Life Sciences Commons^™

Meeting Report Of The Third Annual Tri-Service Microbiome Consortium Symposium, J Philip Karl, Robyn A. Barbato, Laurel A. Doherty, Aarti Gautam, Sarah M. Glaven, Robert J. Kokoska, Dagmar Leary, Rebecca Mickol, Matthew A. Perisin, Andrew J. Hoisington, Edward J. Van Opstal, Vanessa Varaljay, Nancy Kelley-Loughnane, Camilla A. Mauzy, Michael S. Goodson, Jason W. Soares

Faculty Publications

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations and to facilitate resource, material and information sharing among consortium members. The 2019 annual symposium was held 22–24 October 2019 at Wright-Patterson Air Force Base in Dayton, OH. Presentations and discussions centered on microbiome-related topics within five broad thematic areas: 1) human microbiomes; 2) transitioning products into Warfighter solutions; 3) environmental microbiomes; 4) engineering microbiomes; and 5) microbiome simulation and characterization. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted …

Nutrient Timing: A Garage Door Of Opportunity?, Shawn M. Arent, Harry P. Cintineo, Bridget A. Mcfadden, Alexa Jenny Chandler, Michelle Angelique Arent

Faculty Publications

Nutrient timing involves manipulation of nutrient consumption at specific times in and around exercise bouts in an effort to improve performance, recovery, and adaptation. Its historical perspective centered on ingestion during exercise and grew to include pre- and post-training periods. As research continued, translational focus remained primarily on the impact and outcomes related to nutrient consumption during one specific time period to the exclusion of all others. Additionally, there seemed to be increasing emphasis on outcomes related to hypertrophy and strength at the expense of other potentially more impactful performance measures. As consumption of nutrients does not occur at only …

Combined And Independent Effects On Hypoxia And Tributylin On Mrna Expression And Physiology Of The Eastern Oyster (Crassostrea Virginica), Ann Fairly Barnett, James H. Gledhill, Robert J. Griffitt, Marc Slattery, Deborah J. Gochfeld, Kristine L. Willett

Faculty Publications

Oyster reefs are vital to estuarine health, but they experience multiple stressors and globally declining populations. This study examined effects of hypoxia and tributyltin (TBT) on adult Eastern oysters (Crassostrea virginica) exposed either in the laboratory or the field following a natural hypoxic event. In the laboratory, oysters were exposed to either hypoxia followed by a recovery period, or to hypoxia combined with TBT. mRNA expression of HIF1-α and Tβ-4 along with hemocyte counts, biomarkers of hypoxic stress and immune health, respectively, were measured. In field-deployed oysters, HIF1-α and Tβ-4 expression increased, while no effect on hemocytes was …

Heterometallic Multinuclear Nodes Directing Mof Electronic Behavior, Otega A. Ejegbavwo, Anna A. Berseneva, Corey R. Martin, Gabrielle A. Leith, Shubham Pandey, Amy J. Brandt, Kyoung Chul Park, Abhijai Mathur, Sharfa Farzandh, Vladislav V. Klepov, Brittany J. Heiser, Mvs Chandrashekhar, Stavros G. Karakalos, Mark D. Smith, Simon R. Phillpot, Sophya Garashchuk, Donna A. Chen, Natalia B. Shustova Prof. Dr.

Faculty Publications

Metal node engineering in combination with modularity, topological diversity, and porosity of metal–organic frameworks (MOFs) could advance energy and optoelectronic sectors. In this study, we focus on MOFs with multinuclear heterometallic nodes for establishing metal−property trends, i.e., connecting atomic scale changes with macroscopic material properties by utilization of inductively coupled plasma mass spectrometry, conductivity measurements, X-ray photoelectron and diffuse reflectance spectroscopies, and density functional theory calculations. The results of Bader charge analysis and studies employing the Voronoi–Dirichlet partition of crystal structures are also presented. As an example of frameworks with different nodal arrangements, we have chosen MOFs with mononuclear, …

Effects Of Temperature And Antioxidants On The Oxidation Of Biodiesel Derived From Waste Vegetable Oil, Randy L. Maglinao, Torrey J. Wagner, Keegan Duff

