43 results on '"Iyer LK"'
Search Results
2. A program for real-time surveillance of SARS-CoV-2 genetics.
- Author
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Brochu HN, Song K, Zhang Q, Zeng Q, Shafi A, Robinson M, Humphrey J, Croy B, Peavy L, Perera M, Parker S, Pruitt J, Munroe J, Ghatti R, Urban TJ, Harris AB, Alfego D, Norvell B, Levandoski M, Krueger B, Williams JD, Boles D, Nye MB, Dale SE, Sapeta M, Petropoulos CJ, Meltzer J, Eisenberg M, Cohen O, Letovsky S, and Iyer LK
- Subjects
- Humans, United States epidemiology, Mutation, SARS-CoV-2 genetics, SARS-CoV-2 isolation & purification, COVID-19 epidemiology, COVID-19 virology, Genome, Viral
- Abstract
The COVID-19 pandemic brought forth an urgent need for widespread genomic surveillance for rapid detection and monitoring of emerging SARS-CoV-2 variants. It necessitated design, development, and deployment of a nationwide infrastructure designed for sequestration, consolidation, and characterization of patient samples that disseminates de-identified information to public authorities in tight turnaround times. Here, we describe our development of such an infrastructure, which sequenced 594,832 high coverage SARS-CoV-2 genomes from isolates we collected in the United States (U.S.) from March 13th 2020 to July 3rd 2023. Our sequencing protocol ('Virseq') utilizes wet and dry lab procedures to generate mutation-resistant sequencing of the entire SARS-CoV-2 genome, capturing all major lineages. We also characterize 379 clinically relevant SARS-CoV-2 multi-strain co-infections and ensure robust detection of emerging lineages via simulation. The modular infrastructure, sequencing, and analysis capabilities we describe support the U.S. Centers for Disease Control and Prevention national surveillance program and serve as a model for rapid response to emerging pandemics at a national scale., (© 2024. The Author(s).)
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- 2024
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3. An In Silico Analysis of PCR-Based Monkeypox Virus Detection Assays: A Case Study for Ongoing Clinical Surveillance.
- Author
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Song K, Brochu HN, Zhang Q, Williams JD, and Iyer LK
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- Humans, Consensus, Disease Outbreaks, Polymerase Chain Reaction, Biological Assay, Monkeypox virus genetics
- Abstract
The 2022 global Mpox outbreak swiftly introduced unforeseen diversity in the monkeypox virus (MPXV) population, resulting in numerous Clade IIb sublineages. This propagation of new MPXV mutations warrants the thorough re-investigation of previously recommended or validated primers designed to target MPXV genomes. In this study, we explored 18 PCR primer sets and examined their binding specificity against 5210 MPXV genomes, representing all the established MPXV lineages. Our results indicated that only five primer sets resulted in almost all perfect matches against the targeted MPXV lineages, and the remaining primer sets all contained 1-2 mismatches against almost all the MPXV lineages. We further investigated the mismatched primer-genome pairs and discovered that some of the primers overlapped with poorly sequenced and assembled regions of the MPXV genomes, which are consistent across multiple lineages. However, we identified 173 99% genome-wide conserved regions across all 5210 MPXV genomes, representing 30 lineages/clades with at least 80% lineage-specific consensus for future primer development and primer binding evaluation. This exercise is crucial to ensure that the current detection schemes are robust and serve as a framework for primer evaluation in clinical testing development for other infectious diseases.
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- 2023
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4. In-Process Vapor Composition Monitoring in Application to Lyophilization of Ammonium Salt Formulations.
- Author
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Strongrich AD, Tobyn M, Iyer LK, Park Y, Hong J, and Alexeenko AA
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- Ammonia, Freeze Drying methods, Drug Compounding methods, Gases, Temperature, Chemistry, Pharmaceutical methods, Ammonium Compounds
- Abstract
Quality control is of critical importance in manufacturing of lyophilized drug product, which is accomplished by monitoring the process parameters. The residual gas analyzer has emerged as a useful tool in determination of endpoint for primary and secondary drying in lyophilization process as well as leak detection in vacuum systems. This study presents the application of in situ RGA to quantify outgassing rates of species released from aqueous inorganic and organic ammonium salt formulations throughout the freeze-drying process. The determination of ammonia outgassing conditions aids in ensuring product quality where ammonia release is an indication for loss of co-solvent or degradation of active pharmaceutical ingredients (APIs). Data analysis methods are developed to determine ammonia presence under various process conditions. In-situ real time monitoring of vapor dynamics enables RGA to be used as a tool to characterize counter-ion loss throughout the freeze-drying cycle., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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5. Pulse Proteolysis: An Orthogonal Tool for Protein Formulation Screening.
- Author
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Iyer LK, Phanse R, Xu M, Lan W, Krause ME, Bolgar M, and Hart S
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- Calorimetry, Differential Scanning, Drug Compounding, Excipients chemistry, Protein Aggregates, Protein Conformation, Protein Stability, Protein Unfolding, Proteolysis, Recombinant Fusion Proteins chemistry, Antibodies, Monoclonal chemistry
- Abstract
Protein formulation stability is difficult to predict a priori and generally involves long-term stability studies. It is of interest to develop an analytical method that can predict stability trends reliably. Here, pulse proteolysis was evaluated as an analytical tool to predict solution-state stability in different formulations. Four proteins formulated in different buffer and excipient compositions were subjected to urea-induced unfolding and brief enzymatic digestion ("pulse" proteolysis), and relative resistance to proteolysis was measured by microfluidics-based capillary electrophoresis-sodium dodecyl sulfate. Biophysical properties of each formulation were measured using orthogonal biophysical techniques such as differential scanning fluorimetry, differential scanning calorimetry, dynamic light scattering, circular dichroism, and fluorescence spectroscopy. Protein stability in all formulations was monitored by size exclusion chromatography on storage at 5°C and 40°C. For all 4 proteins, formulations with greater proteolytic resistance also showed higher monomer content on thermal stability. In contrast, standard biophysical techniques showed reasonable-to-no correlation with size exclusion chromatography data. The data support the use of pulse proteolysis as an orthogonal, quantitative, and predictive tool to measure protein conformational stability and rank-order formulations., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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6. Comparison of imaged capillary isoelectric focusing and cation exchange chromatography for monitoring dextrose-mediated glycation of monoclonal antibodies in infusion solutions.
- Author
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Demirdirek B, Lan W, Qiu D, Ding W, Iyer LK, Bolgar MS, and Valente JJ
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- Administration, Intravenous, Antibodies, Monoclonal administration & dosage, Chromatography, Ion Exchange methods, Glycosylation, Solutions administration & dosage, Solutions chemistry, Antibodies, Monoclonal analysis, Antibodies, Monoclonal chemistry, Electrophoresis, Capillary methods, Glucose chemistry, Isoelectric Focusing methods
- Abstract
Intravenous (IV) infusion of therapeutic proteins typically involves dilution of the formulated product into infusion media such as normal saline or dextrose, 5% m/v in water. We report results from a rigorous evaluation of imaged capillary isoelectric focusing (iCIEF) for monitoring dextrose-mediated glycation of proteins in IV infusion solutions. In addition to detecting stable Amadori glycation products, iCIEF was able to detect the labile Schiff base (SB) glycation adducts since the equilibrium with free dextrose is maintained on capillary. Method parameters such as sample dilution factor and ampholyte composition (but not urea) were found to influence the observed level of SB glycation adducts. The impacts of dextrose and urea on the apparent pI values are also reported. iCIEF results were compared with results from cation exchange chromatography, which was found to preferentially detect the more stable Amadori glycation products due to the on-column decomposition of the SB adducts resulting from the separation of the protein from free dextrose which in turn altered the SB adduct- free dextrose equilibrium. These results demonstrate the need for careful consideration when selecting the analytical methodology to investigate protein sensitivity to dextrose and to monitor protein stability in dextrose-containing infusion solutions., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2019
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7. Molecular and Functional Properties of Regional Astrocytes in the Adult Brain.
- Author
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Morel L, Chiang MSR, Higashimori H, Shoneye T, Iyer LK, Yelick J, Tai A, and Yang Y
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- Animals, Astrocytes classification, Astrocytes cytology, Brain cytology, Female, Gene Expression Profiling, Humans, Male, Mice, Inbred C57BL, Mice, Transgenic, Astrocytes metabolism, Brain metabolism, Gene Expression Regulation, Mice metabolism, Nerve Tissue Proteins genetics, Transcription Factors metabolism
- Abstract
The molecular signature and functional properties of astroglial subtypes in the adult CNS remain largely undefined. By using translational ribosome affinity purification followed by RNA-Seq, we profiled astroglial ribosome-associated (presumably translating) mRNAs in major cortical and subcortical brain regions (cortex, hippocampus, caudate-putamen, nucleus accumbens, thalamus, and hypothalamus) of BAC aldh1l1 -translational ribosome affinity purification (TRAP) mice (both sexes). We found that the expression of astroglial translating mRNAs closely follows the dorsoventral axis, especially from cortex/hippocampus to thalamus/hypothalamus posteriorly. This region-specific expression pattern of genes, such as synaptogenic modulator sparc and transcriptional factors ( emx2 , lhx2 , and hopx ), was validated by qRT-PCR and immunostaining in brain sections. Interestingly, cortical or subcortical astrocytes selectively promote neurite growth and synaptic activity of neurons only from the same region in mismatched cocultures, exhibiting region-matched astrocyte to neuron communication. Overall, these results generated new molecular signature of astrocyte types in the adult CNS, providing insights into their origin and functional diversity. SIGNIFICANCE STATEMENT We investigated the in vivo molecular and functional heterogeneity of astrocytes inter-regionally from adult brain. Our results showed that the expression pattern of ribosome-associated mRNA profiles in astrocytes closely follows the dorsoventral axis, especially posteriorly from cortex/hippocampus to thalamus/hypothalamus. In line with this, our functional results further demonstrated region-selective roles of cortical and subcortical astrocytes in regulating cortical or subcortical neuronal synaptogenesis and maturation. These in vivo studies provide a previously uncharacterized and important molecular atlas for exploring region-specific astroglial functions., (Copyright © 2017 the authors 0270-6474/17/378706-12$15.00/0.)
