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Accelerating structure-function mapping using the ViVa webtool to mine natural variation
Hamm, Morgan; Moss, Britney; Leydon, Alexander; Gala, Hardik; Lanctot, Amy; Ramos, Román; Klaeser, Hannah; Lemmex, Andrew; Zahler, Mollye; Nemhauser, Jennifer L.; Wright, R. Clay (Wiley, 2018-12-05)
Thousands of sequenced genomes are now publicly available capturing a significant amount of natural variation within plant species; yet, much of this data remains inaccessible to researchers without significant bioinformatics experience. Here, we present a webtool called ViVa (Visualizing Variation) which aims to empower any researcher to take advantage of the amazing genetic resource collected in the Arabidopsis thaliana 1001 Genomes Project (http://1001genomes.org). ViVa facilitates data mining on the gene, gene family or gene network level. To test the utility and accessibility of ViVa, we assembled a team with a range of expertise within biology and bioinformatics to analyze the natural variation within the well-studied nuclear auxin signaling pathway. Our analysis has provided further confirmation of existing knowledge and has also helped generate new hypotheses regarding this well studied pathway. These results highlight how natural variation could be used to generate and test hypotheses about less studied gene families and networks, especially when paired with biochemical and genetic characterization. ViVa is also readily extensible to databases of interspecific genetic variation in plants as well as other organisms, such as the 3,000 Rice Genomes Project (http://snp-seek.irri.org/) and human genetic variation (https://www.ncbi.nlm.nih.gov/clinvar/).
Advancements in the development of HIF-1α-activated protein switches for use in enzyme prodrug therapy
Wright, R. Clay; Khakhar, Arjun; Eshleman, James R.; Ostermeier, Marc (2014-01)
While gene-directed enzyme prodrug therapy has shown potential as a cancer therapeutic in animal and clinical trials, concerns over the efficacy, selectivity, and safety of gene delivery vehicles have restricted its advance. In an attempt to relieve some of the demands on targeted gene delivery vehicles and achieve the full potential of enzyme prodrug therapy, cancer-targeted activity can be engineered into the enzyme itself. We previously engineered a switchable prodrug-activating enzyme that selectively kills human cancer cells accumulating the cancer marker hypoxia-inducible factor-1α (HIF-1α). This HIF-1α-activated protein switch (Haps59) is designed to increase its ability to convert the prodrug 5-fluorocytosine into the chemotherapeutic 5-fluorouracil in a HIF-1α-dependent manner. However, in cancer cell lines expressing Haps59 the 5FC sensitivity difference between the presence and absence of HIF-1α was not as large as desired. In this work, we aimed to improve the cancer specificity of this switch via a directed evolution approach utilizing random mutagenesis, linker mutagenesis, and random insertion and circular permutation. We identified improved HIF-1α-activated protein switches that confer E. coli with modest increases in HIF-1α-dependent 5FC toxicity. Additionally, the current bottleneck in the development of improved HIF-1α-activated protein switches is screening switch candidates in mammalian cells. To accommodate higher throughput and reduce experimental variability, we explored the use of Flp recombinase-mediated isogenic integration in 293 cells. These experiments raised the possibility that Haps59 can be activated by other interactors of the CH1 domain, and experiments in E. coli indicated that CITED2 can also activate Haps59. Although many CH1 binding partners are also oncogenes, CH1's promiscuous binding and subsequent off-target activation of Haps59 needs to be examined under normal physiological conditions to identify off-target activators. With aberrant activating molecules identified, further directed evolution can be performed to improve the cancer specificity of HIF-1α-activated protein switches.
