N,N,N-Trimethyl chitosan as a permeation enhancer for inhalation drug delivery: Interaction with a model pulmonary surfactant.

N,N,N-Trimethyl chitosan (TMC), a biocompatible and biodegradable derivative of chitosan, is currently used as a permeation enhancer to increase the translocation of drugs to the bloodstream in the lungs. This article discusses the effect of TMC on a mimetic pulmonary surfactant, Curosurf®, a low-viscosity lipid formulation administered to preterm infants with acute respiratory distress syndrome. Curosurf® exhibits a strong interaction with TMC, resulting in the formation of aggregates at electrostatic charge stoichiometry. At nanoscale, Curosurf® undergoes a profound reorganization of its lipid vesicles in terms of size and lamellarity. The initial micron-sized vesicles (average size 4.8 µm) give way to a froth-like network of unilamellar vesicles about 300 nm in size. Under such conditions, neutralization of the cationic charges by pulmonary surfactant may inhibit TMC permeation enhancer capacity, especially as electrostatic charge complexation is found at low TMC content. The permeation properties of pulmonary surfactant-neutralized TMC should then be evaluated for its applicability as a permeation enhancer for inhalation in the alveolar region.

Liposomal form of erlotinib for local inhalation administration and efficiency of its transport to the lungs.

This contribution is focused on the preparation of a liposomal drug delivery system of erlotinib resisting the nebulization process that could be used for local treatment of non-small-cell lung cancer. Liposomes with different compositions were formulated to reveal their influence on the encapsulation efficiency of erlotinib. An encapsulation efficiency higher than 98 % was achieved for all vesicles containing phosphatidic acid (d ≈ 100 nm, ζ = - 43 mV) even in the presence of polyethylene glycol (d ≈ 150 nm, ζ = - 17 mV) which decreased this value in all other formulas. The three most promising formulations were nebulized by two air-jet and two vibrating mesh nebulizers, and the aerosol deposition in lungs was calculated by tools of computational fluid and particle mechanics. According to the numerical simulations and measurements of liposomal stability, air-jet nebulizers generated larger portion of the aerosol able to penetrate deeper into the lungs, but the delivery is likely to be more efficient when the formulation is administered by Aerogen Solo vibrating mesh nebulizer because of a higher portion of intact vesicles after the nebulization. The leakage of encapsulated drug from liposomes nebulized by this nebulizer was lower than 2 % for all chosen vesicles.

An unusual fusion gene EML4-ALK in a patient with congenital mesoblastic nephroma.

Congenital mesoblastic nephroma (CMN), the most common renal tumor of infancy, is a mesenchymal neoplasm histologically classified into classic, cellular, or mixed types. Most cellular CMNs harbor a characteristic ETV6-NTRK3 fusion. Here, we report an unusual congenital mesoblastic nephroma presenting in a newborn boy with a novel EML4-ALK gene fusion revealed by Anchored Multiplex RNA Sequencing Assay. The EML4-ALK gene fusion expands the genetic spectrum implicated in the pathogenesis of congenital mesoblastic nephroma, with yet another example of kinase oncogenic activation through chromosomal rearrangement. The methylation profile of the tumor corresponds with infantile fibrosarcoma showing the biological similarity of these two entities.

Interactions between Cationic Ion Pair Amphiphile Vesicles and Hyaluronan-A Physicochemical Study.

High-resolution ultrasound spectroscopy (HR-US), size and ζ-potential titrations, and isothermal titration calorimetry (ITC) were used to characterize the interactions between hyaluronan and catanionic ion pair amphiphile vesicles composed of hexadecyltrimethylammonium-dodecylsulphate (HTMA-DS), dioctadecyldimethylammonium chloride (DODAC), and cholesterol. In addition to these methods, visual observations were performed with the selected molecular weight of hyaluronan. A very good correlation was obtained between data from size titration, HR-US, and visual observation, which indicated in lower charge ratios the formation of hyaluronan-coated vesicles. On the contrary, at higher charge ratios, coated vesicles disintegrated to a size of around 2000 nm. The intensity of these interactions and the disaggregation were dependent on the molecular weight of hyaluronan. All interactions studied by ITC showed strong exothermic behavior, and these interactions between vesicles and hyaluronan were confirmed from the first addition, independently of the molecular weight of hyaluronan.

Heterotrophic euglenid Rhabdomonas costata resembles its phototrophic relatives in many aspects of molecular and cell biology.

