Constraints on the evolution of toxin-resistant Na,K-ATPases have limited dependence on sequence divergence.

A growing body of theoretical and experimental evidence suggests that intramolecular epistasis is a major determinant of rates and patterns of protein evolution and imposes a substantial constraint on the evolution of novel protein functions. Here, we examine the role of intramolecular epistasis in the recurrent evolution of resistance to cardiotonic steroids (CTS) across tetrapods, which occurs via specific amino acid substitutions to the α-subunit family of Na,K-ATPases (ATP1A). After identifying a series of recurrent substitutions at two key sites of ATP1A that are predicted to confer CTS resistance in diverse tetrapods, we then performed protein engineering experiments to test the functional consequences of introducing these substitutions onto divergent species backgrounds. In line with previous results, we find that substitutions at these sites can have substantial background-dependent effects on CTS resistance. Globally, however, these substitutions also have pleiotropic effects that are consistent with additive rather than background-dependent effects. Moreover, the magnitude of a substitution's effect on activity does not depend on the overall extent of ATP1A sequence divergence between species. Our results suggest that epistatic constraints on the evolution of CTS-resistant forms of Na,K-ATPase likely depend on a small number of sites, with little dependence on overall levels of protein divergence. We propose that dependence on a limited number sites may account for the observation of convergent CTS resistance substitutions observed among taxa with highly divergent Na,K-ATPases (See S1 Text for Spanish translation).

Epistatic Effects Between Amino Acid Insertions and Substitutions Mediate Toxin resistance of Vertebrate Na+,K+-ATPases.

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino acid substitutions in the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work revealed that hystricognath rodents (e.g., chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino acid insertions in the H1-H2 loop, but their functional significance was not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance in homologs of each of the two species that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla NKA unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse NKA, the amino acid insertion and substitution Q111R both contribute to an augmented CTS resistance without compromising ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific mutational effects on CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

Macronutrients, trace metals and health risk assessment in agricultural soil and edible plants of Mahshahr City, Iran.

In this study, we evaluate the geochemistry of macro- (Fe, P, Ca, S, K, Na, Mg) and micronutrients (Mo, Cu, Pb, Zn, Co, Mn, Cd, Sr, Cr, Hg, Se), along with possible health risks of heavy metals contamination in agricultural soils and vegetables of the Mahshahr industrial port in Iran. Calculation of geochemical coefficients revealed the low pollution load of Mahshahr agricultural soils. Most of the investigated elements exhibited lower concentrations in soil than international standards. Element concentrations in plant samples were far below the permissible values set by environmental agencies. Based on permissible values, there was deficiency of several soil elements, including Cu, in vegetables because they are mostly present in the soil residual phase. An exception was Mn, which is the most mobile element in soil. The transfer factor (TF) of elements showed the following trend: K > Na > P > S > Mo > Hg > Se > Zn > Cd > Cu > Mg > Mn > Ca > Cr > Co ≈ Fe = Pb. There was high transfer of major elements from soil to plants, and lower values for micronutrients and heavy metals. The calculated daily intake (DIM) and health risk index (HRI) for ten plant species for adults were < 1, while the HRI was larger than 1 for Mn via radish consumption. According to geochemical investigations and statistical tests such as principal component analysis, Kruskal-Wallis, and correlation coefficient calculations, plant species play the most important role in elemental uptake by plants.

Effects of dioxin on testicular histopathology, sperm parameters, and CatSper2 gene and protein expression in Naval Medical Research Institute male mice.

The CatSper gene family is known to be solely expressed in sperm cells and is possibly associated with sperm motility and penetration through the zona pellucida. Despite its vital role in male fertility, factors regulating its expression are not widely known. The present study aimed at evaluating the effects of dioxin on CatSper2 gene and protein expression, testicular histopathology, sperm quality and biochemical parameters in a mice model. The experiments were performed on 32 Naval Medical Research Institute male mice (2-3 months). The animals were divided into four groups in a random manner: (a) control; (b) dioxin 1; (c) dioxin 2; and (d) dioxin 3. The treatment groups received 0.1, 0.5 and 1 µg/kg of dioxin intraperitoneally every day for 2 weeks. Administration of dioxin significantly downregulated the CatSper2 gene and protein expression. A greater reduction in gene and protein expression was found at higher doses of dioxin. At the same time, sperm parameters, especially sperm motility and count, decreased in mice exposed to dioxin. The results of testicular histopathology showed necrotic degeneration and epithelium thickness reduction in the dioxin groups in comparison with the controls. Besides, oxidative stress increased in seminiferous tubules.

