Enhancing the compatibility of BioCaRGOS silica sol-gel technology with ctDNA extraction and droplet digital PCR (ddPCR) analysis.

Previously, our group had demonstrated long term stabilization of protein biomarkers using BioCaRGOS, a silica sol-gel technology. Herein, we describe workflow modifications to allow for extraction of cell free DNA (cfDNA) from primary samples containing working concentrations of BioCaRGOS, as well as the compatibility of BioCaRGOS with droplet digital PCR (ddPCR) analysis for pancreatic cancer biomarkers i.e., KRAS circulating tumor DNA (ctDNA). Preliminary attempts to extract ctDNA from BioCaRGOS containing samples demonstrated interference in the extraction of primary samples and the interference with ddPCR analysis when BioCaRGOS was directly introduced to stabilize sample extracts. In our modified technique, we have minimized the interference caused by methanol with ddPCR by complete removal of methanol from the activated BioCaRGOS formulation prior to addition to the biospecimen or ctDNA extract. Interference of the silica matrix present in BioCaRGOS with ctDNA extraction was eliminated through the introduction of invert filtration of the sample prior to extraction. These modifications to the workflow of BioCaRGOS containing samples allow for use of BioCaRGOS for stabilization of trace quantities of nucleic acid biomarkers such as plasma ctDNA, while retaining the capability to extract the biomarker and quantify based on ddPCR.

Radiation-induced curcumin release from curcumin-chitosan polymer films.

The probability of human exposure to damaging radiation is increased in activities associated with long-term space flight, medical radiation therapies, and responses to nuclear accidents. However, the development of responsive countermeasures to combat radiation damage to biological tissue is lagging behind rates of human exposure. Herein, we report a radiation-responsive drug delivery system that releases doses of curcumin from a chitosan polymer/film in response to low level gamma radiation exposure. As a fibrous chitosan-curcumin polymer, 1 Gy gamma irradiation (137Cs) released 5 ± 1% of conjugated curcumin, while 6 Gy exposure releases 98 ± 1% of conjugated curcumin. The same polymer was formed into a film through solvent casting. The films showed similar, albeit attenuated behavior in water (100% released) and isopropyl alcohol (32% released) with statistically significant drug release following 2 Gy irradiation. ATR FT-IR studies confirmed glycosidic bond cleavage in the chitosan-curcumin polymer in response to gamma radiation exposure. Similar behavior was noted upon exposure of the polymer to 20 cGy (1 GeV amu-1, at 20 cGy min-1) high linear energy transfer (LET) 56Fe radiation based on FTIR studies. Density Functional Theory calculations indicate homolytic bond scission as the primary mechanism for polymer disintegration upon radiation exposure. Films did not change in thickness during the course of radiation exposure. The successful demonstration of radiation-triggered drug release may lead to new classes of radio-protective platforms for developing countermeasures to biological damage from ionizing radiation.

Dataset for dose and time-dependent transcriptional response to ionizing radiation exposure.

Exposure to ionizing radiation associated with highly energetic and charged heavy particles is an inherent risk astronauts face in long duration space missions. We have previously considered the transcriptional effects that three levels of radiation (0.3 Gy, 1.5 Gy, and 3.0 Gy) have at an immediate time point (1 hr) post-exposure [1]. Our analysis of these results suggest effects on transcript levels that could be modulated at lower radiation doses [2]. In addition, a time dependent effect is likely to be present. Therefore, in order to develop a lab-on-a-chip approach for detection of radiation exposure in terms of both radiation level and time since exposure, we developed a time- and dose-course study to determine appropriate sensitive and specific transcript biomarkers that are detectable in blood samples. The data described herein was developed from a study measuring exposure to 0.15 Gy, 0.30 Gy, and 1.5 Gy of radiation at 1 hr, 2 hr, and 6 hr post-exposure using Affymetrix® GeneChip® PrimeView™ microarrays. This report includes raw gene expression data files from the resulting microarray experiments representing typical radiation exposure levels an astronaut may experience as part of a long duration space mission. The data described here is available in NCBI's Gene Expression Omnibus (GEO), accession GSE63952.

Targeted Gold Nanoparticle⁻Oligonucleotide Contrast Agents in Combination with a New Local Voxel-Wise MRI Analysis Algorithm for In Vitro Imaging of Triple-Negative Breast Cancer.

