Cytotoxic effect of carbohydrate derivatives of digitoxigenin involves modulation of plasma membrane Ca2+ -ATPase.

Cardiac glycosides, such as digoxin and digitoxin, are compounds that interact with Na+ /K+ -ATPase to induce anti-neoplastic effects; however, these cardiac glycosides have narrow therapeutic index. Thus, semi-synthetic analogs of digitoxin with modifications in the sugar moiety has been shown to be an interesting approach to obtain more selective and more effective analogs than the parent natural product. Therefore, the aim of this study was to assess the cytotoxic potential of novel digitoxigenin derivatives, digitoxigenin-α-L-rhamno-pyranoside (1) and digitoxigenin-α-L-amiceto-pyranoside (2), in cervical carcinoma cells (HeLa) and human diploid lung fibroblasts (Wi-26-VA4). In addition, we studied the anticancer mechanisms of action of these compounds by comparing its cytotoxic effects with the potential to modulate the activity of three P-type ATPases; Na+ /K+ -ATPase, sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), and plasma membrane Ca2+ -ATPase (PMCA). Briefly, the results showed that compounds 1 and 2 were more cytotoxic and selectivity for HeLa tumor cells than the nontumor cells Wi-26-VA4. While the anticancer cytotoxicity in HeLa cells involves the modulation of Na+ /K+ -ATPase, PMCA and SERCA, the modulation of these P-type ATPases was completely absent in Wi-26-VA4 cells, which suggest the importance of their role in the cytotoxic effect of compounds 1 and 2 in HeLa cells. Furthermore, the compound 2 inhibited directly erythrocyte ghosts PMCA and both compounds were more cytotoxic than digitoxin in HeLa cells. These results provide a better understanding of the mode of action of the synthetic cardiac glycosides and highlights 1 and 2 as potential anticancer agents.

Evaluation of Cardiotonic Steroid Modulation of Cellular Cholesterol and Phospholipid.

We have previously shown that 21-benzylidene digoxin (21-BD) increases the total cholesterol and phospholipid content on the membrane of HeLa cells. Lipid modulation caused by cardiotonic steroids (CTS) is still unexplored. Therefore, the aim of the present study was to evaluate the cholesterol and phospholipid modulation of the cell membrane caused by ouabain and 21-BD and the possible involvement of the caveolae on this modulation. For this, one cell line containing caveolae (HeLa) and other not containing (Caco-2) were used. The modulation of the lipid profile was evaluated by total cholesterol and phospholipids measurements, and identification of membrane phospholipids by HPTLC. The cholesterol distribution was evaluated by filipin staining. The caveolin-1 expression was evaluated by Western Blotting. Ouabain had no effect on the total membrane lipid content in both cell lines. However, 21-BD increased total membrane phospholipid content and had no effect on the membrane cholesterol content in Caco-2 cells. CTS were not able to alter the specific phospholipids content. In the filipin experiments, 21-BD provoked a remarkable redistribution of cholesterol to the perinuclear region of HeLa cells. In Caco-2 cells, it was observed only a slight increase in cholesterol, especially as intracellular vesicles. The caveolin-1 expression was not altered by any of the compounds. Our data mainly show different effects of two cardiotonic steroids. Ouabain had no effect on the lipid profile of cells, whereas 21-BD causes important changes in cholesterol and phospholipid content. Therefore, the modulation of cholesterol content in the plasma membrane of HeLa cells is not correlated with the expression of caveolin-1.

Implications of Synthetic Modifications of the Cardiotonic Steroid Lactone Ring on Cytotoxicity.

