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Michelle Lacey

Tulane University, Mathematics, Faculty Member

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Jayanth Krishnan

Broad Institute

St Jude Childrens research Hospital

Ivanela Kondova

University of Oslo

The Hebrew University of Jerusalem

Universidad de Santander

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Papers by Michelle Lacey

Promoter-adjacent DNA hypermethylation can downmodulate gene expression:TBX15in the muscle lineage

TBX15, which encodes a differentiation-related transcription factor, displays promoter-adjacent D... more TBX15, which encodes a differentiation-related transcription factor, displays promoter-adjacent DNA hypermethylation in myoblasts and skeletal muscle (psoas) that is absent from non-expressing cells in other lineages. By whole-genome bisulfite sequencing (WGBS) and enzymatic methyl-seq (EM-seq), these hypermethylated regions were found to border both sides of a constitutively unmethylated promoter. To understand the functionality of this DNA hypermethylation, we cloned the differentially methylated sequences (DMRs) in CpG-free reporter vectors and tested them for promoter or enhancer activity upon transient transfection. These cloned regions exhibited strong promoter activity and, when placed upstream of a weak promoter, strong enhancer activity specifically in myoblast host cells.In vitroCpG methylation targeted to the DMR sequences in the plasmids resulted in 86 - 100% loss of promoter or enhancer activity, depending on the insert sequence. These results as well as chromatin epige...

Additional file 1 of The SEQC2 epigenomics quality control (EpiQC) study

Additional file 1. contains the supplementary figures (Supplementary Figure 1–14).

Author Correction: The SEQC2 epigenomics quality control (EpiQC) study

Genome Biology, 2021

Statistical Modeling of Methylation Patterns in Ovarian Carcinomas

Changes in cytosine methylation at CpG nucleotides are observed in many cancers, but the biologic... more Changes in cytosine methylation at CpG nucleotides are observed in many cancers, but the biological mechanisms responsible for these changes are not yet fully understood. Previously developed stochastic models for cancer-related methylation change have either treated CpG sites independently or employed a context dependent approach to adjust model parameters according to regional methylation levels. However, our analyses of double-stranded methylation patterns in 0.2 kb regions of the tandem repeats Sat2 and NBL2 have detected small clusters of identically methylated sites in close proximity that could not be explained by random variation. These findings suggest a high degree of site-to-site dependence, and we have developed a neighboring sites model for methylation change as an alternative approach. We have compared the independent sites, context dependent, and neighboring sites models in their ability to generate simulated sequences statistically similar to our Sat2 and NBL2 carcin...

Multivariate analysis of food consumption profiles in crisis settings

PLOS ONE

Preventing malnutrition is one of the primary objectives of many humanitarian agencies, and house... more Preventing malnutrition is one of the primary objectives of many humanitarian agencies, and household surveys are regularly employed to monitor food insecurity caused by political, economic, or environmental crises. Consumption frequencies for standard food groups are often collected to characterize the depth of food insecurity in a community and measure the impact of food assistance programs, producing a vector of bounded, correlated counts for each household. While aggregate indicators are typically used to summarize these results with a single statistic, they can be difficult to interpret and provide insufficient detail to judge the effectiveness of assistance programs. To address these limitations, we have developed a multivariate modeling framework for consumption frequency data. We introduce methods to update baseline models for the analysis of the smaller and more variable surveys typically collected in crisis settings, and we present an application of our approach to nationa...

The SEQC2 Epigenomics Quality Control (EpiQC) Study: Comprehensive Characterization of Epigenetic Methods, Reproducibility, and Quantification

Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmen... more Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and a global resource for epigenetics research from the FDA’s Epigenomics Quality Control (EpiQC) Group. The study design leverages seven human cell lines that are designated as reference materials and publicly available from the National Institute of Standards and Technology (NIST) and Genome in a Bottle (GIAB) consortium. These samples were subject to a variety of genome-wide methylation interrogation approaches across six independent laboratories, with a primary focus was on 5-methyl...

Plasma flow distal to tourniquet placement provides a physiological mechanism for tissue salvage

PLOS ONE, 2020

Military literature has demonstrated the utility and safety of tourniquets in preventing mortalit... more Military literature has demonstrated the utility and safety of tourniquets in preventing mortality for some time, paving the way for increased use of tourniquets in civilian settings, including perioperatively to provide a bloodless surgical field. However, tourniquet use is not without risk and the subsequent effects of tissue ischemia can impede downstream rehabilitative efforts to regenerate and salvage nerve, muscle, tissue and bone in the limb. Limb ischemia studies in both the mouse and pig models have indicated not only that there is residual flow past the tourniquet by means of microcirculation, but also that recovery from tissue ischemia is dependent upon this microcirculation. Here we expand upon these previous studies using portable Near-Infrared Imaging to quantify residual plasma flow distal to the tourniquet in mice, pigs, and humans and leverage this flow to show that plasma can be supersaturated with oxygen to reduce intracellular hypoxia and promote tissue salvage f...

