Browsing by Author "Zhong, Weiwei"
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Item A strategy to apply quantitative epistasis analysis on developmental traits(BioMed Central, 2017) Labocha, Marta K.; Yuan, Wang; Aleman-Meza, Boanerges; Zhong, WeiweiAbstract Background Genetic interactions are keys to understand complex traits and evolution. Epistasis analysis is an effective method to map genetic interactions. Large-scale quantitative epistasis analysis has been well established for single cells. However, there is a substantial lack of such studies in multicellular organisms and their complex phenotypes such as development. Here we present a method to extend quantitative epistasis analysis to developmental traits. Methods In the nematode Caenorhabditis elegans, we applied RNA interference on mutants to inactivate two genes, used an imaging system to quantitatively measure phenotypes, and developed a set of statistical methods to extract genetic interactions from phenotypic measurement. Results Using two different C. elegans developmental phenotypes, body length and sex ratio, as examples, we showed that this method could accommodate various metazoan phenotypes with performances comparable to those methods in single cell growth studies. Comparing with qualitative observations, this method of quantitative epistasis enabled detection of new interactions involving subtle phenotypes. For example, several sex-ratio genes were found to interact with brc-1 and brd-1, the orthologs of the human breast cancer genes BRCA1 and BARD1, respectively. We confirmed the brc-1 interactions with the following genes in DNA damage response: C34F6.1, him-3 (ortholog of HORMAD1, HORMAD2), sdc-1, and set-2 (ortholog of SETD1A, SETD1B, KMT2C, KMT2D), validating the effectiveness of our method in detecting genetic interactions. Conclusions We developed a reliable, high-throughput method for quantitative epistasis analysis of developmental phenotypes.Item An automated system for quantitative analysis of Drosophila larval locomotion(BioMed Central Lt, 2015) Aleman-Meza, Boanerges; Jung, Sang-Kyu; Zhong, WeiweiBackground: Drosophila larvae have been used as a model to study to genetic and cellular circuitries modulating behaviors. One of the challenges in behavioral study is the quantification of complex phenotypes such as locomotive behaviors. Experimental capability can be greatly enhanced by an automatic single-animal tracker that records an animal at a high resolution for an extended period, and analyzes multiple behavioral parameters. Results: Here we present MaggotTracker, a single-animal tracking system for Drosophila larval locomotion analysis. This system controls the motorized microscope stage while taking a video, so that the animal remains in the viewing center. It then reduces the animal to 13 evenly distributed points along the midline, and computes over 20 parameters evaluating the shape, peristalsis movement, stamina, and track of the animal. To demonstrate its utility, we applied MaggotTracker to analyze both wild-type and mutant animals to identify factors affecting locomotive behaviors. Each animal was tracked for four minutes. Our analysis on Canton-S third-instar larvae revealed that the distance an animal travelled was correlated to its striding speed rather than the percentage of time the animal spent striding, and that the striding speed was correlated to both the distance and the duration of one stride. Sexual dimorphism was observed in body length but not in locomotive parameters such as speed. Locomotive parameters were affected by animal developmental stage and the crawling surface. No significant changes in movement speed were detected in mutants of circadian genes such as period (per), timeout, and timeless (tim). The MaggotTracker analysis showed that ether a go-go (eag), Shaker (Sh), slowpoke (slo), and dunce (dnc) mutant larvae had severe phenotypes in multiple locomotive parameters such as stride distance and speed, consistent with their function in neuromuscular junctions. Further, the phenotypic patterns of the K+ channel genes eag, Sh and slo are highly similar. Conclusions: These results showed that MaggotTracker is an efficient tool for automatic phenotyping. The MaggotTracker software as well as the data presented here can be downloaded from our open-access site www.WormLoco.org/MagItem Differences in transcription between free-living and CO2-activated third-stage larvae ofᅠHaemonchus contortus(BioMed Central, 2010) Cantacessi, Cinzia; Campbell, Bronwyn E.; Young, Neil D.; Jex, Aaron R.; Hall, Ross