Corneae from body donors in anatomy department: valuable use for clinical transplantation and experimental research

Background: Explanted corneae are highly needed for the surgical management of patients with severe corneal diseases. The aim of this study was to determine whether the body donors from the Institute of Anatomy are a suitable source of donor corneae. Methods: At the Institute of Anatomy at Saarland University Medical Center in Homburg, corneae are prelevated from body donors who had consented to the removal of tissues for transplantation purposes during their lifetime. Following the report of death, the LIONS Eye Bank is informed and the contraindications of corneal explantation are clarified. Obtaining a blood sample within 24 h postmortem is mandatory. Results: The Institute of Anatomy had 150 body donors in the time period from January 2018 to June 2019. Out of these, 68 (45.3%) were reported to the Eye Bank. The age of the donors (median 82 years (range: 57–96)) is not critical since the quality of the corneae depends on the number of endothelial cells (mean: 2109 ± 67 cells/mm2 (range: 511–2944 cells/mm2)). Contraindications were present in 19 (12.6%) cases. The corneae were extracted from 49 (32.7%) body donors. Out of these 98 corneae, 46 (46.9%) were successfully transplanted. Of all non-transplanted corneae, 6 (6.1%) were microbiologically contaminated, 10 (10.2%) had a positive serology, 22 (22.5%) had an endothelial cell count < 2000 cells/mm2 and 6 (6.1%) are at time of this analysis still in culture medium. The non-transplanted tissues were used for research. Conclusions: Explanted corneae from the Institute of Anatomy are a valuable option in obtaining grafts for corneal transplantation, which is why we are working toward on expanding cooperation with this department

Characterizing, modelling and understanding the climate variability of the deep water formation in the North-Western Mediterranean Sea

Observing, modelling and understanding the climate-scale variability of the deep water formation (DWF) in the North-Western Mediterranean Sea remains today very challenging. In this study, we first characterize the interannual variability of this phenomenon by a thorough reanalysis of observations in order to establish reference time series. These quantitative indicators include 31 observed years for the yearly maximum mixed layer depth over the period 1980–2013 and a detailed multi-indicator description of the period 2007–2013. Then a 1980–2013 hindcast simulation is performed with a fully-coupled regional climate system model including the high-resolution representation of the regional atmosphere, ocean, land-surface and rivers. The simulation reproduces quantitatively well the mean behaviour and the large interannual variability of the DWF phenomenon. The model shows convection deeper than 1000 m in 2/3 of the modelled winters, a mean DWF rate equal to 0.35 Sv with maximum values of 1.7 (resp. 1.6) Sv in 2013 (resp. 2005). Using the model results, the winter-integrated buoyancy loss over the Gulf of Lions is identified as the primary driving factor of the DWF interannual variability and explains, alone, around 50 % of its variance. It is itself explained by the occurrence of few stormy days during winter. At daily scale, the Atlantic ridge weather regime is identified as favourable to strong buoyancy losses and therefore DWF, whereas the positive phase of the North Atlantic oscillation is unfavourable. The driving role of the vertical stratification in autumn, a measure of the water column inhibition to mixing, has also been analyzed. Combining both driving factors allows to explain more than 70 % of the interannual variance of the phenomenon and in particular the occurrence of the five strongest convective years of the model (1981, 1999, 2005, 2009, 2013). The model simulates qualitatively well the trends in the deep waters (warming, saltening, increase in the dense water volume, increase in the bottom water density) despite an underestimation of the salinity and density trends. These deep trends come from a heat and salt accumulation during the 1980s and the 1990s in the surface and intermediate layers of the Gulf of Lions before being transferred stepwise towards the deep layers when very convective years occur in 1999 and later. The salinity increase in the near Atlantic Ocean surface layers seems to be the external forcing that finally leads to these deep trends. In the future, our results may allow to better understand the behaviour of the DWF phenomenon in Mediterranean Sea simulations in hindcast, forecast, reanalysis or future climate change scenario modes. The robustness of the obtained results must be however confirmed in multi-model studies

Polyamine Sharing between Tubulin Dimers Favours Microtubule Nucleation and Elongation via Facilitated Diffusion