Na,K-ATPase (NKA) and cardiotonic steroids (CTS) have shown potent cytotoxic and anticancer effects. Here, we have synthesized a series of CTS digoxin derivatives (γ-benzylidene) with substitutions in the lactone ring and evaluated the cytotoxicity caused by digoxin derivatives in tumor and non-tumor cells lines, as well as their effects on NKA. The cytotoxicity assay was determined in HeLa, A549, and WI-26 VA4 after they were treated for 48 h with increased concentrations of CTS. The effects of CTS on NKA activity and immunoblotting of α1 and ß1 isoforms were evaluated at IC50 concentrations in A549 cell membrane. NKA activity from mouse brain cortex was also measured. The majority of CTS exhibited low cytotoxicity in tumor and non-tumor cells, presenting IC50 values at micromolar concentrations, while digoxin showed cytotoxicity at nanomolar concentrations. BD-15 presented the lowest IC50 value (8 µM) in A549 and reduced its NKA activity in 28%. In contrast, BD-7 was the compound that most inhibited NKA (56% inhibition) and presented high IC50 value for A549. In mouse cortex, only BD-15 modulated the enzyme activity in a concentration-dependent inhibition curve. These results demonstrate that the cytotoxicity of these compounds is not related to NKA inhibition. The substitutions in the lactone ring of digoxin led to an increase in the cytotoxic concentration in tumor cells, which may not be interesting for cancer, but it has the advantage of increasing the therapeutic margin of these molecules when compared to classic CTS, and can be used safely in research for other diseases.

Tumor Tissue Oxidative Stress Changes and Na, K-ATPase Evaluation in Head and Neck Squamous Cell Carcinoma.

Changes in metabolism are mechanisms that are largely implicated in the development, progression, and metastasis of head and neck squamous cell carcinoma (HNSCC) and also in resistance to different anticancer therapies. Identification of biomarkers for differentiation between cancerous and normal epithelium, treatment design and prognosis remain a vital issue in the field of head and neck cancer. The present study analyzed the main biochemical changes that occur in HNSCC tumors by through mechanisms involving oxidative stress. The release of substances reactive to thiobarbituric acid was significantly lower in HNSCC tumor tissue as compared to healthy tissue. The assays related to the lipid profile assays showed changes in membrane biophysics of tumor cells due to an increase in total phospholipids and total cholesterol, as well as an increased activity and expression of the α1 subunit of Na, K-ATPase, which is fundamental in the process of carcinogenesis. The modulation of the antioxidant system was also affected, with a decrease in the catalytic activity of the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), as well as a reduction of glutathione (GSH) content and an increase in H2O2 content. A reduction in catalase (CAT) activity was observed. The data presented here are in accordance with important findings described by us in a previous study, involving the same individuals, but with a focus on the damage generated in red blood cells, resulting from tumor installation. Therefore, it was possible to conclude that the biochemical alterations found in HNSCC cells are fundamental for transformation and maintenance of the tumor cell and once it is installed, it is also capable of generating injuries in the patients' red blood cells. Our data demonstrate that this could be a promising biomarker for HNSCC.

The γ-Benzylidene Digoxin Derivative BD-15 Increases the α3-Na, K-ATPase Activity in Rat Hippocampus and Prefrontal Cortex and no Change on Heart.

Our study aimed to investigate the effects of the new cardiotonic steroid BD-15 (γ-benzylidene derivatives) in the behavioral parameters, oxidative stress and the Na, K-ATPase activity in the hippocampus, prefrontal cortex and heart from rats to verify the safety and possible utilization in brain disorders. For this study, groups of male Wistar rats were used after intraperitoneal injection of 20, 100 and 200 µg/Kg with BD-15. The groups were treated for three consecutive days and the control group received 0.9% saline. BD-15 did not alter behavior of rats treated with different doses. An increase in the specific α2,3-Na, K-ATPase activity was observed for all doses of BD-15 tested in the hippocampus. However, in the prefrontal cortex, only the dose of 100 µg/Kg increased the activity of all Na, K-ATPase isoforms. BD-15 did not cause alteration in the lipid peroxidation levels in the hippocampus, but in the prefrontal cortex, a decrease of lipid peroxidation (~ 25%) was observed. In the hippocampus, GSH levels increased with all doses tested, while in the prefrontal cortex no changes were found. Subsequently, when the effect of BD-15 on cardiac tissue was analyzed, no changes were observed in the tested parameters. BD-15 at a dosage of 100 µg/Kg proved to be promising because it is considered therapeutic for brain disorders, since it increases the activity of the α3-Na, K-ATPase in the hippocampus and prefrontal cortex, as well as decreasing the oxidative stress in these brain regions. In addition, this drug did not cause changes in the tissues of the heart and kidneys, preferentially demonstrating specificity for the brain.

