Bauer, Jack et al. (2021) MicroPubl Biol

Labb, Jean-Claude, Lacroix, La, Bauer, Jack

[MicroPubl Biol, 2021]

To perturb actomyosin function in the primordial germ line, we first monitored germ line organization in L1-stage animals bearing temperature-sensitive (ts) alleles in genes encoding actomyosin regulators and that were reported to interfere with cytokinesis during embryogenesis (Davies et al. 2014). Previous work demonstrated that the initial stages of germline expansion occur normally in cyk-4(ts) and zen-4(ts) animals raised at restrictive temperature from the L1 stage (Lee et al. 2018). We found that primordial germ line organization in cyk-1(ts), nmy-2(ts), cyk-4(ts) or zen-4(ts) L1 larvae maintained at restrictive temperature for 12h was no different than control (Figure 1A-B). Furthermore, the first primordial germ cell (PGC) division occurred normally upon feeding these animals at restrictive temperature with typical bacterial food (E. coli OP50). As noted previously (Lee et al. 2018), germ line disorganization and sterility were observed in all cases when animals reached adulthood (Figure 1B).

Bauer, Jack et al. (2021) International Worm Meeting "Cytokinesis incompletion drives the initial expansion of the C. elegans syncytial germline"

Nguyen, Ken C. Q., Hall, David H., Poupart, Vincent, Bauer, Jack, Labbe, Jean-Claude

[International Worm Meeting, 2021]

While syncytial architectures are common among animal germlines and required for fertility, the mechanisms leading to syncytium formation and expansion remain largely unknown. C. elegans constitutes a powerful in vivo model to study syncytium regulation throughout development. The adult germline is organized as a syncytium in which each germ cell possesses an intercellular bridge that is maintained by a stable actomyosin ring and that connects it to a common pool of cytoplasm, termed the rachis. How germ cells undergo cytokinesis while maintaining this syncytial architecture is not completely understood. We characterized the organization of the primordial germ cells (PGCs) in C. elegans first stage larvae and studied the first PGC division to better understand how the syncytial structure expands. Using confocal and electron microscopy, we found that each PGCs possesses a stable intercellular bridge that connects it to a common pool of cytoplasm, which we term the proto-rachis. We found that as in the adult syncytium these intercellular bridges are stabilized by actomyosin rings and enable cytoplasmic exchange between the germ cells. These results indicate that the organization of the primordial germline in first stage larvae is fundamentally the same as in adult animals. Using live imaging and fluorescence photobleaching approaches, we further found that the first PGC cytokinesis is incomplete and that the stabilized cytokinetic ring progressively moves toward the proto-rachis and eventually integrates into it. In addition, we found that cytoplasmic exchange maintained as PGCs undergo cytokinesis, indicating that the connection to the proto-rachis remains effective during PGC division. Our results support a model in which the initial expansion of the C. elegans syncytial germline occurs by incomplete cytokinesis, where one daughter germ cell inherits the actomyosin ring that was newly formed by stabilization of the cytokinetic ring, while the other inherits the pre-existing stable actomyosin ring. We propose that such mechanism of iterative cytokinesis incompletion underpins C. elegans germline expansion and maintenance.

Takeuchi T et al. (2011) Biol Pharm Bull "-galactosidases from jack bean and Streptococcus have different cleaving abilities towards fucose-containing sugars."

Kasai K, Natsuka S, Takeuchi T, Arata Y, Sugiura K, Nishiyama K, Takahashi H, Natsugari H

[Biol Pharm Bull, 2011]

We examined the sugar-cleaving abilities of -galactosidases from jack bean and Streptococcus towards sugars containing fucose residues, and found that jack bean -galactosidase has an ability to cleave the 1-3 linkage between galactose (Gal) and fucose (Fuc) residues, but not 1-4 linkage. On the other hand, streptococcal -galactosidase was found to cleave the linkage in both Gal1-4Fuc and Gal1-3Fuc disaccharide units. Such a difference in sugar-cleaving abilities between these 2 -galactosidases will be useful for structural analysis of glycans, especially those from species belonging to Protostomia, such as Caenorhabditis elegans.

Dosoky NS et al. (2016) Medicines (Basel) "Composition and Biological Activities of Murraya paniculata (L.) Jack Essential Oil from Nepal."

Satyal P, Setzer WN, Gautam TP, Dosoky NS

[Medicines (Basel), 2016]

Murraya paniculata (L.) Jack, a small tropical evergreen shrub growing in Nepal, has numerous uses in traditional medicine for treatment of abdominal pain, diarrhea, stomach ache, headache, edema, thrombosis, and blood stasis. The present study investigated the chemical composition and bioactivities of the leaf essential oil from M. paniculata from Nepal. The essential oil from leaves was obtained by hydrodistillation and a detailed chemical analysis was conducted by gas chromatography-mass spectrometry (GC-MS). The essential oil was screened for antimicrobial activity using the microbroth dilution test, for nematicidal activity against Caenorhabditis elegans, and for lethality against brine shrimp (Artemia salina). A total of 76 volatile components were identified from the essential oil. The major components were methyl palmitate (11.1%), isospathulenol (9.4%), (E,E)-geranyl linalool (5.3%), benzyl benzoate (4.2%), selin-6-en-4-ol (4.0%), -caryophyllene (4.0%), germacrene B (3.6%), germacrene D (3.4%), and -elemene (3.2%). The essential oil showed no antibacterial activity, marginal antifungal activity against Aspergillus niger (MIC = 313 g/mL), a moderate activity against A. salina (LC50 = 41 g/mL), and a good nematicidal activity against C. elegans (LC50 = 37 g/mL).

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.

