MRE11-RAD50-NBS1 is a critical regulator of FANCD2 stability and function during DNA double-strand break repair.

Monoubiquitination of the Fanconi anaemia protein FANCD2 is a key event leading to repair of interstrand cross-links. It was reported earlier that FANCD2 co-localizes with NBS1. However, the functional connection between FANCD2 and MRE11 is poorly understood. In this study, we show that inhibition of MRE11, NBS1 or RAD50 leads to a destabilization of FANCD2. FANCD2 accumulated from mid-S to G2 phase within sites containing single-stranded DNA (ssDNA) intermediates, or at sites of DNA damage, such as those created by restriction endonucleases and laser irradiation. Purified FANCD2, a ring-like particle by electron microscopy, preferentially bound ssDNA over various DNA substrates. Inhibition of MRE11 nuclease activity by Mirin decreased the number of FANCD2 foci formed in vivo. We propose that FANCD2 binds to ssDNA arising from MRE11-processed DNA double-strand breaks. Our data establish MRN as a crucial regulator of FANCD2 stability and function in the DNA damage response.

Anchoring of both PKA and 14-3-3 inhibits the Rho-GEF activity of the AKAP-Lbc signaling complex.

A-kinase anchoring proteins (AKAPs) target the cAMP-regulated protein kinase (PKA) to its physiological substrates. We recently identified a novel anchoring protein, called AKAP-Lbc, which functions as a PKA-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha subunit of the heterotrimeric G protein G12. Here, we identified 14-3-3 as a novel regulatory protein interacting with AKAP-Lbc. Elevation of the cellular concentration of cAMP activates the PKA holoenzyme anchored to AKAP-Lbc, which phosphorylates the anchoring protein on the serine 1565. This phosphorylation event induces the recruitment of 14-3-3, which inhibits the Rho-GEF activity of AKAP-Lbc. AKAP-Lbc mutants that fail to interact with PKA or with 14-3-3 show a higher basal Rho-GEF activity as compared to the wild-type protein. This suggests that, under basal conditions, 14-3-3 maintains AKAP-Lbc in an inactive state. Therefore, while it is known that AKAP-Lbc activity can be stimulated by Galpha12, in this study we demonstrated that it is inhibited by the anchoring of both PKA and 14-3-3.

Multimodal MRI analysis of structural and functional networks in the human brain : application to two clinical studies : driving abilities under THC intoxication and early white matter changes in FXTAS

