF10 positive patient samples (Dik et al., 2005). The changes in the expression levels of some selected genes were confirmed using a Taqman® Low Density Array (LDA). This analysis showed a statistically significant correlation (r = 0.78, p = 0.001) between the real time PCR results and the expression levels obtained from the Affymetrix arrays. Our results demonstrate that the expression of the leukemogenic CALM/AF10 fusion protein leads to a severe deregulation of critical cellular processes giving a first hint at the direct target genes of this fusion protein.
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Chavadi, Manjunath, Rahul Narasanna, Ashajyothi Chavan, Ajay Kumar Oli, and Chandrakanth Kelmani. R. "Prevalence of Methicillin Resistant and Virulence Determinants in Clinical Isolates of Staphylococcus aureus." Open Infectious Diseases Journal 10, no. 1 (August 13, 2018): 108–15. http://dx.doi.org/10.2174/1874279301810010108.
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Introduction:Methicillin-resistantStaphylococcus aureus(MRSA) is the major threat that is a result of the uncontrolled use of antibiotics causing a huge loss in health, so understanding their prevalence is necessary as a public health measure.Objective:The aim of this study was to determine the prevalence of methicillin-resistant MRSA and virulence determinant among associatedS. aureusfrom the clinical samples obtained from various hospital and health care centers of the Gulbarga region in India.Materials and Methods:All the collected samples were subjected for the screening ofS. aureusand were further characterized by conventional and molecular methods including their antibiotic profiling. Further, the response of methicillin antibiotic on cell morphology was studied using scanning electron microscopy.Results:A total 126S. aureuswas isolated from the clinical samples which showed, 100% resistant to penicillin, 55.5% to oxacillin, 75.3% to ampicillin, 70.6% to streptomycin, 66.6% to gentamicin, 8.7% to vancomycin and 6.3% to teicoplanin. The selected MRSA strains were found to possessmecA(gene coding for penicillin-binding protein 2A) andfemA(factor essential for methicillin resistance)genetic determinants in their genome with virulence determinants such as Coagulase (coa) and the X region of the protein A (spa)gene. Further, the methicillin response in resistantS. aureusshowed to be enlarged and malformed on cell morphology.Conclusion:The molecular typing of clinical isolates ofS. aureusin this study was highly virulent and also resistant to methicillin; this will assist health professionals to control, exploration of alternative medicines and new approaches to combat Staphylococcal infections more efficiently by using targeted therapy.
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Samandari, Nasim, Aashiq Mirza, Simranjeet Kaur, Philip Hougaard, Lotte Nielsen, Siri Fredheim, Henrik Mortensen, and Flemming Pociot. "Influence of Disease Duration on Circulating Levels of miRNAs in Children and Adolescents with New Onset Type 1 Diabetes." Non-Coding RNA 4, no. 4 (November 21, 2018): 35. http://dx.doi.org/10.3390/ncrna4040035.
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Circulating microRNAs (miRNAs) have been implicated in several pathologies including type 1 diabetes. In the present study, we aimed to identify circulating miRNAs affected by disease duration in children with recent onset type 1 diabetes. Forty children and adolescents from the Danish Remission Phase Cohort were followed with blood samples drawn at 1, 3, 6, 12, and 60 months after diagnosis. Pancreatic autoantibodies were measured at each visit. Cytokines were measured only the first year. miRNA expression profiling was performed by RT-qPCR. The effect of disease duration was analyzed by mixed models for repeated measurements adjusted for sex and age. Eight miRNAs (hsa-miR-10b-5p, hsa-miR-17-5p, hsa-miR-30e-5p, hsa-miR-93-5p, hsa-miR-99a-5p, hsa-miR-125b-5p, hsa-miR-423-3p, and hsa-miR-497-5p) were found to significantly change in expression (adjusted p-value < 0.05) with disease progression. Three pancreatic autoantibodies, ICA, IA-2A, and GAD65A, and four cytokines, IL-4, IL-10, IL-21, and IL-22, were associated with the miRNAs at different time points. Pathway analysis revealed associations with various immune-mediated signaling pathways. Eight miRNAs that were involved in immunological pathways changed expression levels during the first five years after diagnosis and were associated with variations in cytokine and pancreatic antibodies, suggesting a possible effect on the immunological processes in the early phase of the disease.
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Everett, S. A., V. M. McErlane, K. F. McLeod, F. M. Daley, P. R. Barber, B. Vojnovic, P. D. Nathan, et al. "Profiling cytochrome P450 CYP1 enzyme expression in primary melanoma and disseminated disease utilizing spectral imaging microscopy (SIM)." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 8556. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.8556.
