Electrical microarrays for highly sensitive detection of multiplex PCR products from biological agents

For the sensitive detection of amplicons derived from diagnostic PCR, a novel electrical low-density microarray is applied and compared to state-of-the-art quantitative real-time PCR. The principle of the electrochemical method and the effective use for analysis are described. Interdigitated array gold electrodes (IDA-E) embedded into a silicon chip are the core technology of the fully automated compact biosensor system, basing on enzyme coupled electrochemical detection. The biointerface is built up with thiol-modified capture oligonucleotides on gold and mediates the specific recognition of hybridised target DNA amplified with uniplex or multiplex PCR. In here we show the potential of the designed electrical microarray to function as an advanced screening method for the parallel detection of a panel of the four pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and ortho pox viruses (genus), which are among the most relevant biowarfare agents. PCR products, generated from 10 to 50 gene equivalents, have been detected reproducibly. The experiments with varying pathogen amounts showed the good reliability and the high sensitivity of the method, equivalent to optical real-time PCR detection systems. Without PCR the total assay time amounts to 27 min. The advantage of the combination of multiplex-PCR with electrical microarray detection avoiding intensive PCR probe labelling strategies is illustrated.     

Does vacuum-packaging or co-delivered amendments enhance shelf-life of Striga-mycoherbicidal products containing Fusarium oxysporum f. sp. strigae during storage?

The effect of vacuum packaging on the shelf-life and handling of Pesta granules and seed treatment made with chlamydospores of Fusarium oxysporum strains Foxy2, PSM197 or their mixture was studied at 4°C and 22±3°C over 1 year. In addition, the effects of co-incorporated amendments [urea in Pesta or co-delivered fungicides (Ridomil Gold®, Apron XL®) on coated sorghum seeds], and coating material (Arabic gum ‘AG’, SUET Binder ‘SB’) on the viability of Striga-mycoherbicides were evaluated. Storage under vacuum packaging did not enhance shelf-life of the formulated Striga-mycoherbicidal products after 12 months of storage regardless of the treatment used. The co-incorporated urea into Pesta granules significantly reduced the viability of mycoherbicides, but less so at 4°C (58% strain-stability after 12 months). No significant differences between the coating materials in maintaining the viability of mycoherbicides were observed. The shelf-life of isolates on coated seeds significantly decreased when adding Ridomil Gold®. However, at 4°C, the fungicide Apron XL® allowed better survival of Foxy2 and PSM197 by maintaining their averaged half-lives (t _0.5) by an additional 6 months compared to Ridomil Gold®. In general, Striga-mycoherbicidal product combinations exhibited a significantly higher shelf-life when stored at 4°C than at 22±3°C. The absence of a positive effect of vacuum packaging on shelf-life of Striga-mycoherbicidal products reflects the tolerance of the formulated fungal propagules (chlamydospores) to withstand an oxygen enriched environment and allows their handling and distribution through ordinary packaging systems in Africa. The high compatibility between Striga-mycoherbicides and the co-delivered fungicide Apron XL®, and the fungal storage stability allows simultaneous control of Striga and fungal cereal diseases within an integrated pest management (IPM).     

Chilean Bromeliaceae: diversity, distribution and evaluation of conservation status

Chile is home to 23 species of Bromeliaceae, including 2 subspecies and 4 varieties. Twenty species are endemic to the country. We examined 883 herbarium specimens from 27 herbaria for our treatment of the Bromeliaceae for the “Flora de Chile”. These data and field observations resulted in a comprehensive database that we used to generate distribution maps for each species. We applied ecological niche modelling to reveal distribution areas and centers of Bromeliaceae diversity. We further analysed the collecting dates of the herbarium specimens to assess possible changes in species abundance. In this study we assess the conservation status of the bromeliad species in Chile. IUCN categories were assigned to the 27 bromeliad taxa as follows: Critically endangered: 4, Endangered: 6, Vulnerable: 11, Near threatened: 2, Least concern: 4. No species has become “Extinct” up to now. We also put forth a hypothesis about their biogeographic history.     

Neural development in Onychophora (velvet worms) suggests a step-wise evolution of segmentation in the nervous system of Panarthropoda

A fundamental question in biology is how animal segmentation arose during evolution. One particular challenge is to clarify whether segmental ganglia of the nervous system evolved once, twice, or several times within the Bilateria. As close relatives of arthropods, Onychophora play an important role in this debate since their nervous system displays a mixture of both segmental and non-segmental features. We present evidence that the onychophoran “ventral organs,” previously interpreted as segmental anlagen of the nervous system, do not contribute to nerve cord formation and therefore cannot be regarded as vestiges of segmental ganglia. The early axonal pathways in the central nervous system arise by an anterior-to-posterior cascade of axonogenesis from neuronal cell bodies, which are distributed irregularly along each presumptive ventral cord. This pattern contrasts with the strictly segmental neuromeres present in arthropod embryos and makes the assumption of a secondary loss of segmentation in the nervous system during the evolution of the Onychophora less plausible. We discuss the implications of these findings for the evolution of neural segmentation in the Panarthropoda (Arthropoda + Onychophora + Tardigrada). Our data best support the hypothesis that the ancestral panarthropod had only a partially segmented nervous system, which evolved progressively into the segmental chain of ganglia seen in extant tardigrades and arthropods.     

