<p><b>A</b>. Cells were treated with 50 and 100 µM of inhibitor VII, and protei... more <p><b>A</b>. Cells were treated with 50 and 100 µM of inhibitor VII, and protein was harvested in RIPA buffer to analyze active b-catenin expression. <b>B</b>. Chromatin immunoprecipitation (ChIP) for β-catenin at the promoter of FZD7 gene in T-47D cells, followed by qPCR with primers that target the domains through -7 kb region of FZD7 promoter/enhancer (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098861#pone-0098861-t001" target="_blank">Table1</a>). <b>C</b>. ChIP analysis of c-Jun enrichment at the FZD7 promoter/enhancer. T-47D cells treated with inhibitor VII or vehicle for 24 hrs and then ChIP performed as previously described. <b>D</b>. ChIP analysis of Dvl1 enrichment at the FZD7 promoter/enhancer. <b>E, F and G</b>. T-47D cells were treated with 100 µM of inhibitor VII for 18 hrs. Cells were then harvested for ChIP-qPCR analysis. Data plotted are from average or 4 experiments +/− S.E.M. An * indicate a p-value <0.05 versus IgG sample, A ** indicates a p-value <0.05 versus DMSO treated samples. Samples were normalized to non-immune IgG, (n = 4).</p
Abstract: SIRT1, an NAD+-dependent deacetylase, has been described in the literature as a major p... more Abstract: SIRT1, an NAD+-dependent deacetylase, has been described in the literature as a major player in the regulation of cellular stress responses. Its expression has been shown to be altered in cancer cells, and it targets both histone and non-histone proteins for deacetylation and thereby alters metabolic programs in response to diverse physiological stress. Interestingly, many of the metabolic pathways that are influenced by SIRT1 are also altered in tumor development. Not only does SIRT1 have the potential to regulate oncogenic factors, it also orchestrates many aspects of metabolism and lipid regulation and recent reports are beginning to connect these areas. SIRT1 influences pathways that provide an alternative means of deriving energy (such as fatty acid oxidation and gluconeogenesis) when a cell encounters nutritive stress, and can therefore lead to altered lipid metabolism in various pathophysiological contexts. This review helps to show the various connections between S...
Evidence of immunogenic cell death as a predictor of response to cancer therapy has increased int... more Evidence of immunogenic cell death as a predictor of response to cancer therapy has increased interest in the high molecular group box 1 protein (HMGB1). HMGB1 is a nuclear protein associated with chromatin organization and DNA damage repair. HMGB1 is also a damage-associated molecular pattern (DAMP) protein and promotes proinflammatory signaling in a paracrine and autocrine manner. Extracellular HMGB1 can promote activation of NF-kB and is associated with several chronic inflammatory and autoimmune diseases, including sepsis, rheumatoid arthritis, atherosclerosis, chronic kidney disease, systemic lupus erythematosus (SLE), as well as cancer. In this review, we describe studies that demonstrate the use of deacetylase inhibitors and HMGB1 inhibitors to alter the expression and localization of HMGB1 in cancer cells, with a focus on lung cancer. The drugs described herein are well established and frequently used in human and small mammal studies. The main objective of this review is to...
There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, vario... more There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, variously sized surveillance sites across geographic areas. We developed a novel indexing method, Melvin’s Index, that provides a normalized and standardized metric of wastewater pathogen load for qPCR assays that is resilient to surveillance site variation. To demonstrate the utility of Melvin’s Index, we used qRT-PCR to measure SARS-CoV-2 genomic RNA levels in influent wastewater from 19 municipal wastewater treatment facilities (WWTF’s) of varying sizes and served populations across the state of Minnesota during the Summer of 2020. SARS-CoV-2 RNA was detected at each WWTF during the 20-week sampling period at a mean concentration of 8.5 × 104 genome copies/L (range 3.2 × 102–1.2 × 109 genome copies/L). Lag analysis of trends in Melvin’s Index values and clinical COVID-19 cases showed that increases in indexed wastewater SARS-CoV-2 levels precede new clinical cases by 15–17 days at the stat...
