Ul Fatima N et al. (2020) J Dev Biol "Conversion of Germ Cells to Somatic Cell Types in C. elegans."

Tursun B, Ul Fatima N

[J Dev Biol, 2020]

The potential of a cell to produce all types of differentiated cells in an organism is termed totipotency. Totipotency is an essential property of germ cells, which constitute the germline and pass on the parental genetic material to the progeny. The potential of germ cells to give rise to a whole organism has been the subject of intense research for decades and remains important in order to better understand the molecular mechanisms underlying totipotency. A better understanding of the principles of totipotency in germ cells could also help to generate this potential in somatic cell lineages. Strategies such as transcription factor-mediated reprogramming of differentiated cells to stem cell-like states could benefit from this knowledge. Ensuring pluripotency or even totipotency of reprogrammed stem cells are critical improvements for future regenerative medicine applications. The <i>C. elegans</i> germline provides a unique possibility to study molecular mechanisms that maintain totipotency and the germ cell fate with its unique property of giving rise to meiotic cells Studies that focused on these aspects led to the identification of prominent chromatin-repressing factors such as the <i>C. elegans</i> members of the Polycomb Repressive Complex 2 (PRC2). In this review, we summarize different factors that were recently identified, which use molecular mechanisms such as control of protein translation or chromatin repression to ensure maintenance of totipotency and the germline fate. Additionally, we focus on recently identified factors involved in preventing transcription-factor-mediated conversion of germ cells to somatic lineages. These so-called reprogramming barriers have been shown in some instances to be conserved with regard to their function as a cell fate safeguarding factor in mammals. Overall, continued studies assessing the different aspects of molecular pathways involved in maintaining the germ cell fate in <i>C. elegans</i> may provide more insight into cell fate safeguarding mechanisms also in other species.

Yanase S et al. (1998) Worm Breeder's Gazette "One tube RT-PCR assay from single worms"

Ishii N, Okano M, Yanase S

[Worm Breeder's Gazette, 1998]

In case of weakly expressed genes, Northern blot analysis may not be sensitive enough to detect transcription unless poly(A)+ RNA is prepared. We would like to report a simple method of RT-PCR assay from single animals that does not require poly(A)+ RNA preparation. Washed with M9 buffer, each single animal is transferred in 2 ul of TE (pH 8.0) in a 0.5 ml tube and frozen (-80oC, 20 min). 1 ul of lysis buffer [0.5 mg/ml proteinase K in 10 mM Tris (pH 8.0), 50 mM KCl, 2.5 mM MgCl2, 0.45% Tween 20 and 0.05% gelatin] is added and mixed well. After the addition of a mineral oil overlay, the tube is heated at 65oC for 1 hour and the reaction is stopped by the boil for 15 min. After cooling to 4oC, 1 ul of DNase I (7.5 units/ul, Pharmacia Biotech) is added and incubated at 37oC for 30 min. The reaction is stopped by boiling for 15 min. 46 ul of a master mix is then pipetted on top of the mineral oil overlay. The mix is formulated to bring the reaction volume to 50 ul using DEPC H2O with these final conditions: each 10 pmol of sense and anti-sense primers, 25 ul of 2x reaction mix and 1ul of SuperScript II RT/Taq DNA polymerase mix (GIBCO BRL). After a brief microfuge spin, the sample is incubated at 45-55oC for 30 min for cDNA synthesis. Then, 94oC for 2 min followed by amplification of 45 cycles of 94oC for 30 sec, 50-65oC (depending on the primer set) for 30 sec and 72oCo for 0.5-3 min (1 kb/min), followed by 72oC for 5 min. The PCR product (10 ul) is analyzed on 0.8-1.5% agarose gels. Using this method, we have been able to detect a typical sod-3 RT-PCR product of which the expression is lower than that of other genes. To avoid error in different stages, we recommend using some animals at the same developmental stage and duplicate with each primer set. This system is also useful for late-stage embryos. References: 1. Williams, B., D. et al. (1992) Genetics 131: 609-624 2. Andachi, Y. et al. (1993) WM'93: 33 3. Lee, E. H. et al. (1997) Focus 19 (2): 39-42 4. Sitaraman, K. et al. (1997) Focus 19 (2): 43-44

Ge YL et al. (2002) Anticancer Res "Effects of adenoviral gene transfer of C. elegans n-3 fatty acid desaturase on the lipid profile and growth of human breast cancer cells."

