Kristin Loomis - Academia.edu (original) (raw)
Papers by Kristin Loomis
Additional file 3. Quality Assessment of RNA-Sequencing Data. This file shows quality control met... more Additional file 3. Quality Assessment of RNA-Sequencing Data. This file shows quality control metrics for the sequences of each individual sample. This includes the number of reads in the sample, the number and percentage of those reads that were mapped to the transcriptome, the number and percentage of total transcripts covered, and the number and percentage of total genes hit. Additionally, we assessed Pearson correlations of each individual sample within a biological treatment group.
Additional file 4. Differential Gene Expression Data. This file contains the differential gene ex... more Additional file 4. Differential Gene Expression Data. This file contains the differential gene expression analysis for each microbiome treatment relative to the axenic control. Each gene included in the analysis is shown, including the basemean, log2 fold change, log2 fold change unshrunk, p-value, and adjusted p-value, as given by DESeq2.
Additional file 5. Gene Set Overrepresentation Test Data. This file contains input and output inf... more Additional file 5. Gene Set Overrepresentation Test Data. This file contains input and output information important to the Gene Set Overrepresentation Test. Genes used for the background gene list. The output data from the PANTHER Overrepresentation tests is also shown. (PDF 1358 kb)
Soft Matter, 2011
... Drug Delivery Rev., 1995, 16(23), 285294 Article ChemPort . 4, AK Iyer, et al., Exploiting ... more ... Drug Delivery Rev., 1995, 16(23), 285294 Article ChemPort . 4, AK Iyer, et al., Exploiting the enhanced permeability and retention effect for tumor targeting, Drug Discovery Today, 2006, 11(1718), 812818 Article ChemPort . ...
Genetics in Medicine Open
Additional file 1. Microorganism Isolate Information. Table containing information for the bacter... more Additional file 1. Microorganism Isolate Information. Table containing information for the bacterial microorganisms used in this study. Table containing the identifying genus, the isolate identifier used by the collecting study, the 16S rRNA sequence, and the least common ancestors of the bacterial isolate determined by classification to the Greengenes, RDP, and SILVA databases [42].
Additional file 6. Antimicrobial Peptide Gene Expression. This file contains differential gene ex... more Additional file 6. Antimicrobial Peptide Gene Expression. This file contains differential gene expression data (Log2 fold change and the adjusted p-value) for genes that encode for antimicrobial proteins and peptides. Citations for the individual genes are also includes. (XLSX 250 kb)
Additional file 2: Figure S1. Increase in available skin microbiome representatives by this project.
Additional file 1. This file contains supporting tables and information not included in the prima... more Additional file 1. This file contains supporting tables and information not included in the primary text.
ACS Nano, 2015
Spinal cord injury (SCI) can lead to permanent motor and sensory deficits. Following the initial ... more Spinal cord injury (SCI) can lead to permanent motor and sensory deficits. Following the initial traumatic insult, secondary injury mechanisms characterized by persistent heightened inflammation are initiated and lead to continued and pervasive cell death and tissue damage. Anti-inflammatory drugs such as methylprednisolone (MP) used clinically have ambiguous benefits with debilitating side effects. Typically, these drugs are administered systemically at high doses, resulting in toxicity and paradoxically increased inflammation. Furthermore, these drugs have a small time window postinjury (few hours) during which they need to be infused to be effective. As an alternative to MP, we investigated the effect of a small molecule inhibitor (Chicago sky blue, CSB) of macrophage migration inhibitory factor (MIF) for treating SCI. The pleiotropic cytokine MIF is known to contribute to upregulation of several pro-inflammatory cytokines in various disease and injury states. In vitro, CSB administration alleviated endotoxin-mediated inflammation in primary microglia and macrophages. Nanocarriers such as liposomes can potentially alleviate systemic side effects of high-dose therapy by enabling site-specific drug delivery to the spinal cord. However, the therapeutic window of 100 nm scale nanoparticle localization to the spinal cord after contusion injury is not fully known. Thus, we first investigated the ability of nanocarriers of different sizes to localize to the injured spinal cord up to 2 weeks postinjury. Results from the study showed that nanocarriers as large as 200 nm in diameter could extravasate into the injured spinal cord up to 96 h postinjury. We then formulated nanocarriers (liposomes) encapsulating CSB and administered them intravenously 48 h postinjury, within the previously determined 96 h therapeutic window. In vivo, in this clinically relevant contusion injury model in rats, CSB administration led to preservation of vascular and white matter integrity, improved wound healing, and an increase in levels of arginase and other transcripts indicative of a resolution phase of wound healing. This study demonstrates the potential of MIF inhibition in SCI and the utility of nanocarrier-mediated drug delivery selectively to the injured cord.
