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1.
Front Bioeng Biotechnol ; 10: 979497, 2022.
Article in English | MEDLINE | ID: mdl-36277394

ABSTRACT

Bioengineering applies analytical and engineering principles to identify functional biological building blocks for biotechnology applications. While these building blocks are leveraged to improve the human condition, the lack of simplistic, machine-readable definition of biohazards at the function level is creating a gap for biosafety practices. More specifically, traditional safety practices focus on the biohazards of known pathogens at the organism-level and may not accurately consider novel biodesigns with engineered functionalities at the genetic component-level. This gap is motivating the need for a paradigm shift from organism-centric procedures to function-centric biohazard identification and classification practices. To address this challenge, we present a novel methodology for classifying biohazards at the individual sequence level, which we then compiled to distinguish the biohazardous property of pathogenicity at the whole genome level. Our methodology is rooted in compilation of hazardous functions, defined as a set of sequences and associated metadata that describe coarse-level functions associated with pathogens (e.g., adherence, immune subversion). We demonstrate that the resulting database can be used to develop hazardous "fingerprints" based on the functional metadata categories. We verified that these hazardous functions are found at higher levels in pathogens compared to non-pathogens, and hierarchical clustering of the fingerprints can distinguish between these two groups. The methodology presented here defines the hazardous functions associated with bioengineering functional building blocks at the sequence level, which provide a foundational framework for classifying biological hazards at the organism level, thus leading to the improvement and standardization of current biosecurity and biosafety practices.

2.
J Clin Endocrinol Metab ; 103(11): 4135-4145, 2018 11 01.
Article in English | MEDLINE | ID: mdl-30165401

ABSTRACT

Context: Although important advances have been made in understanding the genetics of endocrine tumors, cellular physiology is relatively understudied as a determinant of tumor behavior. Oxidative stress and reactive oxygen species are metabolic factors that may affect tumor behavior, and these are, in part, controlled by manganese-dependent superoxide dismutase (MnSod), the mitochondrial superoxide dismutase (encoded by SOD2). Objective: We sought to understand the role of MnSod in the prognosis of aggressive human endocrine cancers and directly assessed the effect of MnSod under- or overexpression on tumor behavior, using established mouse thyroid cancer models. Methods: We performed transcriptome analysis of human and mouse models of endocrine cancer. To address the role of Sod2 in endocrine tumors, we introduced a Sod2 null allele or a transgenic Sod2 overexpression allele into mouse models of benign thyroid follicular neoplasia or aggressive, metastatic follicular thyroid cancer (FTC) and monitored phenotypic changes in tumor initiation and progression. Results: In the thyroid, SOD2/Sod2 was downregulated in FTC but not papillary thyroid cancer. Reduced expression of SOD2 was correlated with poorer survival of patients with aggressive thyroid or adrenal cancers. In mice with benign thyroid tumors, Sod2 overexpression increased tumor burden. In contrast, in mice with aggressive FTC, overexpression of Sod2 reduced tumor proliferation and improved mortality rates, whereas its deficiency enhanced tumor growth. Conclusion: Overall, our results indicate that SOD2 has dichotomous roles in cancer progression and acts in a context-specific manner.


Subject(s)
Adenocarcinoma, Follicular/pathology , Adrenal Gland Neoplasms/pathology , Superoxide Dismutase/metabolism , Thyroid Cancer, Papillary/pathology , Thyroid Neoplasms/pathology , Adenocarcinoma, Follicular/genetics , Adenocarcinoma, Follicular/mortality , Adrenal Gland Neoplasms/mortality , Animals , Cell Transformation, Neoplastic , Disease Models, Animal , Disease Progression , Down-Regulation , Female , Gene Expression Profiling , Humans , Male , Mice , Mice, Transgenic , Oxidative Stress , Reactive Oxygen Species/metabolism , Superoxide Dismutase/genetics , Survival Analysis , Survival Rate , Thyroid Cancer, Papillary/genetics , Thyroid Cancer, Papillary/mortality , Thyroid Gland/pathology , Thyroid Neoplasms/mortality , Tumor Burden
3.
Thyroid ; 28(9): 1153-1161, 2018 09.
Article in English | MEDLINE | ID: mdl-29882482

