Guo, J. et al. (2019) International Worm Meeting "Toxicity of 1-hydroxyphenazine on ugt-1 mutants of Caenorhabditis elegans."

Guo, J., Edison , A., Asif , M., Rahmatullah, T., Maina, S., VanDerGaag, V.

[International Worm Meeting, 2019]

Caenorhabditis elegans is an ideal model organism for studying xenobiotic detoxification pathways of various toxins1. The family of uridine diphosphate-glucuronosyltransferase (UGT) proteins play a fundamental role in detoxifying xenobiotics1, but little is known about the phenotypes of specific ugt genes. We are studying the effects of 1-hydroxyphenazine (1-HP), a toxin produced by Pseudomonas aeruginosa, on the growth and development of different ugt knockout strains of C. elegans. As part of the Vertically Integrated Projects (VIP) undergraduate research team at UGA2, we have been developing a workflow and assay to determine developmental effects of 1-HP on different ugt mutants. Our initial objective is to discern whether the ugt-1 knockout strain is associated with increased or decreased detoxification phenotypes by evaluating the growth and development of worms when exposed to varying concentrations of 1-HP. Prior research has demonstrated that C. elegans can detoxify 1-HP through the general mechanism of O-glucosylation3. It is widely accepted that UGT proteins in C. elegans transfer glycosyl groups to modify certain small molecules1. Thus, we predict that UGTs play a role in the detoxification of xenobiotic factors, including 1-HP. Of the five available ugt mutant strains from the CGC, we chose ugt-1 since its specific function in detoxification is not yet known. Future experiments will test the toxicity of 1-HP on other available mutant deletion strains, from both the CGC and the CRISPR VIP team in our lab, to determine which ugt genes play a role in the xenobiotic process in nematodes. Understanding which genes are involved in this process will help elucidate the mechanism of xenobiotic detoxification by C. elegans.

Guo J et al. (2023) Nanoscale "Bidirectional near-infrared regulation of motor behavior using orthogonal emissive upconversion nanoparticles."

Zhang Y, Wen Q, Xiong F, Gao S, Chen L, Wang R, Guo J, Zhang X

[Nanoscale, 2023]

Bidirectional optogenetic manipulation enables specific neural function dissection and animal behaviour regulation with high spatial-temporal resolution. It relies on the respective activation of two or more visible-light responsive optogenetic sensors, which inevitably induce signal crosstalk due to their spectral overlap, low photoactivation efficiency and potentially high biotoxicity. Herein, a strategy that combines dual-NIR-excited orthogonal emissive upconversion nanoparticles (OUCNPs) with a single dual-colour sensor, BiPOLES, is demonstrated to achieve bidirectional, crosstalk-free NIR manipulation of motor behaviour in vivo. Core@shell-structured OUCNPs with Tm3+ and Er3+ dopants in isolated layers exhibit orthogonal blue and red emissions in response to excitation at 808 and 980 nm, respectively. The OUCNPs subsequently activate BiPOLES-expressing excitatory cholinergic motor neurons in C. elegans, leading to significant inhibition and excitation of motor neurons and body bends, respectively. Importantly, these OUCNPs exhibit negligible toxicity toward neural development, motor function and reproduction. Such an OUCNP-BiPOLES system not only greatly facilitates independent, bidirectional NIR activation of a specific neuronal population and functional dissection, but also greatly simplifies the bidirectional NIR optogenetics toolset, thus endowing it with great potential for flexible upconversion optogenetic manipulation.

Guo J et al. (2003) J Biol Chem "The ubiquinone-binding site of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase is a source of superoxide."

Lemire BD, Guo J

[J Biol Chem, 2003]

The mitochondrial succinate dehydrogenase (SDH) is a tetrameric iron-sulfur flavoprotein of the Krebs cycle and of the respiratory chain. A number of mutations in human SDH genes are responsible for the development of paragangliomas, cancers of the head and neck region. The mev-1 mutation in the Caenorhabditis elegans gene encoding the homolog of the SDHC subunit results in premature aging and hypersensitivity to oxidative stress. It also increases the production of superoxide radicals by the enzyme. In this work, we used the yeast succinate dehydrogenase to investigate the molecular and catalytic effects of paraganglioma- and mev-1-like mutations. We mutated Pro-190 of the yeast Sdh2p subunit to Gln (P190Q) and recreated the C. elegans mev-1 mutation by converting Ser-94 in the Sdh3p subunit into a glutamate residue (S94E). The P190Q and S94E mutants have reduced succinate-ubiquinone oxidoreductase activities and are hypersensitive to oxygen and paraquat. Although the mutant enzymes have lower turnover numbers for ubiquinol reduction, larger fractions of the remaining activities are diverted toward superoxide production. The P190Q and S94E mutations are located near the proximal ubiquinone-binding site, suggesting that the superoxide radicals may originate from a ubisemiquinone intermediate formed at this site during the catalytic cycle. We suggest that certain mutations in SDH can make it a significant source of superoxide production in mitochondria, which may contribute directly to disease progression. Our data also challenge the dogma that superoxide production by SDH is a flavin-mediated event rather than a quinone-mediated one.

