Regulation of fatty acid desaturase- and immunity gene-expression by mbk-1/DYRK1A in Caenorhabditis elegans.

BACKGROUND: In the nematode Caenorhabditis elegans, longevity in response to germline ablation, but not in response to reduced insulin/IGF1-like signaling, is strongly dependent on the conserved protein kinase minibrain-related kinase 1 (MBK-1). In humans, the MBK-1 ortholog DYRK1A is associated with a variety of disorders, most prominently with neurological defects observed in Down syndrome. To better understand mbk-1's physiological roles and their dependence on genetic background, we analyzed the influence of mbk-1 loss on the transcriptomes of wildtype and long-lived, germline-deficient or insulin-receptor defective, C. elegans strains by RNA-sequencing. RESULTS: mbk-1 loss elicited global changes in transcription that were less pronounced in insulin-receptor mutant than in germline-deficient or wildtype C. elegans. Irrespective of genetic background, mbk-1 regulated genes were enriched for functions in biological processes related to organic acid metabolism and pathogen defense. qPCR-studies confirmed mbk-1 dependent induction of all three C. elegans Δ9-fatty acid desaturases, fat-5, fat-6 and fat-7, in wildtype, germline-deficient and insulin-receptor mutant strains. Conversely, mbk-1 dependent expression patterns of selected pathogen resistance genes, including asp-12, dod-24 and drd-50, differed across the genetic backgrounds examined. Finally, cth-1 and cysl-2, two genes which connect pathogen resistance to the metabolism of the gaseous messenger and lifespan regulator hydrogen sulfide (H2S), were commonly suppressed by mbk-1 loss only in wildtype and germline-deficient, but not in insulin-receptor mutant C. elegans. CONCLUSION: Our work reveals previously unknown roles of C. elegans mbk-1 in the regulation of fatty acid desaturase- and H2S metabolic-genes. These roles are only partially dependent on genetic background. Considering the particular importance of fatty acid desaturation and H2S for longevity of germline-deficient C. elegans, we propose that these processes at least in part account for the previous observation that mbk-1 preferentially regulates lifespan in these worms.

Expression of ß-1,4-galactosyltransferases during Aging in Caenorhabditis elegans.

BACKGROUND: Altered plasma activity of ß-1,4-galac-tosyl-transferases (B4GALTs) is a novel candidate biomarker of human aging. B4GALT1 is assumed to be largely responsible for this activity increase, but how it modulates the aging process is unclear at present. OBJECTIVES: To determine how expression of B4GALT1 and other B4GALT enzymes changes during aging of an experimentally tractable model organism, Caenorhabditis elegans. METHODS: Targeted analysis of mRNA levels of all 3 C. elegans B4GALT family members was performed by qPCR in wild-type and in long-lived daf-2 (insulin/IGF1-like receptor)-deficient or germline-deficient animals. RESULTS: bre-4 (B4GALT1/2/3/4) is the only B4GALT whose expression increases during aging in wild-type worms. In addition, bre-4 levels also rise during aging in long-lived daf-2-deficient worms, but not in animals that are long-lived due to the lack of germline stem cells. On the other hand, expression of sqv-3 (B4GALT7) and of W02B12.11 (B4GALT5/6) appears decreased or constant, respectively, in all backgrounds during aging. CONCLUSIONS: The age-dependent bre-4 mRNA increase in C. elegans parallels the age-dependent B4GALT activity increase in humans and is consistent with C. elegans being a suitable experimental organism to define potentially conserved roles of B4GALT1 during aging.

Further Extension of Lifespan by Unc-43/CaMKII and Egl-8/PLCß Mutations in Germline-Deficient Caenorhabditis elegans.

Reduction of insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) promotes longevity across species. In the nematode Caenorhabditis elegans, ablation of germline stem cells (GSCs) and activity changes of the conserved signaling mediators unc-43/CaMKII (calcium/calmodulin-dependent kinase type II) and egl-8/PLCß (phospholipase Cß) also increase lifespan. Like IIS, these pathways depend on the conserved transcription factor daf-16/FOXO for lifespan extension, but how they functionally interact is unknown. Here, we show that altered unc-43/egl-8 activity further increases the lifespan of long-lived GSC-deficient worms, but not of worms that are long-lived due to a strong reduction-of-function mutation in the insulin/IGF1-like receptor daf-2. Additionally, we provide evidence for unc-43 and, to a lesser extent, egl-8 modulating the expression of certain collagen genes, which were reported to be dispensable for longevity of these particular daf-2 mutant worms, but not for other forms of longevity. Together, these results provide new insights into the conditions and potential mechanisms by which CaMKII- and PLCß-signals modulate C. elegans lifespan.

Cell-Free Prototyping of AND-Logic Gates Based on Heterogeneous RNA Activators.

RNA-based devices controlling gene expression bear great promise for synthetic biology, as they offer many advantages such as short response times and light metabolic burden compared to protein-circuits. However, little work has been done regarding their integration to multilevel regulated circuits. In this work, we combined a variety of small transcriptional activator RNAs (STARs) and toehold switches to build highly effective AND-gates. To characterize the components and their dynamic range, we used an Escherichia coli (E. coli) cell-free transcription-translation (TX-TL) system dispensed via nanoliter droplets. We analyzed a prototype gate in vitro as well as in silico, employing parametrized ordinary differential equations (ODEs), for which parameters were inferred via parallel tempering, a Markov chain Monte Carlo (MCMC) method. On the basis of this analysis, we created nine additional AND-gates and tested them in vitro. The functionality of the gates was found to be highly dependent on the concentration of the activating RNA for either the STAR or the toehold switch. All gates were successfully implemented in vivo, offering a dynamic range comparable to the level of protein circuits. This study shows the potential of a rapid prototyping approach for RNA circuit design, using cell-free systems in combination with a model prediction.