Epigenetics of a tandem DNA repeat: chromatin

DNaseI sensitivity and opposite methylation changes in cancers

Evaluating Differences in Elastic Modulus of Regenerated and Uninjured Mouse Digit Bone through microCT Density-Elasticity Calculation and Nanoindentation Testing

Bone is an essential, healing structure in vertebrates that ensures daily function. However, the ... more Bone is an essential, healing structure in vertebrates that ensures daily function. However, the regenerative capacity of bone declines with age, compromising quality of life in the elderly and increasing cost of care. Here, for the first time, the elasticity of regenerated bone in a mouse digit amputation model is evaluated in order to better investigate biomechanics of skeletal regeneration. Amputation of the distal one third of the digit (third phalangeal element – P3) results in de novo regeneration of the digit, where analyzing the structural quality of this regenerated bone is a challenging task due to its small scale and triangular shape. To date, the evaluation of structural quality of the P3 bone has primarily focused on mineral density and bone architecture. This work describes an image-processing based method for assessment of elasticity in the whole P3 bone by using microcomputed tomography-generated mineral density data to calculate spatially discrete elastic modulus va...

Age-Dependent Changes in Bone Architecture, Patterning, and Biomechanics During Skeletal Regeneration

Frontiers in Cell and Developmental Biology, 2021

Mouse digit amputation provides a useful model of bone growth after injury, in that the injury pr... more Mouse digit amputation provides a useful model of bone growth after injury, in that the injury promotes intramembranous bone formation in an adult animal. The digit tip is composed of skin, nerves, blood vessels, bones, and tendons, all of which regenerate after digit tip amputation, making it a powerful model for multi-tissue regeneration. Bone integrity relies upon a balanced remodeling between bone resorption and formation, which, when disrupted, results in changes to bone architecture and biomechanics, particularly during aging. In this study, we used recently developed techniques to evaluate bone patterning differences between young and aged regenerated bone. This analysis suggests that aged mice have altered trabecular spacing and patterning and increased mineral density of the regenerated bone. To further characterize the biomechanics of regenerated bone, we measured elasticity using a micro-computed tomography image-processing method combined with nanoindentation. This analy...

Epigenetics and expression of key genes associated with cardiac fibrosis: NLRP3, MMP2, MMP9, CCN2/CTGF and AGT

Epigenomics, 2021

Aims: Excessive inflammatory signaling and pathological remodeling of the extracellular matrix dr... more Aims: Excessive inflammatory signaling and pathological remodeling of the extracellular matrix drive cardiac fibrosis and require changes in gene expression. Materials and methods: Using bioinformatics, both tissue-specific expression profiles and epigenomic profiles of some genes critical for cardiac fibrosis were examined, namely, NLRP3, MMP2, MMP9, CCN2/CTGF, AGT (encodes angiotensin II precursors) and hsa-mir-223 (post-transcriptionally regulates NLRP3). Results: In monocytes, neutrophils, fibroblasts, venous cells, liver and brain, enhancers or super-enhancers were found that correlate with high expression of these genes. One enhancer extended into a silent gene neighbor. These enhancers harbored tissue-specific foci of DNA hypomethylation, open chromatin and transcription factor binding. Conclusions: This study identified previously undescribed enhancers containing hypomethylated transcription factor binding subregions that are predicted to regulate expression of these cardiac...

Tissue-specific epigenetics of atherosclerosis-related ANGPT and ANGPTL genes

Epigenomics, 2019

Aim: To understand tissue-specific regulation of angiopoietin/angiopoietin-like (ANGPT/ANGPTL) ge... more Aim: To understand tissue-specific regulation of angiopoietin/angiopoietin-like (ANGPT/ANGPTL) genes (especially the five genes embedded in introns of host genes) and their association with atherosclerosis. Methods: Transcription and epigenomic databases from various normal tissues were examined in the vicinity of ANGPT1, ANGPT2, ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4 and ANGPTL8. Results: We identified tissue-specific enhancer chromatin regions that are likely to regulate transcription of ANGPT/ANGPTL genes and were intragenic, intergenic or host gene-linked. In addition, we found atherosclerosis-linked differentially methylated regions associated with ANGPT2 and with sequences encoding miR-145, a microRNA that targets ANGPT2 mRNA in cancers. Conclusion: Our findings implicate enhancers as major contributors to tissue-specific expression of ANGPT/ANGPTL genes, which play critical roles in angiogenesis, atherosclerosis, cancer, and inflammatory and metabolic diseases.

Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription

Epigenetics, 2018

DNA methylation can affect tissue-specific gene transcription in ways that are difficult to disce... more DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development.

Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers

Atherosclerosis, 2018

Background and aims: Atherosclerosis is a widespread and complicated disease involving phenotypic... more Background and aims: Atherosclerosis is a widespread and complicated disease involving phenotypic modulation and transdifferentiation of vascular smooth muscle cells (SMCs), the predominant cells in aorta, as well as changes in endothelial cells and infiltrating monocytes. Alterations in DNA methylation are likely to play central roles in these phenotypic changes, just as they do in normal differentiation and cancer. Methods: We examined genome-wide DNA methylation changes in atherosclerotic aorta using more stringent criteria for differentially methylated regions (DMRs) than in previous studies and compared these DMRs to tissue-specific epigenetic features. Results: We found that disease-linked hypermethylated DMRs account for 85% of the total atherosclerosis-associated DMRs and often overlap aorta-associated enhancer chromatin. These hypermethylated DMRs were associated with functionally different sets of genes compared to atherosclerosis-linked hypomethylated DMRs. The extent and nature of the DMRs could not be explained as direct effects of monocyte/macrophage infiltration. Among the known atherosclerosis-and contractile SMC-related genes that exhibited hypermethylated DMRs at aorta enhancer chromatin were ACTA2 (aorta α2 smooth muscle actin), ELN (elastin), MYOCD (myocardin), C9orf3 (miR-23b and miR-27b host gene), and MYH11 (smooth muscle myosin). Our analyses also suggest a role in atherosclerosis for developmental transcription factor genes having little or no previous association with atherosclerosis, such as NR2F2 (COUP-TFII) and TBX18. Conclusions: We provide evidence for atherosclerosis-linked DNA methylation changes in aorta SMCs that might help minimize or reverse the standard contractile character of many of these cells by down-modulating aorta SMC-related enhancers, thereby facilitating pro-atherosclerotic phenotypic changes in many SMCs.

304 Toxoplasma Gondii Expresses Two Distinct Mitogen-Activated Protein Kinase Genes That Are Differentially Regulated

Journal of Investigative Medicine, 2005

Purpose of Study Toxoplasma gondii causes significant morbidity and mortality in immunocompromise... more Purpose of Study Toxoplasma gondii causes significant morbidity and mortality in immunocompromised patients such as those co-infected with HIV. We have cloned and sequenced two mitogen-activated protein kinase (MAPK) genes which encode novel MAP kinases that are predicted to have distinctly different properties. These predictions are based on examining differences in their primary amino acid sequences and differential gene expression in context with available functional data. Both MAPKs are druggable targets, and we predict that reducing or abolishing their expression in T. gondii will prove to be detrimental to the parasite's ability to proliferate and/or differentiate. Current experiments are underway in this regard. Methods The T. gondii strain Me-49 genome has been completely sequenced, but individual genes from this avirulent strain are still in the process of being annotated. Using information available at this genomic database (www.toxodb.org), along with our nucleotide sequencing data obtained from the virulent RH strain (acquired using an ABI-7700 automated sequencer), we have identified two MAPK genes, which we have designated tgMAPK-1 and tgMAPK-2. Expression, purification, and functional assays performed on TgMAPK-1 have been previously described (Brumlik et al, 2004). Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out in order to examine the differential expression of both these genes. Results Based on the deduced amino acid sequences encoded by these genes (which share only 33% identity), each MAP kinase has unique characteristics. RT-PCR profiles suggest that although both genes encode stress-response MAPKs, tgMAPK-1 appears to be expressed in the bradyzoite (latent) form while tgMAPK-2 seems to be more constitutively expressed. ClustalW alignment of these MAPKs has been performed, along with a phylogenetic analysis. Strain variations between both MAPKs are also discussed. Conclusions Pyridinylimidazoles designed to block the human stress-response p38 MAPK also inhibit TgMAPK-1 in vitro, block T. gondii replication, and protect T. gondii-infected mice. Both TgMAPK-1 and TgMAPK-2 are druggable targets, although it remains to be seen the effect that pyridinylimidazoles have on TgMAPK-2. Understanding the relationship between TgMAPK-1 and TgMAPK-2 and their ability to mediate stress-response and/or stage-differentiation will be important to advancing treatment strategies for T. gondii-infected patients.