S.; Presidente, Paul J.A.; Zawadzki, Jodi L.; Zhong, Weiwei; Aleman-Meza, Boanerges; Loukas, Alex; Sternberg, Paul W.; Gasser, Robin B.Background: The disease caused by Haemonchus contortus, a blood-feeding nematode of small ruminants, is of major economic importance worldwide. The infective third-stage larva (L3) of this gastric nematode is enclosed in a cuticle (sheath) and, once ingested with herbage by the host, undergoes an exsheathment process that marks the transition from the free-living (L3) to the parasitic (xL3) stage. This study explored changes in gene transcription associated with this transition and predicted, based on comparative analysis, functional roles for key transcripts in the metabolic pathways linked to larval development. Results: Totals of 101,305 (L3) and 105,553 (xL3) expressed sequence tags (ESTs) were determined using 454 sequencing technology, and then assembled and annotated; the most abundant transcripts encoded transthyretin-like, calcium-binding EF-hand, NAD(P)-binding and nucleotide-binding proteins as well as homologues of Ancylostoma -secreted proteins (ASPs). Using an in silico -subtractive analysis, 560 and 685 sequences were shown to be uniquely represented in the L3 and xL3 stages, respectively; the transcripts encoded ribosomal proteins, collagens and elongation factors (in L3), and mainly peptidases and other enzymes of amino acid catabolism (in xL3). Caenorhabditis elegans orthologues of transcripts that were uniquely transcribed in each L3 and xL3 were predicted to interact with a total of 535 other genes, all of which were involved in embryonic development. Conclusion: The present study indicated that some key transcriptional alterations taking place during the transition from the L3 to the xL3 stage of H. contortus involve genes predicted to be linked to the development of neuronal tissue (L3 and xL3), formation of the cuticle (L3) and digestion of host haemoglobin (xL3). Future efforts using next-generation sequencing and bioinformatic technologies should provide the efficiency and depth of coverage required for the determination of the complete transcriptomes of different developmental stages and/or tissues of H. contortus as well as the genome of this important parasitic nematode. Such advances should lead to a significantly improved understanding of the molecular biology of H. contortus and, from an applied perspective, to novel methods of intervention.Item High-content behavioral profiling reveals neuronal genetic network modulating Drosophila larval locomotor program(BioMed Central, 2017) Aleman-Meza, Boanerges; Loeza-Cabrera, Mario; Peña-Ramos, Omar; Stern, Michael; Zhong, WeiweiAbstract Background Two key questions in understanding the genetic control of behaviors are: what genes are involved and how these genes interact. To answer these questions at a systems level, we conducted high-content profiling of Drosophila larval locomotor behaviors for over 100 genotypes. Results We studied 69 genes whose C. elegans orthologs were neuronal signalling genes with significant locomotor phenotypes, and conducted RNAi with ubiquitous, pan-neuronal, or motor-neuronal Gal4 drivers. Inactivation of 42 genes, including the nicotinic acetylcholine receptors nAChRα1 and nAChRα3, in the neurons caused significant movement defects. Bioinformatic analysis suggested 81 interactions among these genes based on phenotypic pattern similarities. Comparing the worm and fly data sets, we found that these genes were highly conserved in having neuronal expressions and locomotor phenotypes. However, the genetic interactions were not conserved for ubiquitous profiles, and may be mildly conserved for the neuronal profiles. Unexpectedly, our data also revealed a possible motor-neuronal control of body size, because inactivation of Rdl and Gαo in the motor neurons reduced the larval body size. Overall, these data established a framework for further exploring the genetic control of Drosophila larval locomotion. Conclusions High content, quantitative phenotyping of larval locomotor behaviours provides a framework for system-level understanding of the gene networks underlying such behaviours.Item Impact of Sunlight and Natural Organic Matter on the Fate, Transport, and Toxicity of Carbon Based Nanomaterials(2013-09-16) Qu, Xiaolei; Li, Qilin; Alvarez, Pedro J.; Zhong, Weiwei; Weisman, R. BruceThe fast growing production of carbon based nanomaterials (CNMs) and their potential widespread use in consumer products raise concerns regarding their potential risks to human health and ecosystems. The