Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: Key role of granulocytes

IntroductionCell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34⁺ quantification; (ii) percentage of CD34⁺ viability and (iii) evaluation of HSC functional ability to form colony forming unit–granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34⁺, using a Fluorescent Labeled Inhibitor of CAspases (FLICA).MethodsCaspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34⁺, CD3⁺, CD14⁺ and CD56⁺ cells. Viable 7AAD^?/FLICA⁺ cells were then correlated with various parameters.ResultsWe showed a significant caspase pathway activation, with 23% CD34⁺/7AAD^?/FLICA⁺ cells after thawing, compared with the 2% described in fresh CD34⁺ cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3⁺, CD56⁺ and CD14⁺ cells. We also report a significant correlation between the rate of CD34⁺/7AAD^?/FLICA⁺ cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004).DiscussionOur results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3⁺ and CD56⁺ subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).     

Dissolved humic substances – ecological driving forces from the individual to the ecosystem level?

1. This review focuses on direct and indirect interactions between dissolved humic substances (HS) and freshwater organisms and presents novel opinions and hypotheses on their ecological significance. Despite their abundance in freshwaters, the role of HS is still inadequately understood. These substances have been considered too large to be taken up by freshwater organisms. On the contrary, here we present evidence that dissolved HS are indeed taken up and interact directly and/or indirectly with freshwater organisms. 2. We show that dissolved HS exert a mild chemical stress upon aquatic organisms in many ways; they induce molecular chaperones (stress shock proteins), induce and modulate biotransformation enzymes and modulate (mainly inhibiting) the photosynthetic release of oxygen by freshwater plants. Furthermore, they produce an oxidative stress, which may lead to membrane oxidation. HS modulate the multixenobiotic resistance activity and probably other membrane‐bound pumps. This property may lead to the increased bioaccumulation of xenobiotic chemicals. Furthermore, they can modulate the numbers of offspring in a nematode and feminise fish and amphibians. The ecological consequences of this potential remain obscure at present. HS also have the potential to act as chemical attractants (as shown with a nematode). 3. In some macrophytes and algae we show that HS interfere with photosynthesis and growth. For instance, the presence of HS suppresses cyanobacteria more than eukaryotic algae. By applying a quantitative structure activity relationship approach, we show that quinones in the HS interfere with photosynthetic electron transport. We show that even Phragmites leachate can act as a kind of phytotoxin. HS also have the potential to suppress fungal growth, as shown with the water mould Saprolegnia parasitica and force the fungus to respond by spore production. 4. In very soft, humic freshwaters, such as the Rio Negro, Brazil, HS stimulate the uptake of essential ions, such as Na and Ca, at extremely low pH (3.5–4.0) and prevent the ionoregulatory disturbance induced by acid waters, thereby enabling fish to survive in these environments. 5. We discuss whether or not HS are directly utilised by aquatic microorganisms or via exoenzymes, which may be washed in from the terrestrial catchment. There is accumulating evidence that the quality of the HS controls microbial growth. In total, net‐heterotrophy may result from HS‐mediated suppression of primary production by the quinone structures and/or from HS‐mediated support of microbial growth. As there is also evidence that HS have the potential to support photoautotrophic growth and suppress microbial growth, the opposite community effect could result. Consequently, dissolved organic carbon (DOC) has to be chemically characterised, rather than simply measuring bulk DOC concentration. 6. In sum, dissolved HS interact with freshwater organisms in a variety of ways in unenriched humic lakes. In addition to the well known effects of HS on light regime, for example, and the direct and indirect supply with carbon (energy), other interactions may be much more subtle. For instance, HS may induce internal biochemical stress defence systems and have the potential to cause acclimatisation and even adaptation. We are just at the beginning of understanding these interactions between dissolved HS and freshwater organisms.     

The importance of landscape and habitat properties in explaining instantaneous and long‐term distributions of large branchiopods in subtropical temporary pans

1. The notion that the spatial configuration of habitat patches has to be taken into account to understand the structure and dynamics of ecological communities is the starting point of metacommunity ecology. One way to assess metacommunity structure is to investigate the relative importance of environmental heterogeneity and spatial structure in explaining community patterns over different spatial and temporal scales. 2. We studied metacommunity structure of large branchiopod assemblages characteristic of subtropical temporary pans in SE Zimbabwe using two community data sets: a community snapshot and a long‐term data set covering 4 years. We assessed the relative importance of environmental heterogeneity and dispersal (inferred from patch occupancy patterns) as drivers of community structure. Furthermore, we contrasted metacommunity patterns in pans that occasionally connect to the river (floodplain pans) and pans that lack such connections altogether (endorheic pans) using redundancy models. 3. Echoes of species sorting and dispersal limitation emerge from our data set, suggesting that both local and regional processes contribute to explaining branchiopod assemblages in this system. Relative importance of local and regional factors depended on the type of data set considered. Overall, habitat characteristics that vary in time, such as conductivity, hydroperiod and vegetation cover, best explained the instantaneous species composition observed during a snapshot sampling while long‐term species composition appeared to be linked to more constant intrinsic habitat properties such as river connectivity and spatial location.     