- Published
- 2017
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8. Unliganded estrogen receptor alpha regulates vascular cell function and gene expression.
- Author
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Lu Q, Schnitzler GR, Vallaster CS, Ueda K, Erdkamp S, Briggs CE, Iyer LK, Jaffe IZ, and Karas RH
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- Animals, Aorta metabolism, Cell Movement physiology, Cell Proliferation physiology, Cells, Cultured, Endothelial Cells metabolism, Estradiol metabolism, Estrogens metabolism, Female, Mice, Mice, Knockout, Myocytes, Smooth Muscle metabolism, Estrogen Receptor alpha metabolism, Gene Expression physiology, Muscle, Smooth, Vascular metabolism
- Abstract
The unliganded form of the estrogen receptor is generally thought to be inactive. Our prior studies, however, suggested that unliganded estrogen receptor alpha (ERα) exacerbates adverse vascular injury responses in mice. Here, we show that the presence of unliganded ERα decreases vascular endothelial cell (EC) migration and proliferation, increases smooth muscle cell (SMC) proliferation, and increases inflammatory responses in cultured ECs and SMCs. Unliganded ERα also regulates many genes in vascular ECs and mouse aorta. Activation of ERα by E2 reverses the cell physiological effects of unliganded ERα, and promotes gene regulatory effects that are predicted to counter the effects of unliganded ERα. These results reveal that the unliganded form of ERα is not inert, but significantly impacts gene expression and physiology of vascular cells. Furthermore, they indicate that the cardiovascular protective effects of estrogen may be connected to its ability to counteract these effects of unliganded ERα., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2017
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9. TRAP-seq Profiling and RNAi-Based Genetic Screens Identify Conserved Glial Genes Required for Adult Drosophila Behavior.
- Author
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Ng FS, Sengupta S, Huang Y, Yu AM, You S, Roberts MA, Iyer LK, Yang Y, and Jackson FR
- Abstract
Although, glial cells have well characterized functions in the developing and mature brain, it is only in the past decade that roles for these cells in behavior and plasticity have been delineated. Glial astrocytes and glia-neuron signaling, for example, are now known to have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, we have conducted cell-specific, genome-wide expression profiling of adult Drosophila astrocyte-like brain cells and performed RNA interference (RNAi)-based genetic screens to identify glial factors that regulate behavior. Importantly, our studies demonstrate that adult fly astrocyte-like cells and mouse astrocytes have similar molecular signatures; in contrast, fly astrocytes and surface glia-different classes of glial cells-have distinct expression profiles. Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). Of interest, 1 of the relevant genes encodes a vesicle recycling factor, 4 encode secreted proteins and 3 encode membrane transporters. These results strongly support the idea that glia-neuron communication is vital for adult behavior.
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- 2016
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10. MicroRNA-Offset RNA Alters Gene Expression and Cell Proliferation.
- Author
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Zhao J, Schnitzler GR, Iyer LK, Aronovitz MJ, Baur WE, and Karas RH
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- Animals, Argonaute Proteins genetics, Mice, MicroRNAs genetics, Muscle, Smooth, Vascular cytology, Myocytes, Smooth Muscle cytology, 3' Untranslated Regions, Argonaute Proteins biosynthesis, Cell Proliferation, Down-Regulation, MicroRNAs metabolism, Muscle, Smooth, Vascular metabolism, Myocytes, Smooth Muscle metabolism
- Abstract
MicroRNA-offset RNAs (moRs) were first identified in simple chordates and subsequently in mouse and human cells by deep sequencing of short RNAs. MoRs are derived from sequences located immediately adjacent to microRNAs (miRs) in the primary miR (pri-miR). Currently moRs are considered to be simply a by-product of miR biosynthesis that lack biological activity. Here we show for the first time that a moR is biologically active. We demonstrate that endogenous or over-expressed moR-21 significantly alters gene expression and inhibits the proliferation of vascular smooth muscle cells (VSMC). In addition, we find that miR-21 and moR-21 may regulate different genes in a given pathway and can oppose each other in regulating certain genes. We report that there is a "seed region" of moR-21 as well as a "seed match region" in the target gene 3'UTR that are indispensable for moR-21-mediated gene down-regulation. We further demonstrate that moR-21-mediated gene repression is Argonaute 2 (Ago2) dependent. Taken together, these findings provide the first evidence that microRNA offset RNA alters gene expression and is biologically active.
- Published
- 2016
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11. Process and Formulation Effects on Protein Structure in Lyophilized Solids Using Mass Spectrometric Methods.
- Author
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Iyer LK, Sacha GA, Moorthy BS, Nail SL, and Topp EM
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- Animals, Drug Compounding, Freeze Drying methods, Horses, Mass Spectrometry methods, Protein Structure, Secondary, X-Ray Diffraction methods, Myoglobin analysis, Myoglobin chemistry, Tandem Mass Spectrometry methods
- Abstract
Myoglobin (Mb) was lyophilized in the absence (Mb-A) and presence (Mb-B) of sucrose in a pilot-scale lyophilizer with or without controlled ice nucleation. Cake morphology was characterized using scanning electron microscopy, and changes in protein structure were monitored using solid-state Fourier-transform infrared spectroscopy, solid-state hydrogen-deuterium exchange-mass spectrometry, and solid-state photolytic labeling-mass spectrometry (ssPL-MS). The results showed greater variability in nucleation temperature and irregular cake structure for formulations lyophilized without controlled nucleation. Controlled nucleation resulted in nucleation at ∼(-5°C) and uniform cake structure. Formulations containing sucrose showed better retention of protein structure by all measures than formulations without sucrose. Samples lyophilized with and without controlled nucleation were similar by most measures of protein structure. However, ssPL-MS showed the greatest photoleucine incorporation and more labeled regions for Mb-B lyophilized with controlled nucleation. The data support the use of solid-state hydrogen-deuterium exchange-mass spectrometry and ssPL-MS to study formulation and process-induced conformational changes in lyophilized proteins., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)
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- 2016
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12. Correction: Paternal B Vitamin Intake Is a Determinant of Growth, Hepatic Lipid Metabolism and Intestinal Tumor Volume in Female Apc1638N Mouse Offspring.
- Author
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Sabet JA, Park LK, Iyer LK, Tai AK, Koh GY, Pfalzer AC, Parnell LD, Mason JB, Liu Z, Byun AJ, and Crott JW
- Abstract
[This corrects the article DOI: 10.1371/journal.pone.0151579.].
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- 2016
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13. ER Alpha Rapid Signaling Is Required for Estrogen Induced Proliferation and Migration of Vascular Endothelial Cells.
- Author
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Lu Q, Schnitzler GR, Ueda K, Iyer LK, Diomede OI, Andrade T, and Karas RH
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- Animals, COS Cells, Chlorocebus aethiops, Endothelium, Vascular cytology, Humans, Mutation, Transcription Factors metabolism, Cell Movement physiology, Cell Proliferation physiology, Endothelium, Vascular metabolism, Estradiol physiology, Estrogen Receptor alpha metabolism, Signal Transduction
- Abstract
Estrogen promotes the proliferation and migration of vascular endothelial cells (ECs), which likely underlies its ability to accelerate re-endothelialization and reduce adverse remodeling after vascular injury. In previous studies, we have shown that the protective effects of E2 (the active endogenous form of estrogen) in vascular injury require the estrogen receptor alpha (ERα). ERα transduces the effects of estrogen via a classical DNA binding, "genomic" signaling pathway and via a more recently-described "rapid" signaling pathway that is mediated by a subset of ERα localized to the cell membrane. However, which of these pathways mediates the effects of estrogen on endothelial cells is poorly understood. Here we identify a triple point mutant version of ERα (KRR ERα) that is specifically defective in rapid signaling, but is competent to regulate transcription through the "genomic" pathway. We find that in ECs expressing wild type ERα, E2 regulates many genes involved in cell migration and proliferation, promotes EC migration and proliferation, and also blocks the adhesion of monocytes to ECs. ECs expressing KRR mutant ERα, however, lack all of these responses. These observations establish KRR ERα as a novel tool that could greatly facilitate future studies into the vascular and non-vascular functions of ERα rapid signaling. Further, they support that rapid signaling through ERα is essential for many of the transcriptional and physiological responses of ECs to E2, and that ERα rapid signaling in ECs, in vivo, may be critical for the vasculoprotective and anti-inflammatory effects of estrogen.
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- 2016
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14. Paternal B Vitamin Intake Is a Determinant of Growth, Hepatic Lipid Metabolism and Intestinal Tumor Volume in Female Apc1638N Mouse Offspring.
