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Introduction: Distinct, disease-associated intracellular miRNA (miR) expression profiles have been observed in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematous (SLE) patients. Additionally, we have identified novel estrogenic responses in PBMCs from SLE patients and demonstrated that estrogen upregulates toll-like receptor (TLR)7 and TLR8 expression. TLR7 and TLR8 bind viral-derived single-stranded RNA to stimulate innate inflammatory responses, but recent studies have shown that miR-21, mir-29a, and miR-29b can also bind and activate these receptors when packaged and secreted in extracellular vesicles (EVs). The objective of this study was to evaluate the association of EV-encapsulated small RNA species in SLE and examine the therapeutic approach of miR inhibition in humanized mice. Methods: Plasma-derived EVs were isolated from SLE patients and quantified. RNA was then isolated and bulk RNA-sequencing reads were analyzed. Also, PBMCs from active SLE patients were injected into immunodeficient mice to produce chimeras. Prior to transfer, the PBMCs were incubated with liposomal EVs containing locked nucleic acid (LNA) antagonists to miR-21, mir-29a, and miR-29b. After three weeks, blood was collected for both immunophenotyping and cytokine analysis; tissue was harvested for histopathological examination. Results: EVs were significantly increased in the plasma of SLE patients and dif to healthy controls, including miR-21, mir-29a, and miR-29b. LNA antagonists significantly reduced proinflammatory cytokines and histopathological infiltrates in the small intestine, liver, and kidney, as demonstrated by H&E-stained tissue sections and immunohistochemistry measuring human CD3. Discussion: These data demonstrate distinct EV-derived small RNA signatures representing SLE-associated biomarkers. Moreover, targeting upregulated EVencapsulated miR signaling by antagonizing miRs that may bind to TLR7 and TLR8 reveals a novel therapeutic opportunity to suppress autoimmune-mediated inflammation and pathogenesis in SLE.ferentially expressed EV-derived small RNA profiles were detected compared. [ABSTRACT FROM AUTHOR]

IntroductionDistinct, disease-associated intracellular miRNA (miR) expression profiles have been observed in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematous (SLE) patients. Additionally, we have identified novel estrogenic responses in PBMCs from SLE patients and demonstrated that estrogen upregulates toll-like receptor (TLR)7 and TLR8 expression. TLR7 and TLR8 bind viral-derived single-stranded RNA to stimulate innate inflammatory responses, but recent studies have shown that miR-21, mir-29a, and miR-29b can also bind and activate these receptors when packaged and secreted in extracellular vesicles (EVs). The objective of this study was to evaluate the association of EV-encapsulated small RNA species in SLE and examine the therapeutic approach of miR inhibition in humanized mice.MethodsPlasma-derived EVs were isolated from SLE patients and quantified. RNA was then isolated and bulk RNA-sequencing reads were analyzed. Also, PBMCs from active SLE patients were injected into immunodeficient mice to produce chimeras. Prior to transfer, the PBMCs were incubated with liposomal EVs containing locked nucleic acid (LNA) antagonists to miR-21, mir-29a, and miR-29b. After three weeks, blood was collected for both immunophenotyping and cytokine analysis; tissue was harvested for histopathological examination.ResultsEVs were significantly increased in the plasma of SLE patients and differentially expressed EV-derived small RNA profiles were detected compared to healthy controls, including miR-21, mir-29a, and miR-29b. LNA antagonists significantly reduced proinflammatory cytokines and histopathological infiltrates in the small intestine, liver, and kidney, as demonstrated by H&E-stained tissue sections and immunohistochemistry measuring human CD3.DiscussionThese data demonstrate distinct EV-derived small RNA signatures representing SLE-associated biomarkers. Moreover, targeting upregulated EV-encapsulated miR signaling by antagonizing miRs that may bind to TLR7 and TLR8 reveals a novel therapeutic opportunity to suppress autoimmune-mediated inflammation and pathogenesis in SLE.

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3′ untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections. [ABSTRACT FROM AUTHOR]

Small interfering RNA (siRNA) is a novel class of double-stranded oligonucleotide therapeutics rapidly growing in drug research and development. Accurate, sensitive, and reliable quantification of siRNA analytes in biological samples is required to study their pharmacokinetics, toxicokinetics, and biodistribution. Hybridization LC-MS/MS can achieve highly sensitive and specific bioanalysis of single-stranded oligonucleotides, e.g., antisense oligonucleotides (ASOs); however, its application for bioanalysis of siRNA or other double-stranded oligonucleotides is limited. The detailed rationale and principles for assay development are still not well understood. In this work, we systematically evaluated key steps and parameters of hybridization LC-MS/MS assays, including probes (five different types compared), hybridization procedure and temperature, elution temperature, and column temperature using patisiran, an approved siRNA drug, as the test siRNA. Based on the evaluation, a practical and efficient melting temperature (Tm) guided strategy was developed for fast and reliable method development of hybridization LC-MS/MS assays for siRNA bioanalysis. The strategy was successfully applied to siRNA-A, a test siRNA, in mouse plasma over the range of 1.00-1000 ng/mL and the resulting method has been used to support multiple mouse studies. This method-development strategy showed great value as a general approach for other siRNAs or double-stranded oligonucleotides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected £ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.

Multiplex real‐time quantitative polymerase chain reaction (mRT‐qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one‐tube nested real‐time PCR (mOTNRT‐PCR) assay consisting of five separate internally controlled RT‐qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT‐PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT‐PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real‐time PCR (RT‐qPCR) assay. The analytical sensitivities of mOTNRT‐PCR panel ranged from 2 to 20 copies/reaction, and no cross‐reaction with common respiratory viruses was observed. The coefficients of variation of intra‐assay and inter‐assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT‐PCR assay panel were missed by RT‐qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT‐PCR assay panel provides a more sensitive and high‐throughput method for the detection of 14 respiratory viruses. Research Highlights: A panel of mOTNRT‐PCR consisted of five separate internally controlled RT‐qPCR assay.Locked nucleic acid was used to develop the mOTNRT‐PCR assay panel.The sensitivity of mOTNRT‐PCR panel ranged from 2‐20copies/reaction using plasmid.mOTNRT‐PCR was more sensitive than reported individual RT‐qPCR assay. [ABSTRACT FROM AUTHOR]

Peptide-oligonucleotide conjugates (POCs) represent one of the increasingly successful albeit costly approaches to increasing the cellular uptake, tissue delivery, bioavailability, and, thus, overall efficiency of therapeutic nucleic acids, such as, antisense oligonucleotides and small interfering RNAs. This review puts the subject of chemical synthesis of POCs into the wider context of therapeutic oligonucleotides and the problem of nucleic acid drug delivery, cell-penetrating peptide structural types, the mechanisms of their intracellular transport, and the ways of application, which include the formation of non-covalent complexes with oligonucleotides (peptide additives) or covalent conjugation. The main strategies for the synthesis of POCs are viewed in detail, which are conceptually divided into (a) the stepwise solid-phase synthesis approach and (b) post-synthetic conjugation either in solution or on the solid phase, especially by means of various click chemistries. The relative advantages and disadvantages of both strategies are discussed and compared.

