The dynamic equilibrium of human telomeric G-quadruplexes.

G-quadruplexes are thought to have biological importance, with studies based on small molecule interactions and quadruplex-interactive antibodies demonstrating their potential for formation in vivo. One potential biological function of quadruplex structures is the regulation and maintenance of telomeres. Telomeres are nucleoprotein complexes involved in chromosome stability. Human telomeres are composed of the repeated DNA sequence, 5\u27- d(TIAGGG), that terminates in a 3\u27 single-stranded overhang. DNA sequences with homology to the human telomere are capable of quadruplex formation in vitro. Specifically, sequences containing four-guanine stretches (e.g. 5\u27-d(AGGG(TIAGGGb)) are capable of forming at least five distinct unimolecular structures. Which structure is favored is believed to be linked to solvent composition and the addition of 3\u27- and 5\u27-flanking residues. This dissertation provides an essential biophysical investigation of the polymorphic equilibrium displayed by human telomeric quadruplexes. Multiple biophysical techniques are utilized to assemble a thermodynamic description of the influences of hydration and molecular crowding on conformational selection and elucidate complex unfolding mechanisms with unique intermediate states. This dissertation provides the first application of phasor diagrams in the study of quadruplexes. Phasor diagrams are shown to be sensitive to alterations in quadruplex structure (i.e. folding and unfolding) by monitoring changes in the complex lifetime distribution of 2-aminopurine. This dissertation contains the first multi-faceted biophysical investigation of the underlying mechanism of co-solvent driven conformational changes of human telomeric quadruplexes. The thermodynamic study illustrates that quadruplexes are stabilized by dehydration, a behavior opposite that of canonical duplex structures. Additionally, the ability of PEGs to drive the conformational selection of a parallel quadruplex through differential binding is clarified, addressing unsubstantiated claims that the propeller form is the most biologically relevant conformation. Finally, an in-depth thermodynamic investigation of the thermal unfolding of human telomeric quadruplexes is conducted. Multiple spectroscopic techniques are used to evaluate the thermal unfolding process and characterize potential intermediates states. This dissertation work is the first to apply spectroscopic deconvolutions to demonstrate that human telomeric quadruplexes unfold through sequential mechanisms requiring intermediate species. These results are highlighted by the recovery of an intermediate species whose biophysical description is best characterized by an ensemble of triple-helical conformations

2-aminopurine as a probe for quadruplex loop structures

Fluorescent reporter groups have served for many years as sensitive probes of macromolecular structure. Such probes can be especially useful in comparative studies such as detection of conformational changes and discrimination among structural models. Spectroscopic methods such as fluorescence are attractive because they are rapid, require small amounts of material, are nondestructive, can be carried out with commonly available equipment, and are relatively inexpensive. In addition, there is a rich library of theoretical and practical materials available to aid in data interpretation.The intrinsic fluorescence of most nucleic acids is too low to be useful in structural studies. Thus, it is necessary to incorporate a suitable reporter group to utilize fluorescence methods involving polynucleotide structure. A highly fluorescent adenine analog, 2-aminopurine, has long served in this capacity. The present article describes our use of 2-aminopurine as a probe of loop structures in quadruplex DNA. In particular, we show how knowledge of the relative intensity of 2-aminopurine emission as well as its sensitivity to exogenous quenching molecules such as acrylamide can aid in comparing crystal and solution structures of an oligonucleotide model of the human telomere and in discrimination among models containing tandem repeats of the telomeric quadruplex

G-quadruplex structure and stability illuminated by 2-aminopurine phasor plots

The use of time-resolved fluorescence measurements in studies of telomeric G-quadruplex folding and stability has been hampered by the complexity of fluorescence lifetime distributions in solution. The application of phasor diagrams to the analysis of time-resolved fluorescence measurements, collected from either frequency-domain or time-domain instrumentation, allows for rapid characterization of complex lifetime distributions. Phasor diagrams are model-free graphical representations of transformed time-resolved fluorescence results. Simplification of complex fluorescent decays by phasor diagrams is demonstrated here using a 2-aminopurine substituted telomeric G-quadruplex sequence. The application of phasor diagrams to complex systems is discussed with comparisons to traditional non-linear regression model fitting. Phasor diagrams allow for the folding and stability of the telomeric G-quadruplex to be monitored in the presence of either sodium or potassium. Fluorescence lifetime measurements revealed multiple transitions upon folding of the telomeric G-quadruplex through the addition of potassium. Enzymatic digestion of the telomeric G-quadruplex structure, fluorescence quenching and Förster resonance energy transfer were also monitored through phasor diagrams. This work demonstrates the sensitivity of time-resolved methods for monitoring changes to the telomeric G-quadruplex and outlines the phasor diagram approach for analysis of complex time-resolved results that can be extended to other G-quadruplex and nucleic acid systems