Cytotoxicity of glucoevatromonoside alone and in combination with chemotherapy drugs and their effects on Na+,K+-ATPase and ion channels on lung cancer cells.

Cardiac glycosides (CGs) are useful drugs to treat cardiac illnesses and have potent cytotoxic and anticancer effects in cultured cells and animal models. Their receptor is the Na+,K+ ATPase, but other plasma membrane proteins might bind CGs as well. Herein, we evaluated the short- and long-lasting cytotoxic effects of the natural cardenolide glucoevatromonoside (GEV) on non-small-cell lung cancer H460 cells. We also tested GEV effects on Na+,K+ -ATPase activity and membrane currents, alone or in combination with selected chemotherapy drugs. GEV reduced viability, migration, and invasion of H460 cells spheroids. It also induced cell cycle arrest and death and reduced the clonogenic survival and cumulative population doubling. GEV inhibited Na+,K+-ATPase activity on A549 and H460 cells and purified pig kidney cells membrane. However, it showed no activity on the human red blood cell plasma membrane. Additionally, GEV triggered a Cl-mediated conductance on H460 cells without affecting the transient voltage-gated sodium current. The administration of GEV in combination with the chemotherapeutic drugs paclitaxel (PAC), cisplatin (CIS), irinotecan (IRI), and etoposide (ETO) showed synergistic antiproliferative effects, especially when combined with GEV + CIS and GEV + PAC. Taken together, our results demonstrate that GEV is a potential drug for cancer therapy because it reduces lung cancer H460 cell viability, migration, and invasion. Our results also reveal a link between the Na+,K+-ATPase and Cl- ion channels.

Molecular evolutionary insights from PRLR in mammals.

Prolactin (PRL) is a pleiotropic neurohormone secreted by the mammalian pituitary gland into the blood, thus reaching many tissues and organs beyond the brain. PRL binds to its receptor, PRLR, eliciting a molecular signaling cascade. This system modulates essential mammalian behaviors and promotes notable modifications in the reproductive female tissues and organs. Here, we explore how the intracellular domain of PRLR (PRLR-ICD) modulates the expression of the PRLR gene. Despite differences in the reproductive strategies between eutherian and metatherian mammals, there is no clear distinction between PRLR-ICD functional motifs. However, we found selection signatures that showed differences between groups, with many conserved functional elements strongly maintained through purifying selection across the class Mammalia. We observed a few residues under relaxed selection, the levels of which were more pronounced in Eutheria and particularly striking in primates (Simiiformes), which could represent a pre-adaptive genetic element protected from purifying selection. Alternative, new motifs, such as YLDP (318-321) and others with residues Y283 and Y290, may already be functional. These motifs would have been co-opted in primates as part of a complex genetic repertoire related to some derived adaptive phenotypes, but these changes would have no impact on the primordial functions that characterize the mammals as a whole and that are related to the PRL-PRLR system.

Free combination of dutasteride plus tamsulosin for the treatment of benign prostatic hyperplasia in South Korea: analysis of drug utilization and adverse events using the National Health Insurance Review and Assessment Service database.

