Evolutionary pattern in the OXT-OXTR system in primates: Coevolution and positive selection footprints

Oxytocin is a nonapeptide involved in a wide range of physiologic and behavioral functions. Until recently, it was believed that an unmodified oxytocin sequence was present in all placental mammals. This study analyzed oxytocin (OXT) in 29 primate species and the oxytocin receptor (OXTR) in 21 of these species. We report here three novel OXT forms in the New World monkeys, as well as a more extensive distribution of a previously described variant (Leu8Pro). In structural terms, these OXTs share the same three low-energy conformations in solution during molecular dynamic simulations, with subtle differences in their side chains. A consistent signal of positive selection was detected in the Cebidae family, and OXT position 8 showed a statistically significant (P = 0.013) correlation with litter size. Several OXTR changes were identified, some of them promoting gain or loss of putative phosphorylation sites, with possible consequences for receptor internalization and desensitization. OXTR amino acid sites are under positive selection, and intramolecular and intermolecular coevolutionary processes with OXT were also detected. We suggest that some New World monkey OXT-OXTR forms can be correlated to male parental care through the increase of cross-reactivity with its correlated vasopressin system.Oxytocin has crucial functions related to physiological processes and social behaviors in primates and other placental mammals. A nonapeptide (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly) (1), oxytocin (OXT-8Leu) is both a neurotransmitter released by neuronal cells in synapses and a hormone, activating receptors distant from the site of its synthesis through the circulatory system (2). In mammals, OXT acts as a hormone in uterine contraction during parturition and in milk ejection while lactating. It is also a key central nervous system neurotransmitter, regulating/modulating complex social and reproductive behaviors (i.e., pair bonding and parental care) (3–7).Until recently, it was believed that the OXT amino acid chain was the same in all placental mammals. However, Lee and colleagues (8) reported a T > C change in four New World monkeys (NWms), Saimiri sciureus, Cebus apella, Callithrix jacchus, and Aotus nancimae, substituting leucine to proline at position 8 (OXT-8Pro). This form was also found in Tupaia belangeri, a tree shrew species of Southeast Asia (8). OXT differs from its paralog vasopressin (AVP; Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly) at positions 3 and 8. Variation at position 8 also identifies nonplacental OXT/AVP-like nonapeptides, such as mesotocin, present in some marsupials (7, 9). These findings dispel the notion of a universal OXT amino acid sequence for placental mammals. They also suggest that residue variability at position 8, in some cases associated with variations at positions 2–5, may be connected with the recognition, binding, and activation of receptors, potentially leading to species-specific functional changes (7, 10).OXT activity depends on adequate interaction with its unique receptor, OXTR, although it can also bind to the vasopressin receptors (AVPR1a, AVPR1b, and AVPR2) with lower affinity (11–13). Similar to other receptors that use G proteins as transducer signals across the cell membranes, OXTR is composed of seven transmembrane (TM1–TM7), four extracellular (N-terminal tail-ECL3), and four intracellular (ICL1-C-terminal tail) domains. ECL and ICL are important for the interaction with OXT and G proteins, respectively, whereas TMs are connected with both functions (7, 11).In contrast to what is observed for placental mammal OXT, OXTR presents hundreds of variants in regulatory and coding regions, including at the intraspecific level. In humans, OXTR single-nucleotide polymorphisms have been associated with several social behavioral phenotypes (14).The presence of OXT-OXTR-related systems throughout the animal kingdom indicates that their typical roles in placental mammals are likely exaptations of ancient functions, such as regulation of fluid balance and egg-laying (15, 16). Studies have attempted to investigate both the interaction of OXT-OXTR-like systems and their coevolution (11, 17). However, our knowledge about this nonapeptide-receptor system, including the extent of its variability in the primate order, remains limited.NWm emerged ∼30 million years ago. They are classified into 16 genera and ∼75 species and present a wide range of reproductive and social behaviors (18, 19), but little is known about their genetic variability and concurrent phenotypic variation (20).The present study reports results about OXT and OXTR diversity in 29 primate species, including 20 NWm species. These analyses include original OXT and OXTR sequences for 16 and 12 NWm species, respectively. We discuss details about the coevolution of these systems, as well as possible connections among reported genetic variability, positive selection, and some key species-specific biologic traits.     

