SNP high-throughput screening in grapevine using the SNPlex™ genotyping system

Background

Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP) discovery and genotyping in grapevine (Vitis vinifera L.). However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs) thus providing a valuable source for high-throughput genotyping methods.

Results

Herein we report the first application of the SNPlex? genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA) methods were used for preparation of genomic DNA for the SNPlex assay.

Conclusion

Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA), is a good solution for future applications in well-equipped laboratories.     

High Efficiency Up‐Converting Single Phase Elastomers for Photon Managing Applications

Optically active materials able to up‐convert the frequency of the incident radiation can be used to enhance the performance of photovoltaic and photocatalityc cells, recovering sub‐bandgap photons not directly absorbed by the devices. Actually, sensitized up‐conversion (SUC) based on multi‐component organic systems is the most promising approach for these photon energy managing processes, being efficient also at the solar irradiance. However, applications of SUC on real devices have not been yet accomplished because its conversion yield usually drops dramatically in the solid state where the low dye mobility inhibits the diffusion controlled mechanisms ruling SUC photophysics. To overcome this limit, we prepared a single‐phase elastomer (poly‐butylacrilate) doped with proper dyes (platinum (II) octaetyl‐porphyrin and 9,10‐diphenylanthracene) to fabricate an efficient photon up‐converting material. Thanks to the residual molecular diffusion provided by the soft host, and to the quenching reduction of involved metastable electronic excited‐states in a solid environment compared to a liquid one, we obtained a record SUC yield of 17% at the solid state. SUC efficiency has been studied as function of the excitation power and sample temperature, elucidating the photophysical processes at the base of the high observed yield and assessing the guidelines for the fabrication of technologically appealing low power up‐converting materials.     

Modulation of MicroRNA-194 and cell migration by HER2-targeting trastuzumab in breast cancer

Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER2 oncoprotein, can effectively target HER2-positive breast cancer through several mechanisms. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis and apoptosis have been investigated in depth, the effect of trastuzumab on microRNA (miRNA) has not been extensively studied. We have performed miRNA microarray profiling before and after trastuzumab treatment in SKBr3 and BT474 human breast cancer cells that overexpress HER2. We found that trastuzumab treatment of SKBr3 cells significantly decreased five miRNAs and increased three others, whereas treatment of BT474 cells significantly decreased two miRNAs and increased nine. The only change in miRNA expression observed in both cell lines following trastuzumab treatment was upregulation of miRNA-194 (miR-194) that was further validated in vitro and in vivo. Forced expression of miR-194 in breast cancer cells that overexpress HER2 produced no effect on apoptosis, modest inhibition of proliferation, significant inhibition of cell migration/invasion in vitro and significant inhibition of xenograft growth in vivo. Conversely, knockdown of miR-194 promoted cell migration. Increased miR-194 expression markedly reduced levels of the cytoskeletal protein talin2 and specifically inhibited luciferase reporter activity of a talin2 wild-type 3'-untranslated region, but not that of a mutant reporter, indicating that talin2 is a direct downstream target of miR-194. Trastuzumab treatment inhibited breast cancer cell migration and reduced talin2 expression in vitro and in vivo. Knockdown of talin2 inhibited cell migration/invasion. Knockdown of trastuzumab-induced miR-194 expression with a miR-194 inhibitor compromised trastuzumab-inhibited cell migration in HER2-overexpressing breast cancer cells. Consequently, trastuzumab treatment upregulates miR-194 expression and may exert its cell migration-inhibitory effect through miR-194-mediated downregulation of cytoskeleton protein talin2 in HER2-overexpressing human breast cancer cells.     

The Amplitude and Inactivation Properties of the Delayed Potassium Currents Are Regulated by Protein Kinase Activity in Hair Cells of the Frog Semicircular Canals

In hair cells dissected from the frog crista ampullaris, the combination of a calcium-dependent (IKCa) and a purely voltage-dependent component (IKV) gives rise to the delayed potassium current complex (IKD). These currents have been recently reported to display slow depolarization-induced inactivation and biphasic inactivation removal by hyperpolarization. The amplitude and inactivation kinetics of both IKCa and IKV are drastically modulated by a previously unrecognized mechanism of protein phosphorylation (sensitive to kinase inhibitors H89 and KT5823), which does not interfere with the transient potassium current (IA) or the calcium current (ICa). IKD amplitude was stable in cells patched with pipettes containing 8 mM ATP or under perforated-patch; under these conditions, a 10 min treatment with 10 µM H89 or 1–10 µM KT5823 reduced IKD amplitude by a mean of 67% at +40 mV. Similarly affected was the isolated IKV component (ICa blocked with Cd²⁺). Thus, a large potassium conductance can be activated by depolarization, but it is made available to the cell to a variable extent that depends on membrane potential and protein kinase activity. The total gKD ranged 4.6–44.0 nS in control cells, according to the level of steady-state inactivation, and was reduced to 1.4–2.7 nS after protein kinase inhibition. When sinusoidal membrane potential changes in the −70/−10 mV range were applied, to mimic receptor response to hair bundle deflection, IKD proved the main current dynamically activated and the only one regulated by PK: H89 decreased the total outward charge during each cycle by 60%. Phosphorylation appears to control both the amount of IKCa and IKV conductance activated by depolarization and the fraction thereof which can be rescued by removal of inactivation. The balance between the depolarizing transduction current and the repolarizing potassium current, and eventually the transmitter release at the cytoneural junction, are therefore modulated by a phosphorylation-mediated process.     