Kang ZB, Chen Z, Leaf A, Kang JX, Cluette-Brown J, Laposata M, Ge YL

[Anticancer Res, 2002]

Background: Current evidence from both experimental and human studies indicates that omega-6 polyunsaturated fatty acids (n-6 PUFAs) promote breast tumor development, whereas long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) exert suppressive effects. The ratio of n-6 to n-3 fatty acids appears to be an important factor in controlling tumor development. Human cells usually have a very high n-6/n-3 fatty acid ratio because they cannot convert n-6 PUFAs to n-3 PUFAs due to lack of an n-3 desaturase found in C. elegans. Materials and Methods: Adenoviral strategies were used to introduce the C. elegans fat-1 gene encoding an n-3 fatty acid desaturase into human breast cancer cells followed by examination of the n-6/n-3 fatty acid ratio and growth of the cells. Results: Infection of MCF-7 cells with an adenovirus carrying the fat-1 gene resulted in a high expression of the n-3 fatty acid desaturase. Lipid analysis indicated a remarkable increase in the levels of n-3 PUFAs accompanied with a large decrease in the contents of n-6 PUFAs, leading to a change of the n-6/n-3 ratio from 12.0 to 0.8. Accordingly, production of the eicosanoids derived from n-6 PUFA was reduced significantly in cells expressing the fat-1 gene. Importantly, the gene transfer induced mass cell death and inhibited cell proliferation. Conclusion: The gene transfer of the n-3 fatty acid desaturase, as a novel approach, can effectively modify the n-6/n-3 fatty acid ratio of human tumor cells and provide an anticancer effect, without the need of exogenous n-3 PUFA supplementation. These data also increase the understanding of the effects of n-3 fatty acids and the n-6/n-3 ratio on cancer prevention and treatment.

Baytek, G. et al. (2019) International Worm Meeting "The impact of sumoylation on MRG-1's role in germline safeguarding."

Popp, O., Tursun, B., ul Fatima, N., Mertins, P., Bulut, S., Wolfram, T., Baytek, G.

[International Worm Meeting, 2019]

Direct reprogramming of cellular identities by ectopically-expressed transcription factors (TFs) is highly regulated by a variety of factors including post-translational modifications. In a recent RNAi screen to identify reprogramming barriers in C. elegans, we found that the conserved chromatin regulator MRG-1 safeguards germ cells against ectopic fate induction and conversion to neuron-like cells (Hajduskova and Baytek et al., 2018). In order to reveal whether MRG-1 requires other interacting proteins to protect the germ cell identity, we established a robust Immunoprecipitation protocol followed by Mass Spectrometry (IP-MS). In addition to using the N2 background with specific MRG-1 antibodies, we generated a 3xHA knock-in using CRISPR to obtain a confident set of MRG-1-interacting proteins upon comparing anti-MRG-1 and 3xHA antibody-based IP-MS results. Consistently, SMO-1 was one the most robust interactors of MRG-1 and further protein-biochemical analysis revealed that MRG-1 is post-translationally modified by sumoylation. Interestingly, smo-1 and mrg-1 mutants display defects in related pathways including DNA repair (Psakhye and Jentsch, 2012 ; Bleuyard et al. 2017), repressing X-linked genes (Meyer et al 2010; Dombecki et al. 2011; Xu et al. 2012), chromosome pairing (Cheng et al., 2006; Fujita et al. 2002), and germ cell proliferation (Jones et al, 2002; Gupta et al. 2015). Furthermore, we revealed that MRG-1 associates predominantly with genomic sites that carry the histone modification H3K36me3 and SUMO has been shown to enforce distinct chromatin states to protect cell identities in other species (Cossec et al. 2018). We therefore analyzed the impact of sumoylation on the DNA-binding pattern of MRG-1 and performed ChIP-seq upon smo-1 depletion. Since the lack of MRG-1 sumoylation appears to affect the genomic MRG-1 binding pattern, we mutated lysine residues of MRG-1 that are predicted as sumoylation sites. Overall, our findings indicate that sumoylation impacts MRG-1's function in regulating chromatin in order to protect germ cell identity, which might be a conserved mechanism to post-translationally regulate also the MRG-1 homolog MRG15 in other species.

