Molecular phylogenies disprove a hypothesized C4 reversion in Eragrostis walteri (Poaceae)

Background and Aims

The main assemblage of the grass subfamily Chloridoideae is the largest known clade of C₄ plant species, with the notable exception of Eragrostis walteri Pilg., whose leaf anatomy has been described as typical of C₃ plants. Eragrostis walteri is therefore classically hypothesized to represent an exceptional example of evolutionary reversion from C₄ to C₃ photosynthesis. Here this hypothesis is tested by verifying the photosynthetic type of E. walteri and its classification.

Methods

Carbon isotope analyses were used to determine the photosynthetic pathway of several E. walteri accessions, and phylogenetic analyses of plastid rbcL and ndhF and nuclear internal transcribed spacer DNA sequences were used to establish the phylogenetic position of the species.

Results

Carbon isotope analyses confirmed that E. walteri is a C₃ plant. However, phylogenetic analyses demonstrate that this species has been misclassified, showing that E. walteri is positioned outside Chloridoideae in Arundinoideae, a subfamily comprised entirely of C₃ species.

Conclusions

The long-standing hypothesis of C₄ to C₃ reversion in E. walteri is rejected, and the classification of this species needs to be re-evaluated.     

Soluble T cell immunoglobulin and mucin domain (TIM)-1 and -4 generated by A Disintegrin And Metalloprotease (ADAM)-10 and -17 bind to phosphatidylserine

T cell immunoglobulin and mucin domain 1 and 4 (TIM-1 and -4) proteins serve as phosphatidylserine receptors to engulf apoptotic cells. Here we show that human TIM-1 and TIM-4 proteins are targets of A Disintegrin And Metalloprotease (ADAM)-mediated ectodomain shedding resulting in soluble forms of TIM-1 and TIM-4. We identified ADAM10 and ADAM17 as major sheddases of TIM-1 and TIM-4 as shown by protease-specific inhibitors, the ADAM10 prodomain, siRNA and ADAM10/ADAM17 deficient murine embryonic fibroblasts (MEFs). TIM-1 and TIM-4 lacking the intracellular domain were efficiently cleaved after ionomycin- and PMA-treatment, indicating that the intracellular domain was not necessary for ectodomain shedding. Soluble TIM-1 and -4 were able to bind to phosphatidylserine, suggesting that soluble TIM-1 and -4 might act as negative regulators of cellular TIM-1 and -4. In summary, we describe TIM-1 and TIM-4 as novel targets for ADAM10- and ADAM17-mediated ectodomain shedding.     

The Sphagnum microbiome supports bog ecosystem functioning under extreme conditions

Sphagnum‐dominated bogs represent a unique yet widely distributed type of terrestrial ecosystem and strongly contribute to global biosphere functioning. Sphagnum is colonized by highly diverse microbial communities, but less is known about their function. We identified a high functional diversity within the Sphagnum microbiome applying an Illumina‐based metagenomic approach followed by de novo assembly and MG‐RAST annotation. An interenvironmental comparison revealed that the Sphagnum microbiome harbours specific genetic features that distinguish it significantly from microbiomes of higher plants and peat soils. The differential traits especially support ecosystem functioning by a symbiotic lifestyle under poikilohydric and ombrotrophic conditions. To realise a plasticity–stability balance, we found abundant subsystems responsible to cope with oxidative and drought stresses, to exchange (mobile) genetic elements, and genes that encode for resistance to detrimental environmental factors, repair and self‐controlling mechanisms. Multiple microbe–microbe and plant–microbe interactions were also found to play a crucial role as indicated by diverse genes necessary for biofilm formation, interaction via quorum sensing and nutrient exchange. A high proportion of genes involved in nitrogen cycle and recycling of organic material supported the role of bacteria for nutrient supply. 16S rDNA analysis indicated a higher structural diversity than that which had been previously detected using PCR‐dependent techniques. Altogether, the diverse Sphagnum microbiome has the ability to support the life of the host plant and the entire ecosystem under changing environmental conditions. Beyond this, the moss microbiome presents a promising bio‐resource for environmental biotechnology – with respect to novel enzymes or stress‐protecting bacteria.     