Guo J et al. (2020) J Struct Biol "An optimized approach using cryofixation for high-resolution 3D analysis by FIB-SEM."

Wang G, Zhang Z, Hong J, Wang X, Tang W, Guo J, Song D

[J Struct Biol, 2020]

Compared with conventional two-dimensional transmission electron microscopy (TEM), focused ion beam scanning electron microscopy (FIB-SEM) can provide more comprehensive 3D information on cell substructures at the nanometer scale. Biological samples prepared by cryofixation using high-pressure freezing demonstrate optimal preservation of the morphology of cellular structures, as these are arrested instantly in their near-native states. However, samples from cryofixation often show a weak back-scatter electron signal and bad image contrast in FIB-SEM imaging. In addition, it is impossible to do large amounts of heavy metal staining. This is commonly achieved via established osmium impregnation (OTO) en bloc staining protocols. Here, we compared the FIB-SEM image quality of brain tissues prepared using several common freeze-substitution media, and we developed an approach that overcomes these limitations through a combination of osmium tetroxide, uranyl acetate, tannic acid, and potassium permanganate at proper concentrations, respectively. Using this optimized sample preparation protocol for high-pressure freezing and freeze-substitution, perfect smooth membrane morphology, even of the lipid bilayers of the cell membrane, was readily obtained using FIB-SEM. In addition, our protocol is broadly applicable and we demonstrated successful application to brain tissues, plant tissues, Caenorhabditis elegans, Candida albicans, and chlorella. This approach combines the potential of cryofixation for 3D large volume analysis of subcellular structures with the high-resolution capabilities of FIB-SEM.

Guo X et al. (2013) J Virol "Characterization of virus-encoded RNA interference suppressors in Caenorhabditis elegans."

Guo X, Lu R

[J Virol, 2013]

In fungi, plants, and invertebrates, antiviral RNA interference (RNAi) directed by virus-derived small interfering RNAs (siRNAs) represents a major antiviral defense that the invading viruses have to overcome in order to establish infection. As a counterdefense mechanism, viruses of these hosts produce diverse classes of proteins capable of suppressing the biogenesis and/or function of viral siRNAs. This RNA-directed viral immunity (RDVI) in the nematode Caenorhabditis elegans is known to exhibit some unique features. Currently, little is known about viral suppression of RNAi in C. elegans. Here, we show that ectopic expression of the B2 protein encoded by Flock House virus (FHV) suppresses RNAi induced by either long double-stranded RNA (dsRNA) or an FHV-based replicon and facilitates the natural infection of C. elegans by Orsay virus but is not active against RNA silencing mediated by microRNAs. We report the development of an assay for the identification of viral suppressor of RNAi (VSR) in C. elegans based on the suppression of a viral replicon-triggered RDVI by ectopic expression of candidate proteins. No VSR activity was detected for either of the two Orsay viral proteins proposed previously as VSRs. We detected, among the known heterologous VSRs, VSR activity for B2 of Nodamura virus but not for 2b of tomato aspermy virus, p29 of fungus-infecting hypovirus, or p19 of tomato bushy stunt virus. We further show that, unlike that in plants and insects, FHV B2 suppresses worm RDVI mainly by interfering with the function of virus-derived primary siRNAs.

Guo X et al. (1995) J Parasitol "Hemocyte alterations during melanotic encapsulation of Brugia malayi in the mosquito Armigeres subalbatus."

