Butterfly seed predation: effects of landscape characteristics,plant ploidy level and population structure

Polyploidization has been suggested as one of the most common mechanisms for plant diversification. It is often associated with changes in several morphological, phenological and ecological plant traits, and therefore has the potential to alter insect–plant interactions. Nevertheless, studies evaluating the effect of plant polyploidy on interspecific interactions are still few. We investigated pre-dispersal seed predation by the butterfly Anthocharis cardamines in 195 populations of two ploidy levels of the herb Cardamine pratensis (tetraploid ssp. pratensis, 2n = 30 vs. octoploid ssp. paludosa, 2n = 56–64). We asked if differences in incidence and intensity of predation among populations were related to landscape characteristics, plant ploidy level and population structure. The incidence of the seed predator increased with increasing plant population size and decreasing distance to nearest population occupied by A. cardamines. The intensity of predation decreased with increasing plant population size and was not affected by isolation. Probability of attack decreased with increasing shading, and intensity of predation was higher in grazed than in non-grazed habitats. The attack intensity increased with increasing mean flower number of plant population, but was not affected by flowering phenology. Individuals in tetraploid populations suffered on average from higher levels of seed predation, had higher mean flower number, were less shaded and occurred more often in grazed habitats than octoploid populations. When accounting for differences in habitat preferences between ploidy levels there was no longer a difference in intensity of predation, suggesting that the observed differences in attack rates among populations of the two ploidy levels are mediated by the habitat. Overall, our results suggest that polyploidization is associated with differentiation in habitat preferences and phenotypic traits leading to differences in interspecific interaction among plant populations. This, in turn, may facilitate further divergence of ploidy levels.     

A new non-equilibrium enzyme linked immunosorbent assay for a glycogen-derived urinary tetrasaccharide

The tetrasaccharide, Glc1-6Glc1-4Glc1-4Glc, denoted (Glc)₄, is a limit dextrin formed by amylolytic degradation of glycogen. In order to evaluate the possible clinical importance of (Glc)₄ excretion as an indicator of certain physiological and pathological conditions, we have developed a new rapid and inexpensive immunoassay using a monoclonal antibody raised against (Glc)₄ glycosidically-linked to a carrier protein. As the antibody is highly specific, it can be used to measure native (Glc)₄ directly, without the chemical reduction step required in previous methods. A new type of non-equilibrium ELISA inhibition test was developed based on the capacity of free (Glc)₄ to decrease initial rates of antibody binding to (Glc)₄-coated microtiter wells. The method is highly reproducible and is as sensitive and accurate as the gas chromatography method or radioimmunoassay used previously.Abbreviations (Glc)₄ Glc1-6Glc1-4Glc1-4Glc - KLH keyhole limpet hemacyanin - BSA bovine serum albumin - PEG polyethylene glycol     

A novel assay for monoacylglycerol hydrolysis suitable for high-throughput screening

A simple assay for monoacylglycerol hydrolysis suitable for high-throughput screening is described. The assay uses [(3)H]2-oleoylglycerol as substrate, with the tritium label in the glycerol part of the molecule and the use of phenyl sepharose gel to separate the hydrolyzed product ([(3)H]glycerol) from substrate. Using cytosolic fractions derived from rat cerebella as a source of hydrolytic activity, the assay gives the appropriate pH profile and sensitivity to inhibition with compounds known to inhibit hydrolysis of this substrate. The assay could also be adapted to a 96-well plate format, using C6 cells as the source of hydrolytic activity. Thus the assay is simple and appropriate for high-throughput screening of inhibitors of monoacylglycerol hydrolysis.     

Interaction with Grb14 results in site-specific regulation of tyrosine phosphorylation of the insulin receptor

The dynamics of interaction of the insulin receptor (IR) with Grb14 was monitored, in real time, in living human embryonic kidney cells, using bioluminescence resonance energy transfer (BRET). We observed that insulin rapidly and dose-dependently stimulated this interaction. We also observed that insulin-induced BRET between the IR and protein tyrosine phosphatase 1B (PTP1B) was markedly reduced by Grb14, suggesting that Grb14 regulated this interaction in living cells. Using site-specific antibodies against phosphorylated tyrosines of the IR, we showed that Grb14 protected the three tyrosines of the kinase loop from dephosphorylation by PTP1B, while favouring dephosphorylation of tyrosine 972. This resulted in decreased IRS-1 binding to the IR and decreased activation of the extracellular signal-regulated kinase pathway. Increased Grb14 expression in human liver-derived HuH7 cells also seemed to specifically decrease the phosphorylation of Y972. Our work therefore suggests that Grb14 may regulate signalling through the IR by controlling its tyrosine dephosphorylation in a site-specific manner.     

Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation

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Process optimization for increased yield of surface-expressed protein in Escherichia coli

The autotransporter family of Gram-negative protein exporters has been exploited for surface expression of recombinant passenger proteins. While the passenger in some cases was successfully translocated, a major problem has been low levels of full-length protein on the surface due to proteolysis following export over the cytoplasmic membrane. The aim of the present study was to increase the surface expression yield of the model protein SefA, a Salmonella enterica fimbrial subunit with potential for use in vaccine applications, by reducing this proteolysis through process design using Design of Experiments methodology. Cultivation temperature and pH, hypothesized to influence periplasmic protease activity, as well as inducer concentration were the parameters selected for optimization. Through modification of these parameters, the total surface expression yield of SefA was increased by 200 %. At the same time, the yield of full-length protein was increased by 300 %, indicating a 33 % reduction in proteolysis.     

Identification of New Differentially Methylated Genes That Have Potential Functional Consequences in Prostate Cancer

Many differentially methylated genes have been identified in prostate cancer (PCa), primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19) and adjacent normal tissues (n = 4) and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05), defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa). Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.     

Characterization of EVL-I as a protein kinase D substrate

EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPβ and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell–cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, filopodia and cell–cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domain.     

Folding into a beta-hairpin can prevent amyloid fibril formation

The tetrapeptide KFFE is one of the shortest amyloid fibril-forming peptides described. Herein, we have investigated how the structural environment of this motif affects polymerization. Using a turn motif (YNGK) or a less rigid sequence (AAAK) to fuse two KFFE tetrapeptides, we show by several biophysical methods that the amyloidogenic properties are strongly dependent on the structural environment. The dodecapeptide KFFEAAAKKFFE forms abundant thick fibril bundles. Freshly dissolved KFFEAAAKKFFE is monomeric and shows mainly disordered secondary structure, as evidenced by circular dichroism, NMR spectroscopy, hydrogen/deuterium exchange measurements, and molecular modeling studies. In sharp contrast, the dodecapeptide KFFEYNGKKFFE does not form fibrils but folds into a stable beta-hairpin. This structure can oligomerize into a stable 12-mer and multiples thereof, as shown by size exclusion chromatography, sedimentation analysis, and electrospray mass spectrometry. These data indicate that the structural context in which a potential fibril forming sequence is present can prevent fibril formation by favoring self-limiting oligomerization over polymerization.