The adult Göttingen Minipig is an acknowledged model for safety assessment of antisense oligonucleotide (ASO) drugs developed for adult indications. To assess whether the juvenile Göttingen Minipig is also a suitable nonclinical model for pediatric safety assessment of ASOs, we performed an 8-week repeat-dose toxicity study in different age groups of minipigs ranging from 1 to 50 days of age. The animals received a weekly dose of a phosphorothioated locked-nucleic-acid-based ASO that was assessed previously for toxicity in adult minipigs. The endpoints included toxicokinetic parameters, in-life monitoring, clinical pathology, and histopathology. Additionally, the ontogeny of key nucleases involved in ASO metabolism and pharmacologic activity was investigated using quantitative polymerase chain reaction and nuclease activity assays. Similar clinical chemistry and toxicity findings were observed; however, differences in plasma and tissue exposures as well as pharmacologic activity were seen in the juvenile minipigs when compared with the adult data. The ontogeny study revealed a differential nuclease expression and activity, which could affect the metabolic pathway and pharmacologic effect of ASOs in different tissues and age groups. These data indicate that the juvenile Göttingen Minipig is a promising nonclinical model for safety assessment of ASOs intended to treat disease in the human pediatric population.

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.

This article covers the triazole-linked nucleic acids where the triazole linkage (TL) replaces the natural phosphate backbone. The replacement is done at either a few selected linkages or all the phosphate linkages. Two triazole linkages, the four-atom TL1 and the six-atom TL2, have been discussed in detail. These triazole-modified oligonucleotides have found a wide range of applications, from therapeutics to synthetic biology. For example, the triazole-linked oligonucleotides have been used in the antisense oligonucleotide (ASO), small interfering RNA (siRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology as therapeutic agents. Due to the ease of the synthesis and a wide range of biocompatibility, the triazole linkage TL2 has been used to assemble a functional 300-mer DNA from alkyne- and azide-functionalized 100-mer oligonucleotides as well as an epigenetically modified variant of a 335 base-pair gene from ten short oligonucleotides. These outcomes highlight the potential of triazole-linked nucleic acids and open the doors for other TL designs and artificial backbones to fully exploit the vast potential of artificial nucleic acids in therapeutics, synthetic biology and biotechnology., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Abstract Respiratory syncytial virus (RSV) causes serious respiratory tract infection worldwide. The relatively low RSV load makes it difficult to detect in frail, elderly, and severely immune-compromised patients. In the present study, we developed a locked nucleic acid–-based 1-tube nested real-time RT-PCR (OTNRT-PCR) assay with the advantages of extremely high sensitivity, facile operability, and less likelihood of cross-contamination. The sensitivity, specificity, and clinical performance of the OTNRT-PCR assay were compared in parallel with a conventional TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional 2-step nested RT-PCR assay. The limit of detection of the OTNRT-PCR assay was 1.02 × 10−1 TCID50/mL, equivalent to the traditional 2-step nested RT-PCR assay and 25-fold lower than the qRT-PCR assay. Of 616 nasopharyngeal aspirates tested, 143 RSV-negative samples by qRT-PCR were confirmed as positive by sequencing the OTNRT-PCR products. We therefore conclude that OTNRT-PCR is more sensitive than qRT-PCR for detection of RSV in clinical samples. [ABSTRACT FROM AUTHOR]

Highlights • The effect of humid conditions was evaluated based on two degradation ratios. • At 25 °C, the humidity significantly decreased 129:41- and 305:41-bp ratios after 12 months. • The degradation rapidly progressed under the 40 °C/humid conditions. • DOP-PCR using LNAs improved success rate of genotyping in slightly degraded and low-copy DNA. Abstract We analyzed the degradation level of DNA from buccal cells under humid conditions using quantitative PCR analysis. Gauze samples with buccal cells were incubated for up to 12 months under three different conditions (25 °C/dry, 25 °C/humid, or 40 °C/humid). The degradation was evaluated based on two degradation ratios (129:41 and 305:41 bp). DNA degraded slowly under the 25 °C/humid condition, and significant differences in the two degradation ratios were detected between 25 °C/dry and 25 °C/humid conditions after 12 months. Moreover, the degradation rapidly progressed under the 40 °C/humid condition, and the two degradation ratios in this condition were much lower than those from 25 °C/dry and 25 °C/humid conditions after a short incubation period (3 months). To evaluate the effect of DNA repair on low-copy degraded DNA, degenerate oligonucleotide-primed PCR (DOP-PCR) was performed before short tandem repeats (STR) genotyping. As a standard DOP-PCR, we used a 22-base primer with 10 degenerate sequences (5′-CTCGAGNNNNNNNNNNATGTGG-3′), and additionally designed DOP-PCR primers with 2, 4, 6, or 8 locked nucleic acids (LNAs). When slightly degraded DNA (305:41-bp ratio = 0.60) was used, DOP-PCR significantly increased the fluorescent intensity and success rate of genotyping using Identifiler and Globalfiler kits. In particular, the reaction with four LNAs produced the highest value. However, such benefits were not observed in the analysis of moderately degraded DNA (305:41-bp ratio = 0.13). Although the recovery rates of STR profiles by DOP-PCR were dependent on the degradation level of low-copy DNA, the effectiveness of DOP-PCR highlights the potential of LNA for degenerate sequences. [ABSTRACT FROM AUTHOR]

