Adeyemi, Kayode et al. (2021) International Worm Meeting "Isolation, Characterization, and Antibiotic Resistance Profile of Staphylococci from the Indoor Air of the Students' Halls of Residence at the Obafemi Awolowo University, Ile Ife, Nigeria"

Ezekwem, Favour, Babawale, Omolayo, Adeyemi, Kayode

[International Worm Meeting, 2021]

It has become a public health concern to evaluate the safety of the only readily consumable and unfiltered part of the biosphere, the air, most especially from the densely populated region like the students' hostel. This study was conducted to investigate the colony-forming unit per unit volume of staphylococci in the indoor air of students' halls of residence, the biochemical characteristics, and antibiotic resistance profile of the isolates. Using the open-plate method to collect samples from the indoor air as described by Omeliansky, identification of the isolates was based on standard methods such as colonial morphology, Gram stain reaction, catalase, and deoxyribonuclease tests. The antibiotic susceptibility profile was on the standard disk diffusion method using commercially available antibiotic discs (Abtek, Habdiscs) including ciprofloxacin 5 mug/mL, gentamicin 10 mug/mL, and tetracycline 30 mug/mL. A total of 26 staphylococci isolates were obtained from which none (0%), 4 (15%), and 10 (37%) of the isolates were resistant to ciprofloxacin, gentamicin, and tetracycline, respectively with no intermediate. Using a mathematical model, it is deduced that the year of release of the antibiotics has a non-linear correlation with the recorded rate of resistance of the staphylococci to the antibiotics, which predicts an efficacy duration window of 104 years for each of the antibiotics. These findings predict a future increasing pattern of antimicrobial resistance, which may lead to serious public health threats if antimicrobial stewardship measures are not put in place.

Morten K Larsen et al. (2005) International Worm Meeting "MAA-1, a novel acyl-CoA binding protein involved in vesicle transport in C. elegans"

Nils Fargeman, Jens Knudsen, Simon Tuck, Morten K Larsen

[International Worm Meeting, 2005]

Membrane trafficking between cellular compartments is a complex process involving a variety of factors including coat proteins, small GTPases, adaptors and acyl-CoA. The lipid modifying accessory proteins CtBP/BARS and endophilin, that use acyl-CoA as substrate, are also implicated. Here we present evidence that a novel, membrane-associated acyl-CoA binding protein, MAA-1, functions in maintaining normal morphology of the endosomal recycling compartments in C. elegans. Mutants lacking maa-1 activity, accumulate multiple protrusions from the endosomal recycling compartment consistent with a requirement for vesicle fusion or fission. MAA-1 binds acyl-CoA in vitro and the function is dependent on its acyl-CoA binding ability. We have also investigated the involvement of the homologues of endophilin B, erp-1, and CtBP/BARS, ctbp-1, in vesicular trafficking. Mutants lacking erp-1 and ctbp-1 display a mildly uncoordinated phenotype and furthermore, like maa-1, show defects in endosomal morphology. These results taken together favour a model, in which MAA-1 functions together with either ERP-1 or CTBP-1 during vesicle biogenesis.

Roubin R et al. (2000) European Worm Meeting "Expression of VER-1 (Vascular Endothelial growth factor receptor Related) in amphid and phasmid sheath cells"

Popovici C, Roubin R, Birnbaum D

[European Worm Meeting, 2000]

An overall analysis of Tyrosine Kinase Receptors (RTKs) encoded by the C. elegansgenome has unveiled a family of four RTKs structurally related to Vascular Endothelial Growth Factor Receptors (VEGFRs) (Popovici et al, 1999). These genes are carried by the F59F3 and T17A3 cosmids. One EST for the gene F59F3.1 already existed in the Yugi Kohara's collection, and we were able to amplify two others (F59F3.5 and T17A3.1) by RT-PCR. Gene silencing done by RNAi on 2 of them indicated that these genes were not redundant. We focused on the T17A3.1 gene that we tentatively named ver-1 and studied its pattern of expression using a GFP fusion reporter gene construct. Ver-1::gfp expression was found mainly in amphid and phasmid sheath cells which have glial cells features. To overcome the absence of mutants, we made a construct devoid of the transmembrane and tyrosine kinase domains which was expected, when injected into wild type animals, to play a role as a soluble receptor for endogenous ligands. Overexpression of this soluble receptor led to less fertile animals than wild type, laying few eggs which often stop developing as oocytes. Surviving adults exhibited morphologic and functional alterations in their sheath cells and chemosensory neurons. When this construct was coinjected with ver-1::gfp, the sheath cells did not fluoresce anymore and their ability to be stained with ANEPS (a dye which preferentially colours sheath cells) was lost. In addition, the dye-filling of the chemosensory neurons was affected. We observed a posterior displacement of the lipophilic DiO filled amphid neurons and the appearance of additional neurons in the middle of the isthmus capable to be filled with the dye. The lack of ANEPS staining associated with the inability of visualising GFP positive cells are in favour of a loss of the sheath cell integrity in these animals. These features, related to the modifications in the dye-filling of chemosensorial neurons, are in favour of VER-1 playing a role in guidance and/or migration of sensory neurons. Thus, as already described in mammals for another related RTK family with 5 instead of 7 immunoglobulin domains (Platelet Derived Growth Factor Receptors, PDGFRs), VER-1 is produced by glial cells and may play a role in neurogenesis. It is one of the first observations in invertebrates of angiogenesis being related to neurogenesis. Studying VEGFR like in C. elegans, an animal devoid of a circulatory blood system, may help understand VEGFR function in mammals. Popovici C., Roubin R., Coulier F., Pontarotti P., Birnbaum D. (1999) - The Family of C. elegansTyrosine Kinase Receptors: Similarities and differences with Mammalian Receptors. Genome Res 9:1026-1039

