Maeda I et al. (2001) Tanpakushitsu Kakusan Koso "[Systematic functional analysis of the C. elegans genome]"

Maeda I, Sugimoto A

[Tanpakushitsu Kakusan Koso, 2001]

Maeda I et al. (2000) Midwest Worm Meeting "RNAi Screening with a non-redundant cDNA set"

Kohara Y, Yamamoto M, Maeda I, Sugimoto A

[Midwest Worm Meeting, 2000]

We have established a systematic reverse-genetic screening method based on phenotypes, to identify genes involved in specific developmental processes. In this method, we have combined RNAi and cDNA project. Namely, we use a tag-sequenced, non-redundant cDNA set as templates for preparing dsRNA and perform RNAi systematically. To introduce dsRNA into worms, we use the "soaking method" instead of microinjection, so that we can handle multiple samples concurrently. The soaking method had been known to be less potent, but we optimized the condition and achieved enough efficiency and sensitivity to perform screening (Maeda et al., 1999 International Worm Meeting #565). In this method, four L4 hermaphrodites are soaked in each RNA solution derived from single cDNA clone, then incubated for 24 hours. The worms are then recovered to a new plate and phenotypes of both P0s and F1 progeny are observed. All steps of RNA synthesis, purification and worm soaking are performed in 96-well format. To date, we examined 1728 RNA species. Of those, 1.0% (17/1728) caused sterility of F1s and 15.2% (262/1728) caused embryonic lethality. In total, about 28% (491/1728) of RNA species caused detectable phenotypes. We are now screening at the rate of 200 clones/month. Since we are interested in germ line development and differentiation, we are currently focusing on RNA species that cause sterility with no somatic phenotypes. Among such RNA species, various kinds of translational regulators were included. This is consistent with the fact that translational regulation plays an important role in germ line development. The sterile-only RNA species also included several known genes which have never been reported to be involved in germ line development. We plan to make RNAi data available to the public and report all detectable phenotypes at the NEXTDB WWW server at NIG. We will present the result and the status of our screening.

Maeda I et al. (2001) Curr Biol "Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi."

Maeda I, Sugimoto A, Kohara Y, Yamamoto M

[Curr Biol, 2001]

Genome-wide analysis of gene function is essential for the post-genome era, and development of efficient and economical technology suitable for it has been in demand. Here we report a large-scale inactivation of the expressed genes in the nematode Caenorhabditis elegans, For this purpose, we have established a high-throughput "RNAi-by-soaking" methodology by modifying the conventional RNAi method [1, 2], A set of tag-sequenced, nonredundant cDNAs corresponding to approximately 10,000 genes [3] (representing half of the predicted genes [4]) was used for the systematic RNAi analysis. We have processed approximately 2500 genes to date. In development, 27% of them showed detectable phenotypes, such as embryonic lethality, postembryonic lethality, sterility, and morphological abnormality. Of these, we analyzed the phenotypes of Fl sterility in detail, and we have identified 24 genes that might play important roles in germline development. Combined with the ongoing analysis of expression patterns of these cDNAs [3, 5], the functional information obtained in this work will provide a starting point for the further analysis of each gene. Another finding from this screening is that the incidence of essential genes is significantly lower in the X chromosome than in the autosomes.

I Maeda et al. (1999) International C. elegans Meeting "RNAi Screening with a non-redundant cDNA set"

I Maeda, Y Kohara, A Sugimoto, M Yamamoto

[International C. elegans Meeting, 1999]

RNA interference (RNAi) is a powerful tool for the transient inactivation of specific gene expression. We aim to establish a new screening method in which RNAi is performed systematically, to identify genes involved in specific developmental processes. To maximize the efficiency, we use a tag-sequenced, non-redundant cDNA set (made by Y. K.) as templates for preparing dsRNA. To deliver dsRNA into worms, we use the "soaking method" (in which worms are soaked in dsRNA solution) instead of microinjection, so that multiple RNAi can be performed concurrently. Although the soaking method has been known to be less potent and to give variable results, we optimized the condition to achieve a satisfactory efficacy to be used for the screen. In the control experiments, all nine genes tested ( unc-22 , glp-1 , mei-1 , mes-3 , fem-1 , glh-1 , glh-2 , pgl-1 and emo-1 ) with this method exhibited expected phenotypes with high penetrance. We are currently screening for the RNA species that cause sterility or embryonic lethality, however, we will record any other noticeable phenotypes. We plan to make the RNAi data available to the public at the NEXTDB WWW server at NIG (See the abstract by Shin-i et al. ). In the pilot screen, 10/64 gave embryonic lethality of the F1s, and 6/64 resulted in sterility of the P0s. Progress on the screen will be presented.

