Molecular Detection of 10 of the Most Unwanted Alien Forest Pathogens in Canada Using Real-Time PCR

Invasive alien tree pathogens can cause significant economic losses as well as large-scale damage to natural ecosystems. Early detection to prevent their establishment and spread is an important approach used by several national plant protection organizations (NPPOs). Molecular detection tools targeting 10 of the most unwanted alien forest pathogens in Canada were developed as part of the TAIGA project (http://taigaforesthealth.com/). Forest pathogens were selected following an independent prioritization. Specific TaqMan real-time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96- or 384-well plate format arrays for high-throughput testing of large numbers of samples against multiple targets. Assays were validated for 1) specificity, 2) sensitivity, 3) precision, and 4) robustness on environmental samples. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely-related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and ten target gene region copies. Precision or repeatability of each assay revealed a mean coefficient of variation of 3.4%. All assays successfully allowed detection of target pathogen on positive environmental samples, without any non-specific amplification. These molecular detection tools will allow for rapid and reliable detection of 10 of the most unwanted alien forest pathogens in Canada.     

Bullying and Victimization in Overweight and Obese Outpatient Children and Adolescents: An Italian Multicentric Study

Objective

Being overweight or obese is one of the most common reasons that children and adolescents are teased at school. We carried out a study in order to investigate: i) the relation between weight status and school bullying and ii) the relation between weight status categories and types of victimization and bullying in an outpatient sample of Italian children and adolescents with different degrees of overweight from minimal overweight up to severe obesity.

Participants/Methods

Nine-hundred-forty-seven outpatient children and adolescents (age range 6.0–14.0 years) were recruited in 14 hospitals distributed over the country of Italy. The participants were classified as normal-weight (N = 129), overweight (N = 126), moderately obese (N = 568), and severely obese (N = 124). The nature and extent of verbal, physical and relational bullying and victimization were assessed with an adapted version of the revised Olweus bully-victim questionnaire. Each participant was coded as bully, victim, bully-victim, or not involved.

Results

Normal-weight and overweight participants were less involved in bullying than obese participants; severely obese males were more involved in the double role of bully and victim. Severely obese children and adolescents suffered not only from verbal victimization but also from physical victimization and exclusion from group activities. Weight status categories were not directly related to bullying behaviour; however severely obese males perpetrated more bullying behaviour compared to severely obese females.

Conclusions

Obesity and bullying among children and adolescents are of ongoing concern worldwide and may be closely related. Common strategies of intervention are needed to cope with these two social health challenges.     

Isoelectric points of multi-domain proteins

Although the distribution of protein isoelectric points is multi-modal, large proteins show isoelectric points less variable than small proteins and their isoelectric points tend to converge to a unique value, close to the pH of the milieu in which the proteins are functional, as far as the protein dimension increases. This study demonstrates that large proteins, which contain more than a single domain, do have isoelectric points less variable than small proteins, which contains a single domain. However, the distribution of the isoelectric points of the single domains, contained in large proteins, resembles that of small proteins, which contain a single domain. Thus, large proteins can be soluble even if their pI is very close to the pH of the milieu, in which they perform their function, since they can contain several domains, the electrostatic properties of each of which mirror those of small proteins.     

Quaternary structure change as a mechanism for the regulation of thymidine kinase 1-like enzymes

The human cytosolic thymidine kinase (TK) and structurally related TKs in prokaryotes play a crucial role in the synthesis and regulation of the cellular thymidine triphosphate pool. We report the crystal structures of the TK homotetramer from Thermotoga maritima in four different states: its apo-form, a binary complex with thymidine, as well as the ternary structures with the two substrates (thymidine/AppNHp) and the reaction products (TMP/ADP). In combination with fluorescence spectroscopy and mutagenesis experiments, our results demonstrate that ATP binding is linked to a substantial reorganization of the enzyme quaternary structure, leading to a transition from a closed, inactive conformation to an open, catalytic state. We hypothesize that these structural changes are relevant to enzyme function in situ as part of the catalytic cycle and serve an important role in regulating enzyme activity by amplifying the effects of feedback inhibitor binding.     