Christensen BM, Guo X, Zhao X, Beerntsen BT

[J Parasitol, 1995]

The involvement of hemocytes in melanotic encapsulation reactions against Brugia malayi was assessed in Armigeres subalbatus. Hemocyte populations, epitope changes, phenol oxidase (PO) activity, and the presence of an 84-kDa polypeptide were investigated in mosquitoes exposed to a B. malayi-infective bloodmeal (= immune-activated), in mosquitoes given a noninfective bloodmeal (= controls), in nonbloodfed mosquitoes (= naive), or in some combination of these. Total hemocyte populations in immune-activated mosquitoes significantly decreased at 24 hr postbloodmeal (PB) as compared with controls. At 48 and 72 hr PB, hemocyte population levels in immune-activated mosquitoes increased to control levels. Epitope changes, as indicated by wheat germ agglutinin (WGA) binding, also were observed. There was a significant increase in the percentage of hemocytes binding WGA in immune-activated mosquitoes at 24 hr PB as compared with controls. Furthermore, the activity of hemocyte PO, an enzyme involved in the melanotic encapsulation pathway, was significantly elevated at 12 hr PB in immune-activated mosquitoes as compared with controls. Analysis for the presence of an 84-kDa polypeptide in A. subalbatus indicates that a 2.0-kb message in total RNA hybridized to D6.12, an Aedes aegypti cDNA encoding an 84-kDa polypeptide that is associated with melanotic encapsulation responses. The hybridization of D6.12 to RNA was not greater in immune-activated as compared to control A. subalbatus, as has been observed in A. aegypti. Results indicate that these hemocyte changes correspond in time with the melanotic encapsulation reactions of A. subalbatus against filarial worms.

da Silveira TL et al. (2019) Neuroscience "Guanosine Prevents against Glutamatergic Excitotoxicity in C. elegans."

da Silveira TL, Arantes LP, Tassi CL, Zamberlan DC, Soares FAA, Machado ML, Obetine FBB, Cordeiro LM, da Silva AF

[Neuroscience, 2019]

Glutamatergic neurotransmission is present in most mammalian excitatory synapses and plays a key role in central nervous system homeostasis. When over-activated, it can induce excitotoxicity, which is present in several neuropathologies. The nucleoside guanosine (GUO), is a guanine-based purine known to have neuroprotective effects by modulating glutamatergic system during glutamate excitotoxicity in mammals. However, GUO action in Caenorhabditis elegans, as well as on C. elegans glutamatergic excitotoxicity model, is not known. The GUO effects on behavioral parameters in Wild Type (WT) and knockouts worms for glutamate transporters (GLT-3, GLT-1), glutamate vesicular transporter (EAT-4), and NMDA and non-NMDA receptors were used to evaluate the GUO modulatory effects. The GUO tested concentrations did not alter the animals' development, but GUO reduced pharyngeal pumps in WT animals in a dose-dependent manner. The same effect was observed in pharyngeal pumps, when the animals were treated with 4mM of GUO in glr-1, nmr-1 and eat-4, but not in glt-3 and glt-3;glt-1 knockouts. The double mutant glt-3; glt-1 for GluTs had decreased body bends and an increased number of reversions. This effect was reverted after treatment with GUO. Furthermore, GUO did not alter the sensory response in worms with altered glutamatergic signaling. Thus, GUO seems to modulate the worm's glutamatergic system in situations of exacerbated glutamatergic signaling, which are represented by knockout strains to glutamate transporters. However, in WT animals, GUO appears to reinforce glutamatergic signaling in specific neurons. Our findings indicate that C. elegans strains are useful models to study new compounds that could be used in glutamate-associated neurodegenerative diseases.

Guo YR et al. (2020) J Virol "Orsay CP- adopts a novel -bracelet structural fold and incorporates into virions as a head fiber."

Zhou Y, Holt MV, Fan Y, Wang D, Wang T, Guo YR, Zhong W, Tao YJ, Zhang JL, Jin M, Young NL, Jiang H

[J Virol, 2020]

