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At least 235 records · Page 13

Polaritonic Chern Insulators in Monolayer Semiconductors

Systems with strong light-matter interaction open up new avenues for studying topological phases of matter. Examples include exciton polaritons, mixed light-matter quasiparticles, where the topology of the polaritonic band structure arises from the collective coupling between matter wave and optical fields strongly confined in periodic dielectric structures. Distinct from light-matter interaction in a uniform environment, the spatially varying nature of the optical fields leads to a fundamental modification of the well-known optical selection rules, which were derived under the plane wave approximation. Here, in this study, we identify polaritonic Chern insulators by coupling valley excitons in transition metal dichalcogenides to photonic Bloch modes in a dielectric photonic crystal slab. We show that polaritonic Dirac points, which are markers for topological phase transition points, can be constructed from the collective coupling between valley excitons and photonic Dirac cones in the presence of both time-reversal and inversion symmetries. Lifting exciton valley degeneracy by breaking time-reversal symmetry leads to gapped polaritonic bands with nonzero Chern numbers. Through numerical simulations, we predict polaritonic chiral edge states residing inside the topological gaps. Our Letter paves the way for the further study of strong exciton-photon interaction in nanophotonic structures and for exploring polaritonic topological phases and their practical applications in polaritonic devices.

74 ATOMIC AND MOLECULAR PHYSICS↗

Influence of Microgravity on Bacterial Pathogen Virulence and Immune Cell Function—Relevance for Infectious Disease Risk During Spaceflight

Spaceflight has measurable impacts on astronaut immune profiles as well as the virulence patterns of bacterial pathogens. Data with respect to human immunity indicate diminished T and NK cell function, altered cytokine profiles, persistent inflammation, and latent herpesvirus reactivation. Furthermore, evaluation of International Space Station (ISS) crewmembers gives evidence of compromised immunity, including atypical allergy, infectious disease, and dermatitis. Data with respect to certain human bacterial pathogens suggest modified virulence that may be enhanced. It is therefore critical to examine this interaction of immune dysregulation and increased microbial virulence and whether it might synergistically increase the risk of infectious disease to crew members. The goal of this study is to use modeled microgravity to study the impact of medically significant ISS bacteria that may have altered virulence on the immune response of the host. This study consists of two primary aims to assess this relationship. First, immune cells will be collected from healthy test subjects and cultured in static or in modeled microgravity conditions together with either control pathogens or with microgravity conditioned pathogens that were grown in bioreactors. Second, immune cells will be collected from astronauts before, during, and after flight and co-cultured with the control or the microgravity conditioned bacteria. Three pathogens were selected for this investigation: Pseudomonas aeruginosa , Salmonella enterica serovar Enteritidis and Burkholderia cepacia . Previously, the optimal cell to bacteria ratios that produce the greatest immune cell responses have been derived for these three bacteria. Cellular activation, as determined by the induction of cell surface activation markers and cytokine profiles, will be measured. Interactions between cells and bacteria will be assessed using fluorescent and electron microscopy techniques. This study will provide critical information to help understand how microgravity alters microbial virulence and the associated infectious disease risk to crewmembers during spaceflight missions. Over the past year, the Immunology and Microbiology laboratories at NASA Johnson Space Center have collaborated to process the astronaut subject samples to complete the last year of the flight portion of this study. This included the completion of four astronaut subjects full mission sampling sets. Sample processing included innate and adaptive cell flow cytometry as well as analysis of cytokine concentrations in the supernatant. The ground control segment of the study will take place during FY25 which involves parallel infections run under static and clinostat conditions. The acquired data sets over this 3-year study are now being analyzed to provide a comprehensive set of results and conclusions that will contribute to a more effective risk assessment for astronauts during spaceflight regarding this host-pathogen context.

Immunology↗

Could FDG-PET imaging play a role in the detection of progressing atherosclerosis in HIV-infected patients?