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8556 Background: The aim of this study was to profile cytochrome CYP1 family (CYP1A1/1A2, and CYP1B1) mono-oxygenase enzymes during the malignant progression of primary melanoma and metastatic disease. Methods: Tissue microarrays of primary (n = 75), and metastatic (n = 104) melanoma were constructed with the patient demographics: (1) primary melanoma; age 22 to 93 (median 59); sex M/F 36/44; Breslow thickness 0.4 to 15 mm (median 2.5 mm); ulceration 25/80, and (2) metastatic melanoma; age 26 to 92 (median 60 mm); sex M/F 54/49; ulceration 30/104; number of nodes 1 to 15 (median 2); extra-capsular spread 20/95. CYP1 protein was detected by IHC using validated selective poly- and monoclonal antibodies. Vector SG (grey) stain for CYP1 was used with nuclear fast red counterstain to aid spectral resolution from background melanin. Staining intensity was scored visually (negative 0, weak 1, moderate 2, strong 3) and using SIM at every pixel of a captured image of each melanoma core. Reference spectra of individual chromophores were used to spectrally ‘un-mix’ CYP1 staining before the mean normalised absorbance intensity was determined. Grading was by the 2002 AJCC classification system: primary stage I n = 27 (1A 8, 1B 19), and stage II n = 48 (2A 22, 2B 16, 2C 10), lymph node metastasis stage III n = 98 (3B 53, 3C 45), visceral metastasis stage IV n = 6. Normal skin (n = 27), benign naevi (n = 14), and dysplastic naevi (n = 21) were also included. Results: CYP1B1 was not in normal skin but was over-expressed in both primary and metastatic melanoma (visual: 71% & 65%, SIM: 91% & 83%). Primary melanoma (stage I & II) was significantly greater (p = 0.004) than metastasis (stage III & IV). CYP1B1 did not correlate with ulceration or Breslow thickness but did correlate with N stage lymph node metastasis (p = 0.005). CYP1B1 expression in dysplastic naevi indicated up-regulation at an early stage of melanoma progression. CYP1A1/1A2 was not expressed in normal skin nor primary/metastatic melanoma. Conclusions: CYP1B1 protein expression is maintained with advancing AJCC stage from primary through to visceral metastasis. Future work will seek to correlate protein expression with functionality with a view to exploiting CYP1B1 in the enzyme/prodrug therapy of malignant melanoma. No significant financial relationships to disclose.
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Danese, S., M. Klopocka, E. J. Scherl, J. Romatowski, J. R. Allegretti, E. Peeva, M. S. Vincent, et al. "DOP72 Safety, tolerability and efficacy of anti-TL1A antibody PF-06480605 in treatment of ulcerative colitis: The open-label, multicentre, Phase 2a TUSCANY study." Journal of Crohn's and Colitis 14, Supplement_1 (January 2020): S108—S110. http://dx.doi.org/10.1093/ecco-jcc/jjz203.111.
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Abstract Background Widespread disruption of the mucosal immune system is central to the pathogenesis of ulcerative colitis (UC). One component, tumour necrosis factor (TNF) α-like ligand 1A (TL1A), is upregulated at the site of active disease in UC. Treatment of preclinical rodent models with anti-TL1A antibodies decreases disease activity, highlighting their therapeutic potential for UC. PF-06480605 is a first-in-class fully human immunoglobulin G1 monoclonal antibody targeting TL1A. Methods The Phase 2a, open-label, multicentre, single-arm TUSCANY study (NCT02840721) evaluated the safety, tolerability and efficacy of PF-06480605 in treatment of moderate to severe UC. Participants received 500 mg intravenous (IV) PF-06480605 every 2 weeks (Q2W) for a total of 7 doses, with a 14-week follow-up period. Primary safety and efficacy endpoints were incidence of treatment-emergent adverse events (TEAEs) and endoscopic improvement (EI) (Mayo endoscopic subscore [centrally read] of ≤1 without friability) at Week 14, respectively. Secondary efficacy endpoints included remission (total Mayo score ≤2, with no individual subscore >1) and endoscopic remission (Mayo endoscopic subscore = 0) at Week 14. Transcriptomic profiling on intestinal biopsies was performed. Results Of the 50 participants who received PF-06480605, 42 completed the study. The majority were male (56.0%) and white (96.0%), with a mean age of 40.0 years and prior experience of anti-TNF inhibitors (72.0%). Pancolitis (48.0%) and left-sided colitis (32.0%) were the most common forms of UC at baseline. There were 109 TEAEs, of which 18 were treatment-related. Aside from worsening UC, the most common TEAE by system organ class was arthralgia, which occurred in 6 participants, and 1 was treatment-related. Treatment-emergent serious adverse events were reported in 3 participants, and considered treatment-related in 1 participant (Table 1). No malignancies or deaths were reported. At Week 14, statistically significant EI was observed in 38.2% of participants (Table 2). The proportions of participants achieving remission and endoscopic remission at Week 14 were 24.0% and 10.0%, respectively. Transcriptomic analyses demonstrated normalisation towards a non-inflamed transcriptome in participants with EI. Conclusion PF-06480605 exhibited an acceptable safety and tolerability profile and statistically significant EI in participants with moderate to severe UC. These results warrant further evaluation in subsequent studies.
