RAD51AP1 regulates ALT-HDR through chromatin-directed homeostasis of TERRA.

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates. We also show that RAD51AP1 binds to and might stabilize TERRA-containing R-loops as RAD51AP1 depletion reduces R-loop formation at telomere DNA breaks. Proteomic analyses uncover a role for RAD51AP1-mediated TERRA R-loop homeostasis in a mechanism of chromatin-directed suppression of TERRA and prevention of transcription-replication collisions (TRCs) during ALT-HDR. Intriguingly, we find that both TERRA binding and this non-canonical function of RAD51AP1 require its intrinsic SUMO-SIM regulatory axis. These findings provide insights into the multi-contextual functions of RAD51AP1 within the ALT mechanism and regulation of TERRA.

RAD51AP1 Is an Essential Mediator of Alternative Lengthening of Telomeres.

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.

Effectiveness of adding virtual reality to physiotherapeutic treatment in patients with total hip arthroplasty. A randomized controlled trial.

OBJECTIVE: To determine the clinical effectiveness of adding virtual reality via the Nintendo Wii console and its Wii Balance Board to physiotherapy treatment in patients with total hip arthroplasty. DESIGN: Randomized controlled trial. SETTING: Clinical Hospital San Borja Arriaran, Santiago, Chile. PARTICIPANTS: A total of 73 patients over 50 years of age with total hip arthroplasty were randomly allocated to two groups. INTERVENTIONS: The control group (n = 37) received 6 weeks of physiotherapy treatment; the intervention group (n = 36) received the same treatment plus virtual reality exercises with the Nintendo Wii console. OUTCOME MEASURES: The two groups were assessed at baseline and after the 6 weeks of treatment. The primary outcome assessed was the function with the WOMAC questionnaire. The secondary outcomes were the Berg Balance Scale, distance covered with the six-minute walk test, and difference in weight load on the lower extremities. RESULTS: A total of 73 patients, 37 patients in the control group (20 women; mean age of 70.9 ± 9.16 years) and 36 patients in the intervention group (18 women; mean age of 70.39 ± 9.02 years) were analyzed. At the end of the treatment, the difference between groups for the total WOMAC score was -10.4 points (p = 0.00), 4.7 points (p = 0.00) for the Berg Balance Scale, and 45.2 mt (p = 0.00) for the six-minute walk test All differences were in favor of the intervention group. CONCLUSIONS: In the short term, the addition of virtual reality via the Nintendo Wii and its Wii Balance Board platform showed statistically significant differences in the function of patients with total hip replacement, but these differences were not minimally clinically important.Trial registration: This research was registered in the Clinical Trials Registry of Australia and New Zealand, with reference ACTRN12618001252202.

GDF11 restricts aberrant lipogenesis and changes in mitochondrial structure and function in human hepatocellular carcinoma cells.

Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features. Recently, it has been reported that GDF11 exerts tumor-suppressive properties in hepatocellular carcinoma cells, decreasing clonogenicity, proliferation, spheroid formation, and cellular function, all associated with a decrement in stemness features, resulting in mesenchymal to epithelial transition and loss of aggressiveness. The aim of the present work was to investigate the mechanism associated with the tumor-suppressive properties displayed by GDF11 in liver cancer cells. Hepatocellular carcinoma-derived cell lines were exposed to GDF11 (50 ng/ml), RNA-seq analysis in Huh7 cell line revealed that GDF11 exerted profound transcriptomic impact, which involved regulation of cholesterol metabolic process, steroid metabolic process as well as key signaling pathways, resembling endoplasmic reticulum-related functions. Cholesterol and triglycerides determination in Huh7 and Hep3B cells treated with GDF11 exhibited a significant decrement in the content of these lipids. The mTOR signaling pathway was downregulated, and this was associated with a reduction in key proteins involved in the mevalonate pathway. In addition, real-time metabolism assessed by Seahorse technology showed abridged glycolysis as well as glycolytic capacity, closely related to an impaired oxygen consumption rate and decrement in adenosine triphosphate production. Finally, transmission electron microscopy revealed mitochondrial abnormalities, such as cristae disarrangement, consistent with metabolic changes. Results provide evidence that GDF11 impairs cancer cell metabolism targeting lipid homeostasis, glycolysis, and mitochondria function and morphology.

