Rare earth thiocyanate complexes with tripiperidinophosphine oxide: synthesis and characterization. Crystal structure of Pr(SCN)3(tpppO)3

The synthesis and characterization of rare-earth (La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and Y) thiocyanate adducts with tripiperidinophosphine oxide (tpppO) with general formula (RE)(SCN)₃(tpppO)₃ are reported. Conductance measurements in acetonitrile indicate the non-electrolytic nature of the complexes. Infrared absorption spectra evidence that the SCN⁻ ion coordinates through the nitrogen atom (isothiocyanate form) and that tpppO coordinates through the phosphoryl oxygen. X-ray powder patterns suggest the existence of three different crystal forms: (1) La; (2) an isomorphous series including Ce, Nd and Pr; and (3) another isomorphous series, including Sm, Gd, Eu, Ho, Er, Tb, Lu and Y. The visible spectra of the Nd adduct and the calculated parameters β = 0.98, b^1/2 = 0.072 and δ = 1.06 indicate that the metal-ligand bonds are essentially electrostactic. The emission spectra of the Eu compound showed ⁵D₀ → ⁷F_J bands (J = 0, 1, 2), suggesting a C_3v symmetry for the coordination polyhedron. The lifetime of the ⁵D₀ state is 1.28 ms. The emission spectra of the Tb complex presented ⁵D₄ → ⁷F_J bands (J = 4, 5, 6) and the Dy complex showed the ⁴F_9/2 → ⁶H_13/2 band. The structure of the Pr complex showed that the coordination polyhedron is a trigonal antiprism, with the isothiocyanate anions in one base and three tpppO ligands in the other. Thermal analyses (TG-DTG) were carried out for the Ce, Nd and Gd adducts. Mass losses start between 250 and 334 °C. The final residues at 1300 °C are the corresponding phosphates.     

Time course changes to structural,mechanical and material properties of bone in rats after complete spinal cord injury

Objective:Characterise the spatiotemporal trabecular and cortical bone responses to complete spinal cord injury (SCI) in young rats.Methods:8-week-old male Wistar rats received T9-transection SCI and were euthanised 2-, 6-, 10- or 16-weeks post-surgery. Outcome measures were assessed using micro-computed tomography, mechanical testing, serum markers and Fourier-transform infrared spectroscopy.Results:The trabecular and cortical bone responses to SCI are site-specific. Metaphyseal trabecular BV/TV was 59% lower, characterised by fewer and thinner trabeculae at 2-weeks post-SCI, while epiphyseal BV/TV was 23% lower with maintained connectivity. At later-time points, metaphyseal BV/TV remained unchanged, while epiphyseal BV/TV increased. The total area of metaphyseal and mid-diaphyseal cortical bone were lower from 2-weeks and between 6- and 10-weeks post-SCI, respectively. This suggested that SCI-induced bone changes observed in the rat model were not solely attributable to bone loss, but also to suppressed bone growth. No tissue mineral density differences were observed at any time-point, suggesting that decreased whole-bone mechanical properties were primarily the result of changes to the spatial distribution of bone.Conclusion:Young SCI rat trabecular bone changes resemble those observed clinically in adult and paediatric SCI, while cortical bone changes resemble paediatric SCI only.     

The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton

Maintaining physical connections between the nucleus and the cytoskeleton is important for many cellular processes that require coordinated movement and positioning of the nucleus. Nucleo-cytoskeletal coupling is also necessary to transmit extracellular mechanical stimuli across the cytoskeleton to the nucleus, where they may initiate mechanotransduction events. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, formed by the interaction of nesprins and SUN proteins at the nuclear envelope, can bind to nuclear and cytoskeletal elements; however, its functional importance in transmitting intracellular forces has never been directly tested. This question is particularly relevant since recent findings have linked nesprin mutations to muscular dystrophy and dilated cardiomyopathy. Using biophysical assays to assess intracellular force transmission and associated cellular functions, we identified the LINC complex as a critical component for nucleo-cytoskeletal force transmission. Disruption of the LINC complex caused impaired propagation of intracellular forces and disturbed organization of the perinuclear actin and intermediate filament networks. Although mechanically induced activation of mechanosensitive genes was normal (suggesting that nuclear deformation is not required for mechanotransduction signaling) cells exhibited other severe functional defects after LINC complex disruption; nuclear positioning and cell polarization were impaired in migrating cells and in cells plated on micropatterned substrates, and cell migration speed and persistence time were significantly reduced. Taken together, our findings suggest that the LINC complex is critical for nucleo-cytoskeletal force transmission and that LINC complex disruption can result in defects in cellular structure and function that may contribute to the development of muscular dystrophies and cardiomyopathies.     

