Uptake,Transport and Storage of Cardenolides in Foxglove. Cardenolide Sinks and Occurrence of Cardenolides in the Sieve Tubes of Digitalis lanata1

Cardiac glycoside transport was investigated on the organ and whole plant level. Uptake experiments were carried out with shoot and root cultures of Digitalis lanata. In both systems primary cardenolides, i.e., those with a terminal glucose in their oligosaccharide side chain, were taken up against their concentration gradient, whereas the glucose-free secondary cardenolides were not. Active uptake of primary cardenolides was further evidenced by KCN inhibition of uptake. Using plantlets grown in vitro the long-distance transport of primary cardenolides from the leaves to the roots was demonstrated. Cardenolides were also detected in etiolated leaves, induced on plants with green leaves, which are supposed to be unable to synthezise cardenolides de novo, providing further evidence for long-distance transport. Several primary cardenolides were detected in the honeydew excreted by aphids fed on Digitalis lanata leaves, indicating that the phloem is a transporting tissue for cardenolides. On the other hand, the xylem sap obtained by applying the pressure-chamber technique was cardenolide-free. It was concluded that in Digitalis primary cardenolides serve as both the transport and the storage form of cardenolides. After their synthesis they are either stored in the vacuoles of the source tissue or loaded into the sieve tubes, from which they are unloaded at other sites where they are trapped in the vacuoles of the respective sink tissue.     

Natural flavonoids for the prevention of colon cancer: A comprehensive review of preclinical and clinical studies

Flavonoids comprise a group of natural polyphenols consisting of more than 5,000 subtypes mostly existing in fruits and vegetables. Flavonoids consumption could potentially attenuate the incidence and recurrence risk of colorectal cancers through their antiperoxidative, antioxidant, and anti-inflammatory effects. In addition, these compounds regulate the mitochondrial function, balance the bacterial flora and promote the apoptosis process in cancerous cells. However, some previous data failed to show the effectiveness of flavonoids in reducing the risk of colorectal cancer. In this study, we have reviewed the efficacy of different flavonoids subtypes on the risk of colon cancer and molecular mechanisms involved in this process in both clinical and animal studies. In addition, we tried to elucidate the potential synergy between these compounds and current colorectal cancer treatments.     

Range optimization for target and organs at risk in dynamic adaptive passive scattering proton beam therapy – A proof of concept

PurposeThe purpose of this study was to design and develop a new range optimization for target and organs at risk (OARs) in dynamic adaptive proton beam therapy (PBT).MethodsThe new range optimization for target and OARs (RO-TO) was optimized to maintain target dose coverage but not to increase the dose exposure of OARs, while the other procedure, range optimization for target (RO-T), only focused on target dose coverage. A retrospective analysis of a patient who received PBT for abdominal lymph node metastases was performed to show the effectiveness of our new approach. The original plan (OP), which had a total dose of 60 Gy (relative biological effectiveness; RBE), was generated using six treatment fields. Bone-based registration (BR) and tumor-based registration (TR) were performed on each pretreatment daily CT image dataset acquired once every four fractions, to align the isocenter.ResultsBoth range adaptive approaches achieved better coverage (D_95%) and homogeneity (D_5%−D_95%) than BR and TR only. However, RO-T showed the greatest increases in D_2cc and D_mean values of the small intestine and stomach and exceeded the limitations of dose exposure for those OARs. RO-TO showed comparable or superior dose sparing compared with the OP for all OARs.ConclusionsOur results suggest that BR and TR alone may reduce target dose coverage, and that RO-T may increase the dose exposure to the OARs. RO-TO may achieve the planned dose delivery to the target and OARs more efficiently than the OP. The technique requires testing on a large clinical dataset.     

Design,synthesis and biological evaluation of 4-(pyridin-4-yloxy)benzamide derivatives bearing a 5-methylpyridazin-3(2H)-one fragment

A series of 4-(pyridin-4-yloxy)benzamide derivatives bearing a 5-methylpyridazin-3(2H)-one fragment were designed, synthesized, and evaluated for their biological activity. Most compounds showed effective inhibitory activity against cancer cell lines of A549, HeLa and MCF-7. Among them, the most promising compound 40 showed excellent activity against A549, HeLa and MCF-7 cell lines with IC₅₀ values of 1.03, 1.15 and 2.59 μM, respectively, which was 2.606.95 times more active than that of Golvatinib. The structure-activity relationships (SARs) showed that the introduction of 5-methylpyridazin-3(2H)-one to “5-atom linker” and the modification of the amide with morpholine group were beneficial for enhancing the inhibitory activity of compounds. In addition, the further research on compound 40 mainly include c-Met kinase activity, concentration dependence, apoptosis (acridine orange staining), and molecular docking.     

Phosphorylation of Cdc42 Effector Protein-4 (CEP4) by Protein Kinase C Promotes Motility of Human Breast Cells

Cdc42 effector protein-4 (CEP4) was recently identified by our laboratory to be a substrate of multiple PKC isoforms in non-transformed MCF-10A human breast cells. The significance of phosphorylated CEP4 to PKC-stimulated motility of MCF-10A cells was evaluated. Single site mutants at Ser residues embedded in potential PKC consensus sites (Ser¹⁸, Ser⁷⁷, Ser⁸⁰, and Ser⁸⁶) were individually replaced with Asp residues to simulate phosphorylation. Following expression in weakly motile MCF-10A cells, the S18D and S80D mutants each promoted increased motility, and the double mutant (S18D/S80D) produced a stronger effect. MS/MS analysis verified that Ser¹⁸ and Ser⁸⁰ were directly phosphorylated by PKCα in vitro. Phosphorylation of CEP4 severely diminished its affinity for Cdc42 while promoting Rac activation and formation of filopodia (microspikes). In contrast, the phosphorylation-resistant double mutant S18A/S80A-CEP4 blocked CEP4 phosphorylation and inhibited motility of MCF-10A cells that had been stimulated with PKC activator diacylglycerol lactone. In view of the dissociation of phospho-CEP4 from Cdc42, intracellular binding partners were explored by expressing each CEP4 double mutant from a tandem affinity purification vector followed by affinity chromatography, SDS-PAGE, and identification of protein bands evident only with S18D/S80D-CEP4. One binding partner was identified as tumor endothelial marker-4 (TEM4; ARHGEF17), a guanine nucleotide exchange factor that is involved in migration. In motile cells expressing S18D/S80D-CEP4, knockdown of TEM4 inhibited both Rac activation and motility. These findings support a model in which PKC-mediated phosphorylation of CEP4 at Ser¹⁸ and Ser⁸⁰ causes its dissociation from Cdc42, thereby increasing its affinity for TEM4 and producing Rac activation, filopodium formation, and cell motility.     

Single‐molecule force spectroscopy applied to heparin‐induced thrombocytopenia

Heparin‐induced thrombocytopenia (HIT), occurring up to approximately 1% to 5% of patients receiving the antithrombotic drug heparins, has a complex pathogenesis involving multiple partners ranging from small molecules to cells/platelets. Recently, insights into the mechanism of HIT have been achieved by using single‐molecule force spectroscopy (SMFS), a methodology that allows direct measurements of interactions among HIT partners. Here, the potential of SMFS in unraveling the mechanism of the initial steps in the pathogenesis of HIT at single‐molecule resolution is highlighted. The new findings ranging from the molecular binding strengths and kinetics to the determination of the boundary between risk and non‐risk heparin drugs or platelet‐surface and platelet‐platelet interactions will be reviewed. These novel results together have contributed to elucidate the mechanisms underlying HIT and demonstrate how SMFS can be applied to develop safer drugs with a reduced risk profile.     

A sublethal effect on native Anthocoris nemoralis through competitive interactions with invasive Harmonia axyridis

1. The invasive ladybird Harmonia axyridis Pallas (Coleoptera: Coccinellidae) is implicated in declines of native ladybirds with intraguild predation identified as an underlying mechanism, however, less studied are the effects on non‐coccinellid predators. Intraguild predation between Anthocoris nemoralis Fabricius (Heteroptera: Anthocoridae) and H. axyridis and the relative effects of inter‐ and intraspecific competition on predators on potted Tilia cordata Mill. was investigated, at two aphid densities. 2. Intraguild predation was unidirectional in favour of H. axyridis in Petri dishes although a low level of ladybird egg predation by A. nemoralis occurred. However, on trees intraguild predation was rare. 3. A positive effect of aphid density on H. axyridis weight gain, and increased survival when reared with A. nemoralis on potted trees indicated stronger effects of intraspecific competition on H. axyridis. Consistent suppression of aphids by ladybirds revealed their superior competition for aphids. 4. Anthocoris nemoralis weight gain was reduced when reared with H. axyridis on potted trees, indicating a potential for a sublethal effect through interspecific competition. Survivorship of A. nemoralis did not differ between con‐ and heterospecific treatments, but high aphid density enhanced A. nemoralis survival. Overall, high mortality among nymphs across treatments suggested effects related to both inter‐ and intraspecific competition for resources. 5. Results provide evidence of a sublethal effect by invasive H. axyridis through resource competition, suggesting that where these predators co‐occur, competitive interactions for shared prey are more likely to affect A. nemoralis populations than intraguild predation by H. axyridis.     

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak^−/− MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak^−/− MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak^−/− MEF cells (Ahnak^−/−-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak^−/−-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak^−/− MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.