Low Molecular Weight RNA Research Articles

An Insight into the Proofreading Functions of Multisubunit DNA-Dependent RNA Polymerases and Their Catalytic Mechanism

Aim: To analyze the active sites of the proofreading (PR) functions in the multisubunit DNA-dependent RNA polymerases (MSU RNAPs) from prokaryotes, chloroplasts and eukaryotes, and propose a plausible unified catalytic mechanism for these enzymes.
 Study Design: Data collected on these enzymes from bioinformatics, biochemical, site-directed mutagenesis (SDM), X-ray crystallography and cryo-electron microscopy (cryo-EM) were used for the analyses.
 Methodology: The protein sequence data of MSU RNAPs from prokaryotes, prokaryotic-types (plant chloroplasts) and eukaryotes were obtained from PUBMED and SWISS-PROT databases. The advanced version of Clustal Omega was used for protein sequence analysis. Along with the conserved motifs identified by the bioinformatics analysis, the data already available from biochemical and SDM experiments, and X-ray crystallographic and cryo-EM data on these enzymes are also used to confirm the possible amino acids involved in the active site of the PR function in these MSU RNAPs
 Results: All the seven types of MSU RNAPs (I-VII) reported from prokaryotes to eukaryotes were analyzed by the multiple sequence alignment (MSA) software, Clustal Omega, to find out conservations among them. The MSA analysis showed many conserved amino acid motifs including small and large peptide regions from the MSU RNAPs of prokaryotes, eukaryotes and plant chloroplasts. Interestingly, the catalytic amino acid and template-binding pairs are highly conserved in all these polymerases, with a few exceptions. Most of them use a basic amino acid (R/K/H) for initiating catalysis and an -YG/FG- pair for template-binding. Some odd type of catalytic amino acids and template-binding pairs are observed in human pathogens, parasites and organisms which cannot ferment sugars. In all the MSU RNAPs, the proposed polymerase catalytic region also possessed three invariant Cs and an invariant H within it. The invariant Cs is shown to bind a zinc atom and proposed to involve in the PR function by excising any misincorporated nucleotide during the transcription process. In the plant-specific MSU RNAPs IV and V, which involve in transcriptional gene silencing in plants, the catalytic and template-binding pairs do not follow the regular distance conservations as observed with other five of the MSU RNAPs. Their polymerase/PR active site regions are similar to RNAP III rather than to RNAP II, as all three make only low molecular weight RNAs.
 Conclusions: All the known MSU RNAPs possess three invariant Cs and an invariant H embedded within the polymerase active site itself. The three invariant Cs are shown to bind a zinc atom and the invariant H could act as the proton acceptor from a metal-bound water molecule, for initiating excision of the mismatches by a Zn-mediated hydrolysis. Thus, the PR function in MSU RNAPs is integrated within the polymerase active site itself, which is in sharp contrast to the PR functions reported in DNA-dependent DNA polymerases and RNA-dependent RNA polymerases. Therefore, all the seven MSU RNAPs from prokaryotes and eukaryotes are proposed to follow a unified mechanism to excise the mismatches during transcription. The discovery of intrinsic self-correcting RNA transcription mechanism fulfils the missing link in molecular evolution.

Tumor cell invasion into Matrigel: optimized protocol for RNA extraction.

3D models are increasingly used to study mechanisms driving tumor progression and mimicking in vitro processes such as invasion and migration. However, there is a need to establish more protocols based on 3D culture systems thatallow for downstream molecular biology investigations. Materials & methods: Herewe present a method for optimal RNA extraction from highly aggressive primary glioma cells invading into Matrigel. The method has been established bycomparing previously reported protocols, available commercial kits and optimizing specific steps for matrix dissociation, RNA separation and purification. Results and conclusion: The protocol allows RNA extraction from cells embedded into Matrigel, with optimal yield, purity and integrity suitable for subsequent sequencing analysis of both high and low molecular weight RNA.

