Temperature responses of a plant‐insect system using a food‐web performance approach

Evaluation of the performance of a plant‐herbivore system as a whole is difficult due to the lack of fitness parameters that can be applied to both components. The individual use of traditional measures of performance (e.g., r_m, biomass) can provide useful, but incomplete information on the performance of insect herbivores and seldom incorporates plant performance. We propose the use of the net generational productivity (NGP) to evaluate the fitness of the herbivore, which can then be compared directly with the performance of the plant in biomass units, to obtain the food‐web performance ratio (φ_H/P). We compared three biotypes of the potato aphid, Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae), when raised on three different host plants: potato (Solanum tuberosum L. cv. Norland) and two bell peppers (Capsicum annuum L. cv. Fascinato and cv. Crosby) (all Solanaceae) at temperatures ranging from 8 to 36 °C. The temperature profiles of the potato aphid biotypes suggest that this aphid is better suited to temperate climates, and its performance generally depends on the particular host‐plant/biotype association. Plant growth performance showed that potato has a lower thermal tolerance, but has a faster growth rate than bell peppers, especially in the range of 16–24 °C. Temperature variation in the φ_H/P ratio shows that aphids have a greater performance than plants, especially at lower temperatures, at which they can accumulate biomass up to 148 times faster. Because of the aphid's biological inability to withstand long exposures to temperatures above 28 °C, plants have a slight advantage over aphids. Nonetheless, as the performance of plants is extremely reduced at high temperatures, this advantage cannot withstand long‐term exposures to extreme temperatures. This is the first attempt to obtain a parameter capable of determining the climatic profile and performance of a food web in an inclusive yet simple manner.     

Expression of Gla‐rich protein (GRP) in newly developed cartilage‐derived cell cultures from sturgeon (Acipenser naccarii)

Sturgeons are representative of an ancient fish group, and present mainly an internal cartilaginous skeleton, with bone found essentially in the ganoid plaques forming the exogenous skeleton. Because of its archaic genetics, sturgeon represents an important model organism to understand the role of bone and cartilage‐related Gla proteins and determine if their molecular mechanisms of action were maintained throughout evolution. Of particular relevance is understanding the regulation, in sturgeon, of those proteins known to be involved in tissue mineralization in mammals, as well as unveiling the function of newly identified calcification‐related genes such as the one encoding the recently discovered Gla‐rich protein (GRP), thus contributing to understand the poor calcification observed in sturgeon endoskeleton. However, regulation of gene expression and promoter functional analysis of sturgeon cartilage and bone‐specific genes has been hampered by lack of suitable in vitro cell systems. We have recently developed the first sturgeon vertebra (VAn2H) and branchial arches (BAAn1F) derived cell cultures, and here we report their inability to mineralize their ECM under mineralizing culture conditions, as detected by von Kossa staining. Although a more extensive characterization of these systems is ongoing, our first data indicate that these cells represent a valuable tool for expression analysis of sturgeon bone and cartilage genes.     

Multimodal imaging for tumour characterization from micro‐ to macroscopic level using a newly developed dorsal chamber designed for long‐term follow‐up

Optical imaging of living animals is a unique method of studying the dynamics of physiological and pathological processes at a subcellular level. One‐shot acquisitions at high resolution can be achieved on exteriorized organs before animal euthanasia. For longitudinal follow‐up, intravital imaging can be used and involves imaging windows implanted in cranial, thoracic or dorsal regions. Several imaging window models exist, but none have proven to be applicable for long‐term monitoring and most biological processes take place over several weeks. Moreover, none are compatible with multiple imaging modalities, meaning that different biological parameters cannot be assessed in an individual animal. We developed a new dorsal chamber that was well tolerated by mice (over several months) and allowed individual and collective cell tracking and behaviour analysis by optical imaging, ultrasound and magnetic resonance tomography. This new model broadens potential applications to areas requiring study of long‐term biological processes, as in cancer research.     

