Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples

There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K₂EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2^nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.     

Phyllostomid Bat Assemblage Structure in Amazonian Flooded and Unflooded Forests

In Amazonia, the assemblages of several taxa differ significantly between upland terra firme and white‐water flooded várzea forests, but little is known about the diversity and distribution of bats in these two forest types. We compare the spatio‐temporal patterns of bat assemblage composition and structure in adjacent terra firme and várzea forests in the lower Purus River region of central Brazilian Amazonia. Bats were sampled using mist nets at five sites in each forest type during 40 nights (2400 net‐hours). We captured 1069 bats representing 42 species and Phyllostomidae bats comprised 99.3 percent of all captures. The bat assemblages in várzea and terra firme forests were significantly different, mainly due to a marked dissimilarity in species composition and in the number of captures during high‐water season. In addition, bat assemblages within forest types differed significantly between seasons for both terra firme and várzea. Frugivores dominated the bat assemblages in both forest types. Overall guild structure did not change between várzea and terra firme or between seasons, but frugivore and animalivore abundance increased significantly in várzea forest during the inundation. The difference in assemblage structure observed in the high‐water season is probably caused by the annual várzea flooding, which provides an effective barrier to the persistence of many understory bats. We also hypothesize that some bat species may undertake seasonal movements between forest types in response to fruit abundance, and our results further underline the importance of floodplain habitats for the conservation of species in the Amazon.     

A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD

Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3-methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N-alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily.     

HEHEHE-tagged affibody molecule may be purified by IMAC, is conveniently labeled with [⁹⁹(m)Tc(CO)₃](+), and shows improved biodistribution with reduced hepatic radioactivity accumulation

Affibody molecules are a class of small (ca. 7 kDa) robust scaffold proteins suitable for radionuclide molecular imaging of therapeutic targets in vivo. A hexahistidine tag at the N-terminus streamlines development of new imaging probes by enabling facile purification using immobilized metal ion affinity chromatography (IMAC), as well as convenient [??(m)Tc(CO)?](+)-labeling. However, previous studies in mice have demonstrated that Affibody molecules labeled by this method yield higher liver accumulation of radioactivity, compared to the same tracer lacking the hexahistidine tag and labeled by an alternative method. Two variants of the HER2-binding Affibody molecule Z(HER)?(:)??? were made in an attempt to create a tagged tracer that could be purified by immobilized metal affinity chromatography, yet would not result in anomalous hepatic radioactivity accumulation following labeling with [??(m)Tc(CO)?](+). In one construct, the hexahistidine tag was moved to the C-terminus. In the other construct, every second histidine residue in the hexahistidine tag was replaced by the more hydrophilic glutamate, resulting in a HEHEHE-tag. Both variants, denoted Z(HER)?(:)???-H? and (HE)?-Z(HER)?(:)???, respectively, could be efficiently purified using IMAC and stably labeled with [??(m)Tc(CO)?](+) and were subsequently compared with the parental H?-Z(HER)?(:)??? having an N-terminal hexahistidine tag. All three variants were demonstrated to specifically bind to HER2-expressing cells in vitro. The hepatic accumulation of radioactivity in a murine model was 2-fold lower with [??(m)Tc(CO)?](+)-Z(HER2:342)-H? compared to [??(m)Tc(CO)?](+)-H?-Z(HER)?(:)???, and more than 10-fold lower with [??(m)Tc(CO)?](+)-(HE)?-Z(HER)?(:)???. These differences translated into appreciably superior tumor-to-liver ratio for [??(m)Tc(CO)?](+)-(HE)?-Z(HER)?(:)??? compared to the alternative conjugates. This information might be useful for development of other scaffold-based molecular imaging probes.     

Interspecific primate associations in Amazonian flooded and unflooded forests

Stable associations between two or more primate species are a prominent feature of neotropical forest vertebrate communities and many studies have addressed their prevalence, and their costs and benefits. However, little is known about the influence of different habitat types on the frequency, seasonality, and composition of mixed-species groups in Amazonian forest primates. Here we examine the features of interspecific primate groups in a large mosaic of flooded (várzea and igapó) and unflooded (terra firme) forest in central Amazonia. In total, 12 primate species occurred in the study area, nine of which were observed in mixed-species associations. Primates were more than twice as likely to form associations in várzea forest than in terra firme forest. Squirrel monkeys were most frequently found in mixed-species groups in all forest types, most commonly in association with brown capuchins. Another frequent member of interspecific associations was the buffy saki, which often formed mixed-species groups with tamarins or brown capuchins. There was no seasonality in the frequency of associations in terra firme forest whereas associations in várzea forest were twice as frequent during the late-dry and early-wet seasons than in the late-wet and early-dry seasons. Interspecific primate associations were common in all forest types, but the degrees to which different species associate varied between these environments. We suggest that the temporal variation of várzea forest associations is connected with seasonal changes in habitat structure and resource abundance. However, more work is needed to pinpoint the underlying causes of mixed-species associations in all forest types and their strong seasonality in várzea forest.     