Sorbitol Modulates Resistance to Alternaria alternata by Regulating the Expression of an NLR Resistance Gene in Apple.

In plant-microbe interactions, plant sugars produced by photosynthesis are not only a carbon source for pathogens, but may also act as signals that modulate plant defense responses. Here, we report that decreasing sorbitol synthesis in apple (Malus domestica) leaves by antisense suppression of ALDOSE-6-PHOSPHATE REDUCTASE (A6PR) leads to downregulation of 56 NUCLEOTIDE BINDING/LEUCINE-RICH REPEAT (NLR) genes and converts the phenotypic response to Alternaria alternata from resistant to susceptible. We identified a resistance protein encoded by the apple MdNLR16 gene and a small protein encoded by the fungal HRIP1 gene that interact in both a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. Deletion of HRIP1 in A. alternata enables gain of virulence on the wild-type control plant. Overexpression of MdNLR16 in two antisense A6PR lines increases resistance, whereas RNAi suppression of MdNLR16 in the wild-type control decreases resistance against A. alternata MdWRKY79 transcriptionally regulates MdNLR16 by binding to the promoter of MdNLR16 in response to sorbitol, and exogenous sorbitol feeding partially restores resistance of the antisense A6PR lines to A. alternata These findings indicate that sorbitol modulates resistance to A. alternata via the MdNLR16 protein that interacts with the fungal effector in a classic gene-for-gene manner in apple.

The human Shu complex functions with PDS5B and SPIDR to promote homologous recombination.

RAD51 plays a central role in homologous recombination during double-strand break repair and in replication fork dynamics. Misregulation of RAD51 is associated with genetic instability and cancer. RAD51 is regulated by many accessory proteins including the highly conserved Shu complex. Here, we report the function of the human Shu complex during replication to regulate RAD51 recruitment to DNA repair foci and, secondly, during replication fork restart following replication fork stalling. Deletion of the Shu complex members, SWS1 and SWSAP1, using CRISPR/Cas9, renders cells specifically sensitive to the replication fork stalling and collapse caused by methyl methanesulfonate and mitomycin C exposure, a delayed and reduced RAD51 response, and fewer sister chromatid exchanges. Our additional analysis identified SPIDR and PDS5B as novel Shu complex interacting partners and genetically function in the same pathway upon DNA damage. Collectively, our study uncovers a protein complex, which consists of SWS1, SWSAP1, SPIDR and PDS5B, involved in DNA repair and provides insight into Shu complex function and composition.

In-Vehicle Alcohol Detection Using Low-Cost Sensors and Genetic Algorithms to Aid in the Drinking and Driving Detection.

Worldwide, motor vehicle accidents are one of the leading causes of death, with alcohol-related accidents playing a significant role, particularly in child death. Aiming to aid in the prevention of this type of accidents, a novel non-invasive method capable of detecting the presence of alcohol inside a motor vehicle is presented. The proposed methodology uses a series of low-cost alcohol MQ3 sensors located inside the vehicle, whose signals are stored, standardized, time-adjusted, and transformed into 5 s window samples. Statistical features are extracted from each sample and a feature selection strategy is carried out using a genetic algorithm, and a forward selection and backwards elimination methodology. The four features derived from this process were used to construct an SVM classification model that detects presence of alcohol. The experiments yielded 7200 samples, 80% of which were used to train the model. The rest were used to evaluate the performance of the model, which obtained an area under the ROC curve of 0.98 and a sensitivity of 0.979. These results suggest that the proposed methodology can be used to detect the presence of alcohol and enforce prevention actions.

Septins are required for reproductive propagule development and virulence of the maize pathogen Cochliobolus heterostrophus.

