Comparative next-generation mapping of the Phytophthora infestans resistance gene Rpi-dlc2 in a European accession of Solanum dulcamara

Phytophthora infestans, the causal agent of late blight, remains the main threat to potato production worldwide. Screening of 19 accessions of Solanum dulcamara with P. infestans isolate Ipo82001 in detached leaf assays revealed strong resistance in an individual belonging to accession A54750069-1. This plant was crossed with a susceptible genotype, and an F₁ population consisting of 63 individuals was obtained. This population segregated for resistance in 1:1 ratio, both in detached leaf assays and in an open-field experiment. Presence of the formerly mapped Rpi-dlc1 gene as the cause of the observed segregating resistance could be excluded. Subsequently, AFLP analyses using 128 primer combinations enabled identification of five markers linked to a novel resistance gene named Rpi-dlc2. AFLP markers did not show sequence similarity to the tomato and potato genomes, hampering comparative genetic positioning of the gene. For this reason we used next-generation mapping (NGM), an approach that exploits direct sequencing of DNA (in our case: cDNA) pools from bulked segregants to calculate the genetic distance between SNPs and the locus of interest. Plotting of these genetic distances on the tomato and potato genetic map and subsequent PCR-based marker analysis positioned the gene on chromosome 10, in a region overlapping with the Rpi-ber/ber1 and -ber2 loci from S. berthaultii. Pyramiding of Rpi-dlc2 and Rpi-dlc1 significantly increased resistance to P. infestans, compared with individuals containing only one of the genes, showing the usefulness of this strategy to enhance resistance against Phytophthora.     

Stable isotopes of lakes and precipitation along an altitudinal gradient in the Eastern Alps

Altitude encompasses broad environmental gradients that influence the isotopic composition of lake water. We selected 55 lakes in the Eastern Alps along an altitudinal gradient [214–2,532 m above sea level (a.s.l.)] to model the isotopic signal of surface water dependent on intrinsic (lake geomorphometry) and extrinsic (air temperature, precipitation) factors. Ordinary and generalised least squared regression were used for statistical analysis. The isotope signal of lake water was lower in spring than in summer and decreased with altitude (?0.21 δ¹⁸O ‰/100 m; ?1.5 δ²H ‰/100 m). This pattern largely depended on temperature and a pseudo-latitude effect. The isotopic signal in monthly precipitation (12 stations; altitudinal gradient 90–2,730 m a.s.l.) generally showed the expected pattern of less enriched values with altitude; however, unusual values were related to weather anomalies. The local meteoric water line was similar to the global meteoric water line as shown by overlapping confidence intervals. By discriminating different elevational bands, we could show that high elevation lakes (>1,500 m a.s.l.) experience different patterns of evaporation with respect to low elevation lakes (<1,500 m a.s.l.). Our study showed that lakes have a unique isotopic fingerprint along an altitudinal gradient, potentially useful for tracing ecological processes and for paleoclimatic studies.     

Puccinellia festucaeformis (Host) Parl.: Germinazione e crescita iniziale in funzione della salinità del substrato

Abstract

Puccinellia festucaeformis (Host) Parl.: germination and early growth on different salt substrates. Germination behaviour of Puccinellia festucaeformis seeds and early growth of seedlings at different experimental conditions was analysed. The following growth substrates were utilized: NaCl, KCl, KNO₃, MgCl₂, MgSO₄, Na₂SO₄, NaNO₃, CaCl₂ at the decreasing concentrations of 0.50, 0.25, 0.12, 0.06M. Caryopses were allowed to imbibe and grow at alternating temperatures (10°-20°C or 20°-30°C) in the dark for 3 days. Seedling were grown for 15 days, at controlled light and temperature conditions, in the same nutrient substrates as those used for the germination experiments.

The germination experiments showed a high tolerance to salts up to 0.25M solution and for the whole range of MgSO₄ concentrations. High growth temperatures increased the depressive effects of salt concentrations. Seedling growth was highly reduced when salt concentration was higher than 0.12M. High salt tolerance - maximum shoot and root growth - was showed by seedling allowed to grow on 0.50M MgSO₄.

