Oxidation of methyl α-d-galactopyranoside by galactose oxidase: products formed and optimization of reaction conditions for production of aldehyde

The reaction conditions of galactose oxidase-catalyzed, targeted C-6 oxidation of galactose derivatives were optimized for aldehyde production and to minimize the formation of secondary products. Galactose oxidase, produced in transgenic Pichia pastoris carrying the galactose oxidase gene from Fusarium spp., was used as catalyst, methyl α-d-galactopyranoside as substrate, and reaction medium, temperature, concentration, and combinations of galactose oxidase, catalase, and horseradish peroxidase were used as variables. The reactions were followed by ¹H NMR spectroscopy and the main products isolated, characterized, and identified. An optimal combination of all the three enzymes gave aldehyde (methyl α-d-galacto-hexodialdo-1,5-pyranoside) in approximately 90% yield with a substrate concentration of 70 mM in water at 4 °C using air as oxygen source. Oxygen flushing of the reaction mixture was not necessary. The aldehyde existed as a hydrate in water. The main secondary products, a uronic acid (methyl α-d-galactopyranosiduronic acid) and an α,β-unsaturated aldehyde (methyl 4-deoxy-α-d-threo-hex-4-enodialdo-1,5-pyranoside), were observed for the first time to form in parallel. Formation of uronic acid seemed to be the result of impurities in the galactose oxidase preparation. ¹H and ¹³C NMR data of the products are reported for the α,β-unsaturated aldehyde for the first time, and chemical shifts in DMSO-d₆ for all the products for the first time. Oxidation of d-raffinose (α-d-galactopyranosyl-(1-6)-α-d-glucopyranosyl-(1-2)-β-d-fructofuranoside) in the same optimum conditions also proceeded well, resulting in approximately 90% yield of the corresponding aldehyde.     

Variations in the 16S-23S rRNA internal transcribed spacer of fibrolytic Butyrivibrio isolates from the reindeer rumen

Strains of Butyrivibrio are principal cellulytic bacteria in the rumen of the High Arctic Svalbard reindeer ( Rangifer tarandus platyrhynchus ). According to phylogenetic analysis based on 16S rRNA gene sequencing, Butyrivibrio can be divided into three subgroups within the Clostridia class of the phylum Firmicutes, but the current phenotypic and genotypic differentiation within the family Lachnospiraceae is insufficient. This current study describes the sequence diversity of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Butyrivibrio isolates from reindeer. A total of 17 different ITS sequences with sizes between 449 and 784 nt were obtained. Genes encoding tRNA(Ile) and tRNA(Ala) were identified in four of the sequences. Phylogenetic neighbor-joining trees were constructed based on the ITS sequence and compared with a phylogenetic neighbor-joining tree based on 16S rRNA gene sequences previously obtained for the same isolates. These comparisons indicated a better differentiation between strains in the ITS sequence than the 16S rRNA gene based tree. Through this study, a better means for identifying and tracking fibrolytic and potentially probiotic Butyrivibrio strains in reindeer and other ruminants has been provided.     

Coronary Flow Reserve in Pregnant Rats with Increased Left Ventricular Afterload

Background

Coronary flow reserve (CFR) is used as a measure of coronary endothelial function. We investigated the effect of increased afterload on CFR of pregnant and non-pregnant rats.

Methods

Afterload increase in Wister rats (both pregnant and non-pregnant) was achieved by the infusion of angiotensin II (Ang II) for ∼10 days or by subjecting them to transverse aortic constriction (TAC) for ∼14 days. Control groups were infused with 0.9% NaCl or had sham surgery, respectively. In pregnant rats, the experiments were performed close to term gestation. Doppler velocity waveforms of the left main coronary artery were recorded using a high resolution ultrasound imaging system (Vevo 770, VisualSonics, Canada) at baseline while the animals were anesthetized with 1.5% inhaled isoflurane, and during maximal coronary dilatation obtained by the inhalation of 3.5% of isoflurane. CFR was calculated as the ratio between the peak coronary flow velocities (CFR_peak) and the velocity-time integrals (CFR_VTI) recorded at hyperemia and at baseline.