Evolutionary factors affecting the cross‐species utility of newly developed microsatellite markers in seabirds

Microsatellite loci are ideal for testing hypotheses relating to genetic segregation at fine spatio‐temporal scales. They are also conserved among closely related species, making them potentially useful for clarifying interspecific relationships between recently diverged taxa. However, mutations at primer binding sites may lead to increased nonamplification, or disruptions that may result in decreased polymorphism in nontarget species. Furthermore, high mutation rates and constraints on allele size may also with evolutionary time, promote an increase in convergently evolved allele size classes, biasing measures of interspecific genetic differentiation. Here, we used next‐generation sequencing to develop microsatellite markers from a shotgun genome sequence of the sub‐Antarctic seabird, the thin‐billed prion (Pachyptila belcheri), that we tested for cross‐species amplification in other Pachyptila and related sub‐Antarctic species. We found that heterozygosity decreased and the proportion of nonamplifying loci increased with phylogenetic distance from the target species. Surprisingly, we found that species trees estimated from interspecific F_ST provided better approximations of mtDNA relationships among the studied species than those estimated using D_C, even though F_ST was more affected by null alleles. We observed a significantly nonlinear second order polynomial relationship between microsatellite and mtDNA distances. We propose that the loss of linearity with increasing mtDNA distance stems from an increasing proportion of homoplastic allele size classes that are identical in state, but not identical by descent. Therefore, despite high cross‐species amplification success and high polymorphism among the closely related Pachyptila species, we caution against the use of microsatellites in phylogenetic inference among distantly related taxa.     

Life‐history strategy varies with the strength of competition in a food‐limited ungulate population

Fluctuating population density in stochastic environments can contribute to maintain life‐history variation within populations via density‐dependent selection. We used individual‐based data from a population of Soay sheep to examine variation in life‐history strategies at high and low population density. We incorporated life‐history trade‐offs among survival, reproduction and body mass growth into structured population models and found support for the prediction that different life‐history strategies are optimal at low and high population densities. Shorter generation times and lower asymptotic body mass were selected for in high‐density environments even though heavier individuals had higher probabilities to survive and reproduce. In contrast, greater asymptotic body mass and longer generation times were optimal at low population density. If populations fluctuate between high density when resources are scarce, and low densities when they are abundant, the variation in density will generate fluctuating selection for different life‐history strategies, that could act to maintain life‐history variation.     

A proteomic approach to the bilirubin‐induced toxicity in neuronal cells reveals a protective function of DJ‐1 protein

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long‐term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB‐induced neuronal toxicity, we used the human neuroblastoma cell line SH‐SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ‐1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin‐induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.     

Validation of BIA in Obese Children and Adolescents and Re‐evaluation in a Longitudinal Study

Decrease in fat mass (FM) is a one of the aims of pediatric obesity treatment; however, measurement techniques suitable for routine clinical assessment are lacking. The objective of this study was to validate whole‐body bioelectrical impedance analysis (BIA; TANITA BC‐418MA) against the three‐component (3C) model of body composition in obese children and adolescents, and to test the accuracy of our new equations in an independent sample studied longitudinally. A total of 77 white obese subjects (30 males) aged 5–22 years, BMI‐standard deviation score (SDS) 1.6–3.9, had measurements of weight, height (HT), body volume, total body water (TBW), and impedance (Z). FM and fat‐free mass (FFM) were calculated using the 3C model or predicted from TANITA. FFM was predicted from HT²/Z. This equation was then evaluated in 17 other obese children (5 males) aged 9–13 years. Compared to the 3C model, TANITA manufacturer's equations overestimated FFM by 2.7 kg (P < 0.001). We derived a new equation: FFM = ?2.211 + 1.115 (HT²/Z), with r² of 0.96, standard error of the estimate 2.3 kg. Use of this equation in the independent sample showed no significant bias in FM or FFM (mean bias 0.5 ± 2.4 kg; P = 0.4), and no significant bias in change in FM or FFM (mean bias 0.2 ± 1.8 kg; P = 0.7), accounting for 58% (P < 0.001) and 55% (P = 0.001) of the change in FM and FFM, respectively. Our derived BIA equation, shown to be reliable for longitudinal assessment in white obese children, will aid routine clinical monitoring of body composition in this population.     