Faculty Publications

Biodiesel offers several environmental benefits and improvements to some fuel performance properties, but its poor oxidative stability has been a major concern. Currently, the accepted practice to improve biodiesel oxidative stability is the addition of antioxidants; numerous antioxidants have been studied but their effectiveness in inhibiting biodiesel oxidation is difficult to predict due to variation with resonance stability, solubility, reactivity, and volatility. To improve prediction efforts, this study explored the Rapid Small-Scale Oxidation Test (RSSOT) as a means to investigate how biodiesel oxidation is affected by antioxidant concentration and temperature, and compared its results with the oxidative stability index test. …

The Influence Of Stochasticity, Landscape Structure And Species Traits On Abundant–Centre Relationships, Tad Dallas, Luca Santini

Faculty Publications

Species have been commonly hypothesized to have high population densities in geographic areas which correspond to either the centre of the species geographic range or climatic niche (abundant–centre hypothesis). However, there is mixed empirical support for this relationship, and little theoretical underpinning. We simulate a species spreading across a set of replicated artificial landscapes to examine the expected level of support for abundant–centre relationships in geographic and niche space. Species niche constraints were modeled as a single axis which was related directly to population growth rates. We found strong evidence for abundant–centre relationships when populations follow deterministic growth, dispersal is …

A Highly Elastic And Fatigue-Resistant Natural Protein-Reinforced Hydrogel Electrolyte For Reversible-Compressible Quasi-Solid-State Supercapacitors, Jingya Nan, Gaitong Zhang, Tianyu Zhu, Zhongkai Wang, Lijun Wang, Hongsheng Wang, Fuxiang Chu, Chunpeng Wang, Chuanbing Tang

Faculty Publications

Compressible solid-state supercapacitors are emerging as promising power sources for next-generation flexible electronics with enhanced safety and mechanical integrity. Highly elastic and compressible solid electrolytes are in great demand to achieve reversible compressibility and excellent capacitive stability of these supercapacitor devices. Here, a lithium ion-conducting hydrogel electrolyte by integrating natural protein nanoparticles into polyacrylamide network is reported. Due to the synergistic effect of natural protein nanoparticles and polyacrylamide chains, the obtained hydrogel shows remarkable elasticity, high compressibility, and fatigue resistance properties. More significantly, the supercapacitor device based on this hydrogel electrolyte exhibits reversible compressibility under multiple cyclic compressions, working well …

Evidence Of Pathogen-Induced Immunogenetic Selection Across The Large Geographic Range Of A Wild Seabird, Hila Levy, Stephen R. Fiddaman, Julianna A. Vianna, Daly Noll, Gemma Clucas, Jasmine K. H. Sidhu, Michael J. Polito, Charles A. Bost, Richard A. Phillips, Sarah Crofts, Gary D. Miller, Pierre Pistorius, Francesco Bonnadonna, Celine Le Bohec, Andres Barbosa, Phil Trathan, Andrea Raya Rey, Laurent A. F. Frantz, Tom Hart, Adrian L. Smith

Faculty Publications

Over evolutionary time, pathogen challenge shapes the immunepheno type of the host to better respond to an incipient threat. The extent and direction of this selection pressure depend on the local pathogen composition, which is in turn determined by biotic and abiotic features of the environment. However, little is known about adaptation to local pathogen threats in wild animals. The Gentoo penguin (Pygoscelis papua) is a species complex that lends itself to the study of immune adaptation because of its circumpolar distribution over a large latitudinal range, with little or no admixture between ''different clades. Inthis study, we examine the …

Morphometric Response Of Galaxias Maculatus (Jenyns) To Lake Colonization In Chile, Margaret Mercer, Peter C. Searle, Roberto Cifuentes, Evelyn Habit, Mark C. Belk

Faculty Publications

Body and head shape in fish responds to environmental factors such as water flow rate, food sources, and niche availability. However, the way in which fish respond to these environmental factors varies. In Central Chile, multiple river and lake systems along the coast provide an ideal study site to investigate these types of shape changes. We use geometric morphometrics to characterize shape differences in Galaxias maculatus (Jenyns) between river and lake populations. Lake fish converge on a shape with a more fusiform body, narrower head, and larger eyes, while river fish have a more robust body, rounder head, and smaller …

The Abundant-Centre Is Not All That Abundant: A Comment To Osorio-Olvera Et Al. 2020, Tad Dallas, Samuel Pironon, Luca Santini

Faculty Publications

Species abundance is expected to decrease from the centre towards the edge of their ecological niches (abundant niche-centre hypothesis). Recently, Osorio-Olvera et al. (2020) reported strong support for the abundant niche-centre relationship in North American birds. We demonstrate here that methodological decisions strongly affected perceived support. Avoiding these issues casts doubt on conclusions by Osorio-Olvera et al. and the putative support for the abundant nichecentre hypothesis in North American birds.

