Inhaled tolafentrine reverses pulmonary vascular remodeling via inhibition of smooth muscle cell migration

Background

The aim of the study was to assess the chronic effects of combined phosphodiesterase 3/4 inhibitor tolafentrine, administered by inhalation, during monocrotaline-induced pulmonary arterial hypertension (PAH) in rats.

Methods

CD rats were given a single subcutaneous injection of monocrotaline to induce PAH. Four weeks after, rats were subjected to inhalation of tolafentrine or sham nebulization in an unrestrained, whole body aerosol exposure system. In these animals (i) the acute pulmonary vasodilatory efficacy of inhaled tolafentrine (ii) the anti-remodeling effect of long-term inhalation of tolafentrine (iii) the effects of tolafentrine on the expression profile of 96 genes encoding cell adhesion and extracellular matrix regulation were examined. In addition, the inhibitory effect of tolafentrine on ex vivo isolated pulmonary artery SMC cell migration was also investigated.

Results

Monocrotaline injection provoked severe PAH (right ventricular systolic pressure increased from 25.9 ± 4.0 to 68.9 ± 3.2 after 4 weeks and 74.9 ± 5.1 mmHg after 6 weeks), cardiac output depression and right heart hypertrophy. The media thickness of the pulmonary arteries and the proportion of muscularization of small precapillary resistance vessels increased dramatically, and the migratory response of ex-vivo isolated pulmonary artery smooth muscle cells (PASMC) was increased. Micro-arrays and subsequent confirmation with real time PCR demonstrated upregulation of several extracellular matrix regulation and adhesion genes, such as matrixmetalloproteases (MMP) 2, 8, 9, 10, 11, 12, 20, Icam, Itgax, Plat and serpinb2. When chronically nebulized from day 28 to 42 (12 daily aerosol maneuvers), after full establishment of severe pulmonary hypertension, tolafentrine reversed about 60% of all hemodynamic abnormalities, right heart hypertrophy and monocrotaline-induced structural lung vascular changes, including the proportion of pulmonary artery muscularization. The upregulation of extracellular matrix regulation and adhesion genes was reduced by nearly 80% by inhalation of the tolafentrine. When assessed in vitro, tolafentrine blocked the enhanced PASMC migratory response.

Conclusion

In conclusion, we demonstrate for the first time that inhalation of combined PDE3/4 inhibitor reverses pulmonary hypertension fully developed in response to monocrotaline in rats. This "reverse-remodeling" effect includes structural changes in the lung vascular wall and key molecular pathways of matrix regulation, concomitant with 60% normalization of hemodynamics.     

Childhood Trauma Questionnaire (CTQ) in Brazilian Samples of Different Age Groups: Findings from Confirmatory Factor Analysis

The Childhood Trauma Questionnaire (CTQ) is internationally accepted as a key tool for the assessment of childhood abuse and neglect experiences. However, there are relative few psychometric studies available and some authors have proposed two different factor solutions. We examined the dimensional structure and internal consistency of the Brazilian version of the CTQ. A total of 1,925 participants from eight different clinical and non-clinical samples including adolescents, adults and elders were considered in this study. First, we performed Confirmatory Factor Analysis to investigate the goodness of fit of the two proposed competitive factor structure models for the CTQ. We also investigated the internal consistency of all factors. Second, multi-group analyses were used to investigate measurement invariance and population heterogeneity across age groups and sex. Our findings revealed that the alternative factor structure as opposed to the original factor structure was the most appropriate model within adolescents and adults Brazilian samples. We provide further evidence for the validity and reliability of the CTQ within the Brazilian samples and report that the alternative model showed an improvement in fit indexes and may be a better alternative over the original model.     