present study investigated the role of photochemical transformation and natural organic matter (NOM) in the fate, transport, and toxicity of fullerenes and carbon nanotubes (CNTs) in natural aquatic systems, providing fundamental information for risk assessment and management. Photochemical transformation of aqueous fullerene nanoparticles (nC60) and CNTs occurs at significant rates under UVA irradiation at intensity similar to that in sunlight. The transformation processes are mediated by self-generated ROS, resulting in changes of surface structure depending on the initial surface oxidation state of CNMs. UVA irradiation leads to oxygenation of nC60 surface and decarboxylation of carboxylated multi-walled carbon nanotubes (COOH-MWNTs). The environmental transport of CNMs is significantly affected by their surface chemistry, concentration and species of electrolytes, and concentration and properties of co-existing NOM. In electrolyte solutions without NOM, the mobility of CNMs is largely decided by their surface chemistry, primarily the oxygen-containing functional groups. In NaCl solutions, UVA irradiation remarkably enhanced the mobility of nC60; conversely, it reduced nC60 stability in CaCl2 solutions. The mobility of COOH-MWNTs in NaCl solutions correlated well with the abundance of surface carboxyl groups. Humic acid, once adsorbed on the nC60 surface, can significantly enhance its stability through steric hindrance. The extent of stabilization depends on the amount and properties of humic acid adsorbed. Humic acid has limited adsorption on UVA-irradiated nC60. Soil humic acid is more efficient in stabilizing nC60 than aquatic humic acid due to its higher molecular weight. Humic acid immobilized onto the silica surface can potential enhance or hinder nC60 deposition, depending on the complex interplay of attractive and repulsive forces. MWNTs are more toxicity to bacteria, Escherichia coli, than COOH-MWNTs due to their higher bioavailability and oxidative capacity. Surface oxidation induced by •OH reduced the toxicity of MWNT while reactions with •OH have little effect on the COOH-MWNT toxicity. Antioxidants such as glutathione can effectively inhibit the antibacterial activity of MWNTs.Item Investigating the biological impacts of nanoengineered materials in Caenorhabditis elegans and in vitro(2013-06-05) Contreras, Elizabeth; Colvin, Vicki L.; Wilson, Lon J.; Zhong, WeiweiIn nematode Caenorhabditis elegans, the chronic and multi-generational toxicological effects of commercially relevant engineered nanoparticles (ENPs), such as quantum dots (QDs) and silver (AgNP) caused significant changes in a number of physiological endpoints. The increased water-solubility of ENPs in commercial products, for example, makes them increasingly bioavailable to terrestrial organisms exposed to pollution and waste in the soil. Since 2008, attention to the toxicology of nanomaterials in C. elegans continues to grow. Quantitative data on multiple physiological endpoints paired with metal analysis show the uptake of QDs and AgNPs, and their effects on nematode fitness. First, C. elegans were exposed for four generations through feeding to amphiphilic polymer coated CdSe/ZnS (core-shell QDs), CdSe (core QDs), and different sizes of AgNPs. These ENPs were readily ingested. QDs were qualitatively imaged in the digestive tract using a fluorescence microscopy and their and AgNP uptake quantitatively measured using ICP-MS. Each generation was analyzed for changes in lifespan, reproduction, growth and motility using an automated computer vision system. Core-shell QDs had little impact on C. elegans due to its metal shell coating. In contrast, core QDs lacked a metal shell coating, which caused significant changes to nematode physiology. In the same way, at high concentrations of 100 ppm, AgNP caused the most adverse effect to lifespan and reproduction related to particle size, but its adverse effect to motility had no correlation to particle size. Using C. elegans as an animal model allowed for a better understanding of the negative impacts of ENPs than with cytotoxicity tests. Lastly, to test the toxicity of water-dispersed fullerene (nanoC60) using human dermal fibroblast cells, this thesis investigated a suite of assays and methods in order to establish a standard set of cytotoxicity tests. Ten assays and methods assessed nanoC60 samples of different purities to show differences in cytotoxic effects. Washed samples of fullerenes, with negligible traces of THF and other impurities, rendered the solution nontoxic. Even when