Les approches multimodales dans l'imagerie cérébrale non invasive sont de plus en plus considérées comme un outil indispensable pour la compréhension des différents aspects de la structure et de la fonction cérébrale. Grâce aux progrès des techniques d'acquisition des images de Resonance Magnetique et aux nouveaux outils pour le traitement des données, il est désormais possible de mesurer plusieurs paramètres sensibles aux différentes caractéristiques des tissues cérébraux. Ces progrès permettent, par exemple, d'étudier les substrats anatomiques qui sont à la base des processus cognitifs ou de discerner au niveau purement structurel les phénomènes dégénératifs et développementaux. Cette thèse met en évidence l'importance de l'utilisation d'une approche multimodale pour étudier les différents aspects de la dynamique cérébrale grâce à l'application de cette approche à deux études cliniques: l'évaluation structurelle et fonctionnelle des effets aigus du cannabis fumé chez des consommateurs réguliers et occasionnels, et l'évaluation de l'intégrité de la substance grise et blanche chez des jeunes porteurs de la prémutations du gène FMR1 à risque de développer le FXTAS (Fragile-X Tremor Ataxia Syndrome). Nous avons montré que chez les fumeurs occasionnels de cannabis, même à faible concentration du principal composant psychoactif (THC) dans le sang, la performance lors d'une tâche visuo-motrice est fortement diminuée, et qu'il y a des changements dans l'activité des trois réseaux cérébraux impliqués dans les processus cognitifs: le réseau de saillance, le réseau du contrôle exécutif, et le réseau actif par défaut (Default Mode). Les sujets ne sont pas en mesure de saisir les saillances dans l'environnement et de focaliser leur attention sur la tâche. L'augmentation de la réponse hémodynamique dans le cortex cingulaire antérieur suggère une augmentation de l'activité introspective. Une investigation des ef¬fets au niveau cérébral d'une exposition prolongée au cannabis, montre des changements persistants de la substance grise dans les régions associées à la mémoire et au traitement des émotions. Le niveau d'atrophie dans ces structures corrèle avec la consommation de cannabis au cours des trois mois précédant l'étude. Dans la deuxième étude, nous démontrons des altérations structurelles des décennies avant l'apparition du syndrome FXTAS chez des sujets jeunes, asymptomatiques, et porteurs de la prémutation du gène FMR1. Les modifications trouvées peuvent être liées à deux mécanismes différents. Les altérations dans le réseau moteur du cervelet et dans la fimbria de l'hippocampe, suggèrent un effet développemental de la prémutation. Elles incluent aussi une atrophie de la substance grise du lobule VI du cervelet et l'altération des propriétés tissulaires de la substance blanche des projections afférentes correspondantes aux pédoncules cérébelleux moyens. Les lésions diffuses de la substance blanche cérébrale peu¬vent être un marquer précoce du développement de la maladie, car elles sont liées à un phénomène dégénératif qui précède l'apparition des symptômes du FXTAS. - Multimodal brain imaging is becoming a leading tool for understanding different aspects of brain structure and function. Thanks to the advances in Magnetic Resonance imaging (MRI) acquisition schemes and data processing techniques, it is now possible to measure different parameters sensitive to different tissue characteristics. This allows for example to investigate anatomical substrates underlying cognitive processing, or to disentangle, at a pure structural level degeneration and developmental processes. This thesis highlights the importance of using a multimodal approach for investigating different aspects of brain dynamics by applying this approach to two clinical studies: functional and structural assessment of the acute effects of cannabis smoking in regular and occasional users, and grey and white matter assessment in young FMR1 premutation carriers at risk of developing FXTAS. We demonstrate that in occasional smokers cannabis smoking, even at low concentration of the main psychoactive component (THC) in the blood, strongly decrease subjects' performance on a visuo-motor tracking task, and globally alters the activity of the three brain networks involved in cognitive processing: the Salience, the Control Executive, and the Default Mode networks. Subjects are unable to capture saliences in the environment and to orient attention to the task; the increase in Hemodynamic Response in the Anterior Cingulate Cortex suggests an increase in self-oriented mental activity. A further investigation on long term exposure to cannabis, shows a persistent grey matter modification in brain regions associated with memory and affective processing. The degree of atrophy in these structures also correlates with the estimation of drug use in the three months prior the participation to the study. In the second study we demonstrate structural changes in young asymptomatic premutation carriers decades before the onset of FXTAS that might be related to two different mechanisms. Alteration of the cerebellar motor network and of the hippocampal fimbria/ fornix, may reflect a potential neurodevelopmental effect of the premutation. These include grey matter atrophy in lobule VI and modification of white matter tissue property in the corresponding afferent projections through the Middle Cerebellar Peduncles. Diffuse hemispheric white matter lesions that seem to appear closer to the onset of FXTAS and be related to a neurodegenerative phenomenon may mark the imminent onset of FXTAS.