Septins are highly conserved GTP-binding proteins that function in cell cytokinesis, polarity and morphogenesis. To evaluate the roles of these proteins in inoculum health and disease, mutants deleted for each of five septin proteins (Cdc3, Cdc10, Cdc11, Cdc12, and Cdc100) were characterized in the ascomycete Cochliobolus heterostrophus for ability to develop asexual and sexual spores and for virulence to the host maize. Strains deleted for CDC3, CDC10, CDC11, and CDC12 genes showed significant changes in hyphal growth, and in development of conidia and ascospores compared to the wild-type strain. Conidia had dramatically reduced numbers of septa and rates of germination, while ascospore development was blocked in the meiotic process. Although asci were produced, wild-type ascospores were not. When equal numbers of conidia from wild type and mutants were used to inoculate maize, cdc10 mutants showed reduced virulence compared to the wild-type strain and other mutants. This reduced virulence was demonstrated to be correlated with lower germination rate of cdc10 mutant conidia. When adjusted for germination rate, virulence was equivalent to the wild-type strain. Double mutants (cdc3cdc10, cdc3cdc11) showed augmented reduced growth phenotypes. cdc100 mutants were wild type in all assays. Taken together, these findings indicate that all four conserved septin proteins play a major role in reproductive propagule formation and that mutants with deletions of CDC10 are reduced in virulence to the host maize.

Characterizing environmental asthma triggers and healthcare use patterns in Puerto Rico.

Objective: Asthma carries a high burden of disease for residents of Puerto Rico. We conducted this study to better understand asthma-related healthcare use and to examine potential asthma triggers.Methods: We characterized asthma-related healthcare use in 2013 by demographics, region, and date using outpatient, hospital, and emergency department (ED) insurance claims with a primary diagnostic ICD-9-CM code of 493.XX. We examined environmental asthma triggers, including outdoor allergens (i.e., mold and pollen), particulate pollution, and influenza-like illness. Analyses included descriptive statistics and Poisson time-series regression.Results: During 2013, there were 550,655 medical asthma claims reported to the Puerto Rico Healthcare Utilization database, representing 148 asthma claims/1,000 persons; 71% of asthma claims were outpatient visits, 19% were hospitalizations, and 10% were ED visits. Females (63%), children aged ≤9 years (77% among children), and adults aged ≥45 years (80% among adults) had the majority of asthma claims. Among health regions, Caguas had the highest asthma claim-rate at 142/1,000 persons (overall health region claim-rate = 108). Environmental exposures varied across the year and demonstrated seasonal patterns. Metro health region regression models showed positive associations between increases in mold and particulate matter <10 microns in diameter (PM10) and outpatient asthma claims.Conclusions: This study provides information about patterns of asthma-related healthcare use across Puerto Rico. Increases in mold and PM10 were associated with increases in asthma claims. Targeting educational interventions on exposure awareness and reduction techniques, especially to persons with higher asthma-related healthcare use, can support asthma control activities in public health and clinical settings.

Contribution of a synchronic teleneurology program to decrease the patient number waiting for a first consultation and their waiting time in Chile.

Backround There is a shortage of medical specialists in Chile, including neurologists; currently, there are 56,614 patients waiting for a first adult Neurology appointment in the country. The Teleneurology Program at the Hospital Las Higueras de Talcahuano (HHT) was implemented in 2015 to help reduce both the number of patients waiting for a first consultation and their waiting times. METHODS: This retrospective study analyzed a cohort of 8269 patients referred to the HHT Neurology clinic between 2013 and 2018, from primary, secondary, and tertiary health centers. Cox regression analyses were performed to determine the factors influencing each outcome (number of patients waiting for a consultation and waiting time), such as age, gender, referral health establishment and the type of consultation (whether in situ at the HHT or by synchronic telepresence through the Teleneurology Program). RESULTS: Out of the 8269 patients included in the study, 1743 consulted the neurologist through the Teleneurology Program, while 6526 received a consultation in situ at the HHT. Since its implementation (2015) until the end of 2018, the Teleneurology program contributed to decrease the number of patients waiting for their first appointment from 3084 to 298. Waiting time for the first consultation was 60% shorter for patients enrolled in the Teleneurology program than those with consultation in situ at HHT (6.23 ± 6.82 and 10.47 ± 8.70 months, respectively). Similar differences were observed when sorting patient data according to the referral health center. Cox regression analysis showed that patients waiting for a traditional in situ first adult Neurology consultation at the HHT had a higher risk (OR = 6.74) of waiting 90% longer than patients enrolled in the Teleneurology Program, without significant differences due to gender or age. CONCLUSIONS: Data from this study show a significant contribution of the Teleneurology Program at the HHT to decrease the number of patients waiting for a first consultation with a neurologist, as well as shorter waiting times, when derived from primary and secondary health centers.

