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Biological and molecular characterization of in silico identified putative inhibitors of paraspeckle assembly with potential anti-multiple myeloma activity.
Get PDFIntroduction
Multiple myeloma (MM) is the second most common haematological malignancy, being characterized by abnormal proliferation of plasma cells (PCs) predominantly within the bone marrow.
A new cellular organelle named paraspeckle (PS) has been recently associated with several biological processes, including DNA damage systems regulation and stress response. The assembly of these nuclear bodies requires the presence of the essential long noncoding RNA (lncRNA) NEAT1 and seven PS proteins (PSPs), among which NONO and SFPQ.
The relevance of PSs in MM pathogenesis has been well documented. Indeed, the crucial scaffold NEAT1 has been found to be significantly overexpressed in PCs of MM patients with respect to the normal counterpart and its silencing has been shown to affect malignant PCs proliferation and viability, triggering anti-tumour activity, both in vitro and in vivo. On the contrary, NEAT1 transactivation induced by stressful conditions has been associated with increased PSPs levels and enhanced MM cells viability, suggesting a pro-survival and anti-apoptotic role of PSs in MM.
Similarly, we recently reported higher NONO expression in primary CD138+ MM cells, correlating with poorer OS and PFS.
Aim
Because of the genetic complexity of MM cells, it is still difficult to imagine a universal therapeutic approach able to effectively target all the malignant subclones. As a result, patients inevitably relapse and become resistant to subsequent lines of therapies, highlighting the urgent clinical need to identify novel druggable vulnerabilities. Our hypothesis is that PSs could represent a novel specific vulnerability in MM across genetic subgroups. However, clinical translation of lncRNA targeting may be problematic. On the contrary, protein components of PSs are more amenable to drug design. Therefore, we set out to perform an in silico screening of small molecules targeting crucial interaction points between NEAT1, NONO, and SFPQ and supposed to disrupt PSs structure, thus, at least in part, mimicking the NEAT1 silencing strategy.
The aim of this study is to assess in vitro the on-target activity of the compounds representing the top 6 hits of the screening.
Materials and Methods
The biological activity of the 6 small molecules was evaluated in a panel of 6 MM cell lines (HMCLs), 4 haematological non-HMCLs and 6 healthy donors-derived PBMC samples. Dose-effect curves for the determination of IC50 values were obtained by Trypan Blue exclusion cell counts and CellTiter-Glo assay. Alteration of PSs integrity was evaluated by NEAT1 RNA-FISH and NONO IF by confocal microscopy. Clonogenic potential was evaluated through methylcellulose assay. Cell cycle phases modulation and apoptosis induction were investigated by FACS analysis. The levels of PSPs were assessed by means of WB analysis and IF technique. Transcriptome analysis was performed by means of ClariomTM D arrays on Affymetrix platform.
Results
2 of the 6 compounds demonstrated a significant biological activity after 5 days of treatment in all the tested HMCLs but not in non-MM cells and healthy donors-derived PBMCs (≈75% vs. 25% of affected cellular fraction, respectively), confirming a specific anti-MM effect.
Confocal microscopy experiments showed a ≈45% decrease in the number of PSs per cell in treated HMCLs, validating the expected loss of PS integrity and the on-target activity of both molecules.
In line with biological data, cell cycle analysis revealed a perturbation of the cell distribution upon small compounds treatment of HMCLs. Indeed, HMCLs displayed a significant increase of the cellular population distributed in the Sub-G0/G1 phase, and a downregulation of the percentage of cells in the S phase. FACS data were in agreement with clonogenic potential results showing a median of 50 colonies in vehicle-treated samples vs. 9 and 13 upon treatment with the 2 inhibitors.
Flow cytometry analysis revealed a dose-dependent increase of apoptotic cells in treated HMCLs (2/5-fold increase, depending on the HMCL tested), suggesting a pro-apoptotic effect of both inhibitors on HMCLs.
Additionally, we highlighted a decrease of key and core-localizing PSPs levels after treatment, among which NONO and SFPQ.
Remarkably, in line with previous findings highlighting a MM-specific effect of NEAT1 in a panel of HMCLs, none of the above-mentioned results were observed in non-MM cells, confirming the specificity of both inhibitors for HMCLs.
Finally, preliminary transcriptomic analysis revealed the downregulation of several pathways associated with tumorigenic processes.
Discussion and Conclusions
In the present study, we assessed the biological and molecular activity of the top 6 candidates resulted from an in silico screening of commercially available drug-like small molecules potentially affecting PSs structure integrity. We identified 2 molecules that, by specifically targeting NEAT1 essential protein interactors, exert an anti-MM specific activity resulting in PS structural core impairment and apoptosis induction. These promising preliminary results emphasize the need of a better comprehension of the mechanisms underlying the activity of the novel identified small compounds and suggest PS targeting could represent a possible new druggable vulnerability in MM. As a matter of fact, PSs targeting may have a great translational relevance since cytogenetically different cell lines resulted to be responsive to the selected inhibitors. Additionally, stressful conditions promoting PSs assembly are often associated with more aggressive tumor stages and chemoresistance mechanisms, raising the possibility to use this innovative therapeutic strategy in all the subsets of MM patients
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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