Rodriguez-Perez MA et al. (2010) Am J Trop Med Hyg "Interruption of transmission of Onchocerca volvulus in the Oaxaca focus, Mexico."

Orozco-Algarra ME, Rodriguez-Perez MA, Morales-Castro AL, Dominguez-Vazquez A, Rendon VG, Pena-Flores GP, Arredondo-Jimenez JI, Vasquez-Rodriguez MA, Unnasch TR, Richards F

[Am J Trop Med Hyg, 2010]

All endemic communities of the Oaxaca focus of onchocerciasis in southern Mexico have been treated annually or semi-annually with ivermectin since 1994. In-depth epidemiologic assessments were performed in communities during 2007 and 2008. None of the 52,632 Simulium ochraceum s.l. collected in four sentinel communities was found to contain parasite DNA when tested by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), resulting in an upper bound of the infection rate in the vectors of 0.07/2,000. The prevalence of microfilariae (mf) in the cornea and/or anterior chamber of the eye was also zero (0 of 1,039 residents examined; 95%-UL = 0.35%). Similarly, all 1,164 individuals examined by skin biopsy were mf negative (95%-UL = 0.31%), and sera collected from 3,569 children from 25 communities did not harbor Ov16 IgG4-antibodies (95%-UL = 0.09%). These meet the criteria for absence of morbidity and parasite transmission in the Oaxaca focus. As a result mass treatments with ivermectin were halted in 2009.

Rodriguez-Perez MA et al. (2010) Am J Trop Med Hyg "Lack of active Onchocerca volvulus transmission in the northern Chiapas focus of Mexico."

Pena-Flores GP, Prado-Velasco G, Dominguez-Vazquez A, Morales-Castro AL, Rodriguez-Perez MA, Orozco-Algarra ME, Unnasch TR, Richards F

[Am J Trop Med Hyg, 2010]

The northern Chiapas onchocerciasis focus has undergone 11 years of ivermectin mass treatment. No evidence of microfilariae in the cornea and/or anterior chamber of the eye or in skin snips was seen in residents examined in 2006 in two sentinel communities (upper limit of the 95% confidence interval [UL 95% CI] = 0.5% and 0.3%, respectively). In children 10 and under, 0 of 305 were found to harbor antibodies to Ov16, a marker of parasite exposure; 0 of 4,400 Simulium ochraceum s.l. collected in 2005 contained parasite DNA, giving an UL 95% CI for the infective rate of 0.9/2,000, and an UL 95% CI of the seasonal transmission potential of 1.2 L3/person. These data, assumed to be representative of the focus as a whole, suggest that there is no ongoing transmission of Onchocerca volvulus in the northern Chiapas focus. Community-wide treatments with ivermectin were halted in 2008, and a post-treatment surveillance phase was initiated.

Seo J et al. (2005) Arch Environ Contam Toxicol "Biodegradation of the insecticide N,N-diethyl-m-toluamide by fungi: identification and toxicity of metabolites."

Kim SD, Cha CJ, Hur HG, Lee YG, Seo J, Ahn JH

[Arch Environ Contam Toxicol, 2005]

