Bauer, Rosemary et al. MicroPubl Biol

Golden, Andy, Bauer, Rosemary, Nebenfuehr, Benjamin

[MicroPubl Biol, 2019]

KQT-3 is the C. elegansortholog of human KCNQ1, a voltage-gated potassium channel that is implicated in multiple types of Long Q-T cardiac arrhythmias including Jervell and Lange-Nielsen syndrome and Romano-Ward syndrome. In order to assess the possibility of modeling these syndromes in C. elegans, we obtained the tm542deletion allele, which had previously been associated with changes to the defecation cycle (Nehrke et al., 2008, Kwan et al. 2008). Any visible, easily-scored phenotype would be an indication that we could move forward with creating patient-specific missense alleles by CRISPR. Thetm542deletion, a 1001bp deletion generated by chemical mutagenesis that disrupts exons 2-4, appeared to cause a dramatic reduction in pharyngeal pump frequency (Fig 1B). This was an indication that mutations to kqt-3 might be a reasonable way to model Long Q-T arrhythmias in worms. When three patient missense mutations and two smaller deletions yielded only very subtle changes to pharyngeal pumping, we became increasingly suspicious of the tm542deletion strain. This prompted the creation of a full-length, 9004bp deletion allele by CRISPR that starts 69bp upstream of the start codon, ends 66bp after the stop codon, and inserts stop codons in every frame. Surprisingly, complete deletion of this 621 amino acid protein led to virtually no change in pharyngeal pumping (Fig 1B-D). In order to confirm that the pharyngeal pumping phenotype in the kqt-3(tm542) strain was due to extraneous mutations and not a dominant negative effect, we created the full-length deletion of kqt-3by CRISPR in the tm542 strain. This kqt-3(tm542av187) deletion strain phenocopied the original tm542deletion (Fig 1B-D), indicating that other mutations besides the deletion in kqt-3are responsible for the lack of pharyngeal pumping in the tm542strain. This study should serve as a cautionary tale for any lab studying a deletion allele that was generated by chemical mutagenesis; the strain likely contains numerous other mutations. We now routinely generate a full gene deletion by CRISPR as part of any genetic study.

Bauer, Rosemary et al. (2020) MicroPubl Biol

Golden, Andy, Bauer, Rosemary

[MicroPubl Biol, 2020]

Genetic balancer strains are frequently used to maintain populations of C. elegans mutants that would otherwise die as homozygotes. Additionally, fluorescently-marked strains can be used to identify cross-progeny for use in assays where heterozygotes are of particular interest. In our case, we wanted to utilize a fluorescently-marked but otherwise wildtype strain to obtain marked cross-progeny from a mating. Our mutant of interest displayed altered pharyngeal pumping, so we wanted to assess heterozygotes in the same assay and establish the recessive nature of the phenotype. We happened to have the TY4236 strain growing in the lab, which not only expresses bright pharyngeal and gut GFP from the mIs10 insertion but also contains him-8(e1489), making the experiment quite simple. Due to the bright GFP signal in the pharynx we decided to assess the pharyngeal pumping of the strain itself before using it as a marker in an experiment that heavily implicates the pharynx. To our surprise, this otherwise-healthy strain displayed highly variable pharyngeal pump frequency, decreased average pump duration, and increased average inter-pump interval (IPI) when compared to N2 (Fig 1A-C). This prompted us to test multiple other options, including some that also express GFP in the pharynx (tmC5::Venus, hT2::gfp, nT1::gfp) as well as two that dont [myo-3p::gfp::GFP(mit), tdTomato::H2B]. Two balancers with bright pharyngeal GFP, hT2::gfp and nT1::gfp, displayed moderate pumping phenotypes even as heterozygotes (Fig 1A-C). Interestingly, the pharyngeal pumping phenotypes do not correlate exactly with fluorescence localization; tmC5::Venus was completely unaffected and myo-3p::gfp(mit) myo-3::GFP(mito) had slightly but significantly reduced mean pump duration (Fig 1B). Besides N2, each strain was only assayed once and therefore these results should be treated as preliminary. Additionally, since these strains were all created in different labs across the world and in some cases across the span of many years, it is possible that the pumping phenotype is due, in part, to the genetic background of the strain and not entirely due to the insertion of a fluorescent tag. Pharyngeal pumping phenotypes can indicate upstream nervous system or muscular alterations and can lead to downstream outcomes like altered metabolism and lifespan (Raizen et al., 1995). Regardless of the experimenters interest in characterizing pharyngeal pumping, these fluorescent strains should be assessed for possible subtle phenotypes that could potentially confound the main phenotype of interest.

Lanzerstorfer P et al. (2020) Arch Toxicol "Acute, reproductive, and developmental toxicity of essential oils assessed with alternative in vitro and in vivo systems."