Advances in Biochemical Engineering-Biotechnology
Zhang, Y. H. Percival; Rollin, Joseph A.; Ye, Xinhao; Del Campo, Julia S. Martin; Adams, Michael W. W. (Springer, 2014-07-15)
In vitro hydrogen generation represents a clear opportunity for novel bioreactor and system design. Hydrogen, already a globally important commodity chemical, has the potential to become the dominant transportation fuel of the future. Technologies such as in vitro synthetic pathway biotransformation (SyPaB)—the use of more than 10 purified enzymes to catalyze unnatural catabolic pathways—enable the storage of hydrogen in the form of carbohydrates. Biohydrogen production from local carbohydrate resources offers a solution to the most pressing challenges to vehicular and bioenergy uses: small-size distributed production, minimization of CO2 emissions, and potential low cost, driven by high yield and volumetric productivity. In this study, we introduce a novel bioreactor that provides the oxygen-free gas phase necessary for enzymatic hydrogen generation while regulating temperature and reactor volume. A variety of techniques are currently used for laboratory detection of biohydrogen, but the most information is provided by a continuous low-cost hydrogen sensor. Most such systems currently use electrolysis for calibration; here an alternative method, flow calibration, is introduced. This system is further demonstrated here with the conversion of glucose to hydrogen at a high rate, and the production of hydrogen from glucose 6-phosphate at a greatly increased reaction rate, 157 mmol/L/h at 60 [degrees] C.
Analysis of crab meat volatiles as possible spoilage indicators for blue crab (Callinectes sapidus) meat by gas chromatography-mass spectrometry
Sarnoski, Paul J.; O'Keefe, Sean F.; Jahncke, Michael L.; Mallikarjunan, Parameswarakumar; Flick, George J. Jr. (Elsevier, 2010-10-01)
Traditionally crab meat spoilage has been evaluated using sensory panels. A method was developed using solid-phase microextraction–gas chromatography–mass spectrometry (SPME–GC–MS) to examine the aroma profile of blue crab (Callinectes sapidus) for chemical indicators of spoilage. The chemicals found to correlate best with spoilage were trimethylamine (TMA), ammonia, and indole over a period of 7 days. In addition, chemicals previously not identified in the aroma profile of blue crab were tentatively detected. Scan mode of the mass spectrometer was used to qualitatively determine compounds extracted from the volatile profile of spoiling blue crab by the SPME fiber. Selected ion monitoring (SIM) mode of the mass spectrometer improved resolution, identified compounds at low concentrations, and allowed spoilage related compounds to be detected in one chromatographic run without sample heating. TMA increased linearly. A significant difference in TMA concentrations were found for day 0 and day 4 samples. Indole concentrations corresponded well with sensory and microbial evaluations, in early, mid, and highly spoiled crab meat samples.
Armored kinorhynch-like scalidophoran animals from the early Cambrian.
Zhang, H.; Xiao, S.; Liu, Y.; Yuan, X.; Wan, B.; Muscente, A. D.; Shao, T.; Gong, H.; Cao, G. (2015-11-26)
Morphology-based phylogenetic analyses support the monophyly of the Scalidophora (Kinorhyncha, Loricifera, Priapulida) and Nematoida (Nematoda, Nematomorpha), together constituting the monophyletic Cycloneuralia that is the sister group of the Panarthropoda. Kinorhynchs are unique among living cycloneuralians in having a segmented body with repeated cuticular plates, longitudinal muscles, dorsoventral muscles, and ganglia. Molecular clock estimates suggest that kinorhynchs may have diverged in the Ediacaran Period. Remarkably, no kinorhynch fossils have been discovered, in sharp contrast to priapulids and loriciferans that are represented by numerous Cambrian fossils. Here we describe several early Cambrian (~535 million years old) kinorhynch-like fossils, including the new species Eokinorhynchus rarus and two unnamed but related forms. E. rarus has characteristic scalidophoran features, including an introvert with pentaradially arranged hollow scalids. Its trunk bears at least 20 annuli each consisting of numerous small rectangular plates, and is armored with five pairs of large and bilaterally placed sclerites. Its trunk annuli are reminiscent of the epidermis segments of kinorhynchs. A phylogenetic analysis resolves E. rarus as a stem-group kinorhynch. Thus, the fossil record confirms that all three scalidophoran phyla diverged no later than the Cambrian Period.
