Vertti-Quintero, Nadia et al. (2015) International Worm Meeting "Microfluidic high-throughput fluorescence-based sorter for studying stochastic expression of heat shock proteins on C. elegans."

deMello, Andrew, Gruber, Jan, Vertti-Quintero, Nadia, Dressler, Oliver, Gunawan, Rudiyanto, Casadevall i Solvas, Xavier, Stavrakis, Stavros

[International Worm Meeting, 2015]

Population screening, phenotyping and sorting are among the most common operations in studies involving C. elegans. Manual executions of these tasks often depend on subjective qualitative analysis and are generally time-consuming. Here, we developed a low-cost high-throughput microfluidic fluorescence-based sorting system for quantitative automation of these tasks. We separate worm populations according to three levels of fluorescence intensity and demonstrate the efficacy of our sorting system in the study of the stochastic dynamics of heat shock proteins.Heat shock proteins (HSPs) function as molecular chaperones assisting protein folding and preventing protein aggregation in cells. The regulation of HSPs plays an important role in cellular homeostasis of organisms. Several studies previously reported that the level of heat shock response in worms varies among individuals and that this level correlates with the worm ability to survive lethal thermal stresses and interestingly also with its lifespan. The variability among homogeneous worm population can arise due to intrinsic and extrinsic factors that control stochastic gene expression. Such stochasticity has further been shown to play fundamental functional roles in cell processes, such as in cell decision making. In this study we focused on delineating and quantifying different sources of stochasticity in HSP gene expression in C. elegans. To achieve this we use transgenic C. elegans strain TJ375 [hsp-16.2p::GFP] which allows for an accurate assessment of the amount of expressed native HSP-16.2. Depending on the HSP expression level, isogenic worms are sorted iteratively at different days, so that chronological histories of HSP expression in these worms can be built. Specifically, worms suspended in M9 buffer solution are processed on a microfluidic device in which single worms are screened for fluorescence along their body. The measured intensity is then used in real-time to rapidly sort each worm (by means of on-chip PDMS hydraulic valves) into one of the three different outlets of the device. The threshold parameters for population segmentation can be tailored to collect worms with different levels of HSP expression. We can achieve worm sorting at a rate of 80 ms per individual, corresponding to a maximum throughput of hundreds of worms per minute, a rate that is higher than previously reported microfluidic systems.

Berger, Simon et al. (2017) International Worm Meeting "Long-term C. elegans Immobilization enables High Resolution Developmental Studies in vivo."

Aegarter-Wilmsen, Tinri, Lattmann, Evelyn, Hajnal, Alex, Casadevall i Solvas, Xavier, deMello, Andrew, Berger, Simon, Hengartner, Michael

[International Worm Meeting, 2017]

Owing to its small size, fecundity and genetical tractability C. elegans has become one of the most widely used model organisms in biology. One major challenge when working with C. elegans however, is its high mobility, making immobilization necessary for the study of most cellular and subcellular processes. Immobilization is usually achieved by simply placing the worms of interest on an agar pad, limiting motion through the pressure exerted on the animal and, if necessary, through addition of chemical tranquilizers. Unfortunately, this process has proven to be a major limitation when trying to follow developmental processes over extended periods of time, as it rapidly causes slowdown or arrest of many such processes. Here we present a novel set of microfluidic devices able to trap, feed and image C. elegans in a variety of developmental stages (L1 to adult), without any of the adverse effects encountered when using conventional immobilization methods. Our immobilization platform is based on a simple PDMS microfluidic device mounted on the back of a coverslip. Operation of the device is readily learned and the microfluidic device can easily be integrated with any type of microscope (inverted or upright), all imaging modalities commonly used (brightfield, epifluorescence and confocal microscopy), and any type of objective (high magnification and high numerical aperture). Thus requiring only small changes to existing microscope setups and microscopy protocols prior to adaptation, enabling the study of a great variety of developmental processes, interference free, in vivo. We demonstrate the platforms capabilities in several case studies. First, we studied long-term viability of adult C. elegans on-chip, readily achieving viabilities exceeding 100 hours while immobilized, all while observing normal feeding and egg laying rates. Optimal quality images of the adult gonad were then acquired, showcasing the platforms capabilities of following complex developmental processes over long time periods. Specifically we studied germ cell apoptosis, a process known to arrest on agar pad within 20 minutes, and factors involved in the cell fate decision by tracking 100 cells over the course of 12 hours. For the first time determining an apoptotic rate of 60% in vivo (n = 100). Secondly we demonstrate that crucial developmental process, e.g. anchor cell invasion and distal tip cell migration, occur normally on-chip in all larval stages assessed, and at rates comparable to on plate culture. Compared to development on agar pads, processes occurred 5-7 times faster and more reliable on-chip, with all worms undergoing normal development. This strongly suggests that our immobilization device has minimal negative effects on sensitive developmental processes, thus making it ideally suited for long-term studies of processes so far inaccessible, all while allowing the capture of high resolution images.