Impact Of A Preschool Obesity Program (I-Pop) Program On Nutrition Knowledge Scores Of Head Start Parents And Teachers, Holly Huye, Carol Connell, Caroline Newkirk

Faculty Publications

Objectives: To determine the effectiveness of an evidence-based preschool obesity prevention intervention on rural, low-income parents’ and Head Start teachers’ nutrition knowledge relative to advice from experts, nutrient content of foods, and health benefits of foods.

Methods: Parent-child dyads were recruited from 9 randomized Head Start centers with 5 centers in the experimental group and 4 centers in the control. The intervention consisted of 8 weekly evidence-based nutrition education sessions for children delivered by the Head Start teachers; a teacher workshop on the use of Positive Behavior Interventions and Supports (PBIS) in the classroom; and 8 parent workshops …

Compound Heterozygous Variants In Pediatric Cancers: A Systematic Review, Dustin B. Miller, Stephen R. Piccolo

Faculty Publications

A compound heterozygous (CH) variant is a type of germline variant that occurs when each parent donates one alternate allele and these alleles are located at different loci within the same gene. Pathogenic germline variants have been identified for some pediatric cancer types but in most studies, CH variants are overlooked. Thus, the prevalence of pathogenic CH variants in most pediatric cancer types is unknown. We identified 26 studies (published between 1999 and 2019) that identified a CH variant in at least one pediatric cancer patient. These studies encompass 21 cancer types and have collectively identified 25 different …

The Athletic Gut Microbiota, Alex E. Mohr, Ralf Jäger, Katie C. Carpenter, Chad M. Kerksick, Martin Purpura, Jeremy R. Townsend, Nicholas P. West, Katherine Black, Michael Gleeson, David B. Pyne, Shawn D. Wells, Shawn M. Arent Ph.D., Cscs*D, Fissn, Facsm, Fnak, Richard B. Kreider, Bill I. Campbell, Laurent Bannock, Jonathan Scheiman, Craig J. Wissent, Marco Pane, Douglas S. Kalman, Jamie N. Pugh, Carmen P. Ortega-Santos, Jessica A. Ter Haar, Paul J. Arciero, Jose Antonio

Faculty Publications

The microorganisms in the gastrointestinal tract play a significant role in nutrient uptake, vitamin synthesis, energy harvest, inflammatory modulation, and host immune response, collectively contributing to human health. Important factors such as age, birth method, antibiotic use, and diet have been established as formative factors that shape the gut microbiota. Yet, less described is the role that exercise plays, particularly how associated factors and stressors, such as sport/exercise-specific diet, environment, and their interactions, may influence the gut microbiota. In particular, high-level athletes offer remarkable physiology and metabolism (including muscular strength/power, aerobic capacity, energy expenditure, and heat production) compared to sedentary …

Molecular Properties Are A Primary Control On The Microbial Utilization Of Dissolved Organic Matter In The Ocean, Yuan Shen, Ronald Benner

Faculty Publications

The global ocean sequesters a large amount of reduced carbon in dissolved organic molecules that can persist for centuries to millennia. The persistence of dissolved organic carbon (DOC) in the deep ocean has been attributed to inherently refractory molecules and to low concentrations of molecules, but the relative roles of molecular properties and molecular concentrations remain uncertain. We investigate both of these possibilities using bioas-say experiments with unfiltered seawater collected from five depths (50–1500 m) at the Bermuda Atlantic Time-Series Study site. The microbial utilization of compositionally distinct forms of seawater DOC at in situ and elevated concentrations was determined. …

Life History Divergence In Livebearing Fishes In Response To Predation: Is There A Microevolution To Macroevolution Barrier?, Mark C. Belk, Spencer J. Ingley, Jerald B. Johnson