- Author
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Sabet JA, Park LK, Iyer LK, Tai AK, Koh GY, Pfalzer AC, Parnell LD, Mason JB, Liu Z, Byun AJ, and Crott JW
- Subjects
- Adenomatous Polyposis Coli Protein genetics, Animals, Body Weight drug effects, DNA Methylation drug effects, Female, Gene Expression Regulation drug effects, Humans, Liver metabolism, Male, Mice, Mutation, Reproduction drug effects, Sex Characteristics, Spermatozoa drug effects, Spermatozoa metabolism, Vitamin B Complex blood, Fathers, Growth and Development drug effects, Intestinal Neoplasms pathology, Lipid Metabolism drug effects, Liver drug effects, Tumor Burden drug effects, Vitamin B Complex pharmacology
- Abstract
Background: The importance of maternal nutrition to offspring health and risk of disease is well established. Emerging evidence suggests paternal diet may affect offspring health as well., Objective: In the current study we sought to determine whether modulating pre-conception paternal B vitamin intake alters intestinal tumor formation in offspring. Additionally, we sought to identify potential mechanisms for the observed weight differential among offspring by profiling hepatic gene expression and lipid content., Methods: Male Apc1638N mice (prone to intestinal tumor formation) were fed diets containing replete (control, CTRL), mildly deficient (DEF), or supplemental (SUPP) quantities of vitamins B2, B6, B12, and folate for 8 weeks before mating with control-fed wild type females. Wild type offspring were euthanized at weaning and hepatic gene expression profiled. Apc1638N offspring were fed a replete diet and euthanized at 28 weeks of age to assess tumor burden., Results: No differences in intestinal tumor incidence or burden were found between male Apc1638N offspring of different paternal diet groups. Although in female Apc1638N offspring there were no differences in tumor incidence or multiplicity, a stepwise increase in tumor volume with increasing paternal B vitamin intake was observed. Interestingly, female offspring of SUPP and DEF fathers had a significantly lower body weight than those of CTRL fed fathers. Moreover, hepatic trigylcerides and cholesterol were elevated 3-fold in adult female offspring of SUPP fathers. Weanling offspring of the same fathers displayed altered expression of several key lipid-metabolism genes. Hundreds of differentially methylated regions were identified in the paternal sperm in response to DEF and SUPP diets. Aside from a few genes including Igf2, there was a striking lack of overlap between these genes differentially methylated in sperm and differentially expressed in offspring., Conclusions: In this animal model, modulation of paternal B vitamin intake prior to mating alters offspring weight gain, lipid metabolism and tumor growth in a sex-specific fashion. These results highlight the need to better define how paternal nutrition affects the health of offspring.
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- 2016
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15. Photolytic Cross-Linking to Probe Protein-Protein and Protein-Matrix Interactions in Lyophilized Powders.
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Iyer LK, Moorthy BS, and Topp EM
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- Animals, Chemistry, Pharmaceutical, Chromatography, Liquid methods, Guanidine chemistry, Horses, Mass Spectrometry methods, Myoglobin chemistry, Peptide Fragments chemistry, Water chemistry, Cross-Linking Reagents metabolism, Excipients chemistry, Freeze Drying methods, Myoglobin metabolism, Peptide Fragments metabolism, Photolysis, Powders chemistry
- Abstract
Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic cross-linking with mass spectrometric analysis (ssPC-MS). In order to characterize structure and microenvironment, protein-protein, protein-excipient, and protein-water interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4'-azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography/mass spectrometry (LC-MS). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce cross-linked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to five labels, as detected by LC-MS. Following lyophilization and irradiation, cross-linked peptide-peptide, peptide-water, and peptide-raffinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide-peptide, peptide-water, and peptide-excipient adducts detected. In the absence of excipients, peptide-peptide adducts involving the CD, DE, and EF loops and helix H were common. In the raffinose formulation, peptide-peptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein-protein and protein-water adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC-MS can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution.
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- 2015
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16. Midbrain dopamine neurons in Parkinson's disease exhibit a dysregulated miRNA and target-gene network.
- Author
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Briggs CE, Wang Y, Kong B, Woo TU, Iyer LK, and Sonntag KC
- Subjects
- Female, Gene Expression Profiling, Gene Expression Regulation, Humans, Male, MicroRNAs genetics, Oligonucleotide Array Sequence Analysis, Signal Transduction, Dopaminergic Neurons metabolism, Gene Regulatory Networks, Mesencephalon pathology, MicroRNAs metabolism, Parkinson Disease pathology
- Abstract
The degeneration of substantia nigra (SN) dopamine (DA) neurons in sporadic Parkinson׳s disease (PD) is characterized by disturbed gene expression networks. Micro(mi)RNAs are post-transcriptional regulators of gene expression and we recently provided evidence that these molecules may play a functional role in the pathogenesis of PD. Here, we document a comprehensive analysis of miRNAs in SN DA neurons and PD, including sex differences. Our data show that miRNAs are dysregulated in disease-affected neurons and differentially expressed between male and female samples with a trend of more up-regulated miRNAs in males and more down-regulated miRNAs in females. Unbiased Ingenuity Pathway Analysis (IPA) revealed a network of miRNA/target-gene associations that is consistent with dysfunctional gene and signaling pathways in PD pathology. Our study provides evidence for a general association of miRNAs with the cellular function and identity of SN DA neurons, and with deregulated gene expression networks and signaling pathways related to PD pathogenesis that may be sex-specific., (Copyright © 2015 Elsevier B.V. All rights reserved.)
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- 2015
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17. Diet- and Genetically-Induced Obesity Differentially Affect the Fecal Microbiome and Metabolome in Apc1638N Mice.
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Pfalzer AC, Nesbeth PD, Parnell LD, Iyer LK, Liu Z, Kane AV, Chen CY, Tai AK, Bowman TA, Obin MS, Mason JB, Greenberg AS, Choi SW, Selhub J, Paul L, and Crott JW
- Subjects
- Animals, Female, Intestinal Neoplasms genetics, Intestinal Neoplasms metabolism, Intestinal Neoplasms microbiology, Male, Mice, Mutation, Obesity etiology, Obesity metabolism, Obesity microbiology, Receptors, Leptin deficiency, Diet, High-Fat adverse effects, Feces chemistry, Feces microbiology, Metabolome drug effects, Metabolome genetics, Microbiota drug effects, Microbiota genetics, Obesity genetics, Receptors, Leptin genetics
- Abstract
Obesity is a risk factor for colorectal cancer (CRC), and alterations in the colonic microbiome and metabolome may be mechanistically involved in this relationship. The relative contribution of diet and obesity per se are unclear. We compared the effect of diet- and genetically-induced obesity on the intestinal microbiome and metabolome in a mouse model of CRC. Apc1638N mice were made obese by either high fat (HF) feeding or the presence of the Leprdb/db (DbDb) mutation. Intestinal tumors were quantified and stool microbiome and metabolome were profiled. Genetic obesity, and to a lesser extent HF feeding, promoted intestinal tumorigenesis. Each induced distinct microbial patterns: taxa enriched in HF were mostly Firmicutes (6 of 8) while those enriched in DbDb were split between Firmicutes (7 of 12) and Proteobacteria (5 of 12). Parabecteroides distasonis was lower in tumor-bearing mice and its abundance was inversely associated with colonic Il1b production (p<0.05). HF and genetic obesity altered the abundance of 49 and 40 fecal metabolites respectively, with 5 in common. Of these 5, adenosine was also lower in obese and in tumor-bearing mice (p<0.05) and its concentration was inversely associated with colonic Il1b and Tnf production (p<0.05). HF and genetic obesity differentially alter the intestinal microbiome and metabolome. A depletion of adenosine and P.distasonis in tumor-bearing mice could play a mechanistic role in tumor formation. Adenosine and P. distasonis have previously been shown to be anti-inflammatory in the colon and we postulate their reduction could promote tumorigenesis by de-repressing inflammation.
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- 2015
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18. Effects of oral eicosapentaenoic acid versus docosahexaenoic acid on human peripheral blood mononuclear cell gene expression.
- Author
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Tsunoda F, Lamon-Fava S, Asztalos BF, Iyer LK, Richardson K, and Schaefer EJ
- Subjects
- 1-Alkyl-2-acetylglycerophosphocholine Esterase blood, Administration, Oral, Adult, Biomarkers blood, Boston, Docosahexaenoic Acids blood, Double-Blind Method, Eicosapentaenoic Acid blood, Female, Gene Expression Profiling methods, Gene Expression Regulation drug effects, Gene Regulatory Networks drug effects, Humans, Leukocytes, Mononuclear metabolism, Male, Middle Aged, Oligonucleotide Array Sequence Analysis, Olive Oil administration & dosage, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction drug effects, Signal Transduction genetics, Time Factors, Up-Regulation, Docosahexaenoic Acids administration & dosage, Eicosapentaenoic Acid administration & dosage, Leukocytes, Mononuclear drug effects
- Abstract
Objective: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have beneficial effects on inflammation and cardiovascular disease (CVD). Our aim was to assess the effect of a six-week supplementation with either olive oil, EPA, or DHA on gene expression in peripheral blood mononuclear cells (PBMC)., Methods: Subjects were sampled at baseline and six weeks after receiving either: olive oil 6.0 g/day (n = 16), EPA 1.8 g/day (n = 16), or DHA 1.8 g/day (n = 18). PBMC were subjected to gene expression analysis by microarray with key findings confirmed by quantitative real-time polymerase chain reaction (Q-PCR)., Results: Plasma phospholipid EPA increased 3 fold in the EPA group, and DHA increased 63% in the DHA group (both p < 0.01), while no effects were observed in the olive oil group. Microarray analysis indicated that EPA but not DHA or olive oil significantly affected the gene expression in the following pathways: 1) interferon signaling, 2) receptor recognition of bacteria and viruses, 3) G protein signaling, glycolysis and glycolytic shunting, 4) S-adenosyl-l-methionine biosynthesis, and 5) cAMP-mediated signaling including cAMP responsive element protein 1 (CREB1), as well as many other individual genes including hypoxia inducible factor 1, α subunit (HIF1A). The findings for CREB1 and HIF1A were confirmed by Q-PCR analysis., Conclusions: Our data indicate that EPA supplementation was associated with significant effects on gene expression involving the interferon pathway as well as down-regulation of CREB1 and HIF1A, which may relate to its beneficial effect on CVD risk reduction., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2015
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19. Mass spectrometric approaches to study protein structure and interactions in lyophilized powders.
