Computed tomography dose index determination in dose modulation prospectively involving the third-generation iterative reconstruction and noise index.

PURPOSE: Optimizing CT protocols is challenging in the presence of automatic dose modulation because the CT dose index (CTDIvol) at different patient sizes is unknown to the operator. The task is more difficult when both the image quality index and iterative reconstruction prospectively affect the dose determination. It is of interest in practice to be informed of the CTDIvol during the protocol initialization and evaluation. It was our objective to obtain a predictive relationship between CTDIvol, the image quality index, and iterative reconstruction strength at various patient sizes. METHODS: Dose modulation data were collected on a GE Revolution 256-slice scanner utilizing a Mercury phantom and selections of the noise index (NI) from 8 to 17, the third generation iterative reconstruction (ASIR-V) from 0% to 80%, and phantom diameters from 16 to 36 cm. The fixed parameters were 120 kVp, a pitch of .984, and a collimation of 40 mm with a primary slice width of 2.5 mm. The CTDIvol per diameter was based on the average tube current over three adjacent slices (same or similar diameter) multiplied by a conversion factor between the average mA of the series and the reported CTDIvol. The relationship between CTDIvol, NI, and ASIR-V for each diameter was fitted with a 2nd order polynomial of ASIR-V multiplied by a power law of NI. RESULTS: The ASIR-V fit parameters versus diameter followed a Lorentz function while the NI exponent versus diameter followed an exponential growth function. The CTDIvol predictions were accurate within 15% compared to phantom results on a separate GE Revolution. For clinical relevance, the phantom diameter was converted to an abdomen or chest equivalent diameter and was well matched to patient data. CONCLUSION: The fitted relationship for CTDIvol. for given values of NI and ASIR-V blending for a range of phantom sizes was a good match to phantom and patient data. The results can be of direct help for selecting adequate parameters in CT protocol development.

Dose reduction in digital radiography based on the significance of marginal contrast detectability.

The performance of three digital detectors was measured at two exposure index (EI) levels in terms of the effect on features at the borderline of detectability. The null hypothesis was that there would be no statistically significant difference in the CNR of marginally visible features of a baseline- (2.2 µGy) and reduced dose (1.4 µGy) images. The experiment used three digital detectors and a phantom composed of an aluminum contrast-recovery plate, with features of varying diameters and hole depths, which was placed between the detector/grid and 5-20 cm Lucite. Exposures were made using a kVp between 55 and 110 corresponding to the Lucite thickness and a mAs producing an EI of approximately 220 or 140. Images were acquired for all detectors, EI values, and all Lucite thicknesses, then scored by a team of physicists and technologists in terms of feature visibility for each feature size. Contrast-to-noise ratio (CNR) was calculated for each feature using an ROI over the feature and a local background annulus. The uncertainty in the CNR was determined by sampling the background at each feature size, finding residuals from an overall background fit, and then calculating a standard deviation in the noise for each size. The marginal feature pair for each feature size bracketed the reader score. The difference between the CNR values of corresponding marginal features in EI-paired images was significant (P < 0.05) for one detector and not significant (P > 0.05) for marginal features of the other two. Based on both reader scoring and CNR measurements of phantoms, patient doses can be lowered by 30% for those two detectors without a statistically significant difference in lesion perceptibility of the marginally visible feature, while for the other detector there was a statistically significant change in marginal feature detectability and dose reduction was not recommended.

Dual-Readout Sandwich Immunoassay for Device-Free and Highly Sensitive Anthrax Biomarker Detection.

We report a dual-readout, AuNP-based sandwich immunoassay for the device-free colorimetric and sensitive scanometric detection of disease biomarkers. An AuNP-antibody conjugate serves as a signal transduction and amplification agent by promoting the reduction and deposition of either platinum or gold onto its surface, generating corresponding colorimetric or light scattering (scanometric) signals, respectively. We apply the Pt-based colorimetric readout of this assay to the discovery of a novel monoclonal antibody (mAb) sandwich pair for the detection of an anthrax protective antigen (PA83). The identified antibody pair detects PA83 down to 1 nM in phosphate-buffered saline and 5 nM in human serum, which are physiologically relevant concentrations. Reducing gold rather than platinum onto the mAb-AuNP sandwich enables scanometric detection of subpicomolar PA83 concentrations, over 3 orders of magnitude more sensitive than the colorimetric readout.