Artificial sinks to treat legacy nutrients in agricultural landscapes
Bock, Emily; Stephenson, Stephen Kurt; Easton, Zachary M. (2019-06-05)
Legacy nutrients introduce a critical time lag between changes in nutrient application or implementation of best management practices (BMPs) and observable reductions in loads delivered to downstream waters. Nitrogen and phosphorus leached through soils into groundwater may take decades to eventually be discharged to surface waters and, consequently, often prevent the attainment of water quality improvement goals. For example, the National Resource Council has cautioned that in the Chesapeake Bay watershed legacy nutrients, particularly nitrogen (N), could delay achievement of nutrient load reductions needed to meet Total Maximum Daily Load (TMDL) requirements.. Groundwater discharge transporting legacy N has been identified specifically as a significant nutrient source to the Bay. Unfortunately, most existing BMPs cannot remediate these nutrient reservoirs and the Chesapeake Bay Program has not active policy to address legacy nutrients; better management options are needed...
Assembly of Bio-Nanoparticles for Double Controlled Drug Release
Huang, Wei; Zhang, Jianfei; Dorn, Harry C.; Zhang, Chenming (PLOS, 2013-09-06)
A critical limiting factor of chemotherapy is the unacceptably high toxicity. The use of nanoparticle based drug carriers has significantly reduced the side effects and facilitated the delivery of drugs. Source of the remaining side effect includes (1) the broad final in vivo distribution of the administrated nanoparticles, and (2) strong basal drug release from nanoparticles before they could reach the tumor. Despite the advances in pH-triggered release, undesirable basal drug release has been a constant challenge under in vivo conditions. In this study, functionalized single walled carbon nanohorn supported immunoliposomes were assembled for paclitaxel delivery. The immunoliposomes were formulated with polyethylene glycol, thermal stable and pH sensitive phospholipids. Each nanohorn was found to be encapsulated within one immunoliposome. Results showed a highly pH dependent release of paclitaxel in the presence of serum at body temperature with minimal basal release under physiological conditions. Upon acidification, paclitaxel was released at a steady rate over 30 days with a cumulative release of 90% of the loaded drug. The drug release results proved our hypothesized double controlled release mechanism from the nanoparticles. Other results showed the nanoparticles have doubled loading capacity compared to that of traditional liposomes and higher affinity to breast cancer cells overexpressing Her2 receptors. Internalized nanoparticles were found in lysosomes.
A Bayesian Assignment Method for Ambiguous Bisulfite Short Reads
Tran, H.; Wu, X.; Tithi, S.; Sun, M.-A.; Xie, H.; Zhang, L. (PLOS, 2016-03-24)
DNA methylation is an epigenetic modification critical for normal development and diseases. The determination of genome-wide DNA methylation at single-nucleotide resolution is made possible by sequencing bisulfite treated DNA with next generation high-throughput sequencing. However, aligning bisulfite short reads to a reference genome remains challenging as only a limited proportion of them (around 50–70%) can be aligned uniquely; a significant proportion, known as multireads, are mapped to multiple locations and thus discarded from downstream analyses, causing financial waste and biased methylation inference. To address this issue, we develop a Bayesian model that assigns multireads to their most likely locations based on the posterior probability derived from information hidden in uniquely aligned reads. Analyses of both simulated data and real hairpin bisulfite sequencing data show that our method can effectively assign approximately 70% of the multireads to their best locations with up to 90% accuracy, leading to a significant increase in the overall mapping efficiency. Moreover, the assignment model shows robust performance with low coverage depth, making it particularly attractive considering the prohibitive cost of bisulfite sequencing. Additionally, results show that longer reads help improve the performance of the assignment model. The assignment model is also robust to varying degrees of methylation and varying sequencing error rates. Finally, incorporating prior knowledge on mutation rate and context specific methylation level into the assignment model increases inference accuracy. The assignment model is implemented in the BAM-ABS package and freely available at https://github.com/zhanglabvt/BAM_ABS.
