Stretching DNA origami: effect of nicks and Holliday junctions on the axial stiffness

The axial stiffness of DNA origami is determined as a function of key nanostructural characteristics. Different constructs of two-helix nanobeams with specified densities of nicks and Holliday junctions are synthesized and stretched by fluid flow. Implementing single particle tracking to extract force–displacement curves enables the measurement of DNA origami stiffness values at the enthalpic elasticity regime, i.e. for forces larger than 15 pN. Comparisons between ligated and nicked helices show that the latter exhibit nearly a two-fold decrease in axial stiffness. Numerical models that treat the DNA helices as elastic rods are used to evaluate the local loss of stiffness at the locations of nicks and Holliday junctions. It is shown that the models reproduce the experimental data accurately, indicating that both of these design characteristics yield a local stiffness two orders of magnitude smaller than the corresponding value of the intact double-helix. This local degradation in turn leads to a macroscopic loss of stiffness that is evaluated numerically for multi-helix DNA bundles.     

Cloning,expression, and characterization of sorbitol transporters from developing sour cherry fruit and leaf sink tissues

The acyclic polyol sorbitol is a primary photosynthetic product and the principal photosynthetic transport substance in many economically important members of the family Rosaceace (e.g. almond [Prunus dulcis (P. Mill.) D.A. Webber], apple [Malus pumila P. Mill.], cherry [Prunus spp.], peach [Prunus persica L. Batsch], and pear [Pyrus communis]). To understand key steps in long-distance transport and particularly partitioning and accumulation of sorbitol in sink tissues, we have cloned two sorbitol transporter genes (PcSOT1 and PcSOT2) from sour cherry (Prunus cerasus) fruit tissues that accumulate large quantities of sorbitol. Sorbitol uptake activities and other characteristics were measured by heterologous expression of PcSOT1 and PcSOT2 in yeast (Saccharomyces cerevisiae). Both genes encode proton-dependent, sorbitol-specific transporters with similar affinities (K(m) sorbitol of 0.81 mM for PcSOT1 and 0.64 mM for PcSOT2). Analyses of gene expression of these transporters, however, suggest different roles during leaf and fruit development. PcSOT1 is expressed throughout fruit development, but especially when growth and sorbitol accumulation rates are highest. In leaves, PcSOT1 expression is highest in young, expanding tissues, but substantially less in mature leaves. In contrast, PcSOT2 is mainly expressed only early in fruit development and not in leaves. Compositional analyses suggest that transport mediated by PcSOT1 and PcSOT2 plays a major role in sorbitol and dry matter accumulation in sour cherry fruits. Presence of these transporters and the high fruit sorbitol concentrations suggest that there is an apoplastic step during phloem unloading and accumulation in these sink tissues. Expression of PcSOT1 in young leaves before completion of the transition from sink to source is further evidence for a role in determining sink activity.     

Estrogens, progestins, and coronary artery reactivity in atherosclerotic monkeys

It has been known for many years that sex hormones modulate vasodilator responses of arteries supplying the uterus with blood. Recently, it has been shown that sex hormones such as estrogen modulate vasomotor responses of other arteries, including coronary arteries. It is thought that modulation of vasodilator and constrictor responses of coronary arteries may be one mechanism by which estrogen affects the risk of coronary heart disease. Although several studies have examined the effects (and potential mechanisms) of estrogen on vasodilator responses of nonatherosclerotic arteries, few have focused on estrogen's effects on atherosclerotic coronary arteries. In studies of ovariectomized atherosclerotic female cynomolgus monkeys, both long-term (2 years) and short-term (20 min) estradiol treatment augments dilator responses to acetylcholine, but not nitroglycerin. Presumably, this indicates an effect of estradiol on endothelium-mediated dilator responses of coronary arteries. Addition of the progestin medroxyprogesterone acetate diminishes the beneficial effect of conjugated equine estrogens on these dilator responses. This is significant because a progestin is usually added to estrogen replacement to reduce the risk of endometrial and breast cancer associated with unopposed estrogen therapy. However, it would seem that not all progestins act similarly on vascular reactivity. Studies in monkeys indicate that addition of progesterone or the progestin medroxyprogesterone acetate does not diminish the beneficial effects of estrogen on coronary dilator responses. Thus it would appear that different estrogen/progestin combinations may affect vascular reactivity in different manners, There is also an effort being made to examine the potential of different kinds of estrogens on cardiovascular risk. Studies in monkeys indicate that one of the estrogens found in conjugated equine estrogens (17 alpha-dihydroequilenin) has estrogen effects on vascular reactivity without having detrimental effects on uterine pathology. The isoflavones “plant estrogens” found in soy protein also have estrogenic effects on vascular reactivity and inhibition.     

