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The goal of the Caenorhabditis Intervention Testing Program is to identify robust and reproducible pro-longevity interventions that are efficacious across genetically diverse cohorts in the Caenorhabditis genus. The project design features multiple experimental replicates collected by three different laboratories. Our initial effort employed fully manual survival assays. With an interest in increasing throughput, we explored automation with flatbed scanner-based Automated Lifespan Machines (ALMs). We used ALMs to measure survivorship of 22 Caenorhabditis strains spanning three species. Additionally, we tested five chemicals that we previously found extended lifespan in manual assays. Overall, we found similar sources of variation among trials for the ALM and our previous manual assays, verifying reproducibility of outcome. Survival assessment was generally consistent between the manual and the ALM assays, although we did observe radically contrasting results for certain compound interventions. We found that particular lifespan outcome differences could be attributed to protocol elements such as enhanced light exposure of specific compounds in the ALM, underscoring that differences in technical details can influence outcomes and therefore interpretation. Overall, we demonstrate that the ALMs effectively reproduce a large, conventionally scored dataset from a diverse test set, independently validating ALMs as a robust and reproducible approach toward aging-intervention screening. Electronic supplementary material The online version of this article (10.1007/s11357-019-00108-9) contains supplementary material, which is available to authorized users.

The citrate transporter of Leuconostoc mesenteroides (CitP) and the malate transporter of Lactococcus lactis (MleP) are homologous proteins that catalyze citrate-lactate and malate-lactate exchange, respectively. Both transporters transport a range of substrates that contain the 2-hydroxycarboxylate motif, HO-CR2-COO- [Bandell, M., et al. (1997) J. Biol. Chem. 272, 18140-18146]. In this study, we have analyzed binding and translocation properties of CitP and MleP for a wide variety of substrates and substrate analogues. Modification of the OH or the COO- groups of the 2-hydroxycarboxylate motif drastically reduced the affinity of the transporters for the substrates, indicating their relevance in substrate recognition. Both CitP and MleP were strictly stereoselective when the R group contained a second carboxylate group; the S-enantiomers were efficiently bound and translocated, while the transporters had no affinity for the R-enantiomers. The affinity of the S-enantiomers, and of citrate, was at least 1 order of magnitude higher than for lactate and other substrates with uncharged R groups, indicating a specific interaction between the second carboxylate group and the protein that is responsible for high-affinity binding. MleP was not stereoselective in binding when the R groups are hydrophobic and as large as a benzyl group. However, only the S-enantiomers were translocated by MleP. CitP had a strong preference for binding and translocating the R-enantiomers of substrates with large hydrophobic R groups. These differences between CitP and MleP explain why citrate is a substrate of CitP and not of MleP. The results are discussed in the context of a model for the interaction between sites on the protein and functional groups on the substrates in the binding pockets of the two proteins.