Stair climbing results in more challenging patellofemoral contact mechanics and kinematics than walking at early knee flexion under physiological-like quadriceps loading

The mechanical environment during stair climbing has been associated with patellofemoral pain, but the contribution of loading to this condition is not clearly understood. It was hypothesized that the loading conditions during stair climbing induce higher patellofemoral pressures, a more lateral force distribution on the trochlea and a more lateral shift and tilt of the patella compared to walking at early knee flexion. Optical markers for kinematic measurements were attached to eight cadaveric knees, which were loaded with muscle forces at instances of walking and stair climbing cycles at 12° and 30° knee flexion. Contact mechanics were determined using a pressure sensitive film. At 12° knee flexion, stair climbing loads resulted in higher peak pressure (p=0.012) than walking, more lateral force distribution (p=0.012) and more lateral tilt (p=0.012), whilst mean pressure (p=0.069) and contact area (p=0.123) were not significantly different. At 30° knee flexion, although stair climbing compared to walking loads resulted in significantly higher patellofemoral mean (p=0.012) and peak pressures (p=0.012), contact area (p=0.025), as well as tilt (p=0.017), the medial–lateral force distribution (p=0.674) was not significantly different. No significant differences were observed in patellar shift between walking and stair climbing at either 12° (p=0.093) or 30° (p=0.575) knee flexion. Stair climbing thus leads to more challenging patellofemoral contact mechanics and kinematics than level walking at early knee flexion. The increase in patellofemoral pressure, lateral force distribution and lateral tilt during stair climbing provides a possible biomechanical explanation for the patellofemoral pain frequently experienced during this activity.     

Synthetic LXR agonist attenuates plaque formation in apoE-/- mice without inducing liver steatosis and hypertriglyceridemia

Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism. LXR agonists have been shown to limit the cellular cholesterol content by inducing reverse cholesterol transport, increasing bile acid production, and inhibiting intestinal cholesterol absorption. Most of them, however, also increase lipogenesis via sterol regulatory element-binding protein-1c (SREBP1c) and carbohydrate response element-binding protein activation resulting in hypertriglyceridemia and liver steatosis. We report on the antiatherogenic properties of the steroidal liver X receptor agonist N,N-dimethyl-3beta-hydroxy-cholenamide (DMHCA) in apolipoprotein E (apoE)-deficient mice. Long-term administration of DMHCA (11 weeks) significantly reduced lesion formation in male and female apoE-null mice. Notably, DMHCA neither increased hepatic triglyceride (TG) levels in male nor female apoE-deficient mice. ATP binding cassette transporter A1 and G1 and cholesterol 7alpha-hydroxylase mRNA abundances were increased, whereas SREBP1c mRNA expression was unchanged in liver, and even decreased in macrophages and intestine. Short-term treatment revealed even higher changes on mRNA regulation. Our data provide evidence that DMHCA is a strong candidate as therapeutic agent for the treatment or prevention of atherosclerosis, circumventing the negative side effects of other LXR agonists.     

Structural analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3-kinases

We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1H-1H and 1H-31P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)2-IP4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)2-IP4 ( Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725 ). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.     

Voltametric monitoring of enzyme-mediated indigo reduction in the presence of various fibre materials

A comparative study of enzyme-mediated indigo reduction is presented as an environmentally-friendly alternative to alkaline sodium dithionite reduction. The effect of the mediator 1,8-dihydroxy-9,10-anthraquinone in enzymatic reduction was studied by means of voltammetry, both in the presence and absence of different textile materials (polyamide 6, polyamide 6,6 and cotton), and compared to chemically reduced indigo. It was observed that bio-catalytic formation of leuco indigo and its exhaustion on substrates is inversely proportional to the pH within the range of 7–11. Additionally, substrate coloration was strongly influenced by the mediator, resulting in in situ formation of leuco indigo. This effect was most pronounced for polyamide substrates. The reuse of an enzyme-mediated reduction bath for dyeing was assessed showing that the levelness of the obtained shade was either excellent or good at pH 9 and 11, respectively. The wash, perspiration, and light color fastness properties of all textile materials dyed with enzymatically-reduced indigo were comparable or even better than those obtained with chemically reduced indigo. The use of enzyme-mediated reduction of indigo combined with potential reuse of the reduction bath represents a cost effective and environmentally-friendly dyeing process that can be applied for the dyeing of natural cellulosic and synthetic polyamide fibres.