Global Role of Low Molecular Weight Nucleic Acids in Biological Systems

Some considerations and reports are made regarding personal scientific developments carried out by author V.M. Zemskov in partnership with colleagues team and my close colleague prof. Zemskov A.M. for many years, specifically, 50 years. This is a problem, to which almost entire life has been devoted. It relates to a completely new global consistent pattern that we managed to stumble upon in those distant years, and that is implemented in any biological systems -whether it’s a higher or a lower organism, a human being, or various microbial and cellular populations. Realized by low molecular weight RNA or oligonucleotides of RNA.

Arginine and di-arginine ligands for plasmid DNA purification using negative chromatography

Abstract The increasing number of applications requiring highly purified plasmid DNA (pDNA) generates a corresponding need for simple, scalable, and cost-effective purification processes. Due to the pDNA large size and complex shape, the use of commercial chromatographic beads often results in poor yields and low binding capacities when operated in a positive mode. An alternative to overcome this limitation is the design of chromatographic ligand-resin systems able to efficiently operate in negative mode, where host impurities (especially low molecular weight RNA) are efficiently captured and separated from the target pDNA. In this work, arginine amino acid and di-arginine peptide (arginine-arginine) were immobilized in agarose resins and evaluated for negative chromatographic purification of pDNA from bacterial cell lysates. The results showed that RNA was preferentially bound to the ligands, interfering with the binding of pDNA. The amount of plasmid processed per column volume by arginine and di-arginine, under negative mode, was substantially larger comparing with the conventional positive mode, resulting in pDNA recoveries up to 99%, with a considerable reduction of host impurities. This study shows that negative mode chromatography using arginine-based ligands poses as an interesting alternative for intermediate and polishing pDNA purification operations, with considerable economic and environmental advantages.

Expression profiles of long and short rnas in the cytoplasm and nuclei of growing chicken (Gallus gallus domesticus) oocytes

Maternal RNAs accumulated during oocyte maturation are required not only for zygote formation but also for supporting the first embryonic cell divisions until the activation of the embryo genome. The key stages of transcriptome analysis include the adaptation of RNA extraction procedures and characterization of the RNA expression profile. The ovaries of domestic birds represent an adequate model for the exploration of RNA accumulation during oogenesis. In the present study, we optimized the methodologies for RNA extraction from the oocyte cytoplasm and nucleoplasm of egg-laying chickens (Gallus gallus domesticus), and characterized the changes in the expression profiles of long and short RNAs during oocyte growth. The cytoplasmic RNA fractions contained 28S and 18S ribosomal RNAs (rRNAs), small RNAs, and long RNAs that were heterogeneous in size. The profiles of the total RNAs from growing oocyte nuclei were dominated by low molecular weight RNAs corresponding in size to transport RNAs, small nuclear RNAs, and short regulatory RNAs. Importantly, nuclei from chicken growing oocytes demonstrated trace amounts or the absence of 28S and 18S rRNAs, which was possibly due to the inactivation of the single nucleolus organizer. We identified three groups of short RNAs differing in size (from 20 to 40 nucleotides) in the chicken oocytes. These may correspond to short regulatory RNA classes: microRNA, siRNA and piRNA. Furthermore, we demonstrated that short RNAs accumulate in the cytoplasm during oocyte growth. We propose that the short RNAs accumulated in the avian oocyte cytoplasm are involved in the regulation of genome functions during the early embryogenesis stages.

A new oxygen modification cyclooctaoxygen binds to nucleic acids as sodium crown complex

