Optomagnetic Detection of Rolling Circle Amplification Products.

Rolling circle amplification (RCA) of a synthetic nucleic acid target is detected using magnetic nanoparticles (MNPs) combined with an optomagnetic (OM) readout. Two RCA assays are developed with on-chip detection of rolling circle products (RCPs) either at end-point where MNPs are mixed with the sample after completion of RCA or in real time where MNPs are mixed with the sample during RCA. The plastic chip acts as a cuvette, which is positioned in a setup integrated with temperature control and simultaneous detection of four parallel DNA hybridization reactions between functionalized MNPs and products of DNA amplification. The OM technique probes the small-angle rotation of MNPs bearing oligonucleotide probes complementary to the repeated nucleotide sequence of the RCPs. This rotation is restricted when MNPs bind to RCPs, which can be observed as a turn-off of the signal from MNPs that are free to rotate. The amount of MNPs bound to RCPs is found to increase in response to the amplification time as well as in response to the synthetic DNA target concentration (2-40 pM dynamic range). We report OM real-time results obtained with MNPs present during RCA and compare to relevant end-point OM results for RCPs generated for different RCA times. The real-time approach avoids opening of tubes post-RCA and thus reduces risk of lab contamination with amplification products without compromising the sensitivity and dynamic range of the assay.

Designing immunostimulatory double stranded messenger RNA with maintained translational activity through hybridization with poly A sequences for effective vaccination.

Messenger (m)RNA vaccines require a safe and potent immunostimulatory adjuvant. In this study, we introduced immunostimulatory properties directly into mRNA molecules by hybridizing them with complementary RNA to create highly immunogenic double stranded (ds)RNAs. These dsRNA formulations, comprised entirely of RNA, are expected to be safe and highly efficient due to antigen expression and immunostimulation occurring simultaneously in the same antigen presenting cells. In this strategy, design of dsRNA is important. Indeed, hybridization using full-length antisense (as)RNA drastically reduced translational efficiency. In contrast, by limiting the hybridized portion to the mRNA poly A region, efficient translation and intense immunostimulation was simultaneously obtained. The immune response to the poly U-hybridized mRNAs (mRNA:pU) was mediated through Toll-like receptor (TLR)-3 and retinoic acid-inducible gene (RIG)-I. We also demonstrated that mRNA:pU activation of mouse and human dendritic cells was significantly more effective than activation using single stranded mRNA. In vivo mouse immunization experiments using ovalbumin showed that mRNA:pU significantly enhanced the intensity of specific cellular and humoral immune responses, compared to single stranded mRNA. Our novel mRNA:pU formulation can be delivered using a variety of mRNA carriers depending on the purpose and delivery route, providing a versatile platform for improving mRNA vaccine efficiency.

Highly sensitive electrochemical biosensor for streptavidin detection based on CdSe quantum dots.

An electrochemical biosensor was developed based on a steric hindrance hybridization assay to allow the highly sensitive detection of streptavidin. In the steric hindrance hybridization assay, the signaling strand DNA (sig-DNA) was labeled at the 3' end with CdSe quantum dots (QDs) and at the 5' end with biotin, and capturing strand DNA (the complementary strand of sig-DNA) was labeled at the 5' end with thiol. The steric hindrance effect generated by streptavidin which was bound with the signaling DNA strand. The streptavidin limited the ability of the sig-DNA to hybridize with the cap-DNA, which were linked on the surface of a gold electrode. Therefore, the concentration of streptavidin was detected indirectly based on the concentration of CdSe QDs on the electrode surface. The concentration of CdSe QDs on the electrode surface was detected by differential pulse anodic stripping voltammetry. Under optimal conditions, the streptavidin detection range using the as-prepared biosensor was 1.96pg/mL to 1.96µg/mL and the detection limit was 0.65pg/mL. The experimental results showed that the electrochemical biosensor could detect streptavidin rapidly and accurately.

Microbacterium rhizomatis sp. nov., a ß-glucosidase-producing bacterium isolated from rhizome of Korean mountain ginseng.