Berger, Simon et al. (2015) International Worm Meeting "Microfluidic C. elegans immobilization, enabling long-term studies of germline development at a single-cell resolution."

deMello, Andrew, Casadevall i Solvas, Xavier, Berger, Simon, Aegerter-Wilmsen, Tinri, Hengartner, Michael, Eberhard, Ralf, Stavrakis, Stavros, Hajnal, Alex

[International Worm Meeting, 2015]

The germline of C. elegans has extensively been used to study cell proliferation, differentiation, and death. Even though many such studies can be carried out by simply acquiring steady state images of the animals, some require tracing of individual germ cells for extended periods of time. Thus far, it has been challenging to immobilize adult worms and, at the same time, maintain the conditions required for adequate physiological germline function. Germline health soon declines in animals padded on slides, which has significantly limited germline-related developmental studies.In this study we demonstrate a microfluidic platform for the long-term immobilization of adult C. elegans and its application for the imaging of mitotic and apoptotic germ cells at the single cell level. The immobilization platform allows for the efficient trapping, feeding, and imaging of a single worm over an extended period of time, while not disturbing the physiologically essential and sensitive process of egg-laying. The performance of the platform was validated by bright field microscopy, displaying good immobilization and survival of the worms for up to 150 hours and egg production for up to 60 hours. Fluorescence imaging further showed that fertilization occurs normally and germline health is not affected negatively even over extended periods of time. Subsequently the newly developed microfluidic platform was used to monitor the propagation and development of individual germ cells along the gonad. In the future information gained about cell size and state of the nucleus will be used to determine mitotic and apoptotic cells and reconstruct their development in time.

Vertti-Quintero, Nadia et al. (2017) International Worm Meeting "Study and characterization of dynamic and stochastic expression of HSP-16.2 on C. elegans using a high-throughput microfluidic platform."

Vertti-Quintero, Nadia, deMello, Andrew, Gruber, Jan, Dressler, Oliver, Berger, Simon, Casadevall i Solvas, Xavier, Gunawan, Rudiyanto, Stavrakis, Stavros

[International Worm Meeting, 2017]

Heat shock proteins (HSPs) are essential in the maintenance of cellular proteostasis. Their expression and regulation have been shown to vary even among isogenic C. elegans cohorts, with individuals expressing higher levels of HSPs experiencing higher survival rates, which might reflect an underlying ability of each individual to survive upon exposure to heat challenge[1]. Interestingly, though, only worms that express highly at a specific time after heat shock are significantly longer-lived. This suggests that, rather than the level of expression itself, it is the dynamics of expression which correlate with lifespan. Indeed, the expression of HSPs at the whole organism level follows a dynamical behavior in time. We hypothesize that HSPs expression dynamics correlate with worm survival capability, a relationship that we aim to characterize and study. Towards this end, we use the transgenic C. elegans strain TJ375 [hsp-16.2p::GFP(gpIs1)] and screen the expression of HSP after heat shock. We have built a high-throughput continuous-flow microfluidic platform that enables C. elegans in vivo population screening and sorting based on the quantification of GFP at the whole organism level. Each worm is read in a serial manner while in motion through a straight channel. The quantity of fluorescent proteins within each worm is screened, by means of a laser light sheet which excites the proteins of interest and light is collected by a PMT. Information signals can be processed in real-time for sorting downstream into three possible bins. The sorting mechanism is achieved by hydraulic on-chip valves, which deliver a maximum sorting throughput of 600 worms/min. Worms are then collected in individual vials with no significant loss in viability. To study how HSPs are dynamically regulated in certain subpopulations and to understand the significance of these dynamics in both, stochastic distribution and longevity, we obtain quantitative assessments of the expression of native HSP-16.2 produced by isogenic cohorts at different ages, as well as at different times after exposure to heat shock. Using this data, we use the LASSO technique to infer the existence of singular subpopulations (with dynamics-specific gene expression profiles), which we will then validate with the appropriate sorting of subpopulations of interest and the measuring of their HSPs expression dynamics separately (through subsequent rounds of screening).We will also corroborate the existence of these specific expression dynamics by imaging single individuals immobilized in a microfluidic trap along 48 h after heat shock. Finally, the characterization of the effect of HSPs expression dynamics (i.e. level of transcriptional drift and time profile of expression) on the lifespan of worms will be assessed. 1. Rea, S.L., et al., Nature Genetics, 2005. 37(8): p. 894-898.

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.

Hariri S et al. (2023) MicroPubl Biol "53bp1 mutation enhances brca1 and bard1 embryonic lethality in C. elegans."

Engebrecht J, Hariri S, Li Q

[MicroPubl Biol, 2023]

In mice, mutation of <i>brca1</i> results in embryonic lethality, which is partially suppressed by <i>53bp1</i> mutation. In contrast, mutation of the <i>C. elegans</i> BRCA1 ortholog, <i>brc-1 ,</i> or its binding partner, <i>brd-1</i> , lead to only mild embryonic lethality. We show that in <i>C. elegans</i> , <i>brc-1</i> and <i>brd-1</i> embryonic lethality is enhanced when <i>53bp1</i> ortholog, <i>hsr-9</i> , is also mutated. This is not a consequence of activating <i>polq-1</i> -dependent microhomology-mediated end joining, as <i>polq-1</i> mutation does not suppress embryonic lethality of <i>hsr-9 ; brc-1</i> mutants. Together, these results suggest that BRC-1 - BRD-1 and HSR-9 function in parallel pathways and do not act antagonistically as in mammals.