Encapsulation of Ferritin, Ribosomes, and Ribo-Peptidic Complexes Inside Liposomes: Insights Into the Origin of Metabolism

Here we summarize the main results of our latest investigation on the spontaneous encapsulation of proteins (ferritin) and ribosomes inside lipid vesicles. We show that when vesicles form in a solution containing some macromolecules (even at low concentration), in contrast to the expectations, a few but measurable number of vesicles is able to capture a very high number of solutes, up to 60 times the external concentration. We also show preliminary evidences on the encapsulation of additional solutes (ribo-peptidic complexes, fluorescent proteins and enzymes), and shortly present our current approach aimed at exploiting this phenomenon. In particular, we would like to reveal how the formation of compartments can trigger effective intra-vesicle reactions starting from diluted solutions. Although the mechanistic details for this phenomenon are still missing, we claim that these new evidences are highly relevant for the origin of the first functional cells in primitive times.     

Combination of the antifeedant bean flour and the predator Cheyletus malaccensis suppresses storage mites under laboratory conditions

Stored product mites are pests of serious economic and medical importance. Recently, it has been shown that the biological control of these pests based on the use of natural compounds and predators had a high potential for success. In this study, we investigated the suppression of three pest mites by combination of the predator Cheyletus malaccensis Oudemans, 1913 and bean flour (Phaseolus vulgaris), which is an antifeedant. Wheat grain (100 g) was contaminated with 100 individuals (Acarus siro (Linnaeus, 1758), Aleuroglyphus ovatus (Troupeau, 1878) or Tyrophagus putrescentiae (Schrank, 1781)). We added bean flour in concentrations of 0, 1, 5 and 10 g kg⁻¹ (wt/wt) and/or the predator with initial predator/prey ratios 0, 1/50, 1/25 and 1/10. The experiment was carried out under abiotic conditions that are optimal for pest development. After 21 days, pest and predators numbers were recorded. Application of flour significantly reduced populations of A. siro, slightly reduced populations of T. putrescentiae, the efficiency increased with increasing concentration. A. ovatus was not affect by bean flour. Populations of all mite species were successfully reduced by the sole addition of C. malaccensis, with a higher efficiency at higher Cheyletus ratios. The additive effect of the predator and flour was mainly apparent in the reduction of T. putrescientae. The population of C. malaccensis was not affected by the presence of bean flour. It is therefore recommended to use bean flour for the reduction of A. siro, a combination of bean flour and Cheyletus for the reduction of T. putrescientae, and only Cheyletus for the reduction of A. ovatus. Handling Editor: Arne Janssen.     

Investigation of de novo totally random biosequences, Part II: On the folding frequency in a totally random library of de novo proteins obtained by phage display

We present an investigation on theoretically possible protein structures which have not been selected by evolution and are, therefore, not present on our Earth ('Never Born Proteins' (NBP)). In particular, we attempt to assess whether and to what extent such polypeptides might be folded, thus acquiring a globular protein status. A library (ca. 10(9) clones) of totally random polypeptides, with a length of 50 amino acids, has been produced by phage display. The only structural bias in these sequences is a tripeptide substrate for thrombin: PRG, chosen according to the criteria described in the preceding Part I of this series. The presence of this substrate in an otherwise totally random sequence forms the basis for a qualitative experimental criterion which distinguishes unfolded from folded proteins, as folded proteins are more protected from protease digestion than unfolded ones. The investigation of 79 sequences, randomly selected from the initially large library, shows that over 20% of this population is thrombin-resistant, likely due to folding. Analysis of the amino acid sequences of these clones shows no significant homology to extant proteins, which indicates that they are indeed totally de novo. A few of these sequences have been expressed, and here we describe the structural properties of two thrombin-resistant randomly selected ones. These two de novo proteins have been characterized by spectroscopic methods and, in particular, by circular dichroism. The data show a stable three-dimensional folding, which is temperature-resistant and can be reversibly denatured by urea. The consequences of this finding within a library of 'Never Born Proteins' are discussed in terms of molecular evolution.     

Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method

Small-sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug-containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) then on the novel L-carnitine derivative PUCE (palmitoyl-(R)-carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of "multiple injection method" was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan-containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI-H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no-liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water-insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.     