A novel Gram-staining-positive, rod-shaped bacterium, designated DCY100(T), was isolated from rhizome of mountain ginseng root in Hwacheon mountain, Gangwon province, Republic of Korea. The 16S rRNA gene sequence analysis showed that strain DCY100(T) belonged to the genus Microbacterium and was most closely related to Microbacterium ginsengisoli KCTC 19189(T) (97.9%), Microbacterium lacus JCM 15575(T) (97.2%) and Microbacterium invictum DSM 19600(T) (97.1%). The major menaquinones were MK-11 and MK-12. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol and one unidentified glycolipid. The major fatty acids (>10.0%) were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The cell-wall peptidoglycan contained the amino acids ornithine, alanine, glutamic acid and glycine; whole-cell sugars consisted of glucose, galactose, rhamnose and ribose. The DNA G+C content was 63.6 ± 0.7 mol%. The DNA-DNA hybridization relatedness values between strain DCY100(T) and Microbacterium ginsengisoli KCTC 19189(T), Microbacterium lacus JCM 15575(T) and Microbacterium invictum DSM 19600(T) were 36.2 ± 0.4, 22.0 ± 3.0 and 15.3 ± 1.8%, respectively. On the basis of phenotypic, chemotaxonomic and genotypic analyses, the isolate is classified as a representative of a novel species in the genus Microbacterium, for which the name Microbacterium rhizomatis DCY100(T) is proposed. The type strain is DCY100(T) ( = KCTC 39529(T) = JCM 30598(T)).

Isolation of transcripts overexpressed in the human pathogen Trichophyton rubrum grown in lipid as carbon source.

Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts overexpressed in T. rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-ß-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.

Burkholderia unamae sp. nov., an N2-fixing rhizospheric and endophytic species.

It was shown recently that the genus Burkholderia is rich in N2-fixing bacteria that are associated with plants. A group of these diazotrophic isolates with identical or very similar 16S rDNA restriction patterns [designated amplified rDNA restriction analysis (ARDRA) genotypes 13, 14 and 15] was selected and a polyphasic taxonomic study was performed, which included new isolates that were recovered from rhizospheres, rhizoplanes or internal tissues of maize, sugarcane and coffee plants. Morphological, physiological and biochemical features, as well as multi-locus enzyme electrophoresis profiles and whole-cell protein patterns, of 20 strains were analysed. In addition, analysis of cellular fatty acid profiles, 16S rDNA sequence analysis and DNA-DNA reassociation experiments were performed with representative strains. The taxonomic data indicated that the strains analysed belong to a novel diazotrophic Burkholderia species, for which the name Burkholderia unamae sp. nov. is proposed. Strain MTl-641T (=ATCC BAA-744T=CIP 107921T), isolated from the rhizosphere of maize, was designated as the type strain. B. unamae was found as an endophyte of plants grown in regions with climates ranging from semi-hot subhumid to hot humid, but not from plants grown in regions with semi-hot or hot dry climates. Moreover, B. unamae was isolated from rhizospheres and plants growing in soils with pH values in the range 4.5-7.1, but not from soils with pH values higher than 7.5.

Painting of parental chromatin in Beta hybrids by multi-colour fluorescent in situ hybridization.

Sugar beet (Beta vulgaris L.) is a relatively young crop and has a narrow gene pool. In order to introduce genetic variability into the crop, interspecific hybrids, selected from crosses with wild beets of the sections Corollinae and Procumbentes, have been generated. The introgressed B. procumbens chromatin carries resistance genes to beet cyst nematode Heterodera schachtii Schm. These lines are important for breeding of nematode-resistant sugar beet, while Corollinae species are potential donors of tolerance to biotic and abiotic stresses such as drought or saline soils. We have used in situ hybridization of genomic DNA to discriminate the parental chromosomes in these interspecific hybrids. Suppression of cross-hybridization by blocking DNA was not necessary indicating that the investigated Beta genomes contain sufficient species-specific DNA enabling the unequivocal determination of the genomic composition of the hybrids. Interspecific hybrid lines with an additional chromosome (2n = 18 + 1), chromosome fragment (2n = 18 + fragment) or translocation of B. procumbens (2n = 18) were analysed by genomic in situ hybridization (GISH) at mitosis and meiosis. Species-specific satellites and ribosomal genes used in combination with genomic DNA or in rehybridization experiments served as landmark probes for chromosome identification in hybrid genomes. The detection of a B. procumbens translocation of approx. I Mbp demonstrated the sensitivity and resolution of GISH and showed that this approach is a powerful method in genome analysis projects of the genus Beta.