ABSTRACT

BACKGROUND: Thyroid cancer is an emerging health problem in the United States and worldwide. With incidence rates of thyroid cancer rapidly rising, the need to develop new treatment options is becoming a priority, and understanding the molecular mechanisms of this disease is crucial to furthering these efforts. Thyroid growth is driven by the TSH/cAMP/PKA signaling pathway, and it has previously been shown that activation of PKA through genetic ablation of the regulatory subunit Prkar1a (Prkar1a KO) is sufficient to cause follicular thyroid cancer in mouse models. cAMP also activates the Epac proteins and their downstream effectors, Rap1a and Rap1b. METHODS: Previously, the authors' laboratory generated a mouse model of follicular thyroid cancer by conferring thyroid-specific deletion of Prkar1a (R1a-TpoKO). To probe the roles of other components of the PKA signaling system in the development of thyroid cancer, this study deleted Rap1 and Epac1 in the setting of the Prkar1a knockout. RESULTS: Deletion of Rap1 significantly decreases thyroid size and cancer incidence in Prkar1a KO thyroids. Further, isoform-specific ablation of Rap1a and Rap1b implicates Rap1b as the downstream effector of PKA during thyroid carcinogenesis. In vivo modeling provides definitive evidence that Epac1 plays little role in thyroid proliferation and is dispensable for thyroid carcinogenesis arising from the deletion of Prkar1a. CONCLUSIONS: This study demonstrate that PKA signaling to Rap1b is a key signaling node for follicular thyroid carcinogenesis, while Epac1 activity is not required for tumor development. This work sheds new light on the pathways involved in FTC development and identifies a possible target for the development of new therapies in the treatment of FTC.


Subject(s)
Adenocarcinoma, Follicular/genetics , Carcinogenesis/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Guanine Nucleotide Exchange Factors/genetics , Thyroid Neoplasms/genetics , rap GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/genetics , Adenocarcinoma, Follicular/metabolism , Adenocarcinoma, Follicular/pathology , Animals , Carcinogenesis/metabolism , Carcinogenesis/pathology , Guanine Nucleotide Exchange Factors/metabolism , Mice , Mice, Knockout , Thyroid Gland/metabolism , Thyroid Gland/pathology , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathology , rap GTP-Binding Proteins/metabolism , rap1 GTP-Binding Proteins/metabolism
4.
Horm Behav ; 98: 121-129, 2018 02.
Article in English | MEDLINE | ID: mdl-29289659

ABSTRACT

Alterations in circulating thyroid hormone concentrations are associated with several psychological and behavioral disorders. In humans, behavioral disorders such as anxiety, depression, and attention-deficit hyperactivity disorder can be associated with thyroid disease. The Tpo-Cre;Prkar1aflox/flox;Epac1-/- (R1A-Epac1KO) mice, originally bred to investigate the role of exchange protein directly activated by cAMP (Epac1) in follicular thyroid cancer, displayed self-mutilating and aggressive behaviors during casual observation. To assess these atypical responses, behavioral testing was conducted with the R1A-Epac1KO mice, as well as their single knockout counterparts, the thyroid-specific Prkar1a-/- and global Epac1-/- mice. Mice of all three genotypes demonstrated increased aggressive behavior against an intruder mouse. In addition, Epac1-/- mice increased response to an auditory stimulus, and the Prkar1a-/- and R1A-Epac1KO mice increased swimming behavior in the Porsolt forced swim test. Both Prkar1a-/- mice and R1A-Epac1KO mice have increased circulating thyroxine and corticosterone concentrations. Although hyperthyroidism has not been previously associated with aggression, increased thyroid hormone signaling might contribute to the increased aggressive response to the intruder mouse, as well as the increased swimming response. Mice with a genetic background of Tpo-Cre;Prkar1aflox/flox;Epac1-/- are aggressive, and both the thyroid-specific knockout of Prkar1a and global knockout of Epac1 likely contribute to this aggressive behavior. This study supports the hypothesis that altered thyroid signaling and aggressive behavior are linked.


Subject(s)
Aggression/physiology , Behavior, Animal/physiology , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/genetics , Guanine Nucleotide Exchange Factors/genetics , Thyroid Gland/metabolism , Animals , Anxiety/genetics , Gene Deletion , Genotype , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Organ Specificity/genetics , Signal Transduction/genetics
5.
Endocr Relat Cancer ; 24(11): 579-591, 2017 11.
Article in English | MEDLINE | ID: mdl-28928232

ABSTRACT

Mutations in genes encoding enzymes in the tricarboxylic acid cycle (TCA, also known as the Krebs cycle) have been implicated as causative genetic lesions in a number of human cancers, including renal cell cancers, glioblastomas and pheochromocytomas. In recent studies, missense mutations in the succinate dehydrogenase (SDH) complex have also been proposed to cause differentiated thyroid cancer. In order to gain mechanistic insight into this process, we generated mice lacking the SDH subunit D (Sdhd) in the thyroid. We report that these mice develop enlarged thyroid glands with follicle hypercellularity and increased proliferation. In vitro, human thyroid cell lines with knockdown of SDHD exhibit an enhanced migratory capability, despite no change in proliferative capacity. Interestingly, these cells acquire stem-like features which are also observed in the mouse tumors. The stem-like characteristics are reversed by α-ketoglutarate, suggesting that SDH-associated tumorigenesis results from dedifferentiation driven by an imbalance in cellular metabolites of the TCA cycle. The results of this study reveal a metabolic vulnerability for potential future treatment of SDH-associated neoplasia.


