Synthesis and application of electrochemically active oligonucleotides

Modified oligonucleotides with redox-active functional groups could emerge as attractive tools for sensor development. In principle, changes in oligonucleotide hybridization or conformation may be read out as a change in an electrochemical signal. Monitoring this signal might allow for a direct interface between biology and electronics. This dissertation describes efforts devoted to creating redox active oligonucleotide derivatives designed to allow these application goals to be pursued. The focus is primarily on the synthesis, characterization, and application of oligonucleotides bearing on of two electroactive moieties, namely ferrocene and methylene blue. Chapter 1 provides a brief overview of electrochemically modified oligonucleotides and is designed to provide an historical perspective. Synthetic methodology, fabrication of electrode system, and current applications are introduced. Chapter 2 describes the synthesis of a ferrocene-modified oligonucleotide and its use as a multiplexing signal probe. Included in this chapter are syntheses of a ferrocene subunit bearing alkynes, as well as modified nucleoside phosphoramidites and the oligonucleotide syntheses they permit. A synopsis of electrochemical studies are also provided. Chapter 3 describes a ratiometric electrochemical DNA sensor (a so-called E-Sensor) based on the ferrocene-modified oligonucleotide described in Chapter 2 and its used in the detection of specific genes with greatly improved reproducibility. Oligonucleotide syntheses achieved through enzyme ligation, the fabrication of an E-sensor, and the results of electrochemical assays are provided in this chapter. Chapter 4 describes the design and fabrication of possible wearable devices with the modified electrochemically active oligonucleotides toward real diagnostic applications. This work is being done in collaboration with Dr. Nanshu Lu’s group in the Dept. of Aerospace Engineering and Engineering Mechanics at the University of Texas at Austin. Chapter 5 the details the synthetic procedures, provides characterization of all new products, and contains electrochemical analytical data discussed in this dissertation.Chemistr

RADBIOMOD: A simple program for utilising biological modelling in radiotherapy plan evaluation

Abstract not availableJoe H. Chang, Christopher Gehrke, Ramachandran Prabhakar, Suki Gill, Morikatsu Wada, Daryl Lim Joon, Vincent Kho

Intensity modulated radiation therapy dose painting for localized prostate cancer using(11)C-choline positron emission tomography scans

PURPOSE: To demonstrate the technical feasibility of intensity modulated radiation therapy (IMRT) dose painting using¹¹C-choline positron emission tomography PET scans in patients with localized prostate cancer. METHODS AND MATERIALS: This was an RT planning study of 8 patients with prostate cancer who had ¹¹C-choline PET scans prior to radical prostatectomy. Two contours were semiautomatically generated on the basis of the PET scans for each patient: 60% and 70% of the maximum standardized uptake values (SUV(60%) and SUV(70%)). Three IMRT plans were generated for each patient: PLAN(78), which consisted of whole-prostate radiation therapy to 78 Gy; PLAN(78-90), which consisted of whole-prostate RT to 78 Gy, a boost to the SUV(60%) to 84 Gy, and a further boost to the SUV(70%) to 90 Gy; and PLAN(72-90), which consisted of whole-prostate RT to 72 Gy, a boost to the SUV(60%) to 84 Gy, and a further boost to the SUV(70%) to 90 Gy. The feasibility of these plans was judged by their ability to reach prescription doses while adhering to published dose constraints. Tumor control probabilities based on PET scan-defined volumes (TCP(PET)) and on prostatectomy-defined volumes (TCP(path)), and rectal normal tissue complication probabilities (NTCP) were compared between the plans. RESULTS: All plans for all patients reached prescription doses while adhering to dose constraints. TCP(PET) values for PLAN(78), PLAN(78-90), and PLAN(72-90) were 65%, 97%, and 96%, respectively. TCP(path) values were 71%, 97%, and 89%, respectively. Both PLAN(78-90) and PLAN(72-90) had significantly higher TCP(PET) (P=.002 and .001) and TCP(path) (P<.001 and .014) values than PLAN(78). PLAN(78-90) and PLAN(72-90) were not significantly different in terms of TCP(PET) or TCP(path). There were no significant differences in rectal NTCPs between the 3 plans. CONCLUSIONS: IMRT dose painting for localized prostate cancer using (11)C-choline PET scans is technically feasible. Dose painting results in higher TCPs without higher NTCPs.Joe H. Chang, Daryl Lim Joon, Sze Ting Lee, Sylvia J. Gong, Nigel J. Anderson, Andrew M. Scott, Ian D. Davis, David Clouston, Damien Bolton, Christopher S. Hamilton, Vincent Kho

