Phylogeny and species delineation in the marine diatom Pseudo‐nitzschia (Bacillariophyta) using cox1, LSU,and ITS2 rRNA genes: A perspective in character evolution

Analyses of the mitochondrial cox1, the nuclear‐encoded large subunit (LSU), and the internal transcribed spacer 2 (ITS2) RNA coding region of Pseudo‐nitzschia revealed that the P. pseudodelicatissima complex can be phylogenetically grouped into three distinct clades (Groups I–III), while the P. delicatissima complex forms another distinct clade (Group IV) in both the LSU and ITS2 phylogenetic trees. It was elucidated that comprehensive taxon sampling (sampling of sequences), selection of appropriate target genes and outgroup, and alignment strategies influenced the phylogenetic accuracy. Based on the genetic divergence, ITS2 resulted in the most resolved trees, followed by cox1 and LSU. The morphological characters available for Pseudo‐nitzschia, although limited in number, were overall in agreement with the phylogenies when mapped onto the ITS2 tree. Information on the presence/absence of a central nodule, number of rows of poroids in each stria, and of sectors dividing the poroids mapped onto the ITS2 tree revealed the evolution of the recently diverged species. The morphologically based species complexes showed evolutionary relevance in agreement with molecular phylogeny inferred from ITS2 sequence–structure data. The data set of the hypervariable region of ITS2 improved the phylogenetic inference compared to the cox1 and LSU data sets. The taxonomic status of P. cuspidata and P. pseudodelicatissima requires further elucidation.     

Ecotypes of Planktonic Actinobacteria with Identical 16S rRNA Genes Adapted to Thermal Niches in Temperate, Subtropical, and Tropical Freshwater Habitats

Seven strains with identical 16S rRNA genes affiliated with the Luna2 cluster (Actinobacteria) were isolated from six freshwater habitats located in temperate (Austria and Australia), subtropical (People's Republic of China), and tropical (Uganda) climatic zones. The isolates had sequence differences at zero to five positions in a 2,310-nucleotide fragment of the ribosomal operon, including part of the intergenic spacer upstream of the 16S rRNA gene, the complete 16S rRNA gene, the complete 16S-23S internal transcribed spacer (ITS1), and a short part of the 23S rRNA gene. Most of the few sequence differences found were located in the internal transcribed spacer sequences. Two isolates obtained from habitats in Asia and Europe, as well as two isolates obtained from different habitats in the People's Republic of China, had identical sequences for the entire fragment sequenced. In spite of minimal sequence differences in the part of the ribosomal operon investigated, the strains exhibited significant differences in their temperature response curves (with one exception), as well as pronounced differences in their temperature optima (25.0 to 35.6°C). The observed differences in temperature adaptation were generally in accordance with the thermal conditions in the habitats where the strains were isolated. Strains obtained from temperate zone habitats had the lowest temperature optima, strains from subtropical habitats had intermediate temperature optima, and a strain from a tropical habitat had the highest temperature optimum. Based on the observed temperature responses, we concluded that the strains investigated are well adapted to the thermal conditions in their home habitats. Consequently, these closely related strains represent different ecotypes adapted to different thermal niches.     

Quantitative polymerase chain reaction as a reliable method to determine functional lentiviral titer after ex vivo gene transfer in human mesenchymal stem cells

BACKGROUND: Human mesenchymal stem cells (hMSCs) are a promising target for ex vivo gene therapy and lentiviruses are excellent gene transfer vehicles in hMSCs since they achieve high transduction rates with long-term gene expression. Nevertheless, senescence of hMSCs may limit therapeutic applications due to time-consuming cell selection and viral titration. Here, we describe a fast and reliable method to determine functional lentiviral titer by quantitative polymerase chain reaction (qPCR) after highly efficient ex vivo gene transfer in hMSCs. METHODS: Lentivirus production was tested with different types of packaging systems. Using p24 ELISA remaining viral particles were detected in the cell culture supernatant. The lentiviral gene transfer efficiency was quantified by FACS analysis. Lentiviral titers were determined by qPCR of expressed transgenes. RESULTS: Third-generation self-inactivating vectors showed highly efficient gene transfer in hMSCs. No viral antigen was detected in the cell culture supernatant after four media changes, suggesting the absence of infectious particles after 4 days. We observed a linear correlation between virus dilution and level of transgene expression by qPCR analysis, therefore allowing viral titering by quantification of transgene expression. Finally, we demonstrated that transduced hMSCs retained their stem cell character by differentiation towards adipogenic, osteogenic and chondrogenic lineages. CONCLUSIONS: Quantification of transgene copy numbers by qPCR is a fast and reliable method to determine functional lentiviral titer after ex vivo gene transfer in hMSCs.     