Wastewater-based epidemiology is an important tool for monitoring SARS-CoV-2 and other molecular ... more Wastewater-based epidemiology is an important tool for monitoring SARS-CoV-2 and other molecular targets in populations, using wastewater as a pooled sample. We compared the sensitivity, susceptibility to inhibition, and quantification of reverse transcription quantitative PCR (RT-qPCR), microfluidic well digital RT-PCR (RT-dPCR), and droplet digital RT-PCR (RT-ddPCR) measurements of SARS-CoV-2 (N1 gene target) and Pepper Mild Mottle Virus (PMMoV) RNA in 40 wastewater RNA extracts. All three methods were highly sensitive, but appeared less accurate at very low concentrations. Lower inhibition was observed for RT-ddPCR than RT-qPCR with both SARS-CoV-2 and PMMoV targets, but inhibition appeared to be mitigated by dilution of template RNA. The concentrations of N1 and PMMoV from all three methods were significantly correlated (Pearson’s r=0.97-0.98 for N1 and r=0.89-0.93 for PMMoV), although RT-qPCR reported higher concentrations than digital methods. Taken together, this study provid...
Soft Matter, 2011
... Drug Delivery Rev., 1995, 16(23), 285294 Article ChemPort . 4, AK Iyer, et al., Exploiting ... more ... Drug Delivery Rev., 1995, 16(23), 285294 Article ChemPort . 4, AK Iyer, et al., Exploiting the enhanced permeability and retention effect for tumor targeting, Drug Discovery Today, 2006, 11(1718), 812818 Article ChemPort . ...
To my family who inspire me in so many ways iv ACKNOWLEDGEMENTS It is humbling and heartwarming t... more To my family who inspire me in so many ways iv ACKNOWLEDGEMENTS It is humbling and heartwarming to reflect on all of the help and support that I received throughout my time at Georgia Tech. I was lucky to receive the support and direction from two advisors who helped guide me through the PhD process. First, I would like to thank Dr. Bellamkonda for his support, patience, and encouragement. He provided me with a truly unique laboratory experience, giving me incredible flexibility and freedom, which allowed me to learn first-hand how to approach challenges. I have taken away many lifelong lessons and skills that would not have been possible under other circumstances. Very importantly, Dr. Santangelo, my committee member who turned into an unofficial advisor, has been an incredible force in helping me shape this dissertation. I am extremely grateful that he incorporated me into his lab group. I have learned so much about RNA biology, how to design experiments, ask questions, and solve problems through working with him. I would also like to thank my committee members for advising me throughout my graduate work. They have asked insightful questions and have always steered me in a better direction and forced. They have all helped me to embrace the questions in research to gain a deeper understanding. I had very little molecular biology experience when I began my PhD studies. I would like to thank Balakrishna Pai, Shannon Barker, Lohitash Karumbaiah, and Nalini Mehta for sharing their knowledge with me. Dr. Pai, especially, patiently taught me the basics of DNA cloning, Western blotting, and DNA gels, along with a few tricks and shortcuts along the way. He gave me the technical fundamentals in molecular biology that have enabled future experiments. v Both of the labs that I have been a part of have helped me grow. First, I would like to thank the Neurolab-past and present-for their help, inspiration, and support. The passion and excitement about science in the lab is contagious. I greatly appreciate the questions, support, and scientific conversations we have had over the years. Every single member of the Santangelo lab has directly or indirectly impacted my thesis work in a major way. Many aspects of the work presented here have built upon previous studies in the lab. Daryll Vanover and Jonathan Kirschman have especially helped me with microscopy and general lab support. Sushma Bhosle assisted with experiments and provided company during so much of our time in the PRL. I would also like to acknowledge the undergraduate students who have contributed to this work. Sarah Kutbay started working with me in the very beginning. She was extremely perseverant and cheerful as we struggled to establish protocols for making IVT mRNA and transfection cells. Shahmeer Mirza's enthusiastic questioning and excitement for the research helped me improve my understanding of our experiments. Lastly, Ravi Jindal showed extreme care in performing experiments, even when they were quite monotonous. So many others helped me along the way. I want to thank Brani Vidakovic for his assistance with the statistical analysis of my PLA data. I also want to thank Laura O'Farrell for all of her advice and training. The core-facilities staff have also enabled my studies; Andrew Shaw's technical expertise guided me with some microscopy studies and Dalia Arafat Gulick helped me with the Fluidigm PCR studies. Ketki Patil, Claire McCauley, and Moliehi Mokoroane also provided so much support; they often went out of their way to assist with last minute orders and lab maintenance. My