Key structural factors and intermolecular interactions underlying the formation, functional properties and behaviour in the gastrointestinal tract in vitro of the liposomal form of nutraceuticals coated with whey proteins and chitosan.

The aim of this study was to gain a better understanding of the key structural factors and intermolecular interactions underlying the formation, functionality, and in vitro gastrointestinal behaviour of the liposomal form of nutraceuticals coated with whey proteins (WPI) and chitosan (CHIT). Phosphatidylcholine (PC) liposomes were used to encapsulate a combination of hydrophobic and hydrophilic nutraceuticals. The hydrophobic constituents were long-chain (LC) n-3 PUFAs (DHA and EPA) from fish oil (FO), vitamin D3, and clove essential oil (CEO), while the hydrophilic component was γ-aminobutyric acid (GABA). A combination of physicochemical methods was used to achieve this goal, including electron paramagnetic resonance spectroscopy (EPRS), laser light scattering in dynamic, static, and electrophoretic modes, transmission electron microscopy, spectrophotometry and tensiometry. The efficiency of encapsulating the nutraceuticals in PC liposomes simultaneously was as follows: 100 ± 1% for both FO triglycerides and CEO, 82 ± 2% for vitamin D3, and 50 ± 1% for GABA. According to EPRS data, encapsulating LC PUFA reduced microviscosity at a depth of 20 Å in the PC bilayer. The co-encapsulation of other nutraceuticals in PC liposomes at selected concentrations did not alter this effect. The upper part (8 Å) of PC liposome bilayers showed an increase in rigidity parameter S, indicating the presence of D3, CEO, and partially GABA. The liposome layer-by-layer encapsulation efficiency (EE%) was achieved by using WPI to form the binary complex [WPI-(PC-FO-D3-GABA-CEO)] (EE = 50% at pH 7.0 and I = 0.001 M), followed by coating with chitosan to form the ternary complex [WPI-(PC-FO-D3-GABA-CEO)]-CHIT (EE = 80% at pH 5.1 and I = 0.001 M). The biopolymer-coated liposomes displayed high water solubility owing to their submicron sizes, thermodynamic affinity for the aqueous medium, and 20 mV ζ-potential values. The chitosan shell regulated the release of liposomes from the ternary complex during in vitro gastrointestinal digestion. In the stomach, the hydrolysis of chitosan by pepsin resulted in a 40% release of liposomes. In the small intestine, chitosan was separated from the WPI-liposome core, facilitatig its hydrolysis and resulting in a 60% release of liposomes. The bioavailability of nutraceuticals encapsulated in PC liposomes in the small intestine may be enhanced by the interactions of both non-hydrolysed and hydrolysed liposomes with bile salts and mucin.

Vesicular Release and Uptake of Circular LSD1-RNAs from Non-Cancer and Cancer Lung Cells.

Lysine-specific demethylase 1 (LSD1) is highly expressed in many cancer types and strongly associated with cancer progression and metastasis. Circular RNAs (circRNAs) are produced by back-splicing and influence the interactive RNA network by microRNA and protein sponging. In the present study, we aimedto identify circRNAs that derive from the LSD1-encoding KDM1A gene, and to investigate their potential to be released and uptaken by lung cancer versus non-cancer epithelial cells. We identified four circLSD1-RNAs by RT-PCR with divergent primers, followed by sequencing. The expression level of circLSD1-RNAs was then studied by quantitative PCR on cellular and extracellular fractions of lung cancer (PC9) and non-cancer primary small airway epithelial (PSAE) cells. Moreover, we established the transgenic overexpression of circLSD1-RNAs. We show that circLSD1-RNAs are primarily located in the cytoplasm, but are packaged and released from lung cancer and non-cancer cells by extracellular vesicles (EVs) and ribonucleoprotein (RNP) complexes, respectively. Proteomics demonstrated a different protein pattern of EV fractions released from PC9 versus PSAE cells. Importantly, released circLSD1-RNAs were differently taken up by PSAE and PC9 cells. In conclusion, our findings provide primary evidence that circLSD1-RNAs participate in the intercellular communication of lung cancer cells with the tumor environment.

The relationship between the structure and functionality of essential PUFA delivery systems based on sodium caseinate with phosphatidylcholine liposomes without and with a plant antioxidant: an in vitro and in vivo study.