Study of the physiological role of RasGAP cleavage

Résume Les caspases sont un groupe de protéases à cystéine qui s?activent lors de l'apoptose. Leur activation induit le clivage de nombreuses cibles intracellulaires, conduisant à l'activation de voies pro-apoptotiques et finalement au démantèlement des cellules. Cependant, des caspases ont été décrites dans de nombreux autres processus indépendants de l'apoptose, notamment dans la physiologie des cellules hématopoïétiques, des cellules musculaires, des cellules de la peau et des neurones. Comment est-ce que les cellules réconcilient-elles ces deux fonctions distinctes? Une partie de la réponse réside dans la nature des substrats qu'elles clivent. Certains substrats, une fois clivées, deviennent anti-apoptotiques. RasGAP est une cible des caspases et contient deux sites spécifiques de clivage par les caspases. Lorsque le niveau d?activité des caspases est faible le clivage de RasGAP produit un fragment N-terminal qui active un signal antiapoptotique, relayé par la voie de Ras/PI3K/Akt. Lorsque le niveau d?activité des caspases est plus élevé le fragment RasGAP N-terminal est à nouveau clivé, perdant de ce fait ses propriétés anti-apoptotiques. Dans cette étude, nous avons mis en évidence que l'activation de la voie Ras/PI3K/Akt induite par le fragment RasGAP N-terminal dépend de RasGAP lui-même. Par ailleurs, dans le but d?étudier l?importance du clivage de RasGAP dans un contexte physiologique, nous avons développé un modèle animal exprimant une gêne mutée de RasGAP de sorte que la protéine est devenu insensible a l?action de caspases. Les données préliminaires obtenues montrent que le clivage de RasGAP n'est pas indispensable pour le développement et l?homéostasie chez la souris. Finalement, nous avons développé une souris transgénique surexprimant le fragment de RasGAP N-terminal dans les cellules ß du pancréas. Les animaux obtenus ne montrent pas de symptômes dans les conditions basales bien qu?ils soient plus résistants au diabète induit expérimentalement. Ces résultats montrent que la surexpression du fragment N-terminal de RasGAP protége efficacement les cellules ß du pancréas de l?apoptose induite par le stress sans pourtant affecter d?autres paramètres physiologiques des Ilot de Langerhans.<br/><br/>Caspases are a series of proteases that are activated during apoptosis. Their activation causes the cleavage of numerous intracellular targets, which leads to cell dismantling and activation of pro-apoptotic pathways. Caspases have been found to be involved in the physiology of numerous cell types including haematopoietic cells, muscle cells, skin cells and neurons. How cells conciliate these two opposite functions? Part of the answer lies in the nature of the substrates they cleave. Some substrates become anti-apoptotic once cleaved by caspases. RasGAP is a caspase substrate that possesses two conserved caspase-cleavage sites. At low caspase activity, RasGAP is first cleaved and the generated N-terminal fragment activates a potent anti-apoptotic signal, mediated by the Ras/PI3K/Akt pathway. At higher caspase activity, the N-terminal fragment is further cleaved thereby losing its anti-apoptotic properties. In the present study we show that the activation of the Ras/PI3K/Akt pathway mediated by RasGAP N-terminal fragment is dependent on RasGAP itself. Moreover, to study the role of RasGAP cleavage in a physiological model, we have developed a knock-in mouse model expressing a RasGAP mutant that is not cleavable by caspases. Preliminary data shows that RasGAP cleavage is not required for normal development and homeostasis in mice. Finally, we have developed a transgenic mouse model overexpressing RasGAP N-terminal fragment in the ß-cell of the pancreas. In basal conditions, these mice show no difference with their wt counterparts. However, they are protected against experimentally induced diabetes. These results indicate that fragment N can protect ? cells from stress-induced apoptosis without affecting other physiological parameters of the Islets.

Influence of the substrate in germination of lychee seeds

The lychee is, all over the world, considered as the "queen of the fruits" due to the delicacy of its appearance and flavor. Although it was only recently that it started to have economical importance in Brazil, the lychee is already calling the attention of numerous farmers, mainly those who cultivate citric fruits and/or sugarcane in the State of São Paulo, due to the constant and at the same time growing necessity of finding new alternative crops. Considering that the commercial cultivation of lychee plants in the field requires the previous obtainment of rootstocks viewing to reduce the genetic variability and the length of the juvenile period displayed by plants resulting directly from the seeds, finding ways to improve the germination performance of lychee seeds for the production of rootstock plants is of considerable economic importance. Thus, the objective of this experiment was to test five substrates for the germination of lychee seeds: (1) vermiculite, (2) washed sand, (3) filter paper, (4) carbonized rice hull, and (5) sphagnum. The results showed that the period of time required by a lychee seed to germinate is a short one thus reinforcing the importance of providing a suitable substrate for the germination to take place. It was found that the substrates which apparently allowed the best combinations of water and oxygen availability for the seeds - washed sand and carbonized rice hull - were those leading to the fastest and highest germination values.