OBJECTIVE: To assess the use and safety of free combination therapy (dutasteride and tamsulosin), dutasteride monotherapy, or tamsulosin monotherapy in patients with benign prostatic hyperplasia (BPH). METHODS: This non-interventional retrospective cohort study used claims data from the Korea Health Insurance Review and Assessment-National Patient Sample database. Patients with BPH ≥ 40 years of age receiving combination therapy (dutasteride 0.5 mg and tamsulosin 0.4 mg daily) or dutasteride 0.5 mg, or tamsulosin 0.4 mg daily dose between 2012 and 2017 were included. The frequency, duration of treatment and risk of any adverse event (AE) or serious AE (SAE) was compared for combination therapy versus each monotherapy using non-inferiority testing. RESULTS: Of 14,755 eligible patients, 1529 (10.4%) received combination therapy, 6660 (45.1%) dutasteride monotherapy, and 6566 (44.5%) tamsulosin monotherapy. The proportion of patients treated with combination therapy exceeded the pre-specified 3% threshold for 'frequent' use. Safety results indicated a similar risk of any AE and SAE irrespective of treatment group. The adjusted relative risk for any AE over the treatment observation period comparing combination therapy with dutasteride monotherapy was 1.07 (95% confidence interval [CI] 1.03, 1.12), and with tamsulosin monotherapy was 0.98 (95% CI 0.95, 1.02) demonstrating non-inferiority. The adjusted relative risk for any SAE was 1.07 (95% CI 0.66, 1.74) and 0.90 (95% CI 0.56, 1.45), compared with dutasteride and tamsulosin monotherapy, respectively. Although the SAE results did not statistically demonstrate non-inferiority of combination therapy based on pre-specified margins, the 95% CI for the risk ratio estimates included the null with a lower limit below the non-inferiority margins, indicating no meaningful differences in SAE risk between groups. Absolute SAE risks were low. CONCLUSION: Combination therapy with dutasteride and tamsulosin is frequently used in real-world practice in South Korea for treatment of BPH and demonstrates a safety profile similar to either monotherapy.

Methylphenidate effects on mice odontogenesis and connections with human odontogenesis.

The purpose of this study is to evaluate the effects of Methylphenidate exposure on mice odontogenesis and connect them by bioinformatics with human odontogenesis. Thirty-two pregnant Swiss mice were divided into treated group and control group, which received, respectively, 5 mg/kg of Methylphenidate and saline solution from the 5th to the 17th day of pregnancy. The mouse embryos tooth germs were analyzed through optical microscopy, and the data collected were analyzed statistically by Fisher's exact test. The presence and similarity of Methylphenidate-associated genes (Pharmgkb database) in both organisms and their interaction with dental development genes (AmiGO2 database) were verified on STRING database. Rates of tooth germ malformations were higher in treated than in control group (Control: 18; Treated: 27; p = 0.035). Mouse embryo malformations were connected with 238 interactions between 69 dental development genes with 35 Methylphenidate genes. Fourteen interactions for four Methylphenidate genes with four dental development genes, with human experimental data, were connected with mouse phenotype data. By homology, the interactions and conservation of proteins/genes may indicate similar outcomes for both organisms. The exposure to Methylphenidate during pregnancy affected odontogenesis in mouse embryos and may affect human odontogenesis. The study of malformations in mice, with a bioinformatics approach, could contribute to understanding of the Methylphenidate effect on embryo development. These results may provide novel hypotheses for further testing and reinforce the FDA protocol: as Methylphenidate is included in category C, its use during pregnancy should be considered if the benefits outweigh the risks.

Evaluation of the Erythrocyte Membrane in Head and Neck Cancer Patients.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous and complex disease, both from a clinical and molecular point of view. The prolonged use of alcohol and tobacco, along with the release of tumor secretions can modulate blood cells, such as erythrocytes. Here, this study was conducted with 24 patients diagnosed with HNSCC and an equal number of healthy individuals are matched by age and gender. The levels of lipid peroxidation were measured using the individual plasma, while for lipid concentrations, identification and quantification Na, K-ATPase activity and osmotic fragility, the red blood cell concentrate were used. The release of TBARS was significantly higher in patients with HNSCC. The lipid profile assays demonstrated a rearrangement of the erythrocyte membrane due to a decrease in total phospholipids and phosphatidylethanolamine followed by an increase in total cholesterol and phosphatidylcholine. Na, K-ATPase activity also increased. Erythrocytes were more fragile in patients with HNSCC than in health individuals. Therefore, the membrane of erythrocytes were rearranged and Na, K-ATPase function altered in the HNSCC patients. Our findings suggests that the alcohol, tobacco and tumor secretion modulate in a specific manner that the erythrocytes membranes of these patients making this system a potential tool for HNSCC biomarker of tumor progression.