We suggest for the first time that the action of multivalent cations on microtubule dynamics can result from facilitated diffusion of GTP-tubulin to the microtubule ends. Facilitated diffusion can promote microtubule assembly, because, upon encountering a growing nucleus or the microtubule wall, random GTP-tubulin sliding on their surfaces will increase the probability of association to the target sites (nucleation sites or MT ends). This is an original explanation for understanding the apparent discrepancy between the high rate of microtubule elongation and the low rate of tubulin association at the microtubule ends in the viscous cytoplasm. The mechanism of facilitated diffusion requires an attraction force between two tubulins, which can result from the sharing of multivalent counterions. Natural polyamines (putrescine, spermidine, and spermine) are present in all living cells and are potent agents to trigger tubulin self-attraction. By using an analytical model, we analyze the implication of facilitated diffusion mediated by polyamines on nucleation and elongation of microtubules. In vitro experiments using pure tubulin indicate that the promotion of microtubule assembly by polyamines is typical of facilitated diffusion. The results presented here show that polyamines can be of particular importance for the regulation of the microtubule network in vivo and provide the basis for further investigations into the effects of facilitated diffusion on cytoskeleton dynamics

A Method For Probing Protein Compartmentalization Along The Microtubule Network In The Cells

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High-Resolution Imaging of Microtubules and Cytoskeleton Structures by Atomic Force Microscopy

International audienceAtomic force microscopy (AFM), which combines a nanometer-scale resolution and a unique capacity to image biomolecular interactions in liquid environment, is a promising tool for the investigation of biological samples. In contrast with nucleic acids and nucleoprotein complexes, for which AFM is now of common use and participates in the recent advances in the knowledge of DNA-related biomolecular processes, AFM investigations of cytoskeleton structures and especially microtubules remain rare. The most critical step to observe biomolecules using AFM is the spreading of the biological material on a flat surface. This issue is now better documented concerning DNA but a lot remains to be done concerning microtubules. This is a prerequisite to further document this issue for a proper and large use of AFM to study cytoskeleton structures. We present here an overview of the various procedures previously used to spread microtubules on a flat surface and advance an easy-to-use and efficient experimental protocol for microtubule imaging by AFM in air. We show application of this protocol to observe intermediate structures of microtubule assembly without using any stabilizing agent and the observation of more complex systems like proteins or messenger ribonucleoprotein particles in interaction with microtubules

Preparation and Characterization of the Triflate Complex [Cp*(dppe)FeOSO2CF3]: A Convenient Access to Labile Five- and Six-Coordinate Iron(I) and Iron(II) Complexes

International audienceTreatment of the iron hydride [Cp*(dppe)FeH] (1) with methyl triflate (CH3OSO2CF3) yielded the iron triflate adduct [Cp*(dppe)FeOSO2CF3] (4, 85 %). In the solid state, the triflate is coordinated at the iron center as shown by XRD (d(Fe-O) = 2.118(4) angstrom) and IR spectroscopy (nu(SO) = 1305 cm(-1)). In solution, 4 is in equilibrium with the 16-electron species [Cp*(dppe)Fe]OSO2CF3 (5(OSO2CF3)), 4/5(OSO2CF3) = 21). The CV of 4 displays two waves (E-1 = -0.74 V, E-2 = 0.24 V vs. SCE) assigned to the [Cp*(dppe)Fe(I)]/[Cp*(dppe)Fe(II)](+) and [Cp*(dppe)Fe(II)](+)/[Cp*(dppe)Fe(III)](2+) redox couples. Reduction of 4 with Cp2Co provided the complex [Cp*(dppe)Fe(I)] (6, 95 %) and oxidation of 6 with [Cp2Fe]PF6 gave [Cp*(dppe)Fe]PF6 (5(PF6), 98 %). XRD established the pseudo-trigonal bipyramidal geometry for the five-coordinated cation 5(+). The reactivity of 5(PF6) and 6 toward small molecules (CH2Cl2, H2O, CO, H-2, N-2) is reported

Classical and non-classical iron hydrides: synthesis, NMR characterisation, theoretical investigation and X-ray crystal structure of the iron(IV) dihydride 〚Cp*Fe(dppe)(H)2〛+BF4–

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Effect of SSB binding modes on the slippage error produced by T7 DNA polymerase during replication of DNA repeated sequences