Bleeding and thrombosis represent common complications in myeloproliferative neoplasms (MPN) and significantly contribute to morbidity and mortality. Molecular markers, including CALR mutations, were considered not only as diagnostic markers, but also as risk factors for bleeding and thrombosis associated with MPN, especially for patients in remote primary hospitals. We sought to develop an easy-to-use assay for the rapid detection of CALR type 1 (CALR-1) and type 2 (CALR-2) mutations in Philadelphia chromosome-negative MPN patients. Peptide nucleic acid-locked nucleic acid (PNA-LNA) clamping loop-mediated isothermal amplification (LAMP) assays were established, which were integrated into a centrifugal compact disc (CD) microfluidic platform. A total of 158 clinical blood samples were tested simultaneously by this microfluidic platform and an in-house real time PCR assay. The detection performance of the LAMP arrays was validated and conflicting results were identified by Sanger sequencing. The results suggested that the LAMP methods we developed exhibited good sensitivity, specificity, and precision. By real time fluorescence assay the detection limit for CALR-1 and CALR-2 mutations could reach as low as 1% and 0.5% respectively, and 10% and 5% respectively by visual method. There were no nonspecific background amplifications among different detection systems. For the CALR-1 and CALR-2 LAMP detection systems, intra-batch CV values of 1% mutated plasmid were 10.56% and 10.51% respectively, and the inter-batch CV values were 19.55% and 18.39%, respectively. The products were all analyzed by melting curve analysis and electrophoresis followed by Sanger sequencing analysis, which were consistent with the database sequences. The microfluidic platform could complete rapid detection of CALR-1/2 mutations within 60 min. The results of clinical samples detected by our CD-like microfluidic chipLAMP assay and rtPCR assay suggested that 133 samples were CALR wild type, 15 were CALR-1 mutation type, and 9 were CALR-2 mutation type. The correlation coefficient value (Kendall's tau_b) of the two assays was 0.99. Interestingly, by the newly established detection platform, we were surprised to find that one patient of Chinese origin harbored both CALR-1 and CALR-2 mutations. This result was verified by Sanger sequencing analysis. The LAMP detection systems developed herein displayed good sensitivity, specificity, and stability. Additionally, the detection results could be directly judged by color changes of the reaction systems without any auxiliary equipment. Thus, the platform we developed has the potential of being widely used in remote and economically undeveloped areas in the future. [ABSTRACT FROM AUTHOR]

Background: Mutations in the BRAF gene have been strongly associated with failure in cancer treatment using epidermal growth factor receptor (EGFR) antibodies. To better diagnose and assess the prognosis of cancer patients, mutation screening of the BRAFV600E gene should be performed prior to clinical anti-tumor drug therapy to avoid ineffective treatment. Methods: In our previous study, we developed a real-time wild-type blocking PCR (WTB-PCR), which can amplify the mutant allele at high efficiency while simultaneously inhibiting the amplification of wild-type alleles. In order to reduce base mismatch due to the high number of cycles, as well as to monitor the total quantity of DNA added to the reaction system, an internal reference gene was co-amplified together with the target gene on the basis of WTB-PCR. Results: Our results showed that when 50 - 200 ng of the DNA templates was used, this current built method (realtime quantitative clamp-based PCR technology using wild-type blocker coupled with internal competitive reference to enhance amplification specificities, named wirePCR) completely blocked the amplification of the wild-type BRAFV600E gene with detection of the mutated allele at a mutant/wild-type ratio of 1:10,000, which was in line with the sensitivity requirement for the detection of trace amounts of the mutant gene. In the colorectal biopsies from 50 patients with suspected colorectal cancer, eight patients (16%) with BRAFV600E mutations were detected using wirePCR. The allele percentage of mutations can be obtained directly from the ΔCq between the targeted and reference genes, we demonstrated that among the V600E-positive patient samples, the percentage of BRAF DNA with the V600E mutation ranged from 24.99% to 54.31%. Conclusions: WirePCR is a rapid, simple, and low-cost quantitative analytical technique for the detection of trace amounts of mutant BRAFV600E genes in clinical samples. [ABSTRACT FROM AUTHOR]

Locked nucleic acids (LNA) confer high thermal stability and nuclease resistance to oligonucleotides. The discovery of polymerases that accept LNA triphosphates has led us to propose a scheme for the amplification and re-generation of LNA-containing oligonucleotide libraries. Such libraries could be used for in vitro selection of e.g., native LNA aptamers. We maintained an oligonucleotide library encoding 40 randomized positions with LNA ATP, GTP, CTP, and TTP for 7 rounds of ‘mock’ in vitro selection in the absence of a target and analyzed the sequence composition after rounds 1, 4 and 7. We observed a decrease in LNA-A content from 20.5% in round 1 to 6.6% in round 7. This decrease was accompanied by a substantial bias against successive LNA-As (poly-LNA adenosine tracts) and a relative over-representation of single LNA-As. Maintaining a library with LNA TTP yielded similar results. Together, these results suggest that dispersed LNA monomers are tolerated in our in vitro selection protocol, and that LNA-modified libraries can be sustained for up to at least seven selection rounds, albeit at reduced levels. This enables the discovery of native LNA aptamers and similar oligonucleotide structures.

Nucleic acids are able to adopt a plethora of structures, many of which are of interest in therapeutics, bio- or nanotechnology. However, structural and biochemical stability is a major concern which has been addressed by incorporating a range of modifications and nucleoside derivatives. This review summarizes the use of locked nucleic acid (LNA) and un-locked nucleic acid (UNA) monomers in functional nucleic acids such as aptamers, ribozymes, and DNAzymes.

