Identifying cell and molecular stress after radiation in a three-dimensional (3-D) model of oral mucositis

Mature adipocytes are excellent candidates to influence tumor behavior through heterotypic signaling processes since these cells produce hormones, growth factors, cytokines and other molecules, a heterogeneous group of molecules named adipokines. Using a 2D coculture system, we demonstrate that breast tumor cells previously co-cultivated with mature adipocytes exhibit radioresistance and an earlier and higher increase in the effector kinase Chk1, a phenotype that was associated with decreased cell death as compared to tumor cells grown alone. Interestingly, the adipocytes-induced tumor changes taking place during the coculture time preceding the exposure to IR were sufficient to confer the radioresistant effect. Notorious among the changes brought by adipocytes was the significant increase of IL-6 expression in tumor cells, whose activity may well account for the observed tumor cell protection from IR toxicity. Indeed, our data confirmed the protective role of this cytokine as tumor cells incubated after irradiation with recombinant IL-6 exhibit an increased in Chk1 phosphorylation and a radioresistant phenotype, thus far recapitulating the effects observed in the presence of adipocytes. Our current study sheds light on a new role of tumor-surrounding adipocytes in fostering a radioresistant phenotype in breast tumors, a finding that might have important clinical implications in obese patients that frequently exhibit aggressive diseases.     

Photodynamic effect of novel chlorin e6 derivatives on a single nerve cell

Chlorin e(6) and its derivatives are promising sensitizers for photodynamic therapy (PDT). In order to compare the photodynamic effects of 8 novel derivatives of chlorin e(6) and to explore some mechanisms of their effects at the cellular level, we studied PDT-induced changes in bioelectric activity of crayfish mechanoreceptor neuron that was used as a sensitive experimental model. Neurons were insensitive to red laser irradiation (632.8 nm; 0.3 W/cm(2)) or to photosensitizers alone, but changed firing rate and died under the photodynamic effect of nanomolar concentrations of sensitizers. The dynamics of neuron responses depended on photosensitizer type and concentration. The dependence of neuron lifetime on photosensitizer concentration allowed comparing efficiencies of different photosensitizers. Radachlorin was the most potent photosensitizer comparable with mTHPC. High photodynamic efficiency of some chlorin e(6) derivatives was related to weak dependence of neuron lifetime on sensitizer concentration, indicating to the initiation of 2-3 secondary processes such as free radical membrane damage by one absorbed photon. Photodynamic efficiency of sensitizers depended on amphiphilicity influencing their intracellular localization.     

Conservation of key members in the course of the evolution of the insulin signaling pathway

Our understanding of the evolution of the insulin signaling pathway (ISP) is still incomplete. One intriguing unanswered question is the explanation of the emergence of the glucostatic role of insulin in mammals. To find out whether this is due to the development of new sets of signaling transduction elements in these organisms, or to the establishment of new interactions between pre-existing proteins, we rebuilt putative orthologous ISPs in 17 eukaryotic organisms. Then, we computed the conservation of orthologous ISPs at different levels, from sequence similarity of orthologous proteins to co-evolution of interacting domains. We found that the emergence of glucostatic role in mammals can neither be explained by the development of new sets of signaling elements, nor by the establishment of new interactions between pre-existing proteins. The comparison of orthologous IRS molecules indicates that only in mammals have they acquired their complete functionality as efficient recruiters of effector sub-pathways.     

Long noncoding RNA LINC01234 promoted cell proliferation and invasion via miR-1284/TRAF6 axis in colorectal cancer

Colorectal cancer is one of the most common and leading malignancies globally. Long noncoding RNAs (lncRNAs) function as potentially critical regulator in colorectal cancer. LINC01234, a novel lncRNA in tumor biology, regulates the progression of various tumors. However, the tumorigenic mechanism of LINC01234 in colorectal cancer is still unclear. This study was performed with the aim to prospectively investigate clinical significance, effect, and mechanism of lncRNA LINC01234 in colorectal cancer. First, we found that LINC01234, localized in the cytoplasm, was increased in both colorectal cancer cell lines and tissues. Subsequent functional assays suggested LINC01234 knockdown suppressed cell proliferation, migration, and invasion of colorectal cancer cells, while blocked cell cycle and induced cell apoptosis. Moreover, we identified that miR-1284 was target of LINC01234, we further demonstrated a negative correlation with LINC01234 in colorectal cancer tissues and cells. Furthermore, miR-1284 targeted and suppressed tumor necrosis factor receptor–associated factor 6 (TRAF6). Loss-of-function assay revealed that LINC01234 silencing suppressed colorectal cancer progression through inhibition of miR-1284. In vivo subcutaneous xenotransplanted tumor model indicated LINC01234 knockdown inhibited in vivo tumorigenic ability of colorectal cancer via downregulation of TRAF6. Collectively, this study clarified the biological significance of LINC01234/miR-1284/TRAF6 axis in colorectal cancer progression, providing insights into LINC01234 as novel potential therapeutic target for colorectal cancer therapeutic from bench to clinic.     

Convenient and Efficient Syntheses of Oligodeoxyribonucleotides Containing O 6-(Carboxymethyl)Guanine and O 6-(4-Oxo-4-(3-Pyridyl)Butyl)Guanine

O ⁶-(carboxymethyl)guanine (O ⁶-CMG) and O ⁶-(4-oxo-4-(3-pyridyl)butyl)guanine (O ⁶-pobG) are toxic lesions formed in DNA following exposure to alkylating agents. O ⁶-CMG results from exposure to nitrosated glycine or nitrosated bile acid conjugates and may be associated with diets rich in red meat. O ⁶-pobG lesions are derived from alkylating agents found in tobacco smoke. Efficient syntheses of oligodeoxyribonucleotides (ODNs) containing O ⁶-CMG and O ⁶-pobG are described that involve nucleophilic displacement by the appropriate alcohol on a common synthetic ODN containing the reactive base 2-amino-6-methylsulfonylpurine. ODNs containing O ⁶-pobG and O ⁶-CMG were found to be good substrates for the S. pombe alkyltransferase-like protein Atl1.

[Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids to view the free supplemental file.]     

Role of PCIF1‐mediated 5′‐cap N6‐methyladeonsine mRNA methylation in colorectal cancer and anti‐PD‐1 immunotherapy

Adenosine N6‐methylation (m6A) and N6,2′‐O‐dimethylation (m6Am) are regulatory modifications of eukaryotic mRNAs. m6Am formation is catalyzed by the methyl transferase phosphorylated CTD‐interacting factor 1 (PCIF1); however, the pathophysiological functions of this RNA modification and PCIF1 in cancers are unclear. Here, we show that PCIF1 expression is upregulated in colorectal cancer (CRC) and negatively correlates with patient survival. CRISPR/Cas9‐mediated depletion of PCIF1 in human CRC cells leads to loss of cell migration, invasion, and colony formation in vitro and loss of tumor growth in athymic mice. Pcif1 knockout in murine CRC cells inhibits tumor growth in immunocompetent mice and enhances the effects of anti‐PD‐1 antibody treatment by decreasing intratumoral TGF‐β levels and increasing intratumoral IFN‐γ, TNF‐α levels, and tumor‐infiltrating natural killer cells. We further show that PCIF1 modulates CRC growth and response to anti‐PD‐1 in a context‐dependent mechanism with PCIF1 directly targeting FOS, IFITM3, and STAT1 via m6Am modifications. PCIF1 stabilizes FOS mRNA, which in turn leads to FOS‐dependent TGF‐β regulation and tumor growth. While during immunotherapy, Pcif1‐Fos‐TGF‐β, as well as Pcif1‐Stat1/Ifitm3‐IFN‐γ axes, contributes to the resistance of anti‐PD‐1 therapy. Collectively, our findings reveal a role of PCIF1 in promoting CRC tumorigenesis and resistance to anti‐PD‐1 therapy, supporting that the combination of PCIF1 inhibition with anti‐PD‐1 treatment is a potential therapeutic strategy to enhance CRC response to immunotherapy. Finally, we developed a lipid nanoparticles (LNPs) and chemically modified small interfering RNAs (CMsiRNAs)‐based strategy to silence PCIF1 in vivo and found that this treatment significantly reduced tumor growth in mice. Our results therefore provide a proof‐of‐concept for tumor growth suppression using LNP‐CMsiRNA to silence target genes in cancer.     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine     

Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.