Human pluripotent stem cell registry: Operations,role and current directions

The human plutiripotent stem cell registry (hPSCreg) is a global database for human embryonic and induced pluripotent stem cells (hESC, hiPSC). The publicly accessible Registry (https://hpscreg.eu) was set up to provide a transparent resource of quality‐assessed hPSC lines as well as to increase reproducibility of research and interoperability of data.ObjectivesIn this review, we describe the establishment of the Registry and its mission, its development into a knowledgebase for hPSC and the current status of hPSC‐focussed databases. The data categories available in hPSCreg are detailed. In addition, sharing and hurdles to data sharing on a global level are described.ConclusionsAn outlook is provided on the establishment of digital representatives of donors using hybrids of data and hPSC‐based biological models, and how this can also be used to reposition databases as mediators between donors and researchers.

hPSCreg as a data hub for pluripotent stem cells: Key utility and function.     

A metabolic energy expenditure model with a continuous first derivative and its application to predictive simulations of gait

Whether humans minimize metabolic energy in gait is unknown. Gradient-based optimization could be used to predict gait without using walking data but requires a twice differentiable metabolic energy model. Therefore, the metabolic energy model of Umberger et al. (2003 Umberger BR, Gerritsen KG, Martin PE. 2003. A model of human muscle energy expenditure. Comput Methods Biomech Biomed Eng. 6(2):99–111.[Taylor &; Francis Online] , [Google Scholar]) was adapted to be twice differentiable. Predictive simulations of a reaching task and gait were solved using this continuous model and by minimizing effort. The reaching task simulation showed that energy minimization predicts unrealistic movements when compared to effort minimization. The predictive gait simulations showed that objectives other than metabolic energy are also important in gait.     

Real-time enzyme dynamics illustrated with fluorescence spectroscopy of p-hydroxybenzoate hydroxylase

We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair F?rster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels shows overlap between its fluorescence spectrum and the absorption spectrum of the FAD prosthetic group in the oxidized state, while the other fluorescent probe does not have this spectral overlap. The spectral overlap strongly diminished when the flavin becomes reduced during catalysis. The donor fluorescence properties can then be used as a sensitive antenna for the flavin redox state. Time-resolved fluorescence experiments on ensembles of labeled PHBH molecules were carried out in the absence and presence of enzymatic turnover. Distinct changes in fluorescence decays of spFRET-active PHBH can be observed when the enzyme is performing catalysis using both substrates p-hydroxybenzoate and NADPH. Single-molecule fluorescence correlation spectroscopy on spFRET-active PHBH showed the presence of a relaxation process (relaxation time of 23 micros) that is related to catalysis. In addition, in both labeled PHBH preparations the number of enzyme molecules reversibly increased during enzymatic turnover indicating that the dimer-monomer equilibrium is affected.     

The oligomeric state and stability of the mannitol transporter, EnzymeII(mtl), from Escherichia coli: a fluorescence correlation spectroscopy study

Numerous membrane proteins function as oligomers both at the structural and functional levels. The mannitol transporter from Escherichia coli, EnzymeII(mtl), is a member of the phosphoenolpyruvate-dependent phosphotransferase system. During the transport cycle, mannitol is phosphorylated and released into the cytoplasm as mannitol-1-phosphate. Several studies have shown that EII(mtl) functions as an oligomeric species. However, the oligomerization number and stability of the oligomeric complex during different steps of the catalytic cycle, e.g., substrate binding and/or phosphorylation of the carrier, is still under discussion. In this paper, we have addressed the oligomeric state and stability of EII(mtl) using fluorescence correlation spectroscopy. A functional double-cysteine mutant was site-specifically labeled with either Alexa Fluor 488 or Alexa Fluor 633. The subunit exchange of these two batches of proteins was followed in time during different steps of the catalytic cycle. The most important conclusions are that (1) in a detergent-solubilized state, EII(mtl) is functional as a very stable dimer; (2) the stability of the complex can be manipulated by changing the intermicellar attractive forces between PEG-based detergent micelles; (3) substrate binding destabilizes the complex whereas phosphorylation increases the stability; and (4) substrate binding to the phosphorylated species partly antagonizes the stabilizing effect.     

(Per)chlorate reduction by an acetogenic bacterium, Sporomusa sp., isolated from an underground gas storage

A mesophilic bacterium, strain An4, was isolated from an underground gas storage reservoir with methanol as substrate and perchlorate as electron acceptor. Cells were Gram-negative, spore-forming, straight to curved rods, 0.5–0.8 μm in diameter, and 2–8 μm in length, growing as single cells or in pairs. The cells grew optimally at 37°C, and the pH optimum was around 7. Strain An4 converted various alcohols, organic acids, fructose, acetoin, and H₂/CO₂ to acetate, usually as the only product. Succinate was decarboxylated to propionate. The isolate was able to respire with (per)chlorate, nitrate, and CO₂. The G+C content of the DNA was 42.6 mol%. Based on the 16S rRNA gene sequence analysis, strain An4 was most closely related to Sporomusa ovata (98% similarity). The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell-free extracts.     

Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC (cytosolic regulator of adenylyl cyclase)-GFP) in the cytoplasm of vegetative and chemotaxing Dictyostelium cells has been studied using fluorescence correlation spectroscopy to gain a better understanding of the functioning of the domains and to assess the effect of initiation of chemotaxis on these domains in the cell. PH2-GFP was homogeneously distributed in vegetative as well as chemotaxing cells, whereas PH10-GFP and PH-CRAC-GFP showed translocation to the leading edge of the chemotaxing cell. The diffusion characteristics of PH2-GFP and PH-CRAC-GFP were very similar; however, PH10-GFP exhibited slower diffusion. Photon counting histogram statistics show that this slow diffusion was not due to aggregation. Diffusion of the three PH domains was affected to similar extents by intracellular heterogeneities in vegetative as well as chemotaxing cells. From the diffusion of free cytoplasmic GFP, it was calculated that the viscosity in chemotaxing cells was 1.7 times lower than in vegetative cells. In chemotaxing cells, PH2-GFP showed increased mobility, whereas the mobilities of PH10-GFP and PH-CRAC-GFP remained unchanged.     

Structure and dynamics of egg white ovalbumin adsorbed at the air/water interface

The molecular properties of egg white ovalbumin adsorbed at the air/water interface were studied using infrared reflection absorption spectroscopy (IRRAS) and time-resolved fluorescence anisotropy (TRFA) techniques. Ovalbumin adsorbed at the air/water interface adopts a characteristic partially unfolded conformation in which the content of the beta-sheet is 10% lower compared to that of the protein in bulk solution. Adsorption to the interface leads to considerable changes in the rotational dynamics of ovalbumin. The results indicate that the end-over-end mobility of the ellipsoidal protein becomes substantially restricted. This is likely to reflect a preferential orientation of the protein at the interface. Continuous compression of surface layers of ovalbumin causes local aggregation of the protein, resulting in protein-network formation at the interface. The altered protein-protein interactions contribute to the strong increase in surface pressure observed.     

Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins

Retinal phosducin is known to sequester transducin Gbetagamma, thereby modulating transducin activity. Phosducin is a member of a family of phosducin-like proteins (PhLP) found in eukaryotes. Phylogeny of 33 phosducin-like proteins from metazoa, plants and lower eukaryotes identified three distinct groups named phosducin-I-III. We discovered three phlp genes in Dictyostelium, each encoding a phosducin-like protein of a different group. Disruption of the phlp1 gene strongly impaired G-protein signalling, apparently due to mislocalization of Gbetagamma in phlp1-null cells. GFP-Gbeta and GFP-Ggamma are membrane associated in wild-type cells, but cytosolic in phlp1-null cells. Phlp2 disruption is lethal due to a synchronous collapse of the cells after 16-17 cell divisions. Phlp3 disruptants show no abnormal phenotype. These results establish a role for phosducin-like proteins in facilitating folding, localization or function of proteins, in addition to modulating G-protein signalling.     

Effect of high pressure and reversed micelles on the fluorescent proteins

Two physico-chemical perturbations were applied to ECFP, EGFP, EYFP and DsRed fluorescent proteins: high hydrostatic pressure and encapsulation in reversed micelles. The observed fluorescence changes were described by two-state model and quantified by thermodynamic formalism. ECFP, EYFP and DsRed exhibited similar reaction volumes under pressure. The changes of the chemical potentials of the chromophore in bis(2-ethylhexyl)sulfosuccinate (AOT) micelles caused apparent chromophore protonation changes resulting in a fluorescence decrease of ECFP and EYFP. In contrast to the remarkable stability of DsRed, the highest sensitivity of EYFP fluorescence under pressure and in micelles is attributed to its chromophore structure.     

Exotendons for assistance of human locomotion

Background  

Powered robotic exoskeletons for assistance of human locomotion are currently under development for military and medical applications. The energy requirements for such devices are excessive, and this has become a major obstacle for practical applications. Legged locomotion in many animals, however, is very energy efficient. We propose that poly-articular elastic mechanisms are a major contributor to the economy of locomotion in such specialized animals. Consequently, it should be possible to design unpowered assistive devices that make effective use of similar mechanisms.