Precise construction of oligonucleotide–Fab fragment conjugate for homogeneous immunoassay using HaloTag technology

The use of oligonucleotide–protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide–protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner.     

Delayed density-dependent parasitism of eggs and pupae as a contributor to the cyclic population dynamics of the autumnal moth

Many populations of forest Lepidoptera exhibit 10-year cycles in densities, with impressive outbreaks across large regions. Delayed density-dependent interactions with natural enemies are recognized as key factors driving these cyclic population dynamics, but emphasis has typically been on the larval stages. Eggs, pupae and adults also suffer mortality from predators, parasitoids and pathogens, but little is known about possible density relationships between mortality factors and these non-feeding life stages. In a long-term field study, we experimentally deployed autumnal moth (Epirrita autumnata) eggs and pupae to their natural enemies yearly throughout the 10-year population cycle in northern Norway. The abundance of another geometrid, the winter moth (Operophtera brumata), increased in the study area, permitting comparisons between the two moth species in predation and parasitism. Survival of autumnal moth eggs and pupae was related to the moth abundance in an inverse and delayed manner. Egg and pupal parasitoids dominated as density-dependent mortality factors and predicted the subsequent growth rate of the host population size. In contrast, effects of egg and pupal predators were weakly density dependent, and generally predation remained low. Parasitism rates did not differ between the autumnal and winter moth pupae, whereas predators preferred winter moth pupae over those of the autumnal moth. We conclude that parasitism of the autumnal moth by egg and pupal parasitoids can be related to the changes of the moth density in a delayed density-dependent manner. Furthermore, egg and pupal parasitoids cannot be overlooked as causal factors for the population cycles of forest Lepidoptera in general.     

Fast but ephemeral effects of ecological restoration on forest beetle community

Many protected areas have a long history of human intervention before being protected. In protected forests, the past land use has reduced the amount of natural structures, which are crucial substrates for thousands of species. We evaluate the short-term ecological effect of forest restoration (dead wood creation) on conifer-associated saproxylic (dead-wood dependent) beetles. More specifically, we analyze the effect of dead wood creation on the number of beetle species and individuals 1 and 5 years after restoration in spruce and pine forests, using a large-scale monitoring network over Finland. The number of saproxylic beetle species and individuals was larger at restored than at control plots both 1 and 5 years after restoration in both spruce and pine forests. Community composition in restored plots was different from control plots 1 year after restoration, but had returned towards the control plot composition 5 years after restoration, while control plots remained largely unchanged. Both in spruce and pine forests, there were more red-listed and rare saproxylic beetles in restored than in control plots 1 and 5 years after restoration. Our results indicate that restoration has an overall positive influence on saproxylic beetle diversity immediately after dead wood creation, but this effect is rather short-lived. Long term monitoring of restored dead wood is crucial in investigating successional pathways as well as biotic communities in advanced decay stages, and in fully evaluating the ecological effect of dead wood creation as a forest restoration measure.     

Integrating genetic data and population viability analyses for the identification of harbour seal (Phoca vitulina) populations and management units

Identification of populations and management units is an essential step in the study of natural systems. Still, there is limited consensus regarding how to define populations and management units, and whether genetic methods allow for inference at the relevant spatial and temporal scale. Here, we present a novel approach, integrating genetic, life history and demographic data to identify populations and management units in southern Scandinavian harbour seals. First, 15 microsatellite markers and model‐ and distance‐based genetic clustering methods were used to determine the population genetic structure in harbour seals. Second, we used harbour seal demographic and life history data to conduct population viability analyses (PVAs) in the vortex simulation model in order to determine whether the inferred genetic units could be classified as management units according to Lowe and Allendorf's (Molecular Ecology, 19, 2010, 3038) ‘population viability criterion’ for demographic independence. The genetic analyses revealed fine‐scale population structuring in southern Scandinavian harbour seals and pointed to the existence of several genetic units. The PVAs indicated that the census population size of each of these genetic units was sufficiently large for long‐term population viability, and hence that the units could be classified as demographically independent management units. Our study suggests that population genetic inference can offer the same degree of temporal and spatial resolution as ‘nongenetic’ methods and that the combined use of genetic data and PVAs constitutes a promising approach for delineating populations and management units.     