MRI scans significantly change target coverage decisions in radical radiotherapy for prostate cancer

INTRODUCTION: Conventional clinical staging for prostate cancer has many limitations. This study evaluates the impact of adding MRI scans to conventional clinical staging for guiding decisions about radiotherapy target coverage. METHODS: This was a retrospective review of 115 patients who were treated between February 2002 and September 2005 with radical radiotherapy for prostate cancer. All patients had MRI scans approximately 2 weeks before the initiation of radiotherapy. The T stage was assessed by both conventional clinical methods (cT-staging) as well as by MRI (mT-staging). The radiotherapy target volumes were determined first based on cT-staging and then taking the additional mT staging into account. The number of times extracapsular extension or seminal vesicle invasion was incorporated into target volumes was quantified based on both cT-staging and the additional mT-staging. RESULTS: Extracapsular extension was incorporated into target volumes significantly more often with the addition of mT-staging (46 patients (40%) ) compared with cT-staging alone (37 patients (32%) ) (P = 0.002). Seminal vesicle invasion was incorporated into target volumes significantly more often with the addition of mT-staging (21 patients (18%) ) compared with cT-staging alone (three patients (3%) ) (P < 0.001). A total of 23 patients (20%) had changes to their target coverage based on the mT-staging. CONCLUSIONS: MRI scans can significantly change decisions about target coverage in radical radiotherapy for prostate cancer.Joe H. Chang, Daryl Lim Joon, Brandon T. Nguyen, Chee-Yan Hiew, Stephen Esler, David Angus, Michael Chao, Morikatsu Wada, George Quong, and Vincent Kho

A MATLAB based simulation program for indoor visible light communication system

We report a simulation program for indoor visible light communication environment based on MATLAB and Simulink. The program considers the positions of the transmitters and the reflections at each wall. For visible light communication environment, the illumination light-emitting diode is used not only as a lighting device, but also as a communication device. Using the simulation program, the distributions of illuminance and root-mean-square delay spread are analyzed at bottom surface

Comparison of [(11)C]choline positron emission tomography with T2- and diffusion-weighted magnetic resonance imaging for delineating malignant intraprostatic lesions

Purpose: To compare the accuracy of ¹¹C-choline (CHOL) positron emission tomography (PET) with the combination of T2-weighted (T2W) and diffusion-weighted (DW) magnetic resonance imaging (MRI) for delineating malignant intraprostatic lesions (IPLs) for guiding focal therapies and to investigate factors predicting the accuracy of CHOL-PET. Methods and Materials: This study included 21 patients who underwent CHOL-PET and T2W-/DW-MRI prior to radical prostatectomy. Two observers manually delineated IPL contours for each scan, and automatic IPL contours were generated on CHOL-PET based on varying proportions of the maximum standardized uptake value (SUV). IPLs identified on prostatectomy specimens defined the reference standard contours. The imaging-based contours were compared with the reference standard contours using Dice similarity coefficient (DSC), sensitivity and specificity. Factors that could potentially predict the DSC of the best contouring method were analyzed using linear models. Results: The best automatic contouring method, SUV60, had similar correlations (DSC 0.59) with the manual PET contours (DSC 0.52, P=0.127) and significantly better correlations than the manual MRI contours (DSC 0.37, P<0.001). The sensitivity and specificity values were 72% and 71% for SUV60; 53% and 86% for PET manual contouring; and 28% and 92% for MRI manual contouring. The tumor volume and transition zone pattern could independently predict the accuracy of CHOL-PET. Conclusions: CHOL-PET is superior to the combination of T2W- and DW-MRI for delineating IPLs. The accuracy of CHOL-PET is insufficient for gland-sparing focal therapies, 3 however may be accurate enough for focal boost therapies. The transition zone pattern is a new classification that may predict for how well CHOL-PET delineates IPLs.Joe H. Chang, Daryl Lim Joon, Ian D. Davis, Sze Ting Lee, Chee-Yan Hiew, Stephen Esler, Sylvia J. Gong, Morikatsu Wada, David Clouston, Richard O'Sullivan, Yin P. Goh, Damien Bolton, Andrew M. Scott, Vincent Kho