PhD experience was so much richer because of the friends that I made along the way. Patricia Pacheco's support and friendship cannot be described. She helped me overcome setbacks, and balance my life outside of lab. I also want to thank my labmate vi and classmate Akhil Srinivasan. We shared many late-nights doing homework, lab experiments, and exploring Atlanta. You have been an inspiration to watch navigate the PhD process. Last but far from least, I would like to thank Saujan Sivaram for his incredible love and support throughout my time at Georgia Tech. You have taught me so much about science and about life. I am grateful every day to have you in my life. vii TABLE OF CONTENTS ACKNOWLEDGEMENTS IV LIST OF TABLES xii LIST OF FIGURES XIII LIST OF SYMBOLS AND ABBREVIATIONS XVI SUMMARY XVIII CHAPTER 1 INTRODUCTION 1 1.1 Principles of vaccination 1 1.2 Current vaccine classifications 2 1.2.1 Live attenuated vaccines 2 109 A.4. Analysis of variance on proximity ligation assays 113 A.5. MATLAB scripts 114 APPENDIX B DNA TEMPLATES FOR IVT MRNA 117 xi B.1 DNA Template for Ara h 2 B.2 DNA template for cytoplasmic ovalbumin APPENDIX C PRIMER SEQUENCES FOR QRT-PCR APPENDIX D ANTIVIRAL PCR ARRAY DATA REFERENCES xii LIST OF TABLES 1.1. PRRs known or suspected to respond to exogenous mRNA delivery 11 1.2. Delivery vehicles tested for IVT mRNA 18 4.1 Genes assessed with PCR assay 88 C.1 Primer sequences used in Chapter 2 118 D.1 Fold change of RAW264.7 cells treated with IVT mRNA over Lipofectamine only treatment 119 D.2 Fold regulation of IVT mRNA injection in anterior tibialis over sham injection control 121 xiii LIST OF FIGURES 1.1 IVT mRNA interaction with PRRs. 1.2 Diagram of IVT mRNA and opportunities for engineering its function. 1.3 Strategies for effective delivery of IVT mRNA. 2.1 A point mutation in Ara h 2 IVT mRNA enables transgene expression. 2.2 Transgene expression of mRNA, 5mC/Ψ mRNA, and plasmid DNA 2.3 Transfection of plasmid DNA, but not mRNA, upregulates BMDC maturation markers 2.4 Transfection of plasmid DNA and IVT mRNA downregulate CD11c cell surface expression 2.5 IVT mRNA and plasmid DNA transfection do not lead to appreciable cell death 2.6 Expression of antiviral cytokines in response to nucleic acid transfection. 2.7 NF-КB responses by TLR7-positive and TLR7-negative cells in response to transfection with IVT mRNA 2.8 TLR7 signaling complex formation in response to IVT mRNA 3.1 Schematic illustrating mechanism of PLA 3.2 Experimental overview 3.3 Luciferase expression in the anterior tibialis 16 hours following IVT mRNA injection 3.4 Distribution of IVT mRNA following injection and uptake by CD11b+ and MHCII+ cells 3.5 Cell types containing IVT mRNA 16 hours after i.m. injection 3.6 TLR7, RIG-I, and MDA5 staining in sham injected skeletal muscle tissue 3.7 IVT mRNA uptake by TLR7+, RIG-I+, and MDA5+ cells 3.8 Immunohistochemistry staining controls for TLR7, RIG-I, and MDA5 detection 3.9 Effect of PRR knockdown on cell responses to IVT mRNA transfection 3.10 PLA detection of TLR7-IRAK4 signaling complex following IVT mRNA injection xiv 3.11 PLA detection of RIG-I-IPS-1 signaling complex following IVT mRNA injection 3.12 PLA detection of MDA5-IPS-1 signaling complex following IVT mRNA injection 3.13. IVT mRNA distribution in draining lymph node 3.14 IVT mRNA presence in draining lymph nodes 3.15 PLA assays showing activation of TLR7, RIG-I, and MDA5 signaling pathways 4.1 T cell responses by wild-type (WT) and interferon alpha receptor knockout mice (IFN αR−/−) to an IVT mRNA vaccine 4.2 Approach for building programmable IVT mRNA 4.3 Gene expression of RAW264.7 cells following transfection with ovalbumin IVT mRNA incorporation the modified base pseudouridine or M1Y 4.4 Effect of modified base incorporation on IVT mRNA protein expression 4.5 Effect of tethering the TLR7 adjuvant CL264 to IVT mRNA 4.6 Protein expression of IVT mRNA delivered with and without tethered agonist 4.7 Effect of modified bases on immune response to CL264-tethered IVT mRNA 4.8 Effect of tethering the TLR2 adjuvant Pam2CSK4 to IVT mRNA 4.9 Heat map showing fold change of gene expression over a sham injection control in response to intramuscular injection with the indicated treatment 4.10 Gene expression in the muscle after injection with CL264 or IVT mRNA 4.11 Gene expression after injection of IVT mRNA with CL264 4.12 Gene expression of toll-like receptor responsive genes with more than a threefold increase in 4.11 5.1 Design variables can modulate IVT mRNA characteristics and functionality A.1 Histograms of residuals from data of (A) TLR7, (B) MDA5, and (C) RIG-I PLAs A.2 q-q plots from data from (A) TLR7, (B) MDA5, and (C) RIG-I proximity ligation assays A.3 Calculated JB statistic for each q value from PLAs for (A) TLR7 (B) MDA5 and (C) RIG-I A.4 Histograms of residual plots from data from PLAs of (A) TLR7, (B) MDA5, and (C) RIG-I A.5 q-q plots from data from PLAs of (A) TLR7, (B) MDA5, and (C) RIG-I xv A.6 Plot of y=x1/3 113 A.7 Effect of time on activation of (A) TLR7 (B) MDA-5 and (C) RIG-I