Germination and growth condition of Puccinellia festucaeformis is discussed in relation to the ecological features of this species and to its possible importance as bioindicator of MgSO₄ rich natural substrates.     

ATM kinase enables the functional axis of YAP,PML and p53 to ameliorate loss of Werner protein-mediated oncogenic senescence

Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.     

The design, synthesis and biological evaluation of novel URB602 analogues as potential monoacylglycerol lipase inhibitors

We have synthesised an extensive series of URB602 analogues as inhibitors of monoacylglycerol lipase (MAGL), which is the major enzyme responsible for metabolising the endocannabinoid 2-arachidonylglycerol. The recently identified crystal structure of MAGL was used in the design strategy and revealed three possible binding sites for URB602 and the proposed analogues. A test series of carbamate analogues were docked into the identified sites to predict the most favourable binding location. The synthesised analogues of URB602 explored the biological effects of isosteric replacement, ring size and substitution, para substitution of the biphenyl moiety and the incorporation of a bicyclic element. The compounds were tested for their ability to inhibit human MAGL. The carbamate analogue 16 displayed the most significant inhibitory activity, reducing MAGL activity to 26% of controls at 100 μM compared to 73% for the parent compound URB602.     

When a Text Is Translated Does the Complexity of Its Vocabulary Change? Translations and Target Readerships

In linguistic studies, the academic level of the vocabulary in a text can be described in terms of statistical physics by using a “temperature” concept related to the text''s word-frequency distribution. We propose a “comparative thermo-linguistic” technique to analyze the vocabulary of a text to determine its academic level and its target readership in any given language. We apply this technique to a large number of books by several authors and examine how the vocabulary of a text changes when it is translated from one language to another. Unlike the uniform results produced using the Zipf law, using our “word energy” distribution technique we find variations in the power-law behavior. We also examine some common features that span across languages and identify some intriguing questions concerning how to determine when a text is suitable for its intended readership.     

Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH

The protein PCSK9 (proprotein convertase subtilisin/kexin type 9) is a key regulator of low-density lipoprotein receptor (LDLR) levels and cardiovascular health. We have determined the crystal structure of LDLR bound to PCSK9 at neutral pH. The structure shows LDLR in a new extended conformation. The PCSK9 C-terminal domain is solvent exposed, enabling cofactor binding, whereas the catalytic domain and prodomain interact with LDLR epidermal growth factor(A) and β-propeller domains, respectively. Thus, PCSK9 seems to hold LDLR in an extended conformation and to interfere with conformational rearrangements required for LDLR recycling.     

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.^1,2,3This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.^4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD_260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 μg or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 10⁶ PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 10⁸ plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.⁶ For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 μl of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.Download video file.^{(79M, mov)}     

Chemical proteomics reveals bolinaquinone as a clathrin-mediated endocytosis inhibitor

The emerging field of mass spectrometry-based chemical proteomics provides a powerful instrument in the target discovery of bioactive small-molecules, such as drugs or natural products. The identification of their macromolecular targets is required for a comprehensive understanding of their bio-pharmacological role and for unraveling their mechanism of action. We report the application of a chemical proteomics approach to the analysis of the cellular interactome of the marine metabolite bolinaquinone (BLQ). BLQ was linked to an opportune α,ω-diamino polyethylene glycol chain and then immobilized on a matrix support. The modified beads were then used as a bait for fishing the potential partners of BLQ in a THP-1 macrophage cell lysate. Surprisingly, we identified clathrin, a protein involved in the cell internalization of proteins, viruses and other biologically relevant macromolecules, as a specific and major BLQ partner. In addition, we verified the biochemical role of BLQ testing its ability to inhibit the clathrin-mediated endocytosis of albumin. This finding indicates BLQ as a new biotechnological tool for cell endocytosis studies and paves the way to further investigation on its potential role in modulating internalization process.