exposed to UV-irradiation, washed nanoC60 were not photosensitized and did not cause cellular death. This work characterizes ENPs and investigates their impact in C. elegans and cells to assess toxicity risks to the environment and to human health.Item Multi-endpoint, High-Throughput Study of Nanomaterial Toxicity in Caenorhabditis elegans(American Chemical Society, 2015) Jung, Sang-Kyu; Qu, Xiaolei; Aleman-Meza, Boanerges; Wang, Tianxiao; Riepe, Celeste; Liu, Zheng; Li, Qilin; Zhong, WeiweiThe booming nanotechnology industry has raised public concerns about the environmental health and safety impact of engineered nanomaterials (ENMs). High-throughput assays are needed to obtain toxicity data for the rapidly increasing number of ENMs. Here we present a suite of high-throughput methods to study nanotoxicity in intact animals using Caenorhabditis elegans as a model. At the population level, our system measures food consumption of thousands of animals to evaluate population fitness. At the organism level, our automated system analyzes hundreds of individual animals for body length, locomotion speed, and lifespan. To demonstrate the utility of our system, we applied this technology to test the toxicity of 20 nanomaterials at four concentrations. Only fullerene nanoparticles (nC60), fullerol, TiO2, and CeO2 showed little or no toxicity. Various degrees of toxicity were detected from different forms of carbon nanotubes, graphene, carbon black, Ag, and fumed SiO2 nanoparticles. Aminofullerene and ultraviolet-irradiated nC60 also showed small but significant toxicity. We further investigated the effects of nanomaterial size, shape, surface chemistry, and exposure conditions on toxicity. Our data are publicly available at the open-access nanotoxicity database www.QuantWorm.org/nano.Item Optimization of Proteasomal Degradation Reporter (eDeg-On) System for CRISPR-mediated Whole-genome Knockout Screens(2017-04-21) Liu, Yiwen; Segatori, Laura; Zhong, WeiweiProtein folding and clearance of misfolded proteins are crucial to maintain cellular homeostasis (Jariel-Encontre et al., 2008). Misfolded proteins may associate with other cellular components and possibly impair their functions. They may also self-associate to form insoluble aggregates, which are the hallmarks of a number of neurodegenerative diseases, such as Parkinson’s (Olanow and McNaught, 2006) and Alzheimer’s (Oddo, 2008). The ubiquitin proteasome system (UPS) is the main pathway that catalyzes the degradation of soluble misfolded proteins in mammalian cells. Therefore, enhancing the UPS activity through activation of proteasomal degradation is considered a promising strategy to ameliorate phenotypes associated with the accumulation of misfolded proteins. Modulation of specific UPS components, for instance, results in increased degradation of target proteins (Rechsteiner and Hill, 2005 and Vilchez et al., 2012). However, our current understanding of the molecular mechanism underlying proteasomal degradation is still limited, limiting the rational design of pharmacologic strategies to enhance UPS activity. As a result, proteasome activators are rare and remain poorly characterized (Huang and Chen, 2009). To overcome these limitations, researchers in my group developed a cell-based platform (the eDeg-On system) to monitor changes in UPS activity. This genetic circuit links increase in UPS activity to an increase in fluorescent output, thereby providing a reliable tool for the discovery of proteasome activators. The CRISPR-cas technology has emerged as powerful technique to introduce genetic modifications at the whole-genome scale. I optimized the eDeg-On system and evaluated it for pooled screening of whole-genome CRISPR-mediated knockout library. I replaced the antibiotic resistance gene in the eDeg-On system and assessed the response of HEK293 cells stably expressing the eDeg-On system to modulation of proteasomal degradation. To evaluate the use of a stable cell line expressing the eDeg-On system as a reporter assay in the context of a pooled CRISPR-mediated screen, I conducted mock screens using different ratios of positive and negative controls. The results obtained demonstrate that the eDeg-On system can be used as a reporter assay for CRISPR-mediated whole-genome knockout screens. The use of the eDeg-On system to conduct genetic screen for the discovery of molecules that function as proteasome regulators will contribute to the development of therapeutic strategies for protein misfolding diseases. Further applications of this study include targeting the UPS function