Response of Sphagnum Peatland Testate Amoebae to a 1-Year Transplantation Experiment Along an Artificial Hydrological Gradient

Peatland testate amoebae (TA) are well-established bioindicators for depth to water table (DWT), but effects of hydrological changes on TA communities have never been tested experimentally. We tested this in a field experiment by placing Sphagnum carpets (15 cm diameter) collected in hummock, lawn and pool microsites (origin) at three local conditions (dry, moist and wet) using trenches dug in a peatland. One series of samples was seeded with microorganism extract from all microsites. TA community were analysed at T0: 8–2008, T1: 5–2009 and T2: 8–2009. We analysed the data using conditional inference trees, principal response curves (PRC) and DWT inferred from TA communities using a transfer function used for paleoecological reconstruction. Density declined from T0 to T1 and then increased sharply by T2. Species richness, Simpson diversity and Simpson evenness were lower at T2 than at T0 and T1. Seeded communities had higher species richness in pool samples at T0. Pool samples tended to have higher density, lower species richness, Simpson diversity and Simpson Evenness than hummock and/or lawn samples until T1. In the PRC, the effect of origin was significant at T0 and T1, but the effect faded away by T2. Seeding effect was strongest at T1 and lowest vanished by T2. Local condition effect was strong but not in line with the wetness gradient at T1 but started to reflect it by T2. Likewise, TA-inferred DWT started to match the experimental conditions by T2, but more so in hummock and lawn samples than in pool samples. This study confirmed that TA responds to hydrological changes over a 1-year period. However, sensitivity of TA to hydrological fluctuations, and thus the accuracy of inferred DWT changes, was habitat specific, pool TA communities being least responsive to environmental changes. Lawns and hummocks may be thus better suited than pools for paleoecological reconstructions. This, however, contrasts with the higher prediction error and species’ tolerance for DWT with increasing dryness observed in transfer function models.     

Glutathione Reductase-Mediated Synthesis of Tellurium-Containing Nanostructures Exhibiting Antibacterial Properties

Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells.     

Dual diaminopimelate biosynthesis pathways in Bacteroides fragilis and Clostridium thermocellum

Bacteroides fragilis and Clostridium thermocellum were recently found to synthesize diaminopimelate (DAP) by way of LL-DAP aminotransferase. Both species also contain an ortholog of meso-diaminopimelate dehydrogenase (Ddh), suggesting that they may have redundant pathways for DAP biosynthesis. The B. fragilis Ddh ortholog shows low homology with other examples of Ddh and this species belongs to a phylum, the Bacteriodetes, not previously known to contain this enzyme. By contrast, the C. thermocellum ortholog is well conserved with known examples of Ddh. Using in vitro and in vivo assays both the B. fragilis and C. thermocellum enzymes were found to be authentic examples of Ddh, displaying kinetic properties typical of this enzyme. The result indicates that B. fragilis contains a sequence diverged form of Ddh. Phylogenomic analysis of the microbial genome database revealed that 77% of species with a Ddh ortholog also contain a second pathway for DAP biosynthesis suggesting that Ddh evolved as an ancillary mechanism for DAP biosynthesis.     

Regulatory light chain phosphorylation and N-terminal extension increase cross-bridge binding and power output in Drosophila at in vivo myofilament lattice spacing

The N-terminal extension and phosphorylation of the myosin regulatory light chain (RLC) independently improve Drosophila melanogaster flight performance. Here we examine the functional and structural role of the RLC in chemically skinned fibers at various thick and thin filament lattice spacings from four transgenic Drosophila lines: rescued null or control (Dmlc2⁺), truncated N-terminal extension (Dmlc2^Δ2-46), disrupted myosin light chain kinase phosphorylation sites (Dmlc2^S66A,S67A), and dual mutant (Dmlc2^{Δ2-46; S66A,S67A}). The N-terminal extension truncation and phosphorylation sites disruption mutations decreased oscillatory power output and the frequency of maximum power output in maximally Ca²⁺-activated fibers compressed to near in vivo inter-thick filament spacing, with the phosphorylation sites disruption mutation having a larger affect. The diminished power output parameters with the N-terminal extension truncation and phosphorylation sites disruption mutations were due to the reduction of the number of strongly-bound cross-bridges and rate of myosin force production, with the larger parameter reductions in the phosphorylation sites disruption mutation additionally related to reduced myosin attachment time. The phosphorylation and N-terminal extension-dependent boost in cross-bridge kinetics corroborates previous structural data, which indicate these RLC attributes play a complementary role in moving and orienting myosin heads toward actin target sites, thereby increasing fiber and whole fly power generation.     

Mucosal immunity against Eimeria acervulina infection in broiler chickens following oral immunization with profilin in Montanide™ adjuvants

Leishmaniasis is a disease caused by the unicellular Leishmania parasite. World wide millions of people are affected by this vector born disease. The disease presents itself in different clinical manifestations which are caused by specific Leishmania species. The therapeutic strategy depends on the Leishmania species involved. It is important to detect Leishmania and subsequently type the infecting species in a sensitive way using PCR. Various targets have been proposed but two seem to be best suited, the ITS1 region and the mini-exon. There is, however, no consensus as to which of these two is best. The aim of this study was to compare both targets with our current method, a PCR on the 18S ribosomal RNA gene. The ITS1 PCR proved to be slightly more sensitive and more practical than the mini-exon. Nevertheless, the mini-exon is more polymorphic and is needed in subtyping Leishmania species belonging to the L. Viannia subgenus. The ITS1 method was adapted to use as a real-time PCR for diagnostic purposes. In addition, designing and testing a new primer set improved sensitivity of the PCR on the mini-exon.     