Delineating Geographical Regions with Networks of Human Interactions in an Extensive Set of Countries

Large-scale networks of human interaction, in particular country-wide telephone call networks, can be used to redraw geographical maps by applying algorithms of topological community detection. The geographic projections of the emerging areas in a few recent studies on single regions have been suggested to share two distinct properties: first, they are cohesive, and second, they tend to closely follow socio-economic boundaries and are similar to existing political regions in size and number. Here we use an extended set of countries and clustering indices to quantify overlaps, providing ample additional evidence for these observations using phone data from countries of various scales across Europe, Asia, and Africa: France, the UK, Italy, Belgium, Portugal, Saudi Arabia, and Ivory Coast. In our analysis we use the known approach of partitioning country-wide networks, and an additional iterative partitioning of each of the first level communities into sub-communities, revealing that cohesiveness and matching of official regions can also be observed on a second level if spatial resolution of the data is high enough. The method has possible policy implications on the definition of the borderlines and sizes of administrative regions.     

Changes in coral microbial communities in response to a natural pH gradient

Surface seawater pH is currently 0.1 units lower than pre-industrial values and is projected to decrease by up to 0.4 units by the end of the century. This acidification has the potential to cause significant perturbations to the physiology of ocean organisms, particularly those such as corals that build their skeletons/shells from calcium carbonate. Reduced ocean pH could also have an impact on the coral microbial community, and thus may affect coral physiology and health. Most of the studies to date have examined the impact of ocean acidification on corals and/or associated microbiota under controlled laboratory conditions. Here we report the first study that examines the changes in coral microbial communities in response to a natural pH gradient (mean pH_T 7.3–8.1) caused by volcanic CO₂ vents off Ischia, Gulf of Naples, Italy. Two Mediterranean coral species, Balanophyllia europaea and Cladocora caespitosa, were examined. The microbial community diversity and the physiological parameters of the endosymbiotic dinoflagellates (Symbiodinium spp.) were monitored. We found that pH did not have a significant impact on the composition of associated microbial communities in both coral species. In contrast to some earlier studies, we found that corals present at the lower pH sites exhibited only minor physiological changes and no microbial pathogens were detected. Together, these results provide new insights into the impact of ocean acidification on the coral holobiont.     

Mass‐flowering crops dilute pollinator abundance in agricultural landscapes across Europe

Mass‐flowering crops (MFCs) are increasingly cultivated and might influence pollinator communities in MFC fields and nearby semi‐natural habitats (SNHs). Across six European regions and 2 years, we assessed how landscape‐scale cover of MFCs affected pollinator densities in 408 MFC fields and adjacent SNHs. In MFC fields, densities of bumblebees, solitary bees, managed honeybees and hoverflies were negatively related to the cover of MFCs in the landscape. In SNHs, densities of bumblebees declined with increasing cover of MFCs but densities of honeybees increased. The densities of all pollinators were generally unrelated to the cover of SNHs in the landscape. Although MFC fields apparently attracted pollinators from SNHs, in landscapes with large areas of MFCs they became diluted. The resulting lower densities might negatively affect yields of pollinator‐dependent crops and the reproductive success of wild plants. An expansion of MFCs needs to be accompanied by pollinator‐supporting practices in agricultural landscapes.     

Evaluation of NK-to-target cell binding and evidence for T cell conjugates by flow cytometry

A flow cytometric methodology was set up to assess the binding capability of peripheral blood NK and T cells to the K562 tumor cell line. Differential side scatter characteristics between effectors and targets were used to analyze conjugated and unconjugated cells. The previous labeling of NK and T cells with anti-Leu 11c and anti-Leu 4 monoclonal antibodies, allowed the distinction between unconjugated non-fluorescent and conjugated fluorescent targets and the percentual evaluation of bound anti-Leu 11c⁺ and anti-Leu 4⁺ cells.Abbreviations NK Natural Killer - MoAb Monoclonal Antibody - SSC Side Scatter - FLS Forward Light Scatter - PE Phycoerythrin - MHC Major Histocompatibility Complex - GVHD Graft Versus Host Disease