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• 1. [Article] Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, <i>Austrofundulus limnaeus</i>

  Background: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifsh Austrofundulus limnaeus ...

  Citation × Citation Citation Abstract Copy Title: Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, <i>Austrofundulus limnaeus</i> Author: Romney, Amie L., Podrabsky, Jason E. Year: 2017 Background: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifsh Austrofundulus limnaeus is unique among vertebrates because the embryos have distinct cell movements that separate epiboly from axis formation during early development, can enter into a state of metabolic dormancy known as diapause and can survive extreme environmental conditions. The ability to enter into diapause can be maternally programmed, with young females producing embryos that do not enter into diapause. Alternately, embryos can be programmed to “escape” from diapause and develop directly by both maternal factors and embryonic incubation conditions. Thus, maternally packaged gene products are hypothesized to regulate developmental trajectory and perhaps the other unique developmental characters in this species. Results: Using high-throughput RNA sequencing, we generated transcriptomic profles of mRNAs, long non-coding RNAs and small non-coding RNAs (sncRNAs) in 1–2 cell stage embryos of A. limnaeus. Transcriptomic analyses suggest maternal programming of embryos through alternatively spliced mRNAs and antisense sncRNAs. Comparison of these results to those of comparable studies on zebrafsh and other fshes reveals a surprisingly high abundance of transcripts involved in the cellular response to stress and a relatively lower expression of genes required for rapid transition through the cell cycle. Conclusions: Maternal programming of developmental trajectory is unlikely accomplished by diferential expression of diapause-specifc genes. Rather, evidence suggests a role for trajectory-specifc splice variants of genes expressed in both phenotypes. In addition, based on comparative studies with zebrafsh, the A. limnaeus 1–2 cell stage transcriptome is unique in ways that are consistent with their unique life history. These results not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression during development Title: Transcriptomic Analysis of Maternally Provisioned Cues for Phenotypic Plasticity in the Annual Killifish, <i>Austrofundulus limnaeus</i> Author: Romney, Amie L., Podrabsky, Jason E. Year: 2017 Background: Genotype and environment can interact during development to produce novel adaptive traits that support life in extreme conditions. The development of the annual killifsh Austrofundulus limnaeus is unique among vertebrates because the embryos have distinct cell movements that separate epiboly from axis formation during early development, can enter into a state of metabolic dormancy known as diapause and can survive extreme environmental conditions. The ability to enter into diapause can be maternally programmed, with young females producing embryos that do not enter into diapause. Alternately, embryos can be programmed to “escape” from diapause and develop directly by both maternal factors and embryonic incubation conditions. Thus, maternally packaged gene products are hypothesized to regulate developmental trajectory and perhaps the other unique developmental characters in this species. Results: Using high-throughput RNA sequencing, we generated transcriptomic profles of mRNAs, long non-coding RNAs and small non-coding RNAs (sncRNAs) in 1–2 cell stage embryos of A. limnaeus. Transcriptomic analyses suggest maternal programming of embryos through alternatively spliced mRNAs and antisense sncRNAs. Comparison of these results to those of comparable studies on zebrafsh and other fshes reveals a surprisingly high abundance of transcripts involved in the cellular response to stress and a relatively lower expression of genes required for rapid transition through the cell cycle. Conclusions: Maternal programming of developmental trajectory is unlikely accomplished by diferential expression of diapause-specifc genes. Rather, evidence suggests a role for trajectory-specifc splice variants of genes expressed in both phenotypes. In addition, based on comparative studies with zebrafsh, the A. limnaeus 1–2 cell stage transcriptome is unique in ways that are consistent with their unique life history. These results not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression during development Close

• 2. [Article] Mathematical Model for Bone Mineralization

  Defective bone mineralization has serious clinical manifestations, including deformities and fractures, but the regulation of this extracellular process is not fully understood. We have developed a mathematical ...