Weghuber J, Aumiller T, Mascher B, Sandner G, Lanzerstorfer P, Pitsch J

[Arch Toxicol, 2020]

Essential oils (EOs) have attracted increased interest for different applications such as food preservatives, feed additives and ingredients in cosmetics. Due to their reported variable composition of components, they might be acutely toxic to humans and animals in small amounts. Despite the necessity, rigorous toxicity testing in terms of safety evaluation has not been reported so far, especially using alternatives to animal models. Here, we provide a strategy by use of alternative in vitro (cell cultures) and in vivo (Caenorhabditis elegans, hen's egg test) approaches for detailed investigation of the impact of commonly used rosemary, citrus and eucalyptus essential oil on acute, developmental and reproductive toxicity as well as on mucous membrane irritation. In general, all EOs under study exhibited a comparable impact on measured parameters, with a slightly increased toxic potential of rosemary oil. In vitro cell culture results indicated a concentration-dependent decrease of cell viability for all EOs, with mean IC₅₀ values ranging from 0.08 to 0.17% [v/v]. Similar results were obtained for the C. elegans model when using a sensitized bus-5 mutant strain, with a mean LC₅₀ value of 0.42% [v/v]. In wild-type nematodes, approximately tenfold higher LC₅₀ values were detected. C. elegans development and reproduction was already significantly inhibited at concentrations of 0.5% (wild-type) and 0.1% (bus-5) [v/v] of EO, respectively. Gene expression analysis revealed a significant upregulation of xenobiotic and oxidative stress genes such as cyp-14a3, gst-4, gpx-6 and sod-3. Furthermore, all three EOs under study showed an increased short-time mucous membrane irritation potential, already at 0.5% [v/v] of EO. Finally, GC-MS analysis was performed to quantitate the relative concentration of the most prominent EO compounds. In conclusion, our results demonstrate that EOs can exhibit severe toxic properties, already at low concentrations. Therefore, a detailed toxicological assessment is highly recommended for each EO and single intended application.

Moliner C et al. (2020) Antioxidants (Basel) "Rosemary Flowers as Edible Plant Foods: Phenolic Composition and Antioxidant Properties in Caenorhabditis elegans."

Moliner C, Langa E, Barros L, Gomez-Rincon C, Ferreira ICFR, Lopez V, Dias MI

[Antioxidants (Basel), 2020]

<i>Rosmarinus officinalis</i> L., commonly known as rosemary, has been largely studied for its wide use as food ingredient and medicinal plant; less attention has been given to its edible flowers, being necessary to evaluate their potential as functional foods or nutraceuticals. To achieve that, the phenolic profile of the ethanolic extract of <i>R. officinalis</i> flowers was determined using LC-DAD-ESI/MSn and then its antioxidant and anti-ageing potential was studied through in vitro and in vivo assays using <i>Caenorhabditis elegans</i>. The phenolic content was 14.3 +/- 0.1 mg/g extract, <i>trans</i> rosmarinic acid being the predominant compound in the extract, which also exhibited a strong antioxidant capacity in vitro and increased the survival rate of <i>C. elegans</i> exposed to lethal oxidative stress. Moreover, <i>R. officinalis</i> flowers extended <i>C. elegans</i> lifespan up to 18%. Therefore, these findings support the potential use of <i>R. officinalis</i> flowers as ingredients to develop products with pharmaceutical and/or nutraceutical potential.

Lin CY et al. (2016) Neuropharmacology "Carnosic acid protects SH-SY5Y cells against 6-hydroxydopamine-induced cell death through upregulation of parkin pathway."

Lin CY, Tsai CW

[Neuropharmacology, 2016]

Parkin is a Parkinson's disease (PD)-linked gene that plays an important role in the ubiquitin-proteasome system (UPS). This study explored whether carnosic acid (CA) from rosemary protects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via upregulation of parkin in vivo and in vitro. We found that the reduction in proteasomal activity by 6-OHDA was attenuated in cells pretreated with 1M CA. Immunoblots showed that CA reversed the induction of ubiquitinated protein and the reduction of PTEN-induced putative kinase 1 (PINK1) and parkin protein in 6-OHDA-treated SH-SY5Y cells and rats. Moreover, in a transgenic OW13 Caenorhabditis elegans model of PD that expresses human -synuclein in muscle cells, CA reduced -synuclein accumulation in a dose-dependent manner. In cells pretreated with the proteasome inhibitor MG132, CA no longer reversed the 6-OHDA-mediated induction of cleavage of caspase 3 and poly (ADP)-ribose polymerase and no longer reversed the suppression of proteasome activity. When parkin expression was silenced by use of small inhibitory RNA, the ability of CA to inhibit apoptosis and induce proteasomal activity was significantly reduced. The reduction in 6-OHDA-induced neurotoxicity by CA was associated with the induction of parkin, which in turn upregulated the UPS and then decreased cell death.

Sundaram P et al. (2006) Mol Biol Cell "ATP-binding cassette transporters are required for efficient RNA interference in Caenorhabditis elegans."

