The number of optical coherence tomography (OCT) examinations in substance use disorders is gradually increasing. However, OCT findings in opioid use disorder (OUD) have not yet been investigated. In ...this study, we compared the retinal nerve fiber layer (RNFL), the ganglion cell layer (GCL), the inner plexiform layer (IPL), and choroid thickness (CT) of OUD and control groups. We included 43 male patients and 43 healthy male controls of similar age (p = 0.296) in the study, prospectively. On the day of OCT application, urine toxic screening test results of all OUD patients were positive for opioid use. There was a significant difference between OUD and control groups in terms of CT (p < 0.05), nasal superior (NS), and nasal (N) sectors of the RNFL (p < 0.05) values of both eyes. According to the binary logistic regression analysis, the sensitivity of mean NS (p = 0.001) and mean CT (p = 0.007) related to the diagnosis of OUD was 72.1 percent, and the specificity was 65.1 percent. Receiver operating characteristic (ROC) analysis revealed that the sensitivity and specificity of mean CT for the diagnosis of OUD were 18.6% and 97.7%, respectively. This is the first study to investigate the OCT findings in OUD. Our findings are important in terms of showing thinning in the choroidal layer and an increase in the volume of the NS and N sectors of RNFL while detecting opioids in the body/urine. Further studies are needed to clarify whether these differences are due to the acute and/or chronic effects of opioids.
SPC2996 is a novel locked nucleic acid phosphorothioate antisense molecule targeting the mRNA of the Bcl-2 oncoprotein. We investigated the mechanism of action of SPC2996 and the basis for its ...clinically observed immunostimulatory effects in chronic lymphocytic leukemia (CLL). Patients with relapsed CLL were treated with a maximum of six doses of SPC2996 (0.2-6 mg/kg) in a multicenter phase I trial. Microarray-based transcriptional profiling of circulating CLL cells was carried out before and after the first infusion of SPC2996 in 18 patients. Statistically significant transcriptomic changes were observed at doses 4 mg/kg and occurred as early as 24 h after the first infusion of the oligonucleotide. SPC2996 induced the upregulation of 466 genes including a large number of immune response and apoptotic regulator molecules, which were enriched for Toll-like receptor response genes. Serum measurements confirmed the release of pro-inflammatory cytokines including chemokine (C-C motif) ligand 3 (macrophage inflammatory protein 1α) and tumor necrosis factor-α, thereby validating the in vivo transcriptomic data at the protein level. SPC2996 caused a 50% reduction of circulating lymphocytes in five of 18 (28%) patients, which was found to be independent of its immunostimulatory and anti-Bcl-2 effects.
The binding of a mixed-sequence pentadecamer PNA (peptide nucleic acid) containing all four nucleobases to the fully complementary as well as various singly mismatched RNA and DNA oligonucleotides ...has been systematically investigated using thermal denaturation and BIAcore surface-interaction techniques. The rate constants for association (k a) and dissociation (k d) of the duplex formation as well as the thermal stability (melting temperature, T m) of the duplexes have been determined. Upon binding to PNA tethered via a biotin-linker to streptavidin at the dextran/gold surface, DNA and RNA sequences containing single mismatches at various positions in the center resulted in increased dissociation and decreased association rate constants. T m values for PNA•RNA duplexes are on average 4 °C higher than for PNA•DNA duplexes and follow quantitatively the same variation with mismatches as do the PNA•DNA duplexes. Also a faster k a and a slower k d are found for PNA•RNA duplexes compared to the PNA•DNA duplexes. An overall fair correlation between T m, k a, and k d is found for a series of PNA•DNA and PNA•RNA duplexes although the determination of k a seemed to be prone to artifacts of the method and was not considered capable of providing absolute values representing the association rate constant in bulk solution.
PCR clamping Orum, H
Current Issues in Molecular Biology
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An efficient, PCR based method for the selective amplification of DNA target sequences that differs by a single base pair is described. The method utilises the high affinity and specificity of PNA ...for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.
Aptamers interacting with RNA hairpins through loop−loop (so-called kissing) interactions have been described as an alternative to antisense oligomers for the recognition of RNA hairpins. R06, an RNA ...aptamer, was previously shown to form a kissing complex with the TAR (trans-activating responsive) hairpin of HIV-1 RNA (Ducongé and Toulmé (1999) RNA 5, 1605). We derived a chimeric locked nucleic acid (LNA)/DNA aptamer from R06 that retains the binding properties of the originally selected R06 aptamer. We demonstrated that this LNA/DNA aptamer competes with a peptide of the retroviral protein Tat for binding to TAR, even though the binding sites of the two ligands do not overlap each other. This suggests that upon binding, the aptamer TAR adopts a conformation that is no longer appropriate for Tat association. In contrast, a LNA/DNA antisense oligomer, which exhibits the same binding constant and displays the same base-pairing potential as the chimeric aptamer, does not compete with Tat. Moreover, we showed that the LNA/DNA aptamer is a more specific TAR binder than the LNA/DNA antisense sequence. These results demonstrate the benefit of reading the three-dimensional shape of an RNA target rather than its primary sequence for the design of highly specific oligonucleotides.
One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome ...this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV‐1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV‐spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera–TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets.
Individuals carrying the factor V Leiden mutation have been shown to have an increased risk of developing venous thromboembolism. Our aim was to develop an ELISA-like assay to detect the mutation in ...PCR-amplified genomic DNA using novel, high-affinity DNA analogs, termed locked nucleic acids (LNAs).
LNA octamer probes complementary to the factor V wild-type or mutated sequence were covalently attached to individual wells of a microtiter plate. Biotinylated factor V amplicons were added, and hybridization to the immobilized LNA probes was scored colorimetrically using a horseradish peroxidase-anti-biotin Fab conjugate and tetramethylbenzidine substrate.
In a prospective study of 53 patients, the assay reproducibly scored both factor V homozygotes and heterozygotes with excellent sensitivity and specificity. All results were in complete agreement with the results obtained with the conventional PCR-restriction fragment length polymorphism technique.
The simplicity of the assay and its procedural relatedness to the widely used ELISA format should make it useful for routine factor V testing in the clinical laboratory.
A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and ...bind to their complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding deoxyribooligonucleotides and that they cannot function as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers.
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