Isolation of Extracellular Vesicles of Holothuria (Sea Cucumber Eupentacta fraudatrix).

Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30-100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.

Human Blood Extracellular Vesicles Activate Transcription of NF-kB-Dependent Genes in A549 Lung Adenocarcinoma Cells.

Extracellular vesicles (EVs) produced by various cell types are heterogeneous in size and composition. Changes in the RNA sets of EVs in biological fluids are considered the basis for the development of new approaches to minimally invasive diagnostics and the therapy of human diseases. In this study, EVs were obtained from the blood of healthy donors by centrifugation, followed by ultracentrifugation. It was shown that EVs consist of several populations including small exosome-like vesicles and larger microvesicle-like particles. The composition of EVs' RNAs was determined. A549 lung adenocarcinoma cells were incubated with EV and the NGS analysis of differentially expressed genes was performed. During the incubation of A549 cells with EVs, the levels of mRNA encoding components for the NF-kB signaling pathway increased, as well as the expression of genes controlled by the NF-kB transcription factor. Overall, our results suggest that components of EVs trigger the NF-kB signaling cascade in A549 cells, leading to the transcription of genes including cytokines, adhesion molecules, cell cycle regulators, and cell survival factors. Our data provide insight into the interaction between blood EVs and human cells and can be used for designing new tools for the diagnosis and treatment of human diseases.

Analysis of Proteins and Peptides of Highly Purified CD9+ and CD63+ Horse Milk Exosomes Isolated by Affinity Chromatography.

Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we isolated highly purified vesicle preparations from 18 horse milk samples. Gel filtration of the initial preparations allowed us to remove co-isolating proteins and their complexes and to obtain highly purified vesicles morphologically corresponding to exosomes. Using affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we obtained extra-purified CD9+ and CD63+ exosomes, which simultaneously contain these two tetraspanins, while the CD81 tetraspanin was presented in a minor quantity. SDS-PAGE and MALDI analysis detected several major proteins with molecular masses over 10 kDa: CD9, CD63, CD81, lactadherin, actin, butyrophilin, lactoferrin, and xanthine dehydrogenase. Analysis of extracts by trifluoroacetic acid revealed dozens of peptides with molecular masses in the range of 0.8 to 8.5 kDa. Data on the uneven distribution of tetraspanins on the surface of horse milk exosomes and the presence of peptides open new questions about the biogenesis of these extracellular vesicles.

An Influence of Modification with Phosphoryl Guanidine Combined with a 2'-O-Methyl or 2'-Fluoro Group on the Small-Interfering-RNA Effect.

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2'-O-methyl (2'-OMe) or PG/2'-fluoro (2'-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.

The Effect of Thermomechanical Pretreatment on the Structure and Properties of Lignin-Rich Plant Biomass.

The cooperative thermomechanical properties of plant-derived polymers have been studied insufficiently, although this feedstock has a very high potential. In the present paper, we analyzed the changes in the structure and physicochemical properties of lignin-rich biomass induced by thermomechanical pretreatment. Low-temperature treatment allows one to retain the original supramolecular structure of the cell walls, while an appreciably high disintegration degree is reached. This increases the reactivity of the material in the subsequent heterogeneous reactions. Mechanical pretreatment at medium temperatures (10 °C), when almost all cell wall polymers except for low-molecular-weight lignin are in the glassy state, enhances the mobility of cell wall polymers and causes sufficient cellulose disordering, while the specific surface area is not significantly increased. High-temperature pretreatment of reed biomass is accompanied by pore formation and lignin release from the cell wall structure, which opens up new prospects for using this biomass as a matrix to produce core-shell-structured sorbents of heavy metals. The energy consumed by mechanochemical equipment for the activation of reed biomass was determined.

Raoultella bacteriophage RP180, a new member of the genus Kagunavirus, subfamily Guernseyvirinae.

