The chronological lifespan of eukaryotic organisms is extended by the mutational inactivation of conserved growth-signaling pathways that regulate progression into and through the cell cycle. Here we show that in the budding yeast S. cerevisiae, these and other lifespan-extending conditions, including caloric restriction and osmotic stress, increase the efficiency with which nutrient-depleted cells establish or maintain a cell cycle arrest in G1. Proteins required for efficient G1 arrest and longevity when nutrients are limiting include the DNA replication stress response proteins Mec1 and Rad53. Ectopic expression of CLN3 encoding a G1 cyclin downregulated during nutrient depletion increases the frequency with which nutrient depleted cells arrest growth in S phase instead of G1. Ectopic expression of CLN3 also shortens chronological lifespan in concert with age-dependent increases in genome instability and apoptosis. These findings indicate that replication stress is an important determinant of chronological lifespan in budding yeast. Protection from replication stress by growth-inhibitory effects of caloric restriction, osmotic and other stresses may contribute to hormesis effects on lifespan. Replication stress also likely impacts the longevity of higher eukaryotes, including humans.

To better understand the role of topoisomerase activity in relieving transcription-induced supercoiling, yeast genes encoding rRNA were visualized in cells deficient for either or both of the two major topoisomerases. In the absence of both topoisomerase I (Top1) and topoisomerase II (Top2) activity, processivity was severely impaired and polymerases were unable to transcribe through the 6.7-kb gene. Loss of Top1 resulted in increased negative superhelical density (two to six times the normal value) in a significant subset of rRNA genes, as manifested by regions of DNA template melting. The observed DNA bubbles were not R-loops and did not block polymerase movement, since genes with DNA template melting showed no evidence of slowed elongation. Inactivation of Top2, however, resulted in characteristic signs of slowed elongation in rRNA genes, suggesting that Top2 alleviates transcription-induced positive supercoiling. Together, the data indicate that torsion in front of and behind transcribing polymerase I has different consequences and different resolution. Positive torsion in front of the polymerase induces supercoiling (writhe) and is largely resolved by Top2. Negative torsion behind the polymerase induces DNA strand separation and is largely resolved by Top1.Eukaryotic cells have two major topoisomerases that are capable of efficiently relaxing torsionally stressed DNA: topoisomerase I (Top1) and topoisomerase II (Top2) (75). They are both abundant nuclear proteins with roles in many DNA activities, and since they both can relax positive and negative torsion, they can substitute for each other in most situations (11,28,29,35,62). In spite of this partial functional redundancy, they control DNA topology by very different mechanisms (65). Top1 (a type IB topoisomerase) makes transient single-strand breaks in torsionally stressed DNA (recognizing the torque in such DNA), followed by controlled rotation of the nicked strand and resealing of the DNA in a more relaxed state (38). Top2 (a type IIA topoisomerase) recognizes juxtaposed DNA helices (as in supercoiled DNA) and passes one DNA helix through the other by making a transient doublestrand break in one of the helices (61, 65

p53 and ARF are well-established tumor suppressor proteins that function together in the negative regulation of cancer. Recently, both of these proteins were found to play surprising roles in autophagy. Autophagy (“self-eating”) is a critical response of eukaryotic cells to metabolic and other stress. During this process, portions of the cytosol are sequestered into characteristic double membrane vesicles that are delivered to the lysosome for degradation, leading to the release of free amino acids and subsequent survival. The mechanisms whereby p53 and ARF control autophagy are only now becoming elucidated. An emerging question is whether we can develop metabolic poisons that preferentially destroy tumor cells depending on their reliance on autophagy for survival, and on their p53 and ARF status.