Lake Baikal climatic record between 310 and 50 ky BP: Interplay between diatoms, watershed weathering and orbital forcing

The environmental record from Lake Baikal, Russia, from 310 to 50 ky BP (MIS 9a to MIS 3) was interpreted using rock magnetic, UV–Vis spectral, mineralogical, and diatom analyses. The age model was based on a correlation of the diatom and chemical weathering records and the summer insolation curve at 55°N and checked against an age model based on the proxy of relative palaeointensity of the Earth's magnetic field. Peaks in chemical weathering within the watershed, inferred from maximum concentration of magnetic and coloured minerals and mica, the lowest mean Fe oxidation state in silicates and highs in expandable clay minerals correlated with the Northern Hemisphere summer insolation minima at 55°N. Reconstructed changes in weathering intensity are better correlated to insolation patterns than to global ice volume records. We propose a scheme of yet missing palaeoenvironmental interpretation of the diatom assemblage, including also some extinct species. Aulacoseira baicalensis and Aulacoseira skvortzowii were abundant in the early stages of lake flora recovery immediately after deglaciation and during MIS 7e and MIS 5e; periods of more pronounced continental climate and peak chemical weathering. Stephanodiscus formosus var. minor, Cyclotella minuta and Cyclotella ornata dominated in intervals of decreased seasonality and decreased humidity at the end of most interglacial/interstadial diatom zones. Stephanodiscus grandis, Stephanodiscus carconeiformis and Stephanodiscus formosus were ubiquitous between MIS 8 and MIS 5, an interval marked by high seasonality, i.e., large differences between winter and summer insolation, and low humidity revealed by a low hydrolysis of expandable clay minerals in the watershed. Diatom concentrations peaked in the climatic optima of MIS 7e and MIS 5e and in the short periods marked by shifts to warmer conditions in the upper sections of MIS 5: MIS 5c (103–99 ky BP), MIS 5b (90–88 ky BP), and MIS 5a (84–79 ky BP) in which increased humidity resulted in enhanced hydrolysis of clay minerals. No such short similar climatic optimums were found from MIS 9a to MIS 6. Sharp climate deteriorations recorded in the diatom and clay mineral records at 107, 94, and 87 ky BP, however, occurred within 1–2 ky of cold extremes in North Atlantic sea surface temperature emphasizing the strong teleconnections between the two localities.     

Site-specific protein dynamics in communication pathway from sensor to signaling domain of oxygen sensor protein, HemAT-Bs: Time-resolved Ultraviolet Resonance Raman Study

HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in ～50 μs. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in ～50 μs. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 μs exhibited a pattern similar to that of the static (reduced - CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.     

Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding

Human telomeres are composed of duplex TTAGGG repeats and a 3' single-stranded DNA tail. The telomeric DNA is protected and regulated by the shelterin proteins, including the protection of telomeres 1 (POT1) protein that binds telomeric single-stranded DNA. The single-stranded tail can fold into G-quadruplex (G4) DNA. Both POT1 and G4 DNA play important roles in regulating telomere length homeostasis. To date, most studies have focused on individual quadruplexes formed by four TTAGGG repeats. Telomeric tails in human cells have on average six times as many repeats, and no structural studies have examined POT1 binding in competition with G4 DNA folding. Using single molecule atomic force microscopy imaging, we observed that the majority of the telomeric tails of 16 repeats formed two quadruplexes even though four were possible. The result that physiological telomeric tails rarely form the maximum potential number of G4 units provides a structural basis for the coexistence of G4 and POT1 on the same DNA molecule, which is observed directly in the captured atomic force microscopy images. We further observed that POT1 is significantly more effective in disrupting quadruplex DNA on long telomeric tails than an antisense oligonucleotide, indicating a novel POT1 activity beyond simply preventing quadruplex folding.     