Oxidative stress assessment in sickle cell anemia patients treated with hydroxyurea.

Hydroxyurea (HU) is used as a therapy in sickle cell anemia (SCA). Many studies have established that HU improves patient quality of life by reducing symptoms. However, the effect of HU on erythrocytes is not well-described. We evaluated several parameters related to oxidative stress and total lipid content of erythrocytes in patients with SCA. The patient cohort consisted of 7 SCA patients treated with HU, 17 untreated SCA patients, and 15 healthy subjects. Erythrocytes from patients with SCA displayed increased oxidative stress relative to the control group, including higher thiobarbituric acid reactive substances (TBARS), Fe3+ content, and osmotic fragility, and decreased total cholesterol. We observed that treatment of SCA patients with HU increased Fe3+ content and activity of glutathione peroxidase, and decreased glutathione reductase activity, glutathione levels, total cholesterol, and phospholipid content comaperaded to patients untreated with HU. Thus, HU alters biochemical characteristics of erythrocytes; future studies will determine whether they are beneficial or not.

Persistence and adherence to dutasteride/tamsulosin fixed-dose versus free-combination alpha blocker/5ARI therapy in patients with benign prostate hyperplasia in Germany
.

OBJECTIVE: To evaluate real-world persistence and adherence in patients with benign prostate hyperplasia (BPH) receiving a fixed-dose combination of dutasteride plus tamsulosin (DUT-TAM FDC) versus α-blocker plus 5-α reductase inhibitor (AB/5ARI) free-combination therapy. MATERIALS AND METHODS: This retrospective, observational cohort study utilized the German IMS LRx (IQVIA) database. Patients ≥ 45 years old with BPH receiving DUT-TAM FDC or AB/5ARI free-combination therapy from July 1, 2011 to November 30, 2017 were included. Data were analyzed for 48 months from index date (date of first prescription). Persistence, measured as time to discontinuation (defined as a 90-day gap in therapy), was evaluated using Kaplan-Meier curves (log-rank tests). Adherence, measured as medication possession ratio (MPR), was based on a comparison of mean prescribing duration and expected treatment duration. RESULTS: A total of 141,667 patients were included (DUT-TAM FDC, n = 86,057; free AB/5ARI: n = 55,610). Small differences in persistence were observed between treatment arms. At month 12, 41.8% of DUT-TAM FDC-treated and 41.0% of AB/5ARI free-combination therapy-treated patients were persistent; at month 24, 28.2% and 27.1% were persistent, respectively. A higher proportion of DUT-TAM FDC-treated patients had MPR ≥ 0.80, ≥ 0.75 and ≥ 0.70 compared with AB/5ARI free-combination therapy (p < 0.0001). CONCLUSION: Small differences observed in persistence between treatment arms may not translate to meaningful clinical relevance. Adherence was significantly better in the FDC arm, which may be clinically relevant as improved adherence is associated with better outcomes. Persistence and adherence to BPH therapy in Germany is low; further studies exploring the reasons behind this are required.

Functional New World monkey oxytocin forms elicit an altered signaling profile and promotes parental care in rats.

