DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.
Introduction: LSD1 is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that removes methyl groups from mono- or dimethylated histone H3 lysine 4. LSD1 has been reported to be overexpressed in many malignant tumors, including breast, colon, prostate, lung, gastric cancers and others. Studies have shown that LSD1 may contribute to acute myelogenous leukaemia pathogenesis and patients that are non responders to ATRA have responded to LSD1-ATRA combination treatment in clinic. Recent studies have shown that there is cross-talk between two components of CoREST complex, LSD1 and HDAC which provides an advantage to cancer cell proliferation and survival by regulating key signalling pathways. Accordingly, combined inhibition of LSD1 and HDAC has been shown to be more efficacious in inhibiting the growth of glioblastoma, AML and breast cancer. Therefore, dual inhibitors targeting both LSD1 and HDAC could be useful in treating several cancers effectively without enhancing systemic toxicity mediated by administration of multiple drugs. To test the hypothesis, we have developed a set of molecules that either have LSD1 activity alone or dual activity on LSD1 and HDAC. Methods: Computational chemistry approaches were used to design LSD1 specific and LSD1-HDAC dual inhibitors. To assess in vitro LSD1 potency, TR-FRET assay was used. For assessing in vitro HDAC activity fluorescence based pan-HDAC activity assay was performed. Western blotting was used to assess biomarkers of LSD1 and HDAC inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation. Results: In vitro potency of these compounds on LSD1 ranged between,0.007 to 0.2 μM, which was comparable to literature molecules. Pan-HDAC potency of these molecules in the in vitro assay was comparable to the approved drug, Vorinostat. H3K4 di- and mono-methyl markers and H3K9 acetylation levels respectively, were used as target engagement biomarkers for LSD1and HDAC inhibition. Treatment of leukaemia cells with LSD1or LSD1-HDAC inhibitors resulted in a dose-dependent increase in the levels of H3K4 di-and mono methylation. In addition, compounds with LSD1-HDAC dual activity showed a clear increase in H3K9 acetylation levels as well. In the cell proliferation assay, compounds with LSD1 activity alone did not show any effect up to 72 h of treatment. However, compounds with LSD1-HDAC dual activity showed a strong effect in inhibiting cell proliferation in multiple cell lines, with IC50s ranging from 0.02 to 2 μM, with leukaemia cell lines being generally more sensitive than solid tumors. Conclusion: The data obtained demonstrate that it is feasible to design dual LSD1-HDAC inhibitors that retain individual activity and potently inhibit cell proliferation, and such inhibitors could serve as powerful therapeutic agents for cancer. Further understanding of mechanisms to identify the right tool compound is in progress. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopalan, Sreekala Nair, Purushottam Dewang, Durga Prasanna Kumar, Chandrika Mulakala, Lavanya Mahadevan, Neema Skariah, Venkata Giri Kavuru, Damodara Kuntrapaku, Suchitra Sajja, Mohammad Zainuddin, Krishnakumar V, Ritu Singh, Swarnakumari V, Ramachandraiah Gosu, Jayashree Aiyar, Pravin Iyer, Sriram Rajagopal. Novel dual inhibitors of LSD1-HDAC for treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3509. doi:10.1158/1538-7445.AM2015-3509
Ceramides are known to be involved in various biological processes with their physiological levels elevated in various disease conditions such as diabetes, Alzheimer's, atherosclerosis. To facilitate the rapid screening of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 inhibition in HepG2 cells, a RapidFire coupled to tandem mass spectrometry (RF-MS/MS) method has been developed. The RF platform provides an automated solid-phase extraction system that gave a throughput of 12.6 s per sample to an MS/MS system using electrospray ionization under the positive ion mode. Chromatographic separation of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 was achieved using a ternary gradient on C 8 type E cartridge. The MS/MS ion transitions monitored were 538.2 ! 264. 2, 650.7 ! 264.2, 648.6 ! 264.2, 566.4 ! 264.2, 510.4 ! 264.2, 594.4 ! 264.2, 622.5 ! 264.2, and 552.3 ! 250.2 for Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, d18:1/22:0, and the internal standard (Cer d17:1/18:0), respectively. The RF-MS/MS methodology showed an excellent performance with an average Z 0 value of 0.5-0.7. This is the first report of an RF-MS/MS assay for screening of ceramides which is amenable for high-throughput screening. K E Y W O R D Sceramides, quantification, RapidFire, RF-MS/MS method
The cover image is based on the Short Communication High throughput screening assay for quantification of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0 and d18:1/22:0 ceramides in HepG-2 cells using RapidFire Mass spectrometry Ramesh Mullangi et al., https://doi.org/10.1002/bmc.4790.***.
Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we identified theTrypanosomaPEX1 orthologue using sequence and structural similarities. Using yeast two-hybrid analysis, we demonstrate thatTbPEX1 can bind toTbPEX6. Endogenously taggedTbPEX1 localizes to glycosomes in theT. bruceiparasites. Depletion of PEX1 gene expression by RNA interference causes lethality to bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion.TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptorsTbPEX5 andTbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinatedTbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggests that these parasites possess a highly efficient quality control mechanisms that export the import receptors from glycosomes to the cytosol, in the absence of a functionalTbPEX1-TbPEX6 complex.
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