The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cells.

Growth arrest-specific 5 (GAS5) lncRNA promotes apoptosis, and its expression is down-regulated in breast cancer. GAS5 lncRNA is a decoy of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM), which is essential for the regulation of breast cancer cell apoptosis. This preclinical study aimed to determine if the GAS5 HREM sequence alone promotes the apoptosis of breast cancer cells. Nucleofection of hormone-sensitive and -insensitive breast cancer cell lines with a GAS5 HREM DNA oligonucleotide increased both basal and ultraviolet-C-induced apoptosis, and decreased culture viability and clonogenic growth, similar to GAS5 lncRNA. The HREM oligonucleotide demonstrated similar sequence specificity to the native HREM for its functional activity and had no effect on endogenous GAS5 lncRNA levels. Certain chemically modified HREM oligonucleotides, notably DNA and RNA phosphorothioates, retained pro-apoptotic. activity. Crucially the HREM oligonucleotide could overcome apoptosis resistance secondary to deficient endogenous GAS5 lncRNA levels. Thus, the GAS5 lncRNA HREM sequence alone is sufficient to induce apoptosis in breast cancer cells, including triple-negative breast cancer cells. These findings further suggest that emerging knowledge of structure/function relationships in the field of lncRNA biology can be exploited for the development of entirely novel, oligonucleotide mimic-based, cancer therapies.Breast Cancer No

Targeting long non-coding RNAs (lncRNAs) with oligonucleotides in cancer therapy

This document is the Accepted Manuscript version of a published work that appeared in final form in Translational Cancer Research. To access the final edited and published work see http://dx.doi.org/10.21037/tcr.2016.10.63No abstrac

GAS5 lncRNA Modulates the Action of mTOR Inhibitors in Prostate Cancer Cells

Background There is a need to develop new therapies for castrate-resistant prostate cancer (CRPC) and growth arrest-specific 5 (GAS5) long non-coding RNA (lncRNA), which riborepresses androgen receptor action, may offer novel opportunities in this regard. GAS5 lncRNA expression declines as prostate cancer cells acquire castrate-resistance, and decreased GAS5 expression attenuates the responses of prostate cancer cells to apoptotic stimuli. Enhancing GAS5 lncRNA expression may therefore offer a strategy to improve the effectiveness of chemotherapeutic agents. GAS5 is a member of the 5' terminal oligopyrimidine gene family, and we have therefore examined if mTOR inhibition can enhance cellular GAS5 levels in prostate cancer cells. In addition, we have determined if GAS5 lncRNA itself is required for mTOR inhibitor action in prostate cancer cells, as recently demonstrated in lymphoid cells. Method The effects of mTOR inhibitors on GAS5 lncRNA expression and cell proliferation were determined in a range of prostate cancer cell lines. Transfection of cells with GAS5 siRNA and plasmid constructs was performed to determine the involvement of GAS5 lncRNA in mTOR inhibitor action. Results Treatment with rapamycin and rapalogues increased cellular GAS5 levels and inhibited culture growth in both androgen-dependent (LNCaP) and androgen-sensitive (22Rv1) cell lines, but not in androgen-independent (PC-3 and DU145) cells. GAS5 silencing in both LNCaP and 22Rv1 cells decreased their sensitivity to growth inhibition by mTOR inhibitors. Moreover, transfection of GAS5 lncRNA sensitized PC-3 and DU145 cells to mTOR inhibitors, resulting in inhibition of culture growth. Conclusion mTOR inhibition enhances GAS5 transcript levels in some, but not all, prostate cancer cell lines. This may in part be related to endogenous levels of GAS5 expression, which tend to be lower in prostate cancer cells representative of advanced disease, particularly since current findings demonstrate a role for GAS5 lncRNA in mTOR inhibitor action in prostate cancer cells

Regulation of the cell cycle and cell death by protein phosphatase 4 in breast cancer cell lines