- Author
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Moorthy BS, Iyer LK, and Topp EM
- Subjects
- Amides chemistry, Freeze Drying methods, Kinetics, Peptides chemistry, Protein Conformation, Protein Folding, Protein Structure, Secondary, Deuterium Exchange Measurement methods, Mass Spectrometry methods, Proteins chemistry
- Abstract
Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (N fast, N slow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.
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- 2015
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20. Characterizing Protein Structure, Dynamics and Conformation in Lyophilized Solids.
- Author
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Moorthy BS, Iyer LK, and Topp EM
- Subjects
- Humans, Chemistry, Pharmaceutical, Freeze Drying methods, Mass Spectrometry methods, Protein Conformation, Proteins chemistry, Proteins metabolism
- Abstract
The long-term stability of protein therapeutics in the solid-state depends on the preservation of native structure during lyophilization and in the lyophilized powder. Proteins can reversibly or irreversibly unfold upon lyophilization, acquiring conformations susceptible to degradation during storage. Therefore, characterizing proteins in the dried state is crucial for the design of safe and efficacious formulations. This review summarizes the basic principles and applications of the analytical techniques that are commonly used to characterize protein structure, dynamics and conformation in lyophilized solids. The review also discusses the applications of recently developed mass spectrometry based methods (solid-state hydrogen deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS)) and their ability to study proteins in the solid-state at high resolution.
- Published
- 2015
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21. Genome-wide DNA methylation analysis identifies a metabolic memory profile in patient-derived diabetic foot ulcer fibroblasts.
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Park LK, Maione AG, Smith A, Gerami-Naini B, Iyer LK, Mooney DJ, Veves A, and Garlick JA
- Subjects
- Adult, Aged, Cell Line, Computational Biology, Diabetic Foot metabolism, Female, Humans, Male, Middle Aged, DNA Methylation, Diabetic Foot genetics, Epigenesis, Genetic, Fibroblasts metabolism
- Abstract
Diabetic foot ulcers (DFUs) are a serious complication of diabetes. Previous exposure to hyperglycemic conditions accelerates a decline in cellular function through metabolic memory despite normalization of glycemic control. Persistent, hyperglycemia-induced epigenetic patterns are considered a central mechanism that activates metabolic memory; however, this has not been investigated in patient-derived fibroblasts from DFUs. We generated a cohort of patient-derived lines from DFU fibroblasts (DFUF), and site- and age-matched diabetic foot fibroblasts (DFF) and non-diabetic foot fibroblasts (NFF) to investigate global and genome-wide DNA methylation patterns using liquid chromatography/mass spectrometry and the Illumina Infinium HumanMethylation450K array. DFFs and DFUFs demonstrated significantly lower global DNA methylation compared to NFFs (p = 0.03). Hierarchical clustering of differentially methylated probes (DMPs, p = 0.05) showed that DFFs and DFUFs cluster together and separately from NFFs. Twenty-five percent of the same probes were identified as DMPs when individually comparing DFF and DFUF to NFF. Functional annotation identified enrichment of DMPs associated with genes critical to wound repair, including angiogenesis (p = 0.07) and extracellular matrix assembly (p = 0.035). Identification of sustained DNA methylation patterns in patient-derived fibroblasts after prolonged passage in normoglycemic conditions demonstrates persistent metabolic memory. These findings suggest that epigenetic-related metabolic memory may also underlie differences in wound healing phenotypes and can potentially identify therapeutic targets.
- Published
- 2014
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22. Research resource: Aorta- and liver-specific ERα-binding patterns and gene regulation by estrogen.
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Gordon FK, Vallaster CS, Westerling T, Iyer LK, Brown M, and Schnitzler GR
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- Animals, Base Sequence, Cardiovascular Diseases metabolism, Chromatin metabolism, Consensus Sequence, Estradiol physiology, Female, Gene Expression Regulation, Mice, Inbred C57BL, Organ Specificity, Promoter Regions, Genetic, Protein Binding, Signal Transduction, Aorta metabolism, Estrogen Receptor alpha metabolism, Estrogens physiology, Liver metabolism
- Abstract
Estrogen has vascular protective effects in premenopausal women and in women younger than 60 years who are receiving hormone replacement therapy. However, estrogen also increases the risks of breast and uterine cancers and of venous thromboses linked to up-regulation of coagulation factors in the liver. In mouse models, the vasculoprotective effects of estrogen are mediated by the estrogen receptor α (ERα) transcription factor. Here, through next-generation sequencing approaches, we show that almost all of the genes regulated by 17β-estradiol (E2) differ between mouse aorta and mouse liver, ex vivo, and that this difference is associated with a distinct genomewide distribution of ERα on chromatin. Bioinformatic analysis of E2-regulated promoters and ERα binding site sequences identify several transcription factors that may determine the tissue specificity of ERα binding and E2-regulated genes, including the enrichment of NF-κB, AML1, and AP1 sites in the promoters of E2 down-regulated inflammatory genes in aorta but not liver. The possible vascular-specific functions of these factors suggest ways in which the protective effects of estrogen could be promoted in the vasculature without incurring negative effects in other tissues.
- Published
- 2014
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23. How Gene Networks Can Uncover Novel CVD Players.
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Parnell LD, Casas-Agustench P, Iyer LK, and Ordovas JM
- Abstract
Cardiovascular diseases (CVD) are complex, involving numerous biological entities from genes and small molecules to organ function. Placing these entities in networks where the functional relationships among the constituents are drawn can aid in our understanding of disease onset, progression and prevention. While networks, or interactomes, are often classified by a general term, say lipids or inflammation, it is a more encompassing class of network that is more informative in showing connections among the active entities and allowing better hypotheses of novel CVD players to be formulated. A range of networks will be presented whereby the potential to bring new objects into the CVD milieu will be exemplified.
- Published
- 2014
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- View/download PDF
24. Photolytic labeling to probe molecular interactions in lyophilized powders.
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Iyer LK, Moorthy BS, and Topp EM
- Subjects
- Amino Acid Sequence, Amino Acids chemistry, Apoproteins chemistry, Benzophenones chemistry, Chromatography, High Pressure Liquid methods, Freeze Drying methods, Guanidine chemistry, Mass Spectrometry methods, Myoglobin chemistry, Peptides chemistry, Phenylalanine analogs & derivatives, Phenylalanine chemistry, Photolysis, Sucrose chemistry, Powders chemistry
- Abstract
Local side-chain interactions in lyophilized protein formulations were mapped using solid-state photolytic labeling-mass spectrometry (ssPL-MS). Photoactive amino acid analogues (PAAs) were used as probes and either added to the lyophilized matrix or incorporated within the amino acid sequence of a peptide. In the first approach, apomyoglobin was lyophilized with sucrose and varying concentrations of photoleucine (L-2-amino-4,4'-azipentanoic acid; pLeu). The lyophilized solid was irradiated at 365 nm to initiate photolabeling. The rate and extent of labeling were measured using electrospray ionization/high-performance liquid chromatography/mass spectrometry (ESI-HPLC-MS), with labeling reaching a plateau at ~30 min, forming up to six labeled populations. Bottom-up MS/MS analysis was able to provide peptide-level resolution of the location of pLeu. ssPL-MS was also able to detect differences in side-chain environment between sucrose and guanidine hydrochloride formulations. In the second approach, peptide GCG (1-8)* containing p-benzoyl-L-phenylalanine (pBpA) in the amino acid sequence was lyophilized with various excipients and irradiated. Peptide-peptide and peptide-excipient adducts were detected using MS. Top-down MS/MS on the peptide dimer provided amino acid-level resolution regarding interactions and the cross-linking partner for pBpA in the solid state. The results show that ssPL-MS can provide high-resolution information about protein interactions in the lyophilized environment.
- Published
- 2013
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- View/download PDF
25. Protein aggregation and lyophilization: Protein structural descriptors as predictors of aggregation propensity.
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Roughton BC, Iyer LK, Bertelsen E, Topp EM, and Camarda KV
- Abstract
Lyophilization can induce aggregation in therapeutic proteins, but the relative importance of protein structure, formulation and processing conditions are poorly understood. To evaluate the contribution of protein structure to lyophilization-induced aggregation, fifteen proteins were co-lyophilized with each of five excipients. Extent of aggregation following lyophilization, measured using size-exclusion chromatography, was correlated with computational and biophysical protein structural descriptors via multiple linear regression. Descriptor selection was performed using exhaustive search and forward selection. The results demonstrate that, for a given excipient, extent of aggregation is highly correlated by eight to twelve structural descriptors. Leave-one-out cross validation showed that the correlations were able to successfully predict the aggregation for a protein "left out" of the data set. Selected descriptors varied with excipient, indicating both protein structure and excipient type contribute to lyophilization-induced aggregation. The results show some descriptors used to predict protein aggregation in solution are useful in predicting lyophilized protein aggregation.
- Published
- 2013
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26. Microarrays and microneedle arrays for delivery of peptides, proteins, vaccines and other applications.