According to the National Institutes of Health, clear cell renal cell carcinoma (ccRCC) is the mo... more According to the National Institutes of Health, clear cell renal cell carcinoma (ccRCC) is the most common type of Renal Cell Carcinoma (RCC), making up approximately 75% of total renal carcinoma cases. Clear cell Renal Cell Carcinoma is characterized by a significant accumulation of lipids in the cytoplasm, which allows light from microscopes to pass through giving them a “clear” phenotype. Many of these lipids are in the form of fatty acids, both free and incorporated into lipid droplets. RCC is typically associated with a poor prognosis due to the lack of specific symptoms. Some symptoms include blood in urine, fever, lump on the side, weight loss, fatigue, to name a few; all of which can be associated with non-specific, non-cancerous, health conditions that contribute to difficult diagnosis. Treatment of RCC has typically been centered around radical nephrectomy as the standard of care, but due to the potentially small size of lesions and the possibility of causing surgically in...
Methyl-CpG-binding protein-2 (MeCP2) regulates gene expression by recruiting SWI/SNF DNA helicase... more Methyl-CpG-binding protein-2 (MeCP2) regulates gene expression by recruiting SWI/SNF DNA helicase/ATPase (ATRX) and Histone Deacetylase-1 (HDAC1) to methylated gene regions and modulates heterochromatin association by interacting with Heterochromatin protein-1. As MeCP2 contributes to tumor suppressor gene silencing and its mutation causes Rett Syndrome, we investigated how novel post-translational-modification contributes to its function. Herein we report that upon pharmacological inhibition of SIRT1 in RKO colon and MCF-7 breast cancer cells, endogenous MeCP2 is acetylated at sites critical for binding to DNA and transcriptional regulators. We created an acetylation mimetic mutation in MeCP2 and found it to possess decreased binding to ATRX and HDAC1. Conditions inducing MeCP2 acetylation do not alter its promoter occupancy at a subset of target genes analyzed, but do cause decreased binding to ATRX and HDAC1. We also report here that a specific inhibitor of SIRT1, IV, can be used...
Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early st... more Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 ...
<p><b>A</b>. Semi-quantitative RT-PCR analysis of frizzled receptors 1–9 to det... more <p><b>A</b>. Semi-quantitative RT-PCR analysis of frizzled receptors 1–9 to determine expression pattern in MDA-MB-231 cells and T-47D cells. Samples were resolved on 1% low melting agarose gel.</p
The COVID-19 pandemic has exacerbated the disparities in healthcare delivery in the US. Many comm... more The COVID-19 pandemic has exacerbated the disparities in healthcare delivery in the US. Many communities had, and continue to have, limited access to COVID-19 testing, making it difficult to track the spread and impact of COVID-19 in early days of the outbreak. To address this issue we monitored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA at the population-level using municipal wastewater influent from 19 cities across the state of Minnesota during the COVID-19 outbreak in Summer 2020. Viral RNA was detected in wastewater continually for 20-weeks for cities ranging in populations from 500 to >1, 000, 000. Using a novel indexing method, we were able to compare the relative levels of SARS-CoV-2 RNA for each city during this sampling period. Our data showed that viral RNA trends appeared to precede clinically confirmed cases across the state by several days. Lag analysis of statewide trends confirmed that wastewater SARS-CoV-2 RNA levels preceded new clinical ca...
The Dishevelled gene was first identified in Drosophila mutants with disoriented hair and bristle... more The Dishevelled gene was first identified in Drosophila mutants with disoriented hair and bristle polarity [1-3]. The Dsh gene (Dsh/Dvl, in Drosophila and vertebrates respectively) gained popularity when it was discovered that it plays a key role in segment polarity during early embryonic development in Drosophila [4]. Subsequently, the vertebrate homolog of Dishevelled genes were identified in Xenopus (Xdsh), mice (Dvl1, Dvl2, Dvl3), and in humans (DVL1, DVL2, DVL3) [5-10]. Dishevelled functions as a principal component of Wnt signaling pathway and governs several cellular processes including cell proliferation, survival, migration, differentiation, polarity and stem cell renewal. This review will revisit seminal discoveries and also summarize recent advances in characterizing the role of Dishevelled in both normal and pathophysiological settings.
Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early st... more Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent tran-scriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibi-tors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis.