Background At the molecular level, cell death is often regulated by the level of phosphorylation of particular proteins, i.e. by the balance of between opposing kinase and phosphatase activities on those proteins. Protein phosphatase 4 (PP4) is a PP2A-related serine/threonine phosphatase. PP2A has already been implicated in the control of cell proliferation, cell cycle and tumorigenesis. Using a functional expression cloning strategy, we have previously identified the catalytic subunit of PP4 (PP4c) as an important gene influencing the regulation of both apoptosis and cell proliferation in human leukaemic cell lines and in normal lymphocytes. The aims of this study were to examine the effects of PP4c overexpression and silencing on the cell death and survival of breast cancer cell lines. Method MCF7 and MDA-MB-231 cells were transfected with pcDNA3.1 encoding PP4c (pcDNA3-PP4c) or siRNAs to different PP4c sequences. Cells transfected with scrambled siRNA or empty vector were considered as controls. Culture viability, apoptosis and cell cycle were assessed post transfection. Results In MCF7 and metastatic MDA-MB-231 cells, PP4c over-expression exerted an inhibitory effect on cell proliferation, enhanced spontaneous apoptosis and decreased their colony forming ability. Conversely, siRNA mediated silencing of PP4 enhanced the proliferation and survival of MCF7 and MDA-MB-231 cells, affected cell cycle kinetics by enhancing the proportion of cells in S and G2/M phases, increased the colony forming ability and stimulated the anchorage independent growth. Conclusion PP4c promotes cell death and inhibits proliferation in breast cells, suggestive of a role of PP4c as tumour suppressor gene. Down regulation of PP4c expression increases cell survival, proliferation and anchorage independent growth of breast cancer cells, indicating a potential link between the PP4c expression levels, tumorigenesis and metastasis

The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cell lines – towards oligonucleotide therapies?

Background Growth arrest-specific 5 (GAS5), a non-protein coding gene, encodes snoRNAs and lncRNA; transcript levels are controlled by the mTOR and nonsense-mediated decay pathways. GAS5 lncRNA promotes the apoptosis of breast cells, including triple-negative breast cancer (TNBC) cells, but its expression is down-regulated in breast cancer. Rapalogues enhance GAS5 levels in oestrogen receptor-positive breast cancer cells but not in TNBC cells, so that mTOR inhibitor-independent induction of GAS5 may be more productive in enhancing apoptotic responses to therapies. Notably, GAS5 lncRNA acts by riborepression of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM). The aim of this study was to determine if the GAS5 HREM sequence alone is sufficient to promote the apoptosis of breast cancer cells. Method Cells were nucleofected with a DNA oligonucleotide corresponding to the GAS5 lncRNA HREM; controls received oligonucleotides either with scrambled GAS5 sequence or retaining stem-loop structure but lacking the GAS5 HRE consensus; mock-transfected cells were also studied. Cells were irradiated with ultraviolet-C (UV-C) light at 20 h post-transfection to induce apoptosis. Culture viability and apoptosis were assessed and cellular GAS5 levels were determined by RT-qPCR. Results The basal apoptotic rate almost doubled in MCF7 and MDA-MB-231 cells transfected with the HREM oligonucleotide compared with controls. This effect was apparent at 20 h post­-transfection, and a corresponding decrease was observed in culture viability. The HREM sequence also enhanced UV-C-induced apoptosis in an additive manner in both cell lines. Endogenous GAS5 lncRNA expression was unaffected by transfection of the HREM sequence. Conclusion The GAS5 lncRNA HREM is sufficient to induce apoptosis in breast cancer cells, including TNBC cells. This study serves as an exemplar of how emerging knowledge of biologically important lncRNAs may be exploited towards the development of novel oncotherapeutic agents

Ab initio Random Structure Searching

It is essential to know the arrangement of the atoms in a material in order to compute and understand its properties. Searching for stable structures of materials using first-principles electronic structure methods, such as density functional theory (DFT), is a rapidly growing field. Here we describe our simple, elegant and powerful approach to searching for structures with DFT which we call ab initio random structure searching (AIRSS). Applications to discovering structures of solids, point defects, surfaces, and clusters are reviewed. New results for iron clusters on graphene, silicon clusters, polymeric nitrogen, hydrogen-rich lithium hydrides, and boron are presented.Comment: 44 pages, 23 figure

Hydrogen/silicon complexes in silicon from computational searches

Defects in crystalline silicon consisting of a silicon self-interstitial atom and one, two, three, or four hydrogen atoms are studied within density-functional theory (DFT). We search for low-energy defects by starting from an ensemble of structures in which the atomic positions in the defect region have been randomized. We then relax each structure to a minimum in the energy. We find a new defect consisting of a self-interstitial and one hydrogen atom (denoted by {I,H}) which has a higher symmetry and a lower energy than previously reported structures. We recover the {I,H_2} defect found in previous studies and confirm that it is the most stable such defect. Our best {I,H_3} defect has a slightly different structure and lower energy than the one previously reported, and our lowest energy {I,H_4} defect is different to those of previous studies.Comment: 7 pages, 8 figure

Dysregulated expression of Fau and MELK is associated with poor prognosis in breast cancer.

