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.

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.

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.

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.

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).

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).

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.

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.

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.

Investigation of moral intelligence's predictive components in students of Shahid Beheshti university of medical sciences (SBMU).

This study aimed to investigate dominant predictor components of moral intelligence (MI) based on the Lennick and Kiel's model in students of Shahid Beheshti University of Medical Sciences (SBMU). In this descriptive-analytical study, 322 students of SBMU were chosen through cluster sampling. To collect data, a 40-item questionnaire, whose validity and reliability was confirmed in previous studies, based on the Lennick and Kiel's model was used. The collected data were analyzed by SPSS 21 software using appropriate descriptive and analytical statistics. Of 322 participants, 180 and 142 were female and male, respectively. The mean age of the participants was 22.30±2.69 years. The study's regression analysis revealed that the most and the least direct effects were related to the forgiveness (R2=0.320) and compassion (R2=0.284) components, respectively. Among the inspected components, the responsibility component with an overall effect of R2=0.655 was shown to be the strongest predictor component of MI. Universities play a significant role in students' moral development and enhancement. The present study's findings suggest that developing strategic plans and interventions can enhance MI level (e.g., incentive systems for individuals maintaining high moral responsibility). Since today's students will be tomorrow's medical and healthcare professionals, upgrading of MI level in students studying in various divisions of medical sciences enhances their moral responsibility through setting out strong ethics principles to follow and the quality of care that they will provide to patients, thereby improving health.

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.

Correlation between expression of CatSper1,2 and sperm parameters in the gamma irradiated adult mouse testis.

Propose: CatSper protein channels are responsible for the entry of Ca2+ into sperm cells. These proteins play an important role in motility and male fertility. So it is important to find out whether or not environmental factors, such as gamma radiation, have an effect on the expression of Catsper genes. In this study, we investigated the effects of gamma radiation on the expression of CatSper1 and CatSper2 genes. Materials and methods: Twenty-one male NMRI mice were divided into three groups: a control group without gamma radiation, and two experimental groups; Group 1 treated with 1 Gy of gamma radiation, and Group 2 treated with a higher dose of 2 Gy gamma radiation. Testes were removed from all groups of animals 35 days following irradiation and the testicular tissue, processed and embedded in paraffin blocks for sectioning and histological examination. Sperm samples were also taken from the epididymis for microscopic. Sperm parameters such as sperm count, morphology, motility, and viability rates were analyzed. Expression of CatSper genes was evaluated using Real-time PCR. Data were analyzed using the SPSS software and ANOVA test. Results: Our results showed that after treatment with gamma radiation, testes morphology was changed. Epididymal sperm count, motility, and morphology rates were significantly affected in both experimental groups compared to the control group. The relative expressions of CatSper 1 and 2 genes were significantly reduced in the irradiated mice (1 Gy and 2 Gy) than non-irradiated ones. Conclusions: Gamma radiations not only change testes histology and sperm parameters, but also decrease the expression of CatSper 1 and 2 genes in male mice.

Clinical outcomes of the simultaneous bilateral percutaneous nephrolithotomy (PCNL) in patients with kidney stones: A prospective cohort study.

BACKGROUND: Urinary tract stones is one of the most frequent medical emergencies which leads to life-threatening complications, namely obstructive uropathy as well as renal failure in some situations. Previously, bilateral stones were treated with either open surgery or percutaneous nephrolithotomy (PCNL). However, these treatment options were associated with lengthy operation time, need for more anesthesia, further bleeding, and long hospitalization. Therefore, much effort has been made to treat both sides simultaneously. OBJECTIVE: The aim of this study was an attempt to prospectively review cases undergoing simultaneous bilateral PCNL in terms of the therapy outcomes, complications, and consequences. METHODS: In this prospective cohort study, 39 adult patients with bilateral renal stones were randomly recruited at Imam Reza hospital in Mashhad, Iran between January 2016 and January 2017. Adult patients with bilateral renal stones were included in this study. Exclusion criteria were as follows: Patients with severe heart or lung disease, patients with coagulation disorders, pregnant women, and cases with any contraindications for general anesthesia. After insertion of bilateral ureteral catheters, all patients underwent simultaneous bilateral PCNL in prone position. Transureteral lithotripsy was performed for patients with ureteral stones. The surgery was initially carried out on the symptomatic side and then iterated on the remaining kidney. Major complications including bleeding, fever, pain, urine leakage, and residual stones were recorded. SPSS software was used for data analysis. Data were expressed as percentage and mean ± SD. P value less than 0.05 was considered significant. RESULTS: A total of 39 patients (27 males with mean age of 37.6 years and 12 females with mean age of 45.7 years) were studied. As many as 15 (38%) patients received a unilateral nephrostomy. Three underwent totally tubeless surgery. Bleeding (41.0%) was the most common complication, followed by residual stones (20.5%) and fever (20.5%), urine leakage (15.3%), pain (12.8%), blood transfusion (2.5%) and colon perforation (2.5%). CONCLUSIONS: It was concluded that simultaneous bilateral PCNL was not associated with higher morbidity than the unilateral method.

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.

Snakes exhibit tissue-specific variation in cardiotonic steroid sensitivity of Na+/K+-ATPase.