A novel multiplex PCR assay using non-homologous oligonucleotides with locked nucleic acid (LNA) modifications as universal primers was developed and validated for the simultaneous detection of five swine viruses. The assay utilizes five virus-specific primer pairs modified at the 5′ end through the addition of the universal primer sequence. In the reaction, small amounts of target templates with the 5′ tail were generated and subsequently amplified through the extension of a LNA universal primer set. To validate the specificity of this assay, 27 viral target strains and 12 non-target pathogens were tested. The lower limit of detection of viral nucleic acids was 1.1–1.9 pg per reaction or 11–32 pg in a five-plex viral nucleic acid mixture. The LNA mPCR assay displayed higher analytical sensitivity and efficiency for the detection of plasmid standards compared with the conventional assay, which uses standard primers without the 5′ tail. A total of 207 field samples were tested using both assays. The LNA mPCR assay provided numerically higher detection rates for all pathogens in independent samples. Moreover, the LNA mPCR assay had significantly higher detection rates in independent samples compared with the conventional assay. [ABSTRACT FROM AUTHOR]

This is the first ever attempt to combine anti-cancer therapeutic effects of emerging anticancer biodrug bovine lactoferrin (bLf), and multimodal imaging efficacy of Fe 3 O 4 nanoparticles (NPs) together, as a saturated Fe 3 O 4 -bLf. For cancer stem cell specific uptake of nanocapsules/nanocarriers (NCs), Fe 3 O 4 -bLf was encapsulated in alginate enclosed chitosan coated calcium phosphate (AEC-CP) NCs targeted (Tar) with locked nucleic acid (LNA) modified aptamers against epithelial cell adhesion molecule (EpCAM) and nucleolin markers. The nanoformulation was fed orally to mice injected with triple positive (EpCAM, CD133, CD44) sorted colon cancer stem cells in the xenograft cancer stem cell mice model. The complete regression of tumor was observed in 70% of mice fed on non-targeted (NT) NCs, with 30% mice showing tumor recurrence after 30 days, while only 10% mice fed with Tar NCs showed tumor recurrence indicating a significantly higher survival rate. From tumor tissue analyses of 35 apoptotic markers, 55 angiogenesis markers, 40 cytokines, 15 stem cell markers and gene expression studies of important signaling molecules, it was revealed that the anti-cancer mechanism of Fe 3 O 4 -bLf was intervened through TRAIL, Fas, Fas-associated protein with death domain (FADD) mediated phosphorylation of p53, to induce activation of second mitochondria-derived activator of caspases (SMAC)/DIABLO (inhibiting survivin) and mitochondrial depolarization leading to release of cytochrome C. Induction of apoptosis was observed by inhibition of the Akt pathway and activation of cytokines released from monocytes/macrophages and dendritic cells (interleukin (IL) 27, keratinocyte chemoattractant (KC)). On the other hand, the recurrence of tumor in AEC-CP-Fe 3 O 4 -bLf NCs fed mice mainly occurred due to activation of alternative pathways such as mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) and Wnt signaling leading to an increase in expression of survivin, survivin splice variant (survivin 2B) and other anti-apoptotic proteins Bad, Bcl-2 and XIAP. Apart from the promising anti-cancer efficacy and the exceptional tumor targeting ability observed by multimodal imaging using near-infrared (NIR) imaging, magnetic resonance imaging (MRI) and computerized tomographic (CT) techniques, these NCs also maintained the immunomodulatory benefits of bLf as they were able to increase the RBC, hemoglobin, iron calcium and zinc levels in mice. [ABSTRACT FROM AUTHOR]

Fluorescent in situ hybridization (FISH) is a powerful tool that for more than 30 years has allowed to detect and quantify microorganisms as well as to study their spatial distribution in three-dimensional structured environments such as biofilms. Throughout these years, FISH has been improved in order to face some of its earlier limitations and to adapt to new research objectives. One of these improvements is related to the emergence of Nucleic Acid Mimics (NAMs), which are now employed as alternatives to the DNA and RNA probes that have been classically used in FISH. NAMs such as peptide and locked nucleic acids (PNA and LNA) have provided enhanced sensitivity and specificity to the FISH technique, as well as higher flexibility in terms of applications. In this review, we aim to cover the state-of-the-art of the different NAMs and explore their possible applications in FISH, providing a general overview of the technique advancement in the last decades. [ABSTRACT FROM AUTHOR]

Amplification of fluorescently labeled products is one of the most popular methods for genotyping genetic variations. Two-step amplification using fluorescent universal primers simultaneously produces multiple targeted fragments labeled with fluorescent dyes, and this strategy is applicable to large-scale, cost-effective genotyping. In this study, we developed a fast PCR-based, multiple short tandem repeat (STR) genotyping method using fluorescent universal primers containing locked nucleic acids (LNAs). Four amplification reactions, each assaying six or seven markers and using 0.5–1.0 ng of genomic DNA, produced obvious Fam-labeled peaks in all 26 loci tested (25 autosomal STRs and amelogenin). The overall amplification time was 37 min. Moreover, fluorescent signals for the 25 STRs obtained from LNA-containing primers were 1.5–9.0 fold higher compared to those from non-LNA primers. Using genomic DNA from 120 Japanese individuals, 16 out of the 25 STRs had observed heterozygosity greater than 0.7. Some of these 25 STRs also had high discriminatory power, similar to that of the 13 core STRs in the Combined DNA Index System dataset. The probability of incorrectly assigning a match based on the accumulated matching probability for these 25 STRs is 1.2 × 10 −22 , and their combined use can provide robust information for Japanese forensics. [ABSTRACT FROM AUTHOR]

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). Deficiency of BTK leads to a developmental block in B cell differentiation; hence, the patients essentially lack antibody-producing plasma cells and are susceptible to various infections. A substantial portion of the mutations in BTK results in splicing defects, consequently preventing the formation of protein-coding mRNA. Antisense oligonucleotides (ASOs) are therapeutic compounds that have the ability to modulate pre-mRNA splicing and alter gene expression. The potential of ASOs has been exploited for a few severe diseases, both in pre-clinical and clinical studies. Recently, advances have also been made in using ASOs as a personalized therapy for XLA. Splice-correction of BTK has been shown to be feasible for different mutations in vitro, and a recent proof-of-concept study demonstrated the feasibility of correcting splicing and restoring BTK both ex vivo and in vivo in a humanized bacterial artificial chromosome (BAC)-transgenic mouse model. This review summarizes the advances in splice correction, as a personalized medicine for XLA, and outlines the promises and challenges of using this technology as a curative long-term treatment option. [ABSTRACT FROM AUTHOR]