Johnson TE (1980) Worm Breeder's Gazette "Generation of X-linked Mosaics"

Johnson TE

[Worm Breeder's Gazette, 1980]

We are investigating somatic loss of X-chromosomes as a means of producing mosaic worms at relatively high frequencies. We produce worms heterozygous for the X-linked muscle morphology mutant, unc-78( e1217), which are homozygous for various him's. Only one such mutant, him-2(e1065), generates spontaneous mosaics at a frequency greater than 0.1%. We are still examining two other lines and are exploring other means of inducing high-frequencies of X-linked mosaics . We have observed loss of X-chromosomes from worms irradiated by X- rays from 137Cs. The loss is seen in the F2 of irradiated worms (or the F1 or irradiated embryos). The number of males in this generation is in the range of 4-10%. The frequency of males is not distributed according to a Poisson but rather shows 'jack-pots' as might be expected from events that hit individual X chromosomes. The frequency of males in a jackpot can be as high as 40%. Most hermaphrodite siblings of these males are normal and show no increase in the frequency of male progeny. We asked whether only the irradiated chromosome was lost in an animal heterozygous for a marked irradiated X-chromosome and an unmarked unirradiated X-chromosome. Hermaphrodites marked with the X- linked marker, unc-78, were irradiated and then mated to N2 males. Wild type progeny were picked, cloned and scored for male offspring. Both wild type and unc-78 males were found at approximately equal frequency; apparently either chromosome can be lost.

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.

Saikat Mukhopadhyay et al. (2004) East Coast Worm Meeting "Identification of genes regulating chemosensory neuron-specific morphologies"

Anne Lanjuin, Saikat Mukhopadhyay, Piali Sengupta

[East Coast Worm Meeting, 2004]

Amphid chemosensory neurons sense the external environment via their non-motile cilia, which are specialized for the particular neuron type. For example, AWB has characteristic forked cilia, while AWC has fan-like cilia. How are these structures specified and maintained? FKH-2, a forkhead domain transcription factor is expressed widely in the embryo (Molin et al., 2000) but expression is restricted to the AWB, ASK and ASI amphid neurons in larval stages and adults. In fkh-2 mutants, the ciliary structures of the AWB and AWC neurons but not other neuron types are affected. Dendritic development of AWB and AWC neurons is also affected in fkh-2 mutants, and this effect is manifested as stunting of the dendritic processes as early as the first larval stages. FKH-2 expression in the amphid neurons in early larval stages is regulated by DAF-19, an RFX-type transcription factor regulating general cilia development in all sensory neurons. However, the expression of fkh-2 is partly restored in daf-19 dauers and adults. It is possible that daf-19 could be regulating fkh-2 to determine cell-specific cilia morphology in AWB in addition to its general function in cilia formation. FKH-2 also acts in parallel with the OTX family homeodomain protein CEH-37 to specify the subtype identities of the AWB and ADF sensory neurons. Currently we are screening for additional mutants exhibiting altered cilia morphology specifically in the AWB neurons in order to characterize cell-specific factors necessary for their specialized cilia structure. We are also investigating the role of neuronal activity in the maintenance of ciliary structure. References: Molin L., Mounsey. A., Aslam, S., Bauer, P., Young, J., James, M., Sharma-Oates, A., Hope, I. (2000) Development 127, 4825-4835.

Motran, Lina et al. (2021) International Worm Meeting "Dynamics of nicotine-induced neuroprotection and the ACR-20 receptor"

Cohen Ben-Ami, Hagit, Motran, Lina, Treinin, Millet

[International Worm Meeting, 2021]

Epidemiological studies show that tobacco smoking reduces the prevalence of Parkinson's disease. One explanation for these findings is that tobacco-derived nicotine protects substantia nigra dopaminergic neurons (DNs) from degeneration. To examine this possibility we established, in collaborated with the lab of Guy Caldwell (University of Alabama), a C. elegans model for nicotine-induced protection of DNs. Using this model we have shown that chronic nicotine exposure protects DNs against 6-OHDA toxicity via a signaling pathway involving activation of mitochondrial stress. We also identified four nAChR subunits needed for nicotine-induced protection of DNs (Nourse et al., iScience 2021). ACR-20, one of these "protective" nAChR subunits, was previously shown to form a homomeric receptor (Bauer et al., Mol. Pharmacol. 2014). To examine whether nicotine-induced protection depends on the ACR-20 homomeric receptor we examined the effects of betaine, an agonist of this receptor. We find that chronic betaine exposure like chronic nicotine exposure protects DNs in a nAChR-dependent manner. Protective effects of betaine are, however, distinct from the protective effects of nicotine; pre-exposure to betaine, unlike similar nicotine exposure, is not protective; while, exposure to betaine during and after 6-OHDA treatment, like similar nicotine exposure, is protective. The protective effects of betaine and the differences in their dynamics, relative to the protective effects of nicotine, are consistent with two distinct nAChRs functioning to protect DNs. In addition, differences between effects of nicotine and betaine suggest that the neuroprotective effects of betaine, unlike these of nicotine, do not involve long lasting gene expression changes. This explanation is consistent with the finding that betaine exposure, unlike nicotine exposure, does not enhance expression of mitochondrial stress markers. Of note, efficacy of nicotine as an agonist for ACR-23, closely related to ACR-20, is very low (Rufener et al., PLOS Path. 2013). This raises a question concerning the role of ACR-20 in nicotine-induced protection. We are now examining the possibility that exposure to nicotine, shown to upregulate acr-20 expression (Polli et al., Neurotox. 2015), sensitizes DNs to the protective effects of ACR-20 activation during and after 6-OHDA treatment.