for therapeutic applications as well as for enhancing the production of recombinant proteins in industrial settings.Item Orsay Virus CP-δ Adopts a Novel β-Bracelet Structural Fold and Incorporates into Virions as a Head Fiber(American Society for Microbiology, 2020) Guo, Yusong R.; Fan, Yanlin; Zhou, Ying; Jin, Miao; Zhang, Jim L.; Jiang, Hongbing; Holt, Matthew V.; Wang, Tao; Young, Nicolas L.; Wang, David; Zhong, Weiwei; Tao, Yizhi JaneFiber proteins are commonly found in eukaryotic and prokaryotic viruses, where they play important roles in mediating viral attachment and host cell entry. They typically form trimeric structures and are incorporated into virions via noncovalent interactions. Orsay virus, a small RNA virus which specifically infects the laboratory model nematode Caenorhabditis elegans, encodes a fibrous protein δ that can be expressed as a free protein and as a capsid protein-δ (CP-δ) fusion protein. Free δ has previously been demonstrated to facilitate viral exit following intracellular expression; however, the biological significance and prevalence of CP-δ remained relatively unknown. Here, we demonstrate that Orsay CP-δ is covalently incorporated into infectious particles, the first example of any attached viral fibers known to date. The crystal structure of δ(1–101) (a deletion mutant containing the first 101 amino acid [aa] residues of δ) reveals a pentameric, 145-Å long fiber with an N-terminal coiled coil followed by multiple β-bracelet repeats. Electron micrographs of infectious virions depict particle-associated CP-δ fibers with dimensions similar to free δ. The δ proteins from two other nematode viruses, Le Blanc and Santeuil, which both specifically infect Caenorhabditis briggsae, were also found to form fibrous molecules. Recombinant Le Blanc δ was able to block Orsay virus infection in worm culture and vice versa, suggesting these two viruses likely compete for the same cell receptor(s). Thus, we propose that while CP-δ likely mediates host cell attachment for all three nematode viruses, additional downstream factor(s) ultimately determine the host specificity and range of each virus.Item Orsay δ protein is required for non-lytic viral egress(American Society for Microbiology, 2018) Yuan, Wang; Zhou, Ying; Fan, Yanlin; Tao, Yizhi Jane; Zhong, WeiweiNonenveloped gastrointestinal viruses, such as human rotavirus, can exit infected cells from the apical surface without cell lysis. The mechanism of such nonlytic exit is poorly understood. The nonenveloped Orsay virus is an RNA virus infecting the intestine cells of the nematode Caenorhabditis elegans Dye staining results suggested that Orsay virus exits from the intestine of infected worms in a nonlytic manner. Therefore, the Orsay virus-C. elegans system provides an excellent in vivo model to study viral exit. The Orsay virus genome encodes three proteins: RNA-dependent RNA polymerase, capsid protein (CP), and a nonstructural protein, δ. δ can also be expressed as a structural CP-δ fusion. We generated an ATG-to-CTG mutant virus that had a normal CP-δ fusion but could not produce free δ due to the lack of the start codon. This mutant virus showed a viral exit defect without obvious phenotypes in other steps of viral infection, suggesting that δ is involved in viral exit. Ectopically expressed free δ localized near the apical membrane of intestine cells in C. elegans and colocalized with ACT-5, an intestine-specific actin that is a component of the terminal web. Orsay virus infection rearranged ACT-5 apical localization. Reduction of the ACT-5 level via RNA interference (RNAi) significantly exacerbated the viral exit defect of the δ mutant virus, suggesting that δ and ACT-5 functionally interact to promote Orsay virus exit. Together, these data support a model in which the viral δ protein interacts with the actin network at the apical side of host intestine cells to mediate the polarized, nonlytic egress of Orsay virus.IMPORTANCE: An important step of the viral life cycle is how viruses exit from host cells to spread to other cells. Certain nonenveloped viruses can exit cultured cells in nonlytic ways; however, such nonlytic exit has not been demonstrated in vivo In addition, it is not clear how such nonlytic exit is achieved mechanistically in vivo Orsay virus is a nonenveloped RNA virus that infects the intestine cells of the nematode C. elegans It is currently the only virus known to naturally infect C. elegans Using this in vivo model, we show that the δ protein encoded by Orsay virus facilitates the nonlytic exit of the virus, possibly