Adiposity,inflammation and fat-soluble vitamins in adolescents

ObjectiveEvaluate the association between inflammatory process, adiposity, and vitamins A, D, and E in adolescents, according to gender.MethodsCross-sectional study with adolescents aged 12–19 years old of both genders attending public schools in Recife. A questionnaire was used to collect data on socioeconomic level, lifestyle, and food intake of adolescents. Then, an anthropometric evaluation and a blood sampling were performed to analyze serum concentrations of α-1-acid glycoprotein, retinol, β-carotene, α-tocopherol, and 25-hydroxy-vitamin D.ResultsThe levels of α-1-acid glycoprotein were higher for abdominal obesity in both genders. Male adolescents with insufficient serum α-tocopherol levels had low levels of α-1-acid glycoprotein (p = 0.03) and an increased risk of 25-hydroxy-vitamin D and β-carotene deficiency in relation to total and abdominal fat; female adolescents had an increased risk of insufficient β-carotene with abdominal obesity (PR: 1.33; 95% CI: 1.2–1.5).ConclusionAbdominal adiposity implies a higher risk of inflammation and causes different changes to the levels of fat-soluble vitamins according to gender.     

Azole resistance in Candida albicans from animals: Highlights on efflux pump activity and gene overexpression

This study investigated potential mechanisms of azole resistance among Candida albicans from animals, including efflux pump activity, ergosterol content and gene expression. For this purpose, 30 azole‐resistant C. albicans strains from animals were tested for their antifungal susceptibility, according to document M27‐A3, efflux pump activity by rhodamine 6G test, ergosterol content and expression of the genes CDR1, CDR2, MDR1, ERG11 by RT‐qPCR. These strains were resistant to at least one azole derivative. Resistance to fluconazole and itraconazole was detected in 23 and 26 strains respectively. Rhodamine 6G tests showed increased activity of efflux pumps in the resistant strains, showing a possible resistance mechanism. There was no difference in ergosterol content between resistant and susceptible strains, even after fluconazole exposure. From 30 strains, 22 (73.3%) resistant animal strains overexpressed one or more genes. From this group, 40.9% (9/22) overexpressed CDR1, 18.2% (4/22) overexpressed CDR2, 59.1% (13/22) overexpressed MDR1 and 54.5% (12/22) overexpressed ERG11. Concerning gene expression, a positive correlation was observed only between CDR1 and CDR2. Thus, azole resistance in C. albicans strains from animals is a multifactorial process that involves increased efflux pump activity and the overexpression of different genes.     

Correlations between forced oscillation technique parameters and pulmonary densitovolumetry values in patients with acromegaly

The aims of this study were to evaluate the forced oscillation technique (FOT) andpulmonary densitovolumetry in acromegalic patients and to examine the correlationsbetween these findings. In this cross-sectional study, 29 non-smoking acromegalicpatients and 17 paired controls were subjected to the FOT and quantification of lungvolume using multidetector computed tomography (Q-MDCT). Compared with the controls,the acromegalic patients had a higher value for resonance frequency [15.3 (10.9-19.7)vs 11.4 (9.05-17.6) Hz, P=0.023] and a lower value for meanreactance [0.32 (0.21-0.64) vs 0.49 (0.34-0.96) cmH₂O/L/s², P=0.005]. In inspiratory Q-MDCT, the acromegalicpatients had higher percentages of total lung volume (TLV) for nonaerated and poorlyaerated areas [0.42% (0.30-0.51%) vs 0.25% (0.20-0.32%), P=0.039 and3.25% (2.48-3.46%) vs 1.70% (1.45-2.15%), P=0.001, respectively].Furthermore, the acromegalic patients had higher values for total lung mass in bothinspiratory and expiratory Q-MDCT [821 (635-923) vs 696 (599-769) g,P=0.021 and 844 (650-945) vs 637 (536-736) g, P=0.009,respectively]. In inspiratory Q-MDCT, TLV showed significant correlations with allFOT parameters. The TLV of hyperaerated areas showed significant correlations withintercept resistance (r_s=−0.602, P<0.001) and mean resistance(r_s=−0.580, P<0.001). These data showed that acromegalic patientshave increased amounts of lung tissue as well as nonaerated and poorly aerated areas.Functionally, there was a loss of homogeneity of the respiratory system. Moreover,there were correlations between the structural and functional findings of therespiratory system, consistent with the pathophysiology of the disease.     

Cross‐resistance to fluconazole induced by exposure to the agricultural azole tetraconazole: an environmental resistance school?