Transgenic mice expressing a dual, CRE-inducible reporter for the analysis of axon guidance and synaptogenesis

Improved and modular tools are needed for the neuroanatomical dissection of CNS axonal tracts, and to study the cell-intrinsic and cell-extrinsic cues that govern their assembly and plasticity. Here we describe a general purpose transgenic tracer that can be used to visualize axonal tracts and synaptic terminals in any region of the embryonic neural tube or postnatal CNS, on any wild type or mutant genetic background. The construct permits CRE-inducible expression of a dicistronic axonal marker encoding two surface reporter proteins: a farnesylated GFP and the human Placental Alkaline Phosphatase (PLAP). Both proteins localize alongside the neuronal surface, permitting the concomitant detection of cell body, neurites, and presynaptic and postsynaptic sites in the same neuron. This provides a CRE-inducible dual system for imaging neural circuits in vivo, and to study their assembly and remodeling in cultured neurons, neural stem cells, and tissue explants derived from the reporter line. Unlike existing lines, this reporter does not encode a ubiquitously expressed, floxable LacZ gene, permitting the simultaneous analysis of beta galactosidase activity in mutant lines.     

NADP-Dependent enzymes. I: Conserved stereochemistry of cofactor binding

The ubiquitous redox cofactors nicotinamide adenine dinucleotides [NAD and NADP] are very similar molecules, despite their participation in substantially different biochemical processes. NADP differs from NAD in only the presence of an additional phosphate group esterified to the 2′-hydroxyl group of the ribose at the adenine end and yet NADP is confined with few exceptions to the reactions of reductive biosynthesis, whereas NAD is used almost exclusively in oxidative degradations. The discrimination between NAD and NADP is therefore an impressive example of the power of molecular recognition by proteins. The many known tertiary structures of NADP complexes affords the possibility for an analysis of their discrimination. A systematic analysis of several crystal structures of NAD(P)-protein complexes show that: 1) the NADP coenzymes are more flexible in conformation than those of NAD; 2) although the protein-cofactor interactions are largely conserved in the NAD complexes, they are quite variable in those of NADP; and 3) in both cases the pocket around the nicotinamide moiety is substrate dependent. The conserved and variable interactions between protein and cofactors in the respective binding pockets are reported in detail. Discrimination between NAD and NADP is essentially a consequence of the overall pocket and not of a few residues. A clear fingerprint in NAD complexes is a carboxylate side chain that chelates the diol group at the ribose near the adenine, whereas in NADP complexes an arginine side chain faces the adenine plane and interacts with the phosphomonoester. The latter type of interaction might be a general feature of recognition of nucleotides by proteins. Other features such as strand-like hydrogen bonding between the NADP diphosphate moeties and the protein are also significant. The NADP binding pocket properties should prove useful in protein engineering and design. © 1997 Wiley-Liss Inc.     

Protein Degradation and Apoptotic Death in Lymphocytes during Fiv Infection: Activation of the Ubiquitin–Proteasome Proteolytic System

The movement of a cell through the sequential phases of apoptosis is accompanied by a progressive decrease in cell size with loss in protein mass. In lymphocytes from Hiv-infected persons, protein loss during apoptosis is due to increased protein degradation rather than decreased synthesis. To identify and characterize the proteolytic enzymes or enzyme systems involved in this process, we studied several features of protein turnover in lymphocytes from peripheral blood and lymph nodes during the natural and experimental infection by feline immunodeficiency virus (Fiv). This animal model allowed us to integratein vivoresults within vitroobservations of protein damage. Here we report that protein breakdown in apoptotic cells is concomitant with the activation of the ATP and ubiquitin-dependent multicatalytic system (proteasome). We suggest that proteasome activation is part of the proteolytic cascade in the execution phases of apoptosis in AIDS.     

Insufficient uracil supply in fully aerobic chemostat cultures of Saccharomyces cerevisiae leads to respiro-fermentative metabolism and double nutrient-limitation

A combination of chemostat cultivation and a defined medium was used to demonstrate that uracil limitation leads to a drastic alteration in the physiology of auxotrophic cells of Saccharomyces cerevisiae. Under this condition, the carbon source is dissimilated mainly to ethanol and acetate, even in fully aerobic cultures grown at 0.1 h^?1, which is far below the critical dilution rate. Differently from nitrogen-, sulphur-, or phosphate-limited cultures, uracil limitation leads to residual sugar (either glucose or sucrose) concentrations below 2 mM, which characterizes a situation of double-limitation: by the carbon source and by uracil. Furthermore, the specific rates of CO₂ production and O₂ consumption are increased when compared to the corresponding prototrophic strain. We conclude that when auxotrophic strains are to be used for quantitative physiological studies, special attention must be paid to the cultivation conditions, mainly regarding medium formulation, in order to avoid limitation of growth by the auxotrophic nutrient.