The neurohormone oxytocin is a key player in the modulation of reproductive and social behavioral traits, such as parental care. Recently, a correlation between different forms of oxytocin and behavioral phenotypes has been described in the New World Monkeys (NWMs). Here, we demonstrate that, compared with the Leu8OXT found in most placental mammals, the Cebidae Pro8OXT and Saguinus Val3Pro8OXT taxon-specific variants act as equi-efficacious agonists for the Gq-dependent pathway but are weaker agonists for the ß-arrestin engagement and subsequent endocytosis toward the oxytocin receptor (OXTR). Upon interaction with the AVPR1a, Pro8OXT and the common Leu8OXT yielded similar signaling profiles, being equally efficacious on Gq and ß-arrestin, while Val3Pro8OXT showed reduced relative efficacy toward ß-arrestin. Intranasal treatment with either of the variants increased maternal behavior and also promoted unusual paternal care in rats, as measured by pup-retrieval tests. We therefore suggest that Val3Pro8OXT and Pro8OXT are functional variants, which might have been evolutionarily co-opted as an essential part of the adaptive genetic repertoire that allowed the emergence of taxon-specific complex social behaviors, such as intense parental care in the Cebidae and the genus Saguinus.

Antagonistic anticancer effect of paclitaxel and digoxin combination.

Despite the growing interest in the antitumor effect of cardiotonic steroids, combination treatments with well-established chemotherapy drugs like paclitaxel have been rarely investigated. Moreover, paclitaxel has been suggested as a Na+ /K+ -ATPase inhibitor. Here we investigated the effect of paclitaxel and digoxin alone or in combination on the viability of human lung (A549) and cervical cancer (HeLa) cell lines and the inhibitory effect of paclitaxel on several mammalian Na+ /K+ -ATPases. Although the viability of both tumor cell lines was concentration-dependently affected by digoxin treatment after 48 hours (A549 IC50 = 31 nM and HeLa IC50 = 151 nM), a partial effect was observed for paclitaxel, with a maximal inhibitory effect of 45% at 1000 nM with A549 and around 70% with HeLa cells (IC50 = 1 nM). Although the two drugs were cytotoxic, their combined effect in HeLa cells was revealed to be antagonistic, as estimated by the combination index. No direct inhibitory effect of paclitaxel was detected in human, pig, rat, and mouse Na+ /K+ -ATPase enzymes, but high concentrations of paclitaxel decreased the Na+ /K+ -ATPase activity in HeLa cells after 48 hours without affecting protein expression. Our findings demonstrate that, under our conditions, paclitaxel and digoxin cotreatment produce antagonistic cytotoxic effects in HeLa cells, and the mechanism of action of paclitaxel does not involve a direct inhibition of Na+ /K+ -ATPase. More studies shall be designed to evaluate the consequences of the interaction of cardiotonic steroids and chemotherapy drugs.

Ouabain attenuates oxidative stress and modulates lipid composition in hippocampus of rats in lipopolysaccharide-induced hypocampal neuroinflammation in rats.

Our study aimed to analyze the effect of ouabain (OUA) administration on lipopolysaccharide (LPS)-induced changes in hippocampus of rats. Oxidative parameters were analyzed in Wistar rats after intraperitoneal injection of OUA (1.8 µg/kg), LPS (200 µg/kg), or OUA plus LPS or saline. To reach our goal, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), in addition to levels of reduced glutathione (GSH), protein carbonyl (PCO) and lipid peroxidation (LPO) were evaluated. We also analyzed the membrane lipid profile and some important lipids for the nervous system, such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid and sphingomyelin. The group that received only LPS showed increased oxidative stress, as evidenced by an increase in LPO (about twice), PCO (about three times) levels, and CAT activity (80%). Conversely, administration of LPS decreased GSH levels (55%), and GPx activity (30%), besides a reduction in the amount of PI (60%) and PC (45%). By other side, OUA alone increased the amount of PI (45%), PE (85%), and PC (70%). All harmful effects recorded were attenuated by OUA, suggesting a protective effect against LPS-induced oxidative stress. The relevance of our results extends beyond changes in oxidative parameters induced by LPS, because nanomolar doses of OUA may be useful in neurodegenerative models. Other studies on other cardenolides and substances related issues, as well as the development of new molecules derived from OUA, could also be useful in general oxidative and/or cellular stress, a condition favoring the appearance of neuronal pathologies.

