Forest L. Danford

Demonstration that Drug-targeted Down-regulation of MYC in Non-Hodgkins Lymphoma Is Directly Mediated through the Promoter G-quadruplex

Most transcription of the MYC proto-oncogene initiates in the near upstream promoter, within which lies the nuclease hypersensitive element (NHE) III1 region containing the CT-element. This dynamic stretch of DNA can form at least three different topologies: single-stranded DNA, double-stranded DNA, or higher order secondary structures that silence transcription. In the current report, we identify the ellipticine analog GQC-05 (NSC338258) as a high affinity, potent, and selective stabilizer of the MYC G-quadruplex (G4). In cells, GQC-05 induced cytotoxicity with corresponding decreased MYC mRNA and altered protein binding to the NHE III1 region, in agreement with a G4 stabilizing compound. We further describe a unique feature of the Burkitts lymphoma cell line CA46 that allowed us to clearly demonstrate the mechanism and location of action of GQC-05 within this region of DNA and through the G4. Most importantly, these data present, as far as we are aware, the most direct evidence of intracellular G4-mediated control of a particular promoter.

Differences in the Region- and Depth-Dependent Microstructural Organization in Normal Versus Glaucomatous Human Posterior Sclerae

PURPOSE This study quantitatively investigated differences in the regional- and depth-dependent human posterior scleral microstructure in glaucomatous (G) and nonglaucomatous (NG) donors. METHODS Twenty-five posterior poles from six G and seven NG donors were analyzed using small angle light scattering (SALS) to investigate the organization of scleral fibers around the optic nerve head. Eccentricity (Ecc), fiber splay (FS), and percent equatorial fibers (PEF) were quantified. RESULTS Regional statistically significant differences between G and NG groups existed in Ecc (P < 0.0001), FS (P < 0.005), and PEF (P < 0.005). Distinct and substantial variation through the depth occurred in all three end points. Region-specific differences in Ecc existed at the episcleral surface; however, by 40% into the depth, all regions converged to a similar value. Fiber splay increased in all regions by an average of 0.14 from the episcleral surface to the intraocular surface. The percentage of equatorial fibers decreased universally through the depth from approximately 61% to 33%. Generally, the inferior and superior regions had a lower Ecc and PEF compared to the nasal and temporal regions. CONCLUSIONS Region and depth of the posterior sclera are important factors that should be included when comparing scleral microstructure of G and NG tissue in experimental and computational work. The dramatic changes in the depth of the sclera may represent baseline properties that affect predisposition to primary open angle glaucoma (POAG), and necessitate that further research include depth as a factor in assessing how observed structural differences contribute to or are a result of POAG.

TGFβ2 differentially modulates smooth muscle cell proliferation and migration in electrospun gelatin-fibrinogen constructs.

A main goal of tissue engineering is the development of scaffolds that replace, restore and improve injured tissue. These scaffolds have to mimic natural tissue, constituted by an extracellular matrix (ECM) support, cells attached to the ECM, and signaling molecules such as growth factors that regulate cell function. In this study we created electrospun flat sheet scaffolds using different compositions of gelatin and fibrinogen. Smooth muscle cells (SMCs) were seeded on the scaffolds, and proliferation and infiltration were evaluated. Additionally, different concentrations of Transforming Growth Factor-beta2 (TGFβ2) were added to the medium with the aim of elucidating its effect on cell proliferation, migration and collagen production. Our results demonstrated that a scaffold with a composition of 80% gelatin-20% fibrinogen is suitable for tissue engineering applications since it promotes cell growth and migration. The addition of TGFβ2 at low concentrations (≤ 1 ng/ml) to the culture medium resulted in an increase in SMC proliferation and scaffold infiltration, and in the reduction of collagen production. In contrast, TGFβ2 at concentrations >1 ng/ml inhibited cell proliferation and migration while stimulating collagen production. According to our results TGFβ2 concentration has a differential effect on SMC function and thus can be used as a biochemical modulator that can be beneficial for tissue engineering applications.

Three-Dimensional Segmentation of the Ex-Vivo Anterior Lamina Cribrosa From Second-Harmonic Imaging Microscopy

The lamina cribrosa (LC) is a connective tissue in the posterior eye with a complex mesh-like trabecular microstructure, through which all the retinal ganglion cell axons and central retinal vessels pass. Recent studies have demonstrated that changes in the structure of the LC correlate with glaucomatous damage. Thus, accurate segmentation and reconstruction of the LC is of utmost importance. This paper presents a new automated method for segmenting the microstructure of the anterior LC in the images obtained via multiphoton microscopy using a combination of ideas. In order to reduce noise, we first smooth the input image using a 4-D collaborative filtering scheme. Next, we enhance the beam-like trabecular microstructure of the LC using wavelet multiresolution analysis. The enhanced LC microstructure is then automatically extracted using a combination of histogram thresholding and graph-cut binarization. Finally, we use morphological area opening as a postprocessing step to remove the small and unconnected 3-D regions in the binarized images. The performance of the proposed method is evaluated using mutual overlap accuracy, Tanimoto index, F-score, and Rand index. Quantitative and qualitative results show that the proposed algorithm provides improved segmentation accuracy and computational efficiency compared to the other recent algorithms.