Abstract: In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4–6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4–6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4–5–6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) play an important role in various stages of tumor progression. miR-494, which we had previously identified as a miRNA induced by ionizing radiation (IR) in the glioma cell line U-251, was observed to enhance invasion of U-251 cells by activating MMP-2. The miR-494-induced invasive potential was accompanied by, and dependent on, epidermal growth factor receptor (EGFR) upregulation and the activation of its downstream signaling constituents, Akt and ERK. The upregulation of EGFR by miR-494 involved the suppression of lysosomal protein turnover. Among the putative target proteins tested, p190B RhoGAP (p190B) was downregulated by miR-494, and its reduced expression was responsible for the increase in EGFR expression. A reporter assay using a luciferase construct containing p190B 3′-untranslated region (3′UTR) confirmed that p190B is a direct target of miR-494. Downregulation of p190B by small interfering RNA (siRNA) transfection closely mimicked the outcomes of miR-494 transfection, and showed increased EGFR expression, MMP-2 secretion, and invasion. Ectopic expression of p190B suppressed the miR-494-induced EGFR upregulation and invasion promotion, thereby suggesting that p190B depletion is critical for the invasion-promoting action of miR-494. Collectively, our results suggest a novel function for miR-494 and its potential application as a target to control invasiveness in cancer therapy. [Copyright &y& Elsevier]

A set of real-time PCR based assays using the locked nucleic acid probes from Roche Universal ProbeLibrary were developed for rapid detection of eight bacterial species from positive blood culture bottles. Four duplex real-time PCR reactions targeting to one Gram-positive bacterium and one Gram-negative bacterium were optimized for species identification according to Gram stain results. We also included mecA-specific primers and probes in the assays to indicate the presence of methicillin resistance in the bacterial species. The analytical sensitivity was in the range of 1-10 CFU per PCR reaction mixture. The specificity and cross reactivity of the assay was validated by 28 ATCC reference strains and 77 negative blood culture specimens. No cross-reactivity was observed in these samples thus demonstrating 100 % specificity. 72 previously characterized clinical isolates were tested by the real-time PCR assay and validated the accuracy and feasibility of the real-time PCR assay. Furthermore, 55 positive blood culture samples were tested using real-time PCR and 50 (90.9 %) of them were identified as the same species as judged by biochemical analysis. In total, real-time PCR showed 98.2 % consistent to that of traditional methods. Real-time PCR can be used as a supplement for early detection of the frequently-occurred pathogens from the positive blood cultures. [ABSTRACT FROM AUTHOR]

Highlights: [•] A nuclease-resistant and efficacious LNA/DNA chimeric aptamer probe was developed. [•] The combined use of LNA and 3′-3′-T exhibited a synergistic effect. [•] The optimized probe showed ∼10 times increased half-life for target cells in serum. [•] The in vivo tumor imaging window was extended from <150min to >600min. [Copyright &y& Elsevier]

Highlights: [•] MiR-146a mediates dorsal root ganglion survival in diabetic peripheral neuropathy. [•] MiR-146a targets IRAK1 and TRAF6 in dorsal root ganglion neurons. [•] Upregulation of miR-146a contributes sildenafil-improved diabetic neuropathy. [ABSTRACT FROM AUTHOR]

Abstract: Aberrant regulation in oxidative stress, fibrogenesis, and the epithelial–mesenchymal transition (EMT) in renal cells under hyperglycemic conditions contributes significantly to the onset and progression of diabetic nephropathy. The mechanisms underlying these hyperglycemia-induced dysregulations, however, have not been clearly elucidated. Herein, we report that aldose reductase is capable of regulating the expression of miR-200a-3p/141-3p negatively in renal mesangial cells. MiR-200a-3p/141-3p, in turn, act to target Keap1, Tgfβ2, fibronectin, and Zeb2 directly and regulate Tgfβ1 and Nrf2 indirectly under high-glucose conditions, resulting in profound dysregulations in Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling. In vivo in streptozotocin-induced diabetic mice, we found that aldose reductase deficiency caused significant elevations in miR-200a-3p/141-3p in the renal cortex, which were accompanied by a significant downregulation of Keap1, Tgfβ1/2, and fibronectin but significant upregulation of Nrf2. Moreover, in vivo administration of inhibitors of miR-200a-3p in diabetic animals significantly exacerbated cortical and glomerular fibrogenesis and increased urinary albumin excretion, tightly linking dysregulated miR-200a-3p with the development of diabetic nephropathy. Collectively, our results reveal a novel mechanism whereby hyperglycemia induces aldose reductase to regulate renal expression of miR-200a-3p/141-3p to coordinately control hyperglycemia-induced renal oxidative stress, fibrogenesis, and the EMT. Our novel findings also suggest that inhibition of aldose reductase and in vivo renal cortical restoration of miR-200a-3p/141-3p or their combination are very promising avenues for the development of therapeutic strategies or drugs against diabetic nephropathy. [Copyright &y& Elsevier]

Abstract: Sfmbt2-hosted miR-466a-3p and its close relatives are often among the most significantly up-regulated or down-regulated miRNAs in responses to numerous deleterious environmental stimuli. The exact roles of these miRNAs in cellular stress responses, however, are not clear. Here we showed that many Sfmbt2-hosted miRNAs were highly hypertonic stress responsive in vitro and in vivo. In renal medulla, water deprivation induced alterations in the expression of miR-466(a/b/c/e/p)-3p in a pattern similar to that of miR-200b-3p, a known regulator of osmoresponsive transcription factor Nfat5. Remarkably, exposure of mIMCD3 cells to an arginine vasopressin analog time-dependently down-regulated the expression of miR-466(a/b/c/e/p)-3p and miR-200b-3p, which provides a novel regulatory mechanism for these osmoresponsive miRNAs. In cultured mIMCD3 cells we further demonstrated that miR-466a-3p and miR-466g were capable of targeting Nfat5 by interacting with its 3′UTR. In transgenic mice overexpressing miR-466a-3p, significant down-regulation of Nfat5 and many other osmoregulation-related genes was observed in both the renal cortex and medulla. Moreover, sustained transgenic over-expression of miR-466a-3p was found to be associated with polydipsia, polyuria and disturbed ion homeostasis and kidney morphology. Since the mature sequence of miR-466a-3p is completely equivalent to that of miR-466e-3p and that the seed sequence of miR-466a-3p is completely equivalent to that of miR-297(a/b/c)-3p, miR-466d-3p, miR-467g and miR-669d-3p, and that miR-466a-3p differs from miR-466(b/c/p)-3p only in a 5′ nucleotide, we propose that miR-466a-3p and many of its close relatives are important epigenetic regulators of renal Nfat5 signaling, osmoregulation and urine concentration in mice. [Copyright &y& Elsevier]