Faculty Publications

A central problem in evolutionary biology is to determine whether adaptive phenotypic variation within species (microevolution) ultimately gives rise to new species (macroevolution). Predation environment can select for trait divergence among populations within species. The implied hypothesis is that the selection resulting from predation environment that creates population divergence within species would continue across the speciation boundary such that patterns of divergence after speciation would be a magnified accumulation of the trait variation observed before speciation. In this paper, we test for congruence in the mechanisms of microevolution and macroevolution by comparing the patterns of life history divergence among three …

Tumor Necrosis Factor-Alpha Signaling May Contribute To Chronic West Nile Virus Post-Infectious Proinflammatory State, A. Arturo Leis, Marie F. Grill, Brent P. Goodman, Syed B. Sadiq, David J. Sinclair, Parminder J.S. Vig, Fengwei Bai

Faculty Publications

Background: West Nile virus (WNV) causes a spectrum of human disease ranging from a febrile illness (WNV fever) to severe neuroinvasive disease (meningitis, encephalitis, acute flaccid paralysis). Since WNV gained entry into North America in 1999, clinicians caring for WNV survivors have observed persistent neurological symptoms occurring long-after the production of neutralizing antibodies and clearance of the virus. Accordingly, alternative pathogeneses other than direct viral invasion have been hypothesized to explain these post-infectious symptoms. The dominant hypothesis is that antiviral inflammatory responses triggered initially to clear WNV may persist to promote a post-infectious proinflammatory state.

Methods: In 4 serologically-confirmed …

Development And Characterization Of Genomic Resources For A Non-Model Marine Telost, The Red Snapper (Lutjanus Campechanus, Lutjanidae): Construction Of A High-Density Linkage Map, Anchoring Of Genome Contigs And Comparative Genomic Analysis, Adrienne E. Norrell, Kenneth L. Jones, Eric A. Saillant

Faculty Publications

The red snapper Lutjanus campechanus is an exploited reef fish of major economic importance in the Gulf of Mexico region. Studies of genome wide genetic variation are needed to understand the structure of wild populations and develop breeding programs for aquaculture but interpretation of these genome scans is limited by the absence of reference genome. In this work, the first draft of a reference genome was developed and characterized for the red snapper. P-454 and Illumina sequencing were conducted to produce paired-end reads that were assembled into reference contigs and scaffolds. The current assembly spans over 770 Mb, representing an …

Escherichia Coli Antimicrobial Resistance Variability In Water Runoff And Soil From A Remnant Native Prairie, And Improved Pasture, And A Cultivated Agricultural Watershed, Maitreyee Mukherjee, Terry Gentry, Heidi Mjelde, John P. Brooks, Daren Harmel, Lucas Gregory, Kevin Wagner

Faculty Publications

Although many previous studies have examined patterns of antimicrobial resistance (AMR) and multidrug resistance (MDR) from domestic animals and farm environments, comparatively little is known about the environmental sources and natural reservoirs of AMR and MDR. In this study, we collected stormwater runoff and soil samples from three watersheds in Texas. Escherichia coli (E. coli) were enumerated, isolated, and analyzed for resistance patterns. E. coli from all sites, irrespective of land use, displayed the presence of AMR/MDR. Higher levels of AMR/MDR were observed in water compared to soil. More isolates were resistant to cephalothin than other antibiotics. For …

Nocturnal Copulation In Glaucous-Winged Gulls Larus Glaucescens, Floyd E. Hayes, James L. Hayward

Faculty Publications

Gulls (Laridae) are primarily diurnal, although many species forage opportunistically at night, and several species copulate at night. We used trail cameras to study time-of-day variation in the rate of copulation by Glaucous-winged Gulls Larus glaucescens in a breeding colony (1500+ pairs) at Protection Island, Washington, USA, from 31 May to 07 June 2018. Copulations (n = 353) occurred at a significantly higher rate during the day (0.82/camera-h) than at night (0.51/camera-h), with 76.3 % of copulations during the day and 23.7 % at night (daylight comprised 66.1 % and darkness comprised 33.9 % of the study period). The …

Gomcl: A T Gomcl: A Toolkit T Oolkit To Cluster O Cluster, Evaluate, And Extr Aluate, And Extract Non-R Act Non-Redundant Edundant Associations Of Gene Ontology-Based Functions, Guannan Wang, Dong-Ha Oh, Maheshi Dassanayake