Telomeric co-localization of the modified base J and contingency genes in the protozoan parasite Trypanosoma cruzi

Base J or β-d-glucosylhydroxymethyluracil is a modification of thymine residues within the genome of kinetoplastid parasites. In organisms known to contain the modified base, J is located mainly within the telomeric repeats. However, in Trypanosoma brucei, a small fraction of J is also located within the silent subtelomeric variant surface glycoprotein (VSG) gene expression sites, but not in the active expression site, suggesting a role for J in regulating telomeric genes involved in pathogenesis. With the identification of surface glycoprotein genes adjacent to telomeres in the South American Trypanosome, Trypanosoma cruzi, we became interested in the telomeric distribution of base J. Analysis of J and telomeric repeat sequences by J immunoblots and Southern blots following DNA digestion, reveals ∼25% of J outside the telomeric repeat sequences. Moreover, the analysis of DNA sequences immunoprecipitated with J antiserum, localized J within subtelomeric regions rich in life-stage-specific surface glycoprotein genes involved in pathogenesis. Interestingly, the pattern of J within these regions is developmentally regulated. These studies provide a framework to characterize the role of base J in the regulation of telomeric gene expression/diversity in T. cruzi

Transpapillary drainage has no added benefit on treatment outcomes in patients undergoing EUS-guided transmural drainage of pancreatic pseudocysts: a large multicenter study

Background and Aims The need for transpapillary drainage (TPD) in patients undergoing transmural drainage (TMD) of pancreatic fluid collections (PFCs) remains unclear. The aims of this study were to compare treatment outcomes between patients with pancreatic pseudocysts undergoing TMD versus combined (TMD and TPD) drainage (CD) and to identify predictors of symptomatic and radiologic resolution. Methods This is a retrospective review of 375 consecutive patients with PFCs who underwent EUS-guided TMD from 2008 to 2014 at 15 academic centers in the United States. Main outcome measures included TMD and CD technical success, treatment outcomes (symptomatic and radiologic resolution) at follow-up, and predictors of treatment outcomes on logistic regression. Results A total of 375 patients underwent EUS-guided TMD of PFCs, of which 174 were pseudocysts. TMD alone was performed in 95 (55%) and CD in 79 (45%) pseudocysts. Technical success was as follows: TMD, 92 (97%) versus CD, 35 (44%) (P = .0001). There was no difference in adverse events between the TMD (15%) and CD (14%) cohorts (P = .23). Median long-term (LT) follow-up after transmural stent removal was 324 days (interquartile range, 72-493 days) for TMD and 201 days (interquartile range, 150-493 days) (P = .37). There was no difference in LT symptomatic resolution (TMD, 69% vs CD, 62%; P = .61) or LT radiologic resolution (TMD, 71% vs CD, 67%; P = .79). TPD attempt was negatively associated with LT radiologic resolution of pseudocyst (odds ratio, 0.11; 95% confidence interval, 0.02-0.8; P = .03). Conclusions TPD has no benefit on treatment outcomes in patients undergoing EUS-guided TMD of pancreatic pseudocysts and negatively affects LT resolution of PFCs

Environmentally Relevant Levels of Depleted Uranium Impacts Dermal Fibroblast Proliferation, Viability, Metabolic Activity, and Scratch Closure

Uranium (U) is a heavy metal used in military and industrial settings, with a large portion being mined from the Southwest region of the United States. Uranium has uses in energy and military weaponry, but the mining process has released U into soil and surface waters that may pose threats to human and environmental health. The majority of literature regarding U’s human health concern focuses on outcomes based on unintentional ingestion or inhalation, and limited data are available about its influence via cutaneous contact. Utilizing skin dermis cells, we evaluated U’s topical chemotoxicity. Employing soluble depleted uranium (DU) in the form of uranyl nitrate (UN), we hypothesized that in vitro exposure of UN will have cytotoxic effects on primary dermal fibroblasts by affecting cell viability and metabolic activity and, further, may delay wound healing aspects via altering cell proliferation and migration. Using environmentally relevant levels of U found in water (0.1 μM to 100 μM [UN]; 23.8–23,800 ppb [U]), we quantified cellular mitosis and migration through growth curves and in vitro scratch assays. Cells were exposed from 24 h to 144 h for a time-course evaluation of UN chemical toxicity. The effects of UN were observed at concentrations above and below the Environmental Protection Agency threshold for safe exposure limits. UN exposure resulted in a dose-dependent decrease in the viable cell count; however, it produced an increase in metabolism when corrected for the viable cells present. Furthermore, cellular proliferation, population doubling, and percent closure was hindered at levels ≥10 μM UN. Therefore, inadvertent exposure may exacerbate pre-existing skin diseases in at-risk demographics, and additionally, it may substantially interfere in cutaneous tissue repair processes