Subject(s)
Electron Transport Complex II/genetics , Membrane Proteins/genetics , Thyroid Neoplasms/pathology , Animals , Carcinogenesis , Cell Line , Cell Line, Tumor , Cell Movement , Cell Proliferation , DNA Methylation , Electron Transport Complex II/metabolism , Humans , Membrane Proteins/metabolism , Mice, Transgenic , Phenotype , Protein Subunits/genetics , Protein Subunits/metabolism , Succinate Dehydrogenase , Wound Healing
6.
Arterioscler Thromb Vasc Biol ; 36(2): 328-38, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26634652

ABSTRACT

OBJECTIVE: Aortic valve disease, including calcification, affects >2% of the human population and is caused by complex interactions between multiple risk factors, including genetic mutations, the environment, and biomechanics. At present, there are no effective treatments other than surgery, and this is because of the limited understanding of the mechanisms that underlie the condition. Previous work has shown that valve interstitial cells within the aortic valve cusps differentiate toward an osteoblast-like cell and deposit bone-like matrix that leads to leaflet stiffening and calcific aortic valve stenosis. However, the mechanisms that promote pathological phenotypes in valve interstitial cells are unknown. APPROACH AND RESULTS: Using a combination of in vitro and in vivo tools with mouse, porcine, and human tissue, we show that in valve interstitial cells, reduced Sox9 expression and nuclear localization precedes the onset of calcification. In vitro, Sox9 nuclear export and calcific nodule formation is prevented by valve endothelial cells. However, in vivo, loss of Tgfß1 in the endothelium leads to reduced Sox9 expression and calcific aortic valve disease. CONCLUSIONS: Together, these findings suggest that reduced nuclear localization of Sox9 in valve interstitial cells is an early indicator of calcification, and therefore, pharmacological targeting to prevent nuclear export could serve as a novel therapeutic tool in the prevention of calcification and stenosis.


Subject(s)
Aortic Valve Stenosis/metabolism , Aortic Valve/metabolism , Aortic Valve/pathology , Calcinosis/metabolism , Endothelial Cells/metabolism , Paracrine Communication , SOX9 Transcription Factor/metabolism , Signal Transduction , Transforming Growth Factor beta1/metabolism , Active Transport, Cell Nucleus , Animals , Aortic Valve Stenosis/genetics , Aortic Valve Stenosis/pathology , Aortic Valve Stenosis/prevention & control , Calcinosis/genetics , Calcinosis/pathology , Calcinosis/prevention & control , Cells, Cultured , Collagen Type II/genetics , Collagen Type II/metabolism , Endothelial Cells/pathology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , SOX9 Transcription Factor/genetics , Swine , Time Factors , Tissue Culture Techniques , Transfection , Transforming Growth Factor beta1/genetics , rho-Associated Kinases/metabolism
7.
Arterioscler Thromb Vasc Biol ; 33(2): 285-93, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23202364

ABSTRACT

OBJECTIVE: Calcific aortic valve disease (CAVD) is a major public health problem with no effective treatment available other than surgery. We previously showed that mature heart valves calcify in response to retinoic acid (RA) treatment through downregulation of the SRY transcription factor Sox9. In this study, we investigated the effects of excess vitamin A and its metabolite RA on heart valve structure and function in vivo and examined the molecular mechanisms of RA signaling during the calcification process in vitro. METHODS AND RESULTS: Using a combination of approaches, we defined calcific aortic valve disease pathogenesis in mice fed 200 IU/g and 20 IU/g of retinyl palmitate for 12 months at molecular, cellular, and functional levels. We show that mice fed excess vitamin A develop aortic valve stenosis and leaflet calcification associated with increased expression of osteogenic genes and decreased expression of cartilaginous markers. Using a pharmacological approach, we show that RA-mediated Sox9 repression and calcification is regulated by classical RA signaling and requires both RA and retinoid X receptors. CONCLUSIONS: Our studies demonstrate that excess vitamin A dietary intake promotes heart valve calcification in vivo. Therefore suggesting that hypervitaminosis A could serve as a new risk factor of calcific aortic valve disease in the human population.