Sundaram P, Han W, Echalier B, Hull D, Timmons L

[Mol Biol Cell, 2006]

Monitoring Editor: Marvin P. Wickens RNA interference (RNAi) is a conserved gene-silencing phenomenon that can be triggered by delivery of double-stranded RNA (dsRNA) to cells and is a widely exploited technology in analyses of gene function. While a number of proteins that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here we report that the Caenorhabditis elegans gene haf-6 is required for efficient RNAi. HAF-6 is a member of the ATP binding Cassette (ABC) transporter gene superfamily. ABC transporters utilize ATP to translocate small molecule substrates across the membranes in which they reside, often against a steep concentration gradient. Collectively, ABC transporters are involved in a variety of activities, including protective or barrier mechanisms that export drugs or toxins from cells (Broeks et al., 1996; Bauer et al., 1999; Begley, 2004; Fromm, 2004), in organellar biogenesis (Aubourg, 1994), and in mechanisms that protect against viral infection (Trowsdale et al., 1990; Abele and Tampe, 2004). HAF-6 is expressed predominantly in the intestine and germline and is localized to intracellular reticular organelles. We further demonstrate that nine additional ABC genes from diverse subfamilies are each required for efficient RNAi in C. elegans. Thus, the ability to mount a robust RNAi response to dsRNA depends upon the deployment of two ancient systems that respond to environmental assaults: RNAi mechanisms and membrane transport systems that utilize ABC proteins.

Kumar S et al. Indian J Med Microbiol "In vitro and in vivo fitness of clinical isolates of carbapenem-resistant and -susceptible Acinetobacter baumannii."

Gautam V, Kumar S, Ray P, Singhal L

[Indian J Med Microbiol]

Context: Acinetobacter baumannii is one among the leading nosocomial pathogens in the healthcare settings worldwide. Limited data on relative fitness and virulence of carbapenem-resistant A. baumannii (CRAB) are known. New methods are required to curb the rapidly rising antimicrobial resistance of this bug. Aims: We aimed to study the comparative in vitro and in vivo fitness of clinical isolates of CRAB and carbapenem-susceptible A. baumannii (CSAB). Settings and Design: A total of nine A. baumannii isolates were included in this study. CSAB ATCC-19606 was taken as a reference control strain. Subjects and Methods: PCR were used for species identification. Antimicrobial susceptibility was performed using Kirby-Bauer disk-diffusion method. Minimum inhibitory concentration for carbapenems (imipenem, meropenem and doripenem) was determined using agar dilution method. End point analysis, competitive index (CI), growth kinetics and generation time were determined for CRAB and CSAB isolates. In vivo fitness of CRAB and CSAB was determined using Caenorhabditis elegans host model. Multilocus sequence typing was performed to see the genetic relatedness of the isolates under study. Results: End point analysis, in vitro CI and growth kinetics experiments showed better fitness of clinical isolates of CRAB over CSAB ones. In vivo'nematode fertility assay' using C. elegans also supported the in vitro results. Conclusions: To the best of our knowledge, this is the first study of its kind from India showing difference in fitness of clinical isolates of CRAB and CSAB.

Yao H et al. (2022) Front Pharmacol "Evaluation of in vivo antibacterial drug efficacy using Caenorhabditis elegans infected with carbapenem-resistant Klebsiella pneumoniae as a model host"

Liu J, Yao H, Chen J, Wang F, Xu A

[Front Pharmacol, 2022]

Objective: This study was developed to assess the in vivo antimicrobial activity of specific drugs using a model system consisting of Caenorhabditis elegans (C. elegans) infected with Carbapenem-resistant Klebsiella pneumoniae (CRKP) in an effort to identify promising drugs for CRKP-infected patient treatment. Methods: A C. elegans-CRKP liquid assay platform was developed and used to conduct limited in vivo screening for antimicrobial agents with potential activity against CRKP. Time curves for 10 different concentrations of tested antimicrobial agents were tested in this model system at 0, 2, 6, 8, and 12 h after treatment. The protective effects of these different antimicrobial agents were compared at different time points. Furthermore, ten CRKP strains samples were isolated from clinical specimens to demonstrate the applicability of the nematode model method, and two typical clinical cases are presented. Results: CRKP bacteria were sufficient to induce C. elegans death in a dose- and time-dependent fashion, while effective antimicrobial agents improved the survival of these nematodes in a dose-dependent manner. Notably, PB and TGC exhibited robust antibacterial protection within 12 h even at low tested concentrations, and clear efficacy remained evident for high doses of CAZ at this same time point as mediators of improved nematode survival. The results of C. elegans model method were well consistent with that using the Kirby-Bauer method in 10 CRKP strains samples, and two typical clinical cases showed applicability, reliability and efficacy of C. elegans model method. Conclusion: Overall, nematode models in drug sensitivity testing have shown advantages in clinical settings. Our results highlight the value of C. elegans model systems as tools for the simultaneous screening of different agents for in vivo antibacterial efficacy and are deserved further study.