Results

CFR could be calculated in 60 of 75 (80%) animals. There were no differences in CFR between intervention and control groups irrespective of whether afterload was increased by Ang II or TAC. In the TAC-study CFR_peak (1.54±0.07 vs 1.85±0.17; p = 0.03) was decreased in pregnant compared to non-pregnant shams. When sham animals from both studies were pooled together both CFR_peak (1.42±0.07 vs 1.86±0.16; p = 0.005) as well as CFR_VTI (1.45±0.07 vs 1.78±0.12; p = 0.03) were significantly lower in pregnant rats compared to non-pregnant.

Conclusions

CFR can be measured non-invasively in rats using Doppler echocardiography and high concentrations of inhaled isoflurane as a coronary vasodilator. In pregnant rats, CFR is reduced close to term. CFR is not affected by increased left ventricular afterload caused by chronic Ang II infusion or TAC.     

Evaluation of the Validity of Job Exposure Matrix for Psychosocial Factors at Work

Objective

To study the performance of a developed job exposure matrix (JEM) for the assessment of psychosocial factors at work in terms of accuracy, possible misclassification bias and predictive ability to detect known associations with depression and low back pain (LBP).

Materials and Methods

We utilized two large population surveys (the Health 2000 Study and the Finnish Work and Health Surveys), one to construct the JEM and another to test matrix performance. In the first study, information on job demands, job control, monotonous work and social support at work was collected via face-to-face interviews. Job strain was operationalized based on job demands and job control using quadrant approach. In the second study, the sensitivity and specificity were estimated applying a Bayesian approach. The magnitude of misclassification error was examined by calculating the biased odds ratios as a function of the sensitivity and specificity of the JEM and fixed true prevalence and odds ratios. Finally, we adjusted for misclassification error the observed associations between JEM measures and selected health outcomes.

Results

The matrix showed a good accuracy for job control and job strain, while its performance for other exposures was relatively low. Without correction for exposure misclassification, the JEM was able to detect the association between job strain and depression in men and between monotonous work and LBP in both genders.

Conclusions

Our results suggest that JEM more accurately identifies occupations with low control and high strain than those with high demands or low social support. Overall, the present JEM is a useful source of job-level psychosocial exposures in epidemiological studies lacking individual-level exposure information. Furthermore, we showed the applicability of a Bayesian approach in the evaluation of the performance of the JEM in a situation where, in practice, no gold standard of exposure assessment exists.     

Common variants show predicted polygenic effects on height in the tails of the distribution, except in extremely short individuals

Common genetic variants have been shown to explain a fraction of the inherited variation for many common diseases and quantitative traits, including height, a classic polygenic trait. The extent to which common variation determines the phenotype of highly heritable traits such as height is uncertain, as is the extent to which common variation is relevant to individuals with more extreme phenotypes. To address these questions, we studied 1,214 individuals from the top and bottom extremes of the height distribution (tallest and shortest ∼1.5%), drawn from ∼78,000 individuals from the HUNT and FINRISK cohorts. We found that common variants still influence height at the extremes of the distribution: common variants (49/141) were nominally associated with height in the expected direction more often than is expected by chance (p<5×10⁻²⁸), and the odds ratios in the extreme samples were consistent with the effects estimated previously in population-based data. To examine more closely whether the common variants have the expected effects, we calculated a weighted allele score (WAS), which is a weighted prediction of height for each individual based on the previously estimated effect sizes of the common variants in the overall population. The average WAS is consistent with expectation in the tall individuals, but was not as extreme as expected in the shortest individuals (p<0.006), indicating that some of the short stature is explained by factors other than common genetic variation. The discrepancy was more pronounced (p<10⁻⁶) in the most extreme individuals (height<0.25 percentile). The results at the extreme short tails are consistent with a large number of models incorporating either rare genetic non-additive or rare non-genetic factors that decrease height. We conclude that common genetic variants are associated with height at the extremes as well as across the population, but that additional factors become more prominent at the shorter extreme.     

