Development of molecular tools for the mulundocandin producer<Emphasis Type="Italic"> Aspergillus sydowii</Emphasis>: DNA-mediated transformation and reporter gene expression

The echinocandin-type antimycotic mulundocandin and its derivatives are produced by the filamentous fungus Aspergillus sydowii (strain FH2551). These agents have been considered as a potential drug to treat immunocompromised patients who suffer from severe opportunistic fungal infections. In order to generate strains with a modified mulundocandin biosynthesis, we developed molecular tools for genetic engineering of A. sydowii as an alternative to conventional strain improvement procedures. For our experiments, we used strain FH2551, which was discriminated from other Aspergillus strains by determining the sequence of the two internal transcribed spacers (ITS1 and ITS2) of the rDNA locus. In addition, the electrophoretic karyotype of A. sydowii was established using pulsed-field gel electrophoresis (PFGE), leading to a calculated genomic size of about 40 Mb. For gene mapping, chromosomes were subjected to PFGE either unrestricted or after incubation with rare cutting enzymes and probed with heterologous genes. Using the bacterial hygromycin B phosphotransferase gene as a selectable marker for transformation of A. sydowii, we generated transformants with single and multiple copies of plasmid DNA. Subsequently, the heterologous lacZ and gfp genes were efficiently transferred and expressed in A. sydowii. The majority of lacZ-transformants showed more than 6 pkat beta-galactosidase activity/mg protein, while the control strains had no significant background activity. Fluorescence microscopy of gfp-transformants demonstrated that the green-fluorescent protein is present in a stable and active form in the cytoplasm of vegetative hyphae and conidiospores.     

Desmoid tumors of the abdominal wall: A case report

Background  

Desmoid tumors are slow growing deep fibromatoses with aggressive infiltration of adjacent tissue but without any metastatic potential.     

SH2-containing inositol 5'-phosphatase SHIP2 associates with the p130(Cas) adapter protein and regulates cellular adhesion and spreading

In a previous study, we found that the SHIP2 protein became tyrosine phosphorylated and associated with the Shc adapter protein in response to the treatment of cells with growth factors and insulin (T. Habib, J. A. Hejna, R. E. Moses, and S. J. Decker, J. Biol. Chem. 273:18605-18609, 1998). We describe here a novel interaction between SHIP2 and the p130(Cas) adapter protein, a mediator of actin cytoskeleton organization. SHIP2 and p130(Cas) association was detected in anti-SHIP2 immunoprecipitates from several cell types. Reattachment of trypsinized cells stimulated tyrosine phosphorylation of SHIP2 and increased the formation of a complex containing SHIP2 and a faster-migrating tyrosine-phosphorylated form of p130(Cas). The faster-migrating form of p130(Cas) was no longer recognized by antibodies to the amino terminus of p130(Cas) and appeared to be generated through proteolysis. Interaction of the SHIP2 protein with the various forms of p130(Cas) was mediated primarily through the SH2 domain of SHIP2. Immunofluorescence studies indicated that SHIP2 localized to focal contacts and to lamellipodia. Increased adhesion was observed in HeLa cells transiently expressing exogenous WT-SHIP2. These effects were not seen with SHIP2 possessing a mutation in the SH2 domain (R47G). Transfection of a catalytic domain deletion mutant of SHIP2 (DeltaRV) inhibited cell spreading. Taken together, our studies suggest an important role for SHIP2 in adhesion and spreading.     

A self-perpetuating vicious cycle of tissue damage in human hibernating myocardium

