A Holocene environmental record reflecting vegetation, climate, and fire variability at the Páramo of Quimsacocha, southwestern Ecuadorian Andes

We reconstructed the palaeoenvironmental conditions of the last ca. 8,000 years in the Tres Lagunas region of the Quimsacocha volcanic basin (ca. 3,800 m a.s.l.) in the southwestern Ecuadorian Andes. By means of a pollen and charcoal record, we analysed vegetation, fire, and climate history of this area, which is sensitive to climatic changes of both the Pacific as well as of the eastern Andes and Amazon region. Sediment deposits, pronounced increases of pollen and charcoal concentrations, and pollen taxa reflect warmer and drier conditions in the early to mid-Holocene (~8000 to 3900 cal. b.p.). During the late Holocene (2250 to −57 cal. b.p.), five warm and cold phases occurred at Quimsacocha. The most prominent cold phase possibly corresponds to the globally recognized Little Ice Age (LIA; ~600 to 100 cal. b.p.). The cold phase signal at Quimsacocha was characterized by a higher abundance of Poaceae, Iso?tes and Gentianella, which are favoured by cold and moist conditions. Frequent charcoal particles can be recorded since the early to mid-Holocene (~7600 b.p.). The high Andean tree species Polylepis underwent several phases of degradation and re-establishment in the basin, which could indicate the use of fire by pre-Columbian settlers to enhance the growth of preferred herb species. The Tres Lagunas record suggests that human populations have been influencing the environment around Quimsacocha since the last ca. 8,000 years.     

High-level expression, purification, and characterization of recombinant wheat xylanase inhibitor TAXI-I secreted by the yeast Pichia pastoris

Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11. In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (approximately 75 mg/L). The rTAXI-I protein was purified from the P. pastoris culture medium using cation exchange and gel filtration chromatographic steps. rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation. The recombinant protein fold was confirmed by circular dichroism spectroscopy. Xylanase inhibition by rTAXI-I was optimal at 20-30 degrees C and at pH 5.0. rTAXI-I still showed xylanase inhibition activity at 30 degrees C after a 40 min pre-incubation step at temperatures between 4 and 70 degrees C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively. All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not. Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940IU/mg protein, respectively. The obtained biochemical characteristics of rTAXI-I produced by P. pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli. The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.     

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

We demonstrate the versatility of a collection of insertions of the transposon Minos-mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 integrase attP sites. MiMIC integrates almost at random in the genome to create sites for DNAmanipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase-mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to revert to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp recombinase system. Insertions in coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the D. melanogaster toolkit.     

Successive cambia development in Avicennia marina (Forssk.) Vierh. is not climatically driven in the seasonal climate at Gazi Bay, Kenya

This study is intended to provide early access to recent findings on the formation of the successive cambia of Avicennia marina (Forssk.) Vierh. in Kenya. The non-annual character of the growth layers was demonstrated by using three trees from a cambial marking experiment and three trees from a plantation of known age. The respective number of growth layers produced during one year was on average a half and three. Considering 28 stem disks of trees at three study sites, differing in local site conditions, growth layer development was shown to be strongly correlated with stem diameter (R²=0.84, p<0.0001, n=31). However, an additional influence of the site conditions was also demonstrated (homogeneity-of-slopes model test: F=54.72, p<0.0001, n=28). With increasing salinity and/or inundation class the width of the growth layers decreased. The significance of these variations in growth layer width offer interesting perspectives. The larger proportion of xylem in comparison with phloem in trees with wide as opposed to narrow growth layers may provide extra mechanical strength. On the other hand, the larger fraction of living tissue (phloem and parenchyma) in trees with thin growth layers may be beneficial for the water balance of the tree. Next to the non-annual nature of the growth layers and their networking pattern, more than one cambium was found to be simultaneously active. We conclude that classical dendrochronological methods (ring width measurements) should not be applied to A. marina (from Kenya).     

Detection of Legionellae in Hospital Water Samples by Quantitative Real-Time LightCycler PCR

Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r = 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.