Explore the vast range of titles available.

Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in blood—so-called circulating tumor cells (CTCs)—may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable livecell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy.

Background Transcription elongation is frequently interrupted by pausing signals in DNA, with downstream effects on gene expression. Transcription errors also induce prolonged pausing, which can lead to a destabilized genome by interfering with DNA replication. Mechanisms of pausing associated with translocation blocks and misincorporation have been characterized in vitro , but not in vivo . Results We investigate the pausing pattern of RNA polymerase (RNAP) in Escherichia coli by a novel approach, combining native elongating transcript sequencing (NET-seq) with RNase footprinting of the transcripts (RNET-seq). We reveal that the G-dC base pair at the 5′ end of the RNA-DNA hybrid interferes with RNAP translocation. The distance between the 5′ G-dC base pair and the 3′ end of RNA fluctuates over a three-nucleotide width. Thus, the G-dC base pair can induce pausing in post-translocated, pre-translocated, and backtracked states of RNAP. Additionally, a CpG sequence of the template DNA strand spanning the active site of RNAP inhibits elongation and induces G-to-A errors, which leads to backtracking of RNAP. Gre factors efficiently proofread the errors and rescue the backtracked complexes. We also find that pausing events are enriched in the 5′ untranslated region and antisense transcription of mRNA genes and are reduced in rRNA genes. Conclusions In E. coli , robust transcriptional pausing involves RNAP interaction with G-dC at the upstream end of the RNA-DNA hybrid, which interferes with translocation. CpG DNA sequences induce transcriptional pausing and G-to-A errors.

Contact unmodified antisense DNA biotechnology (CUADb), developed in 2008, employs short antisense DNA oligonucleotides (oligos) as a novel approach to insect pest control. These oligonucleotide-based insecticides target pest mature rRNAs and/or pre-rRNAs and have demonstrated high insecticidal efficacy, particularly against sap-feeding insect pests, which are key vectors of plant DNA viruses and among the most economically damaging herbivorous insects. To further explore the potential of CUADb, this study evaluated the insecticidal efficacy of short 11-mer antisense DNA oligos against Coccus hesperidum, in comparison with long 56-mer single-stranded and double-stranded DNA sequences. The short oligos exhibited higher insecticidal activity. By day 9, the highest mortality rate (97.66 ± 4.04%) was recorded in the Coccus-11 group, while the most effective long sequence was the double-stranded DNA in the dsCoccus-56 group (77.09 ± 6.24%). This study also describes the architecture of the DNA containment (DNAc) mechanism, highlighting the intricate interactions between rRNAs and various types of DNA oligos. During DNAc, the Coccus-11 treatment induced enhanced ribosome biogenesis and ATP production through a metabolic shift from carbohydrates to lipid-based energy synthesis. However, this ultimately led to a ‘kinase disaster’ due to widespread kinase downregulation resulting from insufficient ATP levels. All DNA oligos with high or moderate complementarity to target rRNA initiated hypercompensation, but subsequent substantial rRNA degradation and insect mortality occurred only when the oligo sequence perfectly matched the rRNA. Both short and long oligonucleotide insecticide treatments led to a 3.75–4.25-fold decrease in rRNA levels following hypercompensation, which was likely mediated by a DNA-guided rRNase, such as RNase H1, while crucial enzymes of RNAi (DICER1, Argonaute 2, and DROSHA) were downregulated, indicating fundamental difference in molecular mechanisms of DNAc and RNAi. Consistently, significant upregulation of RNase H1 was detected in the Coccus-11 treatment group. In contrast, treatment with random DNA oligos resulted in only a 2–3-fold rRNA decrease, consistent with the normal rRNA half-life maintained by general ribonucleases. These findings reveal a fundamental new mechanism of rRNA regulation via complementary binding between exogenous unmodified antisense DNA and cellular rRNA. From a practical perspective, this minimalist approach, applying short antisense DNA dissolved in water, offers an effective, eco-friendly and innovative solution for managing sternorrhynchans and other insect pests. The results introduce a promising new concept in crop protection: DNA-programmable insect pest control.

Early biomolecular computer research focused on laboratory-scale, human-operated computers for complex computational problems 1 , 2 , 3 , 4 , 5 , 6 , 7 . Recently, simple molecular-scale autonomous programmable computers were demonstrated 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 allowing both input and output information to be in molecular form. Such computers, using biological molecules as input data and biologically active molecules as outputs, could produce a system for ‘logical’ control of biological processes. Here we describe an autonomous biomolecular computer that, at least in vitro , logically analyses the levels of messenger RNA species, and in response produces a molecule capable of affecting levels of gene expression. The computer operates at a concentration of close to a trillion computers per microlitre and consists of three programmable modules: a computation module, that is, a stochastic molecular automaton 12 , 13 , 14 , 15 , 16 , 17 ; an input module, by which specific mRNA levels or point mutations regulate software molecule concentrations, and hence automaton transition probabilities; and an output module, capable of controlled release of a short single-stranded DNA molecule. This approach might be applied in vivo to biochemical sensing, genetic engineering and even medical diagnosis and treatment. As a proof of principle we programmed the computer to identify and analyse mRNA of disease-related genes 18 , 19 , 20 , 21 , 22 associated with models of small-cell lung cancer and prostate cancer, and to produce a single-stranded DNA molecule modelled after an anticancer drug.

Abstract Background Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion. Methods Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)–specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls. Results Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats. Conclusion Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays.

MicroRNAs (miRNAs) can act not only as tumor suppressor genes but also as oncogenes. Oncogenic miRNAs (oncomiRs) could therefore provide opportunities for the treatment of human malignancies. Here, we aimed to identify oncomiRs present in oral squamous cell carcinoma (OSCC) and addressed whether targeting these miRNAs might be useful in treatment for cancer. Functional screening for oncomiRs in a human OSCC cell line (GFP‐SAS) was carried out using the miRCURY LNA microRNA Knockdown Library – Human version 12.0. We identified a locked nucleic acid (LNA)/DNA antisense oligonucleotide against miR‐361‐3p (LNA‐miR‐361‐3p) which showed the largest degree of growth inhibition of GFP‐SAS cells. Transfection with a synthetic mimic of mature miR‐361‐3p resulted in an approximately 20% increase in the growth of GFP‐SAS cells. We identified odd‐skipped related 2 (OSR2) as a miR‐361‐3p target gene. Transfection of GFP‐SAS cells with LNA‐miR‐361‐3p caused a significant increase in the expression levels of OSR2. Cotransfection of a OSR2 3′‐UTR luciferase reporter plasmid and LNA‐miR‐361‐3p into GFP‐SAS cells produced higher levels of luciferase activity than in cells cotransfected with the LNA‐nontarget. We assessed the effect of LNA‐miR‐361‐3p on the in vivo growth of GFP‐SAS cells. We found that LNA‐miR‐361‐3p significantly reduced the size of s.c. xenografted GFP‐SAS tumors, compared to the control group treated with LNA‐NT. Finally, we observed that miR‐361‐3p is overexpressed in OSCC tissues. These results suggest that miR‐361‐3p supports the growth of human OSCC cells both in vitro and in vivo and that targeting miR‐361‐3p could be a useful therapeutic approach for patients with OSCC. This study shows that microRNA (miR)‐361‐3p supports the growth of human oral squamous cell carcinoma (OSCC) cells both in vitro and in vivo and that targeting miR‐361‐3p could be a useful therapeutic approach for patients with OSCC.

Silencing Y-box binding protein 1 (YB-1) can be an excellent target for cancer therapy and many lung cancer cells express the polysaccharide-recognition receptor Dectin-1. We designed a Dectin-1 targeting vehicle delivering YB-1-antisense DNA. First, we selected five optimal antisense DNA sequences to silence YB-1 from among 153 candidates. We chose the sequence closest to the start codon (AS014), and attached dA40 to the 3′ end; dA40 promotes complex formation with a β-(1→3)-D-glucan called schizophyllan (SPG). The resultant complexes were applied to 12 human-oriented lung cancer cell lines, and cell viability was examined. The cell lines exhibited decreased viability and showed strong affinity to bind SPG, suggesting the AS014/SPG complex entered the cells via the Dectin-1 mediated pathway.

Gold nanobeacons can be used as a powerful tool for cancer theranostics. Here, we proposed a nanomaterial platform based on gold nanobeacons to detect, target and inhibit the expression of a mutant Kras gene in an in vivo murine gastric cancer model. The conjugation of fluorescently-labeled antisense DNA hairpin oligonucleotides to the surface of gold nanoparticles enables using their localized surface plasmon resonance properties to directly track the delivery to the primary gastric tumor and to lung metastatic sites. The fluorescently labeled nanobeacons reports on the interaction with the target as the fluorescent Cy3 signal is quenched by the gold nanoparticle and only emit light following conjugation to the Kras target owing to reorganization and opening of the nanobeacons, thus increasing the distance between the dye and the quencher. The systemic administration of the anti-Kras nanobeacons resulted in approximately 60% tumor size reduction and a 90% reduction in tumor vascularization. More important, the inhibition of the Kras gene expression in gastric tumors prevents the occurrence of metastasis to lung (80% reduction), increasing mice survival in more than 85%. Our developed platform can be easily adjusted to hybridize with any specific target and provide facile diagnosis and treatment for neoplastic diseases.

Increasing numbers of investigations indicate that ethylene response factor (ERF) proteins play important roles in plant stress responses via interacting with GCC box and/dehydration-responsive element/C-repeat to modulate expression of downstream genes, but the detailed regulatory mechanism is not well elucidated. Revealing the modulation pathway of ERF proteins in response to stresses is vital. Previously, we showed that tomato ERF protein TERF2/LeERF2 is ethylene inducible, and ethylene production is suppressed in antisense TERF2/LeERF2 tomatoes, suggesting that TERF2/LeERF2 functions as a positive regulator in ethylene biosynthesis. In this paper, we report that regulation of TERF2/LeERF2 in ethylene biosynthesis is associated with enhanced freezing tolerance of tobacco and tomato. Analysis of gene expression showed that cold slowly induces expression of TERF2/LeERF2 in tomato, implying that TERF2/LeERF2 may be involved in cold response through ethylene modulation. To test the hypothesis, we first observed that overexpressing TERF2/LeERF2 tobaccos not only enhances freezing tolerance via activating expression of cold-related genes, but also significantly reduces electrolyte leakage. In addition, with treatment of ethylene biosynthesis inhibitor or ethylene receptor antagonist, we then showed that blockage of ethylene biosynthesis or the ethylene signaling pathway decreases freezing tolerance of overexpressing TERF2/LeERF2 tobaccos. Moreover, the results from tomatoes showed that overexpressing TERF2/LeERF2 tomatoes enhances while antisense TERF2/LeERF2 transgenic lines decreases freezing tolerance, and application of ethylene precursor 1-aminocyclopropane-1-carboxylic acid restored freezing tolerance of antisense lines. Therefore our results establish that TERF2/LeERF2 enhances freezing tolerance of plants through ethylene biosynthesis and the ethylene signaling pathway.

Fasciclin-like arabinogalactan proteins (FLAs) play important roles in the growth and development of roots, stems, and seeds in Arabidopsis. However, their biological functions in woody plants are largely unknown. In this work, we investigated the possible function of PtFLA6 in poplar. Quantitative real-time PCR, PtFLA6–yellow fluorescent protein (YFP) fusion protein subcellular localization, Western blotting, and immunohistochemical analyses demonstrated that the PtFLA6 gene was expressed specifically in the xylem of mature stem, and PtFLA6 protein was distributed ubiquitous in plant cells and accumulated predominantly in stem xylem fibres. Antisense expression of PtFLA6 in the aspen hybrid clone Poplar davidiana×Poplar bolleana reduced the transcripts of PtFLA6 and its homologous genes. Transgenic plants that showed a significant reduction in the transcripts of PtFLAs accumulated fewer PtFLA6 and arabinogalactan proteins than did the non-transgenic plants, leading to reduced stem flexural strength and stiffness. Further studies revealed that the altered stem biomechanics of transgenic plants could be attributed to the decreased cellulose and lignin composition in the xylem. In addition expression of some xylem-specific genes involved in cell wall biosynthesis was downregulated in these transgenic plants. All these results suggest that engineering the expression of PtFLA6 and its homologues could modulate stem mechanical properties by affecting cell wall composition in trees.