BackgroundOxygen exists in two gaseous and six solid allotropic modifications. An additional allotropic modification of oxygen, the cyclooctaoxygen, was predicted to exist in 1990. MethodsCyclooctaoxygen sodium was synthesized in vitro from atmospheric oxygen, or catalase effect-generated oxygen, under catalysis of cytosine nucleosides and either ninhydrin or eukaryotic low-molecular weight RNA. Thin-layer chromatographic mobility shift assays were applied on specific nucleic acids and the cyclooctaoxygen sodium complex. ResultsWe report the first synthesis and characterization of cyclooctaoxygen as its sodium crown complex, isolated in the form of three cytosine nucleoside hydrochloride complexes. The cationic cyclooctaoxygen sodium complex is shown to bind to nucleic acids (RNA and DNA), to associate with single-stranded DNA and spermine phosphate, and to be essentially non-toxic to cultured mammalian cells at 0.1–1.0mM concentration. ConclusionsWe postulate that cyclooctaoxygen is formed in most eukaryotic cells in vivo from dihydrogen peroxide in a catalase reaction catalyzed by cytidine and RNA. A molecular biological model is deduced for a first epigenetic shell of eukaryotic in vivo DNA. This model incorporates an epigenetic explanation for the interactions of the essential micronutrient selenium (as selenite) with eukaryotic in vivo DNA. General significanceSince the sperminium phosphate/cyclooctaoxygen sodium complex is calculated to cover the active regions (2.6%) of bovine lymphocyte interphase genome, and 12.4% of murine enterocyte mitotic chromatin, we propose that the sperminium phosphate/cyclooctaoxygen sodium complex coverage of nucleic acids is essential to eukaryotic gene regulation and promoted proto-eukaryotic evolution.

Micro RNA in Exosomes from HIV-Infected Macrophages.

Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA) during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM) which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients.

Expression profiles of long and short RNA s in the cytoplasm and nuclei of growing chicken (Gallus gallus domesticus) oocytes

Maternal RNAs accumulated during oocyte maturation are required not only for zygote formation but also for supporting the first embryonic cell divisions until embryo genome activation. Essential stages of transcriptome analysis include adaptation of RNA extraction procedures and characterization of the RNA expression profile. Ovaries of domestic birds represent an adequate model for exploration of RNA accumulation during oogenesis. In the present study, we optimized methods of RNA extraction from chicken (Gallus gallus domesticus) oocyte cytoplasm and nucleoplasm and characterized changes in profiles of long and short RNAs during oocyte growth. Cytoplasmic RNA fractions contained 28S and 18S ribosomal RNAs (rRNAs), small RNAs, and long RNAs heterogeneous in size. The profiles of total RNA from growing oocyte nuclei were dominated by low molecular weight RNAs corresponding in size to transport RNAs, small nuclear RNAs, and short regulatory RNAs. Importantly, oocyte nuclei from chicken egglaying females demonstrated trace amounts or absence of 28S and 18S rRNA, which was due to inactivation of the only nucleolar organizer. Three groups of short RNAs differing in size (from 20 to 40 nucleotides) were recognized in chicken oocytes. They might correspond to short regulatory RNA classes. Furthermore, we demonstrated that short RNAs were accumulated in the cytoplasm during oocyte growth. We suggest that short RNAs accumulated in avian oocyte cytoplasm are involved in the regulation of genome functions at early embryogenesis stages.

MicroRNA-145 promotes differentiation in human urothelial carcinoma through down-regulation of syndecan-1.

BackgroundA new molecular marker of carcinoma in the urinary bladder is needed as a diagnostic tool or as a therapeutic target. Potential markers include microRNAs (miRNAs), which are short, low molecular weight RNAs 19–24 nt long that regulate genes associated with cell proliferation, differentiation, and development in various cancers. In this study, we investigated the molecular mechanisms by which miR-145 promotes survival of urothelial carcinoma cells and differentiation into multiple lineages. We found miR-145 to regulate expression of syndecan-1, a heparin sulfate proteoglycan.MethodsCell proliferation in the human urothelial carcinoma cell lines T24 and KU7 was assessed by MTS assay. Cellular senescence and apoptosis were measured by senescence-associated β-galactosidase (SA-β-gal) and TUNEL assay, respectively. Quantitative RT-PCR was used to measure mRNA expression of various genes, including syndecan-1, stem cell factors, and markers of differentiation into squamous, glandular, or neuroendocrine cells.ResultsOverexpression of miR-145 induced cell senescence, and thus significantly inhibited cell proliferation in T24 and KU7 cells. Syndecan-1 expression diminished, whereas stem cell markers such as SOX2, NANOG, OCT4, and E2F3 increased. miR-145 also up-regulated markers of differentiation into squamous (p63, TP63, and CK5), glandular (MUC-1, MUC-2, and MUC-5 AC), and neuroendocrine cells (NSE and UCHL-1). Finally, expression of miR-145 was down-regulated in high-grade urothelial carcinomas, but not in low-grade tumors.ConclusionsResults indicate that miR-145 suppresses syndecan-1 and, by this mechanism, up-regulates stem cell factors and induces cell senescence and differentiation. We propose that miR-145 may confer stem cell-like properties on urothelial carcinoma cells and thus facilitate differentiation into multiple cell types.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1846-0) contains supplementary material, which is available to authorized users.