Arrest of the replication machinery within a repeated region is associated with the instability of DNA repeat regions. Primer-template misalignment, also known as replication slippage or copy-choice recombination, is one of the mechanisms involved in the generation of deletions or expansions within repeat regions1,2. A direct role for replication slippage in the deletion of repeated sequences at a hairpin structure has been demonstrated both in vivo and in vitro. Blockage of the DNA polymerase at a hairpin structure followed by polymerase dissociation and transient out-of-frame re-annealing of nascent and template strands cause slipped strand mispairing, generating a deletion on the nascent strand1. Several DNA polymerases have been tested for their propensity to slip in vitro while replicating hairpin-containing templates3. Studies on DNA polymerases involved in DNA repair such as E. coli Pol I, E. coli Pol II and replicative DNA polymerases such as E. coli Pol III HE or the T4, T7 and F29 phage DNA polymerases revealed that the strand displacement activity (sda) of a DNA polymerase is inversely related to their propensity to slip3. We have analyzed the role of the SSB protein from E. coli on the slippage performed by T7 DNA polymerase. Slippage is inhibited at increasing SSB concentrations as SSB stimulates specifically the sda of T7 DNA polymerase. Reactions conditions that promote the transition between the (SSB)56 to (SSB)65 binding modes correlate with an specific decrease of the sda of T7 DNA polymerase, thus generating slippage errors. No effect of the SSB binding modes was found on the slippage errors performed by T4 DNA pol on the same DNA template, suggesting that this stimulation seems to be specific for T7 DNA polymerase. References 1. E. Viguera, D. Canceill, and S.D. Ehrlich, Embo J. Replication slippage involves DNA polymerase pausing and dissociation, 2001, 20, 2587-2595. 2. S.T. Lovett, Mol. Microbiol. Encoded errors: mutations and rearrangements mediated by misalignment at repetitive DNA sequences, 2004, 52: 1243-53. 3. D. Canceill, E. Viguera and S.D. Ehrlich, J. Biol. Chem. Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency, 1999, 274, 27481-27490.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Rapid Assembly and Collective Behavior of Microtubule Bundles in the Presence of Polyamines

International audienceMicrotubules (MTs) are cylindrical cytoskeleton polymers composed of α-β tubulin heterodimers whose dynamic properties are essential to fulfill their numerous cellular functions. In response to spatial confinement, dynamic MTs, even in the absence of protein partners, were shown to self-organize into higher order structures (spindle or striped structures) which lead to interesting dynamical properties (MT oscillations). In this study, we considered the assembly and sensitivity of dynamic MTs when in bundles. To perform this study, spermine, a natural tetravalent polyamine present at high concentrations in all eukaryote cells, was used to trigger MT bundling while preserving MT dynamics. Interestingly, we first show that, near physiological ionic strengths, spermine promotes the bundling of MTs whereas it does not lead to aggregation of free tubulin, which would have been detrimental to MT polymerization. Experimental and theoretical results also indicate that, to obtain a high rate of bundle assembly, bundling should take place at the beginning of assembly when rapid rotational movements of short and newly nucleated MTs are still possible. On the other hand, the bundling process is significantly slowed down for long MTs. Finally, we found that short MT bundles exhibit a higher sensitivity to cold exposure than do isolated MTs. To account for this phenomenon, we suggest that a collective behavior takes place within MT bundles because an MT entering into a phase of shortening could increase the probability of the other MTs in the same bundle to enter into shortening phase due to their close proximity. We then elaborate on some putative applications of our findings to in vivo conditions including neurons

Interplay between TDP-43 and FUS in protein assemblies-Influence of mRNA on the aggregation process

International audienceTDP43 and FUS are 2 RNA Binding Proteins (RBP) involved in neurodegenerativediseases. These 2 nuclear proteins are very similar in structure and function; theyboth harbor RRM (RNA Recognitions Motif) to bind to RNA, and a domain of lowcomplexity promoting protein-protein interaction and potentially, aggregation. Theyare involved in several mRNA-related process as RNA biogenesis, transport, andsplicing. TDP-43 targets more than 6000 mRNA thanks to its RRMs with specificitytowards GU motif while FUS interact with broad varieties of mRNA through its ZnF,RGG motifs and RRM without any specific nucleotide sequence. Despite thisdifference in RNA interaction mode, these proteins promote the formation of mRNArich compartments like stress granules, drosha complexes, splicing complexes…via the process of liquid-liquid phase separation (LLPS), and within thesestructures, they accumulate on common RNA targets. Such proximity in functionsand localization point toward an interplay between FUS and TDP-43. Here, weanalyze their ability to mix or phase separate in cellular context using themicrotubule bench technology. Then we reveal the ultrastructure of pure and mixedTDP43 and FUS assemblies as well as the influence of RNA