Evaluation of neuroprotective activity of digoxin and semisynthetic derivatives against partial chemical ischemia.

Recently, cardiotonic steroids (CTS) have been shown to lead to the activation of Na,K-ATPase at low concentrations in brain, promoting neuroprotection against ischemia. We report here the results of the use of digoxin and its semisynthetic derivatives BD-14, BD-15, and BD-16 against partial chemical ischemic induction followed by reperfusion in murine neuroblastoma cells neuro-2a (N2a). For chemical ischemic induction, sodium azide (5 mM) was used for 5 hours, and then reperfusion was induced for 24 hours. Na,K-ATPase activity and protein levels were analyzed in membrane preparation of N2a cells pretreated with the compounds (150 nM), in the controls and in induced chemical ischemia. In the Na,K-ATPase activity and protein levels assays, the steroids digoxin and BD-15 demonstrated a capacity to modulate the activity of the enzyme directly, increasing its levels of expression and activity. Oxidative parameters, such as superoxide dismutase (SOD) activity, lipid peroxidation (thiobarbituric acid reactive substance), glutathione peroxidase (GPx), glutathione (GSH) levels, hydrogen peroxide content, and the amount of free radicals (reactive oxygen species) during induced chemical ischemia were also evaluated. Regarding the redox state, lipid peroxidation, hydrogen peroxide content, and GPx activity, we have observed an increase in the chemical ischemic group, and a reduction in the groups treated with CTS. SOD activity increased in all treated groups when compared to control and GSH levels decreased when treated with sodium azide and did not change with CTS treatments. Regarding the lipid profile, we saw a decrease in the content of phospholipids and cholesterol in the chemical ischemic group, and an increase in the groups treated with CTS. In conclusion, the compounds used in this study demonstrate promising results, since they appear to promote neuroprotection in cells exposed to chemical ischemia.

Evaluation of the effect of cafeteria diet on the kidney Na,K-ATPase activity, and oxidative stress.

In this study, renal tissue, subdivided into the cortex and medulla of Wistar rats subjected to a cafeteria diet (CAF) for 24 days or to normal diet, was used to analyze whether the renal enzyme Na,K-ATPase activity was modified by CAF diet, as well as to analyze the α1 subunit of renal Na,K-ATPase expression levels. The lipid profile of the renal plasma membrane and oxidative stress were verified. In the Na,K-ATPase activity evaluation, no alteration was found, but a significant decrease of 30% in the cortex was detected in the α1 subunit expression of the enzyme. There was a 24% decrease in phospholipids in the cortex of rats submitted to CAF, a 17% increase in cholesterol levels in the cortex, and a 23% decrease in the medulla. Lipid peroxidation was significantly increased in the groups submitted to CAF, both in the cortical region, 29%, and in the medulla, 35%. Also, a reduction of 45% in the glutathione levels was observed in the cortex and medulla with CAF. CAF showed a nearly two-fold increase in glutathione peroxidase (GPX) activity in relation to the control group in the cortex and a 59% increase in the GPx activity in the medulla. In conclusion, although the diet was administered for a short period of time, important results were found, especially those related to the lipid profile and oxidative stress, which may directly affect renal function.

AVPR1b variation and the emergence of adaptive phenotypes in Platyrrhini primates.