Important signals regulating coronary artery angiogenesis.

Angiogenesis is a complex process of budding, the formation of new blood vessels from pre-existing microvessels, via migration, proliferation and survival. Vascular angiogenesis factors include different classes of molecules that have a fundamental role in blood vessel formation. Numerous inducers of angiogenesis, such as the members of the vascular endothelial growth factor (VEGF) family, basic fibroblast growth factor (bFGF), angiopoietin (Ang), hepatocyte growth factor (HGF), and hypoxia inducible factor-1 (HIF-1), have an important role in angiogenesis. However, VEGF, platelet-derived growth factor (PDGF), and transforming growth factor ß (TGF-ß) expression appear to be important in intraplaque angiogenesis. Interaction and combined effects between growth factors is essential in endothelial cell migration, proliferation, differentiation, and endothelial cell-cell communication that ultimately lead to the microvessel formation. Since VEGF has a key role during angiogenesis; it may be considered as a good therapeutic target in the clinic. The essential function of several angiogenic factors involved in coronary angiogenesis and intraplaque angiogenesis in atherosclerosis are carefully considered along with the use of angiogenic factors in clinical practice.

Determine exogenous human DDAH2 gene function in rabbit bone marrow-derived endothelial progenitor cells in vitro.

The in vitro amplification of endothelial progenitor cells (EPCs) is an important method because of its role in gene transferring and regenerative medicine. In this study, we isolated rabbit bone marrow-derived EPCs to further manipulation and overexpression of dimethylarginine dimethylaminohydrolase (DDAH) in EPCs. Isolated EPCs were cultured, expanded in endothelial basal medium. Morphology of EPCs and expression levels of surface markers detected using immunocytochemistry staining and through the use of flow cytometery. Endothelial progenitor cells were transfected with plasmid vectors expressing human DDAH2 (DDAH2-EPCs). Three days after gene transfer, positive transfected-EPCs proliferation and DDAH activity were assayed. We observed colonies conformation and endothelium-like morphology gradually in the third week of culture. Characterization results revealed positive expression of EPC surface markers CD106, Flk-1, vWF, and CD34 using few identification techniques. Overexpression of DDAH2 increased citrulline production after 96 hours of transfection, 235.34 ± 0.69 vs 95.26 ± 5.76 ng/mL; P = .023. These results suggest that cell population with EPC characteristics can be simply isolated from rabbit bone marrow and successfully engineered to overexpress exogenous gene. In this study, we offer a feasible method to isolate and identify EPCs from bone marrow. In addition, an efficient transfection with a plasmid vector (without risk of interference) can be constructed a hybrid structure with EPC and DDAH2 gene to examine their function in vitro.

Corticosteroid responses of snakes to toxins from toads (bufadienolides) and plants (cardenolides) reflect differences in dietary specializations.

Toads are chemically defended by cardiotonic steroids known as bufadienolides. Resistance to the acute effects of bufadienolides in snakes that prey on toads is conferred by target-site insensitivity of the toxin's target enzyme, the Na+/K+-ATPase. Previous studies have focused largely on the molecular mechanisms of resistance but have not investigated the physiological mechanisms or consequences of exposure to the toxins. Adrenal enlargement in snakes often is associated with specialization on a diet of toads. These endocrine glands are partly composed of interrenal tissue, which produces the corticosteroids corticosterone and aldosterone. Corticosterone is the main hormone released in response to stress in reptiles, and aldosterone plays an important role in maintaining ion balance through upregulation of Na+/K+-ATPase. We tested the endocrine response of select species of snakes to acute cardiotonic steroid exposure by measuring circulating aldosterone and corticosterone concentrations. We found that Rhabdophis tigrinus, which specializes on a diet of toads, responds with lower corticosterone and higher aldosterone compared to other species that exhibit target-site resistance to the toxins but do not specialize on toads. We also found differences between sexes in R. tigrinus, with males generally responding with higher corticosterone and aldosterone than females. This study provides evidence of physiological adaptations, beyond target-site resistance, associated with tolerance of bufadienolides in a specialized toad-eating snake.