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Chandrasekhar S, Iyer LK, Panchal JP, Topp EM, Cannon JB, and Ranade VV
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- Animals, Humans, Microarray Analysis instrumentation, Drug Delivery Systems, Peptides administration & dosage, Pharmaceutical Preparations administration & dosage, Protein Array Analysis, Proteins administration & dosage, Vaccines administration & dosage
- Abstract
Introduction: Peptide and protein microarray and microneedle array technology provides direct information on protein function and potential drug targets in drug discovery and delivery. Because of this unique ability, these arrays are well suited for protein profiling, drug target identification/validation and studies of protein interaction, biochemical activity, immune responses, clinical prognosis and diagnosis and for gene, protein and drug delivery., Areas Covered: The aim of this review is to describe and summarize past and recent developments of microarrays in their construction, characterization and production and applications of microneedles in drug delivery. The scope and limitations of various technologies in this respect are discussed., Expert Opinion: This article offers a review of microarray/microneedle technologies and possible future directions in targeting and in the delivery of pharmacologically active compounds for unmet needs in biopharmaceutical research. A better understanding of the production and use of microarrays and microneedles for delivery of peptides, proteins and vaccines is needed.
- Published
- 2013
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27. Estrogen receptor-mediated regulation of microRNA inhibits proliferation of vascular smooth muscle cells.
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Zhao J, Imbrie GA, Baur WE, Iyer LK, Aronovitz MJ, Kershaw TB, Haselmann GM, Lu Q, and Karas RH
- Subjects
- Animals, Aorta metabolism, Aorta pathology, Binding Sites, Cells, Cultured, Estradiol metabolism, Female, Gene Expression Profiling, Gene Expression Regulation, Homeodomain Proteins metabolism, Human Umbilical Vein Endothelial Cells metabolism, Humans, Kruppel-Like Transcription Factors metabolism, Mice, Mice, Inbred C57BL, MicroRNAs genetics, Muscle, Smooth, Vascular pathology, Myocytes, Smooth Muscle pathology, Ovariectomy, Phosphoproteins metabolism, Promoter Regions, Genetic, Proto-Oncogene Proteins c-abl metabolism, RNA Interference, Time Factors, Trans-Activators metabolism, Transcription Factor AP-1 metabolism, Transcription, Genetic, Transfection, Zinc Finger E-box-Binding Homeobox 1, Cell Proliferation, Estrogen Receptor alpha metabolism, MicroRNAs metabolism, Muscle, Smooth, Vascular metabolism, Myocytes, Smooth Muscle metabolism
- Abstract
Objective: Estradiol (E2) regulates gene transcription by activating estrogen receptor-α and estrogen receptor-β. Many of the genes regulated by E2 via estrogen receptors are repressed, yet the molecular mechanisms that mediate E2-induced gene repression are currently unknown. We hypothesized that E2, acting through estrogen receptors, regulates expression of microRNAs (miRs) leading to repression of expression of specific target genes., Methods and Results: Here, we report that E2 significantly upregulates the expression of 26 miRs and downregulates the expression of 6 miRs in mouse aorta. E2-mediated upregulation of one of these miRs, miR-203, was chosen for further study. In cultured vascular smooth muscle cells (VSMC), E2-mediated upregulation of miR-203 is mediated by estrogen receptor-α (but not estrogen receptor-β) via transcriptional upregulation of the primary miR. We demonstrate that the transcription factors Zeb-1 and AP-1 play critical roles in mediating E2-induced upregulation of miR-203 transcription. We show further that miR-203 mediates E2-induced repression of Abl1, and p63 protein abundance in VSMC. Finally, knocking-down miR-203 abolishes E2-mediated inhibition of VSMC proliferation, and overexpression of miR-203 inhibits cultured VSMC proliferation, but not vascular endothelial cell proliferation., Conclusions: Our findings demonstrate that E2 regulates expression of miRs in the vasculature and support the estrogen receptors-dependent induction of miRs as a mechanism for E2-mediated gene repression. Furthermore, our findings demonstrate that miR-203 contributes to E2-induced inhibition of VSMC proliferation and highlight the potential of miR-203 as a therapeutic agent in the treatment of proliferative cardiovascular diseases.
- Published
- 2013
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28. Rapid estrogen receptor signaling is essential for the protective effects of estrogen against vascular injury.
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Bernelot Moens SJ, Schnitzler GR, Nickerson M, Guo H, Ueda K, Lu Q, Aronovitz MJ, Nickerson H, Baur WE, Hansen U, Iyer LK, and Karas RH
- Subjects
- Animals, Aorta cytology, COS Cells, Carotid Artery Injuries genetics, Carotid Artery Injuries pathology, Chlorocebus aethiops, Disease Models, Animal, Female, Human Umbilical Vein Endothelial Cells, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Muscle, Smooth, Vascular cytology, Ovariectomy, Pregnancy, Transcriptome, Carotid Artery Injuries metabolism, Estradiol metabolism, Estrogen Receptor alpha genetics, Estrogen Receptor alpha metabolism, Muscle, Smooth, Vascular physiology, Signal Transduction physiology
- Abstract
Background: Clinical trial and epidemiological data support that the cardiovascular effects of estrogen are complex, including a mixture of both potentially beneficial and harmful effects. In animal models, estrogen protects females from vascular injury and inhibits atherosclerosis. These effects are mediated by estrogen receptors (ERs), which, when bound to estrogen, can bind to DNA to directly regulate transcription. ERs can also activate several cellular kinases by inducing a rapid nonnuclear signaling cascade. However, the biological significance of this rapid signaling pathway has been unclear., Methods and Results: In the present study, we develop a novel transgenic mouse in which rapid signaling is blocked by overexpression of a peptide that prevents ERs from interacting with the scaffold protein striatin (the disrupting peptide mouse). Microarray analysis of ex vivo treated mouse aortas demonstrates that rapid ER signaling plays an important role in estrogen-mediated gene regulatory responses. Disruption of ER-striatin interactions also eliminates the ability of estrogen to stimulate cultured endothelial cell migration and to inhibit cultured vascular smooth muscle cell growth. The importance of these findings is underscored by in vivo experiments demonstrating loss of estrogen-mediated protection against vascular injury in the disrupting peptide mouse after carotid artery wire injury., Conclusions: Taken together, these results support the concept that rapid, nonnuclear ER signaling contributes to the transcriptional regulatory functions of ER and is essential for many of the vasoprotective effects of estrogen. These findings also identify the rapid ER signaling pathway as a potential target for the development of novel therapeutic agents.
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- 2012
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29. Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS.
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Butovsky O, Siddiqui S, Gabriely G, Lanser AJ, Dake B, Murugaiyan G, Doykan CE, Wu PM, Gali RR, Iyer LK, Lawson R, Berry J, Krichevsky AM, Cudkowicz ME, and Weiner HL
- Subjects
- Amyotrophic Lateral Sclerosis drug therapy, Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis pathology, Animals, Antibodies, Monoclonal administration & dosage, Antigens, CD genetics, Antigens, CD metabolism, Antigens, Ly genetics, Antigens, Ly immunology, Antigens, Ly metabolism, Apoptosis, Apyrase genetics, Apyrase metabolism, Cell Proliferation, Chemotaxis, Female, Gene Regulatory Networks, Humans, Inflammation Mediators metabolism, Macrophages, Alveolar metabolism, Male, Metabolic Networks and Pathways, Mice, Mice, Inbred C57BL, Mice, Transgenic, MicroRNAs metabolism, Microglia immunology, Microglia pathology, Monocytes metabolism, Monocytes pathology, Oligonucleotide Array Sequence Analysis, RNA Interference, Rats, Rats, Inbred Lew, Spinal Cord pathology, Spleen immunology, Spleen pathology, Superoxide Dismutase genetics, Superoxide Dismutase-1, Transcription Factors genetics, Transcription Factors metabolism, Transcriptome, Amyotrophic Lateral Sclerosis immunology, Immunomodulation, MicroRNAs genetics, Monocytes immunology, Spinal Cord immunology
- Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with neuronal cell death that is thought to involve aberrant immune responses. Here we investigated the role of innate immunity in a mouse model of ALS. We found that inflammatory monocytes were activated and that their progressive recruitment to the spinal cord, but not brain, correlated with neuronal loss. We also found a decrease in resident microglia in the spinal cord with disease progression. Prior to disease onset, splenic Ly6Chi monocytes expressed a polarized macrophage phenotype (M1 signature), which included increased levels of chemokine receptor CCR2. As disease onset neared, microglia expressed increased CCL2 and other chemotaxis-associated molecules, which led to the recruitment of monocytes to the CNS by spinal cord-derived microglia. Treatment with anti-Ly6C mAb modulated the Ly6Chi monocyte cytokine profile, reduced monocyte recruitment to the spinal cord, diminished neuronal loss, and extended survival. In humans with ALS, the analogous monocytes (CD14+CD16-) exhibited an ALS-specific microRNA inflammatory signature similar to that observed in the ALS mouse model, linking the animal model and the human disease. Thus, the profile of monocytes in ALS patients may serve as a biomarker for disease stage or progression. Our results suggest that recruitment of inflammatory monocytes plays an important role in disease progression and that modulation of these cells is a potential therapeutic approach.
- Published
- 2012
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30. Computational and biological evaluation of quinazolinone prodrug for targeting pancreatic cancer.