Important progresses in the management of patients with human immunodeficiency virus, in particular the advent of new anti-retroviral therapies (ART), have turned this rapidly fatal condition into a controllable chronic disease with a life expectancy that approaches the one from the general population. Cardiovascular diseases have now become one of the leading causes of non-HIV-related mortality in this population. Several factors including the presence of HIV in the vascular wall and the development of dyslipidemia and alteration in body fat distribution under ART might play a role the progression of atherosclerosis in HIV-infected patients. The use of imaging biomarkers may help to identify the factors associated with an increased risk of cardiovascular events and select high-risk patients who will benefit the most from the early implementation of preventive treatments.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Validation of reference genes for real-time PCR of cord blood mononuclear cells, differentiating endothelial progenitor cells, and mature endothelial cells

Highlights: • RT-qPCR was used to assess and follow the differentiation of EPCs. • A set of reliable and non-variable genes is key to obtain accurate RT-qPCR results. • The relative gene expression of CD34+ and differentiating endothelial progenitor cells was determined. • The optimal set of reference for the gene expression studies performed by RT-qPCR was determined. In the last ten years, endothelial progenitor cells (EPCs) have gained interest as an attractive cell population in regenerative medicine for vascular applications. This population is defined as the precursor of endothelial mature cells (ECs) through a process of differentiation. To our knowledge, no single marker can be used to discriminate them from mature ECs. To effectively study their differentiation kinetics, gene expression must be assessed. Quantitative real-time PCR (RT-qPCR) is widely used to analyze gene expression. To minimize the impact of variances from RT-qPCR, a rigorous selection of reference genes must be performed prior to any experiments due to variations in experimental conditions. In this study, CD34+ mononuclear cells were extracted from human cord blood and differentiated into EPCs after seeding for a maximum period of 21 days. To choose the best combinations of reference genes, we compared the results of EPCs, CD34+ mononuclear cells, and mature endothelial cells to ensure that the differentiation kinetics did not affect the expression of our selected reference genes. The expression levels of seven genes, namely, YWHAZ, GAPDH, HPRT1, RPLP0, UBC, B2M, and TBP were thus compared. The algorithms geNorm, NormFinder, BestKeeper, and the Comparative ΔCt method were employed to assess the expression of each candidate gene. Overall results reveal that the expression stability of reference genes may differ depending on the statistical program used. YWHAZ, GAPDH, and UBC composed the optimal set of reference genes for the gene expression studies performed by RT-qPCR in our experimental conditions. This work can thus serve as a starting point for the selection of candidate reference genes to normalize the levels of gene expression in endothelial progenitor cell populations.

60 APPLIED LIFE SCIENCES↗

Substrate oxidation capacity in rodent skeletal muscle: effects of exposure to zero gravity

A study was conducted, as part of the integrated National Aeronautics and Space Administration Space Life Sciences 1 mission flown in June of 1991, to ascertain the effects of 9 days of exposure to zero gravity on the capacity of rodent skeletal muscle fiber types to oxidize either [14C]pyruvate or [14C]palmitate under state 3 metabolic conditions, i.e., nonlimiting amounts of substrate and cofactors. In addition, activity levels of marker enzymes of the tricarboxylic acid cycle, malate shuttle, and beta-oxidation were measured. Results showed that significant differences in muscle weight occurred in both the predominantly slow vastus intermedius and predominantly fast vastus lateralis of flight vs. control groups (P < 0.05). Total protein content of the muscle samples was similar between groups. Both pyruvate oxidation capacity and the marker oxidative enzymes were not altered in the flight relative to control animals. However, the capacity to oxidize long-chain fatty acids was significantly reduced by 37% in both the high- and low-oxidative regions of the vastus muscle (P < 0.05). Although these findings of a selective reduction in fatty acid oxidation capacity in response to spaceflight are surprising, they are consistent with previous findings showing 1) an increased capacity to take up glucose and upregulate glucose transporter proteins and 2) a marked accumulation of triglycerides in the skeletal muscles of rats subjected to states of unloading. Thus, skeletal muscle of animals exposed to non-weight-bearing environments undergo subcellular transformations that may preferentially bias energy utilization to carbohydrates.