Biogeography meets conservation: the genetic structure of the endangered lycaenid butterfly Lycaena helle (Denis & Schiffermüller, 1775)

Cold‐adapted species are thought to have had their largest distribution ranges in central Europe during the glacial periods. Postglacial warming caused severe range shifts of such taxa into higher latitudes and altitudes. We selected the boreomontane butterfly Lycaena helle (Denis & Schiffermüller, 1775) as an example to demonstrate the genetic effects of range changes, and to document the recent status of highly fragmented remnant populations. We analysed five polymorphic microsatellite loci in 1059 individuals sampled at 50 different localities scattered over the European distribution area of the species. Genetic differentiation was strong among the mountain ranges of western Europe, but we did not detect similarly distinct genetic groups following a geographical pattern in the more eastern areas. The Fennoscandian populations form a separate genetic group, and provide evidence for a colonization from southern Finland via northern Scandinavia to south‐central Sweden. Species distribution modelling suggests a large extension of the spatial distribution during the last glacial maximum, but highlights strong retractions to a few mountain areas under current conditions. These findings, combined with our genetic data, suggest a more or less continuous distribution of L. helle throughout central Europe at the end of the last ice age. As a consequence of postglacial warming, the species retreated northwards to Fennoscandia and escaped increasing temperatures through altitudinal shifts. Therefore, the species is today restricted to population remnants located at the mountain tops of western Europe, genetically isolated from each other, and evolved into genetically unique entities. Rising temperatures and advancing habitat destruction threaten this wealth of biodiversity. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 155–168.     

Invertebrate community patterns in Mediterranean temporary wetlands along hydroperiod and salinity gradients

1. Temporary aquatic habitats often are inhabited by a unique fauna and flora and contribute significantly to regional diversity. Temporary wetlands around the world are disappearing rapidly. The individual and interacting impacts of factors influencing community structure and dynamics in temporary wetlands are not always well known.
2. Camargue wetlands are mainly characterized by variable salinity and hydroperiod. The individual and combined impacts of these local factors, together with regional variables, on invertebrate communities remain unknown. We therefore characterized and sampled invertebrates in 30 temporary wetlands along salinity and hydroperiod gradients in the Camargue (Southern France) 3, 5 and 7 months after inundation.
3. Over the three sampling occasions, a total of 17 cladoceran species and 49 macroinvertebrate taxa were identified. Hydroperiod and salinity were the most important variables explaining variation in taxonomic composition and can be considered key factors shaping the invertebrate communities in Camargue wetlands. The impact on taxon richness was significantly positive for hydroperiod but significantly negative for salinity. Regional factors had no significant effect on the structure of the studied invertebrate communities, suggesting that dispersal was not limiting and that species sorting was the most important structuring process.
4. The results of this study suggest that the combined and interacting effects of salinization and hydrological modification of Mediterranean temporary wetlands (due to water management, climate change, etc.) can result in reduced diversity in large numbers of Mediterranean wetlands and induce a considerable decline in regional diversity of aquatic invertebrates.     

Passive external transport of freshwater invertebrates by elephant and other mud‐wallowing mammals in an African savannah habitat

1. Recent findings hint at the potential importance of mammals affecting the spatial dynamics of aquatic organisms in areas where mammals live in close association with water. Perhaps the most iconic example of such an environment is the African savannah. 2. We investigated dispersal patterns of freshwater organisms among a set of temporary ponds in SE Zimbabwe to test the hypothesis that large mammals, and particularly African elephants (Loxodonta africana), can be important vectors of aquatic organisms. Dispersal kernels were reconstructed by hatching mud collected from ‘rubbing’ trees located at increasing distances from a set of isolated ponds. To assess the relative importance of other mammalian vectors, the vertical distribution of mud on rubbing trees was mapped and related to the body size of candidate vector species. 3. Laboratory hatching of mud samples revealed large numbers of propagules of 22 invertebrate taxa as well as some aquatic macrophytes. Dispersing communities reflected source communities and diverged with increasing distance from the source. Both dispersal rates and richness of transported taxa decreased significantly with dispersal distance. No indications for differences in dispersal capacity among propagule types were detected. Instead, common propagules were more likely to travel greater distances. Most mud was attached to trees at heights >1.5 m, implicating elephants as the dominant vector. Vertical distributions of tree mud, however, also revealed clustering at heights up to 50 cm and 90–120 cm corresponding to the height of warthog, rhinoceros and buffalo, respectively. Finally, variation in the vertical distribution of mud on trees in combination with differences in vector vagility suggests that local differences in vector species composition may affect passive dispersal dynamics of aquatic organisms. 4. Based on vagility and vector load, mud‐wallowing mammals emerge as highly effective vectors that, in some areas, may be more important in transporting aquatic organisms than traditionally recognised vectors such as waterbirds. Since most large‐ and medium‐sized mammals currently have restricted geographic distributions, it is likely that mammal‐mediated dispersal was more important in the past.     