Altered structure of the Mn4Ca cluster in the oxygen-evolving complex of photosystem II by a histidine ligand mutation

The effect of replacing a histidine ligand on the properties of the oxygen-evolving complex (OEC) and the structure of the Mn₄Ca cluster in Photosystem II (PSII) is studied by x-ray absorption spectroscopy using PSII core complexes from the Synechocystis sp. PCC 6803 D1 polypeptide mutant H332E. In the x-ray crystallographic structures of PSII, D1-His³³² has been assigned as a direct ligand of a manganese ion, and the mutation of this histidine ligand to glutamate has been reported to prevent the advancement of the OEC beyond the S₂Yz^• intermediate state. The manganese K-edge (1s core electron to 4p) absorption spectrum of D1-H332E shifts to a lower energy compared with that of the native WT samples, suggesting that the electronic structure of the manganese cluster is affected by the presence of the additional negative charge on the OEC of the mutant. The extended x-ray absorption spectrum shows that the geometric structure of the cluster is altered substantially from that of the native WT state, resulting in an elongation of manganese-ligand and manganese-manganese interactions in the mutant. The strontium-H332E mutant, in which calcium is substituted by strontium, confirms that strontium (calcium) is a part of the altered cluster. The structural perturbations caused by the D1-H332E mutation are much larger than those produced by any biochemical treatment or mutation examined previously with x-ray absorption spectroscopy. The substantial structural changes provide an explanation not only for the altered properties of the D1-H332E mutant but also the importance of the histidine ligand for proper assembly of the Mn₄Ca cluster.     

Interaction against different environmental dynamics during a leg extension task is controlled by temporal rather than amplitude scaling of muscular activity

Force exertion against different mechanical environments can affect motor control strategies in order to account for the altered environmental dynamics and to maintain the ability to produce force. Here, we investigated the change of muscular activity of selected muscles of the lower extremities while the participants interacted with an external mechanical device of variable stability. Twenty-five healthy participants exerted force against the device by performing a unilateral ballistic leg extension task under 1 or 3 degrees of freedom (DoF). Directional force data and electromyographic responses from four leg muscles (TA, VM, GM, PL) were recorded. Muscle responses to the altered experimental conditions were analyzed by calculating time to peak electrical activity (TTP), peak electrical activity (PEA), slope of EMG-signal and muscle activity. It was found that neuromuscular system adjustments to the task are expressed mainly by temporal (TTP) rather than amplitude (PEA) scaling of muscular activity. This change was specific for the investigated muscles. Moreover, a selective increase of muscle activity occurred while increasing external DoF. This scheme was accompanied by a significant reduction of applicable force against the device in the unstable 3 DoF condition. The findings suggest that orchestration of movement control is linked to environmental dynamics also affecting the ability to produce force under dynamic conditions. The adjustments of the neuromuscular system are rather temporal in nature being consistent with the impulse timing hypothesis of motor control.     

Supramolecular structure of self-assembling alamethicin analog studied by ESR and PELDOR

Three analogs of alamethicin F50/5, labelled with the TOAC (='2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid') spin label at positions 1 (Alm1), 8 (Alm8), and 16 (Alm16), resp., were studied by Electron-Spin-Resonance (ESR) and Pulsed Electron-Electron Double-Resonance (PELDOR) techniques in solvents of different polarity to investigate the self-assembly of amphipathic helical peptides in membrane-mimicking environments. In polar solvents, alamethicin forms homogeneous solutions. In the weakly polar chloroform/toluene 1 : 1 mixture, however, this peptide forms aggregates that are detectable at 293 K by ESR in liquid solution, as well as by PELDOR in frozen, glassy solution at 77 K. In liquid solution, free alamethicin molecules and their aggregates show rotational-mobility correlation times tau(r) of 0.87 and 5.9 ns, resp. Based on these values and analysis of dipole-dipole interactions of the TOAC labels in the aggregates, as determined by PELDOR, the average number N of alamethicin molecules in the aggregates is estimated to be less than nine. A distance-distribution function between spin labels in the supramolecular aggregate was obtained. This function exhibits two maxima: a broad one at a distance of 3.0 nm, and a wide one at a distance of ca. 7 nm. A molecular-dynamics (MD)-based model of the aggregate, consisting of two parallel tetramers, each composed of four molecules arranged in a 'head-to-tail' fashion, is proposed, accounting for the observed distances and their distribution.     

Single-molecule Force Spectroscopy Approach to Enzyme Catalysis

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.