Euglenids represent a group of protists with diverse modes of feeding. To date, only a partial genomic sequence of Euglena gracilis and transcriptomes of several phototrophic and secondarily osmotrophic species are available, while primarily heterotrophic euglenids are seriously undersampled. In this work, we begin to fill this gap by presenting genomic and transcriptomic drafts of a primary osmotroph, Rhabdomonas costata. The current genomic assembly length of 100 Mbp is 14× smaller than that of E. gracilis. Despite being too fragmented for comprehensive gene prediction it provided fragments of the mitochondrial genome and comparison of the transcriptomic and genomic data revealed features of its introns, including several candidates for nonconventional types. A set of 39,456 putative R. costata proteins was predicted from the transcriptome. Annotation of the mitochondrial core metabolism provides the first data on the facultatively anaerobic mitochondrion of R. costata, which in most respects resembles the mitochondrion of E. gracilis with a certain level of streamlining. R. costata can synthetise thiamine by enzymes of heterogenous provenances and haem by a mitochondrial-cytoplasmic C4 pathway with enzymes orthologous to those found in E. gracilis. The low percentage of green algae-affiliated genes supports the ancestrally osmotrophic status of this species.

Influence of liposomes composition on their stability during the nebulization process by vibrating mesh nebulizer.

In this study, three different molecules (cholesterol, phosphatidic acid, and polyethylene glycol) were used for the stabilization of liposomes during the nebulization process. The purpose of this article is to answer the question of whether the change in the composition of liposomes affected the parameters of generated aerosol and whether the nebulization process affected observed properties of liposomes. Firstly, liposomes with different composition were prepared and their properties were checked by dynamic and electrophoretic light scattering. The membrane properties were measured by fluorescence spectroscopy - especially generalized polarization (Laurdan) and anisotropy (Diphenylhexatriene). The same characteristic of liposomes was measured after the nebulization by vibrating mesh nebulizer. Cholesterol was capable of liposome stabilization because of increased membrane fluidity. The membrane properties of the outer and inner parts were not influenced by the nebulization process. Electrostatic stabilization was successful for the lowest concentration of phosphatidic acid, but after the nebulization process the hydration of the membrane outer part was changed. Higher amount of PEG needs to be added for successful steric stabilization. The nebulization process of the two lowest concentrations of PEG slightly influenced immobilized water and the rigidity of inner part of the membrane (especially around the phase transition temperature).

Cholesterol Effect on Membrane Properties of Cationic Ion Pair Amphiphile Vesicles at Different Temperatures.

This work is focused on the study of the effect of cholesterol on the properties of vesicular membranes of ionic amphiphilic pairs at different temperatures. The hexadecyltrimethylammonium-dodecyl sulfate ionic amphiphilic pair system with the addition of 10 mol % dioctadecyldimethylammonium chloride was chosen for a detailed study of vesicle properties. A large range of cholesterol concentrations (0-73 mol %) in the temperature range 10-80 °C was studied. Under these conditions, the size distribution, the membrane fluidity, and the surface layer were monitored together with the change in the mobility of water in the surface layer. Obtained quantities were correlated with each other and combined into appropriate graphs. It was found that in stable systems that meet the condition of unimodal size distribution with a PDI value lower than 0.3, temperature has virtually no effect on the size of vesicular systems. On the contrary, when studying the hydration and fluidity of the membrane, significant changes in these parameters were found, which, however, do not affect the short-term stability of these vesicular systems. The presented results thus indicate the possibility of adjusting the composition of the vesicular system in terms of fluidity and membrane hydration while maintaining short-term stability and size distribution.

Analyses of environmental sequences and two regions of chloroplast genomes revealed the presence of new clades of photosynthetic euglenids in marine environments.

Euglenophyceae are unicellular algae with the majority of their diversity known from small freshwater reservoirs. Only two dozen species have been described to occur in marine habitats, but their abundance and diversity remain unexplored. Phylogenetic studies revealed marine prasinophyte green alga, Pyramimonas parkeae, as the closest extant relative of the euglenophytes' plastid, but similarly to euglenophytes, our knowledge about the diversity of Pyramimonadales is limited. Here we explored Euglenophyceae and Pyramimonadales phylogenetic diversity in marine environmental samples. We yielded 18S rDNA and plastid 16S rDNA sequences deposited in public repositories and reconstructed Euglenophyceae reference trees. We searched high-throughput environmental sequences from the TARA Oceans expedition and Ocean Sampling Day initiative for 18S rDNA and 16S rDNA, placed them in the phylogenetic context and estimated their relative abundances. To avoid polymerase chain reaction (PCR) bias, we also exploited metagenomic data from the TARA Oceans expedition for the presence of rRNA sequences from these groups. Finally, we targeted these protists in coastal samples by specific PCR amplification of two parts of the plastid genome uniquely shared between euglenids and Pyramimonadales. All approaches revealed previously undetected, but relatively low-abundant lineages of marine Euglenophyceae. Surprisingly, some of those lineages are branching within the freshwater or brackish genera.