Clinically Integrated Molecular Diagnostics in Adenoid Cystic Carcinoma.

BACKGROUND: Adenoid cystic carcinoma (ACC) is an aggressive salivary gland malignancy without effective systemic therapies. Delineation of molecular profiles in ACC has led to an increased number of biomarker-stratified clinical trials; however, the clinical utility and U.S.-centric financial sustainability of integrated next-generation sequencing (NGS) in routine practice has, to our knowledge, not been assessed. MATERIALS AND METHODS: In our practice, NGS genotyping was implemented at the discretion of the primary clinician. We combined NGS-based mutation and fusion detection, with MYB break-apart fluorescent in situ hybridization (FISH) and MYB immunohistochemistry. Utility was defined as the fraction of patients with tumors harboring alterations that are potentially amenable to targeted therapies. Financial sustainability was assessed using the fraction of global reimbursement. RESULTS: Among 181 consecutive ACC cases (2011-2018), prospective genotyping was performed in 11% (n = 20/181; n = 8 nonresectable). Testing identified 5/20 (25%) NOTCH1 aberrations, 6/20 (30%) MYB-NFIB fusions (all confirmed by FISH), and 2/20 (10%) MYBL1-NFIB fusions. Overall, these three alterations (MYB/MYBL1/NOTCH1) made up 65% of patients, and this subset had a more aggressive course with significantly shorter progression-free survival. In 75% (n = 6/8) of nonresectable patients, we detected potentially actionable alterations. Financial analysis of the global charges, including NGS codes, indicated 63% reimbursement, which is in line with national (U.S.-based) and international levels of reimbursement. CONCLUSION: Prospective routine clinical genotyping in ACC can identify clinically relevant subsets of patients and is approaching financial sustainability. Demonstrating clinical utility and financial sustainability in an orphan disease (ACC) requires a multiyear and multidimensional program. IMPLICATIONS FOR PRACTICE: Delineation of molecular profiles in adenoid cystic carcinoma (ACC) has been accomplished in the research setting; however, the ability to identify relevant patient subsets in clinical practice has not been assessed. This work presents an approach to perform integrated molecular genotyping of patients with ACC with nonresectable, recurrent, or systemic disease. It was determined that 75% of nonresectable patients harbor potentially actionable alterations and that 63% of charges are reimbursed. This report outlines that orphan diseases such as ACC require a multiyear, multidimensional program to demonstrate utility in clinical practice.

Genotype-specific effects of ericoid mycorrhizae on floral traits and reproduction in Vaccinium corymbosum.

PREMISE: Most plants interact with mycorrhizal fungi and animal pollinators simultaneously. Yet, whether mycorrhizae affect traits important to pollination remains poorly understood and may depend on the match between host and fungal genotypes. Here, we examined how ericoid mycorrhizal fungi affected flowering phenology, floral traits, and reproductive success, among eight genotypes of highbush blueberry, Vaccinium corymbosum (Ericaceae). We asked three overarching questions: (1) Do genotypes differ in response to inoculation? (2) How does inoculation affect floral and flowering traits? (3) Are inoculated plants more attractive to pollinators and less pollen limited than non-inoculated plants of the same genotype? METHODS: To examine these questions, we experimentally inoculated plants with ericoid mycorrhizal fungi, grew the plants in the field, and measured flowering and floral traits over 2 years. In year 2, we conducted a hand-pollination experiment to test whether plants differed in pollen limitation. RESULTS: Inoculated plants had significantly higher levels of colonization for some genotypes, and there were significant floral trait changes in inoculated plants for some genotypes as well. On average, inoculated plants produced significantly larger floral displays, more fruits per inflorescence, and heavier fruits with lower sugar content, than non-inoculated, control plants. Hand pollination enhanced the production of fruits, and fruit mass, for non-inoculated plants but not for those that were inoculated. CONCLUSIONS: Our results demonstrate that inoculation with ericoid mycorrhizal fungi enhanced flowering and altered investment in reproduction in genotype-specific ways. These findings underscore the importance of examining belowground symbionts and genotype-specific responses in their hosts to fully understand the drivers of aboveground interactions.