Sugimoto A et al. (2000) Japanese Worm Meeting "RNAi Screening with a non-redundant cDNA set"

Yamamoto M, Sugimoto A, Maeda I, Kohara Y

[Japanese Worm Meeting, 2000]

We have established a systematic reverse-genetic screening method based on phenotypes, to identify genes involved in specific developmental processes. In this method, we have combined RNAi and cDNA project. Namely, we use a tag-sequenced, non-redundant cDNA set as templates for preparing dsRNA and perform RNAi systematically. To introduce dsRNA into worms, we use the 'soaking method' instead of microinjection, so that we can handle multiple samples concurrently. The soaking method had been known to be less potent, but we optimized the condition and achieved enough efficiency and sensitivity to perform screening (Maeda et al., 1999 International Worm Meeting #565). In this method, four L4 hermaphrodites are soaked in each RNA solution derived from single cDNA clone, then incubated for 24 hours. The worms are then recovered to a new plate and phenotypes of both P0s and F1 progeny are observed. All steps of RNA synthesis, purification and worm soaking are performed in 96-well format. To date, we examined 2064 RNA species. Of those, 0.9% (19/2064) caused sterility of F1s and 14.3% (295/2064) caused embryonic lethality. In total, about 28% (573/2064) of RNA species caused detectable phenotypes. We are now screening at the rate of 200 clones/month. Since we are interested in germ line development and differentiation, we are currently focusing on RNA species that cause sterility with no somatic phenotypes. Among such RNA species, various kinds of translational regulators were included. This is consistent with the fact that translational regulation plays an important role in germ line development. The sterile-only RNA species also included several known genes which have never been reported to be involved in germ line development. We plan to make RNAi data available to the public and report all detectable phenotypes at the NEXTDB WWW server at NIG. We will present the result and the status of our screening.

Souza ACR et al. (2018) Genes (Basel) "Pathogenesis of the Candida parapsilosis Complex in the Model Host Caenorhabditis elegans."

Colombo AL, Fuchs BB, Jayamani E, Mylonakis E, Souza ACR, Alves VS

[Genes (Basel), 2018]

<i>Caenorhabditis</i><i>elegans</i> is a valuable tool as an infection model toward the study of <i>Candida</i> species. In this work, we endeavored to develop a <i>C</i>. <i>elegans</i>-<i>Candida</i><i>parapsilosis</i> infection model by using the fungi as a food source. Three species of the C. parapsilosis complex (<i>C.</i><i>parapsilosis</i> (<i>sensu</i><i>stricto</i>), <i>Candida</i><i>orthopsilosis</i> and <i>Candida</i><i>metapsilosis</i>) caused infection resulting in <i>C. elegans</i> killing. All three strains that comprised the complex significantly diminished the nematode lifespan, indicating the virulence of the pathogens against the host. The infection process included invasion of the intestine and vulva which resulted in organ protrusion and hyphae formation. Importantly, hyphae formation at the vulva opening was not previously reported in <i>C</i>. <i>elegans</i>-<i>Candida</i> infections. Fungal infected worms in the liquid assay were susceptible to fluconazole and caspofungin and could be found to mount an immune response mediated through increased expression of <i>cnc</i>-<i>4</i>, <i>cnc</i>-<i>7</i>, and <i>fipr</i><i>-</i><i>22</i>/<i>23</i>. Overall, the <i>C</i>. <i>elegans</i>-<i>C</i>. <i>parapsilosis</i> infection model can be used to model <i>C</i>. <i>parapsilosis</i> host-pathogen interactions.

Fourie R et al. (2021) Front Cell Infect Microbiol "Candida albicans SET3 Plays a Role in Early Biofilm Formation, Interaction With Pseudomonas aeruginosa and Virulence in Caenorhabditis elegans."