Gastrointestinal stromal tumors - Summary of mutational status of the primary/secondary KIT/PDGFRA mutations, BRAF mutations and SDH defects.

The most important findings revealing pathogenesis, molecular characteristics, genotyping and targeted therapy of gastrointestinal stromal tumors (GISTs) are activated oncogenic mutations in KIT and PDGFRA genes. Imatinib mesylate (IM), which inhibits both KIT and PDGFRA receptors, significantly improved treatment of advanced (metastatic, recurrent, and/or inoperable) GISTs. However, in a significant number of patients the treatment fails due to the primary or secondary resistance to targeted therapy. Most common cause of secondary resistance is a presence of secondary mutations. Approximately 15% of adult patients with GISTs are negative for mutations in KIT or PDGFRA genes. These so-called wild-type GISTs appear to be characterized by other oncogenetic drivers, including mutations in BRAF, RAS, NF1 genes, and subunits of succinate dehydrogenase (SDH) complex. In the present study we investigated 261 tumour specimens from 239 patients with GIST. Primary mutations were detected in 82 % tumor specimens. 66 of them were in KIT, and 16 % in PDGFRA genes. Remaining 18 % were KIT/PDGFRA wild-type. Secondary KIT mutations were detected in 10 from 133 (7 %) patients treated with IM. We examined secondary KIT mutations in exons 13 and 17 and secondary PDGFRA mutation in exon 18 in sixteen progressive tumors and/or metastasis (from overall 22 samples). We identified BRAF V600E point mutation in 4 % of KIT/PDGFRA wild-type GIST patients. Moreover, we analysed SDH complex mutations in 4 younger patients (15, 33, 37, and 45 years old) from 44 patients without KIT, PDGFRA, and BRAF mutations. Two patients (a 37-year old man, and a 33-year old woman) had defects of the SDH complex. Our findings of mutational status of the primary and secondary KIT/PDGFRA mutations in patients with GIST confirm mechanisms of primary and secondary resistance, and also intralesional and interlesional heterogeneity of secondary mutations within and between progressive lesions. Moreover, detection of V600E BRAF mutation and defects of SDH complex in KIT/PDGFRA wild-type GISTs confirm their activation and allow for a selection of targeted therapy.

The evolution of paralogous enzymes MAT and MATX within the Euglenida and beyond.

BACKGROUND: Methionine adenosyltransferase (MAT) is a ubiquitous essential enzyme that, in eukaryotes, occurs in two relatively divergent paralogues: MAT and MATX. MATX has a punctate distribution across the tree of eukaryotes and, except for a few cases, is mutually exclusive with MAT. This phylogenetic pattern could have arisen by either differential loss of old paralogues or the spread of one of these paralogues by horizontal gene transfer. Our aim was to map the distribution of MAT/MATX genes within the Euglenida in order to more comprehensively characterize the evolutionary history of MATX. RESULTS: We generated 26 new sequences from 23 different lineages of euglenids and one prasinophyte alga Pyramimonas parkeae. MATX was present only in photoautotrophic euglenids. The mixotroph Rapaza viridis and the prasinophyte alga Pyramimonas parkeae, which harbors chloroplasts that are most closely related to the chloroplasts in photoautotrophic euglenids, both possessed only the MAT paralogue. We found both the MAT and MATX paralogues in two photoautotrophic species (Phacus orbicularis and Monomorphina pyrum). The significant conflict between eukaryotic phylogenies inferred from MATX and SSU rDNA data represents strong evidence that MATX paralogues have undergone horizontal gene transfer across the tree of eukaryotes. CONCLUSIONS: Our results suggest that MATX entered the euglenid lineage in a single horizontal gene transfer event that took place after the secondary endosymbiotic origin of the euglenid chloroplast. The origin of the MATX paralogue is unclear, and it cannot be excluded that it arose by a gene duplication event before the most recent common ancestor of eukaryotes.

The plastid genome of Eutreptiella provides a window into the process of secondary endosymbiosis of plastid in euglenids.