From the subarctic to the tropics: effects of 4-month deployment on soldiers' heat stress, heat strain, and physical performance

The aim of the study was to evaluate the heat stress of Finnish male soldiers (N = 20, age 22.0 ± 2.5 years, body mass 78.8 ± 11.5 kg, and height 180.2 ± 5.6 cm) during their 4-month deployment in a hot environment and to find out the effects on physical performance and body composition. The troops moved from 2.5° C (mean monthly temperature) in Finland to 31.9° C in Chad. During the deployment, temperatures varied between 13.5 and 57.0° C outdoors and in the vehicles and tents. During 1-day recording in the middle of the deployment, skin temperatures were 34-35° C during daytime and maximal core temperature remained mainly below 38.0° C. Body mass decreased (78.4 ± 11.5 kg vs. 75.6 ± 8.6, p = 0.007) during the deployment without changes in fat mass. The sit-up performance increased by 10.9% (46 ± 10 reps·min?1 vs. 51 ± 7 reps·min?1, p < 0.01), and the maximal force production of the leg extensor muscles increased (3,042 ± 614 N vs. 3,277 ± 706 N, p < 0.05) without change in the rate of force development. No changes were observed in the push-ups, repeated squats, maximal grip strength, and running distance during the 12-minute test. In conclusion, the soldiers were able to maintain or improve their physical performance during the deployment despite the heat stress. It is important to encourage soldiers to engage in physical training, especially during a thermally appropriate time of the day or in air-conditioned facilities. Monitoring of local heat stress is also recommended.     

Trimerization of collagen IX alpha-chains does not require the presence of the COL1 and NC1 domains

Collagen IX is a heterotrimer of three alpha-chains, which consists of three COL domains (collagenous domains) (COL1-COL3) and four NC domains (non-collagenous domains) (NC1-NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX alpha-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1-NC1 junction. Our results demonstrate that collagen IX alpha-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2-NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1-NC1 region is important for chain specificity.     

Identification of mutations causing temperature-sensitive defects in Semliki Forest virus RNA synthesis

We have sequenced the nonstructural protein coding region of Semliki Forest virus temperature-sensitive (ts) mutant strains ts1, ts6, ts9, ts10, ts11, ts13, and ts14. In each case, the individual amino acid changes uncovered were transferred to the prototype strain background and thereby identified as the underlying cause of the altered RNA synthesis phenotype. All mutations mapping to the protease domain of nonstructural protein nsP2 caused defects in nonstructural polyprotein processing and subgenomic RNA synthesis, and all mutations in the helicase domain of nsP2 affected subgenomic RNA production. These types of defects were not associated with mutations in other nonstructural proteins.     

Modeling and simulation of Streptomyces peucetius var. caesius N47 cultivation and epsilon-rhodomycinone production with kinetic equations and neural networks

This study focuses on comparing different kinetic growth models and the use of neural networks in the batch cultivation of Streptomyces peucetius var. caesius producing epsilon-rhodomycinone. Contois, Monod and Teissier microbial growth models were used as well as the logistic growth modeling approach, which was found best in the simulations of growth and glucose consumption in the batch growth phase. The lag phase was included in the kinetic model with a CO2 trigger and a delay factor. Substrate consumption and product formation were included as Luedeking-Piret and logistic type equations, respectively. Biomass formation was modeled successfully with a 6-8-2 network, and the network was capable of biomass prediction with an R2-value of 0.983. Epsilon-rhodomycinone production was successfully modeled with a recursive 8-3-1 network capable of epsilon-rhodomycinone prediction with an R2-value of 0.903. The predictive power of the neural networks was superior to the kinetic models, which could not be used in predictive modeling of arbitrary batch cultivations.     

COLONIZATION HISTORY OF THE BALTIC HARBOR SEALS: INTEGRATING ARCHAEOLOGICAL, BEHAVIORAL, AND GENETIC DATA

Detailed knowledge about the history of colonization, population dynamics and behavior greatly enhance evaluation of genetic models of population units and migration rates in spatially structured populations. Here, the genetic uniqueness of harbor seals ( Phoca vitulinia ) in the eastern Baltic is evaluated in the light of new information on the distribution and abundance of Baltic and eastern North Sea populations during the last 11,000 yr, recent hunting statistics, and population counts. Archaeological records reveal that the Baltic population of harbor seals was founded about 8,000 yr ago. Adjacent populations in the North Sea areas were either small, or went extinct, and became significant only during the last 300 yr. This information generates the hypothesis that the Baltic population has been isolated during the last 8,000 yr, despite the lack of geographical barriers. We show that stochastic effects, isolation, and a documented recent population bottleneck can account for the low observed genetic variation in Baltic harbor seals.