Epidermal-specific deletion of TC-PTP promotes UVB-induced epidermal cell survival through the regulation of Flk-1/JNK signaling

UVB exposure can contribute to the development of skin cancer by modulating protein tyrosine kinase (PTK) signaling. It has been suggested that UVB radiation increases the ligand-dependent activation of PTKs and induces PTP inactivation. Our recent studies have shown that T-cell protein tyrosine phosphatase (TC-PTP) attenuates skin carcinogenesis induced by chemical regimens, which indicates its critical role in the prevention of skin cancer. In the current work, we report that TC-PTP increases keratinocyte susceptibility to UVB-induced apoptosis via the downregulation of Flk-1/JNK signaling. We showed that loss of TC-PTP led to resistance to UVB-induced apoptosis in vivo epidermis. We established immortalized primary keratinocytes (IPKs) from epidermal-specific TC-PTP-deficient (K14Cre.Ptpn2fl/fl) mice. Immortalized TC-PTP-deficient keratinocytes (TC-PTP/KO IPKs) showed increased cell survival against UVB-induced apoptosis which was concomitant with a UVB-mediated increase in Flk-1 phosphorylation, especially on tyrosine residue 1173. Inhibition of Flk-1 by either its specific inhibitors or siRNA in TC-PTP/KO IPKs reversed this effect and significantly increased cell death after UVB irradiation in comparison with untreated TC-PTP/KO IPKs. Immunoprecipitation analysis using the TC-PTP substrate-trapping mutant TCPTP-D182A indicated that TC-PTP directly interacts with Flk-1 to dephosphorylate it and their interaction was stimulated by UVB. Following UVBmediated Flk-1 activation, the level of JNK phosphorylation was also significantly increased in TC-PTP/KO IPKs compared to control IPKs. Similar to our results with Flk-1, treatment of TC-PTP/KO IPKs with the JNK inhibitor SP600125 significantly increased apoptosis after UVB irradiation, confirming that the effect of TC-PTP on UVB-mediated apoptosis is regulated by Flk-1/JNK signaling. Western blot analysis showed that both phosphorylated Flk-1 and phosphorylated JNK were significantly increased in the epidermis of TC-PTP-deficient mice compared to control mice following UVB. Our results suggest that TC-PTP plays a protective role against UVB-induced keratinocyte cell damage by promoting apoptosis via negative regulation of Flk-1/JNK survival signaling

Diffusion-weighted MRI, (11)C-choline PET and (18)F-fluorodeoxyglucose PET for predicting the Gleason score in prostate carcinoma

Objectives To evaluate the accuracy of transrectal ultrasoundguided (TRUS) biopsy, diffusion-weighted (DW) magnetic resonance imaging (MRI), ¹¹C-choline (CHOL) positron emission tomography (PET), and 18F-fluorodeoxyglucose (FDG) PET in predicting the prostatectomy Gleason risk (GR). Methods The study included 21 patients who underwent TRUS biopsy and multi-technique imaging before radical prostatectomy. Values from five different tests (TRUS biopsy, DW MRI, CHOL PET, FDG PET, and combined DW MRI/ CHOL PET) were correlated with the prostatectomy GR using Spearman’s ρ. Tests that were found to have significant correlations were used to classify patients into GR groups. Results The following tests had significant correlations with prostatectomy GR: TRUS biopsy (ρ=0.617, P =0.003), DW MRI (ρ=–0.601, P =0.004), and combined DW MRI/CHOL PET (ρ=–0.623, P =0.003). CHOL PET alone and FDG PET only had weak correlations. The correct GR classification rates were 67 % with TRUS biopsy, 67 % with DW MRI, and 76 % with combined DW MRI/CHOL PET. Conclusions DW MRI and combined DW MRI/CHOL PET have significant correlations and high rates of correct classification of the prostatectomy GR, the strength and accuracy of which are comparable with TRUS biopsy. Key Points • Accurate determination of the Gleason score is essential for prostate cancer management. • DW MRI ± CHOL PET correlated significantly with prostatectomy Gleason score. • These correlations are similar to that between TRUS biopsy and prostatectomyJoe H. Chang, Daryl Lim Joon, Sze Ting Lee, Chee-Yan Hiew, Stephen Esler, Sylvia J. Gong, Morikatsu Wada, David Clouston, Richard O, Sullivan, Yin P. Goh, Henri Tochon-Danguy, J. Gordon Chan, Damien Bolton, Andrew M. Scott, Vincent Khoo, Ian D. Davi