Effect of kinetin on nucleic acid synthesis in senescing and young leaves in Brassica oleracea L. var. gongylodes L.

In detached kohlrabi leaves senescing in the dark, the decrease in chlorophyll to was more pronounced than in chlorophyll a. The retardation by kinetin of the chlorophyll loss was also markedly stronger in the case of chlorophyll b. Using the fractionation of nucleic acids on polyacrylamide gels it has been shown that during leaf senescence the level of all RNA species decreased, whereas the amount of DNA was more or less constant. In the presence of kinetin, the loss of RNA was inhibited and the incorporation of precursor into the cytoplasmic rRNA as well as into low molecular weight RNA species was supported. Chloroplast rRNA synthesis has not been detected in mature leaves and kinetin showed no effect in this respect. In young expanding leaves detached and kept in light, the synthesis of cytoplasmic rRNA was strongly stimulated by kinetin, whereas in the case of Chloroplast rRNA only an inhibitory effect of kinetin could be found. The results suggest that the cytokinins are primarily involved in processes of the synthesis of cytoplasmic rRNA and low molecular RNA fractions, and in this way affect the development of plastids, in particular the course of their senescence.

Uncovering RNA Editing Sites in Long Non-Coding RNAs

RNA editing is an important co/post-transcriptional molecular process able to modify RNAs by nucleotide insertions/deletions or substitutions. In human, the most common RNA editing event involves the deamination of adenosine (A) into inosine (I) through the adenosine deaminase acting on RNA proteins. Although A-to-I editing can occur in both coding and non-coding RNAs, recent findings, based on RNA-seq experiments, have clearly demonstrated that a large fraction of RNA editing events alter non-coding RNAs sequences including untranslated regions of mRNAs, introns, long non-coding RNAs (lncRNAs), and low molecular weight RNAs (tRNA, miRNAs, and others). An accurate detection of A-to-I events occurring in non-coding RNAs is of utmost importance to clarify yet unknown functional roles of RNA editing in the context of gene expression regulation and maintenance of cell homeostasis. In the last few years, massive transcriptome sequencing has been employed to identify putative RNA editing changes at genome scale. Despite several efforts, the computational prediction of A-to-I sites in complete eukaryotic genomes is yet a challenging task. We have recently developed a software package, called REDItools, in order to simplify the detection of RNA editing events from deep sequencing data. In the present work, we show the potential of our tools in recovering A-to-I candidates from RNA-Seq experiments as well as guidelines to improve the RNA editing detection in non-coding RNAs, with specific attention to the lncRNAs.

Syndecan-1 responsive microRNA-126 and 149 regulate cell proliferation in prostate cancer