Fiber 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 non-covalent interactions. The small RNA virus Orsay, which specifically infects the laboratory model <i>Caenorhabditis elegans</i>, encodes a fibrous protein that can be expressed as a free protein and as a capsid protein- (CP-) fusion protein. Free has previously been demonstrated to facilitate viral exit following intracellular expression; however, the biological significance and prevalence of CP- remained relatively unknown. Here, we demonstrate that Orsay CP- is covalently incorporated into infectious particles, the first example of any attached viral fibers known to date. The crystal structure of (1-101) (<i>i.e.</i> a deletion mutant containing the first 101 amino acid (aa) residues of ) reveals a pentameric, 145-A long fiber with an N-terminal coiled coil followed by multiple -bracelet repeats. Electron micrographs of infectious virions depict particle-associated CP- fibers with dimensions similar to free . proteins from two other nematode viruses Le Blanc and Santeuil, which both specifically infect <i>Caenorhabditis briggsae,</i> were also found to form fibrous molecules. Recombinant Le Blanc 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- likely mediates host cell attachment for all three nematode viruses, additional downstream factor(s) ultimately determine the host specificity and range of each virus.<b>IMPORTANCE</b> Viruses often have extended fibers to mediate host cell recognition and entry, serving as promising targets for antiviral drug development. Unlike other known viral fibers, the proteins from the three recently discovered nematode viruses are incorporated into infectious particles as protruding fibers covalently linked to the capsid. Crystal structures of revealed novel pentameric folding repeats, which we term -bracelets, in the intermediate shaft region. Based on sequence analysis, the -bracelet motif of is conserved in all three nematode viruses and could account for 60% of the total length of the fiber. Our study indicated that plays important roles in cell attachment for this group of nematode viruses. In addition, the tightly knitted -bracelet fold, which presumably allows to survive harsh environments in the worm gut, could serve applicable to bioengineering applications given its potentially high stability.

Guo X et al. (2012) J Virol "Silencing of host genes directed by virus-derived short interfering RNAs in Caenorhabditis elegans."

Li WX, Lu R, Guo X

[J Virol, 2012]

Small interfering RNAs (siRNAs) processed from viral replication intermediates by RNase III-like enzyme Dicer guide sequence-specific antiviral silencing in fungi, plants, and invertebrates. In plants, virus-derived siRNAs (viRNAs) can target and silence cellular transcripts and, in some cases, are responsible for the induction of plant diseases. Currently it remains unclear whether viRNAs are also capable of modulating the expression of cellular genes in the animal kingdom, although animal virus-encoded microRNAs (miRNAs) are known to guide efficient silencing of host genes, thereby facilitating virus replication. In this report, we showed that viRNAs derived from a modified nodavirus triggered potent silencing of homologous cellular transcripts produced by the endogenous gene or transgene in the nematode worm Caenorhabditis elegans. Like that found in plants, virus-induced gene silencing (VIGS) in C. elegans also involves RRF-1, a worm RNA-dependent RNA polymerase (RdRP) that is known to produce single-stranded secondary siRNAs in a Dicer-independent manner. We further demonstrated that VIGS in C. elegans is inheritable, suggesting that VIGS has the potential to generate profound epigenetic consequences in future generations. Altogether, these findings, for the first time, confirmed that viRNAs have the potential to modulate host gene expression in the animal kingdom. Most importantly, the success in uncoupling the trigger and the target of the antiviral silencing would allow for the exploration of novel features of virus-host interactions mediated by viRNAs in the animal kingdom.

Guo X et al. (2013) J Virol "Antiviral RNA silencing initiated in the absence of RDE-4, a double-stranded RNA binding protein, in Caenorhabditis elegans."

Zhang R, Lu R, Guo X, Wang J

[J Virol, 2013]

Small interfering RNAs (siRNAs) processed from double-stranded RNA (dsRNA) of virus origins mediate potent antiviral defense through a process referred to as RNA interference (RNAi) or RNA silencing in diverse organisms. In the simple invertebrate Caenorhabditis elegans, the RNAi process is initiated by a single Dicer, which partners with the dsRNA binding protein RDE-4 to process dsRNA into viral siRNAs (viRNAs). Notably, in C. elegans this RNA-directed viral immunity (RDVI) also requires a number of worm-specific genes for its full antiviral potential. One such gene is rsd-2 (RNAi spreading defective 2), which was implicated in RDVI in our previous studies. In the current study, we first established an antiviral role by showing that rsd-2 null mutants permitted higher levels of viral RNA accumulation, and that this enhanced viral susceptibility was reversed by ectopic expression of RSD-2. We then examined the relationship of rsd-2 with other known components of RNAi pathways and established that rsd-2 functions in a novel pathway that is independent of rde-4 but likely requires the RNA-dependent RNA polymerase RRF-1, suggesting a critical role for RSD-2 in secondary viRNA biogenesis, likely through coordinated action with RRF-1. Together, these results suggest that RDVI in the single-Dicer organism C. elegans depends on the collective actions of both RDE-4-dependent and RDE-4-independent mechanisms to produce RNAi-inducing viRNAs. Our study reveals, for the first time, a novel siRNA-producing mechanism in C. elegans that bypasses the need for a dsRNA-binding protein.