  Citation × Citation Citation Abstract Copy Title: Mathematical Model for Bone Mineralization Author: Komarova, Svetlana V., Safranek, Lee, Gopalakrishnan, Jay, Ou, Miao-jung Yvonne, McKee, Marc D., Murshed, Monzur, Rauch, Frank, Zuhr, Erica Year: 2015 Defective bone mineralization has serious clinical manifestations, including deformities and fractures, but the regulation of this extracellular process is not fully understood. We have developed a mathematical model consisting of ordinary differential equations that describe collagen maturation, production and degradation of inhibitors, and mineral nucleation and growth. We examined the roles of individual processes in generating normal and abnormal mineralization patterns characterized using two outcome measures: mineralization lag time and degree of mineralization. Model parameters describing the formation of hydroxyapatite mineral on the nucleating centers most potently affected the degree of mineralization, while the parameters describing inhibitor homeostasis most effectively changed the mineralization lag time. Of interest, a parameter describing the rate of matrix maturation emerged as being capable of counter-intuitively increasing both the mineralization lag time and the degree of mineralization. We validated the accuracy of model predictions using known diseases of bone mineralization such as osteogenesis imperfecta and X-linked hypophosphatemia. The model successfully describes the highly nonlinear mineralization dynamics, which includes an initial lag phase when osteoid is present but no mineralization is evident, then fast primary mineralization, followed by secondary mineralization characterized by a continuous slow increase in bone mineral content. The developed model can potentially predict the function for a mutated protein based on the histology of pathologic bone samples from mineralization disorders of unknown etiology. Title: Mathematical Model for Bone Mineralization Author: Komarova, Svetlana V., Safranek, Lee, Gopalakrishnan, Jay, Ou, Miao-jung Yvonne, McKee, Marc D., Murshed, Monzur, Rauch, Frank, Zuhr, Erica Year: 2015 Defective bone mineralization has serious clinical manifestations, including deformities and fractures, but the regulation of this extracellular process is not fully understood. We have developed a mathematical model consisting of ordinary differential equations that describe collagen maturation, production and degradation of inhibitors, and mineral nucleation and growth. We examined the roles of individual processes in generating normal and abnormal mineralization patterns characterized using two outcome measures: mineralization lag time and degree of mineralization. Model parameters describing the formation of hydroxyapatite mineral on the nucleating centers most potently affected the degree of mineralization, while the parameters describing inhibitor homeostasis most effectively changed the mineralization lag time. Of interest, a parameter describing the rate of matrix maturation emerged as being capable of counter-intuitively increasing both the mineralization lag time and the degree of mineralization. We validated the accuracy of model predictions using known diseases of bone mineralization such as osteogenesis imperfecta and X-linked hypophosphatemia. The model successfully describes the highly nonlinear mineralization dynamics, which includes an initial lag phase when osteoid is present but no mineralization is evident, then fast primary mineralization, followed by secondary mineralization characterized by a continuous slow increase in bone mineral content. The developed model can potentially predict the function for a mutated protein based on the histology of pathologic bone samples from mineralization disorders of unknown etiology. Close

• 3. [Article] Extrahepatic 25-Hydroxylation of Vitamin D3 in an Engineered Osteoblast Precursor Cell Line Exploring the Influence on Cellular Proliferation and Matrix Maturation during Bone Development

  Osteoblastic precursors experience distinct stages during differentiation and bone development, which include proliferation, extracellular matrix (ECM) maturation, and ECM mineralization. It is well known ...

  Citation × Citation Citation Abstract Copy Title: Extrahepatic 25-Hydroxylation of Vitamin D3 in an Engineered Osteoblast Precursor Cell Line Exploring the Influence on Cellular Proliferation and Matrix Maturation during Bone Development Author: Mason, Shelley S., Kohles, Sean S., Winn, Shelley R., Zelick, Randy D. Year: 2013 Osteoblastic precursors experience distinct stages during differentiation and bone development, which include proliferation, extracellular matrix (ECM) maturation, and ECM mineralization. It is well known that vitamin D plays a large role in the regulation of bone mineralization and homeostasis via the endocrine system. The activation of vitamin D requires two sequential hydroxylation steps, first in the kidney and then in the liver, in order to carry out its role in calcium homeostasis. Recent research has demonstrated that human-derived mesenchymal stem cells (MSCs) and osteoblasts can metabolize the immediate vitamin D precursor 25- dihydroxyvitamin D₃(25OH₂D₃) to the active steroid 1α,25-dihydroxyvitamin D₃ (1,25OH₂D₃) and elicit an osteogenic response. However, reports of extrahepatic metabolism of vitamin D₃, the parental vitamin D precursor, have been limited. In this study, we investigated whether osteoblast precursors have the capacity to convert vitamin D₃to 1,25OH₂D₃ and examined the potential of vitamin D3 to induce 1,25OH₂D₃ associated biological activities in osteoblast precursors. It was demonstrated that the engineered osteoblast precursor derived from human marrow (OPC1) is capable of metabolizing vitamin D₃ to 1,25OH₂D₃ in a dose- dependent manner. It was also demonstrated that administration of vitamin D₃ leads to the increase in alkaline phosphatase (ALP) activity associated with osteoblast ECM maturation and calcium deposits and a decrease in cellular proliferation in both osteoblast precursor cell lines OPC1 and MC3T3-E1. These findings provide a two-dimensional culture foundation for future three- dimensional engineered tissue studies using the OPC1 cell line. Title: Extrahepatic 25-Hydroxylation of Vitamin D3 in an Engineered Osteoblast Precursor Cell Line Exploring the Influence on Cellular Proliferation and Matrix Maturation during Bone Development Author: Mason, Shelley S., Kohles, Sean S., Winn, Shelley R., Zelick, Randy D. Year: 2013 Osteoblastic precursors experience distinct stages during differentiation and bone development, which include proliferation, extracellular matrix (ECM) maturation, and ECM mineralization. It is well known that vitamin D plays a large role in the regulation of bone mineralization and homeostasis via the endocrine system. The activation of vitamin D requires two sequential hydroxylation steps, first in the kidney and then in the liver, in order to carry out its role in calcium homeostasis. Recent research has demonstrated that human-derived mesenchymal stem cells (MSCs) and osteoblasts can metabolize the immediate vitamin D precursor 25- dihydroxyvitamin D₃(25OH₂D₃) to the active steroid 1α,25-dihydroxyvitamin D₃ (1,25OH₂D₃) and elicit an osteogenic response. However, reports of extrahepatic metabolism of vitamin D₃, the parental vitamin D precursor, have been limited. In this study, we investigated whether osteoblast precursors have the capacity to convert vitamin D₃to 1,25OH₂D₃ and examined the potential of vitamin D3 to induce 1,25OH₂D₃ associated biological activities in osteoblast precursors. It was demonstrated that the engineered osteoblast precursor derived from human marrow (OPC1) is capable of metabolizing vitamin D₃ to 1,25OH₂D₃ in a dose- dependent manner. It was also demonstrated that administration of vitamin D₃ leads to the increase in alkaline phosphatase (ALP) activity associated with osteoblast ECM maturation and calcium deposits and a decrease in cellular proliferation in both osteoblast precursor cell lines OPC1 and MC3T3-E1. These findings provide a two-dimensional culture foundation for future three- dimensional engineered tissue studies using the OPC1 cell line. Close