Cyclic heptapeptides from the soil-derived fungus Clonostachys rosea

Three new cyclic heptapeptides (1–3) together with three known compounds (4–6) were isolated from a solid rice culture of the soil-derived fungus Clonostachys rosea. Fermentation of the fungus on white beans instead of rice afforded a new γ-lactam (7) and a known γ-lactone (8) that were not detected in the former extracts. The structures of the new compounds were elucidated on the basis of 1D and 2D NMR spectra as well as by HRESIMS data. Compounds 1 and 4 exhibited significant cytotoxicity against the L5178Y mouse lymphoma cell line with IC₅₀ values of 4.1 and 0.1 µM, respectively. Compound 4 also displayed cytotoxicity against the A2780 human ovarian cancer cell line with an IC₅₀ value of 3.5 µM. The preliminary structure-activity relationships are discussed.     

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.     

Comparison of Bird Communities in Primary vs. Young Secondary Tropical Montane Cloud Forest in Guatemala

Cloud forests in central Guatemala are fragmented and decreasing in area due to slash-and-burn agricultural activities. We studied bird species composition, abundance, guild composition, and site tenacity of a 102 ha plot located in a cloud forest region of the Sierra Yalijux in Guatemala, half of which was primary forest and half young secondary forest (<7-years-old). Of the 100 species present 14 were restricted to the Endemic Bird Area ‘Northern Central American highlands’ (i.e. 66% of a total of 21 endemics). Five of the 100 analysed species, including one of the restricted-range species (Troglodytes rufociliatus), had a significantly different abundance in primary and secondary forests. Theoretical analysis suggests that seven species out of a community comprised of 141 bird species are already extirpated and only three out of the 14 present restricted-range species might survive the current state of deforestation. Insectivores were the dominant guild on the plot in terms of numbers of species, followed by omnivores, frugivores and granivores. However, in terms of individuals, omnivores made up nearly half of the bird individuals in primary forest, but declined by 44% in secondary forest, whereas granivores more than doubled in this habitat type. Numbers of species per guild were not significantly different between habitats, while numbers of individuals per guild were significantly different. In general, individuals per species are significantly different in the two habitats. Results suggest that most of the species that are currently surviving in the remnant forests of the Sierra Yalijux might be fairly well adapted to a range of forest conditions, but that populations of a number of restricted-range species might be small. Even generalists species like the Common Bush Tanager (Chlorospingus ophthalmicus) are less abundant in secondary vegetation than in primary forest of the study plot.     

Neighboring plant influences on arbuscular mycorrhizal fungal community composition as assessed by T-RFLP analysis

Controls on root colonization by arbuscular mycorrhizal fungi (AMF) include host nutrient status, identity of symbionts and soil physico-chemical properties. Here we show, in the field, that the subset of the AMF community colonizing the roots of a common grass species, Dactylis glomerata, was strongly controlled by neighboring roots of a different plant species, Centaurea maculosa, an invasive forb, thus adding a biological spatial component to controls on root colonization. Using an AMF-specific, 18s rDNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis method, significant differences were found between AMF community fingerprints of samples derived from roots of grasses with (G_Cm) and without (G₀) neighboring C. maculosa. There were also significant differences between samples derived from C. maculosa roots (C_mac) and both G_Cm and G₀ roots. Sample ordination indicated three generally distinct groups consisting of C_mac, G_Cm and G₀, with G_Cm samples being of intermediate distance between G₀and C_mac. Our results indicate that, with the presence of C. maculosa, AMF communities of D. glomerata shift to reflect community composition associated with C. maculosa roots. These results highlight the importance of complex spatial distributions of AMF communities at the scale of a root system. An additional dimension to our study is that C. maculosa is an aggressively invasive plant in the intermountain West. Viewed in this light, these results suggest that pervasive influences of this plant on AMF communities, specifically in roots of its competitors, may represent a mechanism contributing to its invasive success. However, further work is clearly required to determine the extent to which AMF genotypic alteration by neighboring plants influences competitive relationships.     

Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system

The identification of mesenchymal stem cells (MSCs) in adult human tissues and the disclosure of their self-renew-al and multi-lineage differentiation capabilities have provided exciting prospects for cell-based regeneration and tis-sue engineering. Although a considerable amount of data is available describing MSCs, there is still lack of information regarding the molecular mechanisms that govern their adhesion and migration. In this work, we will review the current state of knowledge on integrins and other adhesion molecules found to be expressed on MSCs. The discussed topics include the characteristics of MSCs and their clinical applications, integrins and their central role in cell-matrix attachment and migration, and comments on mobilization, differentiation and contribution to tumour development. Finally, by understanding the complex and fundamental pathways by which MSCs attach and migrate, it might be possible to fine-tune the strategies for effective and safe use of MSCs in regenerative therapies.