Clues to an Evolutionary Mystery: The Genes for T-Toxin, Enabler of the Devastating 1970 Southern Corn Leaf Blight Epidemic, Are Present in Ancestral Species, Suggesting an Ancient Origin.

The Southern corn leaf blight (SCLB) epidemic of 1970 devastated fields of T-cytoplasm corn planted in monoculture throughout the eastern United States. The epidemic was driven by race T, a previously unseen race of Cochliobolus heterostrophus. A second fungus, Phyllosticta zeae-maydis, with the same biological specificity, appeared coincidentally. Race T produces T-toxin, while Phyllosticta zeae-maydis produces PM-toxin, both host-selective polyketide toxins necessary for supervirulence. The present abundance of genome sequences offers an opportunity to tackle the evolutionary origins of T- and PM- toxin biosynthetic genes, previously thought unique to these species. Using the C. heterostrophus genes as probes, we identified orthologs in six additional Dothideomycete and three Eurotiomycete species. In stark contrast to the genetically fragmented race T Tox1 locus that encodes these genes, all newly found Tox1-like genes in other species reside at a single collinear locus. This compact arrangement, phylogenetic analyses, comparisons of Tox1 protein tree topology to a species tree, and Tox1 gene characteristics suggest that the locus is ancient and that some species, including C. heterostrophus, gained Tox1 by horizontal gene transfer. C. heterostrophus and Phyllosticta zeae-maydis did not exchange Tox1 DNA at the time of the SCLB epidemic, but how they acquired Tox1 remains uncertain. The presence of additional genes in Tox1-like clusters of other species, although not in C. heterostrophus and Phyllosticta zeae-maydis, suggests that the metabolites produced differ from T- and PM-toxin.

Herbivore removal reduces influence of arbuscular mycorrhizal fungi on plant growth and tolerance in an East African savanna.

The functional relationship between arbuscular mycorrhizal fungi (AMF) and their hosts is variable on small spatial scales. Here, we hypothesized that herbivore exclusion changes the AMF community and alters the ability of AMF to enhance plant tolerance to grazing. We grew the perennial bunchgrass, Themeda triandra Forssk in inoculum from soils collected in the Kenya Long-term Exclosure Experiment where treatments representing different levels of herbivory have been in place since 1995. We assessed AMF diversity in the field, using terminal restriction fragment length polymorphism and compared fungal diversity among treatments. We conducted clipping experiments in the greenhouse and field and assessed regrowth. Plants inoculated with AMF from areas accessed by wild herbivores and cattle had greater biomass than non-inoculated controls, while plants inoculated with AMF from where large herbivores were excluded did not benefit from AMF in terms of biomass production. However, only the inoculation with AMF from areas with wild herbivores and no cattle had a positive effect on regrowth, relative to clipped plants grown without AMF. Similarly, in the field, regrowth of plants after clipping in areas with only native herbivores was higher than other treatments. Functional differences in AMF were evident despite little difference in AMF species richness or community composition. Our findings suggest that differences in large herbivore communities over nearly two decades has resulted in localized, functional changes in AMF communities. Our results add to the accumulating evidence that mycorrhizae are locally adapted and that functional differences can evolve within small geographical areas.

Physical therapy in patients over 60†years of age with a massive and irreparable rotator cuff tear: a case series.

[Purpose] Describe the effect of a Physical Therapy (PT) program in function improvement and pain reduction in patients over 60 years of age with massive and irreparable Rotator Cuff (RC) tear. [Participants and Methods] Ninety-two patients received a 12 weeks PT program that consisted in manual therapy and a specific exercises program. Before the start and at the end of the treatment, the shoulder function was assessed with Constant-Murley, the upper extremity function with DASH, and the pain during activity with the visual analog scale (VAS). [Results] At the end of the treatment, Constant-Murley exhibited an increment of 24.9 points, DASH showed a decrease of 28.7 points, and the VAS, a decrease of 3.6 cm (p=0.00). [Conclusion] A PT program based on manual therapy and specific exercises in a short term improves the function and reduces the pain during activity in patients with a massive and irreparable RC tear.