Ploidy level analysis of apomictic Hieracium (Asteraceae) reveal unexpected patterns and variation

The DNA ploidy level of 673 accessions belonging to 238 species of Hieracium have been analyzed by flow cytometry. 222 of the species were of Scandinavian origin as members of the exclusively apomictic H. sectt. Bifida, Hieracium, Oreadea, Tridentata and Vulgata. For the overwhelming majority of the species, the ploidy level (or chromosme number) has never been investigated before. Approximately 50% of the Scandinavian species, previously believed to be exclusively triploid, were found to be tetraploid. In addition, two pentaploid samples, viz. H. intermarginatum Johanss. & Sam. from Sweden and H. cf. plumbeum Blytt & Fr. from Germany, were found. Although two or more accessions from geographically remote sites were analyzed for approximately 50% of the Scandinavian species, only 2 (<2%) taxonomically undisputed species were found to consist of plants with more than one ploidy level. An intriguing pattern was revealed among Scandinavian members of H. sectt. Bifida and Vulgata when ploidy level and morphometric variation was compared, viz. the most typical or extreme representatives of these sections were found to be exclusively triploid whereas tetraploids dominate among species with intermediate morphology and among species morphologically intermediate between these sections and H. sect. Hieracium. This pattern may indicate that the tetraploids, which tend to have mainly northern distributions, have originated as the result of rare sexual hybridizations between triploid members of different sections, plausibly during or after the northward migration of the parental linages after the last glaciation. The results are believed to be highly relevant for understanding the processes of evolution and speciation within the predominantly apomictic genus Hieracium, but it is emphazised that additional data from e.g. molecular markers and pollen viability analysis are needed before any trustworthy conclusions can be made as far as evolutionary processes are concerned.     

Magnitude and drivers of plant diversity loss differ between spatial scales in Scania,Sweden 1957–2021

Questions

Changed land use, nitrogen deposition, climate change, and the spread of non-native species have repeatedly been reported as the main drivers of recent floristic changes in northern Europe. However, the relevance of the geographical scale at which floristic changes are observed is less well understood and it has only rarely been possible to quantify biodiversity loss. Therefore, we assessed changes in species richness, species composition and mean ecological indicator values (EIVs) at three nested geographic scales during two different time periods, each ca 30 years, since the mid-1900s.

Location

Two parishes in central Scania, southernmost Sweden.

Methods

We analyzed species presence/absence data from three inventories at ca 30-year intervals over 1957–2021 and three geographic scales (157 m², ca 7 km² and ca 45 km²) to document temporal trends and differences between geographic scales in terms of species richness, species composition and mean EIVs.

Results

We found shifts in species composition across all geographical scales. However, the magnitude of biodiversity loss and the main drivers of these changes were scale-dependent. At the smallest spatial scale, we saw a dramatic loss of plant biodiversity with local species richness in 2021 being only 48% of that of 1960. In contrast, at the larger geographic scales no significant changes in species richness were observed because species losses were compensated for by gains of predominantly non-native species, which made up at least 78% of the new species richness. At the smallest spatial scale, changed land use (ceased grazing/mowing and intensified forestry) appeared as the main driver, while an increasing proportion of non-native species, as well as climatic changes and increasing nitrogen loads appeared relatively more important at larger geographic scales.

Conclusion

Our results highlight the precarious situation for biodiversity in the region and at the same time the fundamental importance of geographic scale in studies of biodiversity change. Both the magnitude and drivers of changes may differ depending on the geographic scale and must be considered also when previously published studies are interpreted.     

Effects of drainage and wood ash fertilization on water chemistry at a cutover peatland

Afforestation is one of many different ways to reclaim cutover peatland areas. Among the treatments often required to obtain successful afforestation are supplementary drainage, fertilization and rotovation. This study presents the effects of drainage and wood-ash fertilization on the chemical composition of water in a cutover peatland area, which constitutes 34 hectares of a minerotrophic mire in SW Sweden. Fourteen hectares of the cutover peatland was amended with 23 tonnes of wood ash, 0.4 tonnes of raw phosphate and 0.25 tonnes of superphosphate per hectare, while different doses of a PK-fertilizer were applied on the remaining 20 hectares. The chemical properties of groundwater and stream water in the wood-ash amended area were compared with those in the PK-fertilized area and a control area. Outflowing groundwater emanating from the mineral subsoil affected the chemical composition of peat groundwater and streamwater. Drainage changed chemical composition more than the wood ash fertilization. Drainage led to increases in SO₄ and decreases in alkalinity in both peat groundwater and in stream water. The pH decreased in peat groundwater, whereas the streamwater pH was not affected because the high alkalinity in the mineral soil groundwater buffered the acid peat groundwater. Fertilization increased contents of K, Ca and Mn in both peat groundwater and stream water. Contents of B, o-P and Tot-P increased in peat groundwater but not in stream water, whereas the reverse was true for Mg. The outflow of P did not increase in spite of the large amount of P applied. The high retention of P was caused by large quantities of sesquioxides, especially Fe-oxides in the peat.     

Debates over the Role of the Arctic Council

This article examines the role(s) that the member governments want the Arctic Council to have in Arctic Ocean affairs. The article identifies and examines three determining debates over the role and future of the Arctic Council: The first preceded the Arctic Council's creation in 1996, the second occurred during and as a result of the Ilulissat meeting in 2008, and the third followed the political shift in the United States in 2009.