Browsing by Author "Samuels, David C."

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    An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors
    Wendelsdorf, Katherine V.; Song, Zhuo; Samuels, David C. (Public Library of Science, 2009-01-09)
    Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-gamma hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-gamma) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-gamma with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication.
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    Assessing the Cardiovagal Baroreflex
    Behnam, Abrahm John (Virginia Tech, 2007-02-02)
    Abrupt decreases and increases in systolic arterial blood pressure produce baroreflex mediated shortening and lengthening, respectively, of the R-R interval. This phenomenon, otherwise known as the cardivagal baroreflex, is best described by the sigmoid relationship between R-R interval length and systolic blood pressure. The linear portion of this relationship is used to derive the slope or gain of the cardiovagal baroreflex. Importantly, lower levels of cardiovagal baroreflex have been associated with poor orthostatic tolerance and an increased cardiovascular disease-related mortality. The most commonly used and accepted technique to assess cardiovagal barorelex gain is the modified Oxford techinique. Bolus injections of sodium nitroprusside followed by phenylephrine HCL are used to decrease and raise blood pressure ~15 mmHg, respectively. The baroreflex control of the cardiac vagal outflow can then be assessed by the relation of the R-R interval to systolic blood pressure. However, the modified Oxford technique does not always reveal the nonlinear nature of baroreflex relations. The reasons for this has been unclear. Thus, analysis of baroreflex gain when nonlinearities are not revealed is problematic. Five classifications of baroreflex trials have been identified: acceptable, threshold-heavy, saturation-heavy, linear-heavy, and random trials. A new method of gain estimation was developed that combines the strengths of the current methods of gain estimation with the knowledge of the classifications of baroreflex trials. Using this method, cardiovagal baroreflex gain assessment can be maximized if threshold-heavy, saturation-heavy, and random trials are filtered out of the analysis and the manual method is used to estimate gain on the remaining trials. In addition, a link seems to exist between the variability of delta and the variability in baroreflex gain between different subjects.
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    An Assessment of the Molecular Basis of Toxin-induced Dilated Cardiomyopathy in an Avian Animal Model
    Tian, Xi (Virginia Tech, 2008-12-09)
    Dilated cardiomyopathy (DCM), a disease of the myocardium, causes morbidity and premature death in humans and other domestic animals including turkeys. Though DCM results from many different factors including those that are unknown or idiopathic, genetic factor is a major cause of idiopathic DCM. In this study, I assessed the molecular basis of toxin-induced DCM in turkeys by evaluating the association and effect of mutations in candidate genes in the nucleus and mitochondria on the incidence and severity of this disease. Echocardiographic measurements at 3 weeks of age showed that birds on furazolidone-containing diet exhibited a significant DCM phenotype (increased left ventricular end diastolic dimension and left ventricular end systolic dimension) with a marked decrease in the left ventricular shortening fraction. Pathological phenotype confirmed the dilated heart with extended cell necrosis. Two mutations, both in NADH dehydrogenase genes, were found to be associated with DCM. Real-time RT-PCR quantification indicated that mRNA expression of alpha cardiac actin gene (ACTC) were significantly different between control and treatment birds. While ACTC expression increased, though moderately, in control birds from week 1 to 3, it decreased significantly in treatment birds. These findings suggest that the mitochondrial DNA variation and ACTC expression may be associated with the turkey's response to toxin. Therefore, further research is needed to investigate the molecular mechanism of toxin-induced DCM in the turkey.