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Zhang, Ya, Xinting Hu, Hongzhi Xu, Ying Li, Lingyan Zhang, Lili Feng, Xiangxiang Zhou, et al. "Comprehensive Profiling of the Epitranscriptomic N6-Methyladenosine RNA Methylation in Chronic Lymphocytic Leukemia." Blood 136, Supplement 1 (November 5, 2020): 17–18. http://dx.doi.org/10.1182/blood-2020-141758.
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Introduction N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of eukaryotic mRNA. Accumulating evidence suggests that RNA m6A methylation exerts crucial roles in oncogenesis. However, the mRNA m6A methylation pattern in chronic lymphocytic leukemia (CLL) has not been investigated. Hence, the aim of this study is to perform a comprehensive profiling to identify distinct m6A methylation signatures in CLL patients. Methods Peripheral blood samples from de novo CLL patients were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong First Medical University. CD19+ B cells were isolated with informed consents from healthy donors. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was conduct to profile mRNA m6A methylation of CLL-B cells and normal CD19+ B cells at Novogene (Beijing, China). The library preparations were sequenced on an Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols. The sequencing was carried out with 3 independent biological replicates. After mapping reads to the reference genome, exomePeak R package was used for the m6A peak identification in each anti-m6A immunoprecipitation group with the corresponding input samples serving as a control, and q-value threshold of enrichment of 0.05 was used for all data sets. The m6A-enriched motifs of each group were identified by HOMER. Differential peak calling was performed using exomePeak R package with parameters of p-value < 0.05 and fold_change > 1. Functional enrichment analyses of gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) of differentiated peaks associated genes were performed. All investigators comply with the guiding principles for experimental procedures found in the Declaration of Helsinki of the World Medical Association. Results By MeRIP sequencing, significant distribution of methylation peaks were detected in 5'UTR, 3'UTR and CDS regions of CLL primary cells (Figure 1A) and normal B cells (Figure 1B). Figure 1C-D illustrated the percentage of methylation peaks in the five regions, suggesting distinct m6A patterns in CLL cells. Moreover, Figure 1E-F revealed the positions of m6A methylation peaks in chromosomes of CLL and normal B cells. Furthermore, the compared distributions of m6A methylation peaks in CLL and normal B cells were presented in Figure 2A. Besides, the bean plot visibly displayed the obvious differentiation of methylation peaks in CLL group and normal B cells in read density (Figure 2B). Importantly, a total of 1836 significantly changed peaks, of which 1519 were significantly up-regulated and 317 peaks were significantly down-regulated (p<0.05, |log2Foldchange|>1; Figure 2C). These m6A peaks were located across 1850 genes. Functional enrichment analyses identified that differentiated peaks associated genes were potentially regulate RNA metabolic process via oncogenic pathways in CLL pathogenesis (Figure 3A-B). In addition, HOMER analysis identified 38 significant de novo m6A peak motifs, top 10 most significant peak motifs of which were presented (Figure 3C), illuminating potential detailed mechanism of m6A RNA methylation in the tumorigenesis and progression of CLL. Conclusion Taken together, our investigations explored for the first time the m6A methylation pattern of mRNA in CLL. m6A modifications play crucial roles in the progression and survival of CLL patients,highlighting m6A modifications-targeted intervention formulating a novel treatment paradigm in progressed CLL that warrants clinical investigation. Disclosures No relevant conflicts of interest to declare.
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Kursula, Petri, Inari Kursula, Marzia Massimi, Young-Hwa Song, Joshua Downer, Will A. Stanley, Walter Witke, and Matthias Wilmanns. "High-resolution Structural Analysis of Mammalian Profilin 2a Complex Formation with Two Physiological Ligands: The Formin Homology 1 Domain of mDia1 and the Proline-rich Domain of VASP." Journal of Molecular Biology 375, no. 1 (January 2008): 270–90. http://dx.doi.org/10.1016/j.jmb.2007.10.050.