Context-dependent ‘safekeeping’ of foraging tools in New Caledonian crows

Several animal species use tools for foraging, such as sticks to extract embedded arthropods and honey, or stones to crack open nuts and eggs. While providing access to nutritious foods, these behaviours may incur significant costs, such as the time and energy spent searching for, manufacturing and transporting tools. These costs can be reduced by re-using tools, keeping them safe when not needed. We experimentally investigated what New Caledonian crows do with their tools between successive prey extractions, and whether they express tool ‘safekeeping’ behaviours more often when the costs (foraging at height), or likelihood (handling of demanding prey), of tool loss are high. Birds generally took care of their tools (84% of 176 prey extractions, nine subjects), either trapping them underfoot (74%) or storing them in holes (26%)—behaviours we also observed in the wild (19 cases, four subjects). Moreover, tool-handling behaviour was context-dependent, with subjects: keeping their tools safe significantly more often when foraging at height; and storing tools significantly more often in holes when extracting more demanding prey (under these conditions, foot-trapping proved challenging). In arboreal environments, safekeeping can prevent costly tool losses, removing a potentially important constraint on the evolution of habitual and complex tool behaviour.     

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

Correction to: Predator‑based selection and the impact of edge sympatry on components of coral snake mimicry

Evolutionary Ecology -     

Insights into discharge argon-mediated biofilm inactivation

Formation of bacterial biofilms at solid–liquid interfaces creates numerous problems in biomedical sciences. Conventional sterilization and decontamination methods are not suitable for new and more sophisticated biomaterials. In this paper, the efficiency and effectiveness of gas discharges in the inactivation and removal of biofilms on biomaterials were studied. It was found that although discharge oxygen, nitrogen and argon all demonstrated excellent antibacterial and antibiofilm activity, gases with distinct chemical/physical properties underwent different mechanisms of action. Discharge oxygen- and nitrogen-mediated decontamination was associated with strong etching effects, which can cause live bacteria to relocate thus spreading contamination. On the contrary, although discharge argon at low powers maintained excellent antibacterial ability, it had negligible etching effects. Based on these results, an effective decontamination approach using discharge argon was established in which bacteria and biofilms were killed in situ and then removed from the contaminated biomaterials. This novel procedure is applicable for a wide range of biomaterials and biomedical devices in an in vivo and clinical setting.     

Expression of Eukaryotic Initiation Factor 5A and Hypusine Forming Enzymes in Glioblastoma Patient Samples: Implications for New Targeted Therapies

Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. Despite a broad range of new and more specific treatment strategies, therapy of glioblastomas remains challenging and tumors relapse in all cases. Recent work demonstrated that the posttranslational hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is a crucial regulator of cell proliferation, differentiation and an important factor in tumor formation, progression and maintenance. Here we report that eIF-5A as well as the hypusine-forming enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are highly overexpressed in glioblastoma patient samples. Importantly, targeting eIF-5A and its hypusine modification with GC7, a specific DHS-inhibitor, showed a strong antiproliferative effect in glioblastoma cell lines in vitro, while normal human astrocytes were not affected. Furthermore, we identified p53 dependent premature senescence, a permanent cell cycle arrest, as the primary outcome in U87-MG cells after treatment with GC7. Strikingly, combined treatment with clinically relevant alkylating agents and GC7 had an additive antiproliferative effect in glioblastoma cell lines. In addition, stable knockdown of eIF-5A and DHS by short hairpin RNA (shRNA) could mimic the antiproliferative effects of GC7. These findings suggest that pharmacological inhibition of eIF-5A may represent a novel concept to treat glioblastomas and may help to substantially improve the clinical course of this tumor entity.     

Setting up and running molecular dynamics simulations of membrane proteins

Molecular dynamics simulations have become a popular and powerful technique to study lipids and membrane proteins. We present some general questions and issues that should be considered prior to embarking on molecular dynamics simulation studies of membrane proteins and review common simulation methods. We suggest a practical approach to setting up and running simulations of membrane proteins, and introduce two new (related) methods to embed a protein in a lipid bilayer. Both methods rely on placing lipids and the protein(s) on a widely spaced grid and then 'shrinking' the grid until the bilayer with the protein has the desired density, with lipids neatly packed around the protein. When starting from a grid based on a single lipid structure, or several potentially different lipid structures (method 1), the bilayer will start well-packed but requires more equilibration. When starting from a pre-equilibrated bilayer, either pure or mixed, most of the structure of the bilayer stays intact, reducing equilibration time (method 2). The main advantages of these methods are that they minimize equilibration time and can be almost completely automated, nearly eliminating one time consuming step in MD simulations of membrane proteins.