The Pseudomonas aeruginosa antimetabolite L -2-amino-4-methoxy-trans-3-butenoic acid (AMB) is made from glutamate and two alanine residues via a thiotemplate-linked tripeptide precursor.

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE), and two iron(II)/α-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate. Using a combination of ATP-PPi exchange assays, aminoacylation assays, and mass spectrometry-based analysis of enzyme-bound substrates and pathway intermediates, the AmbB substrate was identified to be L-alanine (L-Ala), while the T1 and T2 domains of AmbE were loaded with L-glutamate (L-Glu) and L-Ala, respectively. Loading of L-Ala at T2 of AmbE occurred only in the presence of AmbB, indicative of a trans loading mechanism. In vitro assays performed with AmbB and AmbE revealed the dipeptide L-Glu-L-Ala at T1 and the tripeptide L-Ala-L-Glu-L-Ala attached at T2. When AmbC and AmbD were included in the assay, these peptides were no longer detected. Instead, an L-Ala-AMB-L-Ala tripeptide was found at T2. These data are in agreement with a biosynthetic model in which L-Glu is converted into AMB by the action of AmbC, AmbD, and tailoring domains of AmbE. The importance of the flanking L-Ala residues in the precursor tripeptide is discussed.

Growth of lulo (Solanum quitoense Lam.) plants affected by salinity and substrate

The effects of 0, 30 and 60 mM NaCl and substrates (red peat, sand or 3:1:1 [w/w] mixture of peat, sand, or soil) on vegetative growth of lulo, an Andean fruit species, during 12 weeks were studied. The experiment was carried out by using 2000 cm³ of polypropylene plastic pots under greenhouse conditions. Plant height, number of leaves and nodes, leaf area, total plant dry matter (DM), and shoot/root ratio were evaluated. With the increase of salt concentration, the plant height, the number of leaves and nodes, the leaf areas and plant dry mass DM decreased, whereas shoot/root ratio increased. Sand grown lulo plants were most affected by salinity and presented total mortality at 60 mM NaCl. On the other hand, plants held either in peat or in substrate mixture developed larger height, greater leaf and node numbers, higher leaf area and dry matter content. Shoot/root ratio in control (soil) and sand-grown plants (30 mM NaCl) was lower.

Assessment of metabolic fluxes in the mouse brain in vivo using 1H-[13C] NMR spectroscopy at 14.1 Tesla.

(13)C magnetic resonance spectroscopy (MRS) combined with the administration of (13)C labeled substrates uniquely allows to measure metabolic fluxes in vivo in the brain of humans and rats. The extension to mouse models may provide exclusive prospect for the investigation of models of human diseases. In the present study, the short-echo-time (TE) full-sensitivity (1)H-[(13)C] MRS sequence combined with high magnetic field (14.1 T) and infusion of [U-(13)C6] glucose was used to enhance the experimental sensitivity in vivo in the mouse brain and the (13)C turnover curves of glutamate C4, glutamine C4, glutamate+glutamine C3, aspartate C2, lactate C3, alanine C3, γ-aminobutyric acid C2, C3 and C4 were obtained. A one-compartment model was used to fit (13)C turnover curves and resulted in values of metabolic fluxes including the tricarboxylic acid (TCA) cycle flux VTCA (1.05 ± 0.04 μmol/g per minute), the exchange flux between 2-oxoglutarate and glutamate Vx (0.48 ± 0.02 μmol/g per minute), the glutamate-glutamine exchange rate V(gln) (0.20 ± 0.02 μmol/g per minute), the pyruvate dilution factor K(dil) (0.82 ± 0.01), and the ratio for the lactate conversion rate and the alanine conversion rate V(Lac)/V(Ala) (10 ± 2). This study opens the prospect of studying transgenic mouse models of brain pathologies.

Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation independent manner

Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs play specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP ribose) polymerase hyperactivation and cell death, and reduced tumor growth and cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. These data predict a novel and totally unexpected biological role for the nucleoside transporter protein hCNT1 that appears to be independent of its role as mediator of nucleoside uptake by cells, thereby suggesting a transceptor function. Cell Death & Disease Anastasis Stephanou Receiving Editor Cell Death & Disease 19th Apr 2013 Dr Perez-Torras Av/ Diagonal 643. Edif. Prevosti, Pl -1 Barcelona 08028 Spain RE: Manuscript CDDIS-13-0136R, 'CDDIS-13-0136R' Dear Dr Perez-Torras, It is a pleasure to inform you that your manuscript has been evaluated at the editorial level and has now been officially accepted for publication in Cell Death & Disease, pending you meet the following editorial requirements: 1) the list of the abbreviations is missing please include Could you send us the revised text as word file via e-mail and we will proceed and transfer the paper onto our typesetters. Please download, print, sign, and return the Licence to Publish Form using the link below. This must be returned via FAX to ++ 39 06 7259 6977 before your manuscript can be published:

In situ spectroellipsometric study of the nucleation and growth of amorphous silicon

A detailed in situ spectroellipsometric analysis of the nucleation and growth of hydrogenated amorphous silicon (a:Si:H) is presented. Photoelectronic quality a‐Si:H films are deposited by plasma‐enhanced chemical vapor deposition on smooth metal (NiCr alloy) and crystalline silicon (c‐Si) substrates. The deposition of a‐Si:H is analyzed from the first monolayer up to a final thickness of 1.2 μm. In order to perform an improved analysis, real time ellipsometric trajectories are recorded, using fixed preparation conditions, at various photon energies ranging from 2.2 to 3.6 eV. The advantage of using such a spectroscopic experimental procedure is underlined. New insights into the nucleation and growth mechanisms of a‐Si:H are obtained. The nucleation mechanism on metal and c‐Si substrates is very accurately described assuming a columnar microstructural development during the early stage of the growth. Then, as a consequence of the incomplete coalescence of the initial nuclei, a surface roughness at the 10-15 Å scale is identified during the further growth of a‐Si:H on both substrates. The bulk a‐Si:H grows homogeneously beneath the surface roughness. Finally, an increase of the surface roughness is evidenced during the long term growth of a‐Si:H. However, the nature of the substrate influenced the film growth. In particular, the film thickness involved in the nucleation‐coalescence phase is found lower in the case of c‐Si (67±8 Å) as compared to NiCr (118±22 Å). Likewise films deposited on c‐Si present a smaller surface roughness even if thick samples are considered (>1 μm). More generally, the present study illustrates the capability of in situ spectroellipsometry to precisely analyze fundamental processes in thin‐film growth, but also to monitor the preparation of complex structures on a few monolayers scale.

