Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors

Plant growth and development are particularly sensitive to changes in the light environment and especially to vegetational shading. The shade-avoidance response is mainly controlled by the phytochrome photoreceptors. In Arabidopsis, recent studies have identified several related bHLH class transcription factors (PIF, for phytochrome-interacting factors) as important components in phytochrome signaling. In addition to a related bHLH domain, most of the PIFs contain an active phytochrome binding (APB) domain that mediates their interaction with light-activated phytochrome B (phyB). Here we show that PIF4 and PIF5 act early in the phytochrome signaling pathways to promote the shade-avoidance response. PIF4 and PIF5 accumulate to high levels in the dark, are selectively degraded in response to red light, and remain at high levels under shade-mimicking conditions. Degradation of these transcription factors is preceded by phosphorylation, requires the APB domain and is sensitive to inhibitors of the proteasome, suggesting that PIF4 and PIF5 are degraded upon interaction with light-activated phyB. Our data suggest that, in dense vegetation, which is rich in far-red light, shade avoidance is triggered, at least partially, as a consequence of reduced phytochrome-mediated degradation of transcription factors such as PIF4 and PIF5. Consistent with this idea, the constitutive shade-avoidance phenotype of phyB mutants partially reverts in the absence of PIF4 and PIF5

Angiofil-mediated visualization of the vascular system by microcomputed tomography: a feasibility study.

Visualization of the vascular systems of organs or of small animals is important for an assessment of basic physiological conditions, especially in studies that involve genetically manipulated mice. For a detailed morphological analysis of the vascular tree, it is necessary to demonstrate the system in its entirety. In this study, we present a new lipophilic contrast agent, Angiofil, for performing postmortem microangiography by using microcomputed tomography. The new contrast agent was tested in 10 wild-type mice. Imaging of the vascular system revealed vessels down to the caliber of capillaries, and the digital three-dimensional data obtained from the scans allowed for virtual cutting, amplification, and scaling without destroying the sample. By use of computer software, parameters such as vessel length and caliber could be quantified and remapped by color coding onto the surface of the vascular system. The liquid Angiofil is easy to handle and highly radio-opaque. Because of its lipophilic abilities, it is retained intravascularly, hence it facilitates virtual vessel segmentation, and yields an enduring signal which is advantageous during repetitive investigations, or if samples need to be transported from the site of preparation to the place of actual analysis, respectively. These characteristics make Angiofil a promising novel contrast agent; when combined with microcomputed tomography, it has the potential to turn into a powerful method for rapid vascular phenotyping.

Pacemaker therapy in patients with neurally mediated syncope and documented asystole: Third International Study on Syncope of Uncertain Etiology (ISSUE-3): a randomized trial.

BACKGROUND: The efficacy of cardiac pacing for prevention of syncopal recurrences in patients with neurally mediated syncope is controversial. We wanted to determine whether pacing therapy reduces syncopal recurrences in patients with severe asystolic neurally mediated syncope. METHODS AND RESULTS: Double-blind, randomized placebo-controlled study conducted in 29 centers in the Third International Study on Syncope of Uncertain Etiology (ISSUE-3) trial. Patients were ≥40 years, had experienced ≥3 syncopal episodes in the previous 2 years. Initially, 511 patients, received an implantable loop recorder; 89 of these had documentation of syncope with ≥3 s asystole or ≥6 s asystole without syncope within 12 ± 10 months and met criteria for pacemaker implantation; 77 of 89 patients were randomly assigned to dual-chamber pacing with rate drop response or to sensing only. The data were analyzed on intention-to-treat principle. There was syncope recurrence during follow-up in 27 patients, 19 of whom had been assigned to pacemaker OFF and 8 to pacemaker ON. The 2-year estimated syncope recurrence rate was 57% (95% CI, 40-74) with pacemaker OFF and 25% (95% CI, 13-45) with pacemaker ON (log rank: P=0.039 at the threshold of statistical significance of 0.04). The risk of recurrence was reduced by 57% (95% CI, 4-81). Five patients had procedural complications: lead dislodgment in 4 requiring correction and subclavian vein thrombosis in 1 patient. CONCLUSIONS: Dual-chamber permanent pacing is effective in reducing recurrence of syncope in patients ≥40 years with severe asystolic neurally mediated syncope. The observed 32% absolute and 57% relative reduction in syncope recurrence support this invasive treatment for the relatively benign neurally mediated syncope. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00359203.

Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.

Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange

The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 muM of acetosyringone. Genetic transformation was confirmed by performing beta-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUS genes.

IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8(+) T cells.

CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.

Rapamycin-mediated FOXO1 inactivation reduces the anticancer efficacy of rapamycin.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors such as rapamycin have shown modest effects in cancer therapy due in part to the removal of a negative feedback loop leading to the activation of the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling pathway. In this report, we have investigated the role of FOXO1, a downstream substrate of the PI3K/Akt pathway in the anticancer efficacy of rapamycin. MATERIALS AND METHODS: Colon cancer cells were treated with rapamycin and FOXO1 phosphorylation was determined by Western blot. Colon cancer cells transfected with a constitutively active mutant of FOXO1 or a control plasmid were treated with rapamycin and the antiproliferative efficacy of rapamycin was monitored. RESULTS: Rapamycin induced the phosphorylation of FOXO1 as well as its translocation from the nucleus to the cytoplasm, leading to FOXO1 inactivation. The expression of an active mutant of FOXO1 in colon cancer cells potentiated the antiproliferative efficacy of rapamycin in vitro and its antitumor efficacy in vivo. CONCLUSION: Taken together these results show that rapamycin-induced FOXO1 inactivation reduces the antitumor efficacy of rapamycin.

Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion.

Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin αvβ5. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvβ5 integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-αvβ5 integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion.

The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes

Nucleotides in the terminal loop of the poliovirus 2C cis-acting replication element (2C(CRE)), a 61 nt structured RNA, function as the template for the addition of two uridylate (U) residues to the viral protein VPg. This uridylylation reaction leads to the formation of VPgpUpU, which is used by the viral RNA polymerase as a nucleotide-peptide primer for genome replication. Although VPg primes both positive- and negative-strand replication, the specific requirement for 2C(CRE)-mediated uridylylation for one or both events has not been demonstrated. We have used a cell-free in vitro translation and replication reaction to demonstrate that 2C(CRE) is not required for the initiation of the negative-sense strand, which is synthesized in the absence of 2C(CRE)-mediated VPgpUpU formation. We propose that the 3' poly(A) tail could serve as the template for the formation of a VPg-poly(U) primer that functions in the initiation of negative-sense strands.

pH-mediated interactions between poly(acrylic acid) and methylcellulose in the formation of ultrathin multilayered hydrogels and spherical nanoparticles

Poly(acrylic acid) forms insoluble hydrogen-bonded interpolymer complexes with methylcellulose in aqueous solutions under acidic conditions. In this work the reaction heats and binding constants were determined for the complexation between poly(acrylic acid) and methylcellulose by isothermal titration calorimetry at different pH and findings are correlated with the aggregation processes occurring in this system. The principal contribution to the complexation heat results from primary polycomplex particle aggregation. Transmission electron microscopy of nanoparticles produced at pH 1.4 and 2.4 demonstrated that they are spherical and dense structures. The nanoparticles ranged from 80 to 200 nm, whereas particles formed at pH 3.2 were 20-30 nm and were stabilized against aggregation by a network of uncomplexed macromolecules. For the first time, multilayered materials were developed on the basis of hydrogen-bonded complexes of poly(acrylic acid) and methylcellulose using layer-by-layer deposition on a glass surface. The thickness of these films was a linear function of the number of deposition cycles. The materials were subsequently cross-linked by thermal treatment, resulting in ultrathin hydrogels which detached from the glass substrate upon swelling. The swelling capacity of ultrathin hydrogels differed from the swelling of the thicker films of a similar chemical composition.

Ligand dissociation from estrogen receptor is mediated by receptor dimerization: Evidence from molecular dynamics simulations

Estrogen Receptor (ER) is an important target for pharmaceutical design. Like other ligand-dependent transcription factors, hormone binding regulates ER transcriptional activity. Nevertheless, the mechanisms by which ligands enter and leave ERs and other nuclear receptors remain poorly understood. Here, we report results of locally enhanced sampling molecular dynamics simulations to identify dissociation pathways of two ER ligands [the natural hormone 17 beta-estradiol (E-2) and the selective ER modulator raloxifene (RAL)] from the human ER alpha ligand-binding domain in monomeric and dimeric forms. E-2 dissociation occurs via three different pathways in ER monomers. One resembles the mousetrap mechanism (Path I), involving repositioning of helix 12 (H12), others involve the separation of H8 and H11 (Path II), and a variant of this pathway at the bottom of the ligand-binding domain (Path II`). RAL leaves the receptor through Path I and a Path I variant in which the ligand leaves the receptor through the loop region between H11 and H12 (Path I`). Remarkably, ER dimerization strongly suppresses Paths II and II` for E-2 dissociation and modifies RAL escape routes. We propose that differences in ligand release pathways detected in the simulations for ER monomers and dimers provide an explanation for previously observed effects of ER quaternary state on ligand dissociation rates and suggest that dimerization may play an important, and hitherto unexpected, role in regulation of ligand dissociation rates throughout the nuclear receptor family.

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. (C) 2011 Elsevier Inc. All rights reserved.

Estimating genomic instability mediated by Alu retroelements in breast cancer

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

ZmPUMP encodes a fully functional monocot plant uncoupling mitochondrial protein whose affinity to fatty acid is increased with the introduction of a His pair at the second matrix loop

Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins that uncouple respiration from ATP synthesis. In this study, cDNA encoding maize uncoupling protein (ZmPUMP) was expressed in Escherichia coli and recombinant ZmPUMP reconstituted in liposomes. ZmPUMP activity was associated with a linoleic acid (LA)-mediated H+ efflux with Km of 56.36 ± 0.27 μM and Vmax of 66.9 μmol H+ min-1 (mg prot)-1. LA-mediated H+ fluxes were sensitive to ATP inhibition with Ki of 2.61 ± 0.36 mM (at pH 7.2), a value similar to those for dicot UCPs. ZmPUMP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in plant UCPs. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism. © 2006 Elsevier Inc. All rights reserved.

Development and validation of a D-loop mtDNA SNP assay for the screening of specimens in forensic casework

Mitochondrial DNA (mtDNA) analysis is usually a last resort in routine forensic DNA casework. However, it has become a powerful tool for the analysis of highly degraded samples or samples containing too little or no nuclear DNA, such as old bones and hair shafts. The gold standard methodology still constitutes the direct sequencing of polymerase chain reaction (PCR) products or cloned amplicons from the HVS-1 and HVS-2 (hypervariable segment) control region segments. Identifications using mtDNA are time consuming, expensive and can be very complex, depending on the amount and nature of the material being tested. The main goal of this work is to develop a less labour-intensive and less expensive screening method for mtDNA analysis, in order to aid in the exclusion of non-matching samples and as a presumptive test prior to final confirmatory DNA sequencing. We have selected 14 highly discriminatory single nucleotide polymorphisms (SNPs) based on simulations performed by Salas and Amigo (2010) [1] to be typed using SNaPShotTM (Applied Biosystems, Foster City, CA, USA). The assay was validated by typing more than 100 HVS-1/HVS-2 sequenced samples. No differences were observed between the SNP typing and DNA sequencing when results were compared, with the exception of allelic dropouts observed in a few haplotypes. Haplotype diversity simulations were performed using 172 mtDNA sequences representative of the Brazilian population and a score of 0.9794 was obtained when the 14 SNPs were used, showing that the theoretical prediction approach for the selection of highly discriminatory SNPs suggested by Salas and Amigo (2010) [1] was confirmed in the population studied. As the main goal of the work is to develop a screening assay to skip the sequencing of all samples in a particular case, a pair-wise comparison of the sequences was done using the selected SNPs. When both HVS-1/HVS-2 SNPs were used for simulations, at least two differences were observed in 93.2% of the comparisons performed. The assay was validated with casework samples. Results show that the method is straightforward and can be used for exclusionary purposes, saving time and laboratory resources. The assay confirms the theoretic prediction suggested by Salas and Amigo (2010) [1]. All forensic advantages, such as high sensitivity and power of discrimination, as also the disadvantages, such as the occurrence of allele dropouts, are discussed throughout the article. © 2013 Elsevier B.V.