Cancer targeting gene-viro-therapy specific for liver cancer by α-fetoprotein-controlled oncolytic adenovirus expression of SOCS3 and IL-24

The combination of gene therapy and virotherapy for cancer treatment has received close attention and has become a trend in the field of cancer biotherapy. A strategy called 'Cancer Targeting Gene-Viro-Therapy' (CTGVT) or 'Gene Armed Oncolytic Viral Therapy' (GAOVT) has been proposed, in which an antitumor gene is inserted into an oncolytic viral vector. In our previous study, a dual-regulated oncolytic adenovirus with enhanced safety for normal cells and strict liver cancer-targeting ability, designated Ad?enAFP?E1A?E1B (Δ55) (briefly Ad?enAFP?D55), was successfully constructed. In the current work, interleukin-24 (IL-24) and suppressor of cytokine signaling 3 (SOCS3) genes were packaged into Ad?enAFP?D55. The new constructs, Ad?enAFP?D55-(IL-24) and Ad?enAFP?D55-(SOCS3), showed improved tumoricidal activity in hepatoma cell lines compared with the oncolytic viral vector Ad?enAFP?D55. The co-administration of Ad?enAFP?D55-(IL-24) and Ad?enAFP?D55-(SOCS3) showed much better antitumor effect than Ad?enAFP?D55-(IL-24) or Ad?enAFP?D55-(SOCS3) alone both in vitro and in a nude mouse xenograft model. Moreover, our results also showed that blockade of the Jak/Stat3 pathway by Ad?enAFP?D55-(SOCS3) infection in HuH-7 cells could down-regulate some anti-apoptosis proteins, such as XIAP, Bcl-xL, and survivin, which might sensitize the cells to Ad?enAFP?D55-(IL-24)-induced apoptosis. These results indicate that co-administration of Ad?enAFP?D55-(IL-24) and Ad?enAFP?D55-(SOCS3) may serve as a candidate therapeutic approach for the treatment of liver cancer.     

Buchnera aphidicola,the endosymbiont of aphids,contains genes for four ribosomal RNA proteins,initiation factor-3, and the α-subunit of RNA polymerase

Buchnera aphidicola is a prokaryotic endosymbiont of the aphidSchizaphis graminum. With the polymerase chain reaction (PCR) and oligonucleotide primers to conserved regions, two DNA fragments of the endosymbiont -operon and L20 operon were amplified, cloned intoEscherichia coli, and their sequences were determined. The results indicated that the organization of the endosymbiont genes on these fragments was identical with that of the corresponding operons ofE. coli. The 1032 base pair (bp) fragment of the -operon contained the genes for small ribosomal subunit proteins S11 and S4, followed by the gene for the -subunit of RNA polymerase (-RNAP). The 702-bp fragment of the L20-operon contained the genes for initiation factor-3 (IF3) and large ribosomal subunit proteins L35 and L20. As in other prokaryotes, the genes of the -operon and the L20-operon were present as single copies in the genome ofB. aphidicola. Comparisons of the amino acid sequences of these proteins were consistent with the previously established close relationship betweenB. aphidicola andE. coli and a distant relationship to species ofBacillus.     

miR-29b ameliorates atrial fibrosis in rats with atrial fibrillation by targeting TGFβRΙ and inhibiting the activation of Smad-2/3 pathway

Objective

Atrial fibrillation (AF) is a major cause of stroke with lifetime risks. microRNAs (miRNAs) are associated with AF attenuation, yet the mechanism remains unknown. This study investigated the functional mechanism of miR-29b in atrial fibrosis in AF.

Methods

The AF rat model was established by a 7-day intravenous injection of Ach-CaCl₂ mixture. AF rats were injected with adeno-associated virus (AAv)-miR-29b and TGFβRΙ overexpression plasmid. AF duration was recorded by electrocardiogram. Atrial fibrosis was observed by Masson staining. Expressions of COL1A1, COL3A1, TGFβRΙ, TGFβΙ, miR-29b and Smad-2/3 pathway-related proteins in atrial tissues were detected by RT-qPCR and Western blot. Binding sites of miR-29b and TGFβRΙ were predicted and their target relationship was verified by dual-luciferase reporter assay.

Results

miR-29b was poorly expressed and expressions of COL1A1, COL3A1, TGFβRΙ, and TGFβ1 were increased in atrial tissues of AF rats. miR-29b overexpression alleviated atrial fibrosis, reduced expressions of COL1A1, COL3A1, and TGFβ1, and shortened AF duration in AF rats. TGFβRΙ was highly expressed in atrial tissues of AF rats. miR-29b targeted TGFβRΙ. TGFβRΙ overexpression overcame the improving effect of miR-29b overexpression on AF. miR-29b overexpression decreased ratios of p-Smad-2/3 and Smad-2/3 and inhibited the Smad-2/3 pathway.

Conclusion

miR-29b might mitigate atrial fibrosis in AF rats by targeting TGFβRΙ and inhibiting the Smad-2/3 pathway.

