Activity Of DNA Topoisomerase Research Articles

Quercetin as a Promising Antiprotozoan Phytochemical: Current Knowledge and Future Research Avenues.

Despite tremendous advances in the prevention and treatment of infectious diseases, only few antiparasitic drugs have been developed to date. Protozoan infections such as malaria, leishmaniasis, and trypanosomiasis continue to exact an enormous toll on public health worldwide, underscoring the need to discover novel antiprotozoan drugs. Recently, there has been an explosion of research into the antiprotozoan properties of quercetin, one of the most abundant flavonoids in the human diet. In this review, we tried to consolidate the current knowledge on the antiprotozoal effects of quercetin and to provide the most fruitful avenues for future research. Quercetin exerts potent antiprotozoan activity against a broad spectrum of pathogens such as Leishmania spp., Trypanosoma spp., Plasmodium spp., Cryptosporidium spp., Trichomonas spp., and Toxoplasma gondii. In addition to its immunomodulatory roles, quercetin disrupts mitochondrial function, induces apoptotic/necrotic cell death, impairs iron uptake, inhibits multiple enzymes involved in fatty acid synthesis and the glycolytic pathways, suppresses the activity of DNA topoisomerases, and downregulates the expression of various heat shock proteins in these pathogens. In vivo studies also show that quercetin is effective in reducing parasitic loads, histopathological damage, and mortality in animals. Future research should focus on designing effective drug delivery systems to increase the oral bioavailability of quercetin. Incorporating quercetin into various nanocarrier systems would be a promising approach to manage localized cutaneous infections. Nevertheless, clinical trials are needed to validate the efficacy of quercetin in treating various protozoan infections.

Targeted repression of topA by CRISPRi reveals a critical function for balanced DNA topoisomerase I activity in the Chlamydia trachomatis developmental cycle.

Chlamydia trachomatis is an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infection. Changes in DNA topology in this pathogen have been linked to its pathogenicity-associated developmental cycle. Here, evidence is provided that the balanced activity of DNA topoisomerases contributes to controlling Chlamydia developmental processes. Utilizing catalytically inactivated Cas12 (dCas12)-based clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomal topA transcription in C. trachomatis without detected toxicity of dCas12. Repression of topA impaired the developmental cycle of C. trachomatis mostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes of C. trachomatis was downregulated, while early genes maintained their expression. Importantly, the developmental defect associated with topA knockdown was rescued by overexpressing topA at an appropriate degree and time, directly linking the growth patterns to the levels of topA expression. Interestingly, topA knockdown had effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency. C. trachomatis with topA knocked down displayed hypersensitivity to moxifloxacin that targets DNA gyrase in comparison with the wild type. These data underscore the requirement of integrated topoisomerase actions to support the essential developmental and transcriptional processes of C. trachomatis.IMPORTANCEWe used genetic and chemical tools to demonstrate the relationship of topoisomerase activities and their obligatory role for the chlamydial developmental cycle. Successfully targeting the essential gene topA with a CRISPRi approach, using dCas12, in C. trachomatis indicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanisms by which well-balanced topoisomerase functions in adaptation of C. trachomatis to unfavorable growth conditions imposed by antibiotics.

Utilizing coordination chemistry through formation of a CuII-quinalizarin complex to manipulate cell biology: An in vitro, in silico approach

Quinalizarin, an analogue of anthracycline anticancer agents, is an anticancer agent itself. A CuII complex was prepared and characterized by elemental analysis, UV-Vis & IR spectroscopy, mass spectrometry, EPR and DFT. The intention behind the preparation of the complex was to increase cellular uptake, compare its binding with DNA against that of quinalizarin, modulation of semiquinone formation, realization of human DNA topoisomerase I & human DNA topoisomerase II inhibition and observation of anticancer activity. While the first two attributes of complex formation lead to increased efficacy, decrease in semiquinone generation could results in a compromise with efficacy. Inhibition of human DNA topoisomerase makes up this envisaged compromise in free radical activity since the complex shows remarkable ability to disrupt activities of human DNA topoisomerase I and II. The complex unlike quinalizarin, does not catalyze flow of electrons from NADH to O2 to the extent known for quinalizarin. Hence, decrease in semiquinone or superoxide radical anion could make modified quinalizarin [as CuII complex] less efficient in free radical pathway. However, it would be less cardiotoxic and that would be advantageous to qualify it as a better anticancer agent. Although binding to calf thymus DNA was comparable to quinalizarin, it was weaker than anthracyclines. Low cost of quinalizarin could justify consideration as a substitute for anthracyclines but the study revealed IC50 of quinalizarin/CuII-quinalizarin was much higher than anthracyclines or their complexes. Even then, there is a possibility that CuII-quinalizarin could be an improved and less costly form of quinalizarin as anticancer agent.