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Pospisil P, Korideck H, Wang K, Yang Y, Iyer LK, and Kassis AI
- Subjects
- Binding Sites, Cell Line, Tumor, Cell Nucleus drug effects, Computer Simulation, Humans, Hydrolysis, Iodine Radioisotopes chemistry, Isotope Labeling, Pancreatic Neoplasms diagnostic imaging, Positron-Emission Tomography, Prodrugs pharmacology, Protein Structure, Tertiary, Quinazolinones pharmacology, Sulfotransferases chemistry, Sulfotransferases metabolism, Prodrugs chemistry, Quinazolinones chemistry
- Abstract
Our concept of enzyme-mediated cancer imaging and therapy aims to use radiolabeled compounds to target hydrolases over-expressed on the extracellular surface of solid tumors. A data mining approach identified extracellular sulfatase 1 (SULF1) as an enzyme expressed on the surface of pancreatic cancer cells. We designed, synthesized, and characterized 2-(2'-sulfooxyphenyl)-6-iodo-4-(3H)-quinazolinone (IQ(2-S)) as well as its radioiodinated form ((125) IQ(2-S)) as a prodrug with potential for hydrolysis by SULF1. IQ(2-S) was successfully docked in silico into three enzymes - homolog of SULF1, alkaline phosphatase, and prostatic acid phosphatase. The incubation of (125) IQ(2-S) and (125) IQ(2-P) with the three enzymes in solution confirms the docking results and enzyme selectivity for the analogs. The hydrolysis of both radioactive compounds produces the water-insoluble, fluorescent product 2-(2'-hydroxyphenyl)-6-[(125) I]iodo-4-(3H)-quinazolinone ((125) IQ(2-OH)). The in vitro incubation of (127) IQ(2-S) and (127) IQ(2-P) with pancreatic, ovarian, and prostate cancer cells expressing studied hydrolases also results in their hydrolysis and the precipitation of (127) IQ(2-OH) fluorescent crystals on the cell surface. To our knowledge, these findings are the first to report the targeting of a radioactive substrate to SULF1 and that this prodrug may be potentially useful in the imaging ((123) I/(124) I/(131) I) and radiotherapy ((131) I) of pancreatic cancer., (© 2012 John Wiley & Sons A/S.)
- Published
- 2012
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31. Aldosterone regulates vascular gene transcription via oxidative stress-dependent and -independent pathways.
- Author
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Newfell BG, Iyer LK, Mohammad NN, McGraw AP, Ehsan A, Rosano G, Huang PL, Mendelsohn ME, and Jaffe IZ
- Subjects
- Animals, Atherosclerosis etiology, Atherosclerosis genetics, Atherosclerosis metabolism, Cells, Cultured, Dactinomycin pharmacology, Endothelium, Vascular drug effects, Endothelium, Vascular injuries, Endothelium, Vascular metabolism, Gene Expression Profiling, Gene Expression Regulation drug effects, Humans, In Vitro Techniques, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mineralocorticoid Receptor Antagonists, Myocytes, Smooth Muscle drug effects, Myocytes, Smooth Muscle metabolism, Oxidative Stress drug effects, RNA, Messenger genetics, RNA, Messenger metabolism, Spironolactone pharmacology, Aldosterone pharmacology, Aorta drug effects, Aorta metabolism
- Abstract
Objective: Aldosterone (Aldo) antagonism prevents cardiovascular mortality by unclear mechanisms. Aldo binds to the mineralocorticoid receptor (MR), a ligand-activated transcription factor, which is expressed in human vascular cells. Here we define the early Aldo-regulated vascular transcriptome and investigate the mechanisms of gene regulation by Aldo in the vasculature that may contribute to vascular disease., Methods and Results: Gene expression profiling of Aldo-treated mouse aortas identified 72 genes regulated by Aldo. These genes are overrepresented in Gene Ontology categories involved in vascular function and disease. Quantitative reverse transcription-polymerase chain reaction was used to confirm and further explore mechanisms of vascular gene regulation by Aldo. Aldo-regulated vascular gene expression was inhibited by actinomycin D and MR antagonists supporting a transcriptional MR-dependent mechanism. Aldo regulation of a subset of genes was enhanced in the setting of vascular endothelial denudation and blocked by the free radical scavenger Tempol, supporting synergy between Aldo and vascular injury that is oxidative stress dependent. In the aortic arch, a region predisposed to atherosclerosis, the injury-enhanced genes also demonstrated enhanced expression compared with the descending aorta, both at baseline and after Aldo exposure. Furthermore, the clinically beneficial MR antagonist spironolactone inhibited expression of the identified genes in aortic tissue from humans with atherosclerosis., Conclusions: This study defines the Aldo-regulated vascular transcriptome and characterizes a subset of proatherogenic genes with enhanced Aldo-stimulated, oxidative stress-dependent expression in the setting of vascular injury and in areas predisposed to atherosclerosis. Inhibition of MR regulation of these genes may play a role in the protective effects of Aldo antagonists in patients with vascular disease, and these pathways may provide novel drug targets to prevent atherosclerosis in humans.
- Published
- 2011
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32. Application of gene network analysis techniques identifies AXIN1/PDIA2 and endoglin haplotypes associated with bicuspid aortic valve.
- Author
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Wooten EC, Iyer LK, Montefusco MC, Hedgepeth AK, Payne DD, Kapur NK, Housman DE, Mendelsohn ME, and Huggins GS
- Subjects
- Axin Protein, Case-Control Studies, Endoglin, Genome-Wide Association Study, Humans, Oligonucleotide Array Sequence Analysis, Polymorphism, Single Nucleotide, Antigens, CD genetics, Aortic Valve abnormalities, Gene Regulatory Networks, Haplotypes, Heart Defects, Congenital genetics, Protein Disulfide-Isomerases genetics, Receptors, Cell Surface genetics, Repressor Proteins genetics
- Abstract
Bicuspid Aortic Valve (BAV) is a highly heritable congenital heart defect. The low frequency of BAV (1% of general population) limits our ability to perform genome-wide association studies. We present the application of four a priori SNP selection techniques, reducing the multiple-testing penalty by restricting analysis to SNPs relevant to BAV in a genome-wide SNP dataset from a cohort of 68 BAV probands and 830 control subjects. Two knowledge-based approaches, CANDID and STRING, were used to systematically identify BAV genes, and their SNPs, from the published literature, microarray expression studies and a genome scan. We additionally tested Functionally Interpolating SNPs (fitSNPs) present on the array; the fourth consisted of SNPs selected by Random Forests, a machine learning approach. These approaches reduced the multiple testing penalty by lowering the fraction of the genome probed to 0.19% of the total, while increasing the likelihood of studying SNPs within relevant BAV genes and pathways. Three loci were identified by CANDID, STRING, and fitSNPS. A haplotype within the AXIN1-PDIA2 locus (p-value of 2.926x10(-06)) and a haplotype within the Endoglin gene (p-value of 5.881x10(-04)) were found to be strongly associated with BAV. The Random Forests approach identified a SNP on chromosome 3 in association with BAV (p-value 5.061x10(-06)). The results presented here support an important role for genetic variants in BAV and provide support for additional studies in well-powered cohorts. Further, these studies demonstrate that leveraging existing expression and genomic data in the context of GWAS studies can identify biologically relevant genes and pathways associated with a congenital heart defect.
- Published
- 2010
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33. Global gene expression patterns in the post-pneumonectomy lung of adult mice.
- Author
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Paxson JA, Parkin CD, Iyer LK, Mazan MR, Ingenito EP, and Hoffman AM
- Subjects
- Actins metabolism, Animals, Cell Proliferation, Female, Fibroblasts metabolism, Gene Regulatory Networks, Immunohistochemistry, Inflammation genetics, Lung metabolism, Lung physiopathology, Mice, Mice, Inbred C57BL, Oligonucleotide Array Sequence Analysis, RNA metabolism, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, S100 Calcium-Binding Protein A4, S100 Proteins metabolism, Signal Transduction genetics, Time Factors, Gene Expression Profiling methods, Gene Expression Regulation, Lung surgery, Pneumonectomy, Regeneration genetics, Thoracotomy
- Abstract
Background: Adult mice have a remarkable capacity to regenerate functional alveoli following either lung resection or injury that exceeds the regenerative capacity observed in larger adult mammals. The molecular basis for this unique capability in mice is largely unknown. We examined the transcriptomic responses to single lung pneumonectomy in adult mice in order to elucidate prospective molecular signaling mechanisms used in this species during lung regeneration., Methods: Unilateral left pneumonectomy or sham thoracotomy was performed under general anesthesia (n = 8 mice per group for each of the four time points). Total RNA was isolated from the remaining lung tissue at four time points post-surgery (6 hours, 1 day, 3 days, 7 days) and analyzed using microarray technology., Results: The observed transcriptomic patterns revealed mesenchymal cell signaling, including up-regulation of genes previously associated with activated fibroblasts (Tnfrsf12a, Tnc, Eln, Col3A1), as well as modulation of Igf1-mediated signaling. The data set also revealed early down-regulation of pro-inflammatory cytokine transcripts and up-regulation of genes involved in T cell development/function, but few similarities to transcriptomic patterns observed during embryonic or post-natal lung development. Immunohistochemical analysis suggests that early fibroblast but not myofibroblast proliferation is important during lung regeneration and may explain the preponderance of mesenchymal-associated genes that are over-expressed in this model. This again appears to differ from embryonic alveologenesis., Conclusion: These data suggest that modulation of mesenchymal cell transcriptome patterns and proliferation of S100A4 positive mesenchymal cells, as well as modulation of pro-inflammatory transcriptome patterns, are important during post-pneumonectomy lung regeneration in adult mice.
- Published
- 2009
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34. Depletion of central BDNF in mice impedes terminal differentiation of new granule neurons in the adult hippocampus.