Relationship between serum cytokines receptors and matrix metalloproteinase 9 levels and coronary artery disease.

BACKGROUND: The prevalence of coronary artery disease (CAD) is increasing globally, supporting the need for the identification of novel biomarkers. Therefore in the present study, we have explored the association of SIL2A, SIL6R, STNFRI, STNFRII, and MMP9 in CAD patients. METHODS: Twenty one patients with angiographically defined CAD with more than 50% occlusion, at least, in one coronary artery and twenty healthy subjects (n=20) without the history of cardiovascular symptoms were enrolled. Demographic and biochemical analysis (e.g. Total Cholesterol (TC), Triglyceride (TG), and HDL-C) were measured in all the subjects. The level of cytokines receptor (SIL2A, SIL6R, SIL6R, STNFRI, STNFRII, and matrix metallopeptidase 9 (MMP9) were evaluated. RESULTS: Our results showed the higher level of MMP9 in patients group compared to the control subjects, while no significant differences were detected for other cytokines. In particular the level of MMP9 was significantly (P=.015) increased from 181.16 ng/mL (95%CI: 112.1-199.2) to 192.0 ng/mL (95%CI: 181.5-265.2). Moreover, the sensitivity and specificity of MMP9 were 95.45% and 45%, respectively, as detected by receiver operating characteristic (ROC) curves. CONCLUSION: We demonstrate the significant correlation of MMP-9 with CAD with sensitivity of 95.45%, suggesting its role as a biomarker in CAD patients. Further studies in larger population - preferably multicenter setting - are warranted to explore the functional role of this marker in coronary artery disease.

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.

Accessory Spleen in the Splenic Hilum: a Cadaveric Study with Clinical Significance.

AIM: An accessory spleen is an additional tissue of the spleen that may be found near the spleen. It is a congenital anomaly of the spleen that its incidence has been reported 10-30% of the population. Hence, the objective of this study was evaluation the incidence of accessory spleen in Iranian cadavers. METHOD: Sixty hundred and ninety three spleens (541 males, 152 females) were excised from cadavers in the dissection hall of Mashhad Forensic Medicine Organization cadavers. Inclusion criteria were as follows: Fresh Iranian cadavers with no history of alcohol, poisoning or drug abuse, and no evidence of pathologic abnormality or injury to the spleen. The presence of accessory spleens, its dimension and weight investigated in cadavers. RESULTS: During routine postmortem examination, five cases with an accessory spleen were found in the autopsy laboratory of Mashhad legal Medicine Organization between June 2014 and July 2015. Of the cases, 3 were male and 2 were female. The accessory spleens were observed at the splenic hilum. The length of the accessory spleens ranged from 2-3.5 cm, while the range of width was between 0.5 and 2.5 cm. The accessory spleens were confirmed by histological examination. CONCLUSION: An accessory spleen has clinical importance in some locations. When an accessory spleen is situated in another site, it may mimic some tumors such as pancreatic tumor and adrenal tumor. In addition, accessory spleen may cause hyperplasia after splenectomy and be responsible for a recurrence of the hematological disorders.

Effects of temperature on embryonic and early larval growth and development in the rough-skinned newt (Taricha granulosa).

We investigated the effects of temperature on the growth and development of embryonic and early larval stages of a western North American amphibian, the rough-skinned newt (Taricha granulosa). We assigned newt eggs to different temperatures (7, 14, or 21°C); after hatching, we re-assigned the newt larvae into the three different temperatures. Over the course of three to four weeks, we measured total length and developmental stage of the larvae. Our results indicated a strong positive relationship over time between temperature and both length and developmental stage. Importantly, individuals assigned to cooler embryonic temperatures did not achieve the larval sizes of individuals from the warmer embryonic treatments, regardless of larval temperature. Our investigation of growth and development at different temperatures demonstrates carry-over effects and provides a more comprehensive understanding of how organisms respond to temperature changes during early development.