Toads are among several groups of organisms chemically defended with lethal concentrations of cardiotonic steroids. As a result, most predators that prey on amphibians avoid toads. However, several species of snakes have gained resistance-conferring mutations of Na+/K+-ATPase, the molecular target of cardiotonic steroids, and can feed on toads readily. Despite recent advances in our understanding of this adaptation at the genetic level, we have lacked functional evidence for how mutations of Na+/K+-ATPase account for cardiotonic steroid resistance in snake tissues. To address this issue, it is necessary to determine how the Na+/K+-ATPases of snakes react to the toxins. Some tissues might have Na+/K+-ATPases that are more susceptible than others and can thus provide clues about how the toxins influence organismal function. Here we provide a mechanistic link between observed Na+/K+-ATPase substitutions and observed resistance using actual snake Na+/K+-ATPases. We used an in vitro approach to determine the tissue-specific levels of sensitivity to cardiotonic steroids in select resistant and non-resistant snakes. We compared the sensitivities of select tissues within and between species. Our results suggest that resistant snakes contain highly resistant Na+/K+-ATPases in their heart and kidney, both of which rely heavily on the enzymes to function, whereas tissues that do not rely as heavily on Na+/K+-ATPases or might be protected from cardiotonic steroids by other means (liver, gut, and brain) contain non-resistant forms of the enzyme. This study reveals functional evidence that tissue-level target-site insensitivity to cardiotonic steroids varies not only among species but also across tissues within resistant taxa.

The predictors of metabolic syndrome based on Walker Health-Promoting lifestyle in Iran 2016.

BACKGROUND: Understanding the metabolic syndrome has been the center of attention in recent years as a major risk factor of many diseases. Given the rising incidence of this disease and as the patient can compete against this disease with choosing healthy lifestyle, in the present study, we investigated the predictors of metabolic syndrome, based on Walker's health-promoting lifestyle in two large cities in Iran, in 2016. METHODS: This cross-sectional study used two-stage cluster sampling to select 1128 patients, among patients who referred to health centers in Isfahan and Yazd. Data were gathered by Walker lifestyle questionnaire that evaluates health-promoting behavior (nutrition, physical activity, health responsibility, spiritual growth, interpersonal relations, and stress management) by self-report that were completed by interviews for illiterate and semi-literate individuals. Data analysis was performed by SPSS statistical software. RESULTS: In this study, 576 women and 454 men participated with mean age of 56.19±12.11years and mean weight of 73.90±14.45kg. RESULTS: showed that the highest mean score for different health-promoting behaviors was related to social responsibility (25.53±5.65) and the lowest was related to nutritional status (15.18±5.79). Regarding the strongest predictor of lifestyle, the results of regression analysis showed that the most direct impact was related to stress management (R2=0.343) and the least direct impact was related to social responsibility (R2=0.243). Among the structures, physical activity with overall effect of R2=0.621 was the strongest predictor of lifestyle based on Walker's pattern. CONCLUSION: Considering the results of this study, the greater emphasis in planning and interventions to reduce the risk of metabolic syndrome should be spotted on physical activity and stress management in patients with the metabolic syndrome.

Toad toxin-resistant snake (Thamnophis elegans) expresses high levels of mutant Na+/K+-ATPase mRNA in cardiac muscle.

Toads are chemically defended by bufadienolides, which are lethal to most predators. These toxins exert their lethal effects by binding to and disabling the Na+/K+-ATPases of cell membranes. Many species of snakes exhibit resistance to the effects of bufadienolides due to target-site insensitivity of the Na+/K+-ATPase. Mutations that confer resistance have previously been identified in ATP1a3, the gene that codes for the Na+/K+-ATPase α-3 paralog. We have found that this mutant gene is expressed at a significantly elevated level in heart tissue compared to gut, kidney, and liver of the bufadienolide-resistant snake, Thamnophis elegans. Furthermore, we found that exposure to bufadienolides elicits a significant increase in the expression levels of ATP1a3 in the heart, but not in the kidneys, liver, or gut 1h after exposure. We suggest that upregulation of ATP1a3 in the heart plays an important role in the physiological processes involved in tolerance of bufadienolides among genetically resistant snakes.

Long-Term Neurodevelopmental Outcome of Neonates with Hypernatremic Dehydration.

BACKGROUND: Neonatal hypernatremic dehydration (NHD) is a dangerous condition that can lead to severe weight loss, renal impairment, and central nervous system complications. We aimed to evaluate the consequences of NHD in infants in their second year of life. MATERIALS AND METHODS: This was a prospective case-control study in Ghaem hospital, Mashhad, Iran. Sixty-five healthy breastfed neonates (serum sodium concentration <150 mmol/L) and 65 hypernatremic (serum sodium concentration ≥150 mmol/L) neonates were followed up from 2008 to 2011. Maternal and neonatal factors were compared between the two groups together with their growth parameters, and developmental milestones (using Denver II developmental assessment scores) were assessed and compared in ages 6, 12, 18, and 24 months, respectively. RESULTS: The weight of infants at 6 months of age was significantly different between the two groups (7,264 ± 1,089 g vs. 7,596 ± 957 g, p = 0.009). Twenty-five percent of infants in the group who had developed NHD had a delay in development at 6 months of age, with corresponding values of 21% at 12 months, 19% at 18 months, and 12% at 24 months of age. Developmental delay was ∼0.3% for the control group at similar ages. The severity of hypernatremia was strongly correlated with poor developmental outcome at 6 months (p = 0.001). Serum sodium concentration of neonates was 153-195 mg/dL in the NHD group. Median peak serum sodium was 158 ± 16 in case group and 141 ± 9 in control group. Serious complications were cerebral edema (five cases), hemorrhage (five cases), and kidney stones (six cases). Hypernatremic dehydration has an adverse effect on child development especially in the first year of life, their prevalence decreases with advanced age. Growth problems are also present during their first year of life. The major signs and symptoms of infants with poor prognosis on admission were poor feeding (8 infants, 61.5%), seizure (3 infants, 23.1%), hyperthermia (1 infant, 7.7%), and lethargy (1 infant, 7.7%). CONCLUSIONS: NHD affects growth parameters and developmental milestones of children. Occasionally the child's weight gain was normalized by the end of first year of life; although developmental delay continued, its severity was reduced, with age.

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.

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.