MicroRNAs (miRNAs) are short (19–24nt), low molecular weight RNAs that play important roles in the regulation of target genes associated with cell proliferation, differentiation, and development, by binding to the 3′-untranslated region of the target mRNAs. In this study, we examined the expression of miRNA-126 (miR-126) and miR-149 in prostate cancer, and investigated the molecular mechanisms by which they affect syndecan-1 in prostate cancer. Functional analysis of miR-126 and miR-149 was conducted in the prostate cancer cell lines, PC3, Du145, and LNCaP. The expression levels of SOX2, NANOG, Oct4, miR-126 and miR-149 were evaluated by quantitative RT-PCR. After silencing syndecan-1, miR-126, and/or miR-149 in the PC3 cells, cell proliferation, senescence, and p21 induction were assessed using the MTS assay, senescence-associated β-galactosidase (SA-β-Gal) assay, and immunocytochemistry, respectively. Compared to the Du145 and LNCaP cells, PC3 cells exhibited higher expression of syndecan-1. When syndecan-1 was silenced, the PC3 cells showed reduced expression of miR-126 and miR-149 most effectively. Suppression of miR-126 and/or miR-149 significantly inhibited cell growth via p21 induction and subsequently, induced senescence. The mRNA expression levels of SOX2, NANOG, and Oct4 were significantly increased in response to the silencing of miR-126 and/or miR-149. Our results suggest that miR-126 and miR-149 are associated with the expression of syndecan-1 in prostate cancer cells. These miRNAs promote cell proliferation by suppressing SOX2, NANOG, and Oct4. The regulation of these factors by miR-126 and miR-149 is essential for syndecan-1-mediated development of androgen-refractory prostate cancer.

A Rapid and Economical Method for Low Molecular Weight RNA Isolation from a Wide Variety of Plant Species

We have developed a time- and cost-effective method for isolating low molecular weight (LMW) RNA from plants. In our protocol, the isolation procedure can be completed within 3 h. Polyethylene glycol (PEG) and absolute ethanol are used to isolate LMW RNA, and the LMW RNA yields were >80 µg/g of fresh-weight tissues for several of the plant species tested.

Pistachio (Pistacia vera L.) is a new natural host of Hop stunt viroid

Besides hop, Hop stunt viroid (HpSVd) infects many woody species including grapevine, citrus, peach, plum, apricot, almond, pomegranate, mulberry and jujube. Here, we report the first detection of HpSVd in pistachio (Pistacia vera L.). Samples corresponding to 16 pistachio cultivars were obtained from a nearby almond collection. From these samples, low molecular weight RNAs were extracted for double polyacrylamide gel electrophoresis, northern-blot analysis and reverse transcription polymerase chain reaction assays. HpSVd was detected in 4 of the 16 pistachio cultivars in the first year and in 6 in the second, being also detected in the almond collection. Examination of the nucleotide sequences of pistachio and almond isolates revealed 13 new sequence variants. Sequences from pistachio shared 92-96% similarity with the first reported HpSVd sequence (GenBank X00009), and multiple alignment and phylogenetic analyses showed that one pistachio isolate (HpSVdPis67Jabari) clustered with the plum group, whereas all the others clustered with the hop, and the recombinants plum-citrus and plum-Hop/cit3 groups. By identifying pistachio as a new natural host, we confirm that HpSVd is an ubiquitous and genetically variable viroid that infects many different fruit trees cultivated worldwide.

Determination of biotic aetiology of tapping panel dryness (TPD) syndrome of rubber tree (Hevea brasiliensis) by return-polyacrylamide gel electrophoresis (R-PAGE) technique

Tapping panel dryness (TPD) syndrome affecting rubber tree (Hevea brasiliensis) is known to reduce natural latex production. Its aetiology remains ambiguous despite long years of research. A low molecular weight RNA similar to viroid RNA was isolated from TPD-affected samples of rubber trees. In the present study, a modified return-polyacrylamide gel electrophoresis procedure was standardised. The viroid-like low molecular weight (LMW) RNA was found associated with leaf, bark and root tissues and rubber seedlings. The technique was employed to detect LMW RNA in different clones of rubber planted in different locations and in bud-grafted plants. The LMW RNA isolated from TPD-affected trees was found infectious on seedlings of tomato cv Pusa Ruby. The LMW RNA was reisolated from symptomatic tomato leaves but not from control plants. This is for the first time that a biotic agent, a viroid RNA, is found consistently associated with the syndrome. The technology developed can be useful to demonstrate the onset of TPD in untapped trees in the absence of other methods such as nucleic acid hybridisation.