by interacting with host actin on the apical side of worm intestine cells.Item Potential Nematode Alarm Pheromone Induces Acute Avoidance in Caenorhabditis elegans(Genetics Society of America, 2017) Zhou, Ying; Loeza-Cabrera, Mario; Liu, Zheng; Aleman-Meza, Boanerges; Nguyen, Julie K.; Jung, Sang-Kyu; Choi, Yuna; Shou, Qingyao; Butcher, Rebecca A.; Zhong, WeiweiIt is crucial for animal survival to detect dangers such as predators. A good indicator of dangers is injury of conspecifics. Here we show that fluids released from injured conspecifics invoke acute avoidance in both free-living and parasitic nematodes. Caenorhabditis elegans avoids extracts from closely related nematode species but not fruit fly larvae. The worm extracts have no impact on animal lifespan, suggesting that the worm extract may function as an alarm instead of inflicting physical harm. Avoidance of the worm extract requires the function of a cGMP signaling pathway that includes the cGMP-gated channel TAX-2/TAX-4 in the amphid sensory neurons ASI and ASK. Genetic evidence indicates that the avoidance behavior is modulated by the neurotransmitters GABA and serotonin, two common targets of anxiolytic drugs. Together, these data support a model that nematodes use a nematode-specific alarm pheromone to detect conspecific injury.Item QuantWorm: A Comprehensive Software Package for Caenorhabditis elegans Phenotypic Assays(Public Library of Science, 2014) Jung, Sang-Kyu; Aleman-Meza, Boanerges; Riepe, Celeste; Zhong, Weiwei; Hendricks, MichaelPhenotypic assays are crucial in genetics; however, traditional methods that rely on human observation are unsuitable for quantitative, large-scale experiments. Furthermore, there is an increasing need for comprehensive analyses of multiple phenotypes to provide multidimensional information. Here we developed an automated, high-throughput computer imaging system for quantifying multiple Caenorhabditis elegans phenotypes. Our imaging system is composed of a microscope equipped with a digital camera and a motorized stage connected to a computer running the QuantWorm software package. Currently, the software package contains one data acquisition module and four image analysis programs: WormLifespan, WormLocomotion, WormLength, and WormEgg. The data acquisition module collects images and videos.The WormLifespan software counts the number of moving worms by using two time-lapse images; the WormLocomotion software computes the velocity of moving worms; the WormLength software measures worm body size; and the WormEgg software counts the number of eggs. To evaluate the performance of our software, we compared the results of our software with manual measurements. We then demonstrated the application of the QuantWorm software in a drug assay and a genetic assay. Overall, the QuantWorm software provided accurate measurements at a high speed. Software source code, executable programs, and sample images are available at www.quantworm.org. Our software package has several advantages over current imaging systems for C. elegans. It is an all-in-one package for quantifying multiple phenotypes. The QuantWorm software is written in Java and its source code is freely available, so it does not require use of commercial software or libraries. It can be run on multiple platforms and easily customized to cope with new methods and requirements.Item Rbt1 is required for Nitric Oxide Stress Resistance in the Human Commensal Fungus Candida albicans(2013-12-03) Dou, Yanru; Gustin, Michael C.; Olson, John S.; Zhong, WeiweiCandida albicans is a human‐resident opportunistic fungal pathogen. Persistence of C. albicans is largely affected by resistance to host‐generate nitric oxide (NO) mediated nitrosative stress. The transcription factor Cta4 was first identified by our group as a regulator of the nitrosative stress resistance gene YHB1. However, little else is known about the molecular mechanism of C. albicans resistance to NO. Here I propose that there are proteins besides Cta4 involved in the regulation of C. albicans nitrosative stress resistance and Rbt1 is one of the positive regulators. Supporting this hypothesis is the observation that Δrbt1 showed significantly inhibited growth when challenged with nitrosative stress producing chemicals nitrite and DPTA Nonoate. Quantitative PCR showed that YHB1 was no longer transcriptionally induced by nitric oxide compared to the wild type strain. Complementation with the plasmid expressing RBT1 gene is needed to confirm the function of Rbt1. But the fact that Δrbt1 mutants generated by other