Matthew Sochor

Validation of sodium magnetic resonance imaging of intervertebral disc.

Study Design. This study demonstrated the diagnostic potential of sodium (Na) magnetic resonance imaging (MRI) for noninvasive quantification of proteoglycan (PG) in the intervertebral discs. Objective. To determine the existence of a linear correlation between intervertebral disc [Na] measured from sodium MRI and [PG] measurement from DMMB assay. Summary of Background Data. Previous studies have shown the possibility of quantifying Na in vivo using sodium MRI, however, none has shown a direct linear correlation between Na measured from sodium MRI and in the invertebral discs. Methods. Three-dimensional sodium MRI images of bovine discs were acquired and converted into [Na] maps. Samples were systematically removed from the discs for DMMB assay. The removal locations were photographically recorded and applied to the [Na] maps to extract the [Na] measurements for comparison. In vivo sodium MRI scans were also carried out on a pair of symptomatic and asymptomatic subjects. Results. The linear regression fit of [Na] versus [PG] data yielded a significant linear correlation coefficient of 0.71. The in vivo sodium MRI image of the symptomatic subject showed significant [Na] decrease when compared to that of the asymptomatic subject. Conclusion. Specificity of sodium MRI for PG in the intervertebral discs makes it a promising diagnostic tool for the earlier phase of disc degeneration.

T1ρ-Prepared Balanced Gradient Echo for Rapid 3D T1ρ MRI

To develop a T1ρ‐prepared, balanced gradient echo (b‐GRE) pulse sequence for rapid three‐dimensional (3D) T1ρ relaxation mapping within the time constraints of a clinical exam (<10 minutes), examine the effect of acquisition on the measured T1ρ relaxation time and optimize 3D T1ρ pulse sequences for the knee joint and spine.

The Hsp104 N-terminal domain enables disaggregase plasticity and potentiation.

The structural basis by which Hsp104 dissolves disordered aggregates and prions is unknown. A single subunit within the Hsp104 hexamer can solubilize disordered aggregates, whereas prion dissolution requires collaboration by multiple Hsp104 subunits. Here, we establish that the poorly understood Hsp104 N-terminal domain (NTD) enables this operational plasticity. Hsp104 lacking the NTD (Hsp104(ΔN)) dissolves disordered aggregates but cannot dissolve prions or be potentiated by activating mutations. We define how Hsp104(ΔN) invariably stimulates Sup35 prionogenesis by fragmenting prions without solubilizing Sup35, whereas Hsp104 couples Sup35 prion fragmentation and dissolution. Volumetric reconstruction of Hsp104 hexamers in ATPγS, ADP-AlFx (hydrolysis transition state mimic), and ADP via small-angle X-ray scattering revealed a peristaltic pumping motion upon ATP hydrolysis, which drives directional substrate translocation through the central Hsp104 channel and is profoundly altered in Hsp104(ΔN). We establish that the Hsp104 NTD enables cooperative substrate translocation, which is critical for prion dissolution and potentiated disaggregase activity.

Thermodynamic Analysis of Mutant lac Repressors

The lactose (lac) repressor is an allosteric protein that can respond to environmental changes. Mutations introduced into the DNA binding domain and the effector binding pocket affect the repressors ability to respond to its environment. We have demonstrated how the observed phenotype is a consequence of altering the thermodynamic equilibrium constants. We discuss mutant repressors, which (1) show tighter repression; (2) induce with a previously noninducing species, orthonitrophenyl-β-D-galactoside; and (3) transform an inducible switch to one that is corepressed. The ability of point mutations to change multiple thermodynamic constants, and hence drastically alter the repressors phenotype, shows how allosteric proteins can perform a wide array of similar yet distinct functions such as that exhibited in the Lac/Gal family of bacterial repressors.

An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications

The future of treating inherited and acquired genetic diseases will be defined by our ability to introduce transgenes into cells and restore normal physiology. Here we describe an autogenous transgene regulatory system (ARES), based on the bacterial lac repressor, and demonstrate its utility for controlling the expression of a transgene in bacteria, eukaryotic cells, and in the retina of mice. This ARES system is inducible by the small non-pharmacologic molecule, Isopropyl β-D-1-thiogalactopyranoside (IPTG) that has no off-target effects in mammals. Following subretinal injection of an adeno-associated virus (AAV) vector encoding ARES, luciferase expression can be reversibly controlled in the murine retina by oral delivery of IPTG over three induction-repression cycles. The ability to induce transgene expression repeatedly via administration of an oral inducer in vivo, suggests that this type of regulatory system holds great promise for applications in human gene therapy.

In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli

A multitude of studies have looked at the in vivo and in vitro behavior of the lac repressor binding to DNA and effector molecules in order to study transcriptional repression, however these studies are not always reconcilable. Here we use in vitro transcription to directly mimic the in vivo system in order to build a self consistent set of experiments to directly compare in vivo and in vitro genetic repression. A thermodynamic model of the lac repressor binding to operator DNA and effector is used to link DNA occupancy to either normalized in vitro mRNA product or normalized in vivo fluorescence of a regulated gene, YFP. An accurate measurement of repressor, DNA and effector concentrations were made both in vivo and in vitro allowing for direct modeling of the entire thermodynamic equilibrium. In vivo repression profiles are accurately predicted from the given in vitro parameters when molecular crowding is considered. Interestingly, our measured repressor–operator DNA affinity differs significantly from previous in vitro measurements. The literature values are unable to replicate in vivo binding data. We therefore conclude that the repressor-DNA affinity is much weaker than previously thought. This finding would suggest that in vitro techniques that are specifically designed to mimic the in vivo process may be necessary to replicate the native system.