The ovaries of aphids (Hemiptera,Sternorrhyncha, Aphidoidea): morphology and phylogenetic implications

The ovaries of aphids belonging to the families Eriosomatidae, Anoeciidae, Drepanosiphidae, Thelaxidae, Aphididae, and Lachnidae were examined at the ultrastructural level. The ovaries of these aphids are composed of several telotrophic ovarioles. The individual ovariole is differentiated into a terminal filament, tropharium, vitellarium, and pedicel (ovariolar stalk). Terminal filaments of all ovarioles join together into the suspensory ligament, which attaches the ovary to the lobe of the fat body. The tropharium houses individual trophocytes and early previtellogenic oocytes termed arrested oocytes. Trophocytes are connected with the central part of the tropharium, the trophic core, by means of broad cytoplasmic processes. One or more oocytes develop in the vitellarium. Oocytes are surrounded by a single layer of follicular cells, which do not diversify into distinct subpopulations. The general organization of the ovaries in oviparous females is similar to that of the ovaries in viviparous females, but there are significant differences in their functioning: (1) in viviparous females, all ovarioles develop, whereas in oviparous females, some of them degenerate; (2) the number of germ cells per ovariole is usually greater in females of the oviparous generation than in females of viviparous generations; (3) in oviparous females, oocytes in the vitellarium develop through three stages (previtellogenesis, vitellogenesis, and choriogenesis), whereas in viviparous females, the development of oocytes stops after previtellogenesis; and (4) in the oocyte cytoplasm of oviparous females, lipid droplets and yolk granules accumulate, whereas in viviparous females, oocytes accrue only lipid droplets. Our results indicate that a large number of germ cells per ovariole represent the ancestral state within aphids. This trait may be helpful in inferring the phylogeny of Aphidoidea.     

Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny,Physiology, and Niche Adaptation on Human Skin

Malassezia is a unique lipophilic genus in class Malasseziomycetes in Ustilaginomycotina, (Basidiomycota, fungi) that otherwise consists almost exclusively of plant pathogens. Malassezia are typically isolated from warm-blooded animals, are dominant members of the human skin mycobiome and are associated with common skin disorders. To characterize the genetic basis of the unique phenotypes of Malassezia spp., we sequenced the genomes of all 14 accepted species and used comparative genomics against a broad panel of fungal genomes to comprehensively identify distinct features that define the Malassezia gene repertoire: gene gain and loss; selection signatures; and lineage-specific gene family expansions. Our analysis revealed key gene gain events (64) with a single gene conserved across all Malassezia but absent in all other sequenced Basidiomycota. These likely horizontally transferred genes provide intriguing gain-of-function events and prime candidates to explain the emergence of Malassezia. A larger set of genes (741) were lost, with enrichment for glycosyl hydrolases and carbohydrate metabolism, concordant with adaptation to skin’s carbohydrate-deficient environment. Gene family analysis revealed extensive turnover and underlined the importance of secretory lipases, phospholipases, aspartyl proteases, and other peptidases. Combining genomic analysis with a re-evaluation of culture characteristics, we establish the likely lipid-dependence of all Malassezia. Our phylogenetic analysis sheds new light on the relationship between Malassezia and other members of Ustilaginomycotina, as well as phylogenetic lineages within the genus. Overall, our study provides a unique genomic resource for understanding Malassezia niche-specificity and potential virulence, as well as their abundance and distribution in the environment and on human skin.     

Raman imaging of changes in the polysaccharides distribution in the cell wall during apple fruit development and senescence

Planta - Du ring on-tree ripening, the pectin distribution changed from polydispersed in cell wall to cumulated in cell wall corners. During apple storage, the pectin distribution returned to...     