DNA Sequence-Specific Ligands. XIX. Synthesis, Spectral Properties, Virological and Biochemical Studies of Fluorescent Dimeric Trisbenzimidazoles DB<sub>3</sub>(<i>n</i>)

In this work, we synthesized and characterized the properties of a series of new fluorescent narrow-groove ligands DB3(n). DB3(n) compounds based on dimeric trisbenzimidazoles have the ability to bind to the AT regions of DNA. The synthesis of DB3(n), trisbenzimidazole fragments of which are linked by oligomethylene linkers of different lengths (n = 1, 5, 9), is based on the condensation of monomeric trisbenzimidazole MB3 with α,ω-alkyldicarboxylic acids. DB3(n) proved to be effective inhibitors of the catalytic activity of HIV-1 integrase at submicromolar concentrations (0.20–0.30 µM). DB3(n) was found to inhibit the catalytic activity of DNA topoisomerase I at low micromolar concentrations.

Antibacterial Activity and Mechanism of Madecassic Acid against Staphylococcus aureus.

Antibacterial resistance has become one of the most serious problems threating global health. To overcome this urgent problem, many scientists have paid great attention to developing new antibacterial drugs from natural products. Hence, for exploring new antibacterial drugs from Chinese medicine, a series of experiments were carried out for verifying and elucidating the antibacterial activity and mechanisms of madecassic acid (MA), which is an active triterpenoid compound isolated from the traditional Chinese medicine, Centella asiatica. The antibacterial activity was investigated through measuring the diameter of the inhibition zone, the minimum inhibitory concentration (MIC), the growth curve, and the effect on the bacterial biofilm, respectively. Meanwhile, the antibacterial mechanism was also discussed from the aspects of cell wall integrity variation, cell membrane permeability, and the activities of related enzymes in the respiratory metabolic pathway before and after the intervention by MA. The results showed that MA had an inhibitory effect on eight kinds of pathogenic bacteria, and the MIC values for Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus megaterium were 31.25, 62.5, 250, 125, 62.5, and 62.5 µg/mL, respectively. For instance, 31.25 µg/mL MA could inhibit the growth of Staphylococcus aureus within 28 h. The antibacterial mechanism experiments confirmed that MA could destroy the integrity of the cell membrane and cell wall of Staphylococcus aureus, causing the leakage of macromolecular substances, inhibiting the synthesis of soluble proteins, reducing the activities of succinate dehydrogenase and malate dehydrogenase, and interacting with DNA, leading to the relaxation and ring opening of supercoiled DNA. Besides, the activities of DNA topoisomerase I and II were both inhibited by MA, which led to the cell growth of Staphylococcus aureus being repressed. This study provides a theoretical basis and reference for the application of MA in the control and inhibition of food-borne Staphylococcus aureus.

DNA Sequence-Specific Ligands. 19. Synthesis, Spectral Properties, Virological and Biochemical Studies of DB3(n) Fluorescent Dimeric Trisbenzimidazoles

In this work, we synthesized and characterized the properties of a series of new fluorescent DB3(n) narrow-groove ligands. DB3(n) compounds based on dimeric trisbenzimidazoles have the ability to bind to the AT regions of DNA. The synthesis of DB3(n), whose trisbenzimidazole fragments are linked by oligomethylene linkers of different lengths (n = 1, 5, 9), is based on the condensation of the MB3 monomeric trisbenzimidazole with α,ω-alkyldicarboxylic acids. DB3 (n) proved to be effective inhibitors of the catalytic activity of HIV-1 integrase at submicromolar concentrations (0.20-0.30 μM). DB3(n) was found to inhibit the catalytic activity of DNA topoisomerase I at low micromolar concentrations.