Sebolai O, Fourie R, Albertyn J, Pohl CH, Gcilitshana O

[Front Cell Infect Microbiol, 2021]

The yeast <i>Candida albicans</i> exhibits multiple morphologies dependent on environmental cues. <i>Candida albicans</i> biofilms are frequently polymicrobial, enabling interspecies interaction through proximity and contact. The interaction between <i>C. albicans</i> and the bacterium, <i>Pseudomonas aeruginosa</i>, is antagonistic <i>in vitro, with P. aeruginosa</i> repressing the yeast-to-hyphal switch in <i>C. albicans</i>. Previous transcriptional analysis of <i>C. albicans</i> in polymicrobial biofilms with <i>P. aeruginosa</i> revealed upregulation of genes involved in regulation of morphology and biofilm formation, including <i>SET3</i>, a component of the Set3/Hos2 histone deacetylase complex (Set3C). This prompted the question regarding the involvement of <i>SET3</i> in the interaction between <i>C. albicans</i> and <i>P. aeruginosa</i>, both <i>in vitro</i> and <i>in vivo.</i> We found that <i>SET3</i> may influence early biofilm formation by <i>C. albicans</i> and the interaction between <i>C. albicans</i> and <i>P. aeruginosa</i>. In addition, although deletion of <i>SET3</i> did not alter the morphology of <i>C. albicans</i> in the presence of <i>P. aeruginosa</i>, it did cause a reduction in virulence in a <i>Caenorhabditis elegans</i> infection model, even in the presence of <i>P. aeruginosa.</i>

Zhu Q et al. (2020) Oxid Med Cell Longev "A Dihydroflavonoid Naringin Extends the Lifespan of C. elegans and Delays the Progression of Aging-Related Diseases in PD/AD Models via DAF-16."

Zhou XG, Qu Y, Luo HR, Zhu Q, Wu GS, Chen JN

[Oxid Med Cell Longev, 2020]

Naringin is a dihydroflavonoid, which is rich in several plant species used for herbal medicine. It has a wide range of biological activities, including antineoplastic, anti-inflammatory, antiphotoaging, and antioxidative activities. So it would be interesting to know if naringin has an effect on aging and aging-related diseases. We examined the effect of naringin on the aging of <i>Caenorhabditis elegans</i> (<i>C</i>. <i>elegans</i>). Our results showed that naringin could extend the lifespan of <i>C</i>. <i>elegans</i>. Moreover, naringin could also increase the thermal and oxidative stress tolerance, reduce the accumulation of lipofuscin, and delay the progress of aging-related diseases in <i>C</i>. <i>elegans</i> models of AD and PD. Naringin could not significantly extend the lifespan of long-lived mutants from genes in insulin/IGF-1 signaling (IIS) and nutrient-sensing pathways, such as <i>daf</i>-<i>2</i>, <i>akt</i>-<i>2</i>, <i>akt</i>-<i>1</i>, <i>eat</i>-<i>2</i>, <i>sir</i>-<i>2</i>.<i>1</i>, and <i>rsks</i>-<i>1</i>. Naringin treatment prolonged the lifespan of long-lived <i>glp</i>-<i>1</i> mutants, which have decreased reproductive stem cells. Naringin could not extend the lifespan of a null mutant of the fox-head transcription factor DAF-16. Moreover, naringin could increase the mRNA expression of genes regulated by <i>daf</i>-<i>16</i> and itself. In conclusion, we show that a natural product naringin could extend the lifespan of <i>C</i>. <i>elegans</i> and delay the progression of aging-related diseases in <i>C</i>. <i>elegans</i> models via DAF-16.

Coomansingh-Springer C et al. (2019) Heliyon "Internal parasitic burdens in brown rats (Rattus norvegicus) from Grenada, West Indies."

Sharma RN, Armstrong E, Vishakha V, Acuna AM, Coomansingh-Springer C

[Heliyon, 2019]

