How to address CPP and AMP translocation? Methods to detect and quantify peptide internalization in vitro and in vivo (Review)

Membrane translocation is a crucial issue when addressing the activity of both cell-penetrating and antimicrobial peptides. Translocation is responsible for the therapeutic potential of cell-penetrating peptides as drug carriers and can dictate the killing mechanisms, selectivity and efficiency of antimicrobial peptides. It is essential to evaluate if the internalization of cell-penetrating peptides is mediated by endocytosis and if it is able to internalize attached cargoes. The mode of action of an antimicrobial peptide cannot be fully understood if it is not known whether the peptide acts exclusively at the membrane level or also at the cytoplasm. Therefore, experimental methods to evaluate and quantify translocation processes are of first importance. In this work, over 20 methods described in the literature for the assessment of peptide translocation in vivo and in vitro, with and without attached macromolecular cargoes, are discussed and their applicability, advantages and disadvantages reviewed. In addition, a classification of these methods is proposed, based on common approaches to detect translocation.     

Mitochondrial Dysfunction and Redox Homeostasis Impairment as Pathomechanisms of Brain Damage in Ethylmalonic Encephalopathy: Insights from Animal and Human Studies

Cellular and Molecular Neurobiology - Ethylmalonic encephalopathy (EE) is a severe intoxication disorder caused by mutations in the ETHE1 gene that encodes a mitochondrial sulfur dioxygenase...     

The mechanism of formate oxidation by metal-dependent formate dehydrogenases

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C–H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.     

Evolution and molecular mechanisms of adaptive developmental plasticity

Aside from its selective role in filtering inter-individual variation during evolution by natural selection, the environment also plays an instructive role in producing variation during development. External environmental cues can influence developmental rates and/or trajectories and lead to the production of distinct phenotypes from the same genotype. This can result in a better match between adult phenotype and selective environment and thus represents a potential solution to problems posed by environmental fluctuation. The phenomenon is called adaptive developmental plasticity. The study of developmental plasticity integrates different disciplines (notably ecology and developmental biology) and analyses at all levels of biological organization, from the molecular regulation of changes in organismal development to variation in phenotypes and fitness in natural populations. Here, we focus on recent advances and examples from morphological traits in animals to provide a broad overview covering (i) the evolution of developmental plasticity, as well as its relevance to adaptive evolution, (ii) the ecological significance of alternative environmentally induced phenotypes, and the way the external environment can affect development to produce them, (iii) the molecular mechanisms underlying developmental plasticity, with emphasis on the contribution of genetic, physiological and epigenetic factors, and (iv) current challenges and trends, including the relevance of the environmental sensitivity of development to studies in ecological developmental biology, biomedicine and conservation biology.     

From research to implementation: Nature conservation in the Eastern Rhodopes mountains (Greece and Bulgaria), European Green Belt

Nature conservation should ideally build on the scientific recommendations that are concluded from applied conservation research, as well as on monitoring schemes that evaluate the effectiveness of recommendations. We considered as a case study a system of six protected areas located in the Eastern Rhodopes mountains in the southern part of the European Green Belt (EGB). To investigate nature conservation effectiveness, we reviewed 196 articles from scientific journals and books, eight doctoral and master theses, and 39 scientific reports regarding the Greek (one protected area, 428 km²) and the Bulgarian (five protected areas, 904 km²) part of the study area. We extracted 743 conservation recommendations, and through questionnaires completed by 10 local experts, we found that 74% of the recommendations were familiar for the experts. In the Greek (GR) and the Bulgarian part (BG) only 52% and 16%, respectively, of the recommendations were implemented, and only 15% (GR) and 3.1% (BG) were implemented and evaluated regarding their effectiveness. According to the experts, the main reasons for non-implementation and non-evaluation were absence or incompetence of the responsible authorities. Some recommendations obtained a remarkable low rate of implementation, such as those regarding agriculture and livestock rearing practices (GR: 29%, BG: 16%) or mammal conservation (GR: 0%, BG: 16%). Some other recommendations obtained rather high rates at least for Greece, such as tourism and environmental education (GR: 57%, BG: 42%) and bird conservation (GR: 57%, BG: 11%). We found that researchers and conservation managers at both sides of the Greek-Bulgarian border face similar implementation problems, related often to the lack of political will for nature conservation and establishment of competent authorities. The role of the EGB is crucial in enhancing the established cross-border collaborations between stakeholders involved in nature conservation.     