Gold nanoparticles (GNPs) have tremendous potential as cancer-targeted contrast agents for diagnostic imaging. The ability to modify the particle surface with both disease-targeting molecules (such as the cancer-specific aptamer AS1411) and contrast agents (such as the gadolinium chelate Gd(III)-DO3A-SH) enables tailoring the particles for specific cancer-imaging and diagnosis. While the amount of image contrast generated by nanoparticle contrast agents is often low, it can be augmented with the assistance of computer image analysis algorithms. In this work, the ability of cancer-targeted gold nanoparticle-oligonucleotide conjugates to distinguish between malignant (MDA-MB-231) and healthy cells (MCF-10A) is tested using a T1-weighted image analysis algorithm based on three-dimensional, deformable model-based segmentation to extract the Volume of Interest (VOI). The gold nanoparticle/algorithm tandem was tested using contrast agent GNP-Gd(III)-DO3A-SH-AS1411) and nontargeted c-rich oligonucleotide (CRO) analogs and control (CTR) counterparts (GNP-Gd(III)-DO3A-SH-CRO/CTR) via in vitro studies. Remarkably, the cancer cells were notably distinguished from the nonmalignant cells, especially at nanomolar contrast agent concentrations. The T1-weighted image analysis algorithm provided similar results to the industry standard Varian software interface (VNMRJ) analysis of T1 maps at micromolar contrast agent concentrations, in which the VNMRJ produced a 19.5% better MRI contrast enhancement. However, our algorithm provided more sensitive and consistent results at nanomolar contrast agent concentrations, where our algorithm produced ~500% better MRI contrast enhancement.

Computer-Aided Diagnostic System for Early Detection of Acute Renal Transplant Rejection Using Diffusion-Weighted MRI.

OBJECTIVE: Early diagnosis of acute renal transplant rejection (ARTR) is critical for accurate treatment. Although the current gold standard, diagnostic technique is renal biopsy, it is not preferred due to its invasiveness, long recovery time (1-2 weeks), and potential for complications, e.g., bleeding and/or infection. METHODS: This paper presents a computer-aided diagnostic (CAD) system for early ARTR detection using (3D + b-value) diffusion-weighted (DW) magnetic resonance imaging (MRI) data. The CAD process starts from kidney tissue segmentation with an evolving geometric (level-set-based) deformable model. The evolution is guided by a voxel-wise stochastic speed function, which follows from a joint kidney-background Markov-Gibbs random field model accounting for an adaptive kidney shape prior and on-going kidney-background visual appearances. A B-spline-based three-dimensional data alignment is employed to handle local deviations due to breathing and heart beating. Then, empirical cumulative distribution functions of apparent diffusion coefficients of the segmented DW-MRI at different b-values are collected as discriminatory transplant status features. Finally, a deep-learning-based classifier with stacked nonnegative constrained autoencoders is employed to distinguish between rejected and nonrejected renal transplants. RESULTS: In our initial "leave-one-subject-out" experiment on 100 subjects, [Formula: see text] of the subjects were correctly classified. The subsequent four-fold and ten-fold cross-validations gave the average accuracy of [Formula: see text] and [Formula: see text], respectively. CONCLUSION: These results demonstrate the promise of this new CAD system to reliably diagnose renal transplant rejection. SIGNIFICANCE: The technology presented here can significantly impact the quality of care of renal transplant patients since it has the potential to replace the gold standard in kidney diagnosis, biopsy.

Transcriptional profile of immediate response to ionizing radiation exposure.

Astronauts participating in long duration space missions are likely to be exposed to ionizing radiation associated with highly energetic and charged heavy particles. Previously proposed gene biomarkers for radiation exposure include phosphorylated H2A Histone Family, Member X (γH2AX), Tumor Protein 53 (TP53), and Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A). However, transcripts of these genes may not be the most suitable biomarkers for radiation exposure due to a lack of sensitivity or specificity. As part of a larger effort to develop lab-on-a-chip methods for detecting radiation exposure events using blood samples, we designed a dose-course microarray study in order to determine coding and non-coding RNA transcripts undergoing differential expression immediately following radiation exposure. The main goal was to elicit a small set of sensitive and specific radiation exposure biomarkers at low, medium, and high levels of ionizing radiation exposure. Four separate levels of radiation were considered: 0 Gray (Gy) control; 0.3 Gy; 1.5 Gy; and 3.0 Gy with four replicates at each radiation level. This report includes raw gene expression data files from the resulting microarray experiments from all three radiation levels ranging from a lower, typical exposure than an astronaut might see (0.3 Gy) to high, potentially lethal, levels of radiation (3.0 Gy). The data described here is available in NCBI's Gene Expression Omnibus (GEO), accession GSE64375.

Enhanced drug delivery via hyperthermal membrane disruption using targeted gold nanoparticles with PEGylated Protein-G as a cofactor.

Gold nanoparticles (GNPs) with near infrared (NIR) plasmon resonance have been promisingly used in photothermal cancer therapy as a less invasive treatment. Recombinant Protein-G (ProG) was PEGylated to act as a cofactor to immobilize immunoglobulins (IgGs) on GNPs by the Fc region, resulting in optimal orientation of IgGs for efficient cancer targeting. In-vitro studies showed that HER-2 overexpressing breast cancer cells, SK-BR-3, were efficiently targeted and ablated at a laser power of 900 J/cm(2) (5 W/cm(2) for 3 min). However, as a means of enhancing treatment efficacy by increasing cellular sensitivity to chemotherapeutic agents, we showed that GNP exposure to lower power laser resulted in small disruptions of cell membrane due to localized hyperthermia. This did not lead to cell death but provided a mechanism for killing cancer cells by providing enhanced uptake of drug molecules thus leading to a new avenue for hyperthermia-anticancer drug combined cancer therapeutics. FROM THE CLINICAL EDITOR: PEGylated recombinant Protein-G was used as a cofactor to optimize the orientation of IgGs providing "target seeking" properties to gold nanoparticles used in photothermal cancer therapy. The system demonstrated excellent properties in cancer therapy, with the hope and expectation of future clinical translation.

