Mechanistic Dissection of the Cop9 Signalosome’s Deneddylation Activity on Cullin-RING Ligases

We set out to understand the precise mechanisms that regulate the activation and deactivation of Cullin-RING Ligases (CRLs). While a great deal of work has already gone into identifying the players involved in these pathways and the cellular consequences associated with the loss of each, the biochemical mechanisms regulating these steps have remained elusive. In this work we sought to gain a better understanding of the mechanisms behind these steps by teasing apart specific their biochemical reactions. By measuring the individual microscopic rate constants of the reactions we have shed light on both the proper sequence of events in the regulation of CRLs as well as how they are in fact controlled.

Prior to this work, it was believed that CSN deactivated CRLs by binding them and enzymatically removing the activating post-translation modification Nedd8. It was believed that CSN could not bind to CRLs while they were active due to the steric hindrance by the CRL substrates, and that they would remain bound to deneddylated CRLs as a sequestering agent until a new substrate could displace it. We now have some insight that substrates themselves cannot inhibit CSN very well, but that the active ubiquitination by an E2 enzyme precludes CSN binding and activity. When the substrate for a CRL becomes depleted, CSN then binds to the CRL in a low affinity, low activity conformation. This triggers a conformational change that pulls the autoinhibitory Ins-1 loop away from the active site in the catalytic subunit Csn5, resulting in a large increase in affinity and cleavage of the isopeptide bond between CRLs and Nedd8. Upon dissociation of Nedd8, CSN rapidly returns to the low affinity state and dissociates from the CRL, allowing it reenter its activation cycle.

Ultrafast transient absorption spectroscopy: principles and applications

The study of photophysical and photochemical processes crosses the interest of many fields of research in physics, chemistry and biology. In particular, the photophysical and photochemical reactions, after light absorption by a photosynthetic pigment-protein complex, are among the fastest events in biology, taking place on timescales ranging from tens of femtoseconds to a few nanoseconds. Among the experimental approaches developed for this purpose, the advent of ultrafast transient absorption spectroscopy has become a powerful and widely used technique.[1,2] Focusing on the process of photosynthesis, it relies upon the efficient absorption and conversion of the radiant energy from the Sun. Chlorophylls and carotenoids are the main players in the process. Photosynthetic pigments are typically arranged in a highly organized fashion to constitute antennas and reaction centers, supramolecular devices where light harvesting and charge separation take place. The very early steps in the photosynthetic process take place after the absorption of a photon by an antenna system, which harvests light and eventually delivers it to the reaction center. In order to compete with internal conversion, intersystem crossing, and fluorescence, which inevitably lead to energy loss, the energy and electron transfer processes that fix the excited-state energy in photosynthesis must be extremely fast. In order to investigate these events, ultrafast techniques down to a sub-100 fs resolution must be used. In this way, energy migration within the system as well as the formation of new chemical species such as charge-separated states can be tracked in real time. This can be achieved by making use of ultrafast transient absorption spectroscopy. The basic principles of this notable technique, instrumentation, and some recent applications to photosynthetic systems[3] will be described. Acknowledgements M. Moreno Oliva thanks the MINECO for a “Juan de la Cierva-Incorporación” research contract. References [1] U. Megerle, I. Pugliesi, C. Schriever, C.F. Sailer and E. Riedle, Appl. Phys. B, 96, 215 – 231 (2009). [2] R. Berera, R. van Grondelle and J.T.M. Kennis, Photosynth. Res., 101, 105 – 118 (2009). [3] T. Nikkonen, M. Moreno Oliva, A. Kahnt, M. Muuronen, J. Helaja and D.M. Guldi, Chem. Eur. J., 21, 590 – 600 (2015).