A novel lytic Raoultella phage, RP180, was isolated and characterized. The RP180 genome has 44,851 base pairs and contains 65 putative genes, 35 of them encoding proteins whose functions were predicted based on sequence similarity to known proteins. The RP180 genome possesses a gene synteny typical of members of the subfamily Guernseyvirinae. Phylogenetic analysis of the RP180 genome and similar phage genomes revealed that phage RP180 is the first member of the genus Kagunavirus, subfamily Guernseyvirinae, that is specific for Raoultella sp. The genome of RP180 encodes a putative protein with similarity to CRISPR-like Cas4 nucleases, which belong to the pfam12705/PDDEXK_1 family. Cas4-like proteins of this family have been shown to interfere with the bacterial host type II-C CRISPR-Cas system.

Extra Purified Exosomes from Human Placenta Contain An Unpredictable Small Number of Different Major Proteins.

Exosomes are nanovesicles (30-100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30-100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins' tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11-13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.

Exosomes from human placenta purified by affinity chromatography on sepharose bearing immobilized antibodies against CD81 tetraspanin contain many peptides and small proteins.

Exosomes are nanovesicles (40-100 nm) containing various RNAs and different proteins. Exosomes are involved in intracellular communication and immune system function. Exosomes from different sources are usually isolated using standard methods-centrifugation and ultracentrifugations. Exosomes isolated by these procedures were reported to contain from a few dozen to thousands of different proteins. Here crude vesicle preparations from five placentas (normal pregnancy) were first obtained using standard centrifugation procedures. According to electron-microscopic studies, these preparations contained vesicles of different size (30-225 nm), particles of round shape of average electron density ("nonvesicles" 20-40 nm) (A), structured clusters of associated proteins and shapeless aggregations (B), as well as ring-shaped 10-14 nm structures formed by ferritin (C). After additional purification of the vesicle preparations by gel filtration on Sepharose 4B, the main part of protein structures was removed; however, the preparations still contained small admixtures of components A-C. Further purification of the preparations by affinity chromatography on Sepharose bearing immobilized antibodies against exosome surface protein CD81 led to isolation of highly purified exosomes (40-100 nm). These exosomes according to electron microscopy data contained tetraspanin embedded in the membrane, which was stained with antibodies against CD81 conjugated with 10-12 nm gold nanoparticles. SDS-PAGE and MALDI MS and MS/MS mass spectrometry of tryptic hydrolysates of proteins contained in these exosomes revealed eleven major proteins (>10 kDa): hemoglobin subunits, CD81, interleukin-1 receptor, annexin A5, cytoplasmic actin, alpha-actin-4, alkaline phosphatase, human serum albumin, serotransferrin, and lactotrasferrin. Using MALDI mass analysis of the highly purified exosomes, we for the first time found that in addition to the large proteins (>10 kDa), exosomes having affinity to CD81 contain more than 27 different peptides and small proteins of 2-10 kDa. This finding can be useful for revealing biological functions of pure exosomes. © 2018 IUBMB Life, 70(11):1144-1155, 2018.

Ameliorative effects of SkQ1 eye drops on cataractogenesis in senescence-accelerated OXYS rats.

BACKGROUND: Antioxidant supplements have been suggested as a strategy to decrease the risk of age-related cataract, but there is no evidence that antioxidants can reduce the signs of the disease. Recently, we showed that the mitochondrial antioxidant SkQ1 can partially reverse cataract signs in senescence-accelerated OXYS rats. The aim of the present study was the histomorphological examination of the influence of SkQ1 eye drops on the cataract development in OXYS rats. METHODS: OXYS rats received SkQ1 eye drops (250 nM) from 9 to 12 months of age. Ophthalmoscopic examination was carried out before and after treatment. Light and electron microscopy were used for histomorphological examination. Expression of the Cryaa and Cryab genes was determined using real-time PCR. αB-crystallin expression was detected using Western blotting. RESULTS: SkQ1 completely prevented the cataract development in OXYS rats, and in some of the animals diminished the signs of the disease. Light and electron microscopy showed that SkQ1 attenuated the (typical for cataract) alterations in the lens capsule and epithelial cells, ameliorated disturbances of the hexagonal packing geometry of lens fibers, and improved ultrastructure of the epithelial cells. The levels of mRNA of α-crystallins genes which encode small heat shock proteins αA- and αB-crystallin that play a central role in maintaining lens transparency were significantly lower in the OXYS rats' lenses than in Wistar rats (control). SkQ1 normalized the level of mRNA of Cryaa, and significantly increased the level of Cryab mRNA as well as αB-crystallin protein in the lens of OXYS rats to the level of the control Wistar rats. CONCLUSION: SkQ1 eye drops hold promise as a treatment of cataract.