Computational Effective Fault Detection by Means of Signature Functions

The paper presents a computationally effective method for fault detection. A system’s responses are measured under healthy and ill conditions. These signals are used to calculate so-called signature functions that create a signal space. The current system’s response is projected into this space. The signal location in this space easily allows to determine the fault. No classifier such as a neural network, hidden Markov models, etc. is required. The advantage of this proposed method is its efficiency, as computing projections amount to calculating dot products. Therefore, this method is suitable for real-time embedded systems due to its simplicity and undemanding processing capabilities which permit the use of low-cost hardware and allow rapid implementation. The approach performs well for systems that can be considered linear and stationary. The communication presents an application, whereby an industrial process of moulding is supervised. The machine is composed of forms (dies) whose alignment must be precisely set and maintained during the work. Typically, the process is stopped periodically to manually control the alignment. The applied algorithm allows on-line monitoring of the device by analysing the acceleration signal from a sensor mounted on a die. This enables to detect failures at an early stage thus prolonging the machine’s life.     

Engineering TaqII bifunctional endonuclease DNA recognition fidelity: the effect of a single amino acid substitution within the methyltransferase catalytic site

The aim of this study was to improve a useful molecular tool—TaqII restriction endonuclease-methyltransferase—by rational protein engineering, as well as to show an application of our novel method of restriction endonuclease activity modulation through a single amino acid change in the NPPY motif of methyltransferase. An amino acid change was introduced using site-directed mutagenesis into the taqIIRM gene. The mutated gene was expressed in Escherichia coli. The protein variant was purified and characterized. Previously, we described a TspGWI variant with an amino acid change in the methyltransferase motif IV. Here, we investigate a complex, pleiotropic effect of an analogous amino acid change on its homologue—TaqII. The methyltransferase activity is reduced, but not abolished, while TaqII restriction endonuclease can be reactivated by sinefungin, with an increased DNA recognition fidelity. The general method for engineering of the IIS/IIC/IIG restriction endonuclease activity/fidelity is developed along with the generation of an improved TaqII enzyme for biotechnological applications. A successful application of our novel strategy for restriction endonuclease activity/fidelity alteration, based on bioinformatics analyses, mutagenesis and the use of cofactor-analogue activity modulation, is presented.     

Effect of temperate forest tree species on soil dehydrogenase and urease activities in relation to other properties of soil derived from loess and glaciofluvial sand

We investigated the effects of several tree species on dehydrogenase and urease activities in soils derived from two different parent materials (glaciofluvial sand and loess) in forested areas in southern Poland. We hypothesized that coniferous forests (pine, spruce) alter the soil cation exchange capacity (CEC) and decrease soil pH and, therefore, might decrease soil enzyme activities compared with broadleaf species growing on similar soils. Eight paired plots (12 × 12 m) were established on glaciofluvial sand in pine (Pinus sylvestris) + oak (Quercus robur) and spruce (Picea abies) + pine stands, as well as on loess-derived soils: beech (Fagus sylvatica) + pine and hornbeam (Carpinus betulus) + pine stands. Each plot was a 4 × 4 m grid with 16 sampling points. In soil samples pH, soil texture, and organic carbon, nitrogen, base cation contents, dehydrogenase and urease activities were determined. On both parent materials, the soil pH was lower under coniferous species than under broadleaf species. The acidifying effect of tree species on sandy soil was in the order of spruce = pine > oak, while that on loess was pine > beech > hornbeam. Hornbeam and oak increased the soil pH and stimulated enzyme activity in the soil. The content of fine fraction enhanced potential enzyme activities in soils, thus the loess soils had greater dehydrogenase and urease activity. The results suggest that pine stores more soil organic C in association with silt increasing the pool of stabilized soil organic C.     

Truncated amplification: a method for high-fidelity template-driven nucleic acid amplification

The error rate of conventional PCR is problematic when amplifying from single cells or amplifying segments for protein functional analysis by in vitro translation. We describe truncated amplification, a method for high-fidelity amplification in which DNA polymerase errors are not propagated efficiently and original DNA templates exert greater influence on the amplification process. Truncated amplification utilizes pairs of oligonucleotides and thermal cycling, but it differs from PCR. Truncated amplification amplifies non-exponentially with one or two chimeric oligonucleotides and produces truncated terminal products that are no more than three rounds of replication from the original template. Exon 6 of the p53 gene was utilized as a model system to demonstrate proof of principle. Chimeric oligonucleotides containing three 3'-->5' reversed-deoxynucleotides or 2'-OMe-ribonucleotides at 6-8 nucleotides from the 3 'terminus retained sequence specificity and primer extension activity. With PfuTurbo but not with Taq or Vent (exo-) DNA polymerases, the modified nucleotides completely truncated the DNA polymerase elongation. The resulting truncated terminal products are not templates for further amplification because of the short length of the 3' complementary region. Truncated amplific ation can amplify quadratically or geometrically depending on whether two or one chimeric oligonucleotides are used. Truncated amplification is a promising approach when template-driven amplification is desired to increase thefrequency of error-free products.     