INTRODUCTION: Programmed cell death through apoptosis plays an essential role in the hormone-regulated physiological turnover of mammary tissue. Failure of this active gene-dependent process is central both to the development of breast cancer and to the appearance of the therapy-resistant cancer cells that produce clinical relapse. Functional expression cloning in two independent laboratories has identified Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed gene (Fau) as a novel apoptosis regulator and candidate tumour suppressor. Fau modifies apoptosis-controller Bcl-G, which is also a key target for candidate oncoprotein maternal embryonic leucine zipper kinase (MELK). METHODS: We have used RNA interference to downregulate Fau and Bcl-G expression, both simultaneously and independently, in breast cancer cells in vitro to determine the importance of their roles in apoptosis. Expression of Fau, Bcl-G and MELK was measured by quantitative RT-PCR in breast cancer tissue and in matched breast epithelial tissue from the same patients. Expression data of these genes obtained using microarrays from a separate group of patients were related to patient survival in Kaplan-Meier analyses. RESULTS: siRNA-mediated downregulation of either Fau or Bcl-G expression inhibited apoptosis, and the inhibition produced by combining the two siRNAs was consistent with control of Bcl-G by Fau. Fau expression is significantly reduced in breast cancer tissue and this reduction is associated with poor patient survival, as predicted for a candidate breast cancer tumour suppressor. In addition, MELK expression is increased in breast cancer tissue and this increase is also associated with poor patient survival, as predicted for a candidate oncogene. Bcl-G expression is reduced in breast cancer tissue but decreased Bcl-G expression showed no correlation with survival, indicating that the most important factors controlling Bcl-G activity are post-translational modification (by Fau and MELK) rather than the rate of transcription of Bcl-G itself. CONCLUSIONS: The combination of in vitro functional studies with the analysis of gene expression in clinical breast cancer samples indicates that three functionally interconnected genes, Fau, Bcl-G and MELK, are crucially important in breast cancer and identifies them as attractive targets for improvements in breast cancer risk prediction, prognosis and therapy.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

RADIOCARBON AND STABLE ISOTOPE EVIDENCE OF DIETARY CHANGE FROM THE MESOLITHIC TO THE MIDDLE AGES IN THE IRON GATES: NEW RESULTS FROM LEPENSKI VIR

This is the published version, also available here: https://journals.uair.arizona.edu/index.php/radiocarbon/article/view/4269.A previous radiocarbon dating and stable isotope study of directly associated ungulate and human bone samples from Late Mesolithic burials at Schela Cladovei in Romania established that there is a freshwater reservoir effect of approximately 500 yr in the Iron Gates reach of the Danube River valley in southeast Europe. Using the d15N values as an indicator of the percentage of freshwater protein in the human diet, the 14C data for 24 skeletons from the site of Lepenski Vir were corrected for this reservoir effect. The results of the paired 14C and stable isotope measurements provide evidence of substantial dietary change over the period from about 9000 BP to about 300 BP. The data from the Early Mesolithic to the Chalcolithic are consistent with a 2-component dietary system, where the linear plot of isotopic values reflects mixing between the 2 end-members to differing degrees. Typically, the individuals of Mesolithic age have much heavier d15N signals and slightly heavier d13C, while individuals of Early Neolithic and Chalcolithic age have lighter d15N and d13C values. Contrary to our earlier suggestion, there is no evidence of a substantial population that had a transitional diet midway between those that were characteristic of the Mesolithic and Neolithic. However, several individuals with Final Mesolithic 14C ages show d15N and d13C values that are similar to the Neolithic dietary pattern. Provisionally, these are interpreted either as incomers who originated in early farming communities outside the Iron Gates region or as indigenous individuals representing the earliest Neolithic of the Iron Gates. The results from Roman and Medieval age burials show a deviation from the linear function, suggesting the presence of a new major dietary component containing isotopically heavier carbon. This is interpreted as a consequence of the introduction of millet into the human food chain

Neurophysiology

Contains reports on sixo research projects.National Institutes of Health (Grant 5 RO1 NB-04985-03)National Institutes of Health (Grant 5 RO1 NB-4897-03)National Institutes of Health (Grant NB-06251-01)U.S. Air Force (Office of Scientific Research) under Grant AF-AFOSR-880-65U.S. Air Force (Research and Technology Division) under Contract AF33(615)-1747The Teagle Foundation, Inc. (Grant)Bell Telephone Laboratories, Inc. (Grant)Instrumentation Laboratory under the auspices of DSR Project 55-257Bioscience Division of National Aeronautics and Space Administratio