Stable iron isotope variations in higher plants : systematics and controls

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Preparation and purification of organic samples for selenium isotope studies

Selenium (Se) is an important micronutrient but also a strong toxin with a narrow tolerance range for many organisms. As such, a globally heterogeneous Se distribution in soils is responsible for various disease patterns (i.e. Se excess and deficiency) and environmental problems, whereby plants play a key role for the Se entrance into the biosphere. Selenium isotope variations were proved to be a powerful tracer for redox processes and are therefore promising for the exploration of the species dependent Se metabolism in plants and the Se cycling within the Critical Zone. Plant cultivation setups enable systematic controlled investigations, but samples derived from them–plant tissue and phytoagar–are particularly challenging and require specific preparation and purification steps to ensure precise and valid Se isotope analytics performed with HG-MC-ICP-MS. In this study, different methods for the entire process from solid tissue preparation to Se isotope measurements were tested, optimized and validated. A particular microwave digestion procedure for plant tissue and a vacuum filtration method for phytoagar led to full Se recoveries, whereby unfavorable organic residues were reduced to a minimum. Three purification methods predominantly described in the literature were systematically tested with pure Se solution, high concentrated multi-element standard solution as well as plant and phytoagar as target matrices. All these methods efficiently remove critical matrix elements, but differ in Se recovery and organic residues. Validation tests doping Se-free plant material and phytoagar with a reference material of known Se isotope composition revealed the high impact of organic residues on the accuracy of MC-ICP-MS measurements. Only the purification method with no detectable organic residues, hydride generation and trapping, results in valid mass bias correction for plant samples with an average deviation to true δ82/76Se values of 0.2 ‰ and a reproducibility (2 SD) of ± 0.2 ‰. For phytoagar this test yields a higher deviation of 1.1 ‰ from the true value and a 2 SD of ± 0.1 ‰. The application of the developed methods to cultivated plants shows sufficient accuracy and precision and is a promising approach to resolve plant internal Se isotope fractionations, for which respective δ82/76Se values of +2.3 to +3.5 ‰ for selenate and +1.2 to +1.9 ‰ for selenite were obtained

Structural Analysis to Determine the Core of Hypoxia Response Network

The advent of sophisticated molecular biology techniques allows to deduce the structure of complex biological networks. However, networks tend to be huge and impose computational challenges on traditional mathematical analysis due to their high dimension and lack of reliable kinetic data. To overcome this problem, complex biological networks are decomposed into modules that are assumed to capture essential aspects of the full network's dynamics. The question that begs for an answer is how to identify the core that is representative of a network's dynamics, its function and robustness. One of the powerful methods to probe into the structure of a network is Petri net analysis. Petri nets support network visualization and execution. They are also equipped with sound mathematical and formal reasoning based on which a network can be decomposed into modules. The structural analysis provides insight into the robustness and facilitates the identification of fragile nodes. The application of these techniques to a previously proposed hypoxia control network reveals three functional modules responsible for degrading the hypoxia-inducible factor (HIF). Interestingly, the structural analysis identifies superfluous network parts and suggests that the reversibility of the reactions are not important for the essential functionality. The core network is determined to be the union of the three reduced individual modules. The structural analysis results are confirmed by numerical integration of the differential equations induced by the individual modules as well as their composition. The structural analysis leads also to a coarse network structure highlighting the structural principles inherent in the three functional modules. Importantly, our analysis identifies the fragile node in this robust network without which the switch-like behavior is shown to be completely absent