Structural and functional assessment of intense therapeutic ultrasound effects on partial Achilles tendon transection

Tendinopathies and tendon tears heal slowly because tendons have a limited blood supply. Intense therapeutic ultrasound (ITU) is a treatment modality that creates very small, focal coagula in tissue, which can stimulate a healing response. This pilot study investigated the effects of ITU on rabbit and rat models of partial Achilles tendon rupture. The right Achilles tendons of 20 New Zealand White rabbits and 118 rats were partially transected. Twenty-four hours after surgery, ITU coagula were placed in the tendon and surrounding tissue, alternating right and left legs. At various time points, the following data were collected: ultrasound imaging, optical coherence tomography (OCT) imaging, mechanical testing, gene expression analysis, histology, and multiphoton microscopy (MPM) of sectioned tissue. Ultrasound visualized cuts and treatment lesions. OCT showed the effect of the interventions on birefringence banding caused by collagen organization. MPM showed inflammatory infiltrate, collagen synthesis and organization. By day 14- 28, all tendons had a smooth appearance and histology, MPM and OCT still could still visualize residual healing processes. Few significant results in gene expression were seen, but trends were that ITU treatment caused an initial decrease in growth and collagen gene expression followed by an increase. No difference in failure loads was found between control, cut, and ITU treatment groups, suggesting that sufficient healing had occurred by 14 days to restore all test tissue to control mechanical properties. These results suggest that ITU does not cause harm to tendon tissue. Upregulation of some genes suggests that ITU may increase healing response.

Analyzing Parallel Programming Models for Magnetic Resonance Imaging

The last several decades have been marked by dramatic increases in the use of diagnostic medical imaging and improvements in the modalities themselves. As such, more data is being generated at an ever increasing rate. However, in the case of Magnetic Resonance Imaging (MRI) analysis and reports remain semi-quantitative, despite reported advantages of quantitative analysis (QA), due to prohibitive execution times. We present a collaborator’s QA algorithm for Dynamic Contrast-Enhanced (DCE) MRI data written in MATLAB as a case study for exploring parallel programming in MATLAB and Julia. Parallelization resulted in a 7.66x speedup in MATLAB and a 72x speedup in Julia. To the best of our knowledge, this comparison of Julia’s performance in a parallel, application-level program is novel. On the basis of these results and our experiences while programming in each language, our collaborator now prototypes in MATLAB and then ports to Julia when performance is critical.

Phosphotungstic Acid Diffusion in Porcine Ocular Tissue for MicroCT Shape Reconstruction

For any ocular research involving finite element analysis, measurement of sample geometry is an important step that determines the accuracy of subsequent results. A shape reconstruction method using diffused Phosphotungstic Acid (PTA) within ocular samples for measurement with a microCT device will be presented here. With all finite element modeling, the material properties of the tissue must be determined using techniques such as digital image correlation (DIC) [1]. With this technique, the limitations for displacement measurement accuracy are the size of non-altering markers and camera resolution. This means that only certain material points can be tracked and the non-tracked material points must be interpolated in finite element models. Along with this interpolation, techniques for thickness measurements across the ocular tissue such as using calipers or a pachymeter [2] typically only give values at a small quantity of points while the thickness changes continuously across the tissue. The PTA staining will provide the advantage of internal geometry as well as external shape to give thickness across the entire sample. While these other techniques provide repeatable results, we believe the PTA method presented here for geometry acquisition provides accurate shape reconstruction of the tissue for finite element applications. The purpose of this study was to quantify the depth of penetration of PTA in porcine scleral samples as a function of PTA concentration and time.Copyright

Abstract 4757: Design and optimization of linked nucleic acid (LNA) probes to detect the c-MYC G-quadruplex

The c-MYC oncogene is upregulated in almost 80% of cancers, and in some cases is the causative oncogenic factor. It is regulated at the transcriptional level with up to 90% initiating at the P1 and P2 promoters. The NHE III1 element regulates this region, which consists of a unique guanine-rich string of DNA capable of adopting at least three topologies: single stranded, double stranded (ds) and the non-B-DNA G-quadruplex (G4) structure. G4s are formed when two or more tetrads stack, each comprised of four guanines bonded by Hoogsteen hydrogen bonds, stabilized with monovalent cations. Putative G4 forming regions have at least four runs of three or more consecutive guanines separated by varying nucleotides that comprise the loop structures. The c-MYC G4 structure has been elucidated to involve guanine runs 1α4 (G41-4) or 2Δ5 (G42-5) (of six total runs) in a parallel formation. Regulatory proteins have been identified, and small molecule compounds are under investigation to stabilize the G4 element and downregulate c-MYC expression. In parallel to these efforts, we are developing linked nucleic acid (LNA)-biotin bridge probes to be used as diagnostic tools to identify these formations in biopsies in order to inform therapeutic regimens. Molecular models of the two G4 isoforms were used to determine the distance occupied by the secondary structure to be between 16.4α16.6 angstroms; various length nucleic acid sequences flanking a 17.7α30 angstrom linker were screened and optimized. Probes were shown to be structure specific - either for the c-MYC G41-4 (probe 1a) or the G42-5 (probe 1b) - by EMSA analysis. The probes were amenable to internal biotinylation for ongoing pull-down assays, but truncation of the 5′-complementary sequence limited affinity. Interestingly, the LNA probes thermally stabilized the c-MYC G4 in a unique pattern and are also being examined for their potential therapeutic efficacy. Experiments are underway to show that these hybrid probes will bind intracellularly and show the frequency of G4 formation in quiescent versus activated cells, and following treatment with c-MYC G4-interactive compounds. Through these works, we highlight for the first time that a ‘FISH’-type probe can be developed and used to selectively detect the formation of the c-MYC G4 in vitro. Up to 80% cancer patients overexpress c-MYC and are candidates for G4-targeted therapies currently under development. The developed probes will be of great utility as companion diagnostics in selecting patients for G4-therapeutics and truly personalized medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4757. doi:1538-7445.AM2012-4757