Biomanufacturing by in vitro biosystems containing cascade enzymes
Zhang, Y. P.; You, C. (2016)
Changes in Fluvial Erosion of Cohesive Streambank Soils with Stream Chemistry
Thompson, Theresa M.; Akinola, Akinrotimi I.; Hoomehr, Siavash; Eick, Matthew J. (2018-06-01)
The goal of this study was to quantify changes in fluvial erosion rates with changes in stream chemistry.
Characterizing Auxin Response Circuits in Saccharomyces cerevisiae by Flow Cytometry.
Pierre-Jerome, Edith; Wright, R. Clay; Nemhauser, Jennifer L. (2017-01)
Recapitulation of the nuclear auxin response pathway in Saccharomyces cerevisiae (yeast) provides a means to functionally assay the contribution of individual signaling components to response dynamics. Here, we describe a time course assay for characterizing auxin response circuits using flow cytometry. This method allows for quantitative measurements of the dynamic response of up to 12 circuits (strains) at once. We also describe a steady-state assay and how to utilize an R package we developed to facilitate data analysis.
Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(+) with Its Application to Biobatteries
Chen, Hui; Zhu, Zhiguang; Huang, Rui; Zhang, Y. H. Percival (Nature Publishing Group, 2016-11-02)
Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP(+) to NAD(+). Through amino acid-sequence alignment of NADP(+)-and NAD(+)-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP(+) were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34I exhibited a x 6.4 x 10(4)-fold reversal of the coenzyme selectivity from NADP(+) to NAD(+). The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm(-2) and 0.255 mA cm(-2), similar to 25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 degrees C, leading to a high power density of 1.75 mW cm(-2). This study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries.
Comparison of kinetic models to describe high pressure and gamma irradiation used to inactivate Vibrio vulnificus and Vibrio parahaemolyticus prepared in buffer solution and whole oysters
Hu, X.; Mallikarjunan, Kumar; Koo, J.; Jahncke, M. L. (2005-03-01)
Current status of the multinational Arabidopsis community
Parry, Geraint; Provart, Nicholas J.; Brady, Siobhan M.; Uzilday, Baris (Wiley, 2020-07-01)
The multinational Arabidopsis research community is highly collaborative and over the past thirty years these activities have been documented by the Multinational Arabidopsis Steering Committee (MASC). Here, we (a) highlight recent research advances made with the reference plant Arabidopsis thaliana; (b) provide summaries from recent reports submitted by MASC subcommittees, projects and resources associated with MASC and from MASC country representatives; and (c) initiate a call for ideas and foci for the "fourth decadal roadmap," which will advise and coordinate the global activities of the Arabidopsis research community.
Determination of quality attributes of blue crab (Callinectes sapidus) meat by electronic nose and Draeger-Tube analysis
Sarnoski, Paul J.; Jahncke, Michael L.; O'Keefe, Sean F.; Mallikarjunan, Parameswarakumar; Flick, George J. Jr. (Haworth Press, 2008-01-01)
In this study, five groups of sequentially spoiled crabmeat were evaluated by a trained sensory panel, and these results were compared with the findings from a Cyranose 320 Electronic Nose and Draeger gas analyzer. Using the electronic nose with filtered compressed breathing air yielded the best results. Although this approach resulted in 100% separation of the known groups, only 30% of the coded unknown samples were correctly identified. All 5 groups of samples analyzed using Draeger-Tubes were found to be significantly different at α=0.05 using a Tukey-Kramer ANOVA statistical procedure. The coded unknown samples were correctly identified at a rate of 83%. The simplicity and precision of this latter procedure may present opportunities for use of Draeger-Tubes by crab processing industries and other food processing industries as an objective method for quality control.
