Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis

BACKGROUND: Outbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary. RESULTS: A robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection. CONCLUSION: The TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions

α-Synuclein Expression Selectively Affects Tumorigenesis in Mice Modeling Parkinson's Disease

Alpha Synuclein (α-Syn) is a protein implicated in mechanisms of neuronal degeneration in Parkinson's disease (PD). α-Syn is primarily a neuronal protein, however, its expression is found in various tumors including ovarian, colorectal and melanoma tumors. It has been hypothesized that neurodegeneration may share common mechanisms with oncogenesis. We tested whether α-Syn expression affects tumorigenesis of three types of tumors. Specifically, B16 melanoma, E0771 mammary gland adenocarcinoma and D122 Lewis lung carcinoma. For this aim, we utilized transgenic mice expression the human A53T α-Syn form. We found that the in vivo growth of B16 and E0771 but not D122 was enhanced in the A53T α-Syn mice. The effect on tumorigenesis was not detected in age-matched APP/PS1 mice, modeling Alzheimer's disease (AD), suggesting a specific effect for α-Syn- dependent neurodegeneration. Importantly, transgenic α-Syn expression was detected within the three tumor types. We further show uptake of exogenously added, purified α-Syn, by the cultured tumor cells. In accord, with the affected tumorigenesis in the young A53T α-Syn mice, over- expression of α-Syn in cultured B16 and E0771 cells enhanced proliferation, however, had no effect on the proliferation of D122 cells. Based on these results, we suggest that certain forms of α-Syn may selectively accelerate cellular mechanisms leading to cancer

Mechanical Stress Induces Remodeling of Vascular Networks in Growing Leaves

International audienceDifferentiation into well-defined patterns and tissue growth are recognized as key processes in organismal development. However, it is unclear whether patterns are passively, homogeneously dilated by growth or whether they remodel during tissue expansion. Leaf vascu-lar networks are well-fitted to investigate this issue, since leaves are approximately two-dimensional and grow manyfold in size. Here we study experimentally and computationally how vein patterns affect growth. We first model the growing vasculature as a network of viscoelastic rods and consider its response to external mechanical stress. We use the so-called texture tensor to quantify the local network geometry and reveal that growth is heterogeneous , resembling non-affine deformations in composite materials. We then apply mechanical forces to growing leaves after veins have differentiated, which respond by anisotropic growth and reorientation of the network in the direction of external stress. External mechanical stress appears to make growth more homogeneous, in contrast with the model with viscoelastic rods. However, we reconcile the model with experimental data by incorporating randomness in rod thickness and a threshold in the rod growth law, making the rods viscoelastoplastic. Altogether, we show that the higher stiffness of veins leads to their reorientation along external forces, along with a reduction in growth heterogeneity. This process may lead to the reinforcement of leaves against mechanical stress. More generally , our work contributes to a framework whereby growth and patterns are coordinated through the differences in mechanical properties between cell types

Effects of different commercial diets on growth performance, health and resistance to Tetrahymena sp. infection in guppies, Poecilia reticulata (Peters)

The effects of feeding guppy fry, Poecilia reticulata (Peters), different commercial diets from BernAqua [MeM (R=Regular, O=Ornamental and P=Premium) and experimental feed (EF] and Ocean Nutrition (ON) on growth was tested for 57days. Health status and resistance to Tetrahymena infection were analysed at the end of the trial. The highest growth was obtained in fish fed MeM Regular and MeM Premium (mean final weights of 427 and 417mg respectively). Fish fed EF had a significantly higher rate of spinal deformity (12.7%) than all other feeding groups (3.05% or less). Histological analysis revealed the accumulation of liver glycogen and/or lipid in fish from all feeding groups; fat accumulation in the abdomen was most pronounced in the EF and MeM Regular-fed fish, and muscle dystrophy was observed in ca. 50% of the fish from all feeding groups except the MeM Ornamental-fed group. The highest mortality following Tetrahymena infection occurred in fish fed EF (87%) and the lowest mortality in the MeM Ornamental-fed fish (58%). Mortalities in all other feeding groups ranged between 69% and 76%. In summary, MeM Ornamental feed provided a moderate weight gain, no muscle dystrophy, negligible deformity and the greatest resistance to parasitic infection

Numerical simulations of a growing network: Anistropy depends on the applied stress.

<p>(A) The effect of external stress. Each vein is represented by a black line. The red box is the unit cell with periodic boundary conditions. For visualization, the texture tensor of each areole is shown in the middle of the areole (blue, not to scale), and the averaged texture tensor in the middle of each figure (green, not to scale). The three tissues grew under turgor pressure; in addition, the stress <i>σ</i>_<i>xx</i> along the <i>x</i> direction or <i>σ</i>_<i>yy</i> along the <i>y</i> direction were set equal to the magnitude of turgor pressure <i>P</i>_<i>tur</i>. (B) The distribution of the main orientation (the orientation of the eigenvector associated with the highest eigenvalue) of the texture tensor in the simulation. The distribution is seen to be relatively widely scattered around 90° when no external stress is present (green). When stress is applied, the distribution becomes sharply concentrated around the direction of the external stress (orange and blue). (C) The anisotropy of the texture tensor field averaged over 40 simulations, as a function of the external stress. The stress is measured in units of the turgor pressure, and the anisotropy is measured when the network has doubled its area from the end of the vein creation stage (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004819#sec013" target="_blank">Methods</a>).</p

Veins in numerical simulations undergo non-affine deformations.

<p>(A) Colormap of the non-affinity index <i>q</i> of each areole, for one realization of the numerical simulation with stretching in the <i>y</i>−direction (<i>σ</i>_<i>yy</i> = <i>P</i>_<i>tur</i> as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004819#pcbi.1004819.g002" target="_blank">Fig 2</a>); in the case of an affine dilation <i>q</i> would be equal to one for all areoles; <i>q</i> > 1 (resp. <i>q</i> < 1) means that the areole grew more (resp. less) than its neighbourhood. (B) Histogram of <i>q</i> over all realizations with stretching in the <i>x</i>−direction and for a stress <i>σ</i>_<i>xx</i> ranging from 0 to 2<i>P</i>_<i>tur</i>. As in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004819#pcbi.1004819.g002" target="_blank">Fig 2</a>, the simulations were stopped when the leaves doubled their area.</p

Model with randomness in vein thickness and threshold in the growth law.

<p>All panels show the stress-dependent distribution of non-affinity index <i>q</i> in all the simulations for a noise amplitude of <i>r</i> = 40%. (A) With no growth threshold (<i>η</i> = 0) the distribution in the presence of anisotropic stress (red) is wider than without it (blue), in contrast to experimental finding (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004819#pcbi.1004819.g006" target="_blank">Fig 6C</a>). (B) When a threshold, <i>η</i> = <i>η</i>₀, is introduced, the trend is reversed, and the stressed tissue features a narrower distribution, in agreement with experiments. (C,D) Sensitivity of the results to the threshold; the normalized threshold <i>η</i>^⋆ = <i>η</i>/<i>η</i>₀ ranges from 0.1 to 1.3. Panels (C) and (D) correspond, respectively, to <i>σ</i>_<i>x</i> = 0 and <i>σ</i>_<i>x</i> = 2<i>P</i>_<i>tur</i>.</p