Preparation and purification of organic samples for selenium isotope studies

<div><p>Selenium (Se) is an important micronutrient but also a strong toxin with a narrow tolerance range for many organisms. As such, a globally heterogeneous Se distribution in soils is responsible for various disease patterns (i.e. Se excess and deficiency) and environmental problems, whereby plants play a key role for the Se entrance into the biosphere. Selenium isotope variations were proved to be a powerful tracer for redox processes and are therefore promising for the exploration of the species dependent Se metabolism in plants and the Se cycling within the Critical Zone. Plant cultivation setups enable systematic controlled investigations, but samples derived from them–plant tissue and phytoagar–are particularly challenging and require specific preparation and purification steps to ensure precise and valid Se isotope analytics performed with HG-MC-ICP-MS. In this study, different methods for the entire process from solid tissue preparation to Se isotope measurements were tested, optimized and validated. A particular microwave digestion procedure for plant tissue and a vacuum filtration method for phytoagar led to full Se recoveries, whereby unfavorable organic residues were reduced to a minimum. Three purification methods predominantly described in the literature were systematically tested with pure Se solution, high concentrated multi-element standard solution as well as plant and phytoagar as target matrices. All these methods efficiently remove critical matrix elements, but differ in Se recovery and organic residues. Validation tests doping Se-free plant material and phytoagar with a reference material of known Se isotope composition revealed the high impact of organic residues on the accuracy of MC-ICP-MS measurements. Only the purification method with no detectable organic residues, hydride generation and trapping, results in valid mass bias correction for plant samples with an average deviation to true δ^82/76Se values of 0.2 ‰ and a reproducibility (2 SD) of ± 0.2 ‰. For phytoagar this test yields a higher deviation of 1.1 ‰ from the true value and a 2 SD of ± 0.1 ‰. The application of the developed methods to cultivated plants shows sufficient accuracy and precision and is a promising approach to resolve plant internal Se isotope fractionations, for which respective δ^82/76Se values of +2.3 to +3.5 ‰ for selenate and +1.2 to +1.9 ‰ for selenite were obtained.</p></div

Se recoveries and external reproducibility tested with purification methods CAE, CTR and HGT depending on sample matrices (reference: CRM-TMDW recovery 101.3 (± 3.6) %).

<p>Se recoveries and external reproducibility tested with purification methods CAE, CTR and HGT depending on sample matrices (reference: CRM-TMDW recovery 101.3 (± 3.6) %).</p

Validation tests with Se-free plant digests and phytoagar extracts doped with 300 ng NIST3149 and purified according to CAE, CTR and HGT (with external reproducibility).

<p>Validation tests with Se-free plant digests and phytoagar extracts doped with 300 ng NIST3149 and purified according to CAE, CTR and HGT (with external reproducibility).</p

Preparation and purification of organic samples for selenium isotope studies - Fig 4

<p>Correlation between Se isotope ratio and β_instr of validation tests performed using CAE (A), CTR (B) and HGT (C) as well as NIST3149 and MH495 standard solutions (*Double Spike added prior to digestion, else directly afterwards) (data Table F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193826#pone.0193826.s001" target="_blank">S1 File</a>). The Double Spike deconvolution assumes the detected signals are purely due to Se ions and that the shift in the abundance of the Se signals is mass-dependent. The β_instr value is thereby calculated by an iterative approach using the 4 Se signals (74, 77, 78, 82).</p

Se recoveries from phytoagar transformation into liquid form in dependence on Se concentration and species (data with external reproducibility Table A in S1 File).

<p>(a) Se recovery after digestion (after [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193826#pone.0193826.ref030" target="_blank">30</a>], average recovery 74.3 ± 5.5%), (b) Se recovery after vacuum filtration (average recovery 103 ± 2.6%).</p

Acquired and transient RBC CD55 deficiency (Inab phenotype) and anti-IFC

Background: Antigens of the Cromer blood group system reside on the glycoprotein CD55 (decay-accelerating factor). The Inab phenotype is the null phenotype of this system. So far, only five propositi have been described who exhibit this phenotype, and single-nucleotide substitutions in the CD55 gene have been found in three of them. This report describes the first example of a patient with an acquired and transient form of the Inab phenotype. Case report: A 54-year-old black patient was admitted to the hospital because of abdominal pain. Multiple splenic infarctions were visualized in the abdominal computerized tomography scan, and a prophylactic splenectomy was performed. The patient's serum reacted by an IAT with all donor RBCs tested. Results: Serologic analysis showed that the patient had the rare Inab phenotype and that his serum contained anti-IFC. Flow cytometry demonstrated the absence of CD55 on his RBCs, whereas lymphocytes, monocytes, granulocytes, and platelets expressed CD55, albeit at a weaker level than cells of common phenotypes. cDNA revealed no differences from the published sequences. Flow cytometry performed 12 months after splenectomy showed reappearance of the CD55 antigen; serologic tests performed after 17 months revealed that the anti-IFC had almost disappeared and that the RBCs were again agglutinated by various Cromer antibodies. Conclusion: A patient with an acquired and transient form of the Inab phenotype is described, in whom the CD55 deficiency is limited to the RBCs and is associated with splenic infarctions.</p