Phylogenetic placement,taxonomic revision and a new species of Nothostele (Orchidaceae), an enigmatic genus endemic to the cerrado of central Brazil

Nothostele is a rare genus endemic to central Brazil. The taxonomy of the genus is controversial and almost every taxonomist has had a different point of view regarding its generic and subtribal classification. After the first collection 138 years ago, N. acianthiformis has been collected again and we report here a phylogenetic analysis of the genus based on nuclear ribosomal internal transcribed spacer and plastid matK and trnL–trnF sequence data. Our results show that Nothostele belongs to subtribe Spiranthinae and is sister to Eltroplectris within the Stenorrhynchos clade. A reanalysis of gynostemium morphology shows that the presence of a hamulus was misinterpreted in Nothostele and that the placement of the genus in Cranichidinae based on morphology of the pollinarium is unwarranted. Furthermore, the flattened, sessile, prostrate leaves of N. acianthiformis, which are described here for the first time, occur in some Spiranthinae but are atypical in Cranichidinae. Specimens from Brasília in the Brazilian central plateau are a distinct new species, which is described here as N. brasiliaënsis. Currently, the genus comprises two disjunct species restricted to the cerrado and rocky field vegetation of central Brazil. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 348–363.     

Short‐term effects of CO2 and O2 on citrate metabolism in illuminated leaves

Although there is now a considerable literature on the inhibition of leaf respiration (CO₂ evolution) by light, little is known about the effect of other environmental conditions on day respiratory metabolism. In particular, CO₂ and O₂ mole fractions are assumed to cause changes in the tricarboxylic acid pathway (TCAP) but the amplitude and even the direction of such changes are still a matter of debate. Here, we took advantage of isotopic techniques, new simple equations and instant freeze sampling to follow respiratory metabolism in illuminated cocklebur leaves (Xanthium strumarium L.) under different CO₂/O₂ conditions. Gas exchange coupled to online isotopic analysis showed that CO₂ evolved by leaves in the light came from ‘old’ carbon skeletons and there was a slight decrease in ¹³C natural abundance when [CO₂] increased. This suggested the involvement of enzymatic steps fractionating more strongly against ¹³C and thus increasingly limiting for the metabolic respiratory flux as [CO₂] increased. Isotopic labelling with ¹³C₂‐2,4‐citrate lead to ¹³C‐enriched Glu and 2‐oxoglutarate (2OG), clearly demonstrating poor metabolism of citrate by the TCAP. There was a clear relationship between the ribulose‐1,5‐bisphosphate oxygenation‐to‐carboxylation ratio (v_o/v_c) and the ¹³C commitment to 2OG, demonstrating that 2OG and Glu synthesis via the TCAP is positively influenced by photorespiration.     

Dispersal of freshwater invertebrates by large terrestrial mammals: a case study with wild boar (Sus scrofa) in Mediterranean wetlands

1. Many invertebrates inhabiting insular aquatic habitats rely on external agents or vectors to disperse. Besides water connections and wind, waterfowl and amphibians are known to mediate passive dispersal of freshwater invertebrates. However, the possibility of dispersal by terrestrial mammals has been largely overlooked. 2. We investigated the potential of both external and internal zoochorous dispersal of aquatic invertebrates by the wild boar (Sus scrofa) in Mediterranean wetlands in the Camargue (France). As wild boar frequently visit wetlands for feeding and wallowing purposes, we hypothesized that they may be important passive dispersal vectors of aquatic invertebrates at a local scale. Dried mud was collected from selected ‘rubbing trees’ used by boars to dispose of parasites. Additionally, faecal pellets were collected from different locations in the wetland area. 3. Seventeen freshwater invertebrate taxa including rotifers, cladocerans, copepods and ostracods hatched from sediment obtained from ‘rubbing trees’, while invertebrates hatching from dried faeces (10 taxa) were mainly rotifers. Dispersing invertebrates were collected up to 318 m from a nearest potential dispersal source. Both abundance and richness of invertebrates significantly decreased with dispersal distance. 4. Our results demonstrate that large mammals such as wild boar can act as dispersal vectors of aquatic invertebrates at a local scale in the wetland area of the Camargue and suggest that external transport may be quantitatively more important than internal transport. As wallowing (mud bathing) is common in many terrestrial mammals, this mode of dispersal may be quite widespread.     

Development and characterization of microsatellite markers for the tropical tree species Tabebuia aurea (Bignoniaceae)

In this study, we report the development and characterization of 21 polymorphic microsatellite loci for Tabebuia aurea, using genomic library enrichment. Number of alleles per locus and expected heterozygosity ranged from nine to 26 and from 0.808 to 0.955. The high combined probability of genetic identity (1.03 × 10^?37) and probability of paternity exclusion (0.9889) showed that multilocus genotypes are likely to be unique and will allow detailed parentage studies in natural populations of T. aurea. Additionally, a high percentage of transferability was achieved for the four species of the same genus studied.