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    An Assessment of the Relationship among Oxidative Stress, Adaptive Immunity and Genetic Variations in the Chicken, Gallus gallus
    Deng, Hui (Virginia Tech, 2010-09-01)
    Oxidative stress (OS) has been associated with aging and age-related diseases in humans, as well as with the decline in economic trait performance in poultry and other domesticated animals. However, the potential effects of OS on the poultry immune system are not well understood. In addition, the impact of bird genetic variation on redox balance remains to be elucidated. Thus, the central hypothesis of this dissertation is: The bird's adaptive immunocompetence is impacted by their OS level, which is not only influenced by environmental factors, but also related to genetic phenotype of either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). In the first phase of this study, White Leghorn chickens were provided ethanol at different concentrations in drinking water to induce OS. Biomarkers including malondialdehyde (MDA), glutathione (GSH), and plasma uric acid (PUA) were measured to assess OS before and after ethanol treatment. The adaptive immune response during an OS event was measured by plasma IgG and IgM levels, major lymphoid organ weights, CD4+/CD8+ cell ratio, and histopathological analysis of the immune organs. Results showed that when OS was induced by 10% ethanol, chicken adaptive immune responses decreased; however, when birds were exposed to 2% ethanol, there was an enhancement in antioxidant defense and immune response; These results would suggest a negative correlation between OS level and chicken adaptive immune response. In the second phase of the study, subsets of chickens were selected based on their high (H)- or low (L)-OS to assess for variations in their genetic phenotypes. Using MDA levels, 36 chickens were chosen to scan a 2734-bp region of mtDNA, but no definitive SNP was detected. In another experiment, 40 chickens were conversely selected according to three biomarkers for OS. Although no variation was found at eight SNP loci tested across the mitochondrial genome, mtDNA damage measured by 8-hydroxy-2′-deoxy-guanosine was shown to increase with time, and at higher levels in the high OS birds (p < 0.05). Thses results suggest that long-term high OS levels in chickens may increase the somatic mutation of their mtDNA. In the final phase of this dissertation, the effect of nDNA on OS, measured via a genome-wide association study was performed with 18 H and 18 L chickens using the latest chicken 60k SNP microarray for genotyping. Among 56,483 SNPs successfully genotyped, 13 SNPs across five independent loci were associated with OS at significance level of p ≤ 0.001, and another 144 SNPs were also associated with OS (p ≤ 0.01). These results indicate new loci and related genes for their genetic influence upon redox balance. In general, experiments carried out on White Leghorn chickens here have shown that adaptive immune response is tightly related to changes of OS. Further, genetic variance in nDNA is associated with the risk of high OS or the ability to better resist it.
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    Discrete Transition System Model and Verification for Mitochondrially Mediated Apoptotic Signaling Pathways
    Lam, Huy Hong (Virginia Tech, 2007-06-20)
    Computational biology and bioinformatics for apoptosis have been gaining much momentum due to the advances in computational sciences. Both fields use extensive computational techniques and modeling to mimic real world behaviors. One problem of particular interest is on the study of reachability, in which the goal is to determine if a target state or protein concentration in the model is realizable for a signaling pathway. Another interesting problem is to examine faulty pathways and how a fault can make a previously unrealizable state possible, or vice versa. Such analysis can be extremely valuable to the understanding of apoptosis. However, these analyses can be costly or even impractical for some approaches, since they must simulate every aspect of the model. Our approach introduces an abstracted model to represent a portion of the apoptosis signaling pathways as a finite state machine. This abstraction allows us to apply hardware testing and verification techniques and also study the behaviors of the system without full simulation. We proposed a framework that is tailor-built to implement these verification techniques for the discrete model. Through solving Boolean constraint satisfaction problems (SAT-based) and with guided stimulation (Genetic Algorithm), we can further extract the properties and behaviors of the system. Furthermore, our model allows us to conduct cause-effect analysis of the apoptosis signaling pathways. By constructing single- and double-fault models, we are able to study what fault(s) can cause the model to malfunction and the reasons behind it. Unlike simulation, our abstraction approach allows us to study the system properties and system manipulations from a different perspective without fully relying on simulation. Using these observations as hypotheses, we aim to conduct laboratory experiments and further refine our model.
