Immunodominant Francisella tularensis antigens identified using proteome microarray

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens.     

Antibody microarray-based profiling of complex specimens: systematic evaluation of labeling strategies

Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined in this study the suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis. N-hydroxysuccinimidyl ester (NHS)- and Universal Linkage System (ULS)-based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labeling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labeling were analyzed and optimized in this study. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labeling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios. Finally, particularly sensitive detection chemistry was applied to monitoring cytokine production of stimulated peripheral T cells. Microarray data were in accord with quantitative cytokine levels measured by ELISA and Luminex, demonstrating comparable reliability and femtomolar range sensitivity of the established microarray approach.     

Exercise affects the gene expression profiles of human white blood cells.

White blood cells (WBCs) express tens of thousands of genes, whose expression levels are modified by genetic and external factors. The purpose of the present study was to investigate the effects of acute exercise on gene expression profiles (GEPs) of WBCs and to identify suitable genes that may serve as surrogate markers for monitoring exercise and training load. Five male participants performed an exhaustive treadmill test (ET) at 80% of their maximal O(2) uptake (Vo(2 max)) and a moderate treadmill test (MT) at 60% Vo(2 max) for exactly the same time approximately 2 wk later. WBCs were isolated by the erythrocyte lysis method. GEPs were measured using the Affymetrix GeneChip technology. After scaling, normalization, and filtering, groupwise comparisons of gene expression intensities were performed, and several measurements were validated by real-time PCR. We found 450 genes upregulated and 150 downregulated (>1.5-fold change; ANOVA with Benjamini-Hochberg correction, P < 0.05) after ET that were closely associated with the gene ontology lists "response to stress" and "inflammatory response". Analysis of mean expression levels after MT showed that the extent of up- and downregulation was workload dependent. The genes for the stress (heat shock) proteins HSPA1A and HSPH1 and for the matrix metalloproteinase MMP-9 showed the most prominent increases, whereas the YES1 oncogene (YES1) and CD160 (BY55) were most strongly reduced. Despite different methodological approaches used, the consistency of our results with the expression data of another study (Connolly PH, Caiozzo VJ, Zaldivar F, Nemet D, Larson J, Hung SP, Heck JD, Hatfield GW, Cooper DM. J Appl Physiol 97: 1461-1469, 2004) suggests that expression fingerprints are useful tools for monitoring exercise and training loads and thereby help to avoid training-associated health risks.     

Melanosome diversity and convergence in the evolution of iridescent avian feathers—Implications for paleocolor reconstruction

Some of the most varied colors in the natural world are created by iridescent nanostructures in bird feathers, formed by layers of melanin‐containing melanosomes. The morphology of melanosomes in iridescent feathers is known to vary, but the extent of this diversity, and when it evolved, is unknown. We use scanning electron microscopy to quantify the diversity of melanosome morphology in iridescent feathers from 97 extant bird species, covering 11 orders. In addition, we assess melanosome morphology in two Eocene birds, which are the stem lineages of groups that respectively exhibit hollow and flat melanosomes today. We find that iridescent feathers contain the most varied melanosome morphologies of all types of bird coloration sampled to date. Using our extended dataset, we predict iridescence in an early Eocene trogon (cf. Primotrogon) but not in the early Eocene swift Scaniacypselus, and neither exhibit the derived melanosome morphologies seen in their modern relatives. Our findings confirm that iridescence is a labile trait that has evolved convergently in several lineages extending down to paravian theropods. The dataset provides a framework to detect iridescence with more confidence in fossil taxa based on melanosome morphology.     

Efficient slow-growth conservation and assessment of clonal fidelity of Ullucus tuberosus Caldas microshoots

Plant Cell, Tissue and Organ Culture (PCTOC) - Slow-growth is a biotechnological tool for medium-term conservation of plant germplasm under in vitro conditions. In the present study, we assessed...     

Fine scale waterbody data improve prediction of waterbird occurrence despite coarse species data

While modelling habitat suitability and species distribution, ecologists must deal with issues related to the spatial resolution of species occurrence and environmental data. Indeed, given that the spatial resolution of species and environmental datasets range from centimeters to hundreds of kilometers, it underlines the importance of choosing the optimal combination of resolutions to achieve the highest possible modelling prediction accuracy. We evaluated how the spatial resolution of land cover/waterbody datasets (meters to 1 km) affect waterbird habitat suitability models based on atlas data (grid cell of 12 × 11 km). We hypothesized that the area, perimeter and number of waterbodies computed from high resolution datasets would explain distributions of waterbirds better because coarse resolution datasets omit small waterbodies affecting species occurrence. Specifically, we investigated which spatial resolution of waterbodies better explain the distribution of seven waterbirds nesting on ponds/lakes with areas ranging from 0.1 ha to hundreds of hectares. Our results show that the area and perimeter of waterbodies derived from high resolution datasets (raster data with 30 m resolution, vector data corresponding with map scale 1:10 000) explain the distribution of the waterbirds better than those calculated using less accurate datasets despite the coarse grain of the species data. Taking into account the spatial extent (global vs regional) of the datasets, we found the Global Inland Waterbody Dataset to be the most suitable for modelling distribution of waterbirds. In general, we recommend using land cover data of a resolution sufficient to capture the smallest patches of the habitat suitable for a given species’ presence for both fine and coarse grain habitat suitability and distribution modelling.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.     

Phosphorus Losses from the Epilimnion in Římov Reservoir

The amount of settling phosphorus was measured in Římov Reservoir using sediment trap technique from April 1986 to April 1987. Sediment traps were placed at three depths near the dam of the reservoir and at the bottom along the reservoir. The highest amount of phosphorus in trapped material was found during the fall turnover in the epilimnion and near the bottom in both spring periods (1986,1997). During the growing season the changes in dry weight and total phosphorus in settling seston were related to changes of phytoplankton biomass in the trophogenic layer. The amount of trapped phosphorus was higher near the bottom than in the upper layers of the reservoir throughout the year.     

CIRCADIAN RHYTHM OF PHOTOSYNTHETIC OXYGEN EVOLUTION IN KAPPAPHYCUS ALVAREZII (RHODOPHYTA): DEPENDENCE ON LIGHT QUANTITY AND QUALITY

The rate of oxygen evolution of the tropical red alga Kappaphycus alvarezii (Doty) Doty was measured for 6 days in the laboratory using a computer-aided method for long-term recording. In cool white light, Kappaphycus exhibited a robust circadian rhythm of O₂ evolution in the irradiance range of 100 to 1000 μmol photons·m ^{− 2}·s ^{− 1}. With increasing irradiance, the period of the free-running rhythm, τ, decreased in blue and increased in red light but did not change significantly in green light. The accelerating or slowing action of blue or red light, respectively, points to two photoreceptors used in the light transduction pathway of the circadian oscillator controlling oxygen evolution or the light reactions of photosynthesis in Kappaphycus. No significant changes of τ were observed with increasing irradiance in cool white light, possibly due to the additive opposing responses caused by blue and red light.