Circulating microRNA signature for the diagnosis of very high-risk prostate cancer.

We report the identification of a molecular signature using the Scano-miR profiling platform based on the differential expression of circulating microRNAs (miRNA, miR) in serum samples specific to patients with very high-risk (VHR) prostate cancer (PCa). Five miRNA PCa biomarkers (miR-200c, miR-605, miR-135a*, miR-433, and miR-106a) were identified as useful for differentiating indolent and aggressive forms of PCa. All patients with VHR PCa in the study had elevated serum levels of miR-200c. Circulating miR-433, which was differentially expressed in patients with VHR versus low-risk (LR) forms of PCa, was not detectable by quantitative real-time PCR in samples from healthy volunteers. In blind studies, the five miRNA PCa biomarkers were able to differentiate patients with VHR PCas from those with LR forms as well as healthy individuals with at least 89% accuracy. Biological pathway analysis showed the predictive capability of these miRNA biomarkers for the diagnosis and prognosis of VHR aggressive PCa.

Universal Biotin-PEG-Linked Gold Nanoparticle Probes for the Simultaneous Detection of Nucleic Acids and Proteins.

Novel biotin-polyethylene glycol (biotin-PEG) gold nanoparticle probes have been synthesized and used as universal constructs for the detection of protein (prostate-specific antigen, PSA) and nucleic acid targets (microRNAs) from a single sample. Microarray assays based upon these probes enabled sensitive detection of biomarker targets (50 fM for nucleic acid targets and 1 pg/µL for the PSA target). Ways of detecting biomarkers, including nucleic acids and proteins, are necessary for the clinical diagnosis of many diseases, but currently available diagnostic platforms rely primarily on the independent detection of proteins or nucleic acids. In addition to the economic benefits associated with the use of a single platform to detect both classes of analytes, studies have shown that the simultaneous identification of multiple classes of biomarkers in the same sample could be useful for the detection and management of early stage diseases, especially when sample amounts are limited. Therefore, these new probes and the assays based upon them open the door for high-sensitivity combination-target assays for studying and tracking biological pathways and diseases.

Evaluation of digital radiography practice using exposure index tracking.

Some digital radiography (DR) detectors and software allow for remote download of exam statistics, including image reject status, body part, projection, and exposure index (EI). The ability to have automated data collection from multiple DR units is conducive to a quality control (QC) program monitoring institutional radiographic exposures. We have implemented such a QC program with the goal to identify outliers in machine radiation output and opportunities for improvement in radiation dose levels. We studied the QC records of four digital detectors in greater detail on a monthly basis for one year. Although individual patient entrance skin exposure varied, the radiation dose levels to the detectors were made to be consistent via phototimer recalibration. The exposure data stored on each digital detector were periodically downloaded in a spreadsheet format for analysis. EI median and stan-dard deviation were calculated for each protocol (by body part) and EI histograms were created for torso protocols. When histograms of EI values for different units were compared, we observed differences up to 400 in average EI (representing 60% difference in radiation levels to the detector) between units nominally cali-brated to the same EI. We identified distinct components of the EI distributions, which in some cases, had mean EI values 300 apart. Peaks were observed at the current calibrated EI, a previously calibrated EI, and an EI representing computed radiography (CR) techniques. Our findings in this ongoing project have allowed us to make useful interventions, from emphasizing the use of phototimers instead of institutional memory of manual techniques to improvements in our phototimer calibration. We believe that this QC program can be implemented at other sites and can reveal problems with radiation levels in the aggregate that are difficult to identify on a case-by-case basis.