Assessing needs and assets for building a regional network infrastructure to reduce cancer related health disparities

Evaluation and Program Planning, 2014

Significant cancer health disparities exist in the United States and Puerto Rico. While numerous ... more

The SEQC2 epigenomics quality control (EpiQC) study

Genome Biology

Background Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of... more Background Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA’s Epigenomics Quality Control Group. Results Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing ...

$Table 2 Summary statistics of mapping and library efficiency per WGBS protocol. Percent CpG capture calculated with call sets normalized to 20x coverage. The total genome-wide CpGs unde consideration were those that could be mapped to uniquely, excluding any CpGs that fall within unresolvable regions methylation call sets for every replicate to a given mean coverage value. Downsampling can be done by either filtering the number of reads in an alignment (BAM files), or by randomly removing a fraction of observed cytosines and observed thymines per CpG within methylation call sets (bedGraph files). Because downsampling at the alignment level can be slow and demanding in terms of disk space and compute time, we set out to evaluate if the signal from downsampling cytosines within bedGraph files recapitu- lated downsampling aligned reads within BAM files. The two approaches yielded simi- lar results in number of CpG sites detected, distribution of read counts, and methylation calls. bedGraph downsampling had the added benefit that the targeted average CpG coverage was more accurately estimated than when downsampling BAMs (Additional file 1: Figure $5).$

Epigenetics of skeletal muscle-associated genes in the ASB, LRRC, TMEM, and OSBPL gene families

Much remains to be discovered about the intersection of tissue-specific transcription control and... more Much remains to be discovered about the intersection of tissue-specific transcription control and the epigenetics of skeletal muscle (SkM), a very complex and dynamic organ. From four gene families, ASB, LRRC, TMEM, and OSBPL, we chose 21 genes that are preferentially expressed in human SkM relative to 52 other tissue types and analyzed relationships between their tissue-specific epigenetics and expression. We also compared their genetics, proteomics, and descriptions in the literature. For this study, we identified genes with little or no previous descriptions of SkM functionality (ASB4, ASB8, ASB10, ASB12, ASB16, LRRC14B, LRRC20, LRRC30, TMEM52, TMEM233, OSBPL6/ORP6, and OSBPL11/ORP11) and included genes whose SkM functions had been previously addressed (ASB2, ASB5, ASB11, ASB15, LRRC2, LRRC38, LRRC39, TMEM38A/TRIC-A, and TMEM38B/TRIC-B). Among the transcription-related SkM epigenetic features that we identified were super-enhancers, promoter DNA hypomethylation, lengthening of co...

Sirtuin 3 deficiency does not impede digit regeneration in mice

Scientific Reports

The mitochondrial deacetylase sirtuin 3 (SIRT3) is thought to be one of the main contributors to ... more The mitochondrial deacetylase sirtuin 3 (SIRT3) is thought to be one of the main contributors to metabolic flexibility–promoting mitochondrial energy production and maintaining homeostasis. In bone, metabolic profiles are tightly regulated and the loss of SIRT3 has deleterious effects on bone volume in vivo and on osteoblast differentiation in vitro. Despite the prominent role of this protein in bone stem cell proliferation, metabolic activity, and differentiation, the importance of SIRT3 for regeneration after bone injury has never been reported. We show here, using the mouse digit amputation model, that SIRT3 deficiency has no impact on the regenerative capacity and architecture of bone and soft tissue. Regeneration occurs in SIRT3 deficient mice in spite of the reduced oxidative metabolic profile of the periosteal cells. These data suggest that bone regeneration, in contrast to homeostatic bone turnover, is not reliant upon active SIRT3, and our results highlight the need to exam...

Expanding Research Capacity in Sub-Saharan Africa Through Informatics, Bioinformatics, and Data Science Training Programs in Mali

Frontiers in Genetics

Shaffer et al. Bioinformatics Training in Mali incremental processes are necessary for engaging h... more Shaffer et al. Bioinformatics Training in Mali incremental processes are necessary for engaging historically underserved countries in bioinformatics and data science research. The multi-tiered enhancement approach laid out here provides a platform for generating bioinformatics and data science technicians, teachers, researchers, and program managers. Increased literature on bioinformatics and data science training approaches and progress is needed to provide a framework for establishing benchmarks on the topics.

