Regulation of HFE expression by poly(ADP-ribose) polymerase-1 (PARP1) through an inverted repeat DNA sequence in the distal promoter.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron overload among Caucasians of northern European descent. Over 85% of all cases with HH are due to mutations in the hemochromatosis protein (HFE) involved in iron metabolism. Although the importance in iron homeostasis is well recognized, the mechanism of sensing and regulating iron absorption by HFE, especially in the absence of iron response element in its gene, is not fully understood. In this report, we have identified an inverted repeat sequence (ATGGTcttACCTA) within 1700bp (-1675/+35) of the HFE promoter capable to form cruciform structure that binds PARP1 and strongly represses HFE promoter. Knockdown of PARP1 increases HFE mRNA and protein. Similarly, hemin or FeCl3 treatments resulted in increase in HFE expression by reducing nuclear PARP1 pool via its apoptosis induced cleavage, leading to upregulation of the iron regulatory hormone hepcidin mRNA. Thus, PARP1 binding to the inverted repeat sequence on the HFE promoter may serve as a novel iron sensing mechanism as increased iron level can trigger PARP1 cleavage and relief of HFE transcriptional repression.

Drying effect creates false assemblies in DNA-coated gold nanoparticles as determined through in situ liquid cell STEM.

The drying effect associated with utilizing transmission electron microscopy to study deoxyribonucleic acid (DNA)-coated gold nanoparticles (AuNPs) remains largely uninvestigated, though this technique is frequently utilized to characterize nanoparticle-DNA interactions. Investigation of the drying effect is essential to the progress of the many fields that utilize AuNPs, including cancer research. In this study, we compare DNA hybridization-directed nanoparticle assemblies with control samples omitting the necessary complementary DNA, effectively blocking directed assembly, in both the liquid state and the dry state, within a scanning transmission electron microscope. We show that the dry samples contain AuNPs spaced at significantly smaller intervals than identical samples measured in situ, with no dependence on the DNA bound to the AuNPs in the dry samples. A partially wet sample, with distances measured along the drying edge, provided an intermediate binding distance, strengthening the conclusion that drastic differences observed between the dry and in situ samples are due to a pronounced drying effect. This drying effect will falsely indicate certain grouping arrangements and will change the impression of the size of the groups formed, providing misinformation for the development of these controlled assemblies that could impact applications such as targeted drug vehicles for cancer treatment.

Liquid-cell scanning transmission electron microscopy and fluorescence correlation spectroscopy of DNA-directed gold nanoparticle assemblies.

In the use of solution-based 3D nanoarchitectures for optics, drug delivery, and cancer treatment, the precise nanoparticle architecture morphologies, architecture sizes, interparticle distances, and the assembly stability are all critical to their functionality. 3D nanoparticle architectures in solution are difficult to characterize, as few techniques can provide individualized information on interparticle spacing (defined by linkage molecule), nanoparticle assembly size, morphology, and identification of false aggregation. Bulk characterization techniques, including small angle x-ray scattering, can provide architecture sizes, though they are unable to precisely measure differences within interparticle spacings for individual architectures and can falsely measure assemblies caused by non-linkage grouped nanoparticles. Two solution-based characterization techniques were used to determine which assembly type and linkage length would produce the fastest assembly rate for large DNA-directed gold nanoparticle assemblies. In-situ liquid-cell scanning transmission electron microscopy (LC-STEM), measured interparticle spacings between DNA-functionalized nanoparticles, and fluorescence correlation spectroscopy provided the bulk volume fraction of large and small assemblies for nanoparticle architectures that were assembled using two different types: (1) the hybrid assemblies join two complementary single-stranded DNA linkages, and (2) the bridged assemblies are comprised of single-stranded DNA (bridging component) that is double the length of two different complementary single-stranded DNA-functionalized gold nanoparticles. Assembly times were tested at 24-hrs intervals over 3 days. Statistics derived from the in-situ LC-STEM images provided data for interparticle distance measurements, which identified the fraction of nanoparticles within the images acquired that were at the expected double-stranded DNA-binding distance of the linkages (varied in three distances for each of the two different architectures). In general, longer linkage lengths assembled in the shortest amount of time. The bridged assemblies formed fewer large architectures at 24-hrs but ultimately assembled a greater fraction of nanoparticles, which was due to the longer functionalized DNA lengths for individual nanoparticles. Fluorescence correlation spectroscopy provided a bulk average of the gold nanoparticle assembly sizes over time, which supported the conclusions drawn from the in-situ LC-STEM data. The microscopy provided sub-2 nm precision in the interparticle distances between gold nanoparticles in a solution environment. This coupled microscopy and spectroscopy characterization approach can provide more detailed information than bulk characterization methods.

Stabilization of measles virus for vaccine formulation.

An attenuated live measles virus (MV) was characterized by several biophysical methods as a function of temperature and pH. Following a method developed previously, the resultant light scattering and spectroscopic data were synthesized into an empirical phase diagram that visually and simultaneously represents the entire data set. Using this empirically-based phase diagram, screening assays were developed to identify potential vaccine stabilizers. Various compounds are shown by these assays to inhibit the temperature-induced aggregation of viral particles, and also to protect the integrity of the viral envelope. Accelerated stability assays show that, upon thermal challenge, MV formulated with these excipients retains its infectivity to a significant extent. Thus, the enhanced physical stability produced by this method is shown to protect the biological activity of this important but labile vaccine.

Imaging the effects of annealing on the polymorphic phases of poly(vinylidene fluoride).

The effect of annealing on the phase transformation and the dielectric properties of poly(vinylidene fluoride) (PVDF) is explored using quasi-single molecule (quasi-SM) microscopy. The solvatochromic properties of nile red (NR) are employed to measure the spatial distribution of the local dielectric constant (ε) in ∼30 µm thick PVDF films before and after annealing at 90 °C. The results presented here demonstrate that nonannealed films exhibit much larger ε distributions, both in terms of magnitude and distribution, when compared to annealed films. The polymorphic phase of PVDF before and after annealing is also confirmed using X-ray diffraction. Nonannealed films are found to be in the γ-phase with annealing promoting the transition to primarily ß-phase. Combining these results, we conclude that the decrease in ε with annealing time is due to the phase transformation from γ- to ß-phase. Using quasi-SM imaging techniques one can readily visualize the ε environments present within different polymorphic phases of PVDF.