Biocompatible infinite-coordination-polymer nanoparticle-nucleic-acid conjugates for antisense gene regulation.

Herein, we report the synthesis of DNA-functionalized infinite-coordination-polymer (ICP) nanoparticles as biocompatible gene-regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3-hydroxy-4-pyridinone (HOPO) ligand bearing a pendant azide. Addition of Fe(III) to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the Fe(III)-HOPO ICP particles by copper-free click chemistry afforded colloidally stable nucleic-acid nanoconstructs. The DNA-ICP particles, when cross-linked through sequence-specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA-ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.

Hollow spherical nucleic acids for intracellular gene regulation based upon biocompatible silica shells.

Cellular transfection of nucleic acids is necessary for regulating gene expression through antisense or RNAi pathways. The development of spherical nucleic acids (SNAs, originally gold nanoparticles functionalized with synthetic oligonucleotides) has resulted in a powerful set of constructs that are able to efficiently transfect cells and regulate gene expression without the use of auxiliary cationic cocarriers. The gold core in such structures is primarily used as a template to arrange the nucleic acids into a densely packed and highly oriented form. In this work, we have developed methodology for coating the gold particle with a shell of silica, modifying the silica with a layer of oligonucleotides, and subsequently oxidatively dissolving the gold core with I(2). The resulting hollow silica-based SNAs exhibit cooperative binding behavior with respect to complementary oligonucleotides and cellular uptake properties comparable to their gold-core SNA counterparts. Importantly, they exhibit no cytotoxicity and have been used to effectively silence the eGFP gene in mouse endothelial cells through an antisense approach.

High-ratio grid considerations in mobile chest radiography.

PURPOSE: Grids are often not used in mobile chest radiography, and when used, they have a low ratio and are often inaccurately aligned. Recently, a mobile radiography automatic grid alignment system (MRAGA) was developed that accurately and automatically aligns the focal spot with the grid. The objective of this study is to investigate high-ratio grid tradeoffs in mobile chest radiography at fixed patient dose when the focal spot lies on the focal axis of the grid. METHODS: The chest phantoms (medium and large) used in this study were modifications of the ANSI (American National Standards Institute) chest phantom and consisted of layers of Lucite™, aluminum, and air. For the large chest phantom, the amount of Lucite and aluminum was increased by 50% over the medium phantom. Further modifications included a mediastinum insert and the addition of contrast targets in the lung and mediastinum regions. Five high-ratio grids were evaluated and compared to the nongrid results at x-ray tube potentials of 80, 90, 100, and 110 kVp for both phantoms. The grids investigated were from two manufacturers: 12:1 and 15:1 aluminum interspace grids from one and 10:1, 13:1, and 15:1 fiber interspace grids from another. MRAGA was employed to align the focal spot with the grid. All exposures for a given kVp and phantom size were made using the same current-time product (CTP). The phantom images were acquired using computed radiography, and contrast-to-noise ratios (CNR) and CNR improvement factors (k(CNR)) were determined from the resultant images. The noise in the targets and the contrast between the targets and their backgrounds were calculated using a local detrending correction, and the CNR was calculated as the ratio of the target contrast to the background noise. k(CNR) was defined as the ratio of the CNR imaged with the grid divided by the CNR imaged without a grid. RESULTS: The CNR values obtained with a high-ratio grid were 4%-65% higher than those obtained without a grid at the same phantom dose. The improvement was greater for the large chest phantom than the medium chest phantom and greater for the mediastinum targets than for the lung targets. In general, the fiber interspace grids performed better than the aluminum interspace grids. In the lung, k(CNR) for both types of grids exhibited little dependence on kVp or grid ratio. In the mediastinum, k(CNR) decreased 4%-10% with increasing kVp, and varied up to 5.3% with grid ratio. CONCLUSIONS: When the focal spot is accurately aligned with the grid, the use of a high-ratio grid in mobile chest radiography improves image quality with no increase in dose to the phantom. For the grids studied, the performance of the fiber interspace grids was superior to the performance of the aluminum interspace grids, with the fiber interspace 13:1 grid producing the best overall results for the medium chest phantom and the fiber interspace 15:1 producing the best overall results for the large chest phantom.

