A multipurpose vector system for the screening of libraries in bacteria, insect and mammalian cells and expression in vivo

We have constructed a novel tetra-promoter vector (pBVboostFG) system that enables screening of gene/cDNA libraries for functional genomic studies. The vector enables an all-in-one strategy for gene expression in mammalian, bacterial and insect cells and is also suitable for direct use in vivo. Virus preparation is based on an improved mini Tn7 transpositional system allowing easy and fast production of recombinant baculoviruses with high diversity and negligible background. Cloning of the desired DNA fragments or libraries is based on the recombination system of bacteriophage lambda. As an example of the utility of the vector, genes or cDNAs of 18 different proteins were cloned into pBVboostFG and expressed in different hosts. As a proof-of-principle of using the vector for library screening, a chromophoric Thr⁶⁵-Tyr-Gly⁶⁷-stretch of enhanced green fluorescent protein was destroyed and subsequently restored by novel PCR strategy and library screening. The pBVboostFG enables screening of genome-wide libraries, thus making it an efficient new platform technology for functional genomics.     

Adaptations to fasting in the American mink (Mustela vison): carbohydrate and lipid metabolism

The aim of this study was to investigate whether the actively wintering American mink Mustela vison is strictly dependent on continuous food availability or if it has evolved physiological adaptations to tolerate nutritional scarcity. Fifty farm-bred male minks were divided into a fed control group and four experimental groups fasted for 2, 3, 5 or 7 days. The rate of weight loss was several-fold higher (1.5-3.2% day(-1)) in the mink than recorded previously in larger carnivores utilizing passive wintering strategies. The minks remained normoglycaemic, although their liver glycogen stores and glucose-6-phosphatase activities decreased during fasting. Adipose tissue constituted approximately 36% of their body mass after 7 days of food deprivation. Intra-abdominal fat, especially retroperitoneal but also mesenteric adipose tissue, were the most important fat depots to be hydrolyzed, but the ability of the mink to utilize its body lipids during fasting may be limited. The increased liver size, hepatic triacylglycerol accumulation and increases in the activities of plasma aminotransferases indicated liver dysfunction. Food deprivation also affected the red blood cell indices, and the blood monocyte and lymphocyte counts decreased suggesting immunosuppression during fasting. The results of the present study suggest that the mink has not evolved sophisticated adaptations to wintertime fasting.     

A Bayesian analysis of multivariate doubly-interval-censored dental data

A Bayesian survival analysis is presented to examine the effect of fluoride-intake on the time to caries development of the permanent first molars in children between 7 and 12 years of age using a longitudinal study conducted in Flanders. Three problems needed to be addressed. Firstly, since the emergence time of a tooth and the time it experiences caries were recorded yearly, the time to caries is doubly interval censored. Secondly, due to the setup of the study, many emergence times were left-censored. Thirdly, events on teeth of the same child are dependent. Our Bayesian analysis is a modified version of the intensity model of Harkanen et al. (2000, Scandinavian Journal of Statistics 27, 577-588). To tackle the problem of the large number of left-censored observations a similar Finnish data set was introduced. Our analysis shows no convincing effect of fluoride-intake on caries development.     

Small Tails Tell Tall Tales – Intra-Individual Variation in the Stable Isotope Values of Fish Fin

Background

Fish fin is a widely used, non-lethal sample material in studies using stable isotopes to assess the ecology of fishes. However, fish fin is composed of two distinct tissues (ray and membrane) which may have different stable isotope values and are not homogeneously distributed within a fin. As such, estimates of the stable isotope values of a fish may vary according to the section of fin sampled.

Methods

To assess the magnitude of this variation, we analysed carbon (δ ¹³C), nitrogen (δ ¹⁵N), hydrogen (δ ²H) and oxygen (δ ¹⁸O) stable isotopes of caudal fin from juvenile, riverine stages of Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). Individual fins were sub-sectioned into tip, mid and base, of which a further subset were divided into ray and membrane.

Findings

Isotope variation between fin sections, evident in all four elements, was primarily related to differences between ray and membrane. Base sections were¹³C depleted relative to tip (~ 1 ‰) with equivalent variation evident between ray and membrane. A similar trend was evident in δ ²H, though the degree of variation was far greater (~ 10 ‰). Base and ray sections were ¹⁸O enriched (~ 2 ‰) relative to tip and membrane, respectively. Ray and membrane sections displayed longitudinal variation in ¹⁵N mirroring that of composite fin (~ 1 ‰), indicating that variation in¹⁵N values was likely related to ontogenetic variation.

Conclusions

To account for the effects of intra-fin variability in stable isotope analyses we suggest that researchers sampling fish fin, in increasing priority, 1) also analyse muscle (or liver) tissue from a subsample of fish to calibrate their data, or 2) standardize sampling by selecting tissue only from the extreme tip of a fin, or 3) homogenize fins prior to analysis.     

Crystal structures of Trichoderma reesei β-galactosidase reveal conformational changes in the active site

We have determined the crystal structure of Trichoderma reesei (Hypocrea jecorina) β-galactosidase (Tr-β-gal) at a 1.2? resolution and its complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4? resolutions, respectively. Tr-β-gal is a potential enzyme for lactose hydrolysis in the dairy industry and belongs to family 35 of the glycoside hydrolases (GH-35). The high resolution crystal structures of this six-domain enzyme revealed interesting features about the structure of Tr-β-gal. We discovered conformational changes in the two loop regions in the active site, implicating a conformational selection-mechanism for the enzyme. In addition, the Glu200, an acid/base catalyst showed two different conformations which undoubtedly affect the pK(a) value of this residue and the catalytic mechanism. The electron density showed extensive glycosylation, suggesting a structure stabilizing role for glycans. The longest glycan showed an electron density that extends to the eighth monosaccharide unit in the extended chain. The Tr-β-gal structure also showed a well-ordered structure for a unique octaserine motif on the surface loop of the fifth domain.     

Eco-evolutionary dynamics of dispersal in spatially heterogeneous environments

Ecology Letters (2011) 14: 1025-1034 ABSTRACT: Evolutionary changes in natural populations are often so fast that the evolutionary dynamics may influence ecological population dynamics and vice versa. Here we construct an eco-evolutionary model for dispersal by combining a stochastic patch occupancy metapopulation model with a model for changes in the frequency of fast-dispersing individuals in local populations. We test the model using data on allelic variation in the gene phosphoglucose isomerase (Pgi), which is strongly associated with dispersal rate in the Glanville fritillary butterfly. Population-specific measures of immigration and extinction rates and the frequency of fast-dispersing individuals among the immigrants explained 40% of spatial variation in Pgi allele frequency among 97 local populations. The model clarifies the roles of founder events and gene flow in dispersal evolution and resolves a controversy in the literature about the consequences of habitat loss and fragmentation on the evolution of dispersal.     

MEG Source Localization Using Invariance of Noise Space

We propose INvariance of Noise (INN) space as a novel method for source localization of magnetoencephalography (MEG) data. The method is based on the fact that modulations of source strengths across time change the energy in signal subspace but leave the noise subspace invariant. We compare INN with classical MUSIC, RAP-MUSIC, and beamformer approaches using simulated data while varying signal-to-noise ratios as well as distance and temporal correlation between two sources. We also demonstrate the utility of INN with actual auditory evoked MEG responses in eight subjects. In all cases, INN performed well, especially when the sources were closely spaced, highly correlated, or one source was considerably stronger than the other.