Assessing radiation dose limits for X-ray fluorescence microscopy analysis of plant specimens

Background and Aims X-ray fluorescence microscopy (XFM) is a powerful technique to elucidate the distribution of elements within plants. However, accumulated radiation exposure during analysis can lead to structural damage and experimental artefacts including elemental redistribution. To date, acceptable dose limits have not been systematically established for hydrated plant specimens. Methods Here we systematically explore acceptable dose rate limits for investigating fresh sunflower (Helianthus annuus) leaf and root samples and investigate the time–dose damage in leaves attached to live plants. Key Results We find that dose limits in fresh roots and leaves are comparatively low (4.1 kGy), based on localized disintegration of structures and element-specific redistribution. In contrast, frozen-hydrated samples did not incur any apparent damage even at doses as high as 587 kGy. Furthermore, we find that for living plants subjected to XFM measurement in vivo and grown for a further 9 d before being reimaged with XFM, the leaves display elemental redistribution at doses as low as 0.9 kGy and they continue to develop bleaching and necrosis in the days after exposure. Conclusions The suggested radiation dose limits for studies using XFM to examine plants are important for the increasing number of plant scientists undertaking multidimensional measurements such as tomography and repeated imaging using XFM.

Synergy of Chemo- and Photodynamic Therapies with C60 Fullerene-Doxorubicin Nanocomplex

A nanosized drug complex was explored to improve the efficiency of cancer chemotherapy, complementing it with nanodelivery and photodynamic therapy. For this, nanomolar amounts of a non-covalent nanocomplex of Doxorubicin (Dox) with carbon nanoparticle C60 fullerene (C60) were applied in 1:1 and 2:1 molar ratio, exploiting C60 both as a drug-carrier and as a photosensitizer. The fluorescence microscopy analysis of human leukemic CCRF-CEM cells, in vitro cancer model, treated with nanocomplexes showed Dox’s nuclear and C60’s extranuclear localization. It gave an opportunity to realize a double hit strategy against cancer cells based on Dox’s antiproliferative activity and C60’s photoinduced pro-oxidant activity. When cells were treated with 2:1 C60-Dox and irradiated at 405 nm the high cytotoxicity of photo-irradiated C60-Dox enabled a nanomolar concentration of Dox and C60 to efficiently kill cancer cells in vitro. The high pro-oxidant and pro-apoptotic efficiency decreased IC50 16, 9 and 7 × 103-fold, if compared with the action of Dox, non-irradiated nanocomplex, and C60’s photodynamic effect, correspondingly. Hereafter, a strong synergy of therapy arising from the combination of C60-mediated Dox delivery and C60 photoexcitation was revealed. Our data indicate that a combination of chemo- and photodynamic therapies with C60-Dox nanoformulation provides a promising synergetic approach for cancer treatment.

Design and In Vitro Biological Evaluation of a Novel Organotin(IV) Complex with 1-(4-Carboxyphenyl)-3-ethyl-3-methylpyrrolidine-2,5-dione

A novel triphenyltin(IV) compound with 1-(4-carboxyphenyl)-3-ethyl-3-methylpyrrolidine-2,5-dione was synthesized and characterized by IR, NMR spectroscopy, mass spectrometry, and elemental analysis. In vitro anticancer activity of ligand precursor and synthesized organotin(IV) compound was determined against tumor cell lines: human adenocarcinoma (HeLa), human myelogenous leukemia (K562), and human breast cancer (MDA-MB-453), using microculture tetrazolium test (MTT) assay. The results indicate that complex exhibited very high antiproliferative activity against all tested cell lines with IC50 values in the range of 0.22 to 0.53 µM. The highest activity organotin(IV) compound expressed against the HeLa cells (IC50 = 0.22 ± 0.04 µM). The ligand precursor did not show anticancer activity (IC50 > 200 µM). Furthermore, fluorescence microscopy analysis of HeLa cells reveal that organotin(IV) complex induced apoptosis as a mode of cell death, which is consistent with the increase of cells in the sub-G1 phase.

Bacteriocin Protein BacL1of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala2-Cross-Bridged Peptidoglycan

Bacteriocin 41 (Bac41) is produced from clinical isolates ofEnterococcus faecalisand consists of two extracellular proteins, BacL1and BacA. We previously reported that BacL1protein (595 amino acids, 64.5 kDa) is a bacteriolytic peptidoglycand-isoglutamyl-l-lysine endopeptidase that induces cell lysis ofE. faecaliswhen an accessory factor, BacA, is copresent. However, the target of BacL1remains unknown. In this study, we investigated the targeting specificity of BacL1. Fluorescence microscopy analysis using fluorescent dye-conjugated recombinant protein demonstrated that BacL1specifically localized at the cell division-associated site, including the equatorial ring, division septum, and nascent cell wall, on the cell surface of targetE. faecaliscells. This specific targeting was dependent on the triple repeat of the SH3 domain located in the region from amino acid 329 to 590 of BacL1. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics rescued bacteria from the bacteriolytic activity of BacL1and BacA. The static growth state also abolished the binding and targeting of BacL1to the cell division-associated site. Furthermore, the targeting of BacL1was detectable among Gram-positive bacteria with anl-Ala-l-Ala-cross-bridging peptidoglycan, includingE. faecalis,Streptococcus pyogenes, orStreptococcus pneumoniae, but not among bacteria with alternate peptidoglycan structures, such asEnterococcus faecium,Enterococcus hirae,Staphylococcus aureus, orListeria monocytogenes. These data suggest that BacL1specifically targets thel-Ala-l-Ala-cross-bridged peptidoglycan and potentially lyses theE. faecaliscells during cell division.