• 4. [Article] Microbial Biomineralization of Iron

  Iron is a common cation in biomineral sand; it is present for example in magnetite produced by magnetotactic bacteria and in iron sulfides produced by sulfate reducing microorganisms. The work presented ...

  Citation × Citation Citation Abstract Copy Title: Microbial Biomineralization of Iron Author: Fang, Wen Year: 2013 Iron is a common cation in biomineral sand; it is present for example in magnetite produced by magnetotactic bacteria and in iron sulfides produced by sulfate reducing microorganisms. The work presented in this thesis focused on two types of microorganisms capable of forming iron biominerals. In the first project I have studied the effect of O2 on the respiratory physiology and the formation of magnetosomes by Magnetospirillum magneticum AMB-1. In the second project I have studied the relationship between olivine and the activity of dissimilatory sulfate reducing (DSR) microorganisms. For the first project, I grew cells of AMB-1 in cultures with various concentrations of O2 and monitored growth and the formation of magnetic mineral particles (MMP). Results have shown that AMB-1 cells grew better at 100-225 uMO2(aq) than at lower [O2], yet the formation of MMP was repressed at ~45 uM O2(aq) and strongly inhibited at >100 uM O2(aq).These results have helped better understand the dissimilarity between the optimal growth conditions of magnetotactic bacteria and the conditions needed for the formation of MMPs. My results have also shown that the reaction between H2S produced by DSRs and olivine is abiotic, not catalyzed and exergonic. The pH did not vary significantly during this reaction and pH variation (in the 5-9 range) did not significantly influence this chemical reaction. Bicarbonate inhibited the reaction between H2S and olivine, but not the chemical equilibrium. Phosphate, a weak iron chelator, influenced the equilibrium of the reaction and it is assumed to help increase the rate of olivine weathering in the presence of DSRs. The activity of DSRs was positively influenced by the presence and abundance of olivine. Based on my results I propose that olivine help DSR obtain energy more efficiently, but does not represent a source of energy or nutrients for the cells. These results helped better understand the formation of iron biominerals and signatures of this activity. Title: Microbial Biomineralization of Iron Author: Fang, Wen Year: 2013 Iron is a common cation in biomineral sand; it is present for example in magnetite produced by magnetotactic bacteria and in iron sulfides produced by sulfate reducing microorganisms. The work presented in this thesis focused on two types of microorganisms capable of forming iron biominerals. In the first project I have studied the effect of O2 on the respiratory physiology and the formation of magnetosomes by Magnetospirillum magneticum AMB-1. In the second project I have studied the relationship between olivine and the activity of dissimilatory sulfate reducing (DSR) microorganisms. For the first project, I grew cells of AMB-1 in cultures with various concentrations of O2 and monitored growth and the formation of magnetic mineral particles (MMP). Results have shown that AMB-1 cells grew better at 100-225 uMO2(aq) than at lower [O2], yet the formation of MMP was repressed at ~45 uM O2(aq) and strongly inhibited at >100 uM O2(aq).These results have helped better understand the dissimilarity between the optimal growth conditions of magnetotactic bacteria and the conditions needed for the formation of MMPs. My results have also shown that the reaction between H2S produced by DSRs and olivine is abiotic, not catalyzed and exergonic. The pH did not vary significantly during this reaction and pH variation (in the 5-9 range) did not significantly influence this chemical reaction. Bicarbonate inhibited the reaction between H2S and olivine, but not the chemical equilibrium. Phosphate, a weak iron chelator, influenced the equilibrium of the reaction and it is assumed to help increase the rate of olivine weathering in the presence of DSRs. The activity of DSRs was positively influenced by the presence and abundance of olivine. Based on my results I propose that olivine help DSR obtain energy more efficiently, but does not represent a source of energy or nutrients for the cells. These results helped better understand the formation of iron biominerals and signatures of this activity. Close