Pumpkin seed oil (prostafit) or prazosin? Which one is better in the treatment of symptomatic benign prostatic hyperplasia.

OBJECTIVE: To assess the efficacy of drugs containing herbal extracts in the treatment of Benign Prostatic Hyperplasia. METHODS: The clinical trial study was performed in 2011-2012 at Imam Reza Hospital, Mashhad, Iran. One hundred patients with Benign Prostatic Hyperplasia were randomly divided into 2 equal groups receiving prostafit and prazosin, respectively. Quality of life and International Prostatic Symptom Score questionnaire were filled and prostate specific antigen level, uroflowmetry and prostate volume were measured at baseline, 3 and 6 months after the medication. The data was analysed using SPSS 15 and repeated measure analysis of variance. RESULTS: No complications were observed during and after the treatment. International Prostatic Symptom Score had significant differences at baseline and 6 months after the treatment in both groups, specially in group 2 18 vs 22 (36% versus 44%). Quality of life was better in group 2, 25.5 vs 31.5 (51% versus 63%). prostate specific antigen level did not change after the treatment and there was no remarkable difference in either group. CONCLUSION: Prostafit is an effective and safe treatment in Benign Prostatic Hyperplasia but not as much as prozasin.

Evaluating the efficacy of protein quantification methods on membrane proteins.

Protein quantification is an important tool for a wide range of biological applications. The most common broadscale methods include the Lowry, bicinchoninic acid (BCA), and Coomassie Bradford assays. Despite their wide applicability, the mechanisms of action imply that these methods may not be ideal for large transmembrane proteins due to the proteins' integration in the plasma membrane. Here, we investigate this problem by assessing the efficacy and applicability of these three common protein quantification methods on a candidate transmembrane protein - the Na,K-ATPase (NKA). We compared these methods to an ELISA, which we newly developed and describe here for the quantification of NKA. The use of a relative standard curve allows this ELISA to be easily adapted to other proteins and across the animal kingdom. Our results revealed that the three conventional methods significantly underestimate the concentration of NKA compared to the ELISA. Further, by applying the protein concentrations determined by the different methods to in vitro assays, we found that variation in the resulting data was consistently low when the assay reactions were prepared based on concentrations determined from the ELISA. Thus, when target protein concentrations vary across samples, the conventional quantification methods cannot produce reliable results in downstream applications. In contrast, the ELISA we describe here consistently provides robust results.

Do all portable cases constructed by caddisfly larvae function in defense?

The portable cases constructed by caddisfly larvae have been assumed to act as a mechanical defense against predatory attacks. However, previous studies have compared the survival of caddisflies with different cases, thereby precluding an analysis of the survival benefits of "weaker" case materials. The level of protection offered by caddisfly cases constructed with rock, stick, or leaf material, as well as a no-case control, was investigated against predatory dragonfly nymphs (Anax junius Drury (Anisoptera: Aeshnidae)). A valid supposition is that the cases made of stronger material are more effective at deterring predators. Yet, observations revealed that there was no difference in survival between the case types. All caddisflies with a case experienced high survival in comparison to caddisflies removed from their case. In addition, larvae with stick-cases experienced fewer attacks and captures by dragonflies. These results showed that the presence of a case, regardless of the material used in its construction, offers survival benefits when faced with predatory dragonfly nymphs.

Virtual Dissection: an Educational Technology to Enrich Medical Students' Learning Environment in Gastrointestinal Anatomy Course.