Drought characterization over India under projected climate scenario
Bisht, D. S.; Sridhar, Venkataramana; Mishra, A.; Chatterjee, C.; Raghuwanshi, N. S. (2019-03-30)
Ecological Engineering Education and Practice in the US
Austin, David; Tilley, David; Thompson, Theresa M. (2016-09-01)
Topics for today:

American Ecological Engineering Society

US ecological engineering education

Example ecological engineering practices in the US
Effects of Large Wood on Floodplain Connectivity in a Headwater Mid-Atlantic Stream
Keys, Tyler A.; Governer, Heather; Jones, C. Nathan; Hession, W. Cully; Hester, Erich T.; Scott, Durelle T. (2018-05-08)
Large wood (LW) plays an essential role in aquatic ecosystem health and function. Traditionally, LW has been removed from streams to minimize localized flooding and increase conveyance efficiency. More recently, LW is often added to streams as a component of stream and river restoration activities. While much research has focused on the role of LW in habitat provisioning, geomorphic stability, and hydraulics at low to medium flows, we know little about the role of LW during storm events. To address this question, we investigated the role of LW on floodplain connectivity along a headwater stream in the Mid-Atlantic region of the United States. Specifically, we conducted two artificial floods, one with and one without LW, and then utilized field measurements in conjunction with hydrodynamic modeling to quantify floodplain connectivity during the experimental floods and to characterize potential management variables for optimized restoration activities. Experimental observations show that the addition of LW increased maximum floodplain inundation extent by 34%, increased floodplain inundation depth by 33%, and decreased maximum thalweg velocity by 10%. Model results demonstrated that different placement of LW along the reach has the potential to increase floodplain flow by up to 40%, with highest flooding potential at cross sections with high longitudinal velocity and shallow depth. Additionally, model simulations show that the effects of LW on floodplain discharge decrease as storm recurrence interval increases, with no measurable impact at a recurrence interval of more than 25 years.
Ex vivo enzymatic conversion of non-food cellulose biomass to starch
Zhang, Y. P.; You, C. (Springer, 2016-12-10)
Fecal Indicator Bacteria and Antibiotic Resistance Genes in Storm Runoff from Dairy Manure and Compost-Amended Vegetable Plots
Jacobs, Kyle; Wind, Lauren L.; Krometis, Leigh-Anne H.; Hession, W. Cully; Pruden, Amy (American Society for Agronomy, 2019-07-01)
Given the presence of antibiotics and resistant bacteria in livestock manures, it is important to identify the key pathways by which land-applied manure-derived soil amendments potentially spread resistance. The goal of this field-scale study was to identify the effects of different types of soil amendments (raw manure from cows treated with cephapirin and pirlimycin, compost from antibiotic-treated or antibiotic-free cows, or chemical fertilizer only) and crop type (lettuce [Lactuca sativa L.] or radish [Raphanus sativus L.]) on the transport of two antibiotic resistance genes (ARGs; sul1 and ermB) via storm runoff from six naturally occurring storms. Concurrent quantification of sediment and fecal indicator bacteria (FIB; Escherichia coli and enterococci) in runoff permitted comparison to traditional agricultural water quality targets that may be driving factors of ARG presence. Storm characteristics (total rainfall volume, storm duration, etc.) significantly influenced FIB concentration (two-way ANOVA, p < 0.05), although both effects from individual storm events (Kruskal-Wallis, p < 0.05) and vegetative cover influenced sediment levels. Composted and raw manure-amended plots both yielded significantly higher sul1 and ermB levels in runoff for early storms, at least 8 wk following initial planting, relative to fertilizer-only or unamended barren plots. There was no significant difference between sul1 or ermB levels in runoff from plots treated with compost derived from antibiotic-treated versus antibiotic-free dairy cattle. Our findings indicate that agricultural fields receiving manure-derived amendments release higher quantities of these two “indicator” ARGs in runoff, particularly during the early stages of the growing season, and that composting did not reduce effects of ARG loading in runoff.

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