'Candidatus Moeniiplasma glomeromycotorum', an endobacterium of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF, subphylum Glomeromycotina) are symbionts of most terrestrial plants. They commonly harbour endobacteria of a largely unknown biology, referred to as MRE (Mollicutes/mycoplasma-related endobacteria). Here, we propose to accommodate MRE in the novel genus 'Candidatus Moeniiplasma.' Phylogeny reconstructions based on the 16S rRNA gene sequences cluster 'Ca.Moeniiplasma' with representatives of the class Mollicutes, whereas phylogenies derived from amino acid sequences of 19 genes indicate that it is a discrete lineage sharing ancestry with the members of the family Mycoplasmataceae. Cells of 'Ca.Moeniiplasma' reside directly in the host cytoplasm and have not yet been cultivated. They are coccoid, ~500 nm in diameter, with an electron-dense layer outside the plasma membrane. However, the draft genomes of 'Ca.Moeniiplasma' suggest that this structure is not a Gram-positive cell wall. The evolution of 'Ca.Moeniiplasma' appears to be driven by an ultrarapid rate of mutation accumulation related to the loss of DNA repair mechanisms. Moreover, molecular evolution patterns suggest that, in addition to vertical transmission, 'Ca.Moeniiplasma' is able to transmit horizontally among distinct Glomeromycotina host lineages and exchange genes. On the basis of these unique lifestyle features, the new species 'Candidatus Moeniiplasma glomeromycotorum' is proposed.

Becoming Overweight Without Gaining a Pound: Weight Evaluations and the Social Integration of Mexicans in the United States.

Mexican women gain weight with increasing duration in the United States. In the United States, body dissatisfaction tends to be associated with depression, disordered eating, and incongruent weight evaluations, particularly among white women and women of higher socioeconomic status. However, it remains unclear how overweight and obesity is interpreted by Mexican women. Using comparable data of women ages 20-64 from both Mexico (the 2006 Encuesta Nacional de Salud y Nutricion; N=17,012) and the United States (the 1999-2009 National Health and Nutrition Examination Surveys; N=8,487), we compare weight status evaluations among Mexican nationals, Mexican immigrants, U.S.-born Mexicans, U.S.-born non-Hispanic Whites, and U.S.-born non-Hispanic blacks. Logistic regression analyses, which control for demographic and social-economic variables and measured body mass index and adjust for the likelihood of migration for Mexican nationals, indicate that the tendency to self-evaluate as overweight among Mexicans converges with levels among non-Hispanic whites and diverges from blacks over time in the United States. Overall, the results suggest a U.S. integration process in which Mexican-American women's less critical self-evaluations originate in Mexico but fade with time in the United States as they gradually adopt U.S. white norms for thinner body sizes. These results are discussed in light of social comparison and negative health assimilation.

Toxin-resistant isoforms of Na+/K+-ATPase in snakes do not closely track dietary specialization on toads.

Toads are chemically defended by bufadienolides, a class of cardiotonic steroids that exert toxic effects by binding to and disabling the Na+/K+-ATPases of cell membranes. Some predators, including a number of snakes, have evolved resistance to the toxic effects of bufadienolides and prey regularly on toads. Resistance in snakes to the acute effects of these toxins is conferred by at least two amino acid substitutions in the cardiotonic steroid binding pocket of the Na+/K+-ATPase. We surveyed 100 species of snakes from a broad phylogenetic range for the presence or absence of resistance-conferring mutations. We found that such mutations occur in a much wider range of taxa than previously believed. Although all sequenced species known to consume toads exhibited the resistance mutations, many of the species possessing the mutations do not feed on toads, much less specialize on that food source. This suggests that either there is little performance cost associated with these mutations or they provide an unknown benefit. Furthermore, the distribution of the mutation among major clades of advanced snakes suggests that the origin of the mutation reflects evolutionary retention more than dietary constraint.