This study aimed to investigate the influence of tetraconazole and malathion, both used in agricultural activities, on resistance to fluconazole, itraconazole and voriconazole in Candida parapsilosis ATCC 22019. The susceptibility to tetraconazole, malathion, fluconazole, itraconazole and voriconazole, through broth microdilution. Then, 12 independent replicates, were separated and exposed to four treatment groups, each one containing three replicates: G1: tetraconazole; G2: malathion; G3: fluconazole (positive control); G4: negative control. Replicates from G1, G2 and G3, were exposed to weekly increasing concentrations of tetraconazole, malathion and fluconazole, respectively, ranging from MIC/2 to 32 × MIC, throughout 7 weeks. The exposure to tetraconazole, but not malathion, decreased susceptibility to clinical azoles, especially fluconazole. The tetraconazole‐induced fluconazole resistance is partially mediated by the increased activity of ATP‐dependent efflux pumps, considering the increase in antifungal susceptibility after the addition of the efflux pump inhibitor, promethazine, and the increase in rhodamine 6G efflux and CDR gene expression in the G1 replicates. Moreover, MDR expression was only detected in G1 and G3 replicates, suggesting that MDR pumps are also involved in tetraconazole‐induced fluconazole resistance. It is noteworthy that tetraconazole and fluconazole‐treated replicates behaved similarly, therefore, resistance to azoles of clinical use may be a consequence of using azoles in farming activities.     

Clinical characteristics of patients with factor V Leiden or prothrombin G20210A and a first episode of venous thromboembolism. Findings from the RIETE Registry

Background

The clinical characteristics of patients with factor V Leiden or prothrombin G20210A presenting with a first episode of venous thromboembolism (VTE) have not been thoroughly studied.

Methods

RIETE is an ongoing registry of consecutive patients with acute VTE. We compared the clinical characteristics of patients with factor V Leiden, prothrombin G20210A, or no thrombophilia, at presentation with a first episode of VTE.

Results

As of May 2009, 22428 patients had been enrolled with a first episode of VTE. Of these, 345 had factor V Leiden, 261 had prothrombin G20210A, and 2399 tested negative. Sixty-two percent of the VTE episodes in women with factor V Leiden or prothrombin G20210A (40% in men) were associated with an acquired risk factor. Among women, pregnancy or contraceptive use accounted for 63% and 67% of such risk factors. Patients with factor V Leiden presented with pulmonary embolism (PE) less likely than those with prothrombin G20210A (31% vs. 51%; p < 0.001) or with negative testing (31% vs. 45%, p < 0.001). In addition, PE patients with Factor V Leiden presented with hypoxaemia (Sat O₂ levels < 90%) less likely than those with prothrombin G20210A (4.5% vs. 17%; p < 0.001) or with no thrombophilia (4.5% vs. 20%; p < 0.001).

Conclusions

Most VTE episodes in women (not men) with factor V Leiden or prothrombin G20210A were associated with an acquired risk factor (mostly pregnancy or contraceptive use). Only 4.5% of patients with factor V Leiden presenting with acute PE had hypoxaemia.     

Coordinated brain development: exploring the synchrony between changes in grey and white matter during childhood maturation

Brain development during childhood and early adolescence is characterized by global changes in brain architecture. Neuroimaging studies have revealed overall decreases in cortical thickness (CT) and increases in fractional anisotropy (FA). Furthermore, previous studies have shown that certain cortical regions display coordinated growth during development. However, there is significant heterogeneity in the timing and speed of these developmental transformations, and it is still unclear whether white and grey matter changes are co-localized. In this multimodal neuroimaging study, we investigated the relationship between grey and white matter developmental changes and asynchronous maturation within brain regions in 249 normally developing children between the ages 7–14. We used structural magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI) to analyze CT and FA, respectively, as well as their covariance across development. Consistent with previous studies, we observed overall cortical thinning with age, which was accompanied by increased FA. We then compared the coordinated development of grey and white matter as indexed by covariance measures. Covariance between grey matter regions and the microstructure of white matter tracts connecting those regions were highly similar, suggesting that coordinated changes in the cortex were mirrored by coordinated changes in their respective tracts. Examining within-brain divergent trajectories, we found significant structural decoupling (decreased covariance) between several brain regions and tracts in the 9- to 11-year-old group, particularly involving the forceps minor and the regions that it connects to. We argue that this decoupling could reflect a developmental pattern within the prefrontal region in 9- and 11-year-old children, possibly related to the significant changes in cognitive control observed at this age.     