In this study, a series of 5′-phosphorylated siRNAs with d-/ l-isonucleotide (isoNA) or locked nucleic acid (LNA) incorporated at the 5′-terminus were synthesized. It was found that after incorporating either isoNA or LNA at the 5′-terminus of the antisense strand or sense strand, the silencing activity of modified strands has been inhibited, which cannot be recovered by phosphorylation at the 5′-terminus; however, the silencing activity of unmodified strand to its own target was increased. This work indicates that the isoNA and LNA modification at 5′-terminus can interfere with the strand selection during the RISC assembling process, and the disturbance of the 5′-phosporylation should not be the only viable mechanism. [ABSTRACT FROM AUTHOR]

Background & Aims: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. Methods: Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. Results: TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. Conclusions: Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are non-coding endogenous RNAs that direct post-transcriptional regulation of gene expression by several mechanisms. Activity is primarily through binding to the 3′ untranslated regions (UTRs) of messenger RNAs (mRNA) resulting in degradation and translation repression. Unlike other small-RNAs, miRNAs do not require perfect base pairing, and thus, can regulate a network of broad, yet specific, genes. Although we have only just begun to gain insights into the full range of biologic functions of miRNA, their involvement in the onset and progression of disease has generated significant interest for therapeutic development. Mounting evidence suggests that miRNA-based therapies, either restoring or repressing miRNAs expression and activity, hold great promise. However, despite the early promise and exciting potential, critical hurdles often involving delivery of miRNA-targeting agents remain to be overcome before transition to clinical applications. Limitations that may be overcome by delivery include, but are not limited to, poor in vivo stability, inappropriate biodistribution, disruption and saturation of endogenous RNA machinery, and untoward side effects. Both viral vectors and nonviral delivery systems can be developed to circumvent these challenges. Viral vectors are efficient delivery agents but toxicity and immunogenicity limit their clinical usage. Herein, we review the recent advances in the mechanisms and strategies of nonviral miRNA delivery systems and provide a perspective on the future of miRNA-based therapeutics. [Copyright &y& Elsevier]

Abstract: Cardiovascular disease is a global scourge to society, with novel therapeutic approaches required in order to alleviate the suffering caused by sustained cardiac damage. MicroRNAs (miRNAs) are being touted as one such approach in the fight against heart disease, acting as possible post-transcriptional molecular triggers responsible for invoking cardiac regeneration. To further ones understanding of miRNAs and cardiac regeneration, it is prudent to learn from organisms that can intrinsically regenerate their hearts following injury. Using the red-spotted newt, an adult chordate capable of cardiac regeneration, we decided to delve deeper into the role miRNAs play during this process. RNA isolated from regenerating newt heart samples, was used in a microarray screen, to identify significantly expressed candidate miRNAs during newt cardiac regeneration. We performed quantitative qPCR analysis on several conserved miRNAs and found one in particular, miR-128, to be significantly elevated when cardiac hyperplasia is at its peak following injury. In-situ hybridisation techniques revealed a localised expression pattern for miR-128 in the cardiomyocytes and non-cardiomyocytes in close proximity to the regeneration zone and in vivo knockdown studies revealed a regulatory role for miR-128 in proliferating non-cardiomyocyte populations and extracellular matrix deposition. Finally, 3′UTR reporter assays revealed Islet1 as a biological target for miR-128, which was confirmed further through in vivo Islet1 transcriptional and translational expression analysis in regenerating newt hearts. From these studies we conclude that miR-128 regulates both cardiac hyperplasia and Islet1 expression during newt heart regeneration and that this information could be translated into future mammalian cardiac studies. [Copyright &y& Elsevier]

Abstract: Background: To date microRNAs and their contribution to the onset and propagation of salivary adenoid cystic carcinoma (SACC) are limited. The objective of this study was to identify miR-181a and its mechanism in the metastasis of SACC. Methods: At first microarray and quantitative RT-PCR were used to investigate microRNA profiles and miR-181a in paired SACC cell lines with different metastatic potential. Then the effect of miR-181a on metastatic potential of SACC was investigated. MiR-181a target genes and Snai2 promoter activity were investigated using luciferase reporter gene assays. Western blot was used to detect MAPK–Snai2 pathway-related protein level. Results: A panel of deregulated microRNAs (including miR-181a) was identified in paired of SACC cell lines. Functional analysis indicated that miR-181a inhibited SACC cell migration, invasion and proliferation in vitro, and it suppressed tumor growth and lung metastasis in vivo. Direct targeting of miR-181a to MAP2K1, MAPK1 and Snai2 was confirmed by luciferase reporter gene assays. MiR-181a mimic inhibited the expression of MAP2K1, MAPK1 and Snai2 in SACC cells. MAP2K1 or MAPK1 siRNA suppressed Snai2 gene promoter activity and reduced Snai2 expression and the metastatic potential of SACC cells. Conclusions: Our results indicate that miR-181a plays an important role in the metastasis of SACC, and may serve as a novel therapeutic target for SACC. MiR-181a regulates the MAPK–Snai2 pathway both through direct cis-regulatory mechanism and through indirect trans-regulatory mechanism. General significance: To our knowledge, this is the first study revealing that miR-181a deregulation mediated the metastasis of SACC by regulating MAPK–Snai2 pathway. [Copyright &y& Elsevier]