Hannes Lans et al. (2001) International C. elegans Meeting "Olfactory G-protein signalling in C. elegans"

Gert Jansen, Hannes Lans

[International C. elegans Meeting, 2001]

We study signal transduction via heterotrimeric G-proteins. C. elegans has 21 G alpha, 2 G beta and 2 G gamma subunits. At least 14 of the G alpha-subunits are involved in sensory signalling in the amphid neurons. With these neurons, C. elegans can sense chemicals from its environment. Two pairs of these neurons, AWA and AWC, can detect and discriminate among many attractive odorants. Some odorants are detected by AWA or AWC only, others can be detected by both. Analysis of GFP-fusion expression patterns and mutant phenotypes indicated that six G alpha subunits play a role in AWA and AWC. These are ODR-3, GPA-2,-3,-5,-6 and -13. ODR-3 provides the main stimulatory signal. The other subunits have redundant stimulatory or inhibitory roles, depending on the odorant. The interactions between the G alpha subunits probably contribute to the ability of the animal to discriminate among odorants. We intend to determine how these different G alpha subunits can function in one cell. We use a forward genetic screen to identify novel components of the GPA-5 signalling cascade. To this end, we make use of the fact that odr-3 mutants are defective in the detection of the odorant diacetyl. This defect, however, is suppressed upon inactivation of the negative regulator GPA-5. Following mutagenization of odr-3 mutants, we can therefore screen for animals that have regained their ability to detect diacetyl. In this way we expect to identify novel components of the GPA-5 signalling cascade. To increase the efficiency of our screen, we use 2,3-pentanedion. odr-3 mutants favour this odorant over diacetyl, while gpa-5,odr-3 double mutants do not. In this way we enrich our mutant population significantly.

Rudolph C et al. (2000) European Worm Meeting "Isolation of Caenorhabditis elegans deletion mutants by a high-throughput method"

Tovar K, Weidner G, Baumeister R, Angelova M, Honer M, Rudolph C

[European Worm Meeting, 2000]

The nematode Caenorhabditis elegans is the first animal whose genome is completely sequenced. Next steps in understanding basic principles of life and diseases are the functional analysis of genes and their interaction. A very elegant way is to create specific knock-out strains in C. elegans, followed by an analysis of the resulting phenotypes. These knock-out strains can then be used to build up model-systems representing individual diseases. The broad sequence information allows deletion mutagenesis of any desired specific target-gene. Instead of the transposon insertion and excision method to create deletion mutants, chemical mutagens were used recently. However, this chemical mutagenesis was so far not appropriate for large-scale isolation of deletion mutants. We scaled up this method and are now able to screen simultaneously 4x106 genomes for deletion mutants by PCR following UV-TMP mutagenesis. For this purpose we substantially simplified the procedures and transformed all steps into 96-well microtiterplates, which involves growth of mutagenized animals and PCR screening in 96-well-form. As our mutagenesis conditions favour deletions of 1500 bp on average, a screening window of 3,2 kb is used. Three people in the lab are now able to generate and isolate a total of 6 mutants per month. At present we are further upscaling our technology. Our preliminary results from currently 16 knock-outs that we have analyzed in greater details indicate that 80% of these mutants display a characteristic phenotype. We do not know whether this is a consequence of the particular genes we selected or whether we apply a more thorough phenotypic analysis on these mutants than others. So far, we were able to isolate a mutant for every gene we addressed. We are therefore confident that our procedure allows the rapid deletion of basically every gene in the C. elegansgenome.