- Author
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Chan JP, Cordeira J, Calderon GA, Iyer LK, and Rios M
- Subjects
- Animals, Brain-Derived Neurotrophic Factor genetics, Cell Movement, Cell Proliferation, Female, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Neurons cytology, Receptor, trkB metabolism, Receptors, GABA-A metabolism, Stem Cells cytology, Stem Cells physiology, Transcription, Genetic, gamma-Aminobutyric Acid metabolism, Brain-Derived Neurotrophic Factor metabolism, Cell Differentiation physiology, Hippocampus cytology, Neurons physiology
- Abstract
Granule neurons generated in the adult mammalian hippocampus synaptically integrate to facilitate cognitive function and antidepressant efficacy. Here, we investigated the role of BDNF in facilitating their maturation in vivo. We found that depletion of central BDNF in mice elicited an increase in hippocampal cell proliferation without affecting cell survival or fate specification. However, new mutant neurons failed to fully mature as indicated by their lack of calbindin, reduced dendritic differentiation and an accumulation of calretinin(+) immature neurons in the BDNF mutant dentate gyrus. Furthermore, the facilitating effects of GABA(A) receptor stimulation on neurogenesis were absent in the mutants, suggesting that defects might be due to alterations in GABA signaling. Transcriptional analysis of the mutant hippocampal neurogenic region revealed increases in markers for immature neurons and decreases in neuronal differentiation facilitators. These findings demonstrate that BDNF is required for the terminal differentiation of new neurons in the adult hippocampus.
- Published
- 2008
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- View/download PDF
35. Integrative genomic data mining for discovery of potential blood-borne biomarkers for early diagnosis of cancer.
- Author
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Yang Y, Pospisil P, Iyer LK, Adelstein SJ, and Kassis AI
- Subjects
- Biomarkers, Tumor genetics, Breast Neoplasms blood, Breast Neoplasms diagnosis, Breast Neoplasms genetics, Carcinoma blood, Carcinoma diagnosis, Carcinoma genetics, Colonic Neoplasms blood, Colonic Neoplasms diagnosis, Colonic Neoplasms genetics, Databases, Genetic, Early Diagnosis, Female, Gene Expression Regulation, Neoplastic, Humans, Lung Neoplasms blood, Lung Neoplasms diagnosis, Lung Neoplasms genetics, Male, Neoplasm Proteins blood, Neoplasms blood, Neoplasms genetics, Neoplastic Cells, Circulating, Ovarian Neoplasms blood, Ovarian Neoplasms diagnosis, Ovarian Neoplasms genetics, Pancreatic Neoplasms blood, Pancreatic Neoplasms diagnosis, Pancreatic Neoplasms genetics, Prostatic Neoplasms blood, Prostatic Neoplasms diagnosis, Prostatic Neoplasms genetics, Up-Regulation, Biomarkers, Tumor blood, Gene Expression Profiling, Microarray Analysis, Neoplasm Proteins genetics, Neoplasms diagnosis
- Abstract
Background: With the arrival of the postgenomic era, there is increasing interest in the discovery of biomarkers for the accurate diagnosis, prognosis, and early detection of cancer. Blood-borne cancer markers are favored by clinicians, because blood samples can be obtained and analyzed with relative ease. We have used a combined mining strategy based on an integrated cancer microarray platform, Oncomine, and the biomarker module of the Ingenuity Pathways Analysis (IPA) program to identify potential blood-based markers for six common human cancer types., Methodology/principal Findings: In the Oncomine platform, the genes overexpressed in cancer tissues relative to their corresponding normal tissues were filtered by Gene Ontology keywords, with the extracellular environment stipulated and a corrected Q value (false discovery rate) cut-off implemented. The identified genes were imported to the IPA biomarker module to separate out those genes encoding putative secreted or cell-surface proteins as blood-borne (blood/serum/plasma) cancer markers. The filtered potential indicators were ranked and prioritized according to normalized absolute Student t values. The retrieval of numerous marker genes that are already clinically useful or under active investigation confirmed the effectiveness of our mining strategy. To identify the biomarkers that are unique for each cancer type, the upregulated marker genes that are in common between each two tumor types across the six human tumors were also analyzed by the IPA biomarker comparison function., Conclusion/significance: The upregulated marker genes shared among the six cancer types may serve as a molecular tool to complement histopathologic examination, and the combination of the commonly upregulated and unique biomarkers may serve as differentiating markers for a specific cancer. This approach will be increasingly useful to discover diagnostic signatures as the mass of microarray data continues to grow in the 'omics' era.
- Published
- 2008
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36. Computational modeling and experimental evaluation of a novel prodrug for targeting the extracellular space of prostate tumors.
- Author
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Pospisil P, Wang K, Al Aowad AF, Iyer LK, Adelstein SJ, and Kassis AI
- Subjects
- Acid Phosphatase, Carcinoma pathology, Humans, Iodine Radioisotopes administration & dosage, Iodine Radioisotopes pharmacokinetics, Male, Models, Biological, Models, Molecular, Prostatic Neoplasms metabolism, Prostatic Neoplasms pathology, Quaternary Ammonium Compounds administration & dosage, Quaternary Ammonium Compounds pharmacokinetics, Quinazolinones administration & dosage, Quinazolinones pharmacokinetics, Tumor Cells, Cultured, Carcinoma drug therapy, Computer Simulation, Drug Delivery Systems, Extracellular Space drug effects, Prodrugs administration & dosage, Prostatic Neoplasms drug therapy, Protein Tyrosine Phosphatases metabolism
- Abstract
We are developing a noninvasive approach for targeting imaging and therapeutic radionuclides to prostate cancer. Our method, Enzyme-Mediated Cancer Imaging and Therapy (EMCIT), aims to use enzyme-dependent, site-specific, in vivo precipitation of a radioactive molecule within the extracellular space of solid tumors. Advanced methods for data mining of the literature, protein databases, and knowledge bases (IT. Omics LSGraph and Ingenuity Systems) identified prostatic acid phosphatase (PAP) as an enzyme overexpressed in prostate cancer and secreted in the extracellular space. Using AutoDock 3.0 software, the prodrug ammonium 2-(2'-phosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone (IQ(2-P)) was docked in silico into the X-ray structure of PAP. The data indicate that IQ(2-P) docked into the PAP active site with a calculated inhibition constant (K(i)) more favorable than that of the PAP inhibitor alpha-benzylaminobenzylphosphonic acid. When (125)IQ(2-P), the radioiodinated form of the water-soluble prodrug, was incubated with PAP, rapid hydrolysis of the compound was observed as exemplified by formation of the water-insoluble 2-(2'-hydroxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone ((125)IQ(2-OH)). Similarly, the incubation of IQ(2-P) with human LNCaP, PC-3, and 22Rv1 prostate tumor cells resulted in the formation of large fluorescent IQ(2-OH) crystals. No hydrolysis was seen in the presence of normal human cells. Autoradiography of tumor cells incubated with (125)IQ(2-P) showed accumulation of radioactive grains ((125)IQ(2-OH)) around the cells. We anticipate that the EMCIT approach will enable the active in vivo entrapment of radioimaging and radiotherapeutic compounds within the extracellular spaces of primary prostate tumors and their metastases.
- Published
- 2007
- Full Text
- View/download PDF
37. In silico design, synthesis, and biological evaluation of radioiodinated quinazolinone derivatives for alkaline phosphatase-mediated cancer diagnosis and therapy.
- Author
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Chen K, Wang K, Kirichian AM, Al Aowad AF, Iyer LK, Adelstein SJ, and Kassis AI
- Subjects
- Animals, Autoradiography methods, Cell Line, Tumor, Crystallography, X-Ray methods, Drug Design, Hydrolysis, Iodine Radioisotopes, Isotope Labeling methods, Mice, Mice, Inbred C3H, Microscopy, Confocal methods, Microscopy, Fluorescence methods, Models, Molecular, Neoplasms diagnosis, Neoplasms drug therapy, Prodrugs chemistry, Prodrugs pharmacology, Quinazolinones chemistry, Quinazolinones pharmacology, Rats, Thermodynamics, Alkaline Phosphatase chemistry, Alkaline Phosphatase metabolism, Neoplasms enzymology, Prodrugs chemical synthesis, Prodrugs pharmacokinetics, Quinazolinones chemical synthesis, Quinazolinones pharmacokinetics
- Abstract
As part of the development of enzyme-mediated cancer imaging and therapy, a novel technology to entrap water-insoluble radioactive molecules within solid tumors, we show that a water-soluble, radioactive quinazolinone prodrug, ammonium 2-(2'-phosphoryloxyphenyl)-6-[125I]iodo-4-(3H)-quinazolinone (125IQ(2-P)), is hydrolyzed by alkaline phosphatase to a water-insoluble, radiolabeled drug, 2-(2'-hydroxyphenyl)-6-[125I]iodo-4-(3H)-quinazolinone (125IQ(2-OH)). Biodistribution data suggest the existence of two isoforms of the prodrug (IQ(2-P(I)) and IQ(2-P)), and this has been confirmed by their synthesis and characterization. Structural differences of the two isoforms have been examined using in silico molecular modeling techniques and docking methods to describe the interaction/binding between the isoforms and human placental alkaline phosphatase (PLAP), a tumor cell, membrane-associated, hydrolytic enzyme whose structure is known by X-ray crystallographic determination. Docking data show that IQ(2-P), but not IQ(2-P(I)), fits the active binding site of PLAP favorably and interacts with the catalytic amino acid Ser(92), which plays an important role in the hydrolytic process. The binding free energies (DeltaG(binding)) of the isoforms to PLAP predict that IQ(2-P) will be the better substrate for PLAP. The in vitro incubation of the isoforms with PLAP leads to the rapid hydrolysis of IQ(2-P) only and confirms the in silico expectations. Fluorescence microscopy shows that in vitro incubation of IQ(2-P) with mouse and human tumor cells causes the extracellular, alkaline phosphatase-mediated hydrolysis of the molecule and precipitation of fluorescent crystals of IQ(2-OH). No hydrolysis is seen in the presence of normal mouse and human cells. Furthermore, the intratumoral injection of 125IQ(2-P) into alkaline phosphatase-expressing solid human tumors grown s.c. in nude rats results in efficient hydrolysis of the compound and retention of approximately 70% of the injected radioactivity, whereas similar injection into normal tissues (e.g., muscle) does not produce any measurable hydrolysis (approximately 1%) or retention of radioactivity at the injected site. These studies support the enzyme-mediated cancer imaging and therapy technology and show the potential of such quinazolinone derivatives in the in vivo radiodetection (123I/124I) and therapy (131I) of solid tumors.