Recently, we proposed the hypothesis that a vicious cycle exists in human hibernating myocardium (HM) between the progression of myocyte degeneration and the development of fibrosis [1]. We now investigated the pathomechanism of this cycle in more detail and established a correlation between the severity of the morphological changes and the degree of postoperative functional recovery of HM.HM was diagnosed by dobutamine echocardiography, thallium-201 scintigraphy and radionuclide ventriculography. Functional recovery was present at 3 months after coronary bypass surgery but remained unchanged at 15 months. Forty patients were subdivided into 2 groups: A with complete and B with incomplete recovery. Biopsies taken during surgery and studied by electron microscopy, immunocytochemistry, rt-PCR, and morphometry revealed myocyte degeneration and inflammatory and fibrinogenic changes in a widened interstitial space. We report here for the first time an upregulation of TGF-₁ evident by a 5-fold increase of fibroblasts and macrophages exhibiting a TGF-₁ content 3-fold larger than in control, and a > 3-fold increase in TGF-₁ mRNA by rt-PCR. The number of angiotensin converting enzyme (ACE) containing structures was increased (n/mm²: control - 11.4, A - 17.6, B - 19.2, control vs. A and B, p < 0.05). Fibrosis was more severe in group B than A or control (%: C - 10.1; A - 21.2; B - 40.6; p < 0.05). Capillary density was significantly reduced (n/mm²: C - 1152; A - 782; B - 579, p < 0.05) and intercapillary distance was widened (m: C - 29.5, A - 36.1, B - 43.3, p < 0.05). The number of CD 3 (n/mm²: C - 5.0; A - 9.6; B - 9.4, ns) and CD 68 positive cells (n/mm²: C - 37.2; A - 80.7; B - 55.0, C vs. A p < 0.05) was elevated in HM as compared to control indicating an inflammatory reaction. Cut-off points for functional recovery are fibrosis > 32%, capillary density < 660/mm² and intercapillary distance > 39.0 m.In HM a self-perpetuating vicious cycle of tissue alterations leads to progressive replacement fibrosis and continuous intracellular degeneration which should be interrupted by early revascularization.     

Temperature adaptation influences the aggregation state of hemocyanin from Astacus leptodactylus

When Astacus leptodactylus were kept at various temperatures for several weeks, different ratios between di-hexameric and hexameric hemocyanins were observed in their hemolymph. The higher the temperature the more hexamers were present. This long-term adaptation to different temperatures or/and to temperature-induced pH-shifts as observed in the hemolymph has different effects on the expression of subunit types building up hexamers and those which covalently link two hexamers within the di-hexamers. The oxygen binding behaviour of di-hexameric hemocyanins from cold and warm adapted animals do not show differences with respect to affinity, Bohr effect and cooperativity.     

Functional electrical stimulation cycling improves body composition,metabolic and neural factors in persons with spinal cord injury

Persons with spinal cord injury (SCI) are at a heightened risk of developing type II diabetes and cardiovascular disease. The purpose of this investigation was to conduct an analysis of metabolic, body composition, and neurological factors before and after 10 weeks of functional electrical stimulation (FES) cycling in persons with SCI. Eighteen individuals with SCI received FES cycling 2–3 times per week for 10 weeks. Body composition was analyzed by dual X-ray absorptiometry. The American Spinal Injury Association (ASIA) neurological classification of SCI test battery was used to assess motor and sensory function. An oral glucose tolerance (OGTT) and insulin-response test was performed to assess blood glucose control. Additional metabolic variables including plasma cholesterol (total-C, HDL-C, LDL-C), triglyceride, and inflammatory markers (IL-6, TNF-α, and CRP) were also measured. Total FES cycling power and work done increased with training. Lean muscle mass also increased, whereas, bone and adipose mass did not change. The ASIA motor and sensory scores for the lower extremity significantly increased with training. Blood glucose and insulin levels were lower following the OGTT after 10 weeks of training. Triglyceride levels did not change following training. However, levels of IL-6, TNF-α, and CRP were all significantly reduced.     

Consider the Source: Adolescents and Adults Similarly Follow Older Adult Advice More than Peer Advice

Individuals learn which of their actions are likely to be rewarded through trial and error. This form of learning is critical for adapting to new situations, which adolescents frequently encounter. Adolescents are also greatly influenced by their peers. The current study tested the extent to which adolescents rely on peer advice to guide their actions. Adolescent and young adult participants completed a probabilistic learning task in which they chose between four pairs of stimuli with different reinforcement probabilities, with one stimulus in each pair more frequently rewarded. Participants received advice about two of these pairs, once from a similarly aged peer and once from an older adult. Crucially, this advice was inaccurate, enabling the dissociation between experience-based and instruction-based learning. Adolescents and adults learned equally well from experience and no age group difference was evident in the overall influence of advice on choices. Surprisingly, when considering the source of advice, there was no evident influence of peer advice on adolescent choices. However, both adolescents and adults were biased toward choosing the stimulus recommended by the older adult. Contrary to conventional wisdom, these data suggest that adolescents may prioritize the advice of older adults over that of peers in certain decision-making contexts.     