How weekly monitoring variables influence players’ and teams’ match performance in elite futsal players

This study aimed to investigate how weekly training load constrains the performance of players and teams in official futsal competitions. Data from a professional male team were collected during two seasons (46 weeks). The applied monitoring system analysed the training load (as measured by session perceived exertion, sRPE), the total recovery status (TQR), the well-being score (WBs) and the variability of neuromuscular performance during each week (CMJ-cv). In addition, the performance was assessed for all the matches. A path analysis model was performed to test the associations across variables. Results from the path analysis model revealed that it explains 31% of the teams’ performance. In general, the results show that previous team performance has no significant effects on the training week. A significant negative relationship was found between CMJ-cv and match performance (β = -.34; CI95% -.359 to -.070), as well as a significant negative relationship between players’ match performance and the team’s match performance (β = -.55; CI95% -.292 to .740). Regarding indirect effects, only a negative association between CMJ-cv and team match performance via players’ match performance (β = -.19; CI95% -.342 to -.049) was identified. The small variation of the weekly CMJ (CMJ-cv) seems to be a key variable to monitor and explain both player and team performance. Based on this model, and only looking at the physical variables, it was possible to explain 31% of the team’s performance. Longitudinal and multi-team studies should be conducted to integrate other technical, tactical and psychological variables that allow the level of understanding of players’ and teams’ performance to be improved.     

Stochastic modeling of T cell receptor gamma gene rearrangement

The mechanisms controlling the recombination process of the gamma genes that encode the gamma chain of the antigen receptor of the gammadelta T lymphocytes are unclear. Based on experimental data on the recombination status of the two major TCR gamma genes expressed in V(gamma)4+ and V(gamma)1+ thymocytes, we tested the plausibility of three possible rearrangement mechanisms: (1) a time window mechanism according to which the two chromosomes are accessible to the recombination machinery during a defined period of time; (2) a feedback mechanism in which recombination stops shortly after the first in-frame rearrangement event anywhere in both chromosomes; and (3) a feedback mechanism with asynchronous chromosome accessibility, in which there is a first period when only one chromosome is accessible for recombination, followed by a second period when both chromosomes are accessible; shortly after the first in-frame rearrangement event, during any of these two periods, recombination will definitely stop. We model the time window mechanism using a pure probabilistic approach and the two feedback mechanisms using a continuous-time Markov chain formalism. We used maximum likelihood methodology to infer the goodness-of-fit of the models showing evidence for the last model, which best fits the data. Further analysis of this model suggests an evolutionary tradeoff between allelic and isotypic exclusion and the probability that a precursor differentiates into a mature gammadelta T lymphocyte.     

Gramicidin D and dithiothreitol effects on erythrocyte exovesiculation

The use of either diphenylhexatriene, trimethylamino-diphenylhexatriene, or heptadecyl-hydroxycoumarin (C17-HC) allows, simultaneously and with the same molecule, the induction of erythrocyte exovesiculation and labeling of the released vesicles with the fluorescent probe. This method was used to evaluate gramicidin D (a channel-forming peptide) and dithiothreitol (a reducing agent) effects on the human erythrocytes vesiculation process. The release of cholesterol and phospholipids in exovesicles at longer incubation times was only detectable in the presence of gramicidin or dithiothreitol. When C17-HC was used to induce the vesiculation, the presence of gramicidin or dithiothreitol lead to a drastic decrease on the [phospholipids]/[cholesterol] ratio. However, in the samples with dithiothreitol, this variation did not result in the expectable decrease of membrane fluidity. These effects can be related with the presence of lipid rafts, the transbilayer lipids reorientation induced by gramicidin or dithiothreitol, and the cholesterol-dependent gramicidin channels inactivation.