The aim of this work was to establish the main relationship between the structure and functionality of supramolecular complexes formed by sodium caseinate (SC) with phosphatidylcholine (PC) liposomes filled with fish oil (FO) to an equal mass ratio of n-3 to n-6 polyunsaturated fatty acids (PUFA) in the absence and presence of one of the most effective plant antioxidants, namely the essential oil of clove buds (EOC). The functionality of the supramolecular complexes (SC-PC-FO and SC-PC-FO-EOC) was considered from the point of view of the possibility of their use as effective delivery systems for long-chain n-3 PUFAs (eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids from FO). The laser light scattering method was used in the static, dynamic and electrophoretic modes to characterize the structure and thermodynamic parameters of the supramolecular complexes in an aqueous medium. It was found that the SC-PC-FO and SC-PC-FO-EOC complex particles had the following similar properties: nanosize; a spherical shape; 100% solubility in an aqueous medium (pH 7.0, ionic strength = 0.001 M); a high encapsulating ability of SC (up to 70%) in relation to the studied liposomes; and a high protective ability relative to lipid autooxidation (up to 96% on the 20th day of storage at room temperature in light). In addition, a sequential transformation of both the structural and thermodynamic parameters has been observed for the complex particles under in vitro simulated gastrointestinal (GI) conditions in accordance with the INFOGEST protocol. A greater release of the encapsulated lipids from the enzymatically hydrolyzed complex particles was observed at the small intestine stage compared to their release at the gastric stage. These data were in good agreement with those on the assessment of the bioavailability of the target PUFAs in in vivo experiments based on the chronic intake of aqueous solutions of the complexes (both SC-PC-FO and SC-PC-FO-EOC) by experimental mice for 92 days. Liver lipid profiles of the mice, obtained by gas-liquid chromatography, showed the following: (i) an almost twofold increase in the DHA content as compared with that of the control; (ii) an almost threefold decrease in the mass ratio of arachidonic acid (AA) (C20:4 n-6) to DHA (C22:6 n-3) compared to that of the control due to both a significant decrease in the AA content and a simultaneous pronounced increase in the DHA content; and (iii) an almost twofold decrease in the mass ratio of the total amounts of n-6 to n-3 PUFAs compared to that of the control.

Mechanisms and biomedical implications of -1 programmed ribosome frameshifting on viral and bacterial mRNAs.

Some proteins are expressed as a result of a ribosome frameshifting event that is facilitated by a slippery site and downstream secondary structure elements in the mRNA. This review summarizes recent progress in understanding mechanisms of -1 frameshifting in several viral genes, including IBV 1a/1b, HIV-1 gag-pol, and SFV 6K, and in Escherichia coli dnaX. The exact frameshifting route depends on the availability of aminoacyl-tRNAs: the ribosome normally slips into the -1-frame during tRNA translocation, but can also frameshift during decoding at condition when aminoacyl-tRNA is in limited supply. Different frameshifting routes and additional slippery sites allow viruses to maintain a constant production of their key proteins. The emerging idea that tRNA pools are important for frameshifting provides new direction for developing antiviral therapies.

Identification of a small molecule inhibitor that stalls splicing at an early step of spliceosome activation.

Small molecule inhibitors of pre-mRNA splicing are important tools for identifying new spliceosome assembly intermediates, allowing a finer dissection of spliceosome dynamics and function. Here, we identified a small molecule that inhibits human pre-mRNA splicing at an intermediate stage during conversion of pre-catalytic spliceosomal B complexes into activated Bact complexes. Characterization of the stalled complexes (designated B028) revealed that U4/U6 snRNP proteins are released during activation before the U6 Lsm and B-specific proteins, and before recruitment and/or stable incorporation of Prp19/CDC5L complex and other Bact complex proteins. The U2/U6 RNA network in B028 complexes differs from that of the Bact complex, consistent with the idea that the catalytic RNA core forms stepwise during the B to Bact transition and is likely stabilized by the Prp19/CDC5L complex and related proteins. Taken together, our data provide new insights into the RNP rearrangements and extensive exchange of proteins that occurs during spliceosome activation.

A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity.

The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5'ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5'ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation.

RNA structure analysis of human spliceosomes reveals a compact 3D arrangement of snRNAs at the catalytic core.

Although U snRNAs play essential roles in splicing, little is known about the 3D arrangement of U2, U6, and U5 snRNAs and the pre-mRNA in active spliceosomes. To elucidate their relative spatial organization and dynamic rearrangement, we examined the RNA structure of affinity-purified, human spliceosomes before and after catalytic step 1 by chemical RNA structure probing. We found a stable 3-way junction of the U2/U6 snRNA duplex in active spliceosomes that persists minimally through step 1. Moreover, the formation of alternating, mutually exclusive, U2 snRNA conformations, as observed in yeast, was not detected in different assembly stages of human spliceosomal complexes (that is, B, B(act), or C complexes). Psoralen crosslinking revealed an interaction during/after step 1 between internal loop 1 of the U5 snRNA, and intron nucleotides immediately downstream of the branchpoint. Using the experimentally derived structural constraints, we generated a model of the RNA network of the step 1 spliceosome, based on the crystal structure of a group II intron through homology modelling. The model is topologically consistent with current genetic, biochemical, and structural data.

Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis.

To better understand the compositional and structural dynamics of the human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior to catalytically active C complexes. By shortening the polypyrimidine tract of the PM5 pre-mRNA, which lacks a 3' splice site and 3' exon, we stalled spliceosome assembly at the activation stage. We subsequently affinity purified human B(act) complexes under the same conditions previously used to isolate B and C complexes, and analyzed their protein composition by mass spectrometry. A comparison of the protein composition of these complexes allowed a fine dissection of compositional changes during the B to B(act) and B(act) to C transitions, and comparisons with the Saccharomyces cerevisiae B(act) complex revealed that the compositional dynamics of the spliceosome during activation are largely conserved between lower and higher eukaryotes. Human SF3b155 and CDC5L were shown to be phosphorylated specifically during the B to B(act) and B(act) to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human B(act) complex was determined by electron microscopy, and a comparison with the B complex revealed that the morphology of the human spliceosome changes significantly during its activation. The overall architecture of the human and S. cerevisiae B(act) complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved.

Exon definition complexes contain the tri-snRNP and can be directly converted into B-like precatalytic splicing complexes.

The first step in splicing of pre-mRNAs with long introns is exon definition, where U1 and U2 snRNPs bind at opposite ends of an exon. After exon definition, these snRNPs must form a complex across the upstream intron to allow splicing catalysis. Exon definition and conversion of cross-exon to cross-intron spliceosomal complexes are poorly understood. Here we demonstrate that, in addition to U1 and U2 snRNPs, cross-exon complexes contain U4, U5, and U6 (which form the tri-snRNP). Tri-snRNP docking involves the formation of U2/U6 helix II. This interaction is stabilized by a 5' splice site (SS)-containing oligonucleotide, which can bind the tri-snRNP and convert the cross-exon complex into a cross-intron, B-like complex. Our data suggest that the switch from cross-exon to cross-intron complexes can occur directly when an exon-bound tri-snRNP interacts with an upstream 5'SS, without prior formation of a cross-intron A complex, revealing an alternative spliceosome assembly pathway.

Isolation of an active step I spliceosome and composition of its RNP core.

Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome's active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of added factors. Comparisons of the composition of the precatalytic versus the catalytic spliceosome revealed a marked exchange of proteins during the transition from the B to the C complex, with apparent stabilization of Prp19-CDC5 complex proteins and destabilization of SF3a/b proteins. Disruption of purified C complexes led to the isolation of a salt-stable ribonucleoprotein (RNP) core that contained both splicing intermediates and U2, U5 and U6 small nuclear RNA plus predominantly U5 and human Prp19-CDC5 proteins and Prp19-related factors. Our data provide insights into the spliceosome's catalytic RNP domain and indicate a central role for the aforementioned proteins in sustaining its catalytically active structure.

Thermodynamic analysis of the impact of the surfactant-protein interactions on the molecular parameters and surface behavior of food proteins.

This paper reports on the thermodynamics of the interactions between surfactants (anionic, CITREM, SSL; nonionic, PGE; zwitterionic, phospholipids) and food proteins (sodium caseinate, legumin) depending on the chemical structure and molecular state (individual molecules, micelles) of the surfactants and the molecular parameters (conformation, molar mass, charge) of the proteins under changes of pH in the range from 7.2 to 5.0 and temperature from 293 to 323 K. The marked effect of the protein-surfactant interactions on the molecular parameters (the weight-average molar mass, the gyration and hydrodynamic radii) and the thermodynamic affinity of the proteins for an aqueous medium were determined by a combination of static and dynamic laser light scattering. Thermodynamically justified schematic sketches of the molecular mechanisms of the complex formation between like-charged proteins and surfactants have been proposed. In response to the complex formation between the proteins and the surfactants, the more stable and fine foams have been detected generally.

Mapping of the second tetracycline binding site on the ribosomal small subunit of E.coli.

Tetracycline blocks stable binding of aminoacyl-tRNA to the bacterial ribosomal A-site. Various tetracycline binding sites have been identified in crystals of the 30S ribosomal small subunit of Thermus thermophilus. Here we describe a direct photo- affinity modification of the ribosomal small subunits of Escherichia coli with 7-[3H]-tetracycline. To select for specific interactions, an excess of the 30S subunits over tetracycline has been used. Primer extension analysis of the 16S rRNA revealed two sites of the modifications: C936 and C948. Considering available data on tetracycline interactions with the prokaryotic 30S subunits, including the presented data (E.coli), X-ray data (T.thermophilus) and genetic data (Helicobacter pylori, E.coli), a second high affinity tetracycline binding site is proposed within the 3'-major domain of the 16S rRNA, in addition to the A-site related tetracycline binding site.