First Report of Coleus blumei viroid 5 from Coleus blumei in India and Indonesia.

Coleus blumei, which was found originally in Indonesia, is an ornamental plant grown worldwide. It can be infected by several viroids of the genus Coleviroid, family Pospiviroidae. Six main viroids that infect coleus have been reported: Coleus blumei viroid 1 through 6 (CbVd-1 ~ CbVd-6). Although CbVd-1 was first reported in a commercial coleus in Brazil in 1989 (1), and then in Germany, Japan, Canada, Korea, China, and India, CbVd-5 was reported only in China in 2009 (2). Symptoms caused by CbVd-5 varied depending on different cultivars, and in case of an unknown cultivar of "Red with dark green edge," are very clear albino symptoms. From 2010 to 2011, 60 and 3 leaf samples of coleus were collected from Hyderabad, India, and Java, Indonesia, respectively, and subjected to low molecular weight RNA extraction according to Li et al. (3). The results of dot-blot hybridization using CbVd-5 cRNA probes and RT-PCR using CbVd-5 specific primers (CbVd-5-PF: 5'-TGACTAGAACAGTAGTAAAG-3' / CbVd-5-PR: 5'-AATTGAGGTCAAACCTCTTT-3') demonstrated that 28 out of the 60 samples from India and all three samples from Indonesia were positive for CbVd-5. The resulting RT-PCR fragments from one sample selected randomly from each country were cloned into the pMD18-T vector (Takara) and transformed into E. coli DH5α competent cells. Five positive clones of each sample were sequenced. The result of sequence analysis revealed that the similarities of CbVd-5 between the sequences we obtained and the reference sequence (GenBank Accession No. NC003683) were 97.8 to 100%. Bioassay using nine viroid-free coleus plants from three cultivars (three from each cultivar), inoculated with CbVd-5 infectious clones by stem slashing, demonstrated that CbVd-5 could induce albino symptom on the leaves of the unknown cultivar "Red with dark green edge" 2 months after inoculation. To our knowledge, this is the first report of CbVd-5 from India and Indonesia, and the second report of CbVd-5 in the world. Considering the effect of CbVd-5 on the appearance of coleus and its recombination ability, a certification program may be needed to control the spread of this viroid.

First Report of Coleus blumei viroid Infecting Coleus in India.