Faculty Publications

Background Functional enrichment of genes and pathways based on Gene Ontology (GO) has been widely used to describe the results of various -omics analyses. GO terms statistically overrepresented within a set of a large number of genes are typically used to describe the main functional attributes of the gene set. However, these lists of overrepresented GO terms are often too large and contains redundant overlapping GO terms hindering informative functional interpretations. Results We developed GOMCL to reduce redundancy and summarize lists of GO terms effectively and informatively. This lightweight python toolkit efficiently identifies clusters within a list of GO terms …

Combined Opportunistic And Equilibrium Life-History Traits Facilitate Successful Invasions Of The Shimofuri Goby (Tridentiger Bifasciatus), Jiao Qin, Miao Xiang, Meixiang Jia, Fei Cheng, Bjorn V. Schmidt, Jian Liu, Songguang Xie

Faculty Publications

The life-history traits of a non-native population of Shimofuri goby (Tridentiger bifasciatus) were investigated in Nansi Lake, which is a storage lake on the East Route of the South-to-North Water Transfer Project, China. The results were analyzed in combination with data from other non-native populations of this species to reveal the life-history traits that caused its successful invasions. Mature gonads in April and May suggest that the species spawns in this period, and cohort tracking and age analysis revealed that individuals had a one-year lifespan. The relative fecundity was 2,825 ± 614 eggs/g. A short lifespan and high fecundity are …

Shinylearner: A Containerized Benchmarking Tool For Machine-Learning Classification Of Tabular Data, Stephen Piccolo, Terry J. Lee, Erica Suh, Kimball Hill

Faculty Publications

Background: Classification algorithms assign observations to groups based on patterns in data. The machine-learning community have developed myriad classification algorithms, which are used in diverse life science research domains. Algorithm choice can affect classification accuracy dramatically, so it is crucial that researchers optimize the choice of which algorithm(s) to apply in a given research domain on the basis of empirical evidence. In benchmark studies, multiple algorithms are applied to multiple datasets, and the researcher examines overall trends. In addition, the researcher may evaluate multiple hyperparameter combinations for each algorithm and use feature selection to reduce data dimensionality. Although software implementations …

Developing Outreach Events That Impact Underrepresented Students: Are We Doing It Right?, Andrew J. Gall, Peter J. Vollbrecht, Tristan Tobias

Faculty Publications

Many outreach programs share the common goals of serving underrepresented groups in STEM and improving public attitudes toward science. To meet these goals, scientists must find ways to both reach the appropriate audience and communicate the importance of science in meaningful and accessible ways. This requires careful consideration of the outreach method being used. Two common outreach methods include in‐school visits (scientist in the classroom) and science fairs or open houses. Here, we compare the effectiveness of these two outreach methods in meeting the goals of reaching underrepresented students and/or students with less initial interest in science. We have found …

Comparative Analysis Of Rhizosphere Microbiomes Of Southern Highbush Blueberry (Vaccinium Corymbosum L.), Darrowâs Blueberry (V. Darrowii Camp), And Rabbiteye Blueberry (V. Virgatum Aiton), Jiangang Li, Olga V. Mavrodi, Jinfeng Hou, Chazden Blackmon, Ebrahiem M. Babiker, Dmitri V. Mavrodi

Faculty Publications

Plants are inhabited by millions of parasitic, commensal, and mutualistic microorganisms that coexist in complex ecological communities, and profoundly affect the plant’s productivity, health, and capacity to cope with environmental stress. Therefore, a better understanding of the rhizosphere microbiome may open a yet untapped avenue for the rational exploitation of beneficial plant–microbe interactions in modern agriculture. Blueberries encompass several wild and cultivated species of shrubs of the genus Vaccinium that are native to North America. They are grown commercially for the production of fruits, which are considered a health food due to the rich content of minerals, trace elements, and …

Listeria Monocytogenes Response To Anaerobic Environments, Brandy N. Roberts, Damayanti Chakravarty, J.C. Gardner Iii, Steven C. Ricke, Janet R. Donaldson