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    Genomic and biochemical analysis of oxidative stress in birds with diverse longevities
    Guan, Xiaojing (Virginia Tech, 2007-05-10)
    The relationship among oxidative stress, mitochondrial DNA integrity, and longevity continues to be without a general consensus. Here, we hypothesize that short- and long-lived birds, including the budgerigar (Melopsittacus undulatus), guineafowl (Numida meleagris), quail (Corturnix japonica), and turkey (Meleagris gallopavo) differ in oxidative stress measured by blood markers and that this difference correlates with mitochondrial genomic integrity both within and among species. In preliminary studies and to establish a reference and standard for the search for single nucleotide polymorphisms (SNPs), we used a combination of experimental and in silico tools for genome analysis to screen selected regions of the chicken (Gallus gallus) mitochondrial genome (mtGenome) for SNPs. A total of 113 SNPs was identified which formed 17 haplotypes. The length of the turkey mtGenome sequence developed was 16,967 bp in length, while that of the budgie was 18,193 bp. Annotation of both sequences revealed a total of 13 genes and 24 RNA (22 tRNA and 2 rRNA). Within the budgie mtGenome sequence, a duplicated control region was observed, and there was an additional nucleotide in the NADH dehydrogenase subunit 3 sequence of both the turkey and budgie. The total number of SNPs within the D-loop and 16S rRNA in each of the four species ranged from zero in the quail to 22 in the budgie. The new mtGenome sequences revealed that the turkey was most closely related to the chicken and quail, and the budgie was closest to kakabo (Strigops habroptilus). Oxidative stress, estimated by biomarkers thiobarbiturate acid reacting substance (TBARS), plasma uric acid (PUA), and glutathione (GSH) and at 10, 30, 55, and 80 wks-of-age within each species, was not consistent. The level of GSH was highest in guineafowl, but lowest in budgie. While PUA, an antioxidant, exhibited a significantly (P<0.05) decrease as birds grew order, TBARS, a lipid peroxidation index, increased with age. In general, oxidant and antioxidant status appeared to vary among species and to be significantly affected by age, unlike mutations in the mtDNA which remained the same in younger and older birds. This primary findings and discoveries of this dissertation research include the large scale SNP discovery in previously described and novel avian mtGenomes including the chicken and turkey, the two main poultry species, and the determination that oxidative stress in birds appears to vary with age but that this does not affect mitochondrial DNA variation. Recent evidence of work in mice appears to support results described in this dissertation that mitochondrial DNA mutations do not increase with age, the central paradigm of the "Free Radical Theory of Aging". The dissertation also described resources and data that will be a foundation for the use of birds, especially the budgie, as a model for testing this theory that remains of interest to both agricultural and biomedical sciences.
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    Insights Into Mitochondrial Genetic and Morphologic Dynamics Gained by Stochastic Simulation
    Rajasimha, Harsha Karur (Virginia Tech, 2007-12-05)
    MtDNA mutations in mammalian cells are implicated in cellular ageing and encephalomyopathies, although mechanisms involved are not completely understood. The mitochondrial genetic bottleneck has puzzled biologists for a long time. Approximate models of genetic bottleneck proposed in the literature do not accurately model underlying biology. Recent studies indicate mitochondrial morphology changes during cellular aging in culture. In particular, the rates of mitochondrial fission and fusion are shown to be in tight balance, though this rate decreases with age. Some proteins involved in mitochondrial morphology maintenance are implicated in apoptosis. Hence, mitochondrial genetic and morphologic dynamics are critical to the life and death of cells. By working closely with experimental collaborators and by utilizing data derived from literature, we have developed stochastic simulation models of mitochondrial genetic and morphologic dynamics. Hypotheses from the mitochondrial genetic dynamics model include: (1) the decay of mtDNA heteroplasmy in blood is exponential and not linear as reported in literature. (2) Blood heteroplasmy measurements are a good proxy for the blood stem cell heteroplasmy. (3) By analyzing our simulation results in tandem with published longitudinal clinical data, we propose for the first time, a way to correct for the patient's age in the analysis of heteroplasmy data. (4) We develop a direct model of the genetic bottleneck process during mouse embryogenesis. (5) Partitioning of mtDNA into daughter cells during blastocyst formation and relaxed replication of mtDNA during the exponential growth phase of primordial germ cells leads to the variation in heteroplasmy inherited by offspring from the same mother. (6) We develop a “simulation control” for experimental studies on mtDNA heteroplasmy variation in cell cultures. Hypothesis from the mitochondrial morphologic dynamics model: (7) A cell adjusts the mitochondrial fusion rate to compensate for the fluctuations in the fission rate, but not vice versa. A deterministic model for this control is proposed. Contributions: extensible simulation models of mitochondrial genetic and morphologic dynamics to aide in the powerful analysis of published and new experimental data. Our results have direct relevance to cell biology and clinical diagnosis. The work also illustrates scientific success by tight integration of theory with practice.