Characteristics of Flowing Film

Työssä tutkittiin uutta teknologiaa pigmenttipäällystykseen. Tämä tekniikka on yleisesti tunnettua eräillä muilla teollisuudenaloilla. Kirjallisuustutkimuksessa on esitelty prosessia ja sen eri osatekijöitä sekä muilla aloilla tunnettuja prosessimuuttujia. Päällystyspastojen ja päällystettävien pintojen teoriaa on selvitetty uuden tekniikan ja pigmenttipäällystyksen valossa. Uuden tekniikan perusmekanismeja tutkittiin kokeellisessa osassa. Valuvan nestefilmin stabiilisuutta tutkittiin minimivirtauksen avulla. Stabiilisuustutkimuksen suorittamiseen käytettiin apuna Taguchi-matriisia DOE-ohjelmalla (Design of Experiments). Kokeiden perusteella minimivirtauksen kannalta päällystyspastalle edullisempi koostumus on kalsiumkarbonaatti- kuin kaoliinipasta. Sideaineella on pienempi osuus lateksia ja polyvinyylialkoholia parempi. Suurempi osuus pinta-aktiivista ainetta ja matala pastan kuiva-ainepitoisuus ovat suositeltuja. Tehokas ilmanpoisto päällystyspastasta on myös tärkeää lopullisen tuloksen kannalta. Koekoneella ajetuissa päällystyskokeissa havaittiin valuvan filmin ominaisuuksien tärkeys. Pienetkin kaasumäärät päällystyspastassa häiritsivät lopullisen päällysteen laatua. Päällystyspastan ilmanpoisto on avainasemassa erityisesti kun päällystetään suurella nopeudella pieniä päällystemääriä. Koeajoissa havaittiin kaikki kirjallisuudessa esitellyt rajoittavat tekijät. Kokeissa päällystettiin 400-1600 m/min nopeudella 5-20 g/m² päällystemääriä. Olosuhteet stabiilille nestefilmille vaativat edelleen kehitystä suurella nopeudella päällystettäessä. Päällysteen eroavaisuuksia verrattiin teräpäällystysmenetelmiin. Terä-päällystyksellä saadaan sileä mutta epätasaisesti peittävä pinta kun taas uuden tekniikan päällyste mukailee päällystettävän alustan topografiaa. Tasapaksun päällysteen etuna on hyvä peittävyys jo pienellä päällystemäärällä.

ZnO nanorods for efficient third harmonic UV generation.

ZnO nanorods grown by both high temperature vapour phase transport and low temperature chemical bath deposition are very promising sources for UV third harmonic generation. Material grown by both methods show comparable efficiencies, in both cases an order of magnitude higher than surface third harmonic generation at the quartz-air interface of a bare quartz substrate. This result is in stark contrast to the linear optical properties of ZnO nanorods grown by these two methods, which show vastly different PL efficiencies. The third harmonic generated signal is analysed using intensity dependent measurements and interferometric frequency resolved optical gating, allowing extraction of the laser pulse parameters. The comparable levels of efficiency of ZnO grown by these very different methods as sources for third harmonic UV generation provides a broad suite of possible growth methods to suit various substrates, coverage and scalability requirements. Potential application areas range from interferometric frequency resolved optical gating characterization of few cycle fs pulses to single cell UV irradiation for biophysical studies.

Motorcycle helmets: what about their coating?

In traffic accidents involving motorcycles, paint traces can be transferred from the rider's helmet or smeared onto its surface. These traces are usually in the form of chips or smears and are frequently collected for comparison purposes. This research investigates the physical and chemical characteristics of the coatings found on motorcycles helmets. An evaluation of the similarities between helmet and automotive coating systems was also performed.Twenty-seven helmet coatings from 15 different brands and 22 models were considered. One sample per helmet was collected and observed using optical microscopy. FTIR spectroscopy was then used and seven replicate measurements per layer were carried out to study the variability of each coating system (intravariability). Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were also performed on the infrared spectra of the clearcoats and basecoats of the data set. The most common systems were composed of two or three layers, consistently involving a clearcoat and basecoat. The coating systems of helmets with composite shells systematically contained a minimum of three layers. FTIR spectroscopy results showed that acrylic urethane and alkyd urethane were the most frequent binders used for clearcoats and basecoats. A high proportion of the coatings were differentiated (more than 95%) based on microscopic examinations. The chemical and physical characteristics of the coatings allowed the differentiation of all but one pair of helmets of the same brand, model and color. Chemometrics (PCA and HCA) corroborated classification based on visual comparisons of the spectra and allowed the study of the whole data set at once (i.e., all spectra of the same layer). Thus, the intravariability of each helmet and its proximity to the others (intervariability) could be more readily assessed. It was also possible to determine the most discriminative chemical variables based on the study of the PCA loadings. Chemometrics could therefore be used as a complementary decision-making tool when many spectra and replicates have to be taken into account. Similarities between automotive and helmet coating systems were highlighted, in particular with regard to automotive coating systems on plastic substrates (microscopy and FTIR). However, the primer layer of helmet coatings was shown to differ from the automotive primer. If the paint trace contains this layer, the risk of misclassification (i.e., helmet versus vehicle) is reduced. Nevertheless, a paint examiner should pay close attention to these similarities when analyzing paint traces, especially regarding smears or paint chips presenting an incomplete layer system.