Background: Virtual dissection provides a digital experience of medical images to visualize anatomy on touchscreen tables. This study aimed to integrate the virtual dissection table (VDT) into the gastrointestinal anatomy course and assess medical students' intended learning outcomes and satisfaction with this educational technology. Methods: This quasi-experimental study enrolled second-year undergraduate medical students who studied anatomical sciences in the autumn semester of 2021-2022 at a single medical school. In the intervention and control groups, the participants were randomized to study anatomy by VDT or topographical anatomy textbooks. The knowledge tests evaluated the students' learning outcomes of gastrointestinal anatomy, and following the course, students completed a satisfaction survey. Results: The findings indicated that a significant gain occurred, and instructional intervention during which the learning environment was enriched with virtual dissection could enhance the students' learning (F = 13.33, df = 2, P < 0.01, partial η2 = 0.20) and satisfaction (T = 6.10, df = 54, P < 0.01, Cohen's d = 1.63, CI95% = 1.02-2.23). Conclusions: This study demonstrates the potential for virtual dissection to augment anatomical science education. Further research is required to consider the contributing features and apply this educational technology to enhance students' anatomy learning. Supplementary Information: The online version contains supplementary material available at 10.1007/s40670-023-01867-z.

Intravenous ketamine compared with diclofenac suppository in suppressing acute postoperative pain in women undergoing gynecologic laparoscopy.

PURPOSE: We aimed to compare the analgesic effects of low-dose intravenous ketamine with the effects of diclofenac suppositories in acute postoperative pain management in women undergoing gynecologic laparoscopic surgery under general anesthesia. METHODS: In a double-blind, randomized clinical trial, 80 patients were selected and entered the study. After the induction of general anesthesia, one group received 0.15 mg/kg intravenous ketamine and the other group received a 100-mg rectal diclofenac suppository. The two groups were compared regarding acute pain scores, postoperative morphine requirements, and untoward complications. RESULTS: Pain scores and morphine requirements were lower in the rectal diclofenac suppository group at the 1st, 3rd, and 6th postoperative hours. Higher incidences of postoperative nausea and vomiting (PONV), delusions, and oral secretions were observed in the ketamine group. CONCLUSIONS: Diclofenac 100-mg suppositories were more effective in suppressing acute pain than 0.15 mg/kg intravenous ketamine in women undergoing elective gynecologic laparoscopy, with fewer untoward complications.

Defence mitigation by predators of chemically defended prey integrated over the predation sequence and across biological levels with a focus on cardiotonic steroids.

Predator-prey interactions have long served as models for the investigation of adaptation and fitness in natural environments. Anti-predator defences such as mimicry and camouflage provide some of the best examples of evolution. Predators, in turn, have evolved sensory systems, cognitive abilities and physiological resistance to prey defences. In contrast to prey defences which have been reviewed extensively, the evolution of predator counter-strategies has received less attention. To gain a comprehensive view of how prey defences can influence the evolution of predator counter-strategies, it is essential to investigate how and when selection can operate. In this review we evaluate how predators overcome prey defences during (i) encounter, (ii) detection, (iii) identification, (iv) approach, (v) subjugation, and (vi) consumption. We focus on prey that are protected by cardiotonic steroids (CTS)-defensive compounds that are found in a wide range of taxa, and that have a specific physiological target. In this system, coevolution is well characterized between specialist insect herbivores and their host plants but evidence for coevolution between CTS-defended prey and their predators has received less attention. Using the predation sequence framework, we organize 574 studies reporting predators overcoming CTS defences, integrate these counter-strategies across biological levels of organization, and discuss the costs and benefits of attacking CTS-defended prey. We show that distinct lineages of predators have evolved dissecting behaviour, changes in perception of risk and of taste perception, and target-site insensitivity. We draw attention to biochemical, hormonal and microbiological strategies that have yet to be investigated as predator counter-adaptations to CTS defences. We show that the predation sequence framework will be useful for organizing future studies of chemically mediated systems and coevolution.

Investigating the effect of macro-scale estimators on worldwide COVID-19 occurrence and mortality through regression analysis using online country-based data sources.

