Comparative flow cytometry-based immunophenotyping analysis of peripheral blood leukocytes before and after fixation with paraformaldehyde.

Flow cytometry based immunophenotyping provides prime insight into cellular population composition and characteristics, and is widely used in basic and clinical research. Challenges in processing peripheral blood samples in a timely manner necessitate protocol adaptations and utilization of fixatives. Fixation, however, may introduce artifacts to the flow cytometry readout. We performed a comparative flow cytometry immunophenotyping analysis of 13 immune cell populations in the whole blood using a staining protocol with and without fixation step. Freshly procured human peripheral blood samples were stained with a panel of 33 fluorochrome-conjugated antibodies. Samples were processed using a protocol with or without a paraformaldehyde-based fixation step, and matching sample pairs were analyzed by flow cytometry. Our results show that paraformaldehyde-based fixation, in comparison to matched unfixed samples, did not significantly affect population distribution and frequency for: B cells, Plasmablasts, Dendritic cells, NK cells, Granulocytes, Neutrophils, Eosinophils, or Hematopoietic Stem/Progenitor Cells. However, fixation led to significant marker shifts in the subpopulation distribution in CD4, T regulatory, CD8, Monocytes, and Basophils. These results indicate the importance of pre-experimental assessment of fixation-introduced artifacts in the flow cytometry output when considering the feasibility of fresh processing. This is especially important for samples analyzed using comprehensive exploratory immunoprofiling panels.

Seasonal variation of leaf thickness: An overlooked component of functional trait variability.

The dry and wet seasons in the Neotropics have strong effects on soil water and nutrient availability, as well as on forest dynamics. Despite these major effects on forest ecology, little is known on how leaf traits vary throughout the seasons in tropical rainforest trees. Here, we investigated the influence of seasonal variations in climate and soil characteristics on leaf trait variation in two tropical tree species. We measured two leaf traits, thickness and water mass per area, in 401 individuals of two species of Symphonia (Clusiaceae) in the Paracou research station in French Guiana tropical lowland rainforest. We found a significant effect of seasonal variation on these two leaf traits. Soil relative extractable water was a strong environmental predictor of leaf trait variation in response to seasonal variation. Reduced soil water availability during the dry season was associated with increased leaf thickness and water mass per area, possibly as a result of stomatal closure. Our findings advocate the need to account for environmental seasonality when studying leaf traits in seasonal ecosystems such as tropical forests.

Long-term decline of the Amazon carbon sink.

Atmospheric carbon dioxide records indicate that the land surface has acted as a strong global carbon sink over recent decades, with a substantial fraction of this sink probably located in the tropics, particularly in the Amazon. Nevertheless, it is unclear how the terrestrial carbon sink will evolve as climate and atmospheric composition continue to change. Here we analyse the historical evolution of the biomass dynamics of the Amazon rainforest over three decades using a distributed network of 321 plots. While this analysis confirms that Amazon forests have acted as a long-term net biomass sink, we find a long-term decreasing trend of carbon accumulation. Rates of net increase in above-ground biomass declined by one-third during the past decade compared to the 1990s. This is a consequence of growth rate increases levelling off recently, while biomass mortality persistently increased throughout, leading to a shortening of carbon residence times. Potential drivers for the mortality increase include greater climate variability, and feedbacks of faster growth on mortality, resulting in shortened tree longevity. The observed decline of the Amazon sink diverges markedly from the recent increase in terrestrial carbon uptake at the global scale, and is contrary to expectations based on models.

MicroRNA-126 inhibits invasion in bladder cancer via regulation of ADAM9.

