Burnaevskiy N et al. (2019) Nat Commun "Chaperone biomarkers of lifespan and penetrance track the dosages of many other proteins."

Burnaevskiy N, Mendenhall A, Yun S, Sands B, Tedesco PM, Johnson TE, Kaeberlein M, Brent R

[Nat Commun, 2019]

Many traits vary among isogenic individuals in homogeneous environments. In microbes, plants and animals, variation in the protein chaperone system affects many such traits. In the animal model C. elegans, the expression level of hsp-16.2 chaperone biomarkers correlates with or predicts the penetrance of mutations and lifespan after heat shock. But the physiological mechanisms causing cells to express different amounts of the biomarker were unknown. Here, we used an in vivo microscopy approach to dissect different contributions to cell-to-cell variation in hsp-16.2 expression in the intestines of young adult animals, which generate the most lifespan predicting signal. While we detected both cell autonomous intrinsic noise and signaling noise, we found both contributions were relatively unimportant. The major contributor to cell-to-cell variation in biomarker expression was general differences in protein dosage. The hsp-16.2 biomarker reveals states of high or low effective dosage for many genes.

Burnaevskiy, N. et al. (2017) International Worm Meeting "Quantitative and functional analyses of the organs and cells of post adult reproductive diapause animals."

Mendenhall, A., Burnaevskiy, N., Kaeberlein, M.

[International Worm Meeting, 2017]

C. elegans have had a fantastical impact on our understanding of the aging process. In addition to ability to extend lifespan upon many pro-longevity treatments, C. elegans will also live a normal, wild-type lifespan after prolonged (weeks) periods in diapuased juvenile states (L1 arrest, dauer). The mechanisms that preserve the soma of these animals during diapaus are therefore attractive targets for understanding and modulating the aging process. Remarkably, it was found that upon starvation even adult animals are able to enter into diapause state, known as adult reproductive diapause (ARD) (Giana Angelo and Marc R. Van Gilst, 2009). Like diapauses in juvenile animals, upon refeeding and exit of the adult reproductive diapause, their lifespan is fully preserved. After prolonged periods in the ARD state, animals show dramatic morphological improvements upon refeeding, and their post-diapause lifespan is indistinguishable from the animals that never experienced starvation. However, it is unclear whether post-diapause animals follow the exact same physiological trajectory as their counterparts that were never starved. To gain a better understanding of the post-diapaused animals we applied quantitative measures of physiology including detailed electrophysiological analysis of pharynx function, 3-dimensional mitochondrial structure examination and measurement of gene expression capacity. Gene expression capacity, the ability to express genes into functional proteins, is a fundamental cellular property that is highly variable between the intestine cells of individual animals, and may affect many aspects of cellular physiology. Our in-depth quantitative analysis provides some mechanistic insights into the cellular processes underlying longevity and healthspan of post-diapause animals.

Burnaevskiy, N et al. (2017) International Worm Meeting "Mechanisms of cell-to-cell and animal-to-animal variation in gene expression in isogenic C. elegans."

Kaeberlein, M., Mendenhall, A., Burnaevskiy, N

[International Worm Meeting, 2017]

Isogenic cells or animals exhibit profound differences in many phenotypes, including drug resistance, penetrance of mutations, lifespan and fertility. These nongenetic differences in physiology occur even in well-controlled, homogeneous environments (Mendenhall et al 2017). Thus interindividual differences in physiology occur independent of genetic differences. This intrinsic, nongenetic physiological variation represents a considerable challenge for understanding the aging process and age-related disease. While some physiological variation in isogenic populations is attributed to epigenetic differences, the actual mechanisms of such variations are largely unknown. Yet, in 2017, we have the tools to identify and quantify sources of nongenetic variation in many quantitative traits, such as variation in the expression level of a particular gene. Here we are focused on quantifying the mechanisms of cell-to-cell variation in gene expression for a small heatshock protein, hsp-16.2. We focused on this gene because prior investigations showed that individual animals express different amounts of hsp-16.2 reporter genes and that these differences predict differences in lifespan and are correlated with differences in the penetrance of mutations. To investigate the source of these physiological differences we developed an approach for in vivo quantitative analysis of gene expression at the single cell level (Mendenhall et al 2015). We measured three types of experimentally tractable noise in gene expression. During the course of our studies, we found strong, cell-specific expression patterns for many reporter genes; that is, for many genes, the ratio of gene A to gene B was fixed in intestine cell type X, and different than intestine cell type Y. We discovered that a fundamental property of cells, gene expression capacity (the ability to express genes into functional protein), accounts for most of the cell-to-cell variation in intestine cell gene expression for the genes we have tested. Our experiments suggest that gene expression capacity affects the ability of animals to produce functional proteins, and thus, may underlie many interindividual differences in physiology. Our discovery highlights the need for careful examination of animal physiology at single cell resolution, as bulk analysis of macromolecules (e.g. RNAseq, mass-spectrometry) alone may fail to capture the physiological heterogeneity that exists within cells in the same tissue or organ.

Burnaevskiy N et al. (2018) Elife "Reactivation of RNA metabolism underlies somatic restoration after adult reproductive diapause in C. elegans."

Mendenhall A, Chen S, Kaeberlein M, Burnaevskiy N, Reynolds A, Karanth S, Mailig M, Van Gilst M

[Elife, 2018]

The mechanisms underlying biological aging are becoming recognized as therapeutic targets to delay the onset of multiple age-related morbidities. Even greater health benefits can potentially be achieved by halting or reversing age-associated changes. <i>C. elegans</i> restore their tissues and normal longevity upon exit from prolonged adult reproductive diapause, but the mechanisms underlying this phenomenon remain unknown. Here, we focused on the mechanisms controlling recovery from adult diapause. Here, we show that functional improvement of post-mitotic somatic tissues does not require germline signaling, germline stem cells, or replication of nuclear or mitochondrial DNA. Instead a large expansion of the somatic RNA pool is necessary for restoration of youthful function and longevity. Treating animals with the drug 5-fluoro-2'-deoxyuridine prevents this restoration by blocking reactivation of RNA metabolism. These observations define a critical early step during exit from adult reproductive diapause that is required for somatic rejuvenation of an adult metazoan animal.

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.

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.

Kang JX (2007) Prostaglandins Leukot Essent Fatty Acids "Fat-1 transgenic mice: a new model for omega-3 research."

Kang JX

[Prostaglandins Leukot Essent Fatty Acids, 2007]

An appropriate animal model that can eliminate confounding factors of diet would be very helpful for evaluation of the health effects of nutrients such as n-3 fatty acids. We recently generated a fat-1 transgenic mouse expressing the Caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase that converts n-6 to n-3 fatty acids (which is absent in mammals). The fat-1 transgenic mice are capable of producing n-3 fatty acids from the n-6 type, leading to abundant n-3 fatty acids with reduced levels of n-6 fatty acids in their organs and tissues, without the need of a dietary n-3 supply. Feeding an identical diet (high in n-6) to the transgenic and wild-type littermates can produce different fatty acid profiles in these animals. Thus, this model allows well-controlled studies to be performed, without the interference of the potential confounding factors of diet. The transgenic mice are now being used widely and are emerging as a new tool for studying the benefits of n-3 fatty acids and the molecular mechanisms of their action.