Proteins are the building blocks of life. While proteins and their localization within cells and sub-cellular compartments are well defined, the proteins predicted to be secreted to form the extracellular matrix - or matrisome - remain elusive in the model organism C. elegans . Here, we used a bioinformatic approach combining gene orthology and protein structure analysis and an extensive curation of the literature to define the C. elegans matrisome. Similar to the human genome, we found that 719 out of ~20,000 genes (~4%) of the C. elegans genome encodes matrisome proteins, including 181 collagens, 35 glycoproteins, 10 proteoglycans, and 493 matrisome-associated proteins. We report that 173 out of the 181 collagen genes are unique to nematodes and are predicted to encode cuticular collagens, which we are proposing to group into five clusters. To facilitate the use of our lists and classification by the scientific community, we developed an automated annotation tool to identify ECM components in large datasets. We also established a novel database of all C. elegans collagens (CeColDB). Last, we provide examples of how the newly defined C. elegans matrisome can be used for annotations and gene ontology analyses of transcriptomic, proteomic, and RNAi screening data. Because C. elegans is a widely used model organism for high throughput genetic and drug screens, and to study biological and pathological processes, the conserved matrisome genes may aid in identifying potential drug targets. In addition, the nematode-specific matrisome may be exploited for targeting parasitic infection of man and crops.

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The demographic shift in the human population reflects an aging society-over 20% of Europeans are predicted to be 65 or over by the year 2025 (Riera & Dillin, 2015). Aging is the major risk factor for developing chronic diseases, such as cancer, Alzheimer's disease, and cardiovascular complications (Partridge et al., 2018).Unfortunately, humans spend on average one-fifth of their lifetime in poor health suffering from one or multiple age-related chronic diseases (Partridge et al., 2018). However, the onset of age-related pathologies is not fixed, and the rate of aging was shown to be malleable. The goal of biomedical research on aging or geroscience is to identify interventions that compress late-life morbidity to increase the period spent healthy and free from disease.

Inhibition of the master growth regulator mTORC1 (mechanistic target of rapamycin complex 1) slows ageing across phyla, in part by reducing protein synthesis. Various stresses globally suppress protein synthesis through the integrated stress response (ISR), resulting in preferential translation of the transcription factor ATF-4. Here we show in C. elegans that inhibition of translation or mTORC1 increases ATF-4 expression, and that ATF-4 mediates longevity under these conditions independently of ISR signalling. ATF-4 promotes longevity by activating canonical anti-ageing mechanisms, but also by elevating expression of the transsulfuration enzyme CTH-2 to increase hydrogen sulfide (H2S) production. This H2S boost increases protein persulfidation, a protective modification of redox-reactive cysteines. The ATF-4/CTH-2/H2S pathway also mediates longevity and increased stress resistance from mTORC1 suppression. Increasing H2S levels, or enhancing mechanisms that H2S influences through persulfidation, may represent promising strategies for mobilising therapeutic benefits of the ISR, translation suppression, or mTORC1 inhibition.

Proper collagen homeostasis is essential for development and aging of any multicellular organism. During aging, two extreme scenarios are commonly occurring: a local excess in collagen deposition, for instance during fibrosis, or a gradual overall reduction of collagen mass. Here, we describe a histological and a colorimetric method to assess collagen levels in mammalian tissues and in the nematode Caenorhabditis elegans. The first method is the polychrome Herovici staining to distinguish between young and mature collagen ratios. The second method is based on hydroxyproline measurements to estimate collagen protein levels. In addition, we show how to decellularize the multicellular organism C. elegans in order to harvest its cuticle, one of two major extracellular matrices, mainly composed of collagen. These methods allow assessing collagen deposition during aging either in tissues or in whole organisms.

Herein we demonstrate the segmentation of alginate solution streams to generate alginate fibers of precisely controllable lengths between 200 and 1000 μm. Moreover, we demonstrate the subsequent encapsulation of the formed fibers within pL-volume microdroplets, produced within the same microfluidic device, in a direct manner. Finally, we show immediate and complete on-chip gelation of alginate fibers in a rapid and reproducible fashion.

Systems biology approaches often use networks of gene expression and metabolite data to identify regulatory factors and pathways connected with phenotypic variance. Separating upstream causal mechanisms, downstream biomarkers, and incidental correlations remains a significant challenge, yet it is essential for designing mechanistic experiments. To address this, we first designed a population following 2157 individual mice from 89 isogenic strains of BXD mice across their lifespans to identify molecular interactions between genotype, environment, age (GxExA) and metabolic fitness. Each strain was separated into two cohorts, fed low fat (6% cal/fat) or high fat (60% cal/fat) diets. One-third of individuals (662) were sacrificed at ~6, 12, 18, or 24 months-of-age, with the remainder monitored until natural death. Transcriptome, proteome, and metabolome profiles were generated from liver samples. These multi-omic measurements were deconvolved into metabolic networks, where we observed varying network connectivity as a function of GxExA. The multiple independent study variables permitted causal inference analysis for the network variants using stability selection. This calculates the strength and directionality of the interactions between molecular measurements and metabolic networks as a function of age, diet, and genotype, and assigns each gene a score for its relative position to the target pathway. At 1% FDR, 94% of novel connections were stable across age and diet, such as the connection between Rdh11 with cholesterol biosynthesis and Mut with mitochondrial translation. 6% of discovered candidate genes were unstable, indicating a clear causal relationship between the segregating independent variable, the gene, and the pathway. For instance, age drives variation in proteasomal genes (e.g. Psmb3, Psmb4), which in turn drive changes in the mitochondrial ribosome. Conversely, COX7A2L malformation drives variation in OXPHOS genes, but both are downstream of changes in mitochondrial translation. Finally, we examined all data for connections with the longevity and known longevity-related pathways, identifying several dozen novel candidate genes. Specific C. elegans orthologs for the top two candidates, Ctsd and St7, were knocked down with RNAi and found to reduce longevity both in wildtype worms and in mutant long-lived strains.

Inhibition of mTORC1 (mechanistic target of rapamycin 1) slows ageing, but mTORC1 supports fundamental processes that include protein synthesis, making it critical to elucidate how mTORC1 inhibition increases lifespan. Under stress conditions, the integrated stress response (ISR) globally suppresses protein synthesis, resulting in preferential translation of the transcription factor ATF-4. Here we show in C. elegans that the ATF-4 transcription program promotes longevity and that ATF-4 upregulation mediates lifespan extension from mTORC1 inhibition. ATF-4 activates canonical anti-ageing mechanisms but also increases expression of transsulfuration enzymes to promote hydrogen sulfide (H2S) production. ATF-4-induced H2S production mediates longevity and stress resistance from C. elegans mTORC1 suppression, and ATF4 drives H2S production in mammalian dietary restriction. This H2S boost increases protein persulfidation, a protective modification of redox-reactive cysteines. Increasing H2S levels, or enhancing mechanisms that H2S modulates through persulfidation, may represent promising strategies for mobilising therapeutic benefits of the ISR or mTORC1 inhibition.