G-quadruplex-containing oligodeoxynucleotides as DNA topoisomerase I inhibitors

DNA topoisomerase I was found to be highly abundant in fast-proliferating tumor cells and is a potential target for anticancer therapy. A series of G-quadruplex-containing oligodeoxynucleotides (ODNs) were designed and used as inhibitors of DNA topoisomerase I. It was demonstrated that ODNs with G-quadruplexes can efficiently inhibit the supercoiled DNA relaxation reaction catalyzed by DNA topoisomerase I. Compared with the other conformations, the parallel propeller-type G-quadruplex was the most efficient DNA topoisomerase I inhibitor. Further studies revealed that integrating G-quadruplexes with duplexes to form quadruplex-duplex hybrids could significantly improve the inhibition efficiency. In addition, a circular ODN that consists of a G-quadruplex motif and DNA topoisomerase I binding site was synthesized and used as a DNA topoisomerase I inhibitor. The results showed that the particularly designed circular ODN displayed high inhibitory efficiency on the activity of DNA topoisomerase I with an IC50 value of 54.8 nM. Moreover, the circular ODN exhibited excellent thermal stability and nuclease resistance. Considering the low cytotoxicity of DNA-based biopharmaceuticals, the design strategy and results reported in this study may shed new light on nucleic acid-based DNA topoisomerase I inhibitor construction for potential anticancer drugs.

Structures of topoisomerase V in complex with DNA reveal unusual DNA-binding mode and novel relaxation mechanism.

Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include 3 AP lyase repair domains. The uncommon architecture of this enzyme bears no resemblance to any other known topoisomerase. Here, we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA-binding mode not observed before and provide information on the way this atypical topoisomerase relaxes DNA. In common with type IB enzymes, topoisomerase V relaxes DNA using a controlled rotation mechanism, but the structures show that topoisomerase V accomplishes this in different manner. Overall, the structures firmly establish that type IC topoisomerases form a distinct type of topoisomerases, with no similarities to other types at the sequence, structural, or mechanistic level. They represent a completely different solution to DNA relaxation.

Multiple gene copy number increases total protein expression and enzyme activity of DNA topoisomerase I in Pichia pastoris

The present study aimed to employ an <em>in vivo</em> strategy for the establishment of multi‑copy gene constructs of human DNA topoisomerase I (hTopI) using the pPIC3.5K vector in <em>Pichia</em>. The clones with multi‑copy inserts (His⁺ transformants) that were able to survive in the highest concentration of Geneticin^® were found to express the highest expression level of total protein and exhibited the highest target enzyme activity. The highest level of total protein found was 1.76 mg/ml in GS115‑pPIC3.5K‑hTop1, which was resistant to 1.00 mg/ml Geneticin at 48 h of incubation. The highest enzyme activity of hTopI was also observed in the culture expressed by GS115‑pPIC3.5K‑hTopI, which was resistant to 1.00 mg/ml Geneticin^® (19.7x104 Ul/OD₆₀₀). On the whole, the present study provides information regarding the production of target protein from recombinant <em>Pichia</em> using only a shaker flask system, which can be further developed as an in‑house resource for screening potential anticancer agents.

Degradation of ciprofloxacin in aqueous solution using ozone microbubbles: spectroscopic, kinetics, and antibacterial analysis