labs showed the same phenotype supported our observation. The screening assay indicated in this thesis yielded several mutants with significant sodium nitrite sensitivity and this suggests that multiple Candida albicans proteins might function synergistically to detoxify in vivo nitric oxide.Item Size-dependent impacts of silver nanoparticles on the lifespan, fertility, growth, and locomotion of Caenorhabditis elegans(Wiley, 2014) Contreras, Elizabeth Q.; Puppala, Hema L.; Escalera, Gabriela; Zhong, Weiwei; Colvin, Vicki L.The increased bioavailability of nanoparticles engineered for good dispersion in water may have biological and environmental impacts. To examine this issue, the authors assessed the biological effects in nematodes as they relate to exposure to silver nanoparticles (AgNPs) of different sizes at low (1 mg/L Ag), medium (10 mg/L Ag), and high concentrations (100 mg/L Ag). Over multiple generations, the authors found that the smallest particle, at 2 nm, had a notable impact on nematode fertility. In contrast, the largest particle, at 10 nm, significantly reduced the lifespan of parent nematodes (P0) by 28.8% and over the span of 3 generations (F1–F3). In addition, a computer vision system automatically measured the adverse effects in body length and motility, which were not size-dependent.Item Structural characterization of a nematode-infecting virus - Orsay(2016-09-13) Guo, Yusong R; Tao, Yizhi Jane; Zhong, WeiweiOrsay virus is the first and so far the only virus known to naturally infects the model organism Caenorhabditis elegans. It has a bipartite, positive-sense RNA genome, encoding the viral polymerase, the capsid protein (CP), and a protein named Delta whose functions are currently unknown. Based upon sequence analysis, the Orsay virus is most closely related to members of the Nodaviridae family, but may define a novel family given its unique features, such as the non-AUG initiation of CP translation and the expression of a CP-Delta fusion protein. To better understand the molecular mechanisms of Orsay virus replication, I performed structural and functional studies of Orsay viral proteins. Recombinant Orsay CP expressed in E. coli spontaneously forms virus-like particle (VLP). Its crystal structure has been determined to 3.25-Å resolution by averaging and phase extension using a 9-Å resolution cryo-EM reconstruction as a phasing model. The Orsay VLP shows T=3 icosahedral symmetry with 60 trimeric spikes. Each CP can be divided into three regions: an N-terminal peptide, a S domain forming the continuous capsid shell, and a P domain forming surface protrusions. The jelly-roll β-barrel in the S domain closely resembles those from small plant viruses, while the P domain is remotely related to betanodaviruses. The N-terminal peptide forms an extensive internal network and may regulate capsid stability and genome packaging. Recombinant CP could also be purified as stable dimers, indicating that Orsay capsid assembly may start from dimer formation. The Delta protein, which has no homology with any other known protein, is rich in β-sheets with high valine content. Under electron microscope, recombinant Delta appeared as a rod-shaped, fibrous protein. The crystal structure of the N-terminal 66 residues at 2.1Å resolution shows a pentameric helical bundle with interesting biological implications. Considering the unique advantage of the C. elegans-Orsay system for studying viral pathogenesis and virus-host interactions and also its promising application in anti-viral drug screening, my studies of the Orsay virus will not only provide important insights into the fundamental biology of RNA viruses in general, but will also facilitate development of the C. elegans-Orsay as a powerful virological model.Item The Plant Circadian Clock: Roles in Jasmonate Accumulation and Postharvest Plant Performance(2015-11-23) Liu, John; Bartel, Bonnie; Gustin, Michael; Matsuda, Seiichi; Zhong, Weiwei; Braam, JanetNearly all organisms have evolved circadian clocks that allow them to anticipate and prepare for cyclical changes in their environment, such as those associated with the transitions from night to day and between seasons. The plant circadian clock functions nearly cell autonomously, regulating rhythmic behaviors in a variety of processes that range from the transcriptional level up to organismal level. Although great progress has been made in determining how the clock functions in plants, not much is known about the clock’s physiological relevance on particular plant processes. Specifically, the clock influences