Immunotherapy of hepatocellular carcinoma with small double-stranded RNA.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, immunotherapy is considered a promising therapeutic approach. Small interfering RNAs (siRNAs), depending on their structure and sequence, can trigger the innate immune system, which can potentially enhance the adaptive anticancer immune response in the tumor-bearing subjects. Immunostimulatory properties of nucleic acids can be applied to develop adjuvants for HCC treatment. METHODS: The transplantable HCC G-29 tumor in male CBA/LacSto (CBA) mice was used to study the effects of immunostimulatory RNA on tumor growth. Tumor size, metastases area in different organs of mice and mouse survival rate were analyzed. Furthermore the mouse serum IFN-α levels were measured using ELISA. RESULTS: In the present study, we found that a 19-bp RNA duplex (ImmunoStimulattory RNA or isRNA) with 3-nt overhangs at the 3'-ends of specific sequence displays immunostimulatory, antitumor, and antimetastatic activities in mice bearing HCC G-29. Our results demonstrate that isRNA strongly increases the level of interferon-α (IFN-α) by up to 25-fold relative to the level in mice injected with Lipofectamine alone (Mock), and to a lesser extent increases the level of proinflammatory cytokine interleukin-6 (IL-6) (by up to 5.5-fold relative to the Mock level), in mice blood serum. We showed that isRNA reliably (P < 0.05) inhibits primary tumor growth in mice compared to the mock group. Furthermore, injections of isRNA significantly enhanced necrotic processes in the center of the primary tumor, and decreased by twofold the width of the undifferentiated peripheral zone and the number of mitotic cells in this zone. The results showed that isRNA efficiently reduces the area of metastases in the liver, kidneys, and heart of CBA/LacSto mice with HCC. CONCLUSIONS: The obtained results clearly demonstrate immunostimulatory and antimetastatic properties of the isRNAs in mice with HCC. Consequently, this short double-stranded RNA can be considered as a potential adjuvant for the therapy of HCC.

Virus nomenclature below the species level: a standardized nomenclature for filovirus strains and variants rescued from cDNA.

Specific alterations (mutations, deletions, insertions) of virus genomes are crucial for the functional characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation of attenuated viruses that could serve as vaccine candidates. Virus genome tailoring can be performed either by using traditionally cloned genomes as starting materials, followed by site-directed mutagenesis, or by de novo synthesis of modified virus genomes or parts thereof. A systematic nomenclature for such recombinant viruses is necessary to set them apart from wild-type and laboratory-adapted viruses, and to improve communication and collaborations among researchers who may want to use recombinant viruses or create novel viruses based on them. A large group of filovirus experts has recently proposed nomenclatures for natural and laboratory animal-adapted filoviruses that aim to simplify the retrieval of sequence data from electronic databases. Here, this work is extended to include nomenclature for filoviruses obtained in the laboratory via reverse genetics systems. The previously developed template for natural filovirus genetic variant naming, (/)///-, is retained, but we propose to adapt the type of information added to each field for cDNA clone-derived filoviruses. For instance, the full-length designation of an Ebola virus Kikwit variant rescued from a plasmid developed at the US Centers for Disease Control and Prevention could be akin to "Ebola virus H.sapiens-rec/COD/1995/Kikwit-abc1" (with the suffix "rec" identifying the recombinant nature of the virus and "abc1" being a placeholder for any meaningful isolate designator). Such a full-length designation should be used in databases and the methods section of publications. Shortened designations (such as "EBOV H.sap/COD/95/Kik-abc1") and abbreviations (such as "EBOV/Kik-abc1") could be used in the remainder of the text, depending on how critical it is to convey information contained in the full-length name. "EBOV" would suffice if only one EBOV strain/variant/isolate is addressed.