Ciprofloxacin (CIP) has been listed in the last version of the surface water due to its ability to kill human cells by inhibiting the activity of DNA topoisomerase IV. Thus, CIP, along with other antibiotic pollution has become a serious threat to the environment and public health. Ozonation has been used as an advanced technique that is applied in wastewater treatment to remove CIP, but the primary limitation of this method is the low solubility of ozone in water. This study is the first report of CIP removal in a scale-up of its aqueous solution using a self-developed aerator pump-enhanced ozonation (APO) system, which only employs a propeller and a zigzag arrangement of meshes. This aerator pump decreased the size of ozone bubbles by 90% and increased the effective ozone solubility to 0.47 ppm. The mechanism of degradation of CIP is attributed to an oxidation reaction of the antibiotic with reactive oxygen species, such as hydroxyl, oxygen, and hydroperoxyl radicals, generated on the surface of the ozone microbubbles. It was found that the rate and efficiency of degradation of CIP using the APO system were 3.64 × 10−3/min and 83.5%, respectively, which is higher compared with those of conventional flow ozonation (FO) systems (1.47 × 10−3/min and 60.9%). The higher degradation efficiency of CIP by the APO system was also revealed by its higher electrical energy efficiency (0.146 g/kWh), compared to that of the FO system (0.106 g/kWh). The degradation of CIP was also monitored by the resulting antibacterial activity against Escherichia coli and Staphylococcus aureus.

Anti-cancer Research on Arnebiae radix-derived Naphthoquinone in Recent Five Years.

In recent years, many naphthoquinone compounds with anticancer activity have been identified in Arnebiae Radix, and some of them have the potential to be developed into anticancer drugs. This article aimed to provide a comprehensive overview of the anticancer effects of naphthoquinone compounds through a detailed review of literature and Chinese patents, and discuss their potential to be developed as anticancer drugs for clinical application. Research papers were collected through the databases of PubMed, Cnki and SciDirect using keyword searches "naphthoquinone compounds" and "anticancer". The keywords of "shikonin" and "shikonin derivatives" were also used in PubMed, Cnki and SciDirect databases to collect research articles. The Chinese patents were collected using the Cnki patent database. Naphthoquinone compounds have been found to possess anti-cancer activity, and their modes of action are associated with inducing apoptosis, inhibiting cancer cell proliferation, promoting autophagy in cancer cells, anti-cancer angiogenesis and inhibition of cell adhesion, invasion and metastasis, inhibiting glycolysis and inhibiting DNA topoisomerase activity. Most of the naphthoquinone compounds show effective anti-cancer activity in vitro. The structure modification of naphthoquinone aims to develop anti-cancer drugs with high efficacy and low toxicity.

A new exploration towardadamantane derivatives as potential anti-MDR agents: Design, synthesis, antimicrobial, and radiosterilization activity as potential topoisomerase IV and DNA gyrase inhibitors.

Developing novel antimicrobial agents has become a necessitate due to the increasing rate of microbial resistance to antibiotics. All the newly adamantane derivatives were evaluated for their antimicrobial activities against six MDR clinical pathogenic isolates. The results exhibited that 13 compounds have from potent to good activity. Among those, five derivatives (6, 7, 9, 14a, and 14b) displayed the potent activities against the different isolates tested (MIC < 0.25 µg/ml with bacteria and <8 µg/ml with fungi) compared with Ciprofloxacin (CIP) and Fluconazole (FCA). Additionally, the potent adamantanes showed bactericidal and fungicidal effects based on (MBCs and MFCs) and the time-kill assay. The most active adamantane derivatives 7 and 14b exhibited a synergistic effect of ΣFIC ≤ 0.5 with CIP and FCA against the bacterial and fungal isolates. Moreover, no antagonistic effect appeared for the tested derivatives. Additionally, the interaction of DNA gyrase and topoisomerase IV enzymes with the compounds 6, 7, 9, 14a, and 14b exhibited potent antimicrobial activity using in vitro biochemical assays and gel-based DNA-supercoiling inhibition method. The activity of DNA gyrase and topoisomerase IV enzymes showed inhibitory activity (IC50 ) of 6.20 µM and 9.40 µM with compound 7 and 10.14 µM and 13.28 µM with compound 14b, respectively. Surprisingly, exposing compound 7 to gamma irradiation sterilized and increased its activity. Finally, the in-silico analysis predicted that the most active derivatives had good drug-likeness and safe properties. Besides, molecular docking and quantum chemical studies revealed several important interactions inside the active sites and showed the structural features necessary for activity.

Interplay between symmetric arginine dimethylation and ubiquitylation regulates TDP1 proteostasis for the repair of topoisomerase I-DNA adducts.