Seasonal changes in neuron numbers in the hippocampal formation of a food‐hoarding bird: The black‐capped chickadee

The volume of the hippocampal formation (HF) in black‐capped chickadees (Poecile atricapillus) varies across the seasons, in parallel with the seasonal cycle in food hoarding. In this study, we estimate cell density and total cell number in the HF across seasons in both juveniles and adults. We find that the seasonal variation in volume is due to an increase in the number of small and large cells (principally neurons) in the fall. Adults also have lower neuron densities than juveniles. Both juveniles and adults show an increase in cell density in the rostral part of the HF in August and a subsequent decrease toward October. This suggests that the net cell addition to the HF may already start in August. We discuss the implications of this early start with respect to the possibility that the seasonal change in HF volume is driven by the experience of food hoarding. We also speculate on the functional significance of the addition of neurons to the HF in the fall. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 414–422, 2000     

Expression of a kallikrein-like protease from the snake venom: engineering of autocatalytic site in the fusion protein to facilitate protein refolding

In order to circumvent the difficulty encountered in the expression and purification of the recombinant products in E. coli system, we have developed a novel and facile method of removing the polyhistidine tag from target proteins after heterologous gene expression. The expression of a serine protease (Tm-5) from Taiwan habu (Trimeresurus mucrosquamatus) is taken as an exemplar to illustrate the basic rationales and protocols involved. In place of an enterokinase recognition site, a polyhistidine tag linked to an autocatalyzed site based on cleavage specificity of the serine protease flanking on the 5'-end of Tm-5 clone sequence was engineered before protein expression in E. coli system. Renaturation of the fusion protein after expression revealed that the recombinant protease had refolded successfully from the inclusion bodies. Upon autocleavage of the expressed protease, the polyhistidine tag with additional amino acid residues appended to the N-terminus of the coding sequence is found to be removed accordingly. The protein expressed and purified by this new strategy possesses a molecular weight of approximately 28,000 in accord with the expected value for this venom protease. Further characterization of the recombinant protein employing a variety of techniques which include immunoblot analysis, RP-HPLC, ESI-MS, and N-terminal amino acid sequencing all shows indistinguishable properties to those of the isolated native protease. Most noteworthy is that the recombinant Tm-5 protease also exhibits amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, a unique and strict substrate for native Tm proteases reported previously.     

From food‐dependent statistics to metabolic parameters,a practical guide to the use of dynamic energy budget theory

The standard model of the dynamic energy budget theory for metabolic organisation has variables and parameters that can be quantified using indirect methods only. We present new methods (and software) to extract food‐independent parameter values of the energy budget from food‐dependent quantities that are easy to observe, and so facilitate the practical application of the theory to enhance predictability and extrapolation. A natural sequence of 10 steps is discussed to obtain some compound parameters first, then the primary parameters, then the composition parameters and finally the thermodynamic parameters; this sequence matches a sequence of required data of increasing complexity which is discussed in detail. Many applications do not require knowledge of all parameters, and we discuss methods to extrapolate parameters from one species to another. The conversion of mass, volume and energy measures of biomass is discussed; these conversions are not trivial because biomass can change in chemical composition in particular ways thanks to different forms of homeostasis. We solve problems like “What would be the ultimate reproduction rate and the von Bertalanffy growth rate at a specific food level, given that we have measured these statistics at abundant food?” and “What would be the maximum incubation time, given the parameters of the von Bertalanffy growth curve?”. We propose a new non‐destructive method for quantifying the chemical potential and entropy of living reserve and structure, that can potentially change our ideas on the thermodynamic properties of life. We illustrate the methods using data on daphnids and molluscs.     