Subject(s)
Aortic Valve/metabolism , Calcinosis/etiology , Dietary Supplements , Heart Valve Diseases/etiology , Hypervitaminosis A/complications , Vitamin A/analogs & derivatives , Vitamins , Animals , Aortic Valve/pathology , Calcinosis/genetics , Calcinosis/metabolism , Calcinosis/pathology , Cell Line , Chick Embryo , Collagen Type II/genetics , Collagen Type II/metabolism , Disease Models, Animal , Diterpenes , Gene Expression Profiling/methods , Gene Expression Regulation , Heart Valve Diseases/genetics , Heart Valve Diseases/metabolism , Heart Valve Diseases/pathology , Hypervitaminosis A/chemically induced , Hypervitaminosis A/genetics , Hypervitaminosis A/metabolism , Hypervitaminosis A/pathology , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Osteogenesis/genetics , Osteopontin/genetics , Osteopontin/metabolism , RNA Interference , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors/genetics , Retinoid X Receptors/metabolism , Retinyl Esters , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Signal Transduction , Time Factors , Tissue Culture Techniques , Transfection , Tretinoin/metabolism , Vitamin A/metabolism , Vitamins/metabolism
8.
J Mol Cell Cardiol ; 53(5): 626-38, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22906538

ABSTRACT

Collagen XIV is a fibril-associated collagen with an interrupted triple helix (FACIT). Previous studies have shown that this collagen type regulates early stages of fibrillogenesis in connective tissues of high mechanical demand. Mice null for Collagen XIV are viable, however formation of the interstitial collagen network is defective in tendons and skin leading to reduced biomechanical function. The assembly of a tightly regulated collagen network is also required in the heart, not only for structural support but also for controlling cellular processes. Collagen XIV is highly expressed in the embryonic heart, notably within the cardiac interstitium of the developing myocardium, however its role has not been elucidated. To test this, we examined cardiac phenotypes in embryonic and adult mice devoid of Collagen XIV. From as early as E11.5, Col14a1(-/-) mice exhibit significant perturbations in mRNA levels of many other collagen types and remodeling enzymes (MMPs, TIMPs) within the ventricular myocardium. By post natal stages, collagen fibril organization is in disarray and the adult heart displays defects in ventricular morphogenesis. In addition to the extracellular matrix, Col14a1(-/-) mice exhibit increased cardiomyocyte proliferation at post natal, but not E11.5 stages, leading to increased cell number, yet cell size is decreased by 3 months of age. In contrast to myocytes, the number of cardiac fibroblasts is reduced after birth associated with increased apoptosis. As a result of these molecular and cellular changes during embryonic development and post natal maturation, cardiac function is diminished in Col14a1(-/-) mice from 3 months of age; associated with dilation in the absence of hypertrophy, and reduced ejection fraction. Further, Col14a1 deficiency leads to a greater increase in left ventricular wall thickening in response to pathological pressure overload compared to wild type animals. Collectively, these studies identify a new role for type XIV collagen in the formation of the cardiac interstitium during embryonic development, and highlight the importance of the collagen network for myocardial cell survival, and function of the working myocardium after birth.


Subject(s)
Collagen/deficiency , Glycoproteins/deficiency , Heart/growth & development , Myocardium/metabolism , Animals , Cell Proliferation , Collagen/genetics , Collagen/physiology , Glycoproteins/genetics , Glycoproteins/physiology , Heart Ventricles/metabolism , Heart Ventricles/pathology , Heart Ventricles/physiopathology , Hypertrophy, Left Ventricular/metabolism , Hypertrophy, Left Ventricular/pathology , Hypertrophy, Left Ventricular/physiopathology , In Vitro Techniques , Male , Mice , Mice, Transgenic , Myocardial Contraction , Myocardium/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/physiology , Stroke Volume , Transcription, Genetic , Ventricular Function, Left , Ventricular Pressure , Ventricular Remodeling
9.
PLoS One ; 6(10): e26769, 2011.
Article in English | MEDLINE | ID: mdl-22046352

ABSTRACT

Sox9 is an SRY-related transcription factor required for expression of cartilaginous genes in the developing skeletal system and heart valve structures. In contrast to positively regulating cartilaginous matrix, Sox9 also negatively regulates matrix mineralization associated with bone formation. While the transcriptional activation of Sox9 target genes during chondrogenesis has been characterized, the mechanisms by which Sox9 represses osteogenic processes are not so clear. Using ChIP-on-chip and luciferase assays we show that Sox9 binds and represses transactivation of the osteogenic glycoprotein Spp1. In addition, Sox9 knockdown in post natal mouse heart valve explants and rib chondrocyte cultures promotes Spp1 expression and matrix mineralization, while attenuating expression of cartilage genes Type II Collagen and Cartilage Link Protein. Further, we show that Spp1 is required for matrix mineralization induced by Sox9 knockdown. These studies provide insights into the molecular mechanisms by which Sox9 prevents pathologic matrix mineralization in tissues that must remain cartilaginous.


Subject(s)
Calcinosis/etiology , Chondrocytes/metabolism , Extracellular Matrix/metabolism , Heart Valves/metabolism , Osteopontin/antagonists & inhibitors , SOX9 Transcription Factor/physiology , Animals , Calcinosis/prevention & control , Chondrogenesis , Extracellular Matrix/pathology , Mice , Osteopontin/genetics , Repressor Proteins , Transcription, Genetic
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