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Han, Jing, Xue Zhang, Yang Yang, Li Feng, Gui-Ying Wang, and Nan Zhang. "Screening and Identification of Differentially Expressed Genes Expressed among Left and Right Colon Adenocarcinoma." BioMed Research International 2020 (January 21, 2020): 1–16. http://dx.doi.org/10.1155/2020/8465068.
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Purpose. Colon adenocarcinoma (COAD) is the third most common malignancy globally and is further categorized as left colon adenocarcinoma (LCOAD) or right colon adenocarcinoma (RCOAD) depending on the location of the primary tumor. The therapeutic outcome and long-term prognosis for patients with COAD are less than satisfactory, and this may be associated with tumor location. Therefore, it is important to investigate the genetic differences in COAD at different sites. Patients and Methods. Public data associated with COAD were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified using R software (version 3.5.3), and functional annotation of DEGs was performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A protein-protein interaction network was constructed, hub genes were identified and analyzed, and data mining using Gene Expression Profiling Interactive Analysis (GEPIA) was conducted. Results. A total of 286 DEGs were identified between LCOAD and RCOAD. Additionally, 10 hub genes associated with COAD at different locations were screened, namely, CDKN2A, IGF1R, MDM2, SMAD3, SLC2A1, GRM5, PLCB4, FGFR1, UBE2V2, and TNFRSF10B. The expression of cyclin-dependent kinase inhibitor 2A (CDKN2A) and solute carrier family 2 member 1 (SLC2A1) was significantly associated with pathological stage P<0.05. COAD patients with high expression levels of CDKN2A exhibited poorer overall survival (OS) times than those with low expression levels P<0.05. Conclusion. CDKN2A expression was significantly different between LCOAD and RCOAD and was closely related to the prognosis of COAD. It is of great value for further understanding of the pathogenesis of LCOAD and RCOAD.
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Weems, Garry Alan, Xiao Hu, Xikui Liu, Hongxiu Li, Madhumita Bogdan, Yilin Gao, Brian Fox, H. Jeffrey Wilkins, and Laura Carter. "Lyc-55716: A novel small-molecule RORγ agonist immuno-oncology agent: Rationale for tumor selection and clinical evaluation of gastric and esophageal carcinoma in phase 2a expansion." Journal of Clinical Oncology 36, no. 4_suppl (February 1, 2018): 67. http://dx.doi.org/10.1200/jco.2018.36.4_suppl.67.
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67 Background: Type 17 effector T cell differentiation and function are regulated by the master transcription factor retinoic acid receptor–related orphan receptor γt (RORγ). Synthetic RORγ agonists, which modulate immune cell gene expression to enhance effector functions and decrease Treg and checkpoint pathways, have shown promise as mono- and combination therapy in syngeneic tumor models. Translational and bioinformatics assessments were performed during Phase 1 testing of the investigational RORγ agonist LYC-55716 to support inclusion of patients with gastric carcinoma (GC) and esophageal carcinoma (EC) in a Phase 2a expansion of clinical trial LYC-55716-1001 (NCT02929862). Methods: Transcriptional profiling of murine and human T cells treated ± RORγ agonists identified a gene signature. Using The Cancer Genome Atlas (TCGA), and other public datasets, data on GC and EC patients were evaluated to determine: (a) RORγ and RORγ-inducing cytokine expression; (b) signature genes associated with RORγ biology, surrogate biomarkers for endogenous RORγ ligands, and correlations with prognosis; (c) tumor microenvironment (TME) immune profiles. Results: Expression: In TCGA RNA sequencing data analysis, 50%-70% of GC and EC samples expressed moderate to high levels of RORγ, indicating infiltration of Type 17 T cells into the tumors. Furthermore, genes that support expression of RORγ were highly expressed in ~75% of GC and EC tumors. Biology: TCGA data showed that GC and EC tumors express low levels of sterol efflux genes, suggesting low levels of endogenous agonists in the TME. In analysis of public datasets, expression of the RORγ target gene IL17A correlated with a favorable prognosis for EC. In addition, some of the other RORγ signature genes also correlated with good prognosis in GC and EC. Immune profile: TCGA showed infiltration of T cells and a high mutational burden in GC and EC, which are positively associated with efficacy of immunotherapy. Conclusions: Bioinformatics assessments of RORγ expression, biology, and tumor immune profiles support the inclusion of GC and EC patients in an ongoing Phase 2 clinical trial of LYC-55716.