Promoter-adjacent DNA hypermethylation can downmodulate gene expression:TBX15in the muscle lineage

TBX15, which encodes a differentiation-related transcription factor, displays promoter-adjacent D... more TBX15, which encodes a differentiation-related transcription factor, displays promoter-adjacent DNA hypermethylation in myoblasts and skeletal muscle (psoas) that is absent from non-expressing cells in other lineages. By whole-genome bisulfite sequencing (WGBS) and enzymatic methyl-seq (EM-seq), these hypermethylated regions were found to border both sides of a constitutively unmethylated promoter. To understand the functionality of this DNA hypermethylation, we cloned the differentially methylated sequences (DMRs) in CpG-free reporter vectors and tested them for promoter or enhancer activity upon transient transfection. These cloned regions exhibited strong promoter activity and, when placed upstream of a weak promoter, strong enhancer activity specifically in myoblast host cells.In vitroCpG methylation targeted to the DMR sequences in the plasmids resulted in 86 - 100% loss of promoter or enhancer activity, depending on the insert sequence. These results as well as chromatin epige...

Additional file 1 of The SEQC2 epigenomics quality control (EpiQC) study

Additional file 1. contains the supplementary figures (Supplementary Figure 1–14).

Author Correction: The SEQC2 epigenomics quality control (EpiQC) study

Genome Biology, 2021

Statistical Modeling of Methylation Patterns in Ovarian Carcinomas

Changes in cytosine methylation at CpG nucleotides are observed in many cancers, but the biologic... more Changes in cytosine methylation at CpG nucleotides are observed in many cancers, but the biological mechanisms responsible for these changes are not yet fully understood. Previously developed stochastic models for cancer-related methylation change have either treated CpG sites independently or employed a context dependent approach to adjust model parameters according to regional methylation levels. However, our analyses of double-stranded methylation patterns in 0.2 kb regions of the tandem repeats Sat2 and NBL2 have detected small clusters of identically methylated sites in close proximity that could not be explained by random variation. These findings suggest a high degree of site-to-site dependence, and we have developed a neighboring sites model for methylation change as an alternative approach. We have compared the independent sites, context dependent, and neighboring sites models in their ability to generate simulated sequences statistically similar to our Sat2 and NBL2 carcin...

Multivariate analysis of food consumption profiles in crisis settings

PLOS ONE

Preventing malnutrition is one of the primary objectives of many humanitarian agencies, and house... more Preventing malnutrition is one of the primary objectives of many humanitarian agencies, and household surveys are regularly employed to monitor food insecurity caused by political, economic, or environmental crises. Consumption frequencies for standard food groups are often collected to characterize the depth of food insecurity in a community and measure the impact of food assistance programs, producing a vector of bounded, correlated counts for each household. While aggregate indicators are typically used to summarize these results with a single statistic, they can be difficult to interpret and provide insufficient detail to judge the effectiveness of assistance programs. To address these limitations, we have developed a multivariate modeling framework for consumption frequency data. We introduce methods to update baseline models for the analysis of the smaller and more variable surveys typically collected in crisis settings, and we present an application of our approach to nationa...

The SEQC2 Epigenomics Quality Control (EpiQC) Study: Comprehensive Characterization of Epigenetic Methods, Reproducibility, and Quantification

Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmen... more Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and a global resource for epigenetics research from the FDA’s Epigenomics Quality Control (EpiQC) Group. The study design leverages seven human cell lines that are designated as reference materials and publicly available from the National Institute of Standards and Technology (NIST) and Genome in a Bottle (GIAB) consortium. These samples were subject to a variety of genome-wide methylation interrogation approaches across six independent laboratories, with a primary focus was on 5-methyl...

Plasma flow distal to tourniquet placement provides a physiological mechanism for tissue salvage

PLOS ONE, 2020

Military literature has demonstrated the utility and safety of tourniquets in preventing mortalit... more Military literature has demonstrated the utility and safety of tourniquets in preventing mortality for some time, paving the way for increased use of tourniquets in civilian settings, including perioperatively to provide a bloodless surgical field. However, tourniquet use is not without risk and the subsequent effects of tissue ischemia can impede downstream rehabilitative efforts to regenerate and salvage nerve, muscle, tissue and bone in the limb. Limb ischemia studies in both the mouse and pig models have indicated not only that there is residual flow past the tourniquet by means of microcirculation, but also that recovery from tissue ischemia is dependent upon this microcirculation. Here we expand upon these previous studies using portable Near-Infrared Imaging to quantify residual plasma flow distal to the tourniquet in mice, pigs, and humans and leverage this flow to show that plasma can be supersaturated with oxygen to reduce intracellular hypoxia and promote tissue salvage f...