The intercalating beta-hairpin of T7 RNA polymerase plays a role in promoter DNA melting and in stabilizing the melted DNA for efficient RNA synthesis

Phage T7 RNA polymerase contains within its single polypeptide all the elements for specific recognition and melting of its promoter DNA. Crystallographic studies indicate that a beta-hairpin (230-245) with an intercalating valine residue plays a role in promoter opening. We mutated V237 to several amino acids, deleted five amino acid residues at the tip of the hairpin, and mutated E242 and D240 at the base of the hairpin to define the roles of the tip and base of the hairpin in DNA strand separation. The affinity of the hairpin mutants for the promoter DNA was not significantly affected. Stopped-flow kinetic studies showed that the bimolecular rate of DNA binding and the observed rate of pre-initiation open complex formation that corresponds to the sum of DNA opening and closing steps were within 20 to 40 % of the wild-type polymerase. Yet, most mutants showed a smaller amount of the pre-initiation open complex at equilibrium, indicating that the individual rates of promoter opening and closing steps were altered in the mutants. The base mutants, E242A and D240A, showed both a lower rate of promoter opening and a higher rate of promoter closing, suggesting their role in stabilization of the open complex. The V237D and the deletion mutant showed mainly a lower rate of promoter opening, suggesting that the tip of the hairpin may nucleate DNA opening. The defect in pre-initiation open complex formation affected downstream steps such as the rate of the first phosphodiester bond formation step, but did not affect significantly the apparent K(d) of initiating GTPs. We propose that D240 and E242 anchor the hairpin to the DNA and position the tip of the hairpin to allow V237 to intercalate and distort the DNA during open complex formation. The interactions of E242 and D240 with the upstream junction of the melted dsDNA promoter also align the template strand within the active site for efficient RNA synthesis.     

Phenotypic integration in a series of trophic traits: tracing the evolution of myrmecophagy in spiders (Araneae)

Several hypotheses have been put forward to explain the evolution of prey specificity (stenophagy). Yet little light has so far been shed on the process of evolution of stenophagy in carnivorous predators. We performed a detailed analysis of a variety of trophic adaptations in one species. Our aim was to determine whether a specific form of stenophagy, myrmecophagy, has evolved from euryphagy via parallel changes in several traits from pre-existing characters. For that purpose, we studied the trophic niche and morphological, behavioural, venomic and physiological adaptations in a euryphagous spider, Selamia reticulata. It is a species that is branching off earlier in phylogeny than stenophagous ant-eating spiders of the genus Zodarion (both Zodariidae). The natural diet was wide and included ants. Laboratory feeding trials revealed versatile prey capture strategies that are effective on ants and other prey types. The performance of spiders on two different diets – ants only and mixed insects – failed to reveal differences in most fitness components (survival and developmental rate). However, the weight increase was significantly higher in spiders on the mixed diet. As a result, females on a mixed diet had higher fecundity and oviposited earlier. No differences were found in incubation period, hatching success or spiderling size. S. reticulata possesses a more diverse venom composition than Zodarion. Its venom is more effective for the immobilisation of beetle larvae than of ants. Comparative analysis of morphological traits related to myrmecophagy in the family Zodariidae revealed that their apomorphic states appeared gradually along the phylogeny to derived prey-specialised genera. Our results suggest that myrmecophagy has evolved gradually from the ancestral euryphagous strategy by integrating a series of trophic traits.     

Multi-Regional Investigation of the Relationship between Functional MRI Blood Oxygenation Level Dependent (BOLD) Activation and GABA Concentration

Several recent studies have reported an inter-individual correlation between regional GABA concentration, as measured by MRS, and the amplitude of the functional blood oxygenation level dependent (BOLD) response in the same region. In this study, we set out to investigate whether this coupling generalizes across cortex. In 18 healthy participants, we performed edited MRS measurements of GABA and BOLD-fMRI experiments using regionally related activation paradigms. Regions and tasks were the: occipital cortex with a visual grating stimulus; auditory cortex with a white noise stimulus; sensorimotor cortex with a finger-tapping task; frontal eye field with a saccade task; and dorsolateral prefrontal cortex with a working memory task. In contrast to the prior literature, no correlation between GABA concentration and BOLD activation was detected in any region. The origin of this discrepancy is not clear. Subtle differences in study design or insufficient power may cause differing results; these and other potential reasons for the discrepant results are discussed. This negative result, although it should be interpreted with caution, has a larger sample size than prior positive results, and suggests that the relationship between GABA and the BOLD response may be more complex than previously thought.     