Acclimatization and leaf anatomy of micropropagated fig plantlets

The survival of micropropagated plants during and after acclimatization is a limiting process to plant establishment. There is little information on how the anatomy of vegetative organs of Ficus carica can be affected by culture conditions and acclimatization. The present research aimed to study the effects of time on culture medium and substrates during the acclimatization of fig tree plantlets produced in vitro, characterizing some leaf anatomy aspects of plantlets cultured in vitro and of fig trees produced in field. Plantlets previously multiplied in vitro were separated and transferred into Wood Plant Medium (WPM) where they were kept for 0, 15, 30, 45 and 60 days. Different substrates were tested and studies on leaf anatomy were performed in order to compare among plantlets grown in vitro, plantlets under 20, 40 and 60 days of acclimatization, and field grown plants. Keeping plantlets for 30 days in WPM allowed better development in Plantmax during acclimatization. Field grown plants presented higher number of stomata, greater epicuticular wax thickness and greater leaf tissue production compared to in vitro ones. The leaf tissues of in vitro plantlets show little differentiation and have great stomata number compared with acclimatized plants, which reduce the number of stomata during the acclimatization process.

Hsp90 inhibition induces both protein-specific and global changes in the ubiquitinome.

Inhibition of the essential chaperone Hsp90 with drugs causes a global perturbation of protein folding and the depletion of direct substrates of Hsp90, also called clients. Ubiquitination and proteasomal degradation play a key role in cellular stress responses, but the impact of Hsp90 inhibition on the ubiquitinome has not been characterized on a global scale. We used stable isotope labeling and antibody-based peptide enrichment to quantify more than 1500 protein sites modified with a Gly-Gly motif, the remnant of ubiquitination, in human T-cells treated with an Hsp90 inhibitor. We observed rapid changes in GlyGly-modification sites, with strong increases for some Hsp90 clients but also decreases for a majority of cellular proteins. A comparison with changes in total protein levels and protein synthesis and decay rates from a previous study revealed a complex picture with different regulatory patterns observed for different protein families. Overall the data support the notion that for Hsp90 clients GlyGly-modification correlates with targeting by the ubiquitin-proteasome system and decay, while for other proteins levels of GlyGly-modification appear to be mainly influenced by their synthesis rates. Therefore a correct interpretation of changes in ubiquitination requires knowledge of multiple parameters. Data are available via ProteomeXchange with identifier PXD001549. BIOLOGICAL SIGNIFICANCE: Proteostasis, i.e. the capacity of the cell to maintain proper synthesis and maturation of proteins, is a fundamental biological process and its perturbations have far-reaching medical implications e.g. in cancer or neurodegenerative diseases. Hsp90 is an essential chaperone responsible for the correct maturation and stability of a number of key proteins. Inhibition of Hsp90 triggers a global stress response caused by accumulation of misfolded chains, which have to be either refolded or eliminated by protein degradation pathways such as the Ubiquitin-Proteasome System (UPS). We present the first global assessment of the changes in the ubiquitinome, the subset of ubiquitin-modified proteins, following Hsp90 inhibition in human T-cells. The results provide clues on how cells respond to a specific proteostasis challenge. Furthermore, our data also suggest that basal ubiquitination levels for most proteins are influenced by synthesis rates. This has broad significance as it implies that a proper interpretation of data on ubiquitination levels necessitates simultaneous knowledge of other parameters.