Background Skin, the largest organ of the human body by weight, hosts a diversity of microorganis... more Background Skin, the largest organ of the human body by weight, hosts a diversity of microorganisms that can influence health. The microbial residents of the skin are now appreciated for their roles in host immune interactions, wound healing, colonization resistance, and various skin disorders. Still, much remains to be discovered in terms of the host pathways influenced by skin microorganisms, as well as the higher-level skin properties impacted through these microbe-host interactions. Towards this direction, recent efforts using mouse models pointed to pronounced changes in the transcriptional profiles of the skin in response to the presence of a microbial community. However, there is a need to quantify the roles of microorganisms at both the individual and community-level in healthy human skin. In this study, we utilize human skin equivalents to study the effects of individual taxa and a microbial community in a precisely controlled context. Through transcriptomics analysis, we i...
Microbiome
Background: The skin micro-environment varies across the body, but all sites are host to microorg... more Background: The skin micro-environment varies across the body, but all sites are host to microorganisms that can impact skin health. Some of these organisms are true commensals which colonize a unique niche on the skin, while open exposure of the skin to the environment also results in the transient presence of diverse microbes with unknown influences on skin health. Culture-based studies of skin microbiota suggest that skin microbes can affect skin properties, immune responses, pathogen growth, and wound healing. Results: In this work, we greatly expanded the diversity of available commensal organisms by collecting > 800 organisms from 3 body sites of 17 individuals. Our collection includes > 30 bacterial genera and 14 fungal genera, with Staphylococcus and Micrococcus as the most prevalent isolates. We characterized a subset of skin isolates for the utilization of carbon compounds found on the skin surface. We observed that members of the skin microbiota have the capacity to metabolize amino acids, steroids, lipids, and sugars, as well as compounds originating from personal care products. Conclusions: This collection is a resource that will support skin microbiome research with the potential for discovery of novel small molecules, development of novel therapeutics, and insight into the metabolic activities of the skin microbiota. We believe this unique resource will inform skin microbiome management to benefit skin health.
Molecular Therapy - Nucleic Acids
The characterization of innate immune activation is crucial for vaccine and therapeutic developme... more The characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNAbased vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA's sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from in vitro-transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile, and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening.
Bioconjugate chemistry, 2018
In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can ... more In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can be manu-factured rapidly at large scale to meet emerging pathogens. However, its performance as a robust vaccine is strengthened by supplemental immune stimulation, which is typically provided by adjuvant formulations that facilitate delivery and stimulate immune responses. Here, we present a strategy for increasing transla-tion of a model IVT mRNA vaccine while simultaneously modulating its immune-stimulatory properties in a programmable fashion, without relying on delivery vehicle formulations. Substitution of uridine with the modified base N1-5-methylpseudouridine reduces the intrinsic immune stimulation of the IVT mRNA and enhances antigen translation. Tethering adjuvants to naked IVT mRNA through antisense nucleotides boosts the immuno-stimulatory properties of adjuvants in vitro, without impairing transgene production or adjuvant activity. In vivo, intramuscular injection of tether...
Nucleic acids research, Jan 26, 2017
The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly p... more The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections,...
J. Mater. Chem. B, 2015
This review discusses the challenges associated with IVT mRNA therapeutics and vaccines as well a... more This review discusses the challenges associated with IVT mRNA therapeutics and vaccines as well as the current strategies employed to overcome these challenges.
Additional file 3. Quality Assessment of RNA-Sequencing Data. This file shows quality control met... more Additional file 3. Quality Assessment of RNA-Sequencing Data. This file shows quality control metrics for the sequences of each individual sample. This includes the number of reads in the sample, the number and percentage of those reads that were mapped to the transcriptome, the number and percentage of total transcripts covered, and the number and percentage of total genes hit. Additionally, we assessed Pearson correlations of each individual sample within a biological treatment group.