P2Y2R Deficiency Attenuates Experimental Autoimmune Uveitis Development

We aimed to study the role of the nucleotide receptor P2Y₂R in the development of experimental autoimmune uveitis (EAU). EAU was induced in P2Y₂^+/+ and P2Y₂^-/- mice by immunization with IRBP peptide or by adoptive transfer of in vitro restimulated semi-purified IRBP-specific enriched T lymphocytes from spleens and lymph nodes isolated from native C57Bl/6 or P2Y₂^+/+ and P2Y₂^-/- immunized mice. Clinical and histological scores were used to grade disease severity. Splenocytes and lymph node cell phenotypes were analyzed using flow cytometry. Semi-purified lymphocytes and MACS-purified CD4⁺ T lymphocytes from P2Y₂^+/+ and P2Y₂^-/- immunized mice were tested for proliferation and cytokine secretion. Our data show that clinical and histological scores were significantly decreased in IRBP-immunized P2Y₂^-/- mice as in P2Y₂^-/- mice adoptively transfered with enriched T lymphocytes from C57Bl/6 IRBP-immunized mice. In parallel, naïve C57Bl/6 mice adoptively transferred with T lymphocytes from P2Y₂^-/- IRBP-immunized mice also showed significantly less disease. No differences in term of spleen and lymph node cell recruitment or phenotype appeared between P2Y₂^-/- and P2Y₂^+/+ immunized mice. However, once restimulated in vitro with IRBP, P2Y₂^-/- T cells proliferate less and secrete less cytokines than the P2Y₂^+/+ one. We further found that antigen-presenting cells of P2Y₂^-/- immunized mice were responsible for this proliferation defect. Together our data show that P2Y₂^-/- mice are less susceptible to mount an autoimmune response against IRBP. Those results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, cell migration and the release of danger signals such as extracellular nucleotides.     

Effect of Chemical Stabilizers on the Thermostability and Infectivity of a Representative Panel of Freeze Dried Viruses

As a partner of the European Virus Archive (EVA) FP7 project, our laboratory maintains a large collection of freeze-dried viruses. The distribution of these viruses to academic researchers, public health organizations and industry is one major aim of the EVA consortium. It is known that lyophilization requires appropriate stabilizers to prevent inactivation of the virus. However, few studies have investigated the influence of different stabilizers and lyophilization protocols on the thermostability of different viruses. In order to identify optimal lyophilization conditions that will deliver maximum retention of viral infectivity titre, different stabilizer formulations containing trehalose, sorbitol, sucrose or foetal bovine serum were evaluated for their efficacy in stabilizing a representative panel of freeze dried viruses at different storage temperatures (-20°C, +4°C and +20°C) for one week, the two latter mimicking suboptimal shipping conditions. The Tissue Culture Infectious Dose 50% (TCID₅₀) assay was used to compare the titres of infectious virus. The results obtained using four relevant and model viruses (enveloped/non enveloped RNA/DNA viruses) still serve to improve the freeze drying conditions needed for the development and the distribution of a large virus collection.     

Antidiabetic effect of green rooibos (Aspalathus linearis) extract in cultured cells and type 2 diabetic model KK-Ay mice

Previous studies have demonstrated antidiabetic effects for rooibos (Aspalathus linearis) and aspalathin (ASP), one of its main polyphenols. Rooibos, an endemic plant of South Africa, is well-known for its use as herbal tea. Green (‘unfermented’) rooibos has been shown to contain more ASP than ‘fermented’ rooibos tea, currently the major product. In the present study, we investigated the antidiabetic effect of green rooibos extract (GRE) through studies on glucose uptake in L6 myotubes and on pancreatic β-cell protective ability from reactive oxygen species (ROS) in RIN-5F cells. Its in vivo effect was also examined using obese diabetic KK-A^y mice. GRE increased glucose uptake under insulin absent condition and induced phosphorylation of 5′-adenosine monophosphate-activated protein kinase (AMPK) in L6 myotubes as previously demonstrated for ASP. In addition to AMPK, GRE also promoted phosphorylation of Akt, another promoter of glucose transporter 4 (GLUT4) translocation, in L6 myotubes unlike ASP, suggesting an involvement of GRE component(s) other than ASP in Akt phosphorylation. Promotion of GLUT4 translocation to the plasma membrane by GRE in L6 myotubes was demonstrated by Western blotting analysis. GRE suppressed the advanced glycation end products (AGEs)-induced increase in ROS levels in RIN-5F pancreatic β-cells. Subchronic feeding with GRE suppressed the increase in fasting blood glucose levels in type 2 diabetic model KK-A^y mice. These in vitro and in vivo results strongly suggest that GRE has antidiabetic potential through multiple modes of action.     

Fast inference of interactions in assemblies of stochastic integrate-and-fire neurons from spike recordings

We present two Bayesian procedures to infer the interactions and external currents in an assembly of stochastic integrate-and-fire neurons from the recording of their spiking activity. The first procedure is based on the exact calculation of the most likely time courses of the neuron membrane potentials conditioned by the recorded spikes, and is exact for a vanishing noise variance and for an instantaneous synaptic integration. The second procedure takes into account the presence of fluctuations around the most likely time courses of the potentials, and can deal with moderate noise levels. The running time of both procedures is proportional to the number S of spikes multiplied by the squared number N of neurons. The algorithms are validated on synthetic data generated by networks with known couplings and currents. We also reanalyze previously published recordings of the activity of the salamander retina (including from 32 to 40 neurons, and from 65,000 to 170,000 spikes). We study the dependence of the inferred interactions on the membrane leaking time; the differences and similarities with the classical cross-correlation analysis are discussed.     

Doxorubicin induces mitochondrial permeability transition and contractile dysfunction in the human myocardium

In human atrial trabeculae, we examined the effects of doxorubicin on the isometric force of contraction, mitochondrial respiration, membrane potential and calcium retention capacity. Compared with untreated controls, doxorubicin induced contractile dysfunction and depression of mitochondrial respiration. Mitochondria isolated from doxorubicin-treated human atrial trabeculae displayed reduced transmembrane potential and calcium retention capacity. Cyclosporine A, a mitochondrial membrane transition pore opening blocker, prevented mitochondrial dysfunction and impaired contractile performance induced by doxorubicin. The study suggests that a mitochondrial membrane transition pore opening is involved in the development of doxorubicin cardiotoxicity in human hearts.