Tyrosyl-DNA phosphodiesterase (TDP1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety and is implicated in the repair of trapped topoisomerase I (Top1)-DNA covalent complexes (Top1cc). Protein arginine methyltransferase 5 (PRMT5) catalyzes arginine methylation of TDP1 at the residues R361 and R586. Here, we establish mechanistic crosstalk between TDP1 arginine methylation and ubiquitylation, which is critical for TDP1 homeostasis and cellular responses to Top1 poisons. We show that R586 methylation promotes TDP1 ubiquitylation, which facilitates ubiquitin/proteasome-dependent TDP1 turnover by impeding the binding of UCHL3 (deubiquitylase enzyme) with TDP1. TDP1-R586 also promotes TDP1-XRCC1 binding and XRCC1 foci formation at Top1cc-damage sites. Intriguingly, R361 methylation enhances the 3'-phosphodiesterase activity of TDP1 in real-time fluorescence-based cleavage assays, and this was rationalized using structural modeling. Together, our findings establish arginine methylation as a co-regulator of TDP1 proteostasis and activity, which modulates the repair of trapped Top1cc.

Design, Synthesis, and Cytotoxicity and Topoisomerase I/IIα Inhibition Activity of Pyrazolo[4,3-f]quinoline Derivatives.

With the several targets of cancer treatment, inhibition of DNA topoisomerase activity is one of the well-known focuses in cancer chemotherapy. Here, we describe the design and synthesis of a novel series of pyrazolo[4,3-f]quinolines with potential anticancer/topoisomerase inhibition activity. Forty newly designed pyrazolo[4,3-f]quinoline derivatives were synthesized via inverse imino Diels–Alder reaction. The antiproliferative activity of the synthesized derivatives was initially measured in the human NUGC-3 cancer cell line. Then, the selected compounds 1B, 1C, 1M, 2A, 2D, 2E, 2F, and 2R with higher activity among tested compounds were screened against six cancer cell lines, including ACHN, HCT-15, MM231, NCI-H23, NUGC-3, and PC-3. The results demonstrated that the compounds 1M, 2E, and 2P were most effective in all cancer cell lines exhibiting GI50 below 8 µM. Among them, 2E showed an equivalent inhibition pattern of topoisomerase IIα activity to that of etoposide, positive control at a 100 µM dose.

Repurposing of the ALK Inhibitor Crizotinib for Acute Leukemia and Multiple Myeloma Cells.

Crizotinib was a first generation of ALK tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small-cell lung carcinoma (NSCLC) patients. COMPARE and cluster analyses of transcriptomic data of the NCI cell line panel indicated that genes with different cellular functions regulated the sensitivity or resistance of cancer cells to crizotinib. Transcription factor binding motif analyses in gene promoters divulged two transcription factors possibly regulating the expression of these genes, i.e., RXRA and GATA1, which are important for leukemia and erythroid development, respectively. COMPARE analyses also implied that cell lines of various cancer types displayed varying degrees of sensitivity to crizotinib. Unexpectedly, leukemia but not lung cancer cells were the most sensitive cells among the different types of NCI cancer cell lines. Re-examining this result in another panel of cell lines indeed revealed that crizotinib exhibited potent cytotoxicity towards acute myeloid leukemia and multiple myeloma cells. P-glycoprotein-overexpressing CEM/ADR5000 leukemia cells were cross-resistant to crizotinib. NCI-H929 multiple myeloma cells were the most sensitive cells. Hence, we evaluated the mode of action of crizotinib on these cells. Although crizotinib is a TKI, it showed highest correlation rates with DNA topoisomerase II inhibitors and tubulin inhibitors. The altered gene expression profiles after crizotinib treatment predicted several networks, where TOP2A and genes related to cell cycle were downregulated. Cell cycle analyses showed that cells incubated with crizotinib for 24 h accumulated in the G2M phase. Crizotinib also increased the number of p-H3(Ser10)-positive NCI-H929 cells illustrating crizotinib’s ability to prevent mitotic exit. However, cells accumulated in the sub-G0G1 fraction with longer incubation periods, indicating apoptosis induction. Additionally, crizotinib disassembled the tubulin network of U2OS cells expressing an α-tubulin-GFP fusion protein, preventing migration of cancer cells. This result was verified by in vitro tubulin polymerization assays. In silico molecular docking also revealed a strong binding affinity of crizotinib to the colchicine and Vinca alkaloid binding sites. Taken together, these results demonstrate that crizotinib destabilized microtubules. Additionally, the decatenation assay showed that crizotinib partwise inhibited the catalytic activity of DNA topoisomerase II. In conclusion, crizotinib exerted kinase-independent cytotoxic effects through the dual inhibition of tubulin polymerization and topoisomerase II and might be used to treat not only NSCLC but also multiple myeloma.

A Type IA DNA/RNA Topoisomerase with RNA Hydrolysis Activity Participates in Ribosomal RNA Processing

Topoisomerases maintain topological homeostasis of bacterial chromosomes by catalysing changes in DNA linking number. The resolution of RNA entanglements occurring in the cell would also require catalytic action of topoisomerases. We describe RNA topoisomerase and hydrolysis activities in DNA topoisomerase I (topo I) from mycobacteria. The interaction of topo I with mRNA, tRNA and rRNA suggested its role in some aspect of RNA metabolism; the enzyme participates in rRNA maturation via its RNA hydrolysis activity. Accumulation of rRNA precursors in a topo I knockdown strain and the rescue of rRNA processing deficiency in RNaseE knockdown cells by topo I expression indicated the enzyme’s back-up support to RNases involved in rRNA processing. We demonstrate that the active-site tyrosine of the enzyme mediates catalytic reactions with both DNA/RNA substrates, and RNA topoisomerase activity can follow two reaction paths in contrast to its DNA topoisomerase activity. Mutation in the canonical proton relay pathway impacts DNA topoisomerase activity whilst retaining activity on RNA substrates. The mycobacterial topo I thus exemplifies the resourcefulness and parsimony of biological catalysis in harnessing the limited chemical repertoire at its disposal to find common solutions to mechanistically related challenges of phosphodiester breakage/exchange reactions in DNA and RNA that are essential for cell survival.

Replication Fork Barriers and Topological Barriers: Progression of DNA Replication Relies on DNA Topology Ahead of Forks.

During replication, the topology of DNA changes continuously in response to well-known activities of DNA helicases, polymerases, and topoisomerases. However, replisomes do not always progress at a constant speed and can slow-down and even stall at precise sites. The way these changes in the rate of replisome progression affect DNA topology is not yet well understood. The interplay of DNA topology and replication in several cases where progression of replication forks reacts differently to changes in DNA topology ahead is discussed here. It is proposed, there are at least two types of replication fork barriers: those that behave also as topological barriers and those that do not. Two-Dimensional (2D) agarose gel electrophoresis is the method of choice to distinguish between these two different types of replication fork barriers.

DNA Double Strand Breaks and Chromosomal Translocations Induced by DNA Topoisomerase II.

DNA double strand breaks (DSBs) are the most cytotoxic lesions of those occurring in the DNA and can lead to cell death or result in genome mutagenesis and chromosomal translocations. Although most of these rearrangements have detrimental effects for cellular survival, single events can provide clonal advantage and result in abnormal cellular proliferation and cancer. The origin and the environment of the DNA break or the repair pathway are key factors that influence the frequency at which these events appear. However, the molecular mechanisms that underlie the formation of chromosomal translocations remain unclear. DNA topoisomerases are essential enzymes present in all cellular organisms with critical roles in DNA metabolism and that have been linked to the formation of deleterious DSBs for a long time. DSBs induced by the abortive activity of DNA topoisomerase II (TOP2) are “trending topic” because of their possible role in genome instability and oncogenesis. Furthermore, transcription associated TOP2 activity appears to be one of the most determining causes behind the formation of chromosomal translocations. In this review, the origin of recombinogenic TOP2 breaks and the determinants behind their tendency to translocate will be summarized.