SESAME: A least-squares approach to the evaluation of protein structures computed from NMR data

Summary A method is proposed for defining a probability distribution on an ensemble of protein conformations from a 2D NOE spectrum, while at the same time back-calculating the experimental spectrum from the ensemble. This enables one to assess the relative quality and significance of the conformations, and to test the consistency of the ensemble as a whole with the experimental spectrum. The method eliminates the need to integrate the cross-peak intensities and is surprisingly insensitive to random noise in the spectrum. In this communication, these advantages are demonstrated by applying the method to simulated data, for which the correct result is already known.Abbreviations NOE nuclear Overhauser effect - BPTI basic pancreatic trypsin inhibitor - rmsd root-mean-square deviation     

Laser‐induced liquid bead ion desorption‐MS of protein complexes from blue‐native gels,a sensitive top‐down proteomic approach

We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN‐PAGE) and laser‐induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent‐tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica. For this complex of 947 kDa, typically 30 μg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small‐scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.     

Estimation of linkage disequilibrium and interspecific gene flow in Ficedula flycatchers by a newly developed 50k single‐nucleotide polymorphism array

With the access to draft genome sequence assemblies and whole‐genome resequencing data from population samples, molecular ecology studies will be able to take truly genome‐wide approaches. This now applies to an avian model system in ecological and evolutionary research: Old World flycatchers of the genus Ficedula, for which we recently obtained a 1.1 Gb collared flycatcher genome assembly and identified 13 million single‐nucleotide polymorphism (SNP)s in population resequencing of this species and its sister species, pied flycatcher. Here, we developed a custom 50K Illumina iSelect flycatcher SNP array with markers covering 30 autosomes and the Z chromosome. Using a number of selection criteria for inclusion in the array, both genotyping success rate and polymorphism information content (mean marker heterozygosity = 0.41) were high. We used the array to assess linkage disequilibrium (LD) and hybridization in flycatchers. Linkage disequilibrium declined quickly to the background level at an average distance of 17 kb, but the extent of LD varied markedly within the genome and was more than 10‐fold higher in ‘genomic islands’ of differentiation than in the rest of the genome. Genetic ancestry analysis identified 33 F₁ hybrids but no later‐generation hybrids from sympatric populations of collared flycatchers and pied flycatchers, contradicting earlier reports of backcrosses identified from much fewer number of markers. With an estimated divergence time as recently as <1 Ma, this suggests strong selection against F₁ hybrids and unusually rapid evolution of reproductive incompatibility in an avian system.     

Development of a picture‐scale‐questionnaire to assess liking for fatty,salty, sweet,and umami seasonings compared to a sensory evaluation for Japanese consumers

PrefQuest is a web‐based questionnaire that measures the liking for sweet, fatty‐sweet, salty, and fatty‐salty sensations using combinations of images of French food items. We adapted the original PrefQuest questionnaire for Japanese respondents (J‐PrefQuest). J‐PrefQuest comprises 17 Japanese food items categorized into four sensations: fatty, salty, sweet, and umami. Participants responded by indicating their preferred level of seasoning for each food item on a 6‐point scale. Segmentation of 161 respondents identified groups who preferred: (a) high amount of umami seasonings; (b) low amount of umami seasonings; (c) high amount of fatty and low amount of salty seasonings. To validate the questionnaire, 70 female university students were recruited for a sensory evaluation of five levels of seasoning used for six food items on a 9‐point hedonic scale before completing the questionnaire. The validity of J‐PrefQuest questionnaire was then analyzed by comparing the two results.

Practical applications

Measuring personal preference or “liking” for the taste of food items is important as such information may contribute to the prevention of various diseases and improve the quality of life of consumers. We developed a simple questionnaire with pictures to evaluate the liking for fatty, salty, sweet, and umami sensations of Japanese respondents. This questionnaire focuses on typical seasonings added to different Japanese food items and provides objective information about liking different food sensations. Thus, it has application as an important screening tool to identify respondents who may need to make their food consumption behavior more healthy. Moreover, following minor modifications, this questionnaire could also be used in other Asian countries where plain white rice is the stable food.     