Peres, Tanara et al. (2021) International Worm Meeting "Nutrient-induced rewiring of microbial metabolic pathways modulate 5-fluorouracil efficacy in C. elegans"

Zimmermman, Johannes, Kaleta, Christoph, Chrysostomou, Despoina, Marchesi, Julian, W. Zatorska, Michalina, Barr, Alexis, Quintaneiro, Leonor, Cabreiro, Filipe, Martinez-Martinez, Daniel, Peres, Tanara, Kramer, Holger, Best, Lena, Marinos, Georgios, B. Mokochinski, Joao

[International Worm Meeting, 2021]

Microbiota derived metabolites have been shown to influence cancer susceptibility, tumour progression and therapy outcome. Diet is a major environmental factor influencing gut microbiota composition, dictating the abundance of potential oncometabolites. The complex interactions between diet and the microbiota need to be clarified so that dietary intake may be shaped to influence disease outcome. Having previously demonstrated the impact of bacterial metabolism in 5-fluorouracil (5-FU) cancer drug efficacy, we set out to investigate the role of nutrition in host-microbe responses to this drug, an antimetabolite widely used for colorectal cancer (CRC) chemotherapy. Host-microbe-drug-nutrient interaction and its influence on drug efficacy were assessed using a high-throughput 4-way screening approach. To understand gene-nutrient interactions at the bacterial level that regulate the effect of a drug on host physiology, genetically modified E. coli from the Keio deletion library were used to probe the role of bacterial metabolic pathways in 5-FU effects in a nutrient-dependent manner. We found that glycolytic nutrients, including sugars, importantly antagonise the toxic effects of 5-FU action on the host. Sugars induce a shift in metabolic pathways that favour the production of nucleotides, specifically UMP and UTP derived from the activation of pyrimidine de novo biosynthesis pathway in bacteria, counteracting pro-drug activation through the salvage pathway. We also found that bacterial mutations in the TCA cycle and pyruvate metabolism decrease drug efficacy in amino acid-based media. A combination of bacterial genetic work and metabolomics has revealed the presence of a novel bacterial metabolite with the capacity to increase 5-FU drug efficacy in both C. elegans and human cancer cells. Importantly, flux balance analysis of the cancer associated microbiota predicts an increased production of this metabolite in several organs. In particular, investigations of the gut microbiome from four independent human CRC cohorts shows an increased production of this metabolite, compared to healthy patients, further implicating this metabolite in drug cancer therapy. These findings highlight the potential of manipulating gut microbiota through diet to improve cancer therapy outcome.

Lionel Pintard et al. (2002) European Worm Meeting "Regulation of Cul-3 Neddylation is essential for SCFCul-3 activity during mitosis in C. elegans"

Sarah Glaser, John Willis, Matthias Peter, Lionel Pintard, Thimo Kurz, Bruce Bowerman

[European Worm Meeting, 2002]

Several cell cycle transitions are regulated by ubiquitin-dependent degradation of specific proteins. This is achieved by a multi-step process whereby ubiquitin is first activated by an ubiquitin-activating enzyme (E1), then passed to an ubiquitin-conjugating enzyme (E2) before being transferred to the target protein by the ubiquitin-ligase (E3). Two types of E3 control cell cycle progression: the APC (Anaphase Promoting Complex) and the SCF (Skp1-Cullin-F-box). The E3 displays substrate specificity and recruits the E2 allowing poly-ubiquitinylation of the substrate and its subsequent degradation by the 26S proteasome. However, little is known about the regulation of E3 ligases. Within the SCF complex, the Cullin subunit was found to be neddylated. In a cascade of reactions similar to ubiquitination, the ubiquitin-like protein Nedd8 is transferred to the Cullin and it is thought that neddylation positively regulates SCF activity by triggering the recruitment of the E2 within the SCF complex. We recently discovered the Nedd8 pathway in C. elegans which is required for activation of the SCFCul-3 complex and subsequent degradation of the Katanin-like protein Mei-1 at the meiosis to mitosis transition. Our results showed that neddylation of Cul-3 is essential for SCFCul-3 activity in vivo. Indeed, Western blot experiments allowed us to confirm that Cul-3 is neddylated in vivo in wildtype but not in a mutant of the neddylation pathway (rfl-1). To better understand the role of neddylation in the regulation of SCF activity, we searched for mutants that were defective in deneddylation. The multiprotein complex COP9/signalosome was found to contain such a deneddylase activity. The COP9/signalosome, originally identified in A. thaliana as a repressor of photomorphogenesis, is widely conserved in metazoans. Searches in the databases allowed us to identify six subunits with significant homology in C. elegans. Interestingly inactivation of these subunits by RNAi leads to an accumulation of the neddylated form of Cul-3 and inhibition of the SCFCul-3 activity. As a result, the SCFCul-3 substrate Mei-1 is not degraded and severs the microtubules during mitosis. Most importantly we found that the lethality observed in neddylation deficient mutant (rf1-1) grown at semi-permissive temperature can be suppressed by the inactivation of deneddylation through RNAi of the signalosome subunits. Our results are in favour of a model where cycles of neddylation-deneddylation are essential for activity of SCF complexes.