The membrane-associated lipoprotein-9 GmpC from Staphylococcus aureus binds the dipeptide GlyMet via side chain interactions

Bacterial dipeptide ABC transporters function to import a wide range of dipeptide substrates. This ability to transport a wide variety of dipeptides is conferred by the cognate substrate binding protein (SBP) of these transporters. SBPs bind dipeptides with little regard for their amino acid content. Here, we report the 1.7 A resolution structure of lipoprotein-9 (SA0422) of Staphylococcus aureus in complex with the dipeptide glycylmethionine. Experimental characterization of the subcellular location of the protein confirmed that SA0422 is an acylated, peripheral membrane protein. This is the first structure determined for an SBP of a Gram-positive dipeptide ABC transporter. Usually, binding of dipeptides occurs in a binding pocket that is largely hydrated and able to accommodate the side chains of several different amino acid residues. Unlike any other known SBP, lipoprotein-9 binds the side chains of the glycylmethionine dipeptide through very specific interactions. Lipoprotein-9 shares significant structural and sequence homology with the MetQ family of methionine SBP. Sequence comparisons between MetQ-like proteins and lipoprotein-9 suggest that the residues forming the tight interactions with the methionine side chains of the ligand are highly conserved between lipoprotein-9 and MetQ homologues, while the residues involved in coordinating the glycine residue are not. Modeling of the Vibrio cholerae MetQ and lipoprotein-9 binding pockets can account for lipoprotein-9 substrate specificity toward glycylmethionine. For this reason, we have designated lipoprotein-9 GmpC, for glycylmethionine binding protein.     

Assessment of fire blight tolerance in apple based on plant inoculations with Erwinia amylovora and DNA markers

Fire blight (Erwinia amylovora) causes serious damage to pome fruit orchards, and identification of germplasm with heritable disease resistance is therefore crucial. Two dominant SCAR (sequence characterised amplified region) marker alleles (AE10-375 and GE-8019), flanking a previously identified QTL (quantitative trait locus) for resistance to fire blight on ‘Fiesta’ linkage group 7 in apple cultivars related to ‘Cox’s Orange Pippin’, were screened on 205 apple cultivars. Both marker alleles were present in 22% of the cultivars, indicating presence of the QTL allele for tolerance, and both were lacking in 25%, indicating homozygosity for absence of the QTL tolerance allele. However, 33% had only the marker allele AE10-375, while 20% had only GE-8019, suggesting that some cultivars with the dominant alleles for both of the flanking markers can carry these on separate chromosomes and may lack the QTL allele for tolerance. In 2009 and 2010, terminal shoots of greenhouse-grown grafted trees of 21 cultivars (only 20 in 2010) were inoculated with Erwinia amylovora. ‘Idared’ (susceptible) and ‘Enterprise’ (tolerant) were included as controls. Disease severity for each cultivar was expressed as percentage of necrosis in relation to entire length of shoot, and the ranking of cultivars in 2009 and 2010 was compared with a Spearman rank correlation test, P < 0.01. A relationship between presence of both flanking marker alleles for tolerance and level of fire blight tolerance was confirmed with a Mann–Whitney U-test, P < 0.01 in 2009, and P < 0.05 in 2010. A PCO (principal coordinate) analysis based on band profiles obtained with 12 SSR (simple sequence repeat) loci produced three loose clusters, two of which contained known offspring of ‘Cox’s Orange Pippin’, and one with cultivars that were either unrelated or had an unknown origin. Cases where DNA markers did not predict level of fire blight damage as expected, were, however, as common among descendants of ‘Cox’s Orange Pippin’ as among apparently unrelated cultivars. Obviously the ‘Fiesta’ LG 7 QTL has some predictive value, both for known ‘Cox’ relatives and others, but more efficient markers would be desirable for marker-assisted selection.