Bacteriophage vB_SepP_134 and Endolysin LysSte_134_1 as Potential Staphylococcus-Biofilm-Removing Biological Agents.

Bacteria of the genus Staphylococcus are significant challenge for medicine, as many species are resistant to multiple antibiotics and some are even to all of the antibiotics we use. One of the approaches to developing new therapeutics to treat staphylococcal infections is the use of bacteriophages specific to these bacteria or the lytic enzymes of such bacteriophages, which are capable of hydrolyzing the cell walls of these bacteria. In this study, a new bacteriophage vB_SepP_134 (St 134) specific to Staphylococcus epidermidis was described. This podophage, with a genome of 18,275 bp, belongs to the Andhravirus genus. St 134 was able to infect various strains of 12 of the 21 tested coagulase-negative Staphylococcus species and one clinical strain from the Staphylococcus aureus complex. The genes encoding endolysin (LysSte134_1) and tail tip lysin (LysSte134_2) were identified in the St 134 genome. Both enzymes were cloned and produced in Escherichia coli cells. The endolysin LysSte134_1 demonstrated catalytic activity against peptidoglycans isolated from S. aureus, S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus warneri. LysSte134_1 was active against S. aureus and S. epidermidis planktonic cells and destroyed the biofilms formed by clinical strains of S. aureus and S. epidermidis.

Changes in the Ultrastructure of Staphylococcus aureus Cells Make It Possible to Identify and Analyze the Injuring Effects of Ciprofloxacin, Polycationic Amphiphile and Their Hybrid.

The purposeful development of synthetic antibacterial compounds requires an understanding of the relationship between effects of compounds and their chemical structure. This knowledge can be obtained by studying changes in bacteria ultrastructure under the action of antibacterial compounds of a certain chemical structure. Our study was aimed at examination of ultrastructural changes in S. aureus cells caused by polycationic amphiphile based on 1,4‒diazabicyclo[2.2.2]octane (DL412), ciprofloxacin and their hybrid (DL5Cip6); the samples were incubated for 15 and 45 min. DL412 first directly interacted with bacterial cell wall, damaging it, then penetrated into the cell and disrupted cytoplasm. Ciprofloxacin penetrated into cell without visually damaging the cell wall, but altered the cell membrane and cytoplasm, and inhibited the division of bacteria. The ultrastructural characteristics of S. aureus cells damaged by the hybrid clearly differed from those under ciprofloxacin or DL412 action. Signs associated with ciprofloxacin predominated in cell damage patterns from the hybrid. We studied the effect of ciprofloxacin, DL412 and their hybrid on S. aureus biofilm morphology using paraffin sections. Clear differences in compound effects on S. aureus biofilm (45 min incubation) were observed. The results obtained allow us to recommend this simple and cheap approach for the initial assessment of antibiofilm properties of synthesized compounds.

Fixation and Visualization of Full Protein Corona on Lipid Surface of Composite Nanoconstruction.

Spontaneous sorption of proteins on the nanoparticles' surface leads to the fact that nanoparticles in biological media are always enveloped by a layer of proteins-the protein corona. Corona proteins affect the properties of nanoparticles and their behavior in a biological environment. In this regard, knowledge about the composition of the corona is a necessary element for the development of nanomedicine. Because proteins have different sorption efficacy, isolating particles with a full corona and characterizing the full corona is challenging. In this study, we propose a photo-activated cross-linker for full protein corona fixation. We believe that the application of our proposed approach will make it possible to capture and visualize the full corona on nanoparticles coated with a lipid shell.

