A yeast surface display platform for plant hormone receptors: Toward directed evolution of new biosensors

Small-molecule biosensors have major applications in biotechnology and medicine but remain difficult to engineer. Plant hormone receptors represent an attractive platform for engineering such biosensors because their chemically induced dimerization architectures naturally decouple small-molecule sensing and sensor actuation. Rapid biosensor engineering will require quantitative high-throughput screening methods. Here we develop a yeast surface display (YSD) platform for the PYR1/HAB1 abscisic acid sensor of Arabidopsis thaliana. We extensively optimized PYR1 surface display, HAB1 purification, and binding reaction conditions. Our system reproduces previous results with wild-type and engineered receptors, and a mathematical analysis of the PYR1/HAB1 system allows us to infer all binding constants. Critically, we find that a previously engineered PYR1 receptor with altered ligand specificity binds HAB1 with identical affinity, suggesting that substantial reengineering of the PYR1 binding pocket does not compromise sensor actuation. This YSD platform for A. thaliana PYR1/HAB1 will facilitate future biosensor engineering efforts.     

A POLE Splice Site Deletion Detected in a Patient with Biclonal CLL and Prostate Cancer: A Case Report

Chronic lymphocytic leukemia (CLL) is considered a clonal B cell malignancy. Sporadically, CLL cases with multiple productive heavy and light-chain rearrangements were detected, thus leading to a bi- or oligoclonal CLL disease with leukemic cells originating either from different B cells or otherwise descending from secondary immunoglobulin rearrangement events. This suggests a potential role of clonal hematopoiesis or germline predisposition in these cases. During the screening of 75 CLL cases for kappa and lambda light-chain rearrangements, we could detect a single case with CLL cells expressing two distinct kappa and lambda light chains paired with two separate immunoglobulin heavy-chain variable regions. Furthermore, this patient also developed a prostate carcinoma. Targeted genome sequencing of highly purified light-chain specific CLL clones from this patient and from the prostate carcinoma revealed the presence of a rare germline polymorphism in the POLE gene. Hence, our data suggest that the detected SNP may predispose for cancer, particularly for CLL.     

Monitoring the degradation of a solid oxide fuel cell stack during 10,000 h via electrochemical impedance spectroscopy

A 5-cell solid oxide fuel cell stack was tested during 10,000 h of continuous operation with simulated reformate gas as fuel (71 vol.% H₂, 20.7 vol.% CO₂ and 8.3 vol.% steam) under high fuel utilization (73%) and constant current load (0.5 A cm^?2 or 25 A) at 750 °C. In situ electrochemical impedance spectroscopy was used to monitor the evolution of ohmic and polarisation resistances of individual cells in the stack without interrupting the current load. Impedance spectra were recorded on each cell periodically (every 1000 h) or after uncontrolled incidents happened with the test setup. It has been found that the stack degradation is mainly attributed to the increased ohmic resistance, pointing to possible causes such as interconnect corrosion and reduced effective contact areas between cells and interconnects. The degradation rate during the first 5000 h was about 1% kh^?1, but increased afterwards up to 1.5% kh^?1 due to the impact of incidents. Both types of incidents (fuel supply fluctuations and overloading failure of the electronic load) were complicated by inhomogeneous fuel distribution among cells, leading to most probably partial re-oxidation of the anode, accelerating the stack degradation.     

Mechanical properties and morphology of papers prepared from single-walled carbon nanotubes functionalized with aromatic amides

Carbon nanotube papers (CNT papers, also referred to as “buckypapers”) prepared from chemically functionalized single-walled CNTs are being investigated for their mechanical tensile properties. While the Young’s moduli are unaffected by the functionalization with diazonium salts of aniline or aromatic mono- and bis-amides tensile strengths of CNT papers are found to increase with a growing degree of functionalization, and more pronounced with a growing number of amide groups capable of hydrogen bonding. The importance of hydrogen bonding becomes evident after its inhibition through N-methylation of the amide groups, resulting in a distinct reduction of strength values. Scanning electron micrography indicates that a high degree of functionalization or a high number of amide group results in the formation of domains with aligned CNTs.     

Cuticular hydrocarbons of Tetramorium ants from central Europe: analysis of GC-MS data with self-organizing maps (SOM) and implications for systematics

Cuticular hydrocarbons were extracted from workers of 63 different nests of five species of Tetramorium ants (Hymenoptera: Formicidae) from Austria, Hungary, and Spain. The GC-MS data were classified (data mining) by self-organizing maps (SOM). SOM neurons derived from primary neuron separation were subjected to hierarchical SOM (HSOM) and were grouped to neuron areas on the basis of vicinity in the hexagonal output grid. While primary neuron separation and HSOM resulted in classifications on a level more sensitive than species differences, neuron areas resulted in chemical phenotypes apparently of the order of species. These chemical phenotypes have implications for systematics: while the chemical phenotypes for T. ferox and T. moravicum correspond to morphological determination, in T. caespitum and T. impurum a total of six chemical phenotypes is found. Three hypotheses are discussed to explain this disparity between morphological and chemical classifications, including in particular the possibility of hybridization and the existence of cryptic species. Overall, the GC-MS profiles classified by SOM prove to be a practical alternative to morphological determination (T. ferox, T. moravicum) and indicate the need to revisit systematics (T. caespitum, T. impurum).     

Structural and functional analogy between pneumolysin and proaerolysin

Pneumolysin and proaerolysin are bacterial toxins that form pores in host cells by oligomerization. We propose that they may have similar structures despite a poor sequence identity. The crystal structure of proaerolysin reveals a protein composed of four domains, arranged in the shape of an elongated comma. Electron microscopy of the pneumolysin monomer shows a similar arrangement of domains. The sequence of pneumolysin recognizes the template of proaerolysin from a library of protein folds. A three-dimensional model of pneumolysin has been constructed by the comparative approach using the structure of proaerolysin. This model, together with results on the activity of site- specific mutants and the positions of antigenic sites, has been used to propose functional roles of individual domains.      

Production of human normal adult and fetal hemoglobins in Escherichia coli

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha- globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma- globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.