plant defense against Trichoplusia ni (T. ni), as well as basal rhythmic accumulation of jasmonic acid (JA), a plant hormone involved in the regulation of resistance against insect herbivores such as T. ni and necrotrophic fungi such as Botrytis cinerea (B. cinerea). Additionally, the clock affects the accumulation of glucosinolates, secondary metabolites involved in plant insect resistance, in both Arabidopsis and postharvest cabbage (Brassica oleracea). Here we show that rhythmic accumulation of basal JA may be transcriptionally regulated through clock controlled JA-biosynthesis gene transcript accumulation. Additionally, the clock may influence rhythmic accumulation of gene transcripts involved in JA biosynthesis independently of JA-positive feedback. Furthermore, while SA and JA act antagonistically, we note that decreased SA does not influence differential accumulation of JA-biosynthesis related gene transcripts. We also show that phase-entrainment dependent plant resistance to T. ni is influenced in part by aliphatic glucosinolates. Finally, we demonstrate that postharvest vegetables kept in light/dark cycles maintain appearance, tissue integrity, chlorophyll levels, and glucosinolate levels for longer periods of time when compared to postharvest vegetables stored in constant light or constant dark. Moreover, postharvest plants exhibit improved resistance to B. cinerea when kept under light/dark cycles when compared to constant light storage.Item Toxicity of Quantum Dots and Cadmium Salt to Caenorhabditis elegans after Multigenerational Exposure(American Chemical Society, 2013) Contreras, Elizabeth Q.; Cho, Minjung; Zhu, Huiguang; Puppala, Hema L.; Escalera, Gabriela; Zhong, Weiwei; Colvin, Vicki L.To fully understand the biological and environmental impacts of nanomaterials requires studies that address both sublethal end points and multigenerational effects. Here, we use a nematode to examine these issues as they relate to exposure to two different types of quantum dots, core (CdSe) and coreヨshell (CdSe/ZnS), and to compare the effect to those observed after cadmium salt exposures. The strong fluorescence of the coreヨshell QDs allowed for the direct visualization of the materials in the digestive track within a few hours of exposure. Multiple end points, including both developmental and locomotive, were examined at QD exposures of low (10 mg/L Cd), medium (50 mg/L Cd), and high concentrations (100 mg/L Cd). While the coreヨshell QDs showed no effect on fitness (lifespan, fertility, growth, and three parameters of motility behavior), the core QDs caused acute effects similar to those found for cadmium salts, suggesting that biological effects may be attributed to cadmium leaching from the more soluble QDs. Over multiple generations, we commonly found that for lower life-cycle exposures to core QDs the parents response was generally a poor predictor of the effects on progeny. At the highest concentrations, however, biological effects found for the first generation were commonly similar in magnitude to those found in future generations.Item WormGender- Open-Source Software for AutomaticCaenorhabditis elegansᅠSex Ratio Measurement(Public Library of Science, 2015) Labocha, Marta K.; Jung, Sang-Kyu; Aleman-Meza, Boanerges; Liu, Zheng; Zhong, WeiweiFast and quantitative analysis of animal phenotypes is one of the major challenges of current biology. Here we report the WormGender open-source software, which is designed for accurate quantification of sex ratio in Caenorhabditis elegans. The software functions include, i) automatic recognition and counting of adult hermaphrodites and males, ii) a manual inspection feature that enables manual correction of errors, and iii) flexibility to use new training images to optimize the software for different imaging conditions. We evaluated the performance of our software by comparing manual and automated assessment of sex ratio. Our data showed that the WormGender software provided overall accurate sex ratio measurements. We further demonstrated the usage of WormGender by quantifying the high incidence of male (him) phenotype in 27 mutant strains. Mutants of nine genes (brc-1, C30G12.6, cep-1, coh-3, him-3, him-5, him-8, skr-1, unc-86) showed significant him phenotype. The WormGender is written in Java and can be installed and run on both Windows and Mac platforms. The source code is freely available together with a user manual and sample data at http://www.QuantWorm.org/. The source code and sample data are also available at http://dx.doi.org/10.6084/m9.figshare.1541248.