Euglenids are a group of protists that comprises species with diverse feeding modes. One distinct and diversified clade of euglenids is photoautotrophic, and its members bear green secondary plastids. In this paper we present the plastid genome of the euglenid Eutreptiella, which we assembled from 454 sequencing of Eutreptiella gDNA. Comparison of this genome and the only other available plastid genomes of photosynthetic euglenid, Euglena gracilis, revealed that they contain a virtually identical set of 57 protein coding genes, 24 genes fewer than the genome of Pyramimonas parkeae, the closest extant algal relative of the euglenid plastid. Searching within the transcriptomes of Euglena and Eutreptiella showed that 6 of the missing genes were transferred to the nucleus of the euglenid host while 18 have been probably lost completely. Euglena and Eutreptiella represent the deepest bifurcation in the photosynthetic clade, and therefore all these gene transfers and losses must have happened before the last common ancestor of all known photosynthetic euglenids. After the split of Euglena and Eutreptiella only one additional gene loss took place. The conservation of gene content in the two lineages of euglenids is in contrast to the variability of gene order and intron counts, which diversified dramatically. Our results show that the early secondary plastid of euglenids was much more susceptible to gene losses and endosymbiotic gene transfers than the established plastid, which is surprisingly resistant to changes in gene content.

Trypanosoma culicavium sp. nov., an avian trypanosome transmitted by Culex mosquitoes.

A novel avian trypanosome, Trypanosoma culicavium sp. nov., isolated from Culex mosquitoes, is described on the basis of naturally and experimentally infected vectors and bird hosts, localization in the vector, morphological characters and molecular data. This study provides the first comprehensive description of a trypanosome species transmitted by mosquitoes, in which parasites form plugs and rosettes on the stomodeal valve. Trypanosomes occurred as long epimastigotes and short trypomastigotes in vectors and culture and as long trypomastigotes in birds. Transmission of parasites to bird hosts was achieved exclusively by ingestion of experimentally infected Culex mosquito females by canaries (Serinus canaria), but not by Japanese quails (Coturnix japonica), nor by the bite of infected vectors, nor by ingestion of parasites from laboratory cultures. Transmission experiments and the identity of isolates from collared flycatchers (Ficedula albicollis) and Culex mosquitoes suggests that the natural hosts of T. culicavium are insectivorous songbirds (Passeriformes). Phylogenetic analyses of small-subunit rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase gene sequences demonstrated that T. culicavium sp. nov. is more related to Trypanosoma corvi than to other avian trypanosomes (e.g. Trypanosoma avium and Trypanosoma bennetti).

Biodiversity of avian trypanosomes.

We have studied the biodiversity of trypanosomes from birds and bloodsucking Diptera on a large number of isolates. We used two molecular approaches, random amplification of polymorphic DNA (RAPD) method, and sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene. RAPD method divided the isolates into 11 separate lineages. Phylogenetic analysis of the SSU rRNA gene was congruent with the RAPD. Morphometric analysis of kinetoplast width and cell length was in agreement with molecular data. Avian trypanosomes appeared polyphyletic on SSU rDNA tree; thus, they do not represent a taxonomic group. We propose that all lineages recovered by SSU analysis probably represent distinct species of avian trypanosomes. We discuss possible transmission ways and geographical distribution of new avian trypanosome lineages. Finally, we recommend methods that should be used for species determination of avian trypanosomes.

Experimental examination of EFL and MATX eukaryotic horizontal gene transfers: coexistence of mutually exclusive transcripts predates functional rescue.

Many eukaryotic genes do not follow simple vertical inheritance. Elongation factor 1α (EF-1α) and methionine adenosyl transferase (MAT) are enzymes with complicated evolutionary histories and, interestingly, the two cases have several features in common. These essential enzymes occur as two relatively divergent paralogs (EF-1α/EFL, MAT/MATX) that have patchy distributions in eukaryotic lineages that are nearly mutually exclusive. To explain such distributions, we must invoke either multiple eukaryote-to-eukaryote horizontal gene transfers (HGTs) followed by functional replacement or presence of both paralogs in the common ancestor followed by long-term coexistence and differential losses in various eukaryotic lineages. To understand the evolution of these paralogs, we have performed in vivo experiments in Trypanosoma brucei addressing the consequences of long-term coexpression and functional replacement. In the first experiment of its kind, we have demonstrated that EF-1α and MAT can be simultaneously expressed with EFL and MATX, respectively, without affecting the growth of the flagellates. After the endogenous MAT or EF-1α was downregulated by RNA interference, MATX immediately substituted for its paralog, whereas EFL was not able to substitute for EF-1α, leading to mortality. We conclude that MATX is naturally capable of evolving patchy paralog distribution via HGTs and/or long- term coexpression and differential losses. The capability of EFL to spread by HGT is lower and so the patchy distribution of EF-1α/EFL paralogs was probably shaped mainly by deep paralogy followed by long-term coexistence and differential losses.