Members of the genus Coleviroid (Coleus blumei viroid [CbVd]), family Pospiviroidae have been reported to infect Coleus (Solenostemon sp.). CbVd-1 was first reported from Brazil, CbVd-2, -3, and -4 were first reported from Germany, whereas CbVd-5 and -6 were recently identified in China (2). In India, Coleus is extensively cultivated as an ornamental plant in home gardens. In March to June 2012, Coleus leaf samples with irregular chlorotic spots/patches were collected from home gardens of two different districts of Karnataka (Kodagu and Mysore districts), India, suspecting the presence of Coleus blumei viroids (CbVd 1 to 6). Low molecular weight RNAs were extracted using 2% CTAB buffer containing 1.4 M NaCl, 20 mM EDTA, pH 8.0 and 100 mM Tris-Cl, pH 8.0 (1). Viroid-like RNA was enriched by fractionation 2M LiCl soluble nucleic acids (4). A DNA fragment with the expected size of CbVd-1 was detected in 10 (including both districts) of 14 analyzed (incident rate of 71%) from reverse transcription (RT)-PCR assay using Coleviroid specific primers (forward 5'-TGGATCCAGCGCTGCAACGGAATCCA-3' and reverse 5'-TTGGATCCGCCAGGGAACCCAGGTAAG-3'). RT was performed at 37°C for 60 min in 25 μl reaction mix containing 5 μl RNA extracts, 1 μl reverse primer, 1× first strand buffer, 10 mM dNTPs, and 200U M-MuLV-RT (Invitrogen, USA). For PCR, 2 μl RT was mixed in 25 μl PCR mix containing 0.2 μM each forward and reverse primers and 2U LA Taq (Takara-Bio, Japan) according to the manufacturer's instruction. PCR parameter was one cycle at 94°C for 2 min, 35 cycles at 94°C for 45 s, 53°C for 30 s, and 72°C for 60 s, followed by final extension at 72°C for 10 min (4). Sequence analysis of cloned amplicons detected CbVd-1 in India. To confirm the sequence of the primer regions, an additional set of tail-tail primers were designed, CbVd1-136F (5'-CTTCGTGGAACGGCTCCGCG-3') and CbVd1-136Rev (5'-GAAGAAGCCGAAGCAACTCTC-3') and were used for RT-PCR. Amplified products were cloned, sequenced and compared with previously obtained data. Further, the presences of CbVd-1 in Coleus samples was confirmed by a RNA gel blot assay using digoxigenin-labeled CbVd-1 cRNA probe (3). Alignment of 19 sequences obtained from four representative Coleus samples found the presence of two sequence variants of CbVd-1, namely Ind-1 (GenBank Accession No. AB740017) and Ind-2 (AB740018). Ind-1 was found to differ from Ind-2 by two nucleotide substitutions at position 40 (C to T) and 211 (T to C). BLAST analysis of Ind-1 showed 100% sequence similarity with CbVd-1 isolates from China (DQ178399) and South Korea (EU 410620), whereas Ind-2 was 99% identical to these two Chinese and Koreans isolates. Furthermore, 97% and 96% sequence identity with CbVd 1-RL RNA (Accession no. X95366) was observed for Ind-1 and Ind-2, respectively. Isolates from India were 88% similar with Coleus blumei viroid 1-RG (X95291). To the best of our knowledge, this is the first molecular evidence for the presence of CbVd-1 infecting Coleus in India. Coleus harbors various viroid species and CbVd-1, reported widely, can transmit efficiently through seed and also could infect the other herbaceous plants (3). This report from India will contribute further understanding of a potential risk of Coleus viroids in ornamental species.

Functional and immunophenotype characteristics of peripheral blood mononuclear leukocytes from healthy donors under sodium nucleinate effect

Event Abstract Back to Event Functional and immunophenotype characteristics of peripheral blood mononuclear leukocytes from healthy donors under sodium nucleinate effect Olga V. Lebedinskaya1*, Elena A. Lebedinskaya1 and Nelly K. Akhmatova2 1 Acad. E.A. Wagner Perm State Medical Academy, Russia 2 I.I. Mechnikov Institute for Vaccines and Sera, RAMS, Russia Aim: investigation of sodium nucleinate (SN) - sodium salt of low-molecular weight RNA produced from baker’s yeast - against functional activity and immunophenotype of cells-effectors of immunity from healthy donor peripheral blood. There were analyzed killer activity relatively to K-562 cell line, proliferative activity and immunophenotype of mononuclear leukocytes isolated from healthy donor blood and cultivated for 48 h with the addition of sodium nucleinate. Investigation demonstrated that lymphocytes obtained from peripheral blood mononuclear leukocytes (PBML) as a result of extracorporal activation with sodium nucleinate were characterized by significantly greater functional activity as compared with the initial cells. Cytotoxic properties of activated cells increased from 49,5% (control) to 69,7-74,7% at SN concentration of 1-100 μg/ml. Proliferative activity increased from 16,1% (control) to 56,3-61,3% after 1-10 μg/ml of SN addition in cultures. Smears made from cultural cell suspension demonstrated large amount of pro-lymphocytes and immunoblasts, some cells in mitotic division that reflected the lymphoid cell blasttransformation as a result of their SN-induced activation. While studying the immunophenotype of SN-activated PBML from donors it was revealed that those expressed activation molecules CD38, CD25, molecules of II-type major histocompatibility complex (HLA-DR), adhesion molecules (CD57, CD58) on their membranes. Thus sodium nucleinate renders promoting effect on natural killers of donors and results in formation of lymphocytes similar to lymphokine-activated killers by their immunophenotype. SN ability to activate PBML evidences for its promising use for immune disease prevention and as nonspecific stimulator of immunity effectors in therapy of oncological patients. Keywords: sodium nucleinate, immunophenotype, immunity effectors, killer activity, proliferative activity Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Lebedinskaya OV, Lebedinskaya EA and Akhmatova NK (2013). Functional and immunophenotype characteristics of peripheral blood mononuclear leukocytes from healthy donors under sodium nucleinate effect. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00843 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Ms. Olga V Lebedinskaya, Acad. E.A. Wagner Perm State Medical Academy, Perm, Russia, lebedinska@mail.ru Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Olga V Lebedinskaya Elena A Lebedinskaya Nelly K Akhmatova Google Olga V Lebedinskaya Elena A Lebedinskaya Nelly K Akhmatova Google Scholar Olga V Lebedinskaya Elena A Lebedinskaya Nelly K Akhmatova PubMed Olga V Lebedinskaya Elena A Lebedinskaya Nelly K Akhmatova Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Plasmid DNA recovery from fermentation broths by a combined process of micro- and ultrafiltration: Modeling and application