Cytotoxic effects of new trans-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines and their interaction with antitumoral drugs gemcitabine and paclitaxel on cellular lines of human breast cancer

Two tetrahydroquinoline compounds, called DM8 and DM12, from a new series of the cis-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines, were selected for cytotoxic effects studies on cellular lines of human breast cancer. The synergistic, additive and antagonistic effects in combination of these compounds with anticancer drugs, such as paclitaxel and gemcitabine, were studied. The isobolograms and their analysis demonstrated models of synergism, additivity and antagonism of these tetrahydroquinolines in the presence of paclitaxel and gemcitabine. Results showed that compounds DM8 and DM12 individually induced growth inhibition on breast cancer cell lines MCF-7 and SKBR3, and the addition of paclitaxel and gemcitabine intensified their cytotoxic activity on both cell lines at conc. below 1 μg/mL. During these studies the compound DM12 was identified as new, perspective and safe agent for adjuvant therapy.     

Protein deiminases: new players in the developmentally regulated loss of neural regenerative ability

Spinal cord regenerative ability is lost with development, but the mechanisms underlying this loss are still poorly understood. In chick embryos, effective regeneration does not occur after E13, when spinal cord injury induces extensive apoptotic response and tissue damage. As initial experiments showed that treatment with a calcium chelator after spinal cord injury reduced apoptosis and cavitation, we hypothesized that developmentally regulated mediators of calcium-dependent processes in secondary injury response may contribute to loss of regenerative ability. To this purpose we screened for such changes in chick spinal cords at stages of development permissive (E11) and non-permissive (E15) for regeneration. Among the developmentally regulated calcium-dependent proteins identified was PAD3, a member of the peptidylarginine deiminase (PAD) enzyme family that converts protein arginine residues to citrulline, a process known as deimination or citrullination. This post-translational modification has not been previously associated with response to injury. Following injury, PAD3 up-regulation was greater in spinal cords injured at E15 than at E11. Consistent with these differences in gene expression, deimination was more extensive at the non-regenerating stage, E15, both in the gray and white matter. As deimination paralleled the extent of apoptosis, we investigated the effect of blocking PAD activity on cell death and deiminated-histone 3, one of the PAD targets we identified by mass-spectrometry analysis of spinal cord deiminated proteins. Treatment with the PAD inhibitor, Cl-amidine, reduced the abundance of deiminated-histone 3, consistent with inhibition of PAD activity, and significantly reduced apoptosis and tissue loss following injury at E15. Altogether, our findings identify PADs and deimination as developmentally regulated modulators of secondary injury response, and suggest that PADs might be valuable therapeutic targets for spinal cord injury.     

Colour-scent associations in a tropical orchid: three colours but two odours

Colour and scent are the major pollinator attractants to flowers, and their production may be linked by shared biosynthetic pathways. Species with polymorphic floral traits are particularly relevant to study the joint evolution of floral traits. We used in this study the tropical orchid Calanthe sylvatica from Réunion Island. Three distinct colour varieties are observed, presenting lilac, white or purple flowers, and named respectively C. sylvaticavar.lilacina (hereafter referred as var. lilacina), C. sylvaticavar. alba (var. alba) and C. sylvatica var. purpurea (var. purpurea). We investigated the composition of the floral scent produced by these colour varieties using the non-invasive SPME technique in the wild. Scent emissions are dominated by aromatic compounds. Nevertheless, the presence of the terpenoid (E)-4,8-dimethylnona-1,3,7-triène (DMNT) is diagnostic of var. purpurea, with the volatile organic compounds (VOC) produced by some individuals containing up to 60% of DMNT. We evidence specific colour-scent associations in C. sylvatica, with two distinct scent profiles in the three colour varieties: the lilacina-like profile containing no or very little DMNT (<2%) and the purpurea-like profile containing DMNT (>2%). Calanthe sylvatica var. alba individuals group with one or the other scent profile independently of their population of origin. We suggest that white-flowered individuals have evolved at least twice, once from var. lilacina and at least once from var. purpurea after the colonisation of la Réunion. White-flowered individuals may have been favoured by the particular pollinator fauna characterising the island. These flowering varieties of C. sylvatica, which display three colours but two scents profiles prove that colour is not always a good indicator of odour and that colour-scent associations may be complex, depending on pollination ecology of the populations concerned.     

Coalescent analysis of mtDNA indicates Pleistocene divergence among three species of howler monkey (<Emphasis Type="Italic">Alouatta</Emphasis> spp.) and population subdivision within the Atlantic Coastal Forest species, <Emphasis Type="Italic">A. guariba</Emphasis>

We have used coalescent analysis of mtDNA cytochrome b (cyt b) sequences to estimate times of divergence of three species of Alouatta—A. caraya, A. belzebul, and A. guariba—which are in close geographic proximity. A. caraya is inferred to have diverged from the A. guariba/A. belzebul clade approximately 3.83  million years ago (MYA), with the later pair diverging approximately 1.55 MYA. These dates are much more recent than previous dates based on molecular-clock methods. In addition, analyses of new sequences from the Atlantic Coastal Forest species A. guariba indicate the presence of two distinct haplogroups corresponding to northern and southern populations with both haplogroups occurring in sympatry within Sao Paulo state. The time of divergence of these two haplogroups is estimated to be 1.2 MYA and so follows quite closely after the divergence of A. guariba and A. belzebul. These more recent dates point to the importance of Pleistocene environmental events as important factors in the diversification of A. belzebul and A. guariba. We discuss the diversification of the three Alouatta species in the context of recent models of climatic change and with regard to recent molecular phylogeographic analyses of other animal groups distributed in Brazil.