Abstract: Acetylcholinesterase expression is modulated in various types of tumor, which suggests it is associated with tumor development; however, the mechanism of acetylcholinesterase gene regulation in tumors remains unclear. Here, we report that acetylcholinesterase is aberrantly expressed in non-small cell lung cancer and is an evolutionarily conserved functional target of miR-212. Acetylcholinesterase expression was negatively regulated by miR-212 in vitro and was inversely correlated with miR-212 expression in vivo. In addition, acetylcholinesterase levels were increased, and miR-212 levels decreased, in non-small cell lung cancer cells during cisplatin-induced apoptosis. We further determined that acetylcholinesterase acted as a pro-apoptotic gene in non-small cell lung cells; and attenuated the growth of xenografts in nude mice when upregulated. In contrast, elevated miR-212 levels preserved the protective effect of acetylcholinesterase silencing by RNA interference against cisplatin-induced apoptosis, whereas restoration of miR-212-resistant synaptic acetylcholinesterase expression inhibited the miR-212 anti-apoptotic function. The results demonstrated that miR-212 exerted an anti-apoptotic effect through direct repression of synaptic acetylcholinesterase expression in non-small cell lung cancer cells. Taken together, our study revealed that synaptic acetylcholinesterase may be a tumor suppressor and is modulated by miR-212 in non-small cell lung cancer. [Copyright &y& Elsevier]

Abstract: Deregulation of miRNAs expression levels has been detected in many human tumor types, and recent studies have demonstrated the critical roles of miRNAs in cancer pathogenesis. Numerous recent studies have shown that miRNAs are rapidly released from tissues into the circulation in many pathological conditions. The high relative stability of miRNAs in biofluids such as plasma and serum, and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNAs as promising non-invasive new tumor biomarkers. In this study, we used liquid bead array technology (Luminex) to profile the expression of 320 mature miRNAs in a pilot testing group of 19 matched fresh frozen cancerous and non-cancerous tissues from NSCLC patients. We further validated our results by RT-qPCR for differentially expressed miRNAs in an independent group of 40 matched fresh frozen tissues, 37 plasma samples from NSCLC patients and 28 healthy donors. We found that eight miRNAs (miR-21, miR-30d, miR-451, miR-10a, miR-30e-5p and miR-126*, miR-126, miR-145) were differentially expressed by three different statistical analysis approaches. Two of them (miR-10a and miR-30e-5p) are reported here for the first time. Bead-array results were further verified in an independent group of 40 matched fresh frozen tissues by RT-qPCR. According to RT-qPCR miR-21 was significantly up-regulated (P =0.010), miR-126* (P =0.002), miR-30d (P =0.012), miR-30e-5p (P <0.001) and miR-451 (P <0.001) were down-regulated, while miR-10a was not differentiated (P =0.732) in NSCLC tissues. However, in NSCLC plasma samples, only three of these miRNAs (miR-21, miR-10a, and miR-30e-5p) displayed differential expression when compared to plasma of healthy donors. High expression of miR-21 was associated with DFI and OS both in NSCLC tissues (P =0.022 and P =0.037) and plasma (P =0.045 and P =0.065), respectively. Moreover, we report for the first time that low expression of miR-10a in NSCLC plasma samples was associated with worse DFI (P =0.050) and high expression of miR-30e-5p was found to be associated with shorter OS (P =0.048). In conclusion, circulating miR-21, miR-10a and miR-30e-5p in plasma should be further evaluated as potential non-invasive biomarkers in NSCLC. [Copyright &y& Elsevier]

Although the causes of diabetic nephropathy are not yet fully known, emerging evidence suggests a role for epigenetic factors in the development of the disease. In particular, microRNAs (miRNAs) are becoming recognized as important mediators of biological processes relevant to diabetic nephropathy. Until recently, investigations of miRNAs in the development of diabetic nephropathy have remained relatively limited; however, the number of reports identifying potential new candidates and mechanisms of impact is presently expanding at a rapid pace. This review seeks to summarize these recent findings, focusing on new candidates and/or novel mechanisms, including the intersection between genetic variation and miRNA function in modulating disease expression, emerging in the field. We also review the latest advances in the diagnostic and therapeutic potential of miRNAs in the treatment of diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Allergic inflammation is accompanied by the coordinated expression of a myriad of genes and proteins that initiate, sustain, and propagate immune responses and tissue remodeling. MicroRNAs (miRNAs) are a class of short single-stranded RNA molecules that posttranscriptionally silence gene expression and have been shown to fine-tune gene transcriptional networks because single miRNAs can target hundreds of genes. Considerable attention has been focused on the key role of miRNAs in regulating homeostatic immune architecture and acquired immunity. Recent studies have identified miRNA profiles in multiple allergic inflammatory diseases, including asthma, eosinophilic esophagitis, allergic rhinitis, and atopic dermatitis. Specific miRNAs have been found to have critical roles in regulating key pathogenic mechanisms in allergic inflammation, including polarization of adaptive immune responses and activation of T cells (eg, miR-21 and miR-146), regulation of eosinophil development (eg, miR-21 and miR-223), and modulation of IL-13–driven epithelial responses (eg, miR-375). This review discusses recent advances in our understanding of the expression and function of miRNAs in patients with allergic inflammation, their role as disease biomarkers, and perspectives for future investigation and clinical utility. [Copyright &y& Elsevier]

Locked nucleic acids (LNA) confer high thermal stability and nuclease resistance to oligonucleotides. The discovery of polymerases that accept LNA triphosphates has led us to propose a scheme for the amplification and re-generation of LNA-containing oligonucleotide libraries. Such libraries could be used for in vitro selection of e.g., native LNA aptamers. We maintained an oligonucleotide library encoding 40 randomized positions with LNA ATP, GTP, CTP, and TTP for 7 rounds of 'mock' in vitro selection in the absence of a target and analyzed the sequence composition after rounds 1, 4 and 7. We observed a decrease in LNA-A content from 20.5% in round 1 to 6.6% in round 7. This decrease was accompanied by a substantial bias against successive LNA-As (poly-LNA adenosine tracts) and a relative over-representation of single LNA-As. Maintaining a library with LNA TTP yielded similar results. Together, these results suggest that dispersed LNA monomers are tolerated in our in vitro selection protocol, and that LNA-modified libraries can be sustained for up to at least seven selection rounds, albeit at reduced levels. This enables the discovery of native LNA aptamers and similar oligonucleotide structures [ABSTRACT FROM AUTHOR]

Three or four aromatic rings interconnected by acetylene bridges form a stiff conjugated system with sufficient conformational freedom to make it useful to link together the two strands of a DNA clamp. Upon targeting a ssDNA, the conformational flexibility allows better stacking of the linker to the underlying non-planar base triplet in the formed triplex. This type of triplexes has a substantially higher thermal melting temperature which can be further improved by inserting locked nucleic acids (LNAs) in the Hoogsteen part of the clamp. An extremely high sensitivity to mismatches is observed in an octamer triplex when placed in the middle of the sequence. [ABSTRACT FROM AUTHOR]