Abstract Microfiltration and ultrafiltration operations were used in tandem to isolate and purify a 6050 bp plasmid DNA (pDNA). To achieve primary isolation of the plasmid from fermentation broths, immediately after cell lysis, a 0.2 μm microfiltration membrane was selected for solid/liquid separation, which was performed in a diafiltration mode, as an alternative to centrifugation. Then to attain plasmid concentration and purification, an ultrafiltration membrane with a pore radius of 4.1 nm was selected. Permeation of pDNA and RNA in the two membrane steps was modeled using recently published mass transfer models applicable to the permeation of closed segmented chains and freely-jointed chains, respectively. The permeation of proteins and genomic DNA (gDNA) was also studied in these operations. The microfiltration operation allowed high plasmid and RNA permeation, as expected. It was observed that significant amounts of gDNA, previously precipitated during the cell lysis step, reappear in solution during the diafiltration step. The effect of the ionic strength on this apparent re-solubilization was investigated, by testing the addition of two different salts to the diafiltration buffer: CH3COOK and CaCl2. The results show that these salts can be used to control gDNA apparent re-solubilization. During the ultrafiltration operation high plasmid retention with low adsorption was obtained under low ionic strength conditions. The results also show that a significant removal proteins and the re-solubilized gDNA is achieved, as well as some purification in respect to low molecular weight RNA, since all these components permeate through the ultrafiltration membrane.

Molecular Identification And Sequence Analysis of the <i>Apple Scar Skin Viroid</i> (ASSVd) Isolated from Four Kinds of Fruit Trees in Xinjiang Province, China

In this paper, low molecular weight RNAs were extracted from tender leaves and shoots of apple, pear, peach and apricot trees, which were collected from Yanqi, Hejing, Bohu, Heshuo, Xinhe, Korla and Aksu in Xinjiang province, China. Then the samples were detected by reverse transcription-polymerase chain reaction (RT-PCR) and in situ RT-PCR technology. The detection results demonstrated that four kinds of fruit trees were infected by Apple scar skin viroid (ASSVd) and the detection rates were 2.1%, 2.1%, 2.8% and 3.3% on apple, pear, peach and apricot trees, respectively. I n situ RT-PCR result further confirmed that the existence of ASSVd was mainly distributed in the nucleus of the leaf tissues. And then the RT-PCR products from the samples were cloned and sequenced, and we obtained 42 ASSVd nucleic acid sequences, which were registered in GenBank and the accession numbers was from EU031455 to EU031496 by using biological software to analyze and the total detection rate was 3.0%. The homology analysis results showed that the 42 isolated ASSVd sequences had 85%~100% nucleotide sequence identity with previously published sequence NC_001340 (Puchta et al.,1990). All this results indicated that the variation of nucleic acid sequence of ASSVd isolate in each host was not obvious and was no significant difference in regions and varieties. In this present study, we established the optimized detection methods of RT-PCR and in situ RT-PCR, which would lay a good foundation for the rapid identification of ASSVd in this four kinds of fruit trees.