Fiber scaffolds of poly (glycerol-dodecanedioate) and its derivative via electrospinning for neural tissue engineering

Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.^

Elemental Analysis and Forensic Comparison of Soils by Laser-Induced Breakdown Spectroscopy (LIBS) and Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS)

The elemental analysis of soil is useful in forensic and environmental sciences. Methods were developed and optimized for two laser-based multi-element analysis techniques: laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and laser-induced breakdown spectroscopy (LIBS). This work represents the first use of a 266 nm laser for forensic soil analysis by LIBS. Sample preparation methods were developed and optimized for a variety of sample types, including pellets for large bulk soil specimens (470 mg) and sediment-laden filters (47 mg), and tape-mounting for small transfer evidence specimens (10 mg). Analytical performance for sediment filter pellets and tape-mounted soils was similar to that achieved with bulk pellets. An inter-laboratory comparison exercise was designed to evaluate the performance of the LA-ICP-MS and LIBS methods, as well as for micro X-ray fluorescence (μXRF), across multiple laboratories. Limits of detection (LODs) were 0.01-23 ppm for LA-ICP-MS, 0.25-574 ppm for LIBS, 16-4400 ppm for µXRF, and well below the levels normally seen in soils. Good intra-laboratory precision (≤ 6 % relative standard deviation (RSD) for LA-ICP-MS; ≤ 8 % for µXRF; ≤ 17 % for LIBS) and inter-laboratory precision (≤ 19 % for LA-ICP-MS; ≤ 25 % for µXRF) were achieved for most elements, which is encouraging for a first inter-laboratory exercise. While LIBS generally has higher LODs and RSDs than LA-ICP-MS, both were capable of generating good quality multi-element data sufficient for discrimination purposes. Multivariate methods using principal components analysis (PCA) and linear discriminant analysis (LDA) were developed for discriminations of soils from different sources. Specimens from different sites that were indistinguishable by color alone were discriminated by elemental analysis. Correct classification rates of 94.5 % or better were achieved in a simulated forensic discrimination of three similar sites for both LIBS and LA-ICP-MS. Results for tape-mounted specimens were nearly identical to those achieved with pellets. Methods were tested on soils from USA, Canada and Tanzania. Within-site heterogeneity was site-specific. Elemental differences were greatest for specimens separated by large distances, even within the same lithology. Elemental profiles can be used to discriminate soils from different locations and narrow down locations even when mineralogy is similar.

Raman spectroscopy of the molybdate minerals chillagite (tungsteinian wulfenite-I4), stolzite, scheelite, wolframite and wulfenite

Raman spectra of chillagite, wulfenite, stolzite, scheelite and wolframite were obtained at 298 and 77 K using a Raman microprobe in combination with a thermal stage. Chillagite is a solid solution of wulfenite and stolzite. The spectra of these molybdate minerals are orientation dependent. The band at 695 cm-1 is interpreted as an antisymmetric bridging mode associated with the tungstate chain. The bands at 790 and 881 cm-1 are associated with the antisymmetric and symmetric Ag modes of terminal WO2 whereas the origin of the 806 cm-1 band remains unclear. The 4(Eg) band was absent for scheelite. The bands at 353 and 401 cm-1 are assigned as either deformation modes or as r(Bg) and (Ag) modes of terminal WO2. The band at 462 cm-1 has an equivalent band in the infrared at 455 cm-1 assigned as as(Au) of the (W2O4)n chain. The band at 508 cm-1 is assigned as sym(Bg) of the (W2O4)n chain.

New dimeric supramolecular structure of mixed (phthalocyaninato)(porphyrinato)-europium(III) sandwiches : preparation and spectroscopic characteristics

The mixed double-decker Eu\[Pc(15C5)4](TPP) (1) was obtained by base-catalysed tetramerisation of 4,5-dicyanobenzo-15-crown-5 using the half-sandwich complex Eu(TPP)(acac) (acac = acetylacetonate), generated in situ, as the template. For comparative studies, the mixed triple-decker complexes Eu2\[Pc(15C5)4](TPP)2 (2) and Eu2\[Pc(15C5)4]2(TPP) (3) were also synthesised by the raise-by-one-story method. These mixed ring sandwich complexes were characterised by various spectroscopic methods. Up to four one-electron oxidations and two one-electron reductions were revealed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). As shown by electronic absorption and infrared spectroscopy, supramolecular dimers (SM1 and SM3) were formed from the corresponding double-decker 1 and triple-decker 3 in the presence of potassium ions in MeOH/CHCl3.