Epigenetics of a tandem DNA repeat: chromatin

DNaseI sensitivity and opposite methylation changes in cancers

Evaluating Differences in Elastic Modulus of Regenerated and Uninjured Mouse Digit Bone through microCT Density-Elasticity Calculation and Nanoindentation Testing

Bone is an essential, healing structure in vertebrates that ensures daily function. However, the ... more Bone is an essential, healing structure in vertebrates that ensures daily function. However, the regenerative capacity of bone declines with age, compromising quality of life in the elderly and increasing cost of care. Here, for the first time, the elasticity of regenerated bone in a mouse digit amputation model is evaluated in order to better investigate biomechanics of skeletal regeneration. Amputation of the distal one third of the digit (third phalangeal element – P3) results in de novo regeneration of the digit, where analyzing the structural quality of this regenerated bone is a challenging task due to its small scale and triangular shape. To date, the evaluation of structural quality of the P3 bone has primarily focused on mineral density and bone architecture. This work describes an image-processing based method for assessment of elasticity in the whole P3 bone by using microcomputed tomography-generated mineral density data to calculate spatially discrete elastic modulus va...

Age-Dependent Changes in Bone Architecture, Patterning, and Biomechanics During Skeletal Regeneration

Frontiers in Cell and Developmental Biology, 2021

Mouse digit amputation provides a useful model of bone growth after injury, in that the injury pr... more Mouse digit amputation provides a useful model of bone growth after injury, in that the injury promotes intramembranous bone formation in an adult animal. The digit tip is composed of skin, nerves, blood vessels, bones, and tendons, all of which regenerate after digit tip amputation, making it a powerful model for multi-tissue regeneration. Bone integrity relies upon a balanced remodeling between bone resorption and formation, which, when disrupted, results in changes to bone architecture and biomechanics, particularly during aging. In this study, we used recently developed techniques to evaluate bone patterning differences between young and aged regenerated bone. This analysis suggests that aged mice have altered trabecular spacing and patterning and increased mineral density of the regenerated bone. To further characterize the biomechanics of regenerated bone, we measured elasticity using a micro-computed tomography image-processing method combined with nanoindentation. This analy...

Epigenetics and expression of key genes associated with cardiac fibrosis: NLRP3, MMP2, MMP9, CCN2/CTGF and AGT

Epigenomics, 2021

Aims: Excessive inflammatory signaling and pathological remodeling of the extracellular matrix dr... more Aims: Excessive inflammatory signaling and pathological remodeling of the extracellular matrix drive cardiac fibrosis and require changes in gene expression. Materials and methods: Using bioinformatics, both tissue-specific expression profiles and epigenomic profiles of some genes critical for cardiac fibrosis were examined, namely, NLRP3, MMP2, MMP9, CCN2/CTGF, AGT (encodes angiotensin II precursors) and hsa-mir-223 (post-transcriptionally regulates NLRP3). Results: In monocytes, neutrophils, fibroblasts, venous cells, liver and brain, enhancers or super-enhancers were found that correlate with high expression of these genes. One enhancer extended into a silent gene neighbor. These enhancers harbored tissue-specific foci of DNA hypomethylation, open chromatin and transcription factor binding. Conclusions: This study identified previously undescribed enhancers containing hypomethylated transcription factor binding subregions that are predicted to regulate expression of these cardiac...

Tissue-specific epigenetics of atherosclerosis-related ANGPT and ANGPTL genes

Epigenomics, 2019

Aim: To understand tissue-specific regulation of angiopoietin/angiopoietin-like (ANGPT/ANGPTL) ge... more Aim: To understand tissue-specific regulation of angiopoietin/angiopoietin-like (ANGPT/ANGPTL) genes (especially the five genes embedded in introns of host genes) and their association with atherosclerosis. Methods: Transcription and epigenomic databases from various normal tissues were examined in the vicinity of ANGPT1, ANGPT2, ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4 and ANGPTL8. Results: We identified tissue-specific enhancer chromatin regions that are likely to regulate transcription of ANGPT/ANGPTL genes and were intragenic, intergenic or host gene-linked. In addition, we found atherosclerosis-linked differentially methylated regions associated with ANGPT2 and with sequences encoding miR-145, a microRNA that targets ANGPT2 mRNA in cancers. Conclusion: Our findings implicate enhancers as major contributors to tissue-specific expression of ANGPT/ANGPTL genes, which play critical roles in angiogenesis, atherosclerosis, cancer, and inflammatory and metabolic diseases.

Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription

Epigenetics, 2018

DNA methylation can affect tissue-specific gene transcription in ways that are difficult to disce... more DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development.

Atherosclerosis-associated differentially methylated regions can reflect the disease phenotype and are often at enhancers

Atherosclerosis, 2018

Background and aims: Atherosclerosis is a widespread and complicated disease involving phenotypic... more Background and aims: Atherosclerosis is a widespread and complicated disease involving phenotypic modulation and transdifferentiation of vascular smooth muscle cells (SMCs), the predominant cells in aorta, as well as changes in endothelial cells and infiltrating monocytes. Alterations in DNA methylation are likely to play central roles in these phenotypic changes, just as they do in normal differentiation and cancer. Methods: We examined genome-wide DNA methylation changes in atherosclerotic aorta using more stringent criteria for differentially methylated regions (DMRs) than in previous studies and compared these DMRs to tissue-specific epigenetic features. Results: We found that disease-linked hypermethylated DMRs account for 85% of the total atherosclerosis-associated DMRs and often overlap aorta-associated enhancer chromatin. These hypermethylated DMRs were associated with functionally different sets of genes compared to atherosclerosis-linked hypomethylated DMRs. The extent and nature of the DMRs could not be explained as direct effects of monocyte/macrophage infiltration. Among the known atherosclerosis-and contractile SMC-related genes that exhibited hypermethylated DMRs at aorta enhancer chromatin were ACTA2 (aorta α2 smooth muscle actin), ELN (elastin), MYOCD (myocardin), C9orf3 (miR-23b and miR-27b host gene), and MYH11 (smooth muscle myosin). Our analyses also suggest a role in atherosclerosis for developmental transcription factor genes having little or no previous association with atherosclerosis, such as NR2F2 (COUP-TFII) and TBX18. Conclusions: We provide evidence for atherosclerosis-linked DNA methylation changes in aorta SMCs that might help minimize or reverse the standard contractile character of many of these cells by down-modulating aorta SMC-related enhancers, thereby facilitating pro-atherosclerotic phenotypic changes in many SMCs.

304 Toxoplasma Gondii Expresses Two Distinct Mitogen-Activated Protein Kinase Genes That Are Differentially Regulated

Journal of Investigative Medicine, 2005

Purpose of Study Toxoplasma gondii causes significant morbidity and mortality in immunocompromise... more Purpose of Study Toxoplasma gondii causes significant morbidity and mortality in immunocompromised patients such as those co-infected with HIV. We have cloned and sequenced two mitogen-activated protein kinase (MAPK) genes which encode novel MAP kinases that are predicted to have distinctly different properties. These predictions are based on examining differences in their primary amino acid sequences and differential gene expression in context with available functional data. Both MAPKs are druggable targets, and we predict that reducing or abolishing their expression in T. gondii will prove to be detrimental to the parasite's ability to proliferate and/or differentiate. Current experiments are underway in this regard. Methods The T. gondii strain Me-49 genome has been completely sequenced, but individual genes from this avirulent strain are still in the process of being annotated. Using information available at this genomic database (www.toxodb.org), along with our nucleotide sequencing data obtained from the virulent RH strain (acquired using an ABI-7700 automated sequencer), we have identified two MAPK genes, which we have designated tgMAPK-1 and tgMAPK-2. Expression, purification, and functional assays performed on TgMAPK-1 have been previously described (Brumlik et al, 2004). Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out in order to examine the differential expression of both these genes. Results Based on the deduced amino acid sequences encoded by these genes (which share only 33% identity), each MAP kinase has unique characteristics. RT-PCR profiles suggest that although both genes encode stress-response MAPKs, tgMAPK-1 appears to be expressed in the bradyzoite (latent) form while tgMAPK-2 seems to be more constitutively expressed. ClustalW alignment of these MAPKs has been performed, along with a phylogenetic analysis. Strain variations between both MAPKs are also discussed. Conclusions Pyridinylimidazoles designed to block the human stress-response p38 MAPK also inhibit TgMAPK-1 in vitro, block T. gondii replication, and protect T. gondii-infected mice. Both TgMAPK-1 and TgMAPK-2 are druggable targets, although it remains to be seen the effect that pyridinylimidazoles have on TgMAPK-2. Understanding the relationship between TgMAPK-1 and TgMAPK-2 and their ability to mediate stress-response and/or stage-differentiation will be important to advancing treatment strategies for T. gondii-infected patients.