NASA Discipline Musculoskeletal↗

Genetics and Genomics of Pathogen Resistance in Switchgrass (Final Report)

This project was funded by DOE under Grant no. DE-SC0016108. Originally approved for the 2016-2019 period, two no-cost extensions were solicited and approved, which prolonged the lifespan through July 2021. This final report informs on the results obtained so far from the research implemented. The research hinged on integrating genomics (genomic selection, RNAseq, virus-plant interactions) with classical genetics (conventional breeding) to incorporate durable resistance to fungal (rust) and viral (mosaic) diseases in switchgrass (Panicum virgatum) populations being bred for bioenergy. Higher biomass yield, higher quality (low lignin content), and durable disease resistance are key features to make lignocellulosic switchgrass feedstocks economically competitive and sustainable. Genomic selection is being applied on three generations of a switchgrass population derived from crossing two ecotypes (Kanlow as lowland female and Summer as upland male) with differential performance in terms of biomass yield and quality, disease resistance, and winter survivability. Target populations were screened for rust and mosaic in field and/or lab and phenotyped for biomass yield and quality traits. Genetic analyses were applied across generations to capture the joint inheritance of the targeted traits and predict breeding values for parents and progeny with greater accuracy. Parental and a panel of different switchgrass populations were genotyped with the DArTseq technology to develop SNP (0, 1, 2) and in-silico (presence/absence) DArT markers. Rust inoculations techniques were developed and applied successfully on switchgrass. The original populations (Kanlow and Summer) were sequenced with RNAseq to capture the gene expression profiles across sequential time-points and appraise the basis of greater resistance in the Kanlow vs the Summer ecotype. Constructs of PMV and sPMV mosaic virus were assembled and tested first on proso millet to find the best protocol to use later on switchgrass. Results from the preliminary analyses indicate that 1) ample additive genetic variation is available for selection and improving this inter-ecotypic population for yield, quality, and disease traits, 2) significant gains are to be expected with the genetic correlations being favorable between yield and lignin content and between yield and disease ratings, 3) substantial differences exist in the genetic regions controlling rust resistance in the two ecotypes, 4) co-infection with PMV isolates from Nebraska and its satellite from Kansas elicit severe mosaic symptoms, and 5) two different genetic systems are responsible for imparting resistance to rust and virus in switchgrass.

59 BASIC BIOLOGICAL SCIENCES↗

The face of epicardial and endocardial derived cells in zebrafish

Highlights: • Growth factors cocktails can to lead a heart regeneration in ex-vivo culture. • Ex-vivo cultures have permitted to characterize the cells with mobility capability. • Epicardial- and endocardial-derived cells can be expanded for months in culture. • Whole heart or cells culture open a new field of translational studies. Zebrafish hearts can regenerate through activation of growth factors and trans-differentiation of fibroblasts, epicardial, myocardial and endocardial cells, all positive for GATA4 during the process. A possible model of regeneration of the whole heart and the regenerating cells in ex-vivo culture is presented here by a stimulation of cocktail of growth factors. In ex-vivo growth-factors-supplemented culture the heart regeneration was quite complete without signs of fibrosis. Epicardial- and endocardial-derived cells have been analyzed by electron microscopy evidencing two main types: 1) larger/prismatic and 2) small/rounded. Type (1) showed on the surface protein-sculptures, while type(2) was smooth with sparse globular proteins. To confirm their nature we have contemporarily analyzed their proliferative capability and markers-positivity. The cells treated by growth factors have at least two-fold more proliferation with GATA4-positivity. The type (1) cell evidenced WT1+(marker of embryonic epicardium); the type (2) showed NFTA2+(marker of embryonic endocardium); whereas cTNT-cardiotroponin was negative. Under growth factors stimulation, GATA4+/WT1+ and GATA4+/NFTA2+ could be suitable candidates to be the cells with capability to move in/out of the tissue, probably by using their integrins, and it opens the possibility to have long term selected culture to future characterization.

60 APPLIED LIFE SCIENCES↗

SU-G-IeP2-06: Evaluation of Registration Accuracy for Cone-Beam CT Reconstruction Techniques