<p><b>A</b>. Cells were treated with 50 and 100 µM of inhibitor VII, and protei... more <p><b>A</b>. Cells were treated with 50 and 100 µM of inhibitor VII, and protein was harvested in RIPA buffer to analyze active b-catenin expression. <b>B</b>. Chromatin immunoprecipitation (ChIP) for β-catenin at the promoter of FZD7 gene in T-47D cells, followed by qPCR with primers that target the domains through -7 kb region of FZD7 promoter/enhancer (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098861#pone-0098861-t001" target="_blank">Table1</a>). <b>C</b>. ChIP analysis of c-Jun enrichment at the FZD7 promoter/enhancer. T-47D cells treated with inhibitor VII or vehicle for 24 hrs and then ChIP performed as previously described. <b>D</b>. ChIP analysis of Dvl1 enrichment at the FZD7 promoter/enhancer. <b>E, F and G</b>. T-47D cells were treated with 100 µM of inhibitor VII for 18 hrs. Cells were then harvested for ChIP-qPCR analysis. Data plotted are from average or 4 experiments +/− S.E.M. An * indicate a p-value <0.05 versus IgG sample, A ** indicates a p-value <0.05 versus DMSO treated samples. Samples were normalized to non-immune IgG, (n = 4).</p
Abstract: SIRT1, an NAD+-dependent deacetylase, has been described in the literature as a major p... more Abstract: SIRT1, an NAD+-dependent deacetylase, has been described in the literature as a major player in the regulation of cellular stress responses. Its expression has been shown to be altered in cancer cells, and it targets both histone and non-histone proteins for deacetylation and thereby alters metabolic programs in response to diverse physiological stress. Interestingly, many of the metabolic pathways that are influenced by SIRT1 are also altered in tumor development. Not only does SIRT1 have the potential to regulate oncogenic factors, it also orchestrates many aspects of metabolism and lipid regulation and recent reports are beginning to connect these areas. SIRT1 influences pathways that provide an alternative means of deriving energy (such as fatty acid oxidation and gluconeogenesis) when a cell encounters nutritive stress, and can therefore lead to altered lipid metabolism in various pathophysiological contexts. This review helps to show the various connections between S...
Evidence of immunogenic cell death as a predictor of response to cancer therapy has increased int... more Evidence of immunogenic cell death as a predictor of response to cancer therapy has increased interest in the high molecular group box 1 protein (HMGB1). HMGB1 is a nuclear protein associated with chromatin organization and DNA damage repair. HMGB1 is also a damage-associated molecular pattern (DAMP) protein and promotes proinflammatory signaling in a paracrine and autocrine manner. Extracellular HMGB1 can promote activation of NF-kB and is associated with several chronic inflammatory and autoimmune diseases, including sepsis, rheumatoid arthritis, atherosclerosis, chronic kidney disease, systemic lupus erythematosus (SLE), as well as cancer. In this review, we describe studies that demonstrate the use of deacetylase inhibitors and HMGB1 inhibitors to alter the expression and localization of HMGB1 in cancer cells, with a focus on lung cancer. The drugs described herein are well established and frequently used in human and small mammal studies. The main objective of this review is to...
There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, vario... more There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, variously sized surveillance sites across geographic areas. We developed a novel indexing method, Melvin’s Index, that provides a normalized and standardized metric of wastewater pathogen load for qPCR assays that is resilient to surveillance site variation. To demonstrate the utility of Melvin’s Index, we used qRT-PCR to measure SARS-CoV-2 genomic RNA levels in influent wastewater from 19 municipal wastewater treatment facilities (WWTF’s) of varying sizes and served populations across the state of Minnesota during the Summer of 2020. SARS-CoV-2 RNA was detected at each WWTF during the 20-week sampling period at a mean concentration of 8.5 × 104 genome copies/L (range 3.2 × 102–1.2 × 109 genome copies/L). Lag analysis of trends in Melvin’s Index values and clinical COVID-19 cases showed that increases in indexed wastewater SARS-CoV-2 levels precede new clinical cases by 15–17 days at the stat...
According to the National Institutes of Health, clear cell renal cell carcinoma (ccRCC) is the mo... more According to the National Institutes of Health, clear cell renal cell carcinoma (ccRCC) is the most common type of Renal Cell Carcinoma (RCC), making up approximately 75% of total renal carcinoma cases. Clear cell Renal Cell Carcinoma is characterized by a significant accumulation of lipids in the cytoplasm, which allows light from microscopes to pass through giving them a “clear” phenotype. Many of these lipids are in the form of fatty acids, both free and incorporated into lipid droplets. RCC is typically associated with a poor prognosis due to the lack of specific symptoms. Some symptoms include blood in urine, fever, lump on the side, weight loss, fatigue, to name a few; all of which can be associated with non-specific, non-cancerous, health conditions that contribute to difficult diagnosis. Treatment of RCC has typically been centered around radical nephrectomy as the standard of care, but due to the potentially small size of lesions and the possibility of causing surgically in...