P1.03-17 Function of Antisense LncRNA RP11-539E17.5 and FAM83A-AS1 Up-Regulating FAM83A in Lung Adenocarcinoma Tumorigenesis and Development

Chromosome 8q24 lies in a gene desert with only a few predicted coding genes, but its variation is associated with multiple tumors. FAM83A in this region activates both PI3K/AKT/TOR and RAS/MAPK signaling cascades downstream of EGFR. The contribution of long noncoding gene in the antisense chain of FAM83A (RP11-539E17.5 and FAM83A-AS1) to the risk of lung adenocarcinoma (LUAD) need to be studied. We use systematic informatics method to identify the differential expression genes of the Cancer Genome Atlas (TCGA) LUAD transcriptome sequencing data. Then use data mining method to analysis the lncATLAS, USCS, Cistrome, and ENCODE database to infer the biological role of these two lncRNAs. Significant analysis of gene ontology (GO) terms enrichment analyzed the co-expression genes of RP11-539E17.5 in the LUAD and adjacent tissues. At last we use cell experiment to verify the lncRNA function. FAM83A-AS1 and RP11-539E17.5 located in Chromosome 8q24 were significantly different expressed between cancer and adjacent tissues (log2foldchange>5, p<0.05). The two lncRNA expression levels had notable correlation with FAM83A expression (coR> 0.8, P<0.005) and were significantly associated with patient overall survival and clinical stage (p<0.05). RP11-539E17.5, located in the enhancer element GH08J123181，belong to the enhancer lncRNA group. lncRNA subcellular localization data (lncATLAS) results showed that RP11-539E17.5 transcripts in K562 cells is mainly distributed in the nucleus, and most around the nucleolus and chromosomes. USCS, Cistrome, and ENCODE data mining identified four transcription factors (FOSL2, CTBP2, CEBPB, and MAFK) have significant correlation with the expression of FAM83A-AS1 and RP11-539E17.5. In K562 cell line with high expression of FAM83A-AS1, RP11-539E17.5 and FAM83A showed that expanded regions of RP11-539E17.5 gene sequence were marked by enhancer-specific modifications (H3K27ac domains and H3K4me3 domains). This region also showed an excellent source of DNase sensitive site, and with several binding sites for CTCF, FOSL2, CTBP2, CEBPB and MAFK. In addition, RP11-539E17.5 gene also with enriched DNA methylation feature. RP11-539E17.5 and FAM83A-AS1 knocked down both inhibited the expression of FAM83A the proliferation of PC9, and RP11-539E17.5 gene owning a more significant effect. GO terms enrichment analysis of the co-expressed genes with RP11-539E17.5 showed that the most significantly activated biological pathways is involved with Cell cycle，and cell mitotic signaling pathway, while the molecular function involved with transcription factor binding, DNA binding, ATP binding, endonuclease activity, DNA topoisomerase activity. We found a LncRNA RP11-539E17.5 with enhancer character up regulate FAM83A expression in LUAD. These results can lay a theoretical foundation for LUAD corresponding countermeasures.

In vitro Activities of Nemonoxacin and Other Antimicrobial Agents Against Human Mycoplasma and Ureaplasmas Isolates and Their Defined Resistance Mechanisms.

Nemonoxacin, a newly developed non-fluorinated quinolone (NFQ), selectively inhibits bacterial DNA topoisomerase activity. However, its activities against Mycoplasmas have rarely been studied to date. Herein, the activities of nemonoxacin were evaluated against clinical isolates of 50 Mycoplasma pneumoniae, 20 Mycoplasma hominis, and 77 Ureaplasma spp., and they were compared to fluoroquinolones, tetracyclines, and macrolides. Nemonoxacin MICs (μg/ml) ranged from 0.03 to 0.25 for M. pneumoniae, 0.25 to 8 for M. hominis, and 0.06 to >16 for Ureaplasma spp., and all of the ranges are similar to those of fluoroquinolones. The activity of nemonoxacin against Mycoplasmas was not affected by resistance to macrolides in the strains tested, but it seems to have the same resistant mechanism as fluoroquinolones. In addition, minimum bactericidal concentrations (MBC) of nemonoxacin to M. pneumoniae were within two dilutions of the MIC values, indicating a bactericidal effect on M. pneumoniae. Nemonoxacin merits further study for treating infections caused by these organisms.