Estradiol and dihydrotestosterone regulate endothelial cell barrier function after hypergravity-induced alterations in MAPK activity.

Postflight orthostatic intolerance (POI) was reported to be higher in female than male astronauts and may result from sex-dependent differences in endothelial cell (EC) barrier permeability. Here the effect of 17beta-estradiol (E(2)) and dihydrotestosterone (DHT) on the expression of the tight junction protein occludin, EC barrier function, and MAPK activation over time was tested after subjecting human umbilical vein EC (HUVEC) to brief hypergravity identical to that experienced by astronauts during liftoff (LO) into space. After LO hypergravity, HUVEC showed a time-dependent decrease in occludin correlating with an increase in paracellular permeability and a decrease in transendothelial electrical resistance, indicating a decrease in EC barrier function. LO hypergravity inhibited MAPK activation, which remained suppressed 4 h after LO. Inhibition of MAPK activation correlated with decreased phosphotyrosine occludin, decreased cytochrome-c oxidase activity, and increased paracellular permeability, suggesting a mechanism by which LO hypergravity decreased EC barrier function. Time-dependent differences in MAPK activation, decreased occludin, and EC barrier function between HUVEC treated with E(2) vs. DHT were observed. HUVEC showed delayed activation of MAPK with DHT, i.e., 4 h rather than 2 h for E(2), which correlated with decreased paracellular permeability and the observed sex differences in POI in astronauts. These data temporally separate E(2) and DHT effects in HUVEC and provide evidence for the possible protective roles of sex steroids on EC function after brief exposure to low hypergravity.

Effect of estradiol and dihydrotestosterone on hypergravity-induced MAPK signaling and occludin expression in human umbilical vein endothelial cells.

Female astronauts have been reported to have a higher incidence of post-flight orthostatic intolerance (POI) compared with that of their male counterparts. POI may result from increased permeability of the endothelial cell (EC) layer in the vasculature. The goal of this study has been to determine whether estradiol (E(2)) and dihydrotesterone (DHT) alter human umbilical vein ECs (HUVECs) responses to short term (10 min) hypergravity (1-3 g) mimicking the g force experienced by astronauts during liftoff. E(2) and DHT rapidly (within 5 min) activated MAPK (mitogen-activated protein kinase) in HUVEC at 1 g in a receptor-dependent manner. Liftoff inhibited MAPK phosphorylation, and rapid E(2) and DHT activation of MAPK was blocked. Liftoff simulation or brief (5-90 min) treatment with E(2) or DHT at 1 g had no effect on the expression of the EC tight-junction protein occludin. However, 24-h pre-treatment of HUVECs with E(2) and DHT prior to liftoff simulation significantly increased occludin expression, and hypergravity exposure did not alter this increase. These data provide evidence for a possible protective effect of E(2) and DHT on EC function as indicated by increased occludin; this may help maintain the integrity of EC tight junction and could thus retard or reduce the incidence of POI.

Resveratrol and estradiol rapidly activate MAPK signaling through estrogen receptors alpha and beta in endothelial cells.

Vascular endothelial cells (EC) are an important target of estrogen action through both the classical genomic (i.e. nuclear-initiated) activities of estrogen receptors alpha and beta (ERalpha and ERbeta) and the rapid "non-genomic" (i.e. membrane-initiated) activation of ER that stimulates intracellular phosphorylation pathways. We tested the hypothesis that the red wine polyphenol trans-resveratrol activates MAPK signaling via rapid ER activation in bovine aortic EC, human umbilical vein EC, and human microvascular EC. We report that bovine aortic EC, human umbilical vein EC, and human microvascular EC express ERalpha and ERbeta. We demonstrate that resveratrol and estradiol (E(2)) rapidly activated MAPK in a MEK-1, Src, matrix metalloproteinase, and epidermal growth factor receptor-dependent manner. Importantly, resveratrol activated MAPK and endothelial nitric-oxide synthase (eNOS) at nm concentrations (i.e. an order of magnitude less than that required for ER genomic activity) and concentrations possibly achieved transiently in serum following oral red wine consumption. Co-treatment with ER antagonists ICI 182,780 or 4-hydroxytamoxifen blocked resveratrol- or E(2)-induced MAPK and eNOS activation, indicating ER dependence. We demonstrate for the first time that ERalpha-and ERbeta-selective agonists propylpyrazole triol and diarylpropionitrile, respectively, stimulate MAPK and eNOS activity. A red but not a white wine extract also activated MAPK, and activity was directly correlated with the resveratrol concentration. These data suggest that ER may play a role in the rapid effects of resveratrol in EC and that some of the atheroprotective effects of resveratrol may be mediated through rapid activation of ER signaling in EC.