Novel homo Yin-Yang probes improve sensitivity in RT-qPCR detection of low copy HIV RNA
Nucleic acids labeled with a fluorophore/quencher pair are widely used as probes in biomedical research and
molecular diagnostics. Here we synthesized novel DNA molecular beacons double labeled with the identical dyes
(R6G, ROX and Cy5) at 5′- and 3′-end and studied their photo physical properties. We demonstrated that
fluorescence quenching by formation of the homo dimer exciton in such molecular beacons allows using them in
homogeneous assays. Further, we developed and evaluated homo Yin-Yang DNA probes labeled with identical
dyes and used them for detection of low copy HIV RNA by RT-qPCR. They demonstrated improved sensitivity
(LLQ: 10 vs 30 copies mL-1) in comparison to commercially available Abbott RealTime HIV-1 kit based on VICBHQ
dyes both for model mixtures (naive human plasma with added deactivated HIV-1 virus) and for preliminarily
confirmed 36 clinical samples (4 vs 1 positive ones for low-copy samples).
Oligonucleotide probes labeled with pyrene pairs that form excimers have a number of applications in hybridization analysis of nucleic acids. A long excited state lifetime, large Stokes shift and chemical stability make pyrene excimer an attractive fluorescent label. Here we report synthesis of a chiral phosphoramidite building blocks based on (R)-4-amino-2,2-dimethylbutane-1,3-diol, easily available from an inexpensive D-(–)-pantolactone. 1-Pyreneacetamide, 1-pyrenecarboxamide and Dabcyl derivatives have been used in preparation of molecular beacon (MB) probes labeled with one or two pyrenes/quenchers. We observed significant difference in the excimer emission maxima (475–510 nm; Stokes shifts 125–160 nm or 7520–8960 cm-1) and excimer/monomer ratio (from 0.5 to 5.9) in fluorescence spectra depending on the structure and position of monomers in the pyrene pair. The pyrene excimer formed by two rigid 1-pyrenecarboxamide residues showed the brightest emission. This is consistent with molecular dynamics data on excimer stability. Increase of the excimer fluorescence for MBs after hybridization with DNA was up to 24-fold.
Quantification and normalization of RT-qPCR data critically depends on the expression of so called reference genes. Our goal was to develop a strategy for the selection of reference genes that utilizes microarray data analysis and combines known approaches for gene stability evaluation and to select a set of appropriate reference genes for research and clinical analysis of breast samples with different receptor and cancer status using this strategy.
A preliminary search of reference genes was based on high-throughput analysis of microarray datasets. The final selection and validation of the candidate genes were based on the RT-qPCR data analysis using several known methods for expression stability evaluation: comparative ∆Ct method, geNorm, NormFinder and Haller equivalence test.
A set of five reference genes was identified: ACTB, RPS23, HUWE1, EEF1A1 and SF3A1. The initial selection was based on the analysis of publically available well-annotated microarray datasets containing different breast cancers and normal breast epithelium from breast cancer patients and epithelium from cancer-free patients. The final selection and validation were performed using RT-qPCR data from 39 breast cancer biopsy samples. Three genes from the final set were identified by the means of microarray analysis and were novel in the context of breast cancer assay. We showed that the selected set of reference genes is more stable in comparison not only with individual genes, but also with a system of reference genes used in commercial OncotypeDX test.
A selection of reference genes for RT-qPCR can be efficiently performed by combining a preliminary search based on the high-throughput analysis of microarray datasets and final selection and validation based on the analysis of RT-qPCR data with a simultaneous examination of different expression stability measures. The identified set of reference genes proved to be less variable and thus potentially more efficient for research and clinical analysis of breast samples comparing to individual genes and the set of reference genes used in OncotypeDX assay.
Many environmental stimuli present a quasi-rhythmic structure at different timescales that the brain needs to decompose and integrate. Cortical oscillations have been proposed as instruments of sensory de-multiplexing, i.e., the parallel processing of different frequency streams in sensory signals. Yet their causal role in such a process has never been demonstrated. Here, we used a neural microcircuit model to address whether coupled theta–gamma oscillations, as observed in human auditory cortex, could underpin the multiscale sensory analysis of speech. We show that, in continuous speech, theta oscillations can flexibly track the syllabic rhythm and temporally organize the phoneme-level response of gamma neurons into a code that enables syllable identification. The tracking of slow speech fluctuations by theta oscillations, and its coupling to gamma-spiking activity both appeared as critical features for accurate speech encoding. These results demonstrate that cortical oscillations can be a key instrument of speech de-multiplexing, parsing, and encoding.
Papers about natural protection territories
Hypoxia of trophoblast cells is an important regulator of normal development of the placenta. However, some pathological states associated with hypoxia, e.g. preeclampsia, impair the functions of placental cells. Oxyquinoline derivative inhibits HIF-prolyl hydroxylase by stabilizing HIF-1 transcription complex, thus modeling cell response to hypoxia. In human choriocarcinoma cells BeWo b30 (trophoblast model), oxyquinoline increased the expression of a core hypoxia response genes along with up-regulation of NOS3, PDK1, and BNIP3 genes and down-regulation of the PPARGC1B gene. These changes in the expression profile attest to activation of the metabolic cell reprogramming mechanisms aimed at reducing oxygen consumption by enabling the switch from aerobic to anaerobic glucose metabolism and the respective decrease in number of mitochondria. The possibility of practical use of the therapeutic properties of oxyquinoline derivatives is discussed.