Genotypic Tannin Levels in Populus tremula Impact the Way Nitrogen Enrichment Affects Growth and Allocation Responses for Some Traits and Not for Others

Plant intraspecific variability has been proposed as a key mechanism by which plants adapt to environmental change. In boreal forests where nitrogen availability is strongly limited, nitrogen addition happens indirectly through atmospheric N deposition and directly through industrial forest fertilization. These anthropogenic inputs of N have numerous environmental consequences, including shifts in plant species composition and reductions in plant species diversity. However, we know less about how genetic differences within plant populations determine how species respond to eutrophication in boreal forests. According to plant defense theories, nitrogen addition will cause plants to shift carbon allocation more towards growth and less to chemical defense, potentially enhancing vulnerability to antagonists. Aspens are keystone species in boreal forests that produce condensed tannins to serve as chemical defense. We conducted an experiment using ten Populus tremula genotypes from the Swedish Aspen Collection that express extreme levels of baseline investment into foliar condensed tannins. We investigated whether investment into growth and phenolic defense compounds in young plants varied in response to two nitrogen addition levels, corresponding to atmospheric N deposition and industrial forest fertilization. Nitrogen addition generally caused growth to increase, and tannin levels to decrease; however, individualistic responses among genotypes were found for height growth, biomass of specific tissues, root:shoot ratios, and tissue lignin and N concentrations. A genotype’s baseline ability to produce and store condensed tannins also influenced plant responses to N, although this effect was relatively minor. High-tannin genotypes tended to grow less biomass under low nitrogen levels and more at the highest fertilization level. Thus, the ability in aspen to produce foliar tannins is likely associated with a steeper reaction norm of growth responses, which suggests a higher plasticity to nitrogen addition, and potentially an advantage when adapting to higher concentrations of soil nitrogen.     

Differences in Energy Balance-Related Behaviours in European Preschool Children: The ToyBox-Study

Background

The aim of the current study was to compare levels of energy balance-related behaviours (physical activity, sedentary behaviour, and dietary behaviours (more specifically water consumption, sugar-sweetened beverage consumption and unhealthy snacking)) in four- to six-year-old preschoolers from six European countries (Belgium, Bulgaria, Germany, Greece, Poland, and Spain) within the ToyBox cross-sectional study.

Methods

A sample of 4,045 preschoolers (4.77 ± 0.43 years; 52.2% boys) had valid physical activity data (steps per day), parents of 8,117 preschoolers (4.78 ± 0.46 years; 53.0% boys) completed a parental questionnaire with questions on sedentary behaviours (television viewing, computer use, and quiet play), and parents of 7,244 preschoolers (4.77 ± 0.44 years; 52.0% boys) completed a food frequency questionnaire with questions on water consumption, sugar-sweetened beverage consumption and unhealthy snacking.

Results

The highest levels of physical activity were found in Spain (12,669 steps/day on weekdays), while the lowest levels were found in Bulgaria and Greece (9,777 and 9,656 steps/day on weekdays, respectively). German preschoolers spent the least amount of time in television viewing (43.3 min/day on weekdays), while Greek preschoolers spent the most time in television viewing (88.5 min/day on weekdays). A considerable amount of time was spent in quiet play in all countries, with the highest levels in Poland (104.9 min/day on weekdays), and the lowest levels in Spain (60.4 min/day on weekdays). Belgian, German, and Polish preschoolers had the lowest intakes of water and the highest intakes of sugar-sweetened beverages. The intake of snacks was the highest in Belgian preschoolers (73.1 g/day) and the lowest in Greek preschoolers (53.3 g/day).

Conclusions

Across six European countries, differences in preschoolers’ energy balance-related behaviours were found. Future interventions should target European preschoolers’ energy balance-related behaviours simultaneously, but should apply country-specific adaptations.