Introduction: KRAS mutations in colon carcinomas are associated with lack of response to anti-EGFR monoclonal antibody treatment. Therefore, patients must undergo genetic testing to be eligible for treatment. Several methods for KRAS mutation analysis exist, but many are not sensitive enough to detect a mutation in samples with low fraction of malignant cells. In the present study, we developed a KRAS mutations detection method that is both simple and sensitive. Methods: Using a locked nucleic acid (LNA) containing oligonucleotide, we developed a PCR clamping method that preferentially amplifies the mutated over wild type KRAS. We evaluated the sensitivity of this method using serial dilutions of plasmids containing wild-type and mutated KRAS fragments. Additionally, KRAS mutation status was evaluated on 60 archived tissue samples of colon carcinoma, and compared to direct sequencing and high resolution melting (HRM) methods. Results: The PCR clamping method could detect as little as 1% mutated DNA in the sample analyzed. Of the 29 KRAS mutations identified by the PCR clamping method, only 23 (79%) were identified by standard direct sequencing. The results of PCR clamping correlated with HRM results. Conclusions: LNA based PCR clamping method is a simple and highly sensitive method for the detection of KRAS mutations. [ABSTRACT FROM AUTHOR]

Nucleic acids are able to adopt a plethora of structures, many of which are of interest in therapeutics, bio- or nanotechnology. However, structural and biochemical stability is a major concern which has been addressed by incorporating a range of modifications and nucleoside derivatives. This review summarizes the use of locked nucleic acid (LNA) and un-locked nucleic acid (UNA) monomers in functional nucleic acids such as aptamers, ribozymes, and DNAzymes. [ABSTRACT FROM AUTHOR]

Objective: OCT4A has been known to play a critical role in the maintenance of pluripotency of embryonic stem cells. Recent research has shown that OCT4A is also expressed in partial tumor cell lines and tissues. This study is aimed to develop a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for relative quantitative detection of OCT4A mRNA and discrimination from OCT4B, pseudogene, and genomic contaminations. Methods: A locked nucleic acid (LNA)-modified probe was designed to discern the single base difference 352A/C to identify OCT4A mRNA. An exon-junction primer was designed to avoid false positive caused by genomic contaminations. In addition, a house keeping gene glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) was measured in parallel to normalize the differences between samples and operations. Results: Experiments showed that the newly established RT-PCR assay amplified the OCT4A mRNA selectively; OCT4A analogues gave negative signals. Cell lines nTERA-2 and HepG2 showed positive results in OCT4A expression, while for HeLa and 293 cell lines, as well as primary peripheral blood mononuclear cells (PBMCs), OCT4A expression was negative. Additionally, the relative quantity of OCT4A mRNA was calculated by cycle threshold ( Ct) method and house keeping gene normalization. Conclusions: This technique proved to be effective for relative quantitation of OCT4A mRNA with high specificity. [ABSTRACT FROM AUTHOR]

Abstract: Appropriately designed 2′-N-(pyren-1-yl)carbonyl-2′-amino-LNA (locked nucleic acid) display large increases in fluorescence intensity and remarkably high quantum yields upon hybridization with nucleic acid targets. Thermal denaturation and fluorescence spectroscopy studies on ONs modified with known thymine monomer X and novel 5-methylcytosine monomer Y provide new insights into the design principles and mechanism of these Glowing LNA nucleic acid detection probes. [ABSTRACT FROM AUTHOR]

We established an in situ hybridization (ISH) technique by modification of hybridization probes with locked nucleic acids (LNAs) and demonstrated isoform-specific localization of transcripts of Brassica rapa nitrilase ( BrNIT-T) genes in clubroot tissue infected with Plasmodiophora brassicae. Chimeric oligo DNA probes containing LNAs demonstrated highly improved specificities and could discriminate between BrNIT-T1 and BrNIT-T2. These LNA-containing probes were applied to ISH. BrNIT-T1 was strongly expressed in cells containing expanding secondary plasmodia of P. brassicae, but not in cells containing resting spores. On the other hand, BrNIT-T2 transcripts were localized in noninfected cells rather than infected cells during the clubroot growth phase but coexisted with mature resting spores at a later phase of clubroot development. Immunostaining for indole-3-acetic acid (IAA) revealed IAA accumulation in cells containing growing plasmodia. IAA immunostaining in infected cells was reduced as the pathogen formed resting spores, but the signal was again enhanced in cells containing mature resting spores at a later phase of infection, suggesting that IAA is involved in both the early growth and the latest maturation phase of clubroot development. Expression of BrNIT-T1 and BrNIT-T2 in turnip roots was upregulated by exogenous treatment with cytokinin and jasmonic acid, respectively. Thus, these two phytohormones are possible triggers of abnormal IAA production in clubroot tissue via induction of the respective nitrilase. Given these results, we propose a model for isoform-specific roles of B. rapa nitrilases in auxin biosynthesis involved in phytohormone crosstalk during development of clubroot disease. [ABSTRACT FROM AUTHOR]

Abstract: We present a novel method using flow cytometry–fluorescence in situ hybridization (flow–FISH) to detect specific messenger RNA (mRNA) in suspended cells using locked nucleic acid (LNA)-modified oligonucleotide probes. β-Actin mRNA was targeted in whole A549 epithelial cells by hybridization with a biotinylated, LNA-modified probe. The LNA bound to β-actin was then stained using phycoerythrin-conjugated streptavidin and detected by flow cytometry. Shifts in fluorescence signal intensity between the β-actin LNA probe and a biotinylated, nonspecific control LNA were used to determine optimal conditions for this type of flow–FISH. Multiple conditions for permeabilization and hybridization were tested, and it was found that conditions using 3μg/ml of proteinase K for permeabilization and 90min hybridization at 60°C with buffer containing 50% formamide allow cells containing the LNA-bound mRNA to be detected and differentiated from the control LNA with high confidence (< 14% overlap between curves). This combined method, called LNA flow–FISH, can be used for detection and quantification of other RNA species as well as for telomerase measurement and detection. [Copyright &y& Elsevier]