OBJECTIVE: To investigate macro-scale estimators of the variations in COVID-19 cases and deaths among countries. DESIGN: Epidemiological study. SETTING: Country-based data from publicly available online databases of international organisations. PARTICIPANTS: The study involved 170 countries/territories, each of which had complete COVID-19 and tuberculosis data, as well as specific health-related estimators (obesity, hypertension, diabetes and hypercholesterolaemia). PRIMARY AND SECONDARY OUTCOME MEASURES: The worldwide heterogeneity of the total number of COVID-19 cases and deaths per million on 31 December 2020 was analysed by 17 macro-scale estimators around the health-related, socioeconomic, climatic and political factors. In 139 of 170 nations, the best subsets regression was used to investigate all potential models of COVID-19 variations among countries. A multiple linear regression analysis was conducted to explore the predictive capacity of these variables. The same analysis was applied to the number of deaths per hundred thousand due to tuberculosis, a quite different infectious disease, to validate and control the differences with the proposed models for COVID-19. RESULTS: In the model for the COVID-19 cases (R2=0.45), obesity (ß=0.460), hypertension (ß=0.214), sunshine (ß=-0.157) and transparency (ß=0.147); whereas in the model for COVID-19 deaths (R2=0.41), obesity (ß=0.279), hypertension (ß=0.285), alcohol consumption (ß=0.173) and urbanisation (ß=0.204) were significant factors (p<0.05). Unlike COVID-19, the tuberculosis model contained significant indicators like obesity, undernourishment, air pollution, age, schooling, democracy and Gini Inequality Index. CONCLUSIONS: This study recommends the new predictors explaining the global variability of COVID-19. Thus, it might assist policymakers in developing health policies and social strategies to deal with COVID-19. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT04486508).

Treatment of atherosclerosis through transplantation of endothelial progenitor cells overexpressing dimethylarginine dimethylaminohydrolase (DDAH) in rabbits.

BACKGROUND: Endothelial dysfunction is a key event in the development of vascular diseases, including atherosclerosis. Endothelial progenitor cells (EPCs) play an important role in vascular repair. Decreased dimethylarginine dimethylaminohydrolase (DDAH) activity is observed in several pathological conditions, and it is associated with an increased risk of vascular disease. We hypothesized that bone marrow-derived EPCs and combination therapy with DDAH2-EPCs could reduce plaque size and ameliorate endothelial dysfunction in an atherosclerosis rabbit model. METHOD: Four groups of rabbits (n = 8 per group) were subjected to a hyperlipidemic diet for a month. After establishing the atherosclerosis model, rabbits received 4 × 106 EPC, EPCs expressing DDAH2, through femoral vein injection, or saline (the control group with basic food and the untreated group). One month after transplantation, plaque thickness, endothelial function, oxidative stress, and inflammatory mRNAs, DDAH, and eNOS function were assessed. RESULTS: DDAH2-EPCs transplantation (p < 0.05) and EPCs transplantation (p < 0.05) were both associated with a reduction in plaque size compared to the control saline injection. The antiproliferative and antiatherogenic effects of EPCs were further enhanced by the overexpression of DDAH2 (p < 0.05, DDAH2-EPCs vs. EPCs). Furthermore, DDAH2-EPCs transplantation significantly increased endothelium integrity compared to the EPCs transplantation. CONCLUSION: Transplantation of EPCs overexpressing DDAH2 may enhance the repair of injured endothelium by reducing inflammation and restoring endothelial function. Therefore, pCMV6-mediated DDAH2 gene-transfected EPCs are a potentially valuable tool for the treatment of atherosclerosis.

Concerted evolution reveals co-adapted amino acid substitutions in Na+K+-ATPase of frogs that prey on toxic toads.

Although gene duplication is an important source of evolutionary innovation, the functional divergence of duplicates can be opposed by ongoing gene conversion between them. Here, we report on the evolution of a tandem duplication of Na+,K+-ATPase subunit α1 (ATP1A1) shared by frogs in the genus Leptodactylus, a group of species that feeds on toxic toads. One ATP1A1 paralog evolved resistance to toad toxins although the other retained ancestral susceptibility. Within species, frequent non-allelic gene conversion homogenized most of the sequence between the two copies but was counteracted by strong selection on 12 amino acid substitutions that distinguish the two paralogs. Protein-engineering experiments show that two of these substitutions substantially increase toxin resistance, whereas the additional 10 mitigate their deleterious effects on ATPase activity. Our results reveal how examination of neo-functionalized gene duplicate evolution can help pinpoint key functional substitutions and interactions with the genetic backgrounds on which they arise.