Purpose: Cone-beam (CB) computed tomography (CT) is used for image guidance during radiotherapy treatment delivery. Conventional Feldkamp and compressed sensing (CS) based CBCT recon-struction techniques are compared for image registration. This study is to evaluate the image registration accuracy of conventional and CS CBCT for head-and-neck (HN) patients. Methods: Ten HN patients with oropharyngeal tumors were retrospectively selected. Each HN patient had one planning CT (CTP) and three CBCTs were acquired during an adaptive radiotherapy proto-col. Each CBCT was reconstructed by both the conventional (CBCTCON) and compressed sens-ing (CBCTCS) methods. Two oncologists manually labeled 23 landmarks of normal tissue and implanted gold markers on both the CTP and CBCTCON. Subsequently, landmarks on CTp were propagated to CBCTs, using a b-spline-based deformable image registration (DIR) and rigid registration (RR). The errors of these registration methods between two CBCT methods were calcu-lated. Results: For DIR, the mean distance between the propagated and the labeled landmarks was 2.8 mm ± 0.52 for CBCTCS, and 3.5 mm ± 0.75 for CBCTCON. For RR, the mean distance between the propagated and the labeled landmarks was 6.8 mm ± 0.92 for CBCTCS, and 8.7 mm ± 0.95 CBCTCON. Conclusion: This study has demonstrated that CS CBCT is more accurate than conventional CBCT in image registration by both rigid and non-rigid methods. It is potentially suggested that CS CBCT is an improved image modality for image guided adaptive applications.

60 APPLIED LIFE SCIENCES↗

VirION2: a short- and long-read sequencing and informatics workflow to study the genomic diversity of viruses in nature

Microbes play fundamental roles in shaping natural ecosystem properties and functions, but do so under constraints imposed by their viral predators. However, studying viruses in nature can be challenging due to low biomass and the lack of universal gene markers. Though metagenomic short-read sequencing has greatly improved our virus ecology toolkit—and revealed many critical ecosystem roles for viruses—microdiverse populations and fine-scale genomic traits are missed. Some of these microdiverse populations are abundant and the missed regions may be of interest for identifying selection pressures that underpin evolutionary constraints associated with hosts and environments. Though long-read sequencing promises complete virus genomes on single reads, it currently suffers from high DNA requirements and sequencing errors that limit accurate gene prediction. Here we introduce VirION2, an integrated short- and long-read metagenomic wet-lab and informatics pipeline that updates our previous method (VirION) to further enhance the utility of long-read viral metagenomics. Using a viral mock community, we first optimized laboratory protocols (polymerase choice, DNA shearing size, PCR cycling) to enable 76% longer reads (now median length of 6,965 bp) from 100-fold less input DNA (now 1 nanogram). Using a virome from a natural seawater sample, we compared viromes generated with VirION2 against other library preparation options (unamplified, original VirION, and short-read), and optimized downstream informatics for improved long-read error correction and assembly. VirION2 assemblies combined with short-read based data (‘enhanced’ viromes), provided significant improvements over VirION libraries in the recovery of longer and more complete viral genomes, and our optimized error-correction strategy using long- and short-read data achieved 99.97% accuracy. In the seawater virome, VirION2 assemblies captured 5,161 viral populations (including all of the virus populations observed in the other assemblies), 30% of which were uniquely assembled through inclusion of long-reads, and 22% of the top 10% most abundant virus populations derived from assembly of long-reads. Viral populations unique to VirION2 assemblies had significantly higher microdiversity means, which may explain why short-read virome approaches failed to capture them. These findings suggest the VirION2 sample prep and workflow can help researchers better investigate the virosphere, even from challenging low-biomass samples. Our new protocols are available to the research community on protocols.io as a ‘living document’ to facilitate dissemination of updates to keep pace with the rapid evolution of long-read sequencing technology.

Long-reads↗

Final-Approach Spacing Aids (FASA) evaluation for terminal-area, time-based air traffic control

A jointly funded (NASA/FAA) real-time simulation study was conducted at NASA Langley Research Center to gather comparative performance data among three candidate final-approach spacing aid (FASA) display formats. Several objective measures of controller performance and their display eye-scan behavior as well as subjective workload and rating questionnaires were used. For each of two representative pattern-speed procedures (a 170-knot procedure and a 210-knot procedure with speed control aiding), data were gathered, via twelve FAA controllers, using four final-controller display format conditions (manual/ARTS 3, graphic marker, DICE countdown, and centerline slot marker). Measured runway separations were more precise with both the graphic marker and DICE countdown formats than with the centerline slot marker and both (graphic and DICE) improved precision relative to the manual/ARTS 3 format. For three separate rating criteria, the subject controllers ranked the FASA formats in the same order: graphic marker, DICE countdown, and centerline slot marker. The increased precision measured with the 210-knot pattern-speed procedure may indicate the potential for the application of speed-control aiding where higher pattern speeds are practical after the base-to-final turn. Also presented are key FASA issues, a rationale for the formats selected for testing, and their description.