Fungi (Cunninghamella elegans ATCC 9245, Mucor ramannianus R-56, Aspergillus niger VKMF-1119, and Phanerochaete chrysosporium BKMF-1767) were tested to elucidate the biologic fate of the topical insect repellent N,N-diethyl-m-toluamide (DEET). The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column, showed that three peaks occurred after incubation of C. elegans, with which 1 mM DEET was combined as a final concentration. The peaks were not detected in the control experiments with either DEET alone or tested fungus alone. The metabolites produced by C. elegans exhibited a molecular mass of 207 with a fragment ion (m/z) at 135, a molecular mass of 179 with an m/z at 135, and a molecular mass of 163 with an m/z at 119, all of which correspond to N,N-diethyl-m-toluamide-N-oxide, N-ethyl-m-toluamide-N-oxide, and N-ethyl-m-toluamide, respectively. M. ramannianus R-56 also produced N, N-diethyl-m-toluamide-N-oxide and N-ethyl-m-toluamide but did not produce N-ethyl-m-toluamide-N-oxide. For the biologic toxicity test with DEET and its metabolites, the freshwater zooplankton Daphnia magna was used. The biologic sensitivity in decreasing order was DEET > N-ethyl-m-toluamide > N,N-diethyl-m-toluamide-N-oxide. Although DEET and its fungal metabolites showed relatively low mortality compared with other insecticides, the toxicity was increased at longer exposure periods. These are the first reports of the metabolism of DEET by fungi and of the biologic toxicity of DEET and its fungal metabolites to the freshwater zooplankton D. magna.

Kang ZB et al. (2001) Proc Natl Acad Sci U S A "Adenoviral gene transfer of Caenorhabditis elegans n--3 fatty acid desaturase optimizes fatty acid composition in mammalian cells."

Leaf A, Laposata M, Kang ZB, Kang JX, Chen Z, Ge Y, Cluette-Brown J

[Proc Natl Acad Sci U S A, 2001]

Omega-3 polyunsaturated fatty acids (PUFAs) are essential components required for normal cellular function and have been shown to exert many preventive and therapeutic actions. The amount of n-3 PUFAs is insufficient in most Western people, whereas the level of n-6 PUFAs is relatively too high, with an n-6/n-3 ratio of >18. These two classes of PUFAs are metabolically and functionally distinct and often have important opposing physiological functions; their balance is important for homeostasis and normal development. Elevating tissue concentrations of n-3 PUFAs in mammals relies on chronic dietary intake of fat rich in n-3 PUFAs, because mammalian cells lack enzymatic activities necessary either to synthesize the precursor of n-3 PUFAs or to convert n-6 to n-3 PUFAs. Here we report that adenovirus-mediated introduction of the Caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase into mammalian cells can quickly and effectively elevate the cellular n - 3 PUFA contents and dramatically balance the ratio of n-6/n-3 PUFAs, Heterologous expression of the fat-1 gene in rat cardiac myocytes rendered cells capable of converting various n-6 PUFAs to the corresponding n-3 PUFAs, and changed the n-6/n-3 ratio from about 15:1 to 1:1. In addition, an eicosanoid derived from n-6 PUFA (i.e., arachidonic acid) was reduced significantly in the transgenic cells. This study demonstrates an effective approach to modifying fatty acid composition of mammalian cells and also provides a basis for potential applications of this gene transfer in experimental and clinical settings.

De Stasio E et al. (1994) Worm Breeder's Gazette "Mutagenesis of C. elegans Using N-ethyl-N-nitrosourea."

De Stasio E, Stanislaus D, Lephoto C

[Worm Breeder's Gazette, 1994]

Mutagenesis of C. elegans using N-ethyl-N-nitrosourea Elizabeth De Stasio, Dinesh Stanislaus and Catherine Lephoto. Department of Biology, Lawrence University, Appleton, Wl 54911

Kang JX et al. (2004) Nature "Transgenic mice: fat-1 mice convert n-6 to n-3 fatty acids."

Kang JX, Wang J, Kang ZB, Wu L

[Nature, 2004]

Mammals cannot naturally produce omega-3 (n-3) fatty acids--beneficial nutrients found mainly in fish oil--from the more abundant omega-6 (n-6) fatty acids and so they must rely on a dietary supply. Here we show that mice engineered to carry a fat-1 gene from the roundworm Caenorhabditis elegans can add a double bond into an unsaturated fatty-acid hydrocarbon chain and convert n-6 to n-3 fatty acids. This results in an abundance of n-3 and a reduction in n-6 fatty acids in the organs and tissues of these mice, in the absence of dietary n-3. As well as presenting an opportunity to investigate the roles played by n-3 fatty acids in the body, our discovery indicates that this technology might be adapted to enrich n-3 fatty acids in animal products such as meat, milk and eggs.