Predictive Model for 82Rb Generator Bolus Times as a Function of Generator Lifetime.

82Rb cardiac PET is largely used to study myocardial perfusion with function and to calculate myocardial blood flow (MBF) and coronary flow reserve or myocardial flow reserve. Although the dosing activity of 82Rb is determined by the patient weight, the infusion volume and activity concentration varies with the age of the 82Rb generator. We sought to predict the needed bolus volume of 82Rb to help evaluate the accuracy of MBF findings. Methods: Data were collected from deidentified tickets of an 82Rb generator, including the instantaneous eluted activity flow rate. The times to reach 4 activity levels-740, 1,110, 1,480, and 1,665 MBq (20, 30, 40, and 45 mCi, respectively)-were also calculated. The activity flow rate for the largest bolus was fitted to determine the functional form. The time to reach each bolus level was fitted as a function of the generator age, and 95% CIs were created. Results: The activity flow rate was fitted with a growth-saturation model, allowing a calculation of bolus volume. The amplitude of the fit was observed to also be influenced by the time since the last elution and possibly other clinical factors. Elution times to reach the 4 activity levels were plotted versus generator age. The linearized data were fitted, and 95% CIs were created symmetrically around the fit. The 95% CI band allowed a prediction of elution time to achieve each bolus size for future generators, as a function only of generator age. Conclusion: A predictive model was created for elution times from this brand of 82Rb generator as a function of generator age. The value of this model is in determining whether the necessary amount of activity can be extracted from a generator before reaching one of the backup infusion settings, such as volume limits per administration, given a generator age. Some sites may also control the bolus duration for better MBF calculations, since predicting the time for the injection to complete may determine whether MBF and coronary flow reserve calculations are meaningful.

Acute in vitro exposure to environmentally relevant atrazine levels perturbs bovine preimplantation embryo metabolism and cell number.

Atrazine is a widely used herbicide known to negatively alter endocrine systems and perturb metabolism. Preimplantation exposure to pesticides may adversely affect long-term health, however few studies examine the effect of environmental levels and whether specific periods of development are particularly sensitive. In this study, the effect of acute, preimplantation atrazine exposure (days 3.5-7.5 post-fertilization) at levels detected and deemed safe in drinking water (0.02 and 20 µg/L respectively) on in vitro bovine embryo development, quality, metabolism, and gene expression was investigated. Atrazine exposure had no effect on development or quality, but significantly reduced blastocyst total cell numbers, attributable to a decrease in trophectoderm cells. Notably, atrazine (20 µg/L) markedly increased carbohydrate metabolism. Therefore, short-term exposure to environmentally relevant atrazine concentrations perturbs bovine preimplantation embryo metabolism and cell number, highlighting a potential mechanism by which atrazine can mediate embryo viability and health.

Spherical nucleic acid targeting microRNA-99b enhances intestinal MFG-E8 gene expression and restores enterocyte migration in lipopolysaccharide-induced septic mice.

Milk fat globule-EGF factor 8 (MFG-E8) maintains the intestinal homeostasis by enhancing enterocyte migration and attenuating inflammation. We previously reported that sepsis is associated with down-regulation of intestinal MFG-E8 and impairment of enterocyte migration. Here, we showed that impairment of intestinal epithelial cell migration occurred in lipopolysaccharide (LPS)-induced septic mice. Treatment of RAW264.7 cells (a murine macrophage-like cell line) with LPS increased expression of miR-99b, a microRNA that is predicted to target mouse MFG-E8 3'UTR. Using a luciferase assay, we showed that miR-99b mimic suppressed the activity of a reporter containing MFG-E8 3'UTR. This suggests the role of miR-99b in inhibition of MFG-E8 gene expression. In addition, we developed an anti-miR99b spherical nucleic acid nanoparticle conjugate (SNA-NC(anti-miR99b)). Treatment of both naïve and LPS-challenged cells with SNA-NC(anti-miR99b) enhanced MFG-E8 expression in the cells. Administration of SNA-NC(anti-miR99b) rescued intestinal MFG-E8 expression in LPS-induced septic mice and attenuated LPS inhibitory effects on intestinal epithelial cell migration along the crypt-villus axis. Collectively, our study suggests that LPS represses MFG-E8 expression and disrupts enterocyte migration via a miR-99b dependent mechanism. Furthermore, this work shows that SNA-NC(anti-miR99b) is a novel nanoparticle-conjugate capable of rescuing MFG-E8 gene expression and maintaining intestinal epithelial homeostasis in sepsis.