In Vitro Effect of Sulfamethoxazole-Trimethoprim against Histoplasma capsulatum var. capsulatum

This study evaluated the in vitro effect of sulfamethoxazole-trimethoprim against Histoplasma capsulatum var. capsulatum isolated from HIV-positive patients. The drugs were tested by microdilution testing in accordance with the CLSI guidelines. All of the strains were inhibited by sulfamethoxazole-trimethoprim, with MIC ranges of 0.039 (sulfamethoxazole)/0.0078 (trimethoprim) mg/ml to 0.625/0.125 mg/ml for mycelial forms and 0.0025/0.0005 to 0.02/0.004 mg/ml for yeast-like forms. However, in vivo studies are necessary to evaluate the significance of these results.Histoplasmosis is an infection caused by the dimorphic fungus Histoplasma capsulatum. It is endemic in the Americas (3) and is currently one of the most important systemic infections in Brazil (6). In a retrospective study carried out in Ceará state (northeastern Brazil) from 1995 to 2004, 164 histoplasmosis cases were found in HIV-positive patients (4).The treatment of histoplasmosis depends on the infection''s severity and clinical manifestation, along with individual risk factors (9, 13). Because of the increase in histoplasmosis, particularly in HIV-positive patients, as well as the development of antifungal resistance associated with refractory and repeated infections (13), there is a need for seeking new therapeutic options for this mycosis. Therefore, this study aimed at testing the in vitro activity of sulfamethoxazole-trimethoprim (SMX-TMP) against H. capsulatum var. capsulatum strains isolated from AIDS patients.A total of 84 clinical strains of H. capsulatum isolated from two different biogeographic regions in Brazil were included in this study. Of them, 68 came from Ceará (northeastern semiarid region) and 16 from southeastern states (a subtropical region). The strains were obtained from the collection of the Specialized Medical Mycology Center of Ceará Federal University and were handled in our level 3 biosecurity laboratory.For each strain, a combined solution of SMX-TMP (Roche, Brazil) was used at a concentration range of 0.0025 mg/ml SMX/0.0005 mg/ml TMP and 20/4 mg/ml. The inocula were prepared as described by Li et al. (10), with some modifications. Briefly, H. capsulatum strains were cultured in the mycelial phase onto brain heart infusion (BHI) agar for 7 days at 28°C, and then 2 ml of sterile saline was added to each culture. The surfaces of the mycelia were harvested with a swab. To obtain yeast cells, isolates were cultured on agar BHI with sheep blood (10%) at 35°C and were maintained through weekly passages (7). The supernatant was read in a spectrophotometer at 530 nm, and the transmittance was adjusted to 90 to 95%. The suspensions then were diluted to obtain an inoculum of 0.5 × 10³ to 2.5 × 10⁴ CFU/ml, as demonstrated by quantitative colony counts on Sabouraud dextrose agar (10). Susceptibility tests were carried out as described by Nakai et al. (11), except that the readings were performed after 7 or 4 days for mycelial and yeast growth, respectively. For SMX-TMP, the MIC was defined as the lowest concentration at which no visible growth was observed (14). The differences in the MICs between northeastern and southeastern strains were evaluated by Student''s t test (P < 0.05). Susceptibility control tests were performed with amphotericin B (AMB) against 84 and 7 H. capsulatum strains in mycelial and yeast-like forms, respectively. SMX-TMP quality control was carried out with Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853.All H. capsulatum strains were inhibited by SMX-TMP, with MICs ranging from 0.039/0.0078 to 0.625/0.125 mg/ml for the mycelial forms and 0.0025/0.0005 to 0.02/0.004 mg/ml for the yeast-like forms. The SMX-TMP MICs for strains from northeastern Brazil presented geometric means of 0.1544 mg/ml for sulfamethoxazole and 0.0309 mg/ml for trimethoprim for the mycelial forms and 0.0141 mg/ml for sulfamethoxazole and 0.0028 mg/ml for trimethoprim for yeast-like forms. The geometric means for the strains from southeast Brazil were 0.1152 mg/ml for sulfamethoxazole and 0.0230 mg/ml for trimethoprim for the mycelial forms and 0.0079 mg/ml for sulfamethoxazole and 0.0016 mg/ml for trimethoprim for yeast-like forms (Table ​(Table11).

TABLE 1.

MICs of SMX/TMP against strains of H. capsulatum var. capsulatum in yeast-like and mycelial forms from northeastern and southeastern Brazil^a