Additional file 4. Differential Gene Expression Data. This file contains the differential gene ex... more Additional file 4. Differential Gene Expression Data. This file contains the differential gene expression analysis for each microbiome treatment relative to the axenic control. Each gene included in the analysis is shown, including the basemean, log2 fold change, log2 fold change unshrunk, p-value, and adjusted p-value, as given by DESeq2.
Additional file 5. Gene Set Overrepresentation Test Data. This file contains input and output inf... more Additional file 5. Gene Set Overrepresentation Test Data. This file contains input and output information important to the Gene Set Overrepresentation Test. Genes used for the background gene list. The output data from the PANTHER Overrepresentation tests is also shown. (PDF 1358 kb)
Soft Matter, 2011
... Drug Delivery Rev., 1995, 16(23), 285294 Article ChemPort . 4, AK Iyer, et al., Exploiting ... more ... Drug Delivery Rev., 1995, 16(23), 285294 Article ChemPort . 4, AK Iyer, et al., Exploiting the enhanced permeability and retention effect for tumor targeting, Drug Discovery Today, 2006, 11(1718), 812818 Article ChemPort . ...
Genetics in Medicine Open
Additional file 1. Microorganism Isolate Information. Table containing information for the bacter... more Additional file 1. Microorganism Isolate Information. Table containing information for the bacterial microorganisms used in this study. Table containing the identifying genus, the isolate identifier used by the collecting study, the 16S rRNA sequence, and the least common ancestors of the bacterial isolate determined by classification to the Greengenes, RDP, and SILVA databases [42].
Additional file 6. Antimicrobial Peptide Gene Expression. This file contains differential gene ex... more Additional file 6. Antimicrobial Peptide Gene Expression. This file contains differential gene expression data (Log2 fold change and the adjusted p-value) for genes that encode for antimicrobial proteins and peptides. Citations for the individual genes are also includes. (XLSX 250 kb)
Additional file 2: Figure S1. Increase in available skin microbiome representatives by this project.
Additional file 1. This file contains supporting tables and information not included in the prima... more Additional file 1. This file contains supporting tables and information not included in the primary text.
ACS Nano, 2015
Spinal cord injury (SCI) can lead to permanent motor and sensory deficits. Following the initial ... more Spinal cord injury (SCI) can lead to permanent motor and sensory deficits. Following the initial traumatic insult, secondary injury mechanisms characterized by persistent heightened inflammation are initiated and lead to continued and pervasive cell death and tissue damage. Anti-inflammatory drugs such as methylprednisolone (MP) used clinically have ambiguous benefits with debilitating side effects. Typically, these drugs are administered systemically at high doses, resulting in toxicity and paradoxically increased inflammation. Furthermore, these drugs have a small time window postinjury (few hours) during which they need to be infused to be effective. As an alternative to MP, we investigated the effect of a small molecule inhibitor (Chicago sky blue, CSB) of macrophage migration inhibitory factor (MIF) for treating SCI. The pleiotropic cytokine MIF is known to contribute to upregulation of several pro-inflammatory cytokines in various disease and injury states. In vitro, CSB administration alleviated endotoxin-mediated inflammation in primary microglia and macrophages. Nanocarriers such as liposomes can potentially alleviate systemic side effects of high-dose therapy by enabling site-specific drug delivery to the spinal cord. However, the therapeutic window of 100 nm scale nanoparticle localization to the spinal cord after contusion injury is not fully known. Thus, we first investigated the ability of nanocarriers of different sizes to localize to the injured spinal cord up to 2 weeks postinjury. Results from the study showed that nanocarriers as large as 200 nm in diameter could extravasate into the injured spinal cord up to 96 h postinjury. We then formulated nanocarriers (liposomes) encapsulating CSB and administered them intravenously 48 h postinjury, within the previously determined 96 h therapeutic window. In vivo, in this clinically relevant contusion injury model in rats, CSB administration led to preservation of vascular and white matter integrity, improved wound healing, and an increase in levels of arginase and other transcripts indicative of a resolution phase of wound healing. This study demonstrates the potential of MIF inhibition in SCI and the utility of nanocarrier-mediated drug delivery selectively to the injured cord.
Wastewater-based epidemiology is an important tool for monitoring SARS-CoV-2 and other molecular ... more Wastewater-based epidemiology is an important tool for monitoring SARS-CoV-2 and other molecular targets in populations, using wastewater as a pooled sample. We compared the sensitivity, susceptibility to inhibition, and quantification of reverse transcription quantitative PCR (RT-qPCR), microfluidic well digital RT-PCR (RT-dPCR), and droplet digital RT-PCR (RT-ddPCR) measurements of SARS-CoV-2 (N1 gene target) and Pepper Mild Mottle Virus (PMMoV) RNA in 40 wastewater RNA extracts. All three methods were highly sensitive, but appeared less accurate at very low concentrations. Lower inhibition was observed for RT-ddPCR than RT-qPCR with both SARS-CoV-2 and PMMoV targets, but inhibition appeared to be mitigated by dilution of template RNA. The concentrations of N1 and PMMoV from all three methods were significantly correlated (Pearson’s r=0.97-0.98 for N1 and r=0.89-0.93 for PMMoV), although RT-qPCR reported higher concentrations than digital methods. Taken together, this study provid...