Chemical Modifications Influence the Number of siRNA Molecules Adsorbed on Gold Nanoparticles and the Efficiency of Downregulation of a Target Protein.

Small interfering RNAs (siRNAs) are a powerful tool for specific suppression of protein synthesis in the cell, and this determines the attractiveness of siRNAs as a drug. Low resistance of siRNA to nucleases and inability to enter into target cells are the most crucial issues in developing siRNA-based therapy. To face this challenge, we designed multilayer nanoconstruct (MLNC) with AuNP core bearing chemically modified siRNAs. We applied chemical modifications 2'-OMe and 2'-F substitutions as well as their combinations with phosphoryl guanidine group in the internucleotide phosphate. The effect of modification on the efficiency of siRNA loading into nanocarriers was examined. The introduction of the internucleotide modifications into at least one of the strands raised the efficiency of siRNA adsorption on the surface of gold core. We also tested the stability of modified siRNA adsorbed on gold core in the presence of serum. Based on loading efficiency and stability, MLNCs with the most siRNA effective cargo were selected, and they showed an increase in biological activity compared to control MLNCs. Our study demonstrated the effect of chemical modifications of siRNA on its binding to the AuNP-based carrier, which directly affects the efficiency of target protein expression inhibition.

Effect of Fluorescent Labels on DNA Affinity for Gold Nanoparticles.

Fluorophore (FD) labeling is widely used for detection and quantification of various compounds bound to nanocarriers. The systems, composed of gold nanoparticles (GNPs) and oligonucleotides (ONs) labeled with FDs, have wide applications. Our work was aimed at a systemic study of how FD structure (in composition of ON-FDs) influenced the efficiency of their non-covalent associates' formation with GNPs (ON-FD/GNPs). We examined ONs of different length and nucleotide composition, and corresponding ON-FDs (FDs from a series of xanthene, polymethine dyes; dyes based on polycyclic aromatic hydrocarbons). Methods: fluorometry, dynamic light scattering, high performance liquid chromatography, gel electrophoresis, molecular modeling and methods of thermodynamic and statistical analysis. We observed significant, differing several times, changes in surface density and Langmuir constant values of ON-FDs vs. ONs, evidence for the critical significance of FD nature for binding of ON-FDs with GNPs. Surface density of ON-FD/GNPs; hydrophobicity and total charge of ON or ON-FD; and charge and surface area of FDs were revealed as key factors determining affinity (Langmuir constant) of ON or ON-FDs for GNPs. These factors compose a specific set, which makes possible the highly reliable prediction of efficiency of ONs and ON-FDs binding with GNPs. The principal possibility of creating an algorithm for predictive calculation of efficiency of ONs and GNPs interaction was demonstrated. We proposed a hypothetical model that described the mechanism of contact interaction between negatively charged nano-objects, such as citrate-stabilized GNPs, and ONs or ON-FDs.

A Lipid-Coated Nanoconstruct Composed of Gold Nanoparticles Noncovalently Coated with Small Interfering RNA: Preparation, Purification and Characterization.

There is an urgent need to develop systems for nucleic acid delivery, especially for the creation of effective therapeutics against various diseases. We have previously shown the feasibility of efficient delivery of small interfering RNA by means of gold nanoparticle-based multilayer nanoconstructs (MLNCs) for suppressing reporter protein synthesis. The present work is aimed at improving the quality of preparations of desired MLNCs, and for this purpose, optimal conditions for their multistep fabrication were found. All steps of this process and MLNC purification were verified using dynamic light scattering, transmission electron microscopy, and UV-Vis spectroscopy. Factors influencing the efficiency of nanocomposite assembly, colloidal stability, and purification quality were identified. These data made it possible to optimize the fabrication of target MLNCs bearing small interfering RNA and to substantially improve end product quality via an increase in its homogeneity and a decrease in the amount of incomplete nanoconstructs. We believe that the proposed approaches and methods will be useful for researchers working with lipid nanoconstructs.

Rational Design of Albumin Theranostic Conjugates for Gold Nanoparticles Anticancer Drugs: Where the Seed Meets the Soil?

Multifunctional gold nanoparticles (AuNPs) may serve as a scaffold to integrate diagnostic and therapeutic functions into one theranostic system, thereby simultaneously facilitating diagnosis and therapy and monitoring therapeutic responses. Herein, albumin-AuNP theranostic agents have been obtained by conjugation of an anticancer nucleotide trifluorothymidine (TFT) or a boron-neutron capture therapy drug undecahydro-closo-dodecaborate (B12H12) to bimodal human serum albumin (HSA) followed by reacting of the albumin conjugates with AuNPs. In vitro studies have revealed a stronger cytotoxicity by the AuNPs decorated with the TFT-tagged bimodal HSA than by the boronated albumin conjugates. Despite long circulation time, lack of the significant accumulation in the tumor was observed for the AuNP theranostic conjugates. Our unique labelling strategy allows for monitoring of spatial distribution of the AuNPs theranostic in vivo in real time with high sensitivity, thus reducing the number of animals required for testing and optimizing new nanosystems as chemotherapeutic agents and boron-neutron capture therapy drug candidates.

Characterization of Powassan viruses from Far Eastern Russia.

We report the isolation and detailed characterization of the novel strain, Partizansk/2006, of Powassan virus (POWV) from a human case of infection, which occurred in Primorsky krai, Russia, in 2006. Comparative complete genome sequence analysis of the Far Eastern strains Spassk-9 (1975), Nadezdinsk-1991 and Partizansk/2006 of POWV revealed that these strains are 99.8% similar to the LB strain, which was isolated in Canada in 1958. Phylogenetic analysis of 5' UTR sequences of five other strains of POWV isolated from 1972 to 1986 in Primorsky krai produced similar results. Presumably, Far Eastern POWV has common putative ancestor with LB strain POWV from North America, and the time of divergence of these POWVs is relatively short. We conclude that POWV has become endemic in Far Eastern Russia.

Long-term stability and scale-up of noncovalently bound gold nanoparticle-siRNA suspensions.

Gold nanoparticles (AuNPs) are a platform for the creation of nanoconstructions that can have a variety of functions, including the delivery of therapeutic nucleic acids. We previously designed a AuNP/small interfering RNA (siRNA) nanoconstruction consisting of siRNA noncovalently bound on the AuNP surface and showed that this construction, when coated with a lipid shell, was an efficient vehicle for the delivery of siRNA into cells. The goal of the present work was to study the possibility of scaling up the synthesis of AuNP-siRNA and its long-term storage without loss of physicochemical characteristics and siRNA duplex integrity as well as siRNA surface density. Dynamic light scattering, transmission electron microscopy, UV-vis spectroscopy, and electrophoresis were used to study the effect of scaling up the AuNP-siRNA synthesis and long term storage of its suspension on physicochemical properties of the samples and integrity of the siRNA duplex. It was shown that a ten-fold increase in the volume of the reaction mixture decreased the surface density of siRNA by about 10%, which influenced the corresponding physicochemical characteristics of the AuNP-siRNA suspension. The storage of the AuNP-siRNA suspension at 4 °C for different times resulted in the formation of particle clusters of high colloidal stability as demonstrated by conventional methods. These clusters completely disintegrated when albumin was added, indicating that they are agglomerates (and not aggregates) of AuNP-siRNA. The AuNPs-siRNA nanoconstruction demonstrated integrity of the siRNA duplex and high stability of the siRNA surface density during storage for seven months at 4 °C. Thus, it can be concluded that it is possible to scale-up the synthesis of noncovalent AuNP-siRNA and to obtain a nanoconstruction possessing high stability in terms of physicochemical characteristics and siRNA surface density for a long period.