Isolation of a cDNA from Arabidopsis thaliana that complements the sec14 mutant of yeast.

The SEC14 gene of Saccharomyces cerevisiae codes for a phosphatidylinositol-transfer protein (Sec14p(sc)) which is capable of transferring both phosphatidylinositol and phosphatidylcholine between membranes in vitro. Genetic and biochemical studies conducted in S. cerevisiae have shown that this protein acts as an inhibitor of phosphatidylcholine biosynthesis via the so-called Kennedy pathway only. This inhibition is controlled by the binding of phospholipids to the Sec14p(sc) protein. Here we describe the isolation of a cDNA from Arabidopsis thaliana by functional complementation of a sec14(ts) mutant of S. cerevisiae. This cDNA, designated AtSEC14, is capable of restoring the growth of the sec14(ts) mutant at the restrictive temperature of 37 degrees C. Extracellular invertase measurements indicated that the cDNA can partly restore protein secretion. In addition, the phosphatidylinositol-transfer activity measured in protein extracts is greatly enhanced in the complemented mutant strain when compared with the sec14(ts) mutant. The best sequence similarity at the amino acid level is found with the Sec14p protein of S. cerevisiae (36.5% similarity), and most of the amino acids that are thought to be involved in the binding of phospholipids in the yeast protein are conserved in the AtSEC14 gene product. Southern analysis suggests the presence of a single gene in the Arabidopsis genome, although the existence of distantly related sequences cannot be excluded. This gene is expressed in roots, leaves, flowers and siliques of Arabidopsis.

NMR studies and restrained-molecular-dynamics calculations of a long A+T-rich stretch in DNA. Effects of phosphate charge and solvent approximations.

The nonamer duplex d(GCAAAAACG).d(CGTTTTTGC) was studied by 1H-NMR at 500 MHz. With the exception of the H5' and H5" sugar protons, all protons were assigned by two-dimensional NMR experiments [NOE spectroscopy (NOESY), double-quantum-filtered J-correlated spectroscopy (DQF-COSY) and total correlation spectroscopy (TOCSY)]. The exchange kinetics of the imino protons of the Watson-Crick base pairing were studied at 15 degrees C by measuring inversion-recovery rates under conditions of extensive ammonia base catalysis. Extrapolation to infinite base concentration gave anomalous long lifetimes for the A-tract in accordance with previous results [Leroy, J.-L., Charettier, E., Kochoyan, M. & Guéron, M. (1988) Biochemistry 27, 8894-8898]. On average, 11 NOESY distance constraints/nucleotide were evaluated using the complete relaxation matrix approach. Deoxyribose coupling constants were obtained from simulations of the DQF-COSY cross-peaks, assuming a rapid two-state equilibrium between a C2'-endo and C3'-endo conformer. The sugars were found to be predominantly in the C2'-endo conformation. The NMR-derived distance and torsion constraints were implemented into three different restrained-molecular-dynamics (rMD) protocols, two in vacuo, with different charges on the phosphate group and the third with the solvent explicitly included. All protocols displayed good convergence from different starting structures. The structures derived from the three protocols satisfied experimental restraints equally well and had similar final energies. Although the overall pattern of sequence dependence of helical parameters shows some resemblance in all structures, we find that the absolute amplitudes of the parameters are largely dependent on the rMD protocols, particularly the twist parameters. The minor groove distance P(n + 2)-P(m + 2) varies from 0.7 nm to 1.2 nm in the three protocols. Still the NOESY-derived anomalously short distances AH2(n)-H1'(m + 1) and AH2(n)-H1'(n + 1), n and m denote complementary residues, which are assumed to be indicative of a compressed minor groove, are kept in all calculated structures.