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    LYSMD3: A mammalian pattern recognition receptor for chitin
    He, Xin; Howard, Brad A.; Liu, Yang; Neumann, Aaron K.; Li, Liwu; Menon, Nidhi; Roach, Tiffany; Kale, Shiv D.; Samuels, David C.; Li, Hongyan; Kite, Trenton; Kita, Hirohito; Hu, Tony Y.; Luo, Mengyao; Jones, Caroline N.; Okaa, Uju Joy; Squillace, Diane L.; Klein, Bruce S.; Lawrence, Christopher B. (2021-07-20)
    Chitin, a major component of fungal cell walls, has been associated with allergic disorders such as asthma. However, it is unclear how mammals recognize chitin and the principal receptor(s) on epithelial cells that sense chitin remain to be determined. In this study, we show that LYSMD3 is expressed on the surface of human airway epithelial cells and demonstrate that LYSMD3 is able to bind chitin, as well as beta-glucan, on the cell walls of fungi. Knockdown or knockout of LYSMD3 also sharply blunts the production of inflammatory cytokines by epithelial cells in response to chitin and fungal spores. Competitive inhibition of the LYSMD3 ecto-domain by soluble LYSMD3 protein, multiple ligands, or antibody against LYSMD3 also blocks chitin signaling. Our study reveals LYSMD3 as a mammalian pattern recognition receptor (PRR) for chitin and establishes its role in epithelial cell inflammatory responses to chitin and fungi.
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    Mitochondrial-encoded membrane protein transcripts are pyrimidine-rich while soluble protein transcripts and ribosomal RNA are purine-rich
    Bradshaw, Patrick C.; Rathi, Anand; Samuels, David C. (2005-09-26)
    Background Eukaryotic organisms contain mitochondria, organelles capable of producing large amounts of ATP by oxidative phosphorylation. Each cell contains many mitochondria with many copies of mitochondrial DNA in each organelle. The mitochondrial DNA encodes a small but functionally critical portion of the oxidative phosphorylation machinery, a few other species-specific proteins, and the rRNA and tRNA used for the translation of these transcripts. Because the microenvironment of the mitochondrion is unique, mitochondrial genes may be subject to different selectional pressures than those affecting nuclear genes. Results From an analysis of the mitochondrial genomes of a wide range of eukaryotic species we show that there are three simple rules for the pyrimidine and purine abundances in mitochondrial DNA transcripts. Mitochondrial membrane protein transcripts are pyrimidine rich, rRNA transcripts are purine-rich and the soluble protein transcripts are purine-rich. The transitions between pyrimidine and purine-rich regions of the genomes are rapid and are easily visible on a pyrimidine-purine walk graph. These rules are followed, with few exceptions, independent of which strand encodes the gene. Despite the robustness of these rules across a diverse set of species, the magnitude of the differences between the pyrimidine and purine content is fairly small. Typically, the mitochondrial membrane protein transcripts have a pyrimidine richness of 56%, the rRNA transcripts are 55% purine, and the soluble protein transcripts are only 53% purine. Conclusion The pyrimidine richness of mitochondrial-encoded membrane protein transcripts is partly driven by U nucleotides in the second codon position in all species, which yields hydrophobic amino acids. The purine-richness of soluble protein transcripts is mainly driven by A nucleotides in the first codon position. The purine-richness of rRNA is also due to an abundance of A nucleotides. Possible mechanisms as to how these trends are maintained in mtDNA genomes of such diverse ancestry, size and variability of A-T richness are discussed.