Soft Matter, 2011
... Drug Delivery Rev., 1995, 16(23), 285294 Article ChemPort . 4, AK Iyer, et al., Exploiting ... more ... Drug Delivery Rev., 1995, 16(23), 285294 Article ChemPort . 4, AK Iyer, et al., Exploiting the enhanced permeability and retention effect for tumor targeting, Drug Discovery Today, 2006, 11(1718), 812818 Article ChemPort . ...
To my family who inspire me in so many ways iv ACKNOWLEDGEMENTS It is humbling and heartwarming t... more To my family who inspire me in so many ways iv ACKNOWLEDGEMENTS It is humbling and heartwarming to reflect on all of the help and support that I received throughout my time at Georgia Tech. I was lucky to receive the support and direction from two advisors who helped guide me through the PhD process. First, I would like to thank Dr. Bellamkonda for his support, patience, and encouragement. He provided me with a truly unique laboratory experience, giving me incredible flexibility and freedom, which allowed me to learn first-hand how to approach challenges. I have taken away many lifelong lessons and skills that would not have been possible under other circumstances. Very importantly, Dr. Santangelo, my committee member who turned into an unofficial advisor, has been an incredible force in helping me shape this dissertation. I am extremely grateful that he incorporated me into his lab group. I have learned so much about RNA biology, how to design experiments, ask questions, and solve problems through working with him. I would also like to thank my committee members for advising me throughout my graduate work. They have asked insightful questions and have always steered me in a better direction and forced. They have all helped me to embrace the questions in research to gain a deeper understanding. I had very little molecular biology experience when I began my PhD studies. I would like to thank Balakrishna Pai, Shannon Barker, Lohitash Karumbaiah, and Nalini Mehta for sharing their knowledge with me. Dr. Pai, especially, patiently taught me the basics of DNA cloning, Western blotting, and DNA gels, along with a few tricks and shortcuts along the way. He gave me the technical fundamentals in molecular biology that have enabled future experiments. v Both of the labs that I have been a part of have helped me grow. First, I would like to thank the Neurolab-past and present-for their help, inspiration, and support. The passion and excitement about science in the lab is contagious. I greatly appreciate the questions, support, and scientific conversations we have had over the years. Every single member of the Santangelo lab has directly or indirectly impacted my thesis work in a major way. Many aspects of the work presented here have built upon previous studies in the lab. Daryll Vanover and Jonathan Kirschman have especially helped me with microscopy and general lab support. Sushma Bhosle assisted with experiments and provided company during so much of our time in the PRL. I would also like to acknowledge the undergraduate students who have contributed to this work. Sarah Kutbay started working with me in the very beginning. She was extremely perseverant and cheerful as we struggled to establish protocols for making IVT mRNA and transfection cells. Shahmeer Mirza's enthusiastic questioning and excitement for the research helped me improve my understanding of our experiments. Lastly, Ravi Jindal showed extreme care in performing experiments, even when they were quite monotonous. So many others helped me along the way. I want to thank Brani Vidakovic for his assistance with the statistical analysis of my PLA data. I also want to thank Laura O'Farrell for all of her advice and training. The core-facilities staff have also enabled my studies; Andrew Shaw's technical expertise guided me with some microscopy studies and Dalia Arafat Gulick helped me with the Fluidigm PCR studies. Ketki Patil, Claire McCauley, and Moliehi Mokoroane also provided so much support; they often went out of their way to assist with last minute orders and lab maintenance. My PhD experience was so much richer because of the friends that I made along the way. Patricia Pacheco's support and friendship cannot be described. She helped me overcome setbacks, and balance my life outside of lab. I also want to thank my labmate vi and classmate Akhil Srinivasan. We shared many late-nights doing homework, lab experiments, and exploring Atlanta. You have been an inspiration to watch navigate the PhD process. Last but far from least, I would like to thank Saujan Sivaram for his incredible love and support throughout my time at Georgia Tech. You have taught me so much about science and about life. I am grateful every day to have you in my life. vii TABLE OF CONTENTS ACKNOWLEDGEMENTS IV LIST OF TABLES xii LIST OF FIGURES XIII LIST OF SYMBOLS AND ABBREVIATIONS XVI SUMMARY XVIII CHAPTER 1 INTRODUCTION 1 1.1 Principles of vaccination 1 1.2 Current vaccine classifications 2 1.2.1 Live attenuated vaccines 2 109 A.4. Analysis of variance on proximity ligation assays 113 A.5. MATLAB scripts 114 APPENDIX B DNA TEMPLATES FOR IVT MRNA 117 xi B.1 DNA Template for Ara h 2 B.2 DNA template for cytoplasmic ovalbumin APPENDIX C PRIMER SEQUENCES FOR QRT-PCR APPENDIX D ANTIVIRAL PCR ARRAY DATA REFERENCES xii LIST OF TABLES 1.1. PRRs known or suspected to respond to exogenous mRNA delivery 11 1.2. Delivery vehicles tested for IVT mRNA 18 4.1 Genes assessed with PCR assay 88 C.1 Primer sequences used in Chapter 2 118 D.1 Fold change of RAW264.7 cells treated with IVT mRNA over Lipofectamine only treatment 119 D.2 Fold regulation of IVT mRNA injection in anterior tibialis over sham injection control 121 xiii LIST OF FIGURES 1.1 IVT mRNA interaction with PRRs. 1.2 Diagram of IVT mRNA and opportunities for engineering its function. 