Platyrrhini (New World monkeys, NWm) are a group of primates characterized by behavioral and reproductive traits that are otherwise uncommon among primates, including social monogamy, direct paternal care, and twin births. As a consequence, the study of Platyrrhine primates is an invaluable tool for the discovery of the genetic repertoire underlying these taxon-specific traits. Recently, high conservation of vasopressin (AVP) sequence, in contrast with high variability of oxytocin (OXT), has been described in NWm. AVP and OXT functions are possible due to interaction with their receptors: AVPR1a, AVPR1b, AVPR2, and OXTR; and the variability in this system is associated with the traits mentioned above. Understanding the variability in the receptors is thus fundamental to understand the function and evolution of the system as a whole. Here we describe the variability of AVPR1b coding region in 20 NWm species, which is well-known to influence behavioral traits such as aggression, anxiety, and stress control in placental mammals. Our results indicate that 4% of AVPR1b sites may be under positive selection and a significant number of sites under relaxed selective constraint. Considering the known role of AVPR1b, we suggest that some of the changes described here for the Platyrrhini may be a part of the genetic repertoire connected with the complex network of neuroendocrine mechanisms of AVP-OXT system in the modulation of the HPA axis. Thus, these changes may have promoted the emergence of social behaviors such as direct paternal care in socially monogamous species that are also characterized by small body size and twin births.

Involvement of Src signaling in the synergistic effect between cisplatin and digoxin on cancer cell viability.

Cisplatin and other platinum-containing drugs have played a crucial role in anticancer treatments for over 30 years. However, treatment with cisplatin may cause serious side effects, such as myelosuppression, nausea, ototoxicity, nephrotoxicity, and cell resistance processes. In addition, cardiotonic steroids, particularly digoxin, have recently been suggested to exert potent anticancer effects. Therefore, it is possible that the combined treatment of HeLa cells with cisplatin and digoxin can ameliorate the cytotoxic effects and decrease the side effects of cisplatin. In this study, we demonstrated that the interaction between cisplatin and digoxin had a synergistic effect on cervical cancer cells and a significantly positive cytotoxic and antiproliferative effect on this cell line compared to the control and single cisplatin treatments. Although a decrease in the Na,K-ATPase α1 subunit expression was observed in total extracts, its expression remains unchanged in the membrane, as does the Na,K-ATPase activity. The antiproliferative effect of the synergistic treatment appears to depend on Src kinase activation, indicating the possible involvement of the Scr-EGFR-ERK1/2 pathway in the antitumor effect. The inhibition of ERK1/2 provoked the same synergism with 1 µM cisplatin as that observed with 1 nM digoxin plus 1 µM cisplatin but not with 1 nM digoxin. Pretreatment with PP2 during combined treatment abolished the synergistic effect on the antiproliferative activity. Cisplatin and digoxin are already used in the clinical setting; therefore, this study opens possibilities for future clinical trials of combined treatments to improve treatment outcomes with a lower incidence of toxicity and side effects.

Differential burden of rare protein truncating variants in Alzheimer's disease patients compared to centenarians.

We compared coding region variants of 53 cognitively healthy centenarians and 45 patients with Alzheimer's disease (AD), all of Ashkenazi Jewish (AJ) ancestry. Despite the small sample size, the known AD risk variant APOE4 reached genome-wide significance, indicating the advantage of utilizing 'super-controls'. We restricted our subsequent analysis to rare variants observed at most once in the 1000 Genomes database and having a minor allele frequency below 2% in our AJ sample. We compared the burden of predicted protein altering variants between cases and controls as normalized by the level of rare synonymous variants. We observed an increased burden among AD subjects for predicted loss-of-function (LoFs) variants defined as stop-gain, frame shift, initiation codon (INIT) and splice site mutations (n = 930, OR = 1.3, P = 1.5×E-5). There was no enrichment across all rare protein altering variants defined as missense plus LoFs, in frame indels and stop-loss variants (n = 13 014, OR = 0.97, P = 0.47). Among LoFs, the strongest burden was observed for INIT (OR = 2.16, P = 0.0097) and premature stop variants predicted to cause non-sense-mediated decay in the majority of transcripts (NMD) (OR = 1.98, P = 0.02). Notably, this increased burden of NMD, INIT and splice variants was more pronounced in a set of 1397 innate immune genes (OR = 4.55, P = 0.0043). Further comparison to additional exomes indicates that the difference in LoF burden originated both from the AD and centenarian sample. In summary, we observed an overall increased burden of rare LoFs in AD subjects as compared to centenarians, and this enrichment is more pronounced for innate immune genes.