Methyl-CpG-binding protein-2 (MeCP2) regulates gene expression by recruiting SWI/SNF DNA helicase... more Methyl-CpG-binding protein-2 (MeCP2) regulates gene expression by recruiting SWI/SNF DNA helicase/ATPase (ATRX) and Histone Deacetylase-1 (HDAC1) to methylated gene regions and modulates heterochromatin association by interacting with Heterochromatin protein-1. As MeCP2 contributes to tumor suppressor gene silencing and its mutation causes Rett Syndrome, we investigated how novel post-translational-modification contributes to its function. Herein we report that upon pharmacological inhibition of SIRT1 in RKO colon and MCF-7 breast cancer cells, endogenous MeCP2 is acetylated at sites critical for binding to DNA and transcriptional regulators. We created an acetylation mimetic mutation in MeCP2 and found it to possess decreased binding to ATRX and HDAC1. Conditions inducing MeCP2 acetylation do not alter its promoter occupancy at a subset of target genes analyzed, but do cause decreased binding to ATRX and HDAC1. We also report here that a specific inhibitor of SIRT1, IV, can be used...
Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early st... more Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent transcriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 ...
<p><b>A</b>. Semi-quantitative RT-PCR analysis of frizzled receptors 1–9 to det... more <p><b>A</b>. Semi-quantitative RT-PCR analysis of frizzled receptors 1–9 to determine expression pattern in MDA-MB-231 cells and T-47D cells. Samples were resolved on 1% low melting agarose gel.</p
The COVID-19 pandemic has exacerbated the disparities in healthcare delivery in the US. Many comm... more The COVID-19 pandemic has exacerbated the disparities in healthcare delivery in the US. Many communities had, and continue to have, limited access to COVID-19 testing, making it difficult to track the spread and impact of COVID-19 in early days of the outbreak. To address this issue we monitored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA at the population-level using municipal wastewater influent from 19 cities across the state of Minnesota during the COVID-19 outbreak in Summer 2020. Viral RNA was detected in wastewater continually for 20-weeks for cities ranging in populations from 500 to >1, 000, 000. Using a novel indexing method, we were able to compare the relative levels of SARS-CoV-2 RNA for each city during this sampling period. Our data showed that viral RNA trends appeared to precede clinically confirmed cases across the state by several days. Lag analysis of statewide trends confirmed that wastewater SARS-CoV-2 RNA levels preceded new clinical ca...
The Dishevelled gene was first identified in Drosophila mutants with disoriented hair and bristle... more The Dishevelled gene was first identified in Drosophila mutants with disoriented hair and bristle polarity [1-3]. The Dsh gene (Dsh/Dvl, in Drosophila and vertebrates respectively) gained popularity when it was discovered that it plays a key role in segment polarity during early embryonic development in Drosophila [4]. Subsequently, the vertebrate homolog of Dishevelled genes were identified in Xenopus (Xdsh), mice (Dvl1, Dvl2, Dvl3), and in humans (DVL1, DVL2, DVL3) [5-10]. Dishevelled functions as a principal component of Wnt signaling pathway and governs several cellular processes including cell proliferation, survival, migration, differentiation, polarity and stem cell renewal. This review will revisit seminal discoveries and also summarize recent advances in characterizing the role of Dishevelled in both normal and pathophysiological settings.
Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early st... more Quiescent CD4+ T cells restrict human immunodeficiency virus type 1 (HIV-1) infection at early steps of virus replication. Low levels of both deoxyribonucleotide triphosphates (dNTPs) and the biosynthetic enzymes required for their de novo synthesis provide one barrier to infection. CD4+ T cell activation induces metabolic reprogramming that reverses this block and facilitates HIV-1 replication. Here, we show that phospholipase D1 (PLD1) links T cell activation signals to increased HIV-1 permissivity by triggering a c-Myc-dependent tran-scriptional program that coordinates glucose uptake and nucleotide biosynthesis. Decreasing PLD1 activity pharmacologically or by RNA interference diminished c-Myc-dependent expression during T cell activation at the RNA and protein levels. PLD1 inhibition of HIV-1 infection was partially rescued by adding exogenous deoxyribonucleosides that bypass the need for de novo dNTP synthesis. Moreover, the data indicate that low dNTP levels that impact HIV-1 restriction involve decreased synthesis, and not only increased catabolism of these nucleotides. These findings uncover a unique mechanism of action for PLD1 inhibi-tors and support their further development as part of a therapeutic combination for HIV-1 and other viral infections dependent on host nucleotide biosynthesis.
Uploads
Papers by Glenn E Simmons Jr