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    Replication Pauses of the Wild-Type and Mutant Mitochondrial DNA Polymerase Gamma: A Simulation Study
    Song, Zhuo; Cao, Yang; Samuels, David C. (Public Library of Science, 2011-11-17)
    The activity of polymerase gamma is complicated, involving both correct and incorrect DNA polymerization events, exonuclease activity, and the disassociation of the polymerase: DNA complex. Pausing of pol-gamma might increase the chance of deletion and depletion of mitochondrial DNA. We have developed a stochastic simulation of pol-gamma that models its activities on the level of individual nucleotides for the replication of mtDNA. This method gives us insights into the pausing of two pol-gamma variants: the A467T substitution that causes PEO and Alpers syndrome, and the exonuclease deficient pol-gamma (exo(-)) in premature aging mouse models. To measure the pausing, we analyzed simulation results for the longest time for the polymerase to move forward one nucleotide along the DNA strand. Our model of the exo(-) polymerase had extremely long pauses, with a 30 to 300-fold increase in the time required for the longest single forward step compared to the wild-type, while the naturally occurring A467T variant showed at most a doubling in the length of the pauses compared to the wild-type. We identified the cause of these differences in the polymerase pausing time to be the number of disassociations occurring in each forward step of the polymerase.
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    Theoretical and Statistical Approaches to Understand Human Mitochondrial DNA Heteroplasmy Inheritance
    Wonnapinij, Passorn (Virginia Tech, 2010-04-09)
    Mitochondrial DNA (mtDNA) mutations have been widely observed to cause a variety of human diseases, especially late-onset neurodegenerative disorders. The prevalence of mitochondrial diseases caused by mtDNA mutation is approximately 1 in 5,000 of the population. There is no effective way to treat patients carrying pathogenic mtDNA mutation; therefore preventing transmission of mutant mtDNA became an important strategy. However, transmission of human mtDNA mutation is complicated by a large intergenerational random shift in heteroplasmy level causing uncertainty for genetic counseling. The aim of this dissertation is to gain insight into how human mtDNA heteroplasmy is inherited. By working closely with our experimental collaborators, the computational simulation of mouse embryogenesis has been developed in our lab using their measurements of mouse mtDNA copy number. This experimental-computational interplay shows that the variation of offspring heteroplasmy level has been largely generated by random partition of mtDNA molecules during pre- and early postimplantation development. By adapting a set of probability functions developed to describe the segregation of allele frequencies under a pure random drift process, we now can model mtDNA heteroplasmy distribution using parameters estimated from experimental data. The absence of an estimate of sampling error of mtDNA heteroplasmy variance may largely affect the biological interpretation drawn from this high-order statistic, thereby we have developed three different methods to estimate sampling error values for mtDNA heteroplasmy variance. Applying this error estimation to the comparison of mouse to human mtDNA heteroplasmy variance reveals the difference of the mitochondrial genetic bottleneck between these organisms. In humans, the mothers who carry a high proportion of m.3243A>G mutation tend to have fewer daughters than sons. This offspring gender bias has been revealed by applying basic statistical tests on the human clinical pedigrees carrying this mtDNA mutation. This gender bias may partially determine the mtDNA mutation level among female family members. In conclusion, the application of population genetic theory, statistical analysis, and computational simulation help us gain understanding of human mtDNA heteroplasmy inheritance. The results of these studies would be of benefit to both scientific research and clinical application.