Raman spectroscopy of some complex arsenate minerals—implications for soil remediation

The application of spectroscopy to the study of contaminants in soils is important. Among the many contaminants is arsenic, which is highly labile and may leach to non-contaminated areas. Minerals of arsenate may form depending upon the availability of specific cations for example calcium and iron. Such minerals include carminite, pharmacosiderite and talmessite. Each of these arsenate minerals can be identified by its characteristic Raman spectrum enabling identification.

Raman spectroscopic characteristics of phthalocyanine and naphthalocyanine in sandwich-type phthalocyaninato and porphyrinato rare earth complexes Part 5. – Raman spectroscopic characteristics of naphthalocyanine in mixed [tetrakis(4-tert-butylphenyl)porphyrinato]-(naphthalocyaninato) rare earth double-deckers

Raman spectra were recorded in the range 400–1800 cm−1 for a series of 15 mixed \[tetrakis(4-tert-butylphenyl)porphyrinato](2,3-naphthalocyaninato) rare earth double-deckers M(TBPP)(Nc) (M = Y; La–Lu except Pm) using laser excitation at 632.8 and 785 nm. Comparisons with bis(naphthalocyaninato) rare earth counterparts reveal that the vibrations of the metallonaphthalocyanine M(Nc) fragment dominate the Raman features of M(TBPP)(Nc). When excited with radiation of 632.8 nm, the most intense vibration appears at about 1595 cm−1, due to the naphthalene stretching. These complexes exhibit the marker Raman band for Nc•− as a medium-intense band in the range 1496–1507 cm−1, attributed to the coupling of pyrrole and aza stretching, while the marker Raman band of Nc2− in intermediate-valence Ce(TBPP)(Nc) appears as a strong band at 1493 cm−1 and is due to the isoindole stretchings. By contrast, when excited with radiation of 785 nm that is in close resonance with the main Q absorption band of the naphthalocyanine ligand, the ring radial vibrations at ca 680 and 735 cm−1 for MIII(TBPP)(Nc) are selectively intensified and are the most intense bands. For the cerium double-decker, the most intense vibration also acting as the marker Raman band of Nc2− appears at 1497 cm−1 with contributions from both pyrrole CC and aza CN stretches. The same vibrational modes show weak to medium intensity scattering at 1506–1509 cm−1 for MIII(TBPP)(Nc) and this is the marker Raman band of Nc•− when thus excited. The scatterings due to the Nc breathings, ring radial vibration, aza group stretchings, naphthalene stretchings, benzoisoindole stretchings and the coupling of pyrrole CC and aza CN stretchings in MIII(TBPP)(Nc) are all slightly blue shifted along with the decrease in rare earth ionic radius, confirming the effects of increased ring–ring interactions on the Raman characteristics of naphthalocyanine in the mixed ring double-deckers.

Raman spectroscopy of kaolinites using different excitation wavelengths

The Raman spectra of both low- and high-defect kaolinites in the hydroxyl stretching and low-wavenumber region were obtained with excitation at three visible wavelengths of 633, 514 and 442 nm and a UV wavelength of 325 nm. The UV-excited spectra were comparable to those excited by the visible wavelengths. The Raman spectra show hydroxyl stretching bands at 3621 cm-1 attributed to the inner hydroxyl, at 3692 and 3684 cm-1 attributed to the longitudinal and transverse optic modes of the inner surface hydroxyls and at 3668 and 3653 cm-1 assigned to the out-of phase vibrations of the inner surface hydroxyls. Two bands were observed in the spectral profile at 3695 cm-1 for the high-defect kaolinite at 3698 and 3691 cm-1 and were assigned to TO/LO splitting. An increase in relative intensity of the transverse optic mode is observed with decrease in laser wavelength. The intensity of the out-of-phase vibrations at 3668 and 3653 cm-1 of the inner surface hydroxyls shows a linear relationship with the longitudinal and transverse optic modes. In the low-wavenumber region excellent correlation was found between the experimentally determined and the calculated band positions.