Credeur, Leonard↗

Using Genotyping by Sequencing to Map Two Novel Anthracnose Resistance Loci in Sorghum bicolor

Colletotrichum sublineola is an aggressive fungal pathogen that causes anthracnose in sorghum [ Sorghum bicolor (L.) Moench]. The obvious symptoms of anthracnose are leaf blight and stem rot. Sorghum, the fifth most widely grown cereal crop in the world, can be highly susceptible to the disease, most notably in hot and humid environments. In the southeastern United States the acreage of sorghum has been increasing steadily in recent years, spurred by growing interest in producing biofuels, bio-based products, and animal feed. Resistance to anthracnose is, therefore, of paramount importance for successful sorghum production in this region. To identify anthracnose resistance loci present in the highly resistant cultivar ‘Bk7’, a biparental mapping population of F 3:4 and F 4:5 sorghum lines was generated by crossing ‘Bk7’ with the susceptible inbred ‘Early Hegari-Sart’. Lines were phenotyped in three environments and in two different years following natural infection. The population was genotyped by sequencing. Following a stringent custom filtering protocol, totals of 5186 and 2759 informative SNP markers were identified in the two populations. Segregation data and association analysis identified resistance loci on chromosomes 7 and 9, with the resistance alleles derived from ‘Bk7’. Both loci contain multiple classes of defense-related genes based on sequence similarity and gene ontologies. In addition, genetic analysis following an independent selection experiment of lines derived from a cross between ‘Bk7’ and sweet sorghum ‘Mer81-4’ narrowed the resistance locus on chromosome 9 substantially, validating this QTL. As observed in other species, sorghum appears to have regions of clustered resistance genes. Further characterization of these regions will facilitate the development of novel germplasm with resistance to anthracnose and other diseases.

59 BASIC BIOLOGICAL SCIENCES↗

TU-H-CAMPUS-TeP2-05: Selective Protection of Normal Tissue by Cerium Oxide Nanoparticles During Radiation Therapy

Purpose: Cerium oxide nanoparticles (CONPs) have unique pH dependent properties such that they act as a radical modulator. These properties may be used in radiation therapy (RT) to protect normal tissue. This work investigates the selective radioprotection of CONPs in-vitro and potential for in-situ delivery of CONPs in prostate cancer RT. Methods: i) Normal human umbilical vein endothelial cells (HUVEC) and human prostate cancer cells (PC-3) were treated with 0 or 2 ng/mL CONPs (NP size: 5 nm). 2 Gy of 100 kVp radiation was delivered to the cells 4 hours after the CONP treatment. Cell viability was checked 48 hours later using MTS assays. ii) A prostate tumor was modeled as a 2-cm diameter sphere. CONPs were proposed to be loaded in a hollow radiotherapy fiducial marker. The concentration profile for the CONPs within the tumor was modeled with a previously validated diffusion equation employed in other studies for nanoparticles 10 nm or less. Results: i) Without radiation, cell viability was above 90% when treated with 2 ng/mL CONPs for both HUVEC and PC-3. After irradiation, a slightly higher viability was observed in HUVEC with CONPs than the ones without CONPs, and this effect was not observed in PC-3. ii) Based on the calculations, 2 ng/mL of CONPs could be delivered to normal cells by diffusion with a 1 µg/mL initial concentration within two weeks. Conclusion: We conclude that CONPs can provide selective radioprotection. The delivery of needed concentrations of CONPs is feasible via in-situ release from radiotherapy biomaterials (e.g. fiducials) loaded with the CONPs.