1.3 Strategies for effective delivery of IVT mRNA. 2.1 A point mutation in Ara h 2 IVT mRNA enables transgene expression. 2.2 Transgene expression of mRNA, 5mC/Ψ mRNA, and plasmid DNA 2.3 Transfection of plasmid DNA, but not mRNA, upregulates BMDC maturation markers 2.4 Transfection of plasmid DNA and IVT mRNA downregulate CD11c cell surface expression 2.5 IVT mRNA and plasmid DNA transfection do not lead to appreciable cell death 2.6 Expression of antiviral cytokines in response to nucleic acid transfection. 2.7 NF-КB responses by TLR7-positive and TLR7-negative cells in response to transfection with IVT mRNA 2.8 TLR7 signaling complex formation in response to IVT mRNA 3.1 Schematic illustrating mechanism of PLA 3.2 Experimental overview 3.3 Luciferase expression in the anterior tibialis 16 hours following IVT mRNA injection 3.4 Distribution of IVT mRNA following injection and uptake by CD11b+ and MHCII+ cells 3.5 Cell types containing IVT mRNA 16 hours after i.m. injection 3.6 TLR7, RIG-I, and MDA5 staining in sham injected skeletal muscle tissue 3.7 IVT mRNA uptake by TLR7+, RIG-I+, and MDA5+ cells 3.8 Immunohistochemistry staining controls for TLR7, RIG-I, and MDA5 detection 3.9 Effect of PRR knockdown on cell responses to IVT mRNA transfection 3.10 PLA detection of TLR7-IRAK4 signaling complex following IVT mRNA injection xiv 3.11 PLA detection of RIG-I-IPS-1 signaling complex following IVT mRNA injection 3.12 PLA detection of MDA5-IPS-1 signaling complex following IVT mRNA injection 3.13. IVT mRNA distribution in draining lymph node 3.14 IVT mRNA presence in draining lymph nodes 3.15 PLA assays showing activation of TLR7, RIG-I, and MDA5 signaling pathways 4.1 T cell responses by wild-type (WT) and interferon alpha receptor knockout mice (IFN αR−/−) to an IVT mRNA vaccine 4.2 Approach for building programmable IVT mRNA 4.3 Gene expression of RAW264.7 cells following transfection with ovalbumin IVT mRNA incorporation the modified base pseudouridine or M1Y 4.4 Effect of modified base incorporation on IVT mRNA protein expression 4.5 Effect of tethering the TLR7 adjuvant CL264 to IVT mRNA 4.6 Protein expression of IVT mRNA delivered with and without tethered agonist 4.7 Effect of modified bases on immune response to CL264-tethered IVT mRNA 4.8 Effect of tethering the TLR2 adjuvant Pam2CSK4 to IVT mRNA 4.9 Heat map showing fold change of gene expression over a sham injection control in response to intramuscular injection with the indicated treatment 4.10 Gene expression in the muscle after injection with CL264 or IVT mRNA 4.11 Gene expression after injection of IVT mRNA with CL264 4.12 Gene expression of toll-like receptor responsive genes with more than a threefold increase in 4.11 5.1 Design variables can modulate IVT mRNA characteristics and functionality A.1 Histograms of residuals from data of (A) TLR7, (B) MDA5, and (C) RIG-I PLAs A.2 q-q plots from data from (A) TLR7, (B) MDA5, and (C) RIG-I proximity ligation assays A.3 Calculated JB statistic for each q value from PLAs for (A) TLR7 (B) MDA5 and (C) RIG-I A.4 Histograms of residual plots from data from PLAs of (A) TLR7, (B) MDA5, and (C) RIG-I A.5 q-q plots from data from PLAs of (A) TLR7, (B) MDA5, and (C) RIG-I xv A.6 Plot of y=x1/3 113 A.7 Effect of time on activation of (A) TLR7 (B) MDA-5 and (C) RIG-I
Background Skin, the largest organ of the human body by weight, hosts a diversity of microorganis... more Background Skin, the largest organ of the human body by weight, hosts a diversity of microorganisms that can influence health. The microbial residents of the skin are now appreciated for their roles in host immune interactions, wound healing, colonization resistance, and various skin disorders. Still, much remains to be discovered in terms of the host pathways influenced by skin microorganisms, as well as the higher-level skin properties impacted through these microbe-host interactions. Towards this direction, recent efforts using mouse models pointed to pronounced changes in the transcriptional profiles of the skin in response to the presence of a microbial community. However, there is a need to quantify the roles of microorganisms at both the individual and community-level in healthy human skin. In this study, we utilize human skin equivalents to study the effects of individual taxa and a microbial community in a precisely controlled context. Through transcriptomics analysis, we i...