Assessing needs and assets for building a regional network infrastructure to reduce cancer related health disparities

Evaluation and Program Planning, 2014

Significant cancer health disparities exist in the United States and Puerto Rico. While numerous ... more

The SEQC2 epigenomics quality control (EpiQC) study

Genome Biology

Background Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of... more Background Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA’s Epigenomics Quality Control Group. Results Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing ...

$Table 2 Summary statistics of mapping and library efficiency per WGBS protocol. Percent CpG capture calculated with call sets normalized to 20x coverage. The total genome-wide CpGs unde consideration were those that could be mapped to uniquely, excluding any CpGs that fall within unresolvable regions methylation call sets for every replicate to a given mean coverage value. Downsampling can be done by either filtering the number of reads in an alignment (BAM files), or by randomly removing a fraction of observed cytosines and observed thymines per CpG within methylation call sets (bedGraph files). Because downsampling at the alignment level can be slow and demanding in terms of disk space and compute time, we set out to evaluate if the signal from downsampling cytosines within bedGraph files recapitu- lated downsampling aligned reads within BAM files. The two approaches yielded simi- lar results in number of CpG sites detected, distribution of read counts, and methylation calls. bedGraph downsampling had the added benefit that the targeted average CpG coverage was more accurately estimated than when downsampling BAMs (Additional file 1: Figure $5).$

Epigenetics of skeletal muscle-associated genes in the ASB, LRRC, TMEM, and OSBPL gene families

Much remains to be discovered about the intersection of tissue-specific transcription control and... more Much remains to be discovered about the intersection of tissue-specific transcription control and the epigenetics of skeletal muscle (SkM), a very complex and dynamic organ. From four gene families, ASB, LRRC, TMEM, and OSBPL, we chose 21 genes that are preferentially expressed in human SkM relative to 52 other tissue types and analyzed relationships between their tissue-specific epigenetics and expression. We also compared their genetics, proteomics, and descriptions in the literature. For this study, we identified genes with little or no previous descriptions of SkM functionality (ASB4, ASB8, ASB10, ASB12, ASB16, LRRC14B, LRRC20, LRRC30, TMEM52, TMEM233, OSBPL6/ORP6, and OSBPL11/ORP11) and included genes whose SkM functions had been previously addressed (ASB2, ASB5, ASB11, ASB15, LRRC2, LRRC38, LRRC39, TMEM38A/TRIC-A, and TMEM38B/TRIC-B). Among the transcription-related SkM epigenetic features that we identified were super-enhancers, promoter DNA hypomethylation, lengthening of co...

Sirtuin 3 deficiency does not impede digit regeneration in mice

Scientific Reports

The mitochondrial deacetylase sirtuin 3 (SIRT3) is thought to be one of the main contributors to ... more The mitochondrial deacetylase sirtuin 3 (SIRT3) is thought to be one of the main contributors to metabolic flexibility–promoting mitochondrial energy production and maintaining homeostasis. In bone, metabolic profiles are tightly regulated and the loss of SIRT3 has deleterious effects on bone volume in vivo and on osteoblast differentiation in vitro. Despite the prominent role of this protein in bone stem cell proliferation, metabolic activity, and differentiation, the importance of SIRT3 for regeneration after bone injury has never been reported. We show here, using the mouse digit amputation model, that SIRT3 deficiency has no impact on the regenerative capacity and architecture of bone and soft tissue. Regeneration occurs in SIRT3 deficient mice in spite of the reduced oxidative metabolic profile of the periosteal cells. These data suggest that bone regeneration, in contrast to homeostatic bone turnover, is not reliant upon active SIRT3, and our results highlight the need to exam...

Expanding Research Capacity in Sub-Saharan Africa Through Informatics, Bioinformatics, and Data Science Training Programs in Mali

Frontiers in Genetics

Shaffer et al. Bioinformatics Training in Mali incremental processes are necessary for engaging h... more Shaffer et al. Bioinformatics Training in Mali incremental processes are necessary for engaging historically underserved countries in bioinformatics and data science research. The multi-tiered enhancement approach laid out here provides a platform for generating bioinformatics and data science technicians, teachers, researchers, and program managers. Increased literature on bioinformatics and data science training approaches and progress is needed to provide a framework for establishing benchmarks on the topics.