BACKGROUND: The miRNA deregulation is commonly observed in human malignancies, where they act as tumour suppressors or oncogenes. Despite the association of several miRNAs with bladder cancer, little is known about the miRNAs that contribute to bladder cancer progression from non-muscle invasive (NMI) to muscle-invasive (MI) disease. METHODS: We first profiled the expression of miRNAs and mRNAs in a cohort of urothelial carcinomas and further characterised the role of miR-126 in invasion, as it emerged as the most downregulated miRNA between MI and NMI tumours. RESULTS: We found that restoration of miR-126 levels attenuated the invasive potential of bladder cancer cells. Mechanistically, we identified the role of miR-126 in invasion through its ability to target ADAM9. Notably, a significant inverse correlation between miR-126 and ADAM9 expression was observed, where ADAM9 was upregulated in MI bladder cancer cells. While knockdown of ADAM9 attenuated the invasiveness of cells with low miR-126 levels, experimental upregulation of ADAM9 recapitulated the invasive phenotype. Furthermore, ADAM9 expression assessed by immunohistochemistry significantly correlated with poor prognosis in patients with urothelial carcinoma. CONCLUSIONS: In this study we describe the role of miR-126 in bladder cancer progression, identifying miR-126 and ADAM9 as potential clinical biomarkers of disease aggressiveness.

Litter mixture effects on tropical tree seedling growth--a greenhouse experiment.

Decomposing litter provides critical nutrients for plants, particularly in nutrient-poor ecosystems such as tropical forests. We hypothesised that decomposing litter improves the performance of a variety of tropical tree seedlings, and that this litter effect varies depending on the species of litter present in litter mixtures. We addressed these hypotheses with a large pot experiment manipulating a range of different litter mixtures of contrasting quality and using seedlings of four tree species from the Amazonian forest of French Guiana. In contrast to our initial hypothesis, decomposing litter had either neutral or negative impacts on seedling growth, despite strongly different growth rates, biomass allocation patterns and leaf and root traits among tree species. Tree species varied in their responses to litter additions, which were further modified by species identity of the added litter. Our data show litter species-specific effects on growth, biomass allocation and leaf and root traits of tropical tree seedlings. These results suggest that a net nutrient release from decomposing litter does not necessarily improve tree seedling growth, even under nutrient-limiting conditions. In conclusion, litter layer composition may affect seedling establishment and recruitment success beyond litter-derived plant nutrient availability, which may contribute to tree species composition and dynamics in the studied tropical forest.

Interspecific variability of δ13C among trees in rainforests of French Guiana: functional groups and canopy integration.

The interspecific variability of sunlit leaf carbon isotope composition (δ13C), an indicator of leaf intrinsic water-use efficiency (WUE, CO2 assimilation rate/leaf conductance for water vapour), was investigated in canopy trees of three lowland rainforest stands in French Guiana, differing in floristic composition and in soil drainage characteristics, but subjected to similar climatic conditions. We sampled leaves with a rifle from 406 trees in total, representing 102 species. Eighteen species were common to the three stands. Mean species δ13C varied over a 6.0‰ range within each stand, corresponding to WUE varying over about a threefold range. Species occurring in at least two stands displayed remarkably stable δ13C values, suggesting a close genetic control of species δ13C. Marked differences in species δ13C values were found with respect to: (1) the leaf phenology pattern (average δ13C=-29.7‰ and -31.0‰ in deciduous-leaved and evergreen-leaved species, respectively), and (2) different types of shade tolerance defined by features reflecting the plasticity of growth dynamics with respect to contrasting light conditions. Heliophilic species exhibited more negative δ13C values (average δ13C=-30.5‰) (i.e. lower WUE) than hemitolerant species (-29.3‰). However, tolerant species (-31.4‰) displayed even more negative δ13C values than heliophilic ones. We could not provide a straightforward ecophysiological interpretation of this result. The negative relationship found between species δ13C and midday leaf water potential (Ψwm) suggests that low δ13C is associated with high whole tree leaf specific hydraulic conductance. Canopy carbon isotope discrimination (Δ A ) calculated from the basal area-weighed integral of the species δ13C values was similar in the three stands (average Δ A =23.1‰), despite differences in stand species composition and soil drainage type, reflecting the similar proportions of the three different shade-tolerance types among stands.