Spherical nucleic acid nanoparticle conjugates as an RNAi-based therapy for glioblastoma.

Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many "undruggable" oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)-based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)--an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas--were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.

Intracranial microcapsule drug delivery device for the treatment of an experimental gliosarcoma model.

Controlled-release drug delivery systems are capable of treating debilitating diseases, including cancer. Brain cancer, in particular glioblastoma multiforme (GBM), is an extremely invasive cancer with a dismal prognosis. The use of drugs capable of crossing the blood-brain barrier has shown modest prolongation in patient survival, but not without unsatisfactory systemic, dose-limiting toxicity. Among the reasons for this improvement include a better understanding of the challenges of delivery of effective agents directly to the brain tumor site. The combination of carmustine delivered by biodegradable polyanhydride wafers (Gliadel(®)), with the systemic alkylating agent, temozolomide, allows much higher effective doses of the drug while minimizing the systemic toxicity. We have previously shown that locally delivering these two drugs leads to further improvement in survival in experimental models. We postulated that microcapsule devices capable of releasing temozolomide would increase the therapeutic capability of this approach. A biocompatible drug delivery microcapsule device for the intracranial delivery of temozolomide is described. Drug release profiles from these microcapsules can be modulated based on the physical chemistry of the drug and the dimensions of the release orifices in these devices. The drug released from the microcapsules in these experiments was the clinically utilized chemotherapeutic agent, temozolomide. In vitro studies were performed in order to test the function, reliability, and drug release kinetics of the devices. The efficacy of the temozolomide-filled microcapsules was tested in an intracranial experimental rodent gliosarcoma model. Immunohistochemical analysis of tissue for evidence of DNA strand breaks via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed. The experimental release curves showed mass flow rates of 36 µg/h for single-orifice devices and an 88 µg/h mass flow rate for multiple-orifice devices loaded with temozolomide. In vivo efficacy results showed that localized intracranial delivery of temozolomide from microcapsule devices was capable of prolonging animal survival and may offer a novel form of treatment for brain tumors.

Small molecule modifier screen for kit-dependent functions in zebrafish embryonic melanocytes.

Zebrafish is gaining popularity as a vertebrate model for screening small molecules that affect specific phenotypes or genetic pathways. In this study, we present a targeted drug screen to identify drug modifiers of the melanocyte migration defect of a temperature-sensitive allele of the Kit receptor tyrosine kinase, kit(ts). We first test two candidate drugs, the phosphatidylinositol-3-kinase kinase inhibitor (LY294002) and the Erk/MAP kinase inhibitor (PD98059), for their effect on melanocyte migration and survival. We find that LY294002 enhances the migration defect of kit(ts), implicating the phosphatidylinositol-3-kinase kinase pathway in promoting kit-dependent melanocyte migration, but not survival. We then used the kit(ts)-sensitized genetic background to screen a panel of 1280 pharmacologically active drugs to identify drug enhancers and suppressors of the kit(ts) melanocyte migration defect. We identified three drug enhancers of migration, two of which, Papaverine and Isoliquiritigenin, specifically enhance the kit(ts) migration defect, while 8-DPAT affected both melanocyte migration and survival. These drugs now provide additional experimental tools for investigating the mechanisms of kit-promoted melanocyte migration and survival in the zebrafish embryo.