Microbiome
Background: The skin micro-environment varies across the body, but all sites are host to microorg... more Background: The skin micro-environment varies across the body, but all sites are host to microorganisms that can impact skin health. Some of these organisms are true commensals which colonize a unique niche on the skin, while open exposure of the skin to the environment also results in the transient presence of diverse microbes with unknown influences on skin health. Culture-based studies of skin microbiota suggest that skin microbes can affect skin properties, immune responses, pathogen growth, and wound healing. Results: In this work, we greatly expanded the diversity of available commensal organisms by collecting > 800 organisms from 3 body sites of 17 individuals. Our collection includes > 30 bacterial genera and 14 fungal genera, with Staphylococcus and Micrococcus as the most prevalent isolates. We characterized a subset of skin isolates for the utilization of carbon compounds found on the skin surface. We observed that members of the skin microbiota have the capacity to metabolize amino acids, steroids, lipids, and sugars, as well as compounds originating from personal care products. Conclusions: This collection is a resource that will support skin microbiome research with the potential for discovery of novel small molecules, development of novel therapeutics, and insight into the metabolic activities of the skin microbiota. We believe this unique resource will inform skin microbiome management to benefit skin health.
Molecular Therapy - Nucleic Acids
The characterization of innate immune activation is crucial for vaccine and therapeutic developme... more The characterization of innate immune activation is crucial for vaccine and therapeutic development, including RNAbased vaccines, a promising approach. Current measurement methods quantify type I interferon and inflammatory cytokine production, but they do not allow for the isolation of individual pathways, do not provide kinetic activation or spatial information within tissues, and cannot be translated into clinical studies. Here we demonstrated the use of proximity ligation assays (PLAs) to detect pattern recognition receptor (PRR) activation in cells and in tissue samples. First, we validated PLA's sensitivity and specificity using well-characterized soluble agonists. Next, we characterized PRR activation from in vitro-transcribed (IVT) mRNAs, as well as the effect of sequence and base modifications in vitro. Finally, we established the measurement of PRR activation in tissue sections via PLA upon IVT mRNA intramuscular (i.m.) injection in mice. Overall, our results indicate that PLA is a valuable, versatile, and sensitive tool to monitor PRR activation for vaccine, adjuvant, and therapeutic screening.
Bioconjugate chemistry, 2018
In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can ... more In vitro transcribed (IVT) mRNA is an appealing platform for next generation vaccines, as it can be manu-factured rapidly at large scale to meet emerging pathogens. However, its performance as a robust vaccine is strengthened by supplemental immune stimulation, which is typically provided by adjuvant formulations that facilitate delivery and stimulate immune responses. Here, we present a strategy for increasing transla-tion of a model IVT mRNA vaccine while simultaneously modulating its immune-stimulatory properties in a programmable fashion, without relying on delivery vehicle formulations. Substitution of uridine with the modified base N1-5-methylpseudouridine reduces the intrinsic immune stimulation of the IVT mRNA and enhances antigen translation. Tethering adjuvants to naked IVT mRNA through antisense nucleotides boosts the immuno-stimulatory properties of adjuvants in vitro, without impairing transgene production or adjuvant activity. In vivo, intramuscular injection of tether...
Nucleic acids research, Jan 26, 2017
The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly p... more The use of synthetic messenger ribonucleic acid (mRNA) to express specific proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections,...
J. Mater. Chem. B, 2015
This review discusses the challenges associated with IVT mRNA therapeutics and vaccines as well a... more This review discusses the challenges associated with IVT mRNA therapeutics and vaccines as well as the current strategies employed to overcome these challenges.