- Published
- 2006
- Full Text
- View/download PDF
38. A combined approach to data mining of textual and structured data to identify cancer-related targets.
- Author
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Pospisil P, Iyer LK, Adelstein SJ, and Kassis AI
- Subjects
- Database Management Systems, Drug Delivery Systems classification, Drug Delivery Systems methods, Gene Targeting classification, Humans, Natural Language Processing, Neoplasm Proteins classification, Neoplasm Proteins genetics, Neoplasms genetics, Systems Integration, Biomarkers, Tumor metabolism, Databases, Protein, Gene Targeting methods, Information Storage and Retrieval methods, Neoplasm Proteins metabolism, Neoplasms metabolism, PubMed
- Abstract
Background: We present an effective, rapid, systematic data mining approach for identifying genes or proteins related to a particular interest. A selected combination of programs exploring PubMed abstracts, universal gene/protein databases (UniProt, InterPro, NCBI Entrez), and state-of-the-art pathway knowledge bases (LSGraph and Ingenuity Pathway Analysis) was assembled to distinguish enzymes with hydrolytic activities that are expressed in the extracellular space of cancer cells. Proteins were identified with respect to six types of cancer occurring in the prostate, breast, lung, colon, ovary, and pancreas., Results: The data mining method identified previously undetected targets. Our combined strategy applied to each cancer type identified a minimum of 375 proteins expressed within the extracellular space and/or attached to the plasma membrane. The method led to the recognition of human cancer-related hydrolases (on average, approximately 35 per cancer type), among which were prostatic acid phosphatase, prostate-specific antigen, and sulfatase 1., Conclusion: The combined data mining of several databases overcame many of the limitations of querying a single database and enabled the facile identification of gene products. In the case of cancer-related targets, it produced a list of putative extracellular, hydrolytic enzymes that merit additional study as candidates for cancer radioimaging and radiotherapy. The proposed data mining strategy is of a general nature and can be applied to other biological databases for understanding biological functions and diseases.
- Published
- 2006
- Full Text
- View/download PDF
39. Functional proteomics of the active cysteine protease content in Drosophila S2 cells.
- Author
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Kocks C, Maehr R, Overkleeft HS, Wang EW, Iyer LK, Lennon-Dumenil AM, Ploegh HL, and Kessler BM
- Subjects
- Amino Acid Sequence, Animals, Biotin chemistry, Cathepsins genetics, Cathepsins metabolism, Cell Line, Cysteine Endopeptidases genetics, Drosophila genetics, Drosophila Proteins genetics, Electrophoresis, Polyacrylamide Gel, Leucine analogs & derivatives, Leucine chemistry, Lysosomes enzymology, Mice, Molecular Probes chemistry, Molecular Sequence Data, Spectrometry, Mass, Electrospray Ionization, Cysteine Endopeptidases metabolism, Drosophila enzymology, Drosophila Proteins metabolism, Phagocytes enzymology
- Abstract
The fruit fly genome is characterized by an evolutionary expansion of proteases and immunity-related genes. In order to characterize the proteases that are active in a phagocytic Drosophila model cell line (S2 cells), we have applied a functional proteomics approach that allows simultaneous detection and identification of multiple protease species. DCG-04, a biotinylated, mechanism-based probe that covalently targets mammalian cysteine proteases of the papain family was found to detect Drosophila polypeptides in an activity-dependent manner. Chemical tagging combined with tandem mass spectrometry permitted retrieval and identification of these polypeptides. Among them was thiol-ester motif-containing protein (TEP) 4 which is involved in insect innate immunity and shares structural and functional similarities with the mammalian complement system factor C3 and the pan-protease inhibitor alpha2-macroglobulin. We also found four cysteine proteases with homologies to lysosomal cathepsin (CTS) L, K, B, and F, which have been implicated in mammalian adaptive immunity. The Drosophila CTS equivalents were most active at a pH of 4.5. This suggests that Drosophila CTS are, similar to their mammalian counterparts, predominantly active in lysosomal compartments. In support of this concept, we found CTS activity in phagosomes of Drosophila S2 cells. These results underscore the utility of activity profiling to address the functional role of insect proteases in immunity.
- Published
- 2003
- Full Text
- View/download PDF
40. Molecular dynamics simulation of alpha-lactalbumin and calcium binding c-type lysozyme.
- Author
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Iyer LK and Qasba PK
- Subjects
- Animals, Computer Simulation, Crystallography, Horses, Humans, Magnetic Resonance Spectroscopy, Protein Conformation, Calcium chemistry, Lactalbumin chemistry, Models, Molecular, Muramidase chemistry
- Abstract
Alpha-lactalbumins (LAs) and c-type lysozymes (LYZs) are two classes of proteins which have a 35-40% sequence homology and share a common three dimensional fold but perform different functions. Lysozymes bind and cleave the glycosidic bond linkage in sugars, where as, alpha-lactalbumin does not bind sugar but participates in the synthesis of lactose. Alpha-lactalbumin is a metallo-protein and binds calcium, where as, only a few of the LYZs bind calcium. These proteins consist of two domains, an alpha-helical and a beta-strand domain, separated by a cleft. Calcium is bound at a loop situated at the bottom of the cleft and is important for the structural integrity of the protein. Calcium is an ubiquitous intracellular signal in higher eukaryotes and structural changes induced on calcium binding have been observed in a number of proteins. In the present study, molecular dynamics simulations of equine LYZ and human LA, with and without calcium, were carried out. We detail the differences in the dynamics of equine LYZ and human LA, and discuss it in the light of experimental data already available and relate it to the behavior of the functionally important regions of both the proteins. These simulations bring out the role of calcium in the conformation and dynamics of these metallo-proteins. In the calcium bound LA, the region of the protein around the calcium binding site is not only frozen but the atomic fluctuations are found to increase away from the binding site and peak at the exposed sites of the protein. This channeling of fluctuations away from the metal binding site could serve as a general mechanism by which the effect of metal binding at a site is transduced to other parts of the protein and could play a key role in protein-ligand and/or protein-protein interaction.
- Published
- 1999
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41. The stability of transmembrane helices: a molecular dynamics study on the isolated helices of bacteriorhodopsin.
- Author
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Iyer LK and Vishveshwara S
- Subjects
- Amino Acid Sequence, Cell Membrane chemistry, Drug Stability, Molecular Sequence Data, Protein Folding, Thermodynamics, Bacteriorhodopsins chemistry, Protein Structure, Secondary
- Abstract
Bacteriorhodopsin (bR) continues to be a proven testing ground for the study of integral membrane proteins (IMPs). It is important to study the stability of the individual helices of bR, as they are postulated to exist as independently stable transmembrane helices (TMHs) and also for their utility as templates for modeling other IMPs with the postulated seven-helix bundle topology. Toward this purpose, the seven helices of bR have been studied by molecular dynamics simulation in this study. The suitability of using the backbone-dependent rotamer library of side-chain conformations arrived at from the data base of globular protein structures in the case TMHs has been tested by another set of 7 helix simulations with the side-chain orientations taken from this library. The influence of the residue's net charge on the helix stability was examined by simulating the helices III, IV, and VI (from both of the above sets of helices) with zero net charge on the side chains. The results of these 20 simulations demonstrate in general the stability of the isolated helices of bR in conformity with the two-stage hypothesis of IMP folding. However, the helices I, II, V, and VII are more stable than the other three helices. The helical nature of certain regions of III, IV, and VI are influenced by factors such as the net charge and orientation of several residues. It is seen that the residues Arg, Lys, Asp, and Glu (charged residues), and Ser, Thr, Gly, and Pro, play a crucial role in the stability of the helices of bR. The backbone-dependent rotamer library for the side chains is found to be suitable for the study of TMHs in IMP.
- Published
- 1996
- Full Text
- View/download PDF
42. A model for transmembrane helix with a cis-proline in the middle.
- Author
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Iyer LK and Vishveshwara S
- Subjects
- Models, Chemical, Protein Conformation, Membrane Proteins chemistry, Proline chemistry
- Abstract
The presence of a higher percentage of Proline in the transmembrane helices of transport proteins indicates that they are involved in the function of these integral membrane proteins (IMPs). In many cases, the possible involvement of cis-trans isomerization in function/folding of IMPs has been suggested. The introduction of cis-Pro in an ideal alpha-helix results in a helix-turn-helix motif. A molecular dynamics (MD) simulation is carried out on the sequence ACE-(ALA)10-cis-Pro-(ALA)10-NME with ideal alpha-helical structure to investigate if and how a straight helix can accommodate a cis-Pro. The analysis of the conformations accessed during MD simulation showed that the residues near cis-Pro can adopt alternate conformations other than the right-handed helical conformation such that an almost straight helix is obtained. This may have implications in the involvement of cis-trans isomerization in folding and/or function of IMPs.
- Published
- 1995
- Full Text
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43. Molecular-dynamics studies of systems of confined dumbbell molecules.
- Author
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Ma WJ, Iyer LK, Vishveshwara S, Koplik J, and Banavar JR
- Published
- 1995
- Full Text
- View/download PDF
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