Faculty Publications

Listeria monocytogenes is a Gram-positive facultative anaerobic bacterium that is responsible for the disease, listeriosis. It is particularly lethal in pregnant women, the fetus, the elderly and the immunocompromised. The pathogen survives and replicates over a wide range of temperatures (4 to 42 °C), pH, salt and oxygen concentrations. Because it can withstand various environments, L. monocytogenes is a major concern in food processing industries, especially in dairy products and ready-to-eat fruits, vegetables and deli meats. The environment in which the pathogen is exposed can influence the expression of virulence genes. For instance, studies have shown that variations in oxygen …

Landscape Performance Of Buck Roses Under Minimal-Input Conditions In North-Central Texas, Derald A. Harp, Gaye Hammond, David C. Zlesak, Greg Church, Mark Chamblee, Steve George

Faculty Publications

Griffith Buck (Iowa State University) bred roses (Rosa sp.) to survive long, cold winters and hot, humid summers yet still retain their foliage without fungicides. Unfortunately, there is little known about the performance of Buck roses in the southern United States. Thirty-eight Buck rose cultivars were evaluated for flowering, disease resistance, drought tolerance, and overall landscape performance in alkaline soils with no fertilizer, no pesticides, and only limited irrigation. Flowering occurred on a bimodal basis, with the highest per plant mean bloom number (16.3 blooms) and bloom coverage (9.7%) in April, and a second flowering in the fall, with …

A New Ectotherm 3d Tracking And Behavior Analytics System Using A Depth-Based Approach With Color Validation, With Preliminary Data On Kihansi Spray Toad (Nectophrynoides Asperginis) Activity, Philip Bal, Damian Lyons, Avishai Shuter

Faculty Publications

The Kihansi spray toad (Nectophrynoides asperginis), classified as Extinct in the Wild by the IUCN, is being bred at the Wildlife Conservation Society’s (WCS) Bronx Zoo as part of an effort to successfully reintroduce the species into the wild. Thousands of toads live at the Bronx Zoo presenting an opportunity to learn more about their behaviors for the first time, at scale. It is impractical to perform manual observations for long periods of time. This paper reports on the development of a RGB-D tracking and analytics approach that allows researchers to accurately and efficiently gather information about the toads’ behavior. …

A 24 Hour Naproxen Dose On Gastrointestinal Distress And Performance During Cycling In The Heat, Dawn M. Emerson, J Mark Davis, Stephen Cl Chen, Toni M. Torres-Mcgehee, Craig E. Pfeifer, Charles C. Emerson, Joseph D. Bivona, Justin V. Stone

Faculty Publications

Using a double-blind, randomized and counterbalanced, cross-over design, we assessed naproxen's effects on gastrointestinal (GI) distress and performance in eleven volunteers (6 male, 5 female). Participants completed 4 trials: 1) placebo and ambient); 2) placebo and heat; 3) naproxen and ambient; and 4) naproxen and heat. Independent variables were one placebo or 220 mg naproxen pill every 8 h (h) for 24 h and ambient (22.7 ± 1.8°C) or thermal environment (35.7 ± 1.3°C). Participants cycled 80 min at a steady heart rate then 10 min for maximum distance. Perceived exertion was measured throughout cycling. Gastrointestinal distress was assessed pre-, …

Hydrogen Peroxide Triggers An Increase In Cell Surface Expression Of System XC− In Cultured Human Glioma Cells, Leah A. Chase, Mary Verheulen Kleyn, Natasha Schiller, Abby Goltz King, Guillermo Flores, Sasha Balcazar Engelsman, Christina Bowles, Sara Lang Smith, Anne E. Robinson, Jeffrey Rothstein

Faculty Publications

System x_c⁻ exchanges extracellular cystine for intracellular glutamate across the plasma membrane of many cell types. One of the physiological roles of System x_c⁻ is to provide cystine for synthesis of the antioxidant glutathione. Here we report that hydrogen peroxide (H₂O₂) triggers the translocation of System x_c⁻ to the plasma membrane within 10 min of the initial exposure. Specifically, we observed a three-fold increase in ³⁵S-L-cystine uptake following a 10 min exposure to 0.3 mM H₂O₂. This effect was dose-dependent with an EC₅₀ for …

Editorial: Deep Learning For Toxicity And Disease Prediction, Ping Gong, Chaoyang Zhang, Minjun Chen

Faculty Publications

No abstract provided.