Direct measurement of the interaction force between immunostimulatory CpG-DNA and TLR9 fusion protein

The specific interaction between human Toll-like receptor 9 (TLR9)-ectodomain (ECD)-fusion protein and immunostimulatory CpG-DNA was measured using force spectroscopy. Flexible tethers were used between receptors and surface as well as between DNA and atomic force microscope tip to make efficient recognition studies possible. The molecular recognition forces detected are in the range of 50 to 150 ± 20 pN at the used force-loading rates, and the molecular interaction probability was much reduced when the receptors were blocked with free CpG-DNA. A linear increase of the unbinding force with the logarithm of the loading rate was found over the range 0.1 to 30 nN/s. This indicates a single potential barrier characterizing the energy landscape and no intermediate state for the unbinding pathway of CpG-DNA from the TLR9-ECD. Two important kinetic parameters for CpG-DNA interaction with TLR9-ECD were determined from the force-loading rate dependency: an off-rate of k(off) = 0.14 ± 0.10 s(-1) and a binding interaction length of x(β) = 0.30 ± 0.03 nm, which are consistent with literature values. Various models for the molecular interaction of this innate immune receptor binding to CpG-DNA are discussed.     

Genetic structure in a fragmented Northern Hemisphere rainforest: large effective sizes and high connectivity among populations of the epiphytic lichen Lobaria pulmonaria

An extraordinary diversity of epiphytic lichens is found in the boreal rainforest of central Norway, the highest-latitude rainforest in the world. These rainforest relicts are located in ravine systems, and clear cutting has increased the distance between remaining patches. We hypothesized that the relatively small lichen populations in the remaining forest stands have suffered a depletion of genetic diversity through bottlenecks and founder events. To test this hypothesis, we assessed genetic diversity and structure in the populations of the tripartite lichen Lobaria pulmonaria using eight SSR loci. We sampled thalli growing on Picea abies branches and propagules deposited in snow at three localities. Contrary to expectations, we found high genetic diversity in lichen and snow samples, and high effective sizes of the studied populations. Also, limited genetic differentiation between populations, high historical migration rates, and a high proportion of first generation immigrants were estimated, implying high connectivity across distances <30km. Almost all genetic variation was attributed to variation within sites; spatial genetic structures within populations were absent or appeared on small scales (5-10m). The high genetic diversity in the remaining old boreal rainforests shows that even relict forest patches might be suitable for conservation of genetic diversity.     

Dichloromethane fermentation by a Dehalobacter sp. in an enrichment culture derived from pristine river sediment

Dichloromethane (DCM) as the sole substrate supported growth of a Dehalobacter sp. in an enrichment culture derived from noncontaminated river sediment. DCM was not reductively dechlorinated, and acetate was produced, indicating DCM fermentation and further suggesting Dehalobacter growth is not limited to organohalide respiration.     

Determining algal-available phosphorus of differing origin: routine phosphorus analyses versus algal assays

Eutrophication of surface waters can be accelerated by anthropogenic P-inputs, provided that P is in a form available to aquatic primary producers. Potentially algal-available P (P_aa) under aerobic conditions was determined with a dual-culture assay from 172 samples representing P in point and nonpoint sources and in lacustrine matter. The availability of P – expressed as the proportion of P_aa in total P (Tot-P) – ranged from 0 to 100%. In the different P sources, the mean availability ranged from 3.4 to 89% in descending order: wastewater of rural population > biologically treated urban wastewater > dairy house wastewater > biologically and chemically treated urban wastewater > field runoff > forest industrial effluent > fish fodder and feces > river water > field surface soil > forest runoff > lake settling matter > lake bottom sediments. Of the P fractions, dissolved reactive P (o-P) was highly available to algae, whereas particulate P (P_part) and dissolved unreactive P (unr-P_diss) contributed to P_aa to a lower but varying degree. An approach based on source-dependent availability coefficients, derived from the algal assays, appeared promising in transforming the load of Tot-P into that of P_aa. Although the values for P_aa obtained by the dual-culture assay probably underestimate the true levels for ultimately available P, they may still give valuable information for eutrophication abatement.