Intraosseous Vascular Access through the Anterior Mandible – A Cadaver Model Pilot Study

Background

Several insertion sites have been described for intraosseous puncture in cases of emergencies when a conventional vascular access cannot be established. This pilot study has been designed to evaluate the feasibility of the mandibular bone for the use of an intraosseous vascular access in a cadaver model.

Methodology/Principal Findings

17 dentistry and 16 medical students participating in a voluntary course received a short introduction into the method and subsequently used the battery powered EZ-IO system with a 15 mm cannula for a puncture of the anterior mandible in 33 cadavers. The time needed to perform each procedure was evaluated. India ink was injected into the accesses and during the anatomy course cadavers were dissected to retrace the success or failure of the puncture. Dental students needed 25.5±18.9(mean±standard deviation)s and medical students 33±20.4 s for the procedure (p = 0.18). Floor of mouth extravasation occurred in both groups in 3 cases. Success rates were 82 and 75% (p = 0.93).

Conclusions/Significance

Despite floor of mouth extravasation of injected fluid into a mandibular intraosseous access might severely complicate this procedure, the anterior mandible may be helpful as an alternative to other intraosseous and intravenous insertion sites when these are not available in medical emergencies.     

Kinetic and spectroscopic studies of Tritrichomonas foetus low-molecular weight phosphotyrosyl phosphatase. Hydrogen bond networks and electrostatic effects

Virtually all of the eukaryotic low-molecular weight protein tyrosine phosphatases (LMW PTPases) studied to date contain a conserved, high-pK(a) histidine residue that is hydrogen bonded to a conserved active site asparagine residue of the phosphate binding loop. However, in the putative enzyme encoded by the genome of the trichomonad parasite Tritrichomonas foetus, this otherwise highly conserved histidine is replaced with a glutamine residue. We have cloned the gene, expressed the enzyme, demonstrated its catalytic activity, and examined the structural and functional roles of the glutamine residue using site-directed mutagenesis, kinetic measurements, and NMR spectroscopy. Titration studies of the two native histidine residues in the T. foetus enzyme as monitored by (1)H NMR revealed that H44 has a pK(a) of 6.4 and H143 has a pK(a) of 5.3. When a histidine residue was introduced in place of the native glutamine at position 67, a pK(a) of 8.2 was measured for this residue. Steady state kinetic methods were employed to study how mutation of the native glutamine to alanine, asparagine, and histidine affected the catalytic activity of the enzyme. Examination of k(cat)/K(m) showed that Q67H exhibits a substrate selectivity comparable to that of the wild-type (WT) enzyme, while Q67N and Q67A show reduced activity. The effect of pH on the reaction rate was examined. Importantly, the pH-rate profile of the WT TPTP enzyme revealed a much more clearly defined acidic limb than that which can be observed for other wild-type LMW PTPases. The pH-rate curve of the Q67H mutant shows a shift to a lower pH optimum relative to that seen for the wild-type enzyme. The Q67N and Q67A mutants showed curves that were shifted to higher pH optima. Although the active site of this enzyme is likely to be similar to that of other LMW PTPases, the hydrogen bonding and electrostatic changes afford new insight into factors affecting the pH dependence and catalysis by this family of enzymes.     

A genetic screen identifies BRCA2 and PALB2 as key regulators of G2 checkpoint maintenance

To identify key connections between DNA-damage repair and checkpoint pathways, we performed RNA interference screens for regulators of the ionizing radiation-induced G2 checkpoint, and we identified the breast cancer gene BRCA2. The checkpoint was also abrogated following depletion of PALB2, an interaction partner of BRCA2. BRCA2 and PALB2 depletion led to premature checkpoint abrogation and earlier activation of the AURORA A-PLK1 checkpoint-recovery pathway. These results indicate that the breast cancer tumour suppressors and homologous recombination repair proteins BRCA2 and PALB2 are main regulators of G2 checkpoint maintenance following DNA-damage.     

Binding of FAD and tryptophan to the tryptophan 6‐halogenase Thal is negatively coupled

Flavin‐dependent halogenases require reduced flavin adenine dinucleotide (FADH₂), O₂, and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6‐halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH₂ by flavin reductases.