Surgical Decision Making for Unstable Thoracolumbar Spine Injuries: Results of a Consensus Panel Review by the Spine Trauma Study Group

Objectives: The optimal surgical approach and treatment of unstable thoracolumbar spine injuries are poorly defined owing to a lack of widely accepted level I clinical literature. This lack of evidence based standards has led to varied practice patterns based on individual surgeon preferences. The purpose of this study was to survey the leaders in the field of spine trauma to define the major characteristics of thoracolumbar injuries that influence their surgical decision making. In the absence of good scientific data, expert consensus opinions may provide surgeons with a practical framework to guide therapy and to conduct future research. Methods: A panel of 22 leading spinal surgeons from 20 level I trauma centers in seven countries met to discuss the indications for surgical approach selection in unstable thoracolumbar injuries. Injuries were presented to the surgeons in a case scenario survey format. Preferred surgical approaches to the clinical scenarios were tabulated and comments weighed. Results: All members of the panel agreed that three independent characteristics of thoracolumbar injuries carry primary importance in surgical decision making: the injury morphology, the neurologic status of the patient, and the integrity of the posterior ligaments. Six clinical scenarios based on the neurologic status of the patient (intact, incomplete, or complete) and on the status of the posterior ligamentous complex (intact or disrupted) were created, and consensus treatment approaches were described. Additional circumstances capable of altering the treatments were acknowledged. Conclusions: Decision making for the surgical treatment of thoracolumbar injuries is largely dependent on three patient characteristics: injury morphology, neurologic status, and posterior ligament integrity. A logical and practical decision-making process based on these characteristics may guide treatment even for the most complicated fracture patterns

Hypoxia-targeted radiotherapy dose painting for head and neck cancer using 18F-FMISO PET: A biological modeling study

BACKGROUND: This study investigates the use of (18)F-fluoromisonidazole (FMISO) PET-guided radiotherapy dose painting for potentially overcoming the radioresistant effects of hypoxia in head and neck squamous cell carcinoma (HNSCC). MATERIAL AND METHODS: The study cohort consisted of eight patients with HNSCC who were planned for definitive radiotherapy. Hypoxic subvolumes were automatically generated on pre-radiotherapy FMISO PET scans. Three radiotherapy plans were generated for each patient: a standard (STD) radiotherapy plan to a dose of 70 Gy, a uniform dose escalation (UDE) plan to the standard target volumes to a dose of 84 Gy, and a hypoxia dose-painted (HDP) plan with dose escalation only to the hypoxic subvolume to 84 Gy. Plans were compared based on tumor control probability (TCP), normal tissue complication probability (NTCP), and uncomplicated tumor control probability (UTCP). RESULTS: The mean TCP increased from 73% with STD plans to 95% with the use of UDE plans (p < 0.001) and to 93% with HDP plans (p < 0.001). The mean parotid NTCP increased from 26% to 44% with the use of UDE plans (p = 0.003), and the mean mandible NTCP increased from 2% to 27% with the use of UDE plans (p = 0.001). There were no statistically significant differences between any of the NTCPs between the STD plans and HDP plans. The mean UTCP increased from 48% with STD plans to 66% with HDP plans (p = 0.016) and dropped to 37% with UDE plans (p = 0.138). CONCLUSION: Hypoxia-targeted radiotherapy dose painting for head and neck cancer using FMISO PET is technically feasible, increases the TCP without increasing the NTCP, and increases the UTCP. This approach is superior to uniform dose escalation.Joe H. Chang, Morikatsu Wada, Nigel J. Anderson, Daryl Lim Joon, Sze Ting Lee, Sylvia J. Gong, Dishan H. Gunawardana, John Sachinidis, Graeme O, Keefe, Hui K. Gan, Vincent Khoo, Andrew M. Scot