Gal‐NCAM is a differentially expressed marker for mature sensory neurons in the rat olfactory system

A new monoclonal antibody, 2E11, was produced by immunizing mice with the microsomal fraction of rat accessory olfactory bulb cells. This IgM recognizes a previously described complex α‐galactosyl containing glycolipid, as well as N‐linked glycoproteins at 170 and 210 kD. These proteins correspond to a new nerve cell adhesion molecule (NCAM) glycoform, Gal‐NCAM, which contains a blood group B‐like oligosaccharide. During embryonic development, the 2E11 epitope is expressed by a subset of mature olfactory sensory neurons randomly dispersed throughout the olfactory epithelium, whereas in the olfactory bulb, immunostaining is restricted to medial areas of the nerve layer. When compared to PSA‐NCAM, another NCAM glycoform, Gal‐NCAM has a mutually exclusive distribution pattern both in the olfactory epithelium and in the olfactory bulb. We propose a model for the hierarchy of neuronal maturation in the olfactory epithelium, including a switch from PSA‐NCAM expression by immature neurons to the expression of Gal‐NCAM by mature neurons. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 173–185, 2000     

Distribution of individual members of the mosquito Anopheles maculipennis complex in Germany identified by newly developed real‐time PCR assays

Owing to their role as vectors of malaria parasites, species of the Anopheles maculipennis complex (Diptera: Culicidae) Meigen were intensively studied in the past, but with the disappearance of malaria in Germany in the middle of the last century, the interest in this field of research declined. A comprehensive ecological analysis of the current species distribution for Germany is lacking. Between 2010 and 2013, a total of 1445 mosquitoes of the An. maculipennis complex were collected at 72 different sites in Germany. The samples comprise 722 single individuals as well as 723 individuals in 90 pools of up to 25 mosquitoes. All samples were analysed with newly developed species‐specific qPCR assays for the identification of the four German species using nucleotide differences within the internal transcribed spacer 2 (ITS2) ribosomal DNA. All gathered data were used for species distribution modelling. The overall prevalence of An. messeae s.l. was highest with 98.89% of all pools; An. daciae with 6.93% of all individuals and An. messeae s.s. with 69.53%. The prevalence of the other two species was relatively low: An. maculipennis s.s. with 13.30% of all individuals (6.67% of all pools) and An. atroparvus with 1.80% of all individuals (1.11% of all pools).     

Characterization of a highly flexible self‐assembling protein system designed to form nanocages

The design of proteins that self-assemble into well-defined, higher order structures is an important goal that has potential applications in synthetic biology, materials science, and medicine. We previously designed a two-component protein system, designated A-(+) and A-(−), in which self-assembly is mediated by complementary electrostatic interactions between two coiled-coil sequences appended to the C-terminus of a homotrimeric enzyme with C₃ symmetry. The coiled-coil sequences are attached through a short, flexible spacer sequence providing the system with a high degree of conformational flexibility. Thus, the primary constraint guiding which structures the system may assemble into is the symmetry of the protein building block. We have now characterized the properties of the self-assembling system as a whole using native gel electrophoresis and analytical ultracentrifugation (AUC) and the properties of individual assemblies using cryo-electron microscopy (EM). We show that upon mixing, A-(+) and A-(−) form only six different complexes in significant concentrations. The three predominant complexes have hydrodynamic properties consistent with the formation of heterodimeric, tetrahedral, and octahedral protein cages. Cryo-EM of size-fractionated material shows that A-(+) and A-(−) form spherical particles with diameters appropriate for tetrahedral or octahedral protein cages. The particles varied in diameter in an almost continuous manner suggesting that their structures are extremely flexible.     

Proteomic analysis of low‐ to high‐grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non‐neoplastic brain tissue as control using 2‐DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up‐regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p<0.05), whereas four proteins were down‐regulated, among them raf kinase inhibitor protein (RKIP) (p<0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real‐time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.