Long-Term Pollen Monitoring in the Benelux: Evaluation of Allergenic Pollen Levels and Temporal Variations of Pollen Seasons.

Airborne pollen is a major cause of allergic rhinitis, affecting between 10 and 30% of the population in Belgium, the Netherlands, and Luxembourg (Benelux). Allergenic pollen is produced by wind pollinating plants and released in relatively low to massive amounts. Current climate changes, in combination with increasing urbanization, are likely to affect the presence of airborne allergenic pollen with respect to exposure intensity, timing as well as duration. Detailed analysis of long-term temporal trends at supranational scale may provide more comprehensive insight into these phenomena. To this end, the Spearman correlation was used to statistically compare the temporal trends in airborne pollen concentration monitored at the aerobiological stations which gathered the longest time-series (30-44 years) in the Benelux with a focus on the allergenic pollen taxa: Alnus, Corylus, Betula, Fraxinus, Quercus, Platanus, Poaceae, and Artemisia. Most arboreal species showed an overall trend toward an increase in the annual pollen integral and peak values and an overall trend toward an earlier start and end of the pollen season, which for Betula resulted in a significant decrease in season length. For the herbaceous species (Poaceae and Artemisia), the annual pollen integral and peak values showed a decreasing trend. The season timing of Poaceae showed a trend toward earlier starts and longer seasons in all locations. In all, these results show that temporal variations in pollen levels almost always follow a common trend in the Benelux, suggesting a similar force of climate change-driven factors, especially for Betula where a clear positive correlation was found between changes in temperature and pollen release over time. However, some trends were more local-specific indicating the influence of other environmental factors, e.g., the increasing urbanization in the surroundings of these monitoring locations. The dynamics in the observed trends can impact allergic patients by increasing the severity of symptoms, upsetting the habit of timing of the season, complicating diagnosis due to overlapping pollen seasons and the emergence of new symptoms due allergens that were weak at first.

Cdk1 restrains NHEJ through phosphorylation of XRCC4-like factor Xlf1.

Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs): homologous recombination (HR) and nonhomologous end-joining (NHEJ). DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1) activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2(Cdk1) provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms.

Evolutionary and functional conservation of the DNA non-homologous end-joining protein, XLF/Cernunnos.

Non-homologous end-joining is a major pathway of DNA double-strand break repair in mammalian cells, deficiency in which confers radiosensitivity and immune deficiency at the whole organism level. A core protein complex comprising the Ku70/80 heterodimer together with a complex between DNA ligase IV and XRCC4 is conserved throughout eukaryotes and assembles at double-strand breaks to mediate ligation of broken DNA ends. In Saccharomyces cerevisiae an additional NHEJ protein, Nej1p, physically interacts with the ligase IV complex and is required in vivo for ligation of DNA double-strand breaks. Recent studies with cells derived from radiosensitive and immune-deficient patients have identified the human protein, XLF (also named Cernunnos), as a crucial NHEJ protein. Here we show that XLF and Nej1p are members of the same protein superfamily and that this family has members in diverse eukaryotes. Indeed, we show that a member of this family encoded by a previously uncharacterized open-reading frame in the Schizosaccharomyces pombe genome is required for NHEJ in this organism. Furthermore, our data reveal that XLF family proteins can bind to DNA and directly interact with the ligase IV-XRCC4 complex to promote DSB ligation. We therefore conclude that XLF family proteins interact with the ligase IV-XRCC4 complex to constitute the evolutionarily conserved enzymatic core of the NHEJ machinery.

Additional vectors for PCR-based gene tagging in Saccharomyces cerevisiae and Schizosaccharomyces pombe using nourseothricin resistance.

The one-step PCR-mediated technique used for modification of chromosomal loci is a powerful tool for functional analysis in yeast. Both Saccharomyces cerevisiae and Schizosaccharomyces pombe are amenable to this technique. However, the scarce availability of selectable markers for Sz. pombe hampers the easy use of this technique in this species. Here, we describe the construction of new vectors deriving from the pFA6a family, which are suitable for tagging in both yeasts owing to the presence of a nourseothricin-resistance cassette. These plasmids allow various gene manipulations at chromosomal loci, viz. N- and C-terminal tagging with 3HA (haemagglutinin) or 13Myc epitopes, GST (glutathione S-transferase), 4TAP (tandem affinity purification) and several GFP (green fluorescent protein) isoforms. For N-terminal modifications, the use of different promoters allows constitutive (PADH1) or regulatable (PGAL1) promoters for S. cerevisiae and derivatives of Pnmt1 for Sz. pombe expression.

Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe.

The ease of construction of multiple mutant strains in Schizosaccharomyces pombe is limited by the number of available genetic markers. We describe here three new cassettes for PCR-mediated gene disruption that can be used in combination with commonly used fission yeast markers to make multiple gene deletions. The natMX6, hphMX6 and bleMX6 markers give rise to resistance towards the antibiotics nourseothricin (NAT), hygromycin B and phleomycin, respectively. The cassettes are composed of exogenous sequences to increase the frequency of integration at targeted loci, and have a structure similar to the commonly used pFA6a-kanMX6 modular plasmid system. This allows a simple exchange of the kanMX6 marker in existing strains with any of the three new cassettes. Alternatively, oligonucleotide primers designed for the modular kanMX6 cassettes can be used to make the transforming PCR fragments for gene disruption. We illustrate the construction of a mutant strain with six independent gene disruptions, using the novel antibiotic cassettes in combination with existing genetic markers.