The effect of stored barley cultivars, temperature and humidity on population increase of Acarus siro, Lepidoglyphus destructor and Tyrophagus putrescentiae

The rate of population increase of three mite species, Acarus siro (L.), Lepidoglyphus destructor (Schrank) and Tyrophagus putrescentiae (Schrank), was studied on various types of barley and at various combinations of temperature and humidity. The mites were added into the chambers and incubated for 21 days on seven different kinds of barley coming from four sites, including six cultivars and a mixture. The population increase of all species was higher on the mixture than on any other cultivar, except for Sebastian and Calgary. The increase of mites was studied at constant temperatures ranging from 5 to 35 °C and relative humidity (RH) ranging from 50 to 90 %. Positive rate of increase was found above 70 % RH for all species. The optimal humidity was at 85 % RH for A. siro and L. destructor and at 90 % RH for T. putrescentiae. As concerns the temperature, positive rate of increase was found at temperatures higher than 10, 15 and 20 °C for A. siro, L. destructor and T. putrescentiae, respectively. The temperature optima were at 23, 25, and 30 °C for A. siro, L. destructor and T. putrescentiae, respectively. Model estimated on laboratory data was then fitted to temperature and humidity records from August to November in the Czech grain store. Estimated population rate of increase was rarely positive: for A. siro it was for 24 %, for L. destructor for only 1 % and for T. putrescentiae for only 7 % days of the study period. It is concluded that in the climatic conditions of the Czech Republic the population increase of three mite pests is negligible during autumn and winter.     

Is the evolution of inaccurate mimicry a result of selection by a suite of predators? A case study using myrmecomorphic spiders

Several hypotheses have been put forward to explain the evolution of inaccurate mimicry. Here we investigated the novel hypothesis that inaccurate mimicry (in color and shape) is maintained by opposing selective pressures from a suite of different predators: model-aversive visually oriented predators and model- and mimic-specialized predators indifferent to mimetic cues. We hypothesize that spiders resembling ants in color and shape escape predators that typically avoid ants but fall prey to ant-eating predators. We tested whether inaccurate myrmecomorphic spiders are perceived as their models by two types of predators and whether they can escape from these predators. We found that model-specialized (ant-eating) predators captured mimics significantly less frequently than their ant models, because mimics changed their behavior by fleeing predatory attacks. The fastest escape was found in less accurate mimics, indicating a negative association between visual resemblance and effectiveness of defenses. In trials with spider-eating predators, mimics were not captured more frequently than their models. The quality of defensive mechanisms appears to result from opposing selection forces exerted by the predator complex: mimics are more accurate (in color and shape) in microhabitats dominated by model-aversive predators and less accurate in microhabitats with model- and mimic-specialized predators.     

Temperature-dependent population growth of three species of stored product mites (Acari: Acaridida)

The pest potential of stored product mites depends on the reproduction rate that is affected by the environmental conditions. In this study we investigated the effect of temperature, ranging from 5 to 35°C, on the population growth of three important mite species, Acarus siro, Tyrophagus putrescentiae and Auleroglyphus ovatus at 85% r.h. Starting with 10 individuals the population increase of mites was observed after 3 weeks of cultivation, or after 6 weeks for those kept at low temperatures (5, 10, 12.5, and 15°C). The rate of increase was calculated for each temperature and species. The obtained data were fitted with polynomial models. The mite population growth rates increased with increasing moderate temperatures until 25°C, when r _m -values were 0.179, 0.177 and 0.190 for A. siro, A. ovatus and T. putrescentiae, respectively. The lower development threshold was 10.2°C in all three species. Estimated upper temperature threshold was higher in T. putrescentiae (49°C) than in A. siro and A. ovatus (38°C). Simulation of the rate of population increase under ideal conditions, using real temperature records obtained from Czech grain stores, showed that the pest mite populations increase only during 3.5 months within a typical 9-month storage season in Central Europe. These results indicate that control of mites, be it chemical, physical or biological, is recommended during the months when allergens and pests are produced, i.e. from September to mid November and in May.