60 APPLIED LIFE SCIENCES↗

Whole-Genome Comparative Analysis of Two Carbapenem-Resistant ST-258 Klebsiella pneumoniae Strains Isolated during a North-Eastern Ohio Outbreak: Differences within the High Heterogeneity Zones

Klebsiella pneumoniae has become one of the most dangerous causative agents of hospital infections due to the acquisition of resistance to carbapenems, one of the last resort families of antibiotics. Resistance is usually mediated by carbapenemases coded for by different classes of genes. A prolonged outbreak of carbapenem-resistant K. pneumoniae infections has been recently described in northeastern Ohio. Most strains isolated from patients during this outbreak belong to MLST sequence type 258 (ST258). To understand more about this outbreak two isolates (strains 140 and 677), one of them responsible for a fatal infection, were selected for genome comparison analyses. Whole genome map and sequence comparisons demonstrated that both strains are highly related showing 99% average nucleotide identity. However, the genomes differ at the so-called high heterogeneity zone (HHZ) and other minor regions. This study identifies the potential value of the HHZ as a potential marker for K. pneumoniae clinical and epidemiological studies.

Evolutionary Biology↗

Developing a High-Quality Linkage Map for the Atlantic Killifish Fundulus heteroclitus

Killifish (Fundulus heteroclitus) are widely distributed among different aquatic environments where they demonstrate an impressive range of highly-plastic and locally adaptive phenotypes. High-throughput sequencing has begun to unravel the mechanisms and evolutionary history of these interesting features by establishing relationships in the genotype-phenotype map. However, some genotype-phenotype analyses require a higher order of contiguity than what initial scaffolded (fragmented genome assembly where contigs have been assemble into scaffolds) genome assemblies can provide. Here, we used 5,685 high-quality RAD-Seq markers from a single mapping family to order 84% of the scaffolded genome assembly to 24 chromosomes. This serves to: 1) expand the killifish genomic toolkit, 2) estimate genome-wide recombination rates, and 3) compare genome synteny to humans and other fishes. After initially building our map, we found that the selection of thresholds for sequence data filtration highly impacted scaffold placement in the map. We outline each step of the approach that dramatically improved our map to help guide others toward more effective linkage mapping for genome assembly. Our final map supports strong conservation of genomic synteny among closely related fish species and reveals previously described chromosomal rearrangements between more distantly related clades. However, we also commonly found minor scaffold misorientations in F. heteroclitus and in other assemblies, suggesting that further mapping (such as optical mapping) is necessary for finer scale resolution of genome structure. Lastly, we discuss the problems that would be expected from misoriented/unplaced scaffolds and stress the importance of a quality mapped genome as a key feature for further investigating population and comparative genomic questions with F. heteroclitus and other taxa.

Genetics & Heredity↗

Identifying Sociomarkers of Pediatric Asthma Patients at Risk of Hospital Revisiting

ObjectiveAsthma is one of the most common chronic childhood diseases in the United States [2, 3]. In addition to its pervasiveness, pediatric asthma shows high sensitivity to the environment. Combining medical-social dataset with machine learning methods we demonstrate how socio-markers play an important role in identifying patients at risk of hospital revisits due to pediatric asthma within a year.IntroductionA socio-marker is a measurable indicator of social conditions where a patient is embedded in and exposed to, being analogous with a biomarker indicating the severity or presence of some disease state. Social factors are one of the most clinical health determinants [1], which play a critical role in explaining health outcomes. Socio-markers can help medical practitioners and researchers to reliably identify high-risk individuals in a timely manner.MethodsWe collected data from three different sources: pediatric asthma encounter records from Jan 1st, 2016 to Dec 31st, 2016 at a children’s hospital, the 2010 U.S census data and neighborhood quality survey data by Memphis Property Hub. After merging these datasets we examine the effect of social features in identifying the patients who visited the hospital more than once during the observation period. We only use the first time visit (3,678 cases) to avoid over-counting of the same patients. In addition to demographic features (age, gender, insurance type, and race (African American and White)), we incorporate the social features such as the proportion of individuals living below the federal poverty level, blight prevalence, neighborhood quality, neighborhood quality inequality, trash dumping presence, the broken window pervasiveness within the zip code area of patients’ residence are included.We then implemented a Support Vector Machine (SVM) based classification model using abovementioned 11 social features. The classification outcome is either patient visits the hospital only one-time (class 0) or revisits the hospital within a year (class 1). Among 3,678 unique patients in the dataset, only 823 pediatric patients revisited hospital with asthma. So, to overcome the class imbalance issue, we have used 823 patients’ data (randomly selected in 1,000 iterations) from each class. Further, to avoid overfitting and ensure generalizability, we divided the dataset as training, test, and validation with a proportion of 60%, 20%, and 20%, respectively. The reported test (5-folds cross-validation using training and testing data) and validation accuracy of the SVM method are averaged over 1,000 iterations to avoid sampling error and bias.ResultsThe proposed socio-marker model resulted in an average classification accuracy of 63.70% for the test set and 63.67 % for the validation set. Further, the average specificity (the total true negative cases divided by the sum of true negative and false positive) and sensitivity (the total number of true positive cases divided by the sum of positive predicted cases) is found to be 62.79% and 64.77%, respectively for the test set and 62.79% and 64.83%, respectively for the validation set. Results of this study suggest that socio-marker features that are not directly related to a patient’s medical conditions can still predict whether the patient will come back to the hospital within a year or not with approximately 64% accuracy.ConclusionsBringing the socio-marker features in the surveillance system may ease the burden of detecting the patients at risk of revisiting the hospital. The results should be interpreted with caution because we only used 12-month period of observation and the visit beyond the observation window is not considered. Also the patients may have visited different hospitals which are not captured in the data.References1. Booske BC, Athens JK, Kindig DA, Park H, Remington PL: Different perspectives for assigning weights to determinants of health. University of Wisconsin: Population Health Institute 2010.2. Subbarao P, Mandhane PJ, Sears MR: Asthma: epidemiology, etiology and risk factors. Canadian Medical Association Journal 2009, 181(9):E181-E190.3. Gold DR, Wright R: Population disparities in asthma. Annu Rev Public Health 2005, 26:89-113.

