Evolutionary adaptation increases the fitness of a species in its environment. It can occur through rewiring of gene regulatory networks, such that an organism responds appropriately to environmental changes. We investigated whether sirtuin deacetylases, which repress transcription and require NAD+ for activity, serve as transcriptional rewiring points that facilitate the evolution of potentially adaptive traits. If so, bringing genes under the control of sirtuins could enable organisms to mount appropriate responses to stresses that decrease NAD+ levels. To explore how the genomic targets of sirtuins shift over evolutionary time, we compared two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, that display differences in cellular metabolism and lifecycle timing in response to nutrient availability. We identified sirtuin-regulated genes through a combination of chromatin immunoprecipitation and RNA expression. In both species, regulated genes were associated with NAD+ homeostasis, mating, and sporulation, but the specific genes differed. In addition, regulated genes in K. lactis were associated with other processes, including utilization of non-glucose carbon sources, detoxification of arsenic, and production of the siderophore pulcherrimin. Consistent with the species-restricted regulation of these genes, sirtuin deletion impacted relevant phenotypes in K. lactis but not S. cerevisiae Finally, sirtuin-regulated gene sets were depleted for broadly-conserved genes, consistent with sirtuins regulating processes restricted to a few species. Taken together, these results are consistent with the notion that sirtuins serve as rewiring points that allow species to evolve distinct responses to low NAD+ stress. Copyright © 2020, Genetics.Neutral genetic diversity across the genome is determined by the complex interplay of mutation, demographic history, and natural selection. While the direct action of natural selection is limited to functional loci across the genome, its impact can have effects on nearby neutral loci due to genetic linkage. These effects of selection at linked sites, referred to as genetic hitchhiking and background selection (BGS), are pervasive across natural populations. However, only recently has there been a focus on the joint consequences of demography and selection at linked sites, and some empirical studies have come to apparently contradictory conclusions as to their combined effects. In order to understand the relationship between demography and selection at linked sites, we conducted an extensive forward simulation study of BGS under a range of demographic models. We found that the relative levels of diversity in BGS and neutral regions vary over time and that the initial dynamics after a population size change are often in the opposite direction of the long-term expected trajectory. Our detailed observations of the temporal dynamics of neutral diversity in the context of selection at linked sites in non-equilibrium populations provides new intuition about why patterns of diversity under BGS vary through time in natural populations and help reconcile previously contradictory observations. Most notably, our results highlight that classical models of BGS are poorly suited for predicting diversity in non-equilibrium populations. Copyright © 2020, Genetics.tRNA genes are widely studied sites of replication-fork pausing and genome instability in the budding yeast Saccharomyces cerevisiae tRNAs are extremely highly transcribed and serve as constitutive condensin binding sites. Manogepix Fungal inhibitor tRNA transcription by RNA polymerase III has previously been identified as stimulating replication-fork pausing at tRNA genes, but the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of candidates to replication-fork progression at tDNAs in yeast transcription factor binding, transcription, topoisomerase activity, condensin-mediated clustering, and Rad18-dependent DNA repair. We show that an asymmetric block to replication is maintained even when tRNA transcription is abolished by depletion of one or more subunits of RNA polymerase III. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication. Therefore, our data suggest that the RNA polymerase III transcription complex itself represents an asymmetric obstacle to replication even in the absence of RNA synthesis. We additionally demonstrate that replication-fork progression past tRNA genes is unaffected by the global depletion of condensin from the nucleus, and can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways. Copyright © 2020, Genetics.The Transgenic RNAi Project (TRiP), a Drosophila melanogaster functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes. Cutting is triggered by co-expression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering. Copyright © 2020, Genetics.INTRODUCTION Compared with fee-for-service systems, prospective payment based on casemix classification is thought to promote more efficient, needs-based care provision. We aim to develop a casemix classification to predict the costs of home care in the Netherlands. METHODS AND ANALYSIS The research is designed as a multicentre, cross-sectional cohort study using quantitative methods to identify the relative cost predictors of home care and combine these into a casemix classification, based on individual episodes of care. The dependent variable in the analyses is the cost of home care utilisation, which is operationalised through various measures of formal and informal care, weighted by the relative wage rates of staff categories. As independent variables, we will use data from a recently developed Casemix Short-Form questionnaire, combined with client information from participating home care providers’ (nursing) classification systems and data on demographics and care category (ie, a classification mandated by health insurers).