The ARF tumor suppressor, encoded by the CDKN2A gene, has a well-defined role regulating TP53 stability; this activity maps to exon 1β of CDKN2A. In contrast, little is known about the function(s) of exon 2 of ARF, which contains the majority of mutations in human cancer. In addition to controlling TP53 stability, ARF also has a role in the induction of autophagy. However, whether the principal molecule involved is full-length ARF, or a small molecular weight variant called smARF, has been controversial. Additionally, whether tumor-derived mutations in exon 2 of CDKN2A affect ARF's autophagy function is unknown. Finally, whereas it is known that silencing or inhibiting TP53 induces autophagy, the contribution of ARF to this induction is unknown. In this report we used multiple autophagy assays to map a region located in the highly conserved 5' end of exon 2 of CDKN2A that is necessary for autophagy induction by both human and murine ARF. We showed that mutations in exon 2 of CDKN2A that affect the coding potential of ARF, but not p16INK4a, all impair the ability of ARF to induce autophagy. We showed that whereas full-length ARF can induce autophagy, our combined data suggest that smARF instead induces mitophagy (selective autophagy of mitochondria), thus potentially resolving some confusion regarding the role of these variants. Finally, we showed that silencing Tp53 induces autophagy in an ARF-dependent manner. Our data indicated that a conserved domain in ARF mediates autophagy, and for the first time they implicate autophagy in ARF's tumor suppressor function.

Zika virus (ZIKV) infections occur in areas where dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and other viruses of the genus Flavivirus cocirculate. The envelope (E) proteins of these closely related flaviviruses induce specific long-term immunity, yet subsequent infections are associated with cross-reactive antibody responses that may enhance disease susceptibility and severity. To gain a better understanding of ZIKV infections against a background of similar viral diseases, we examined serological immune responses to ZIKV, WNV, DENV, and YFV infections of humans and nonhuman primates (NHPs). Using printed microarrays, we detected very specific antibody responses to primary infections with probes of recombinant E proteins from 15 species and lineages of flaviviruses pathogenic to humans, while high cross-reactivity between ZIKV and DENV was observed with 11 printed native viruses. Notably, antibodies from human primary ZIKV or secondary DENV infections that occurred in areas where flavivirus is endemic broadly recognized E proteins from many flaviviruses, especially DENV, indicating a strong influence of infection history on immune responses. A predictive algorithm was used to tentatively identify previous encounters with specific flaviviruses based on serum antibody interactions with the multispecies panel of E proteins. These results illustrate the potential impact of exposure to related viruses on the outcome of ZIKV infection and offer considerations for development of vaccines and diagnostics.

Over half the world’s population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti , thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes -borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito–human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = −9.11) and 12.4% (95% CI 4.2%, ∞); z = −2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

Loss of sample integrity during specimen transport can lead to false-negative diagnostic results. In an effort to improve upon the status quo, we used dengue as a model RNA virus to evaluate the stabilization of RNA and antibodies in three commercially available sample stabilization products: Whatman FTA Micro Cards (GE Healthcare Life Sciences, Pittsburgh, PA), DNAstāble Blood tubes (Biomātrica, San Diego, CA), and ViveST tubes (ViveBio, Alpharetta, GA). Both contrived and clinical dengue-positive specimens were stored on these products at ambient temperature or 37°C for up to 1 month. Antibody and viral RNA levels were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays, respectively, and compared with frozen unloaded controls. We observed reduced RNA and antibody levels between stabilized contrived samples and frozen controls at our earliest time point, and this was particularly pronounced for the FTA cards. However, despite some time and temperature dependent loss, a 94.6–97.3% agreement was observed between stabilized clinical specimens and their frozen controls for all products. Additional considerations such as cost, sample volume, matrix, and ease of use should inform any decision to incorporate sample stabilization products into a diagnostic testing workflow. We conclude that DNAstāble Blood and ViveST tubes are useful alternatives to traditional filter paper for ambient temperature shipment of clinical specimens for downstream molecular and serological testing.

Upstream activating factor (UAF) is a multisubunit complex that functions in the activation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I). Cells lacking the Uaf30 subunit of UAF reduce the rRNA synthesis rate by ϳ70% compared to wild-type cells and produce rRNA using both Pol I and Pol II. Miller chromatin spreads demonstrated that even though there is an overall reduction in rRNA synthesis in uaf30 mutants, the active rDNA genes in such strains are overloaded with polymerases. This phenotype was specific to defects in Uaf30, as mutations in other UAF subunits resulted in a complete absence of rDNA genes with high or even modest Pol densities. The lack of Uaf30 prevented UAF from efficiently binding to the rDNA promoter in vivo, leading to an inability to activate a large number of rDNA genes. The relatively few genes that did become activated were highly transcribed, apparently to compensate for the reduced rRNA synthesis capacity. The results show that Uaf30p is a key targeting factor for the UAF complex that facilitates activation of a large proportion of rDNA genes in the tandem array.