     

Simple and tunable Förster resonance energy transfer-based bioprobes for high-throughput monitoring of caspase-3 activation in living cells by using flow cytometry

Sensing systems based on F?rster resonance energy transfer (FRET) can be used to monitor enzymatic reactions, protein-protein interactions, changes in conformation, and Ca2+ oscillations in studies on cellular dynamics. We developed a series of FRET-based chimeric bioprobes, each consisting of fluorescent protein attached to a fluorescent dye. Green and red fluorescent proteins were used as donors and a series of Alexa Fluor dyes was used as acceptors. The basic fluorescent proteins were substituted with appropriate amino acids for recognition of the target (caspase-3) and subjected to site-directed modification with a fluorescent dye. Variants that retained similar emission profiles to the parent proteins were readily derived for use as FRET-based bioprobes with various fluorescent patterns by incorporating various fluorescent proteins and dyes, the nature of which could be adjusted to experimental requirements. All the constructs prepared functioned as bioprobes for quantitative measurement of caspase-3 activity in vitro. Introduction of the bioprobes into cells was so simple and efficient that activation of caspase-3 upon apoptosis could be monitored by means of cytometric analysis. FRET-based bioprobes are valuable tool for high-throughput flow-cytometric analysis of many cellular events when used in conjunction with other fluorescent labels or markers. Statistical dynamic studies on living cells could provide indications of paracrine signaling.     

CSL311, a novel,potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common β chain of the IL-3, GM-CSF and IL-5 receptors

The β common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared β common (β_c, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human β_c receptor. The binding epitope of CSL311 on the β_c receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human β_c receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 β common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human β_c receptor is central to pathogenesis. The coordinates for the β_c/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).     

Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway

T cell Ig mucin domain-containing molecule 3 (Tim-3) is a glycoprotein found on the surface of a subset of CD8(+) and Th1 CD4(+) T cells. Elevated expression of Tim-3 on virus-specific T cells during chronic viral infections, such as HIV-1, hepatitis B virus, and hepatitis C virus, positively correlates with viral load. Tim-3(+) cytotoxic T cells are dysfunctional and are unable to secrete effector cytokines, such as IFN-γ and TNF-α. In this study, we examined potential inducers of Tim-3 on primary human T cells. Direct HIV-1 infection of CD4(+) T cells, or LPS, found to be elevated in HIV-1 infection, did not induce Tim-3 on T cells. Tim-3 was induced by the common γ-chain (γc) cytokines IL-2, IL-7, IL-15, and IL-21 but not IL-4, in an Ag-independent manner and was upregulated on primary T cells in response to TCR/CD28 costimulation, as well as γc cytokine stimulation with successive divisions. γc cytokine-induced Tim-3 was found on naive, effector, and memory subsets of T cells. Tim-3(+) primary T cells were more prone to apoptosis, particularly upon treatment with galectin-9, a Tim-3 ligand, after cytokine withdrawal. The upregulation of Tim-3 could be blocked by the addition of a PI3K inhibitor, LY 294002. Thus, Tim-3 can be induced via TCR/CD28 costimulation and/or γc cytokines, likely through the PI3K pathway.     

Downregulation of wild-type p53 protein by HER-2／neu mediated PI3K pathway activation in human breast cancer cells： its effect on cell proliferation and implication for therapy

Overexpression and activation of HER-2/neu (also known as c-erbB-2), a proto-oncogene, was found in about 30% of human breast cancers, promoting cancer growth and making cancer cells resistant to chemo- and radio-therapy.Wild-type p53 is crucial in regulating cell growth and apoptosis and is found to be mutated or deleted in 60-70% of human cancers. And some cancers with a wild-type p53 do not have normal p53 function, suggesting that it is implicated in a complex process regulated by many factors. In the present study, we showed that the overexpression of HER-2/neu could decrease the amount of wild-type p53 protein via activating PI3K pathway, as well as inducing MDM2 nuclear translocation in MCF7 human breast cancer cells. Blockage of PI3K pathway with its specific inhibitor LY294002 caused G1-S phase arrest, decreased cell growth rate and increased chemo- and radio-therapeutic sensitivity in MCF7 cells expressing wild-type p53. However, it did not increase the sensitivity to adriamycin in MDA-MB-453 breast cancer cells containing mutant p53. Our study indicates that blocking PI3K pathway activation mediated by HER-2/neu overexpression may be useful in the treatment of breast tumors with HER-2/neu overexpression and wild-type p53.     

Synthesis and evaluation of 6-(11C-methyl(4-(pyridin-2-yl)thiazol-2-yl)amino)benzo[d]thiazol-2(3H)-one for imaging γ-8 dependent transmembrane AMPA receptor regulatory protein by PET

Transmembrane AMPA receptor regulatory proteins (TARPs) are a recently discovered family of proteins that modulate AMPA receptors activity. Based on a potent and selective TARP subtype γ-8 antagonist, 6-(methyl(4-(pyridin-2-yl)thiazol-2-yl)amino)benzo[d]thiazol-2(3H)-one (compound 9), we perform the radiosynthesis of its ¹¹C-isotopologue 1 and conduct preliminary PET evaluation to test the feasibility of imaging TARP γ-8 dependent receptors in vivo.