Shin, Eun Kyong↗

In situ embryo rescue for generation of wide intra‐ and interspecific hybrids of Panicum virgatum L.

Summary Wide crosses have been used for decades as a method for transferring novel genetic material and traits in plant breeding. Historically, many products of wide crosses require tedious and inefficient surgical embryo rescue prior to embryo abortion to recover single plantlets. We have utilized transgenic switchgrass ( Panicum virgatum L. cv Alamo) as a pollen donor in conjunction with antibiotic or herbicide selection for recovery of intra‐and interspecific F 1 crosses by using developing ovules from the female parent and selecting for embryogenic cultures derived from the in situ immature embryo. Using this approach, several intravarietial crosses were generated between transgenic Alamo and the switchgrass varieties Kanlow, Blackwell and Cave ‐ in ‐ Rock as well as an interspecific cross with Atlantic coastal panicgrass. This procedure selected F 1 embryogenic callus produced from the developing embryo contained within isolated immature ovules. Several clonal plants were successfully regenerated from each cross. Southern blot, PCR , phenotypic analyses and genomic analysis confirmed F 1 hybrids. Using genotyping‐by‐sequencing shows the hybridization of the recovered plants by determining the ratio of transgressive markers to total compared markers between parents and their potential offspring. The ratio of transgressive markers to total compared markers was significantly lower between parents and their predicted offspring than between parents and offspring unrelated to them. This approach provides the possibility to move useful transgenes into varieties that are recalcitrant to direct transformation which can be optionally segregated thus useful to create new hybrids, as well as recovery of wide crosses that are either difficult or impossible using traditional techniques.

59 BASIC BIOLOGICAL SCIENCES↗

FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D

Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is called a "lights on" FRET response. The vitamin D aptamer beacon gives a "lights off" or inversely proportional fluorescence response to the amount of vitamin D present in diluted serum. These FRET-aptamer assays are rapid (<30 minutes), sensitive (low ng/mL detection limits), and quite easy to carry out (add sample, mix, and detect in the handheld reader). Benefits include the speed of the assays as well as the small amount of space taken up by the handheld reader and cuvette assays. The aptamer DNA sequences represent novel additional features of the existing (patent-pending) FRET-aptamer assay platform.

Bruno, John G.↗

An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure

Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5 min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC{sub 50} values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC{sub 50} values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6. - Highlights: • Ten P450 isoforms activities assessed simultaneously with only one incubation. • P450 activity levels measured using the metabolic ratio approach. • IC{sub 50} values generated 10-fold faster and cheaper compared to individual assays. • P450 2B6 was the most affected by pesticides with IC{sub 50} in the nanomolar range. • Cumulative inhibition of P450 2B6 by mixtures of four low-dosed insecticides.

60 APPLIED LIFE SCIENCES↗