Cyclodextrin-siRNA conjugates as versatile gene silencing agents

Functional siRNAs (luciferase and PLK1) have been conjugated to β-cyclodextrin and the ability of the conjugates to retain gene knockdown activity has been assessed by delivery to cancer cell lines using various formulations. Initially two formulations used complexation with polycations, namely Lipofectamine 2000 and an amphiphilic polycationic cyclodextrin. Gene knockdown results for human glioblastoma cells (U87) and prostate cancer cells (PC3, DU145) showed that conjugation to the cyclodextrin did not reduce gene silencing by the RNA. A third mode of delivery involved formation of targeted nanoparticles in which the conjugate was first complexed with adamantyl-PEG-ligands (targeting ligand RVG peptide or dianisamide) by adamantyl inclusion in the cyclodextrin cavities of the conjugates, followed by charge neutralisation with the cationic polymer chitosan. Enhanced knockdown was achieved by these ligand-targeted formulations. In summary, while this study illustrated the gene silencing efficacy of a simple cyclodextrin-siRNA conjugate it is envisaged that future studies will explore the use of conjugates with a modified cyclodextrin which would be self-delivering. Detailed data such as stability, lysosomal escape etc. will then be reported for each conjugate, since this will be appropriate for conjugates which are intended to exploit, rather than merely demonstrate, the concept. The present paper was intended to demonstrate the viability and generality of this novel concept

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Reading Orlando with the Mandala Browser: A Case Study in Algorithmic Criticism via Experimental Visualization

This paper describes the preliminary results of combining two complementary technologies: 'Orlando', a semantically-tagged XML collection of born-digital scholarly resources, and the Mandala Browser, an XML visualization tool. 'Orlando's' current delivery system privileges text as an approach to literary historical scholarship. The Mandala browser represents a radically different way of mediating between the user and the text, translating a text or set of texts into a circular visual form and pushing the user towards a more distant, or at least a more selective, reading of the materials than that associated with conventional print or screen rendering. Through experimental visualizations of 'Orlando' content, we began to address questions concerning the participation of Victorian and Renaissance writers in various genres, the relationship between reproduction and literary production, the connection of censorship to the destruction of literary works, and the relationship between suffrage and liberal or conservative political groups. We argue that, just as a postcolonialist or a new historicist needs to learn about the tenets and processes involved in a postcolonial or new historical critical framework, so too an algorithmic critic should expect to invest some time learning the techniques of a given approach and how to apply them to a particular text or body of texts. These investigations may interest other humanities scholars working with online digital collections, as well as those thinking through the question of how to involve computational processes in complex inquiries using large quantities of texts

Substance Use Disorder Visits Among Adolescents at Children\u27s Hospitals During COVID-19

PURPOSE: To assess changes in adolescent visits with substance use disorders (SUDs) at children\u27s hospitals during COVID-19. METHODS: We conducted a retrospective cohort study of adolescents (11-18 years) with SUD diagnoses during a hospital visit in the Pediatric Health Information System. Study periods were defined as spring (03/15-05/31), summer (06/01-08/31), and fall (09/01-12/31), pre-COVID-19 (2017-2019), or during COVID-19 (2020). We summarized the change in weekly median visits and interquartile range (IQR) by characteristics and compared between the two periods (e.g., spring pre-COVID-19 [3/15-05/31/2017-2019] versus spring COVID-19 [3/15-05/31/2020]) using median regression. RESULTS: There were 42,979 SUD visits (10,697 COVID-19; 32,282 pre-COVID-19) and 256 annual weekly median adolescent SUD visits [IQR 235, 280] pre-COVID-19 and 268 [IQR 245, 278] during COVID-19. The median number of weekly SUD visits increased by 14.3% during summer COVID-19 (median visits, [IQR]: 272 [268, 278]) compared to pre-COVID-19 (median visits: 237, IQR [216, 249]; p \u3c .001) but did not significantly differ in spring (p = .091) or fall (p = .65) COVID-19. DISCUSSION: Our findings suggest increased problematic adolescent substance use during summer COVID-19. Efforts to increase the identification and treatment of adolescent SUDs remain critical

NIR Spectroscopy of Selected Iron (II) and Iron (III) Sulphates

A problem exists when closely related minerals are found in paragenetic relationships. The identification of such minerals cannot be undertaken by normal techniques such as X-ray diffraction. Vibrational spectroscopic techniques may be applicable especially when microtechniques or fibre-optic techniques are used. NIR spectroscopy is one technique which can be used for the identification of these paragenetically related minerals and has been applied to the study of selected iron(II) and iron(III) sulphates. The Near–IR spectral regions may be conveniently divided into four regions (a) the high wavenumber region > 7500 cm-1 (b) the high wavenumber region between 6400 and 7400 cm-1 attributed to the first overtone of the fundamental hydroxyl stretching mode (c) the 5500-6300 cm-1 region attributed to water combination modes of the hydroxyl fundamentals of water, and (d) the 4000-5500 cm-1 region attributed to the combination of the stretching and deformation modes of the iron(II) and iron(III) sulphates. The minerals containing iron(II) show a strong, broad band with splitting, around 11000-8000 cm-1 attributed to 5T2g --> 5Eg transition. This shows the ferrous ion has distorted octahedral coordination in some of these sulphate minerals. For each of these regions, the minerals show distinctive spectra which enable their identification and characterisation. NIR spectroscopy is a less used technique which has great application for the study of minerals, particularly minerals which have hydrogen in the structure either as hydroxyl units or as water bonded to the cation as is the case for iron(II) and iron(III) sulphates. The study of minerals on planets is topical and NIR spectroscopy provides a rapid technique for the distinction and identification of iron(II) and iron(III) sulphates minerals

Synthesis and characterization of rabies virus glycoprotein-tagged amphiphilic cyclodextrins for siRNA delivery in human glioblastoma cells: in vitro analysis

In man brain cancer is an aggressive, malignant form of tumour, it is highly infiltrative in nature, is associated with cellular heterogeneity and affects cerebral hemispheres of the brain. Current drug therapies are inadequate and an unmet clinical need exists to develop new improved therapeutics. The ability to silence genes associated with disease progression by using short interfering RNA (siRNA) presents the potential to develop safe and effective therapies. In this work, in order to protect the siRNA from degradation, promote cell specific uptake and enhance gene silencing efficiency, a PEGylated cyclodextrin (CD)-based nanoparticle, tagged with a CNS-targeting peptide derived from the rabies virus glycoprotein (RVG) was formulated and characterized. The modified cyclodextrin derivatives were synthesized and co-formulated to form nanoparticles containing siRNA which were analysed for size, surface charge, stability, cellular uptake and gene-knockdown in brain cancer cells. The results identified an optimised co-formulation prototype at a molar ratio of 1:1.5:0.5 (cationic cyclodextrin:PEGylated cyclodextrin:RVG-tagged PEGylated cyclodextrin) with a size of 281±39.72nm, a surface charge of 26.73±3mV, with efficient cellular uptake and a 27% gene-knockdown ability. This CD-based formulation represents a potential nanocomplex for systemic delivery of siRNA targeting brain cancer