This study identified the endoparasites in Brown rat (<i>Rattus norvegicus)</i> during May to July 2017 in Grenada, West Indies. A total of 162 rats, 76 females and 86 males were trapped from St. George and St. David parishes in Grenada. The collected fecal samples were examined for parasitic eggs and/or oocysts using simple fecal flotation technique. Adult parasites found in the intestinal tract were examined for identification. The overall prevalence of intestinal parasites among rats was 79 %. Ten helminth species were recovered, several of which were reported for the first time in rodents in Grenada. The internal parasites consist of seven nematodes (<i>Angiostrongylus</i> spp., <i>Nippostrongylus braziliensis</i>, <i>Heterakis spumosa</i>, <i>Strongyloides ratti</i>, <i>Aspiculuris tetraptera</i>, <i>Syphacia</i> spp. and <i>Protospirura</i> spp.), one cestode (<i>Hymenolepsis diminuta</i>), one acanthocephalan (<i>Moniliformis moniliformis</i>) and one protozoa species (<i>Eimeria</i> spp.). The most prevalent zoonotic species were <i>Angiostrongylus</i> spp. (35.2%), <i>Hymenolepsis diminuta</i> (7.4%) and <i>Moniliformis moniliformis</i> (3.1%). Several nonzoonotic endoparasites; which included <i>Nippostrongylus braziliensis</i> (50.6%), <i>Heterakis spumosa</i> (15.4%), <i>Strongyloides ratti</i> (43.2%), <i>Aspiculuris tetraptera</i> (2.5%), <i>Syphacia</i> spp<i>.</i> (1.9%), <i>Protospirura</i> spp. (1.2%) and <i>Eimeria</i> spp. (4.7%) were also identified. The most prevalent parasites were <i>Nippostrongylus brasiliensis</i> (50.6%), <i>Strongyloides ratti</i> (43.2%) and <i>Angiostrongylus spp.</i> (35.2%). Co-infections occurred with up to six species per rat showing different combinations of parasitic infections.

Wu CJ et al. (2019) Appl Environ Microbiol "Genotypic and Phenotypic Characteristics of Aeromonas Isolates from Fish and Patients in Tainan City."

Shu CY, Li CW, Ko WC, Su YC, Chen YW, Lee NY, Su SL, Wu CJ, Chen PL, Li MC, Lin YT

[Appl Environ Microbiol, 2019]

The present study aimed to isolate <i>Aeromonas</i> from fish sold in the markets as well as in sushi and seafood shops and compare their virulence factors and antimicrobial characteristics with those of clinical isolates. Among the 128 fish isolates and 47 clinical isolates, <i>A. caviae</i>, <i>A. dhakensis</i>, and <i>A. veronii</i> were the principal species. <i>A. dhakensis</i> isolates carried at least 5 virulence genes, more than other <i>Aeromonas</i> species. The predominant genotype of virulence genes was <i>hlyA/lip/alt/col/el</i> in both <i>A. dhakensis</i> and <i>A. hydrophila</i> isolates, <i>alt/col/ela</i> in <i>A. caviae</i> isolates, and <i>act</i> in <i>A. veronii</i> isolates. <i>A. dhakensis</i>, <i>A. hydrophila</i>, and <i>A. veronii</i> isolates more often exhibited hemolytic and proteolytic activity and showed greater virulence than <i>A. caviae</i> in <i>Caenorhabditis elegans</i> and the C2C12 cell line. However, the link between the genotypes and phenotypes of the studied virulence genes in <i>Aeromonas</i> species is not evident. Among the four major clinical <i>Aeromonas</i> species, nearly all (99.0%) <i>A. dhakensis</i>, <i>A. hydrophila</i>, and <i>A. veronii</i> isolates harbored <i>bla</i>_CphA, which encodes a carbapenemase, but only a minority (6.7%, 7/104) were nonsusceptible to carbapenem. Regarding AmpC -lactamase genes, <i>bla</i>_AQU-1 was exclusively found in <i>A. dhakensis</i> isolates and <i>bla</i>_MOX3 only in <i>A. caviae</i> isolates, but only 7.6% (6) of the 79 <i>Aeromonas</i> isolates carrying <i>bla</i>_AQU-1 or <i>bla</i>_MOX3 exhibited a cefotaxime resistance phenotype. In conclusion, fish <i>Aeromonas</i> isolates carry a variety of combinations of virulence and B-lactamase resistance genes and exhibit virulence phenotypes and antimicrobial resistance profiles similar to those of clinical isolates.<b>IMPORTANCE</b><i>Aeromonas</i> species can cause severe infections in immunocompromised individuals upon exposure to virulent pathogens in the environment, but the characteristics of environmental <i>Aeromonas</i> species remain unclear. Our study showed several pathogenic <i>Aeromonas</i> species possessing virulence traits and antimicrobial resistance similar to those of <i>Aeromonas</i> isolates causing clinical diseases were present in fish intended for human consumption in Tainan City.