Range-wide persistence of the endangered arroyo toad (Anaxyrus californicus) for 20+ years following a prolonged drought.

Prolonged drought due to climate change has negatively impacted amphibians in southern California, U.S.A. Due to the severity and length of the current drought, agencies and researchers had growing concern for the persistence of the arroyo toad (Anaxyrus californicus), an endangered endemic amphibian in this region. Range-wide surveys for this species had not been conducted for at least 20 years. In 2017-2020, we conducted collaborative surveys for arroyo toads at historical locations. We surveyed 88 of the 115 total sites having historical records and confirmed that the arroyo toad is currently extant in at least 61 of 88 sites and 20 of 25 historically occupied watersheds. We did not detect toads at almost a third of the surveyed sites but did detect toads at 18 of 19 specific sites delineated in the 1999 Recovery Plan to meet one of four downlisting criteria. Arroyo toads are estimated to live 7-8 years, making populations susceptible to prolonged drought. Drought is estimated to increase in frequency and duration with climate change. Mitigation strategies for drought impacts, invasive aquatic species, altered flow regimes, and other anthropogenic effects could be the most beneficial strategies for toad conservation and may also provide simultaneous benefits to several other native species that share the same habitat.

Protection of Salmonella by ampicillin-resistant Escherichia coli in the presence of otherwise lethal drug concentrations.

Microbial systems have become the preferred testing grounds for experimental work on the evolution of traits that benefit other group members. This work, based on conceptual and theoretical models of frequency-dependent selection within populations, has proven fruitful in terms of understanding the dynamics of group beneficial or 'public goods' traits within species. Here, we expand the scope of microbial work on the evolution of group-beneficial traits to the case of multi-species communities, particularly those that affect human health. We examined whether beta-lactamase-producing Escherichia coli could protect ampicillin-sensitive cohorts of other species, particularly species that could cause human disease. Both beta-lactamase-secreting E. coli and, surprisingly, those engineered to retain it, allowed for survival of a large number of ampicillin-sensitive cohorts of Salmonella enterica serovar Typhimurium, including both laboratory and clinical isolates. The Salmonella survivors, however, remained sensitive to ampicillin when re-plated onto solid medium and there was no evidence of gene transfer. Salmonella survival did not even require direct physical contact with the resistant E. coli. The observed phenomenon appears to involve increased release of beta-lactamase from the E. coli when present with S. enterica. Significantly, these findings imply that resistant E. coli, that are not themselves pathogenic, may be exploited, even when they are normally selfish with respect to other E. coli. Thus, Salmonella can gain protection against antibiotics from E. coli without gene transfer, a phenomenon not previously known. As a consequence, antibiotic-resistant E. coli can play a decisive role in the survival of a species that causes disease and may thereby interfere with successful treatment.

Evolution of altruists and cheaters in near-isogenic populations of Escherichia coli.

Emergence of antibiotic-resistant bacteria threatens the continued efficacy of many critical drugs used to treat serious infections. What if such resistant organisms could also act as altruists and "share" their resistance with sensitive cohorts without any actual genetic exchange? We competed resistant strains that differ solely in their ability to secrete a plasmid-encoded beta-lactamase. Sensitive strains were otherwise isogenic with their resistant counterparts and were either plasmid-free or contained a "Dummy" plasmid of roughly the same size as that of the resistance plasmids. Absent antibiotic selection, plasmid-free sensitive strains outperformed the plasmid-bearing strains. In the presence of ampicillin, the outcome depended on whether the resistant strain secreted its beta-lactamase (Altruist) or retained it (Selfish). In the latter case, only resistant cells survived. When beta-lactamase was secreted, some sensitive cohorts were also provided protection, with the largest fitness increase provided to plasmid-free cells. However, some Altruist strains appeared to be at a disadvantage, as a great deal of their enzyme broke off cells. Thus, additional variables must be considered when designing microbial competition experiments.

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses.

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.

Cytotoxic and mutagenic effects of tobacco-borne free fatty acids.

Tobacco smoke contains substances capable of binding iron in an aqueous medium and transferring the metal into both organic solvents and intact mammalian red cells. This iron-binding activity is due to free fatty acids which are abundant in tobacco smoke and form 2:1 (free fatty acid:iron) chelates with ferrous iron. These earlier observations suggested that smoke-borne free fatty acids and the associated delocalization of iron within the lung might contribute to both the chronic pulmonary inflammation and the carcinogenesis associated with smoking. We now report that micromolar concentrations of iron or free fatty acid are not toxic to cultured human lung fibroblasts. However, when combined, the same low concentrations of iron and free fatty acid exert synergistic toxicity. Furthermore, the combination of free fatty acid and iron is highly mutagenic, inducing almost as many selectable mutations in the gene for hypoxanthine/guanine phosphoribosyl transferase as does benzo[a]pyrenediolepoxide, a class I carcinogen generated from benzo[a]pyrene present in cigarette smoke. The combination of free fatty acid and iron also promotes transformation of NIH 3T3 cells into an anchorage-independent phenotype. We conclude that free fatty acids in tobacco smoke may be important contributors to both the pulmonary damage and the carcinogenesis associated with smoking.

REV1 accumulates in DNA damage-induced nuclear foci in human cells and is implicated in mutagenesis by benzo[a]pyrenediolepoxide.

The REV1 gene encodes a Y-family DNA polymerase that has been postulated to have both catalytic and structural functions in translesion replication past UV photoproducts in mammalian cells. To examine if REV1 is implicated in DNA damage tolerance mechanisms after exposure of human cells to a chemical carcinogen, we generated a plasmid expressing REV1 protein fused at its C-terminus with green fluorescent protein (GFP). In transient transfection experiments, virtually all of the transfected cells had a diffuse nuclear pattern in the absence of carcinogen exposure. In contrast, in cells exposed to benzo[a]pyrenediolepoxide, the fusion protein accumulated in a focal pattern in the nucleus in 25% of the cells, and co-localized with PCNA. These data support the idea that REV1 is present at stalled replication forks. We also examined the mutagenic response at the HPRT locus of human cells that had greatly reduced levels of REV1 mRNA due to the stable expression of gene-specific ribozymes, and compared them to wild-type cells. The mutant frequency was greatly reduced in the ribozyme-expressing cells. These data indicate that REV1 is implicated in the mutagenic DNA damage tolerance response to BPDE and support the development of strategies to target this protein to prevent such mutations.

Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene.

In yeast, mutations induced by UV radiation are dependent on the function of the Rev1 gene product, a Y-family DNA polymerase that assists in translesion replication with potentially mutagenic consequences. Human REV1 has been cloned, but its role in mutagenesis and carcinogenesis remains obscure. To examine the role of REV1 in UV mutagenesis in human cells and to evaluate its potential as a therapeutic target to prevent such mutations, we developed a ribozyme that cleaves human REV1 mRNA in vitro. Stable expression of the ribozyme in human cells reduced the target REV1 mRNA up to 90%. We examined the cytotoxic and mutagenic response to UV of seven independent clones that had reduced levels of endogenous REV1 mRNA. In each case, the clonogenic survival after UV was not different from that of the parental cell strains. In contrast, the UV-induced mutant frequencies at the endogenous HPRT locus were reduced up to 75% in cells with reduced levels of REV1 mRNA. The data support the idea that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations.