Circadian clock adjustment to plant iron status depends on chloroplast and phytochrome function

Plant chloroplasts are not only the main cellular location for storage of elemental iron (Fe), but also the main site for Fe, which is incorporated into chlorophyll, haem and the photosynthetic machinery. How plants measure internal Fe levels is unknown. We describe here a new Fe‐dependent response, a change in the period of the circadian clock. In Arabidopsis, the period lengthens when Fe becomes limiting, and gradually shortens as external Fe levels increase. Etiolated seedlings or light‐grown plants treated with plastid translation inhibitors do not respond to changes in Fe supply, pointing to developed chloroplasts as central hubs for circadian Fe sensing. Phytochrome‐deficient mutants maintain a short period even under Fe deficiency, stressing the role of early light signalling in coupling the clock to Fe responses. Further mutant and pharmacological analyses suggest that known players in plastid‐to‐nucleus signalling do not directly participate in Fe sensing. We propose that the sensor governing circadian Fe responses defines a new retrograde pathway that involves a plastid‐encoded protein that depends on phytochromes and the functional state of chloroplasts.     

Measuring Symmetry,Asymmetry and Randomness in Neural Network Connectivity

Cognitive functions are stored in the connectome, the wiring diagram of the brain, which exhibits non-random features, so-called motifs. In this work, we focus on bidirectional, symmetric motifs, i.e. two neurons that project to each other via connections of equal strength, and unidirectional, non-symmetric motifs, i.e. within a pair of neurons only one neuron projects to the other. We hypothesise that such motifs have been shaped via activity dependent synaptic plasticity processes. As a consequence, learning moves the distribution of the synaptic connections away from randomness. Our aim is to provide a global, macroscopic, single parameter characterisation of the statistical occurrence of bidirectional and unidirectional motifs. To this end we define a symmetry measure that does not require any a priori thresholding of the weights or knowledge of their maximal value. We calculate its mean and variance for random uniform or Gaussian distributions, which allows us to introduce a confidence measure of how significantly symmetric or asymmetric a specific configuration is, i.e. how likely it is that the configuration is the result of chance. We demonstrate the discriminatory power of our symmetry measure by inspecting the eigenvalues of different types of connectivity matrices. We show that a Gaussian weight distribution biases the connectivity motifs to more symmetric configurations than a uniform distribution and that introducing a random synaptic pruning, mimicking developmental regulation in synaptogenesis, biases the connectivity motifs to more asymmetric configurations, regardless of the distribution. We expect that our work will benefit the computational modelling community, by providing a systematic way to characterise symmetry and asymmetry in network structures. Further, our symmetry measure will be of use to electrophysiologists that investigate symmetry of network connectivity.     

Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation

Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-α–mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-α activity is regulated by Rack1–keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-α activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.     

Mechanisms Underlying the Anti-Tumoral Effects of Citrus bergamia Juice

Based on the growing deal of data concerning the biological activity of flavonoid-rich natural products, the aim of the present study was to explore in vitro the potential anti-tumoral activity of Citrus Bergamia (bergamot) juice (BJ), determining its molecular interaction with cancer cells. Here we show that BJ reduced growth rate of different cancer cell lines, with the maximal growth inhibition observed in neuroblastoma cells (SH-SY5Y) after 72 hs of exposure to 5% BJ. The SH-SY5Y antiproliferative effect elicited by BJ was not due to a cytotoxic action and it did not induce apoptosis. Instead, BJ stimulated the arrest in the G1 phase of cell cycle and determined a modification in cellular morphology, causing a marked increase of detached cells. The inhibition of adhesive capacity on different physiologic substrates and on endothelial cells monolayer were correlated with an impairment of actin filaments, a reduction in the expression of the active form of focal adhesion kinase (FAK) that in turn caused inhibition of cell migration. In parallel, BJ seemed to hinder the association between the neural cell adhesion molecule (NCAM) and FAK. Our data suggest a mechanisms through which BJ can inhibit important molecular pathways related to cancer-associated aggressive phenotype and offer new suggestions for further studies on the role of BJ in cancer treatment.     

Red light imaging for programmed cell death visualization and quantification in plant–pathogen interactions

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.     

Expression of SFRP Family Proteins in Human Keratoconus Corneas

We investigated the expression of the secreted frizzled-related proteins (SFRPs) in keratoconus (KC) and control corneas. KC buttons (∼8 mm diameter) (n = 15) and whole control corneas (n = 7) were fixed in 10% formalin or 2% paraformaldehyde and subsequently paraffin embedded and sectioned. Sections for histopathology were stained with hematoxylin and eosin, or Periodic Acid Schiff’s reagent. A series of sections was also immunolabelled with SFRP 1 to 5 antibodies, visualised using immunofluorescence, and examined with a Zeiss LSM700 scanning laser confocal microscope. Semi-quantitative grading was used to compare SFRP immunostaining in KC and control corneas. Overall, KC corneas showed increased immunostaining for SFRP1 to 5, compared to controls. Corneal epithelium in all KC corneas displayed heterogeneous moderate to strong immunoreactivity for SFRP1 to 4, particularly in the basal epithelium adjacent to cone area. SFRP3 and 5 were localised to epithelial cell membranes in KC and control corneas, with increased SFRP3 cytoplasmic expression observed in KC. Strong stromal expression of SFRP5, including extracellular matrix, was seen in both KC and control corneas. In control corneas we observed differential expression of SFRP family proteins in the limbus compared to more central cornea. Taken together, our results support a role for SFRPs in maintaining a healthy cornea and in the pathogenesis of epithelial and anterior stromal disruption observed in KC.     

β-Alanine containing cyclic peptides with predetermined turned structure. V

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and solution structural characterization by nmr spectroscopy, combined with restrained molecular dynamic simulations, of the cyclic hexapeptide cyclo-(Pro-Phe-β-Ala-Phe-Phe-β-Ala). The peptide was synthesized by classical solution methods and the cyclization of the free hexapeptide was accomplished in good yields in diluted methylenechloride solution using N, N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2₁ from methanol/ethyl acetate. The molecule adopts in the solid state a conformation characterized by cis β-Ala⁶-Pro¹ peptide bond. The α-amino acid residues are at the corner positions of turned structures. The Pro¹-Phe² segment is incorporated in a pseudo type I β-turn, while Phe⁴-Phe⁵ is in a typical type I β-turn. Assignment of all ¹H and ¹³C resonances was achieved by homo- and heteronuclear two-dimensional techniques in dimethylsulfoxide (DMSO) solutions. The conformational analysis was based on inter-proton distances derived from rotating frame nuclear Overhauser effect spectroscopy spectra and homonuclear coupling constants. Restrained molecular dynamic simulation in vacuo was also performed to built refined molecular models. The molecule is present in DMSO solution as two slowly interconverting conformers, characterized by a cis-tran isomerism around the β-Ala⁶-Pro¹ peptide bond. This work confirms our expectations on the low propensity of β-alanyl residues to be positioned at the corners of turned structure. © 1994 John Wiley & Sons, Inc.     

Effects of Adenine Derivatives and Auxin-related Compounds on Mitochondrial Membrane Permeability

Adenine derivatives and auxin-related compounds, 2,4-dichlorophenoxyaceticacid (2,4-D) and 2,3,5-triiodobenzoic acid (TIBA), did not inhibitthe transport systems for succinate or malate into mitochondria.In iso-osmotic KC1 medium, some of these compounds increasedion fluxes moderately. TIBA and 2,4-D inhibited the mitochondrialshrinkage induced by the substrates. In contrast, adenine derivativesinhibited only the shrinkage induced by the substrate whoseoxidation they were able to block specifically. (Received February 18, 1987; Accepted June 29, 1987)     

Regulation of stem cell therapies under attack in Europe: for whom the bell tolls

At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non‐medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context—social, financial, medical, legal—in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of ‘translational medicine’. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients.