Targeting of 3D oral cancer spheroids by αVß6 integrin using near-infrared peptide-conjugated IRDye 680.

BACKGROUND: In the treatment of oral cavity cancer, margin status is one of the most critical prognostic factors. Positive margins are associated with higher local recurrence and lower survival rates. Therefore, the universal goal of oral surgical oncology is to achieve microscopically clear margins. Near-infrared fluorescence guided surgery (FGS) could improve surgical resection using fluorescent probes. αVß6 integrin has shown great potential for cancer targeting due to its overexpression in oral cancers. Red fluorescent contrast agent IRDye 680 coupled with anti-αVß6 peptide (IRDye-A20) represents an asset to improve FGS of oral cancer. This study investigates the potential of IRDye-A20 as a selective imaging agent in 3D three-dimensional tongue cancer cells. METHODS: αVß6 integrin expression was evaluated by RT-qPCR and Western Blotting in 2D HSC-3 human tongue cancer cells and MRC-5 human fibroblasts. Targeting ability of IRDye-A20 was studied in both cell lines by flow cytometry technique. 3D tumor spheroid models, homotypic (HSC-3) and stroma-enriched heterotypic (HSC-3/MRC-5) spheroids were produced by liquid overlay procedure and further characterized using (immuno)histological and fluorescence-based techniques. IRDye-A20 selectivity was evaluated in each type of spheroids and each cell population. RESULTS: αVß6 integrin was overexpressed in 2D HSC-3 cancer cells but not in MRC-5 fibroblasts and consistently, only HSC-3 were labelled with IRDye-A20. Round shaped spheroids with an average diameter of 400 µm were produced with a final ratio of 55%/45% between HSC-3 and MRC-5 cells, respectively. Immunofluorescence experiments demonstrated an uniform expression of αVß6 integrin in homotypic spheroid, while its expression was restricted to cancer cells only in heterotypic spheroid. In stroma-enriched 3D model, Cytokeratin 19 and E-cadherin were expressed only by cancer cells while vimentin and fibronectin were expressed by fibroblasts. Using flow cytometry, we demonstrated that IRDye-A20 labeled the whole homotypic spheroid, while in the heterotypic model all cancer cells were highly fluorescent, with a negligible fluorescence in fibroblasts. CONCLUSIONS: The present study demonstrated an efficient selective targeting of A20FMDV2-conjugated IRDye 680 in 3D tongue cancer cells stroma-enriched spheroids. Thus, IRDye-A20 could be a promising candidate for the future development of the fluorescence-guided surgery of oral cancers.

Photon correlations in the collective emission of hybrid gold-(CdSe/CdS/CdZnS) nanocrystal supraparticles.

We investigate the photon statistics of the light emitted by single self-assembled hybrid gold-CdSe/CdS/CdZnS colloidal nanocrystal supraparticles through the detailed analysis of the intensity autocorrelation functiong(2)(τ). We first reveal that, despite the large number of nanocrystals involved in the supraparticle emission, antibunching can be observed. We then present a model based on non-coherent Förster energy transfer and Auger recombination that well captures photon antibunching. Finally, we demonstrate that some supraparticles exhibit a bunching effect at short time scales corresponding to coherent collective emission.

Fluorescence decay enhancement and FRET inhibition in self-assembled hybrid gold CdSe/CdS/CdZnS colloidal nanocrystal supraparticles.

We report on the synthesis of hybrid light emitting particles with a diameter ranging between 100 and 500 nm, consisting in a compact semiconductor CdSe/CdS/CdZnS nanocrystal aggregate encapsulated by a controlled nanometric size silica and gold layers. We first characterize the Purcell decay rate enhancement corresponding to the addition of the gold nanoshell as a function of the particle size and find a good agreement with the predictions of numerical simulations. Then, we show that the contribution corresponding to Förster resonance energy transfer is inhibited.

Enhancing fluorescence in vivo imaging using inorganic nanoprobes.

Fluorescence imaging is a versatile tool for biological and preclinical studies with steady improvements in performance thanks to instrumentation and probe developments. The sensitive detection and imaging of deep targets in vivo is especially challenging due to the diffusion and absorption of light by the tissues and to the emission of autofluorescence from intrinsic chromophores. Fluorescent inorganic nanoparticles present interesting optical properties that may significantly differ from organic dyes. In this short review, we present recent developments in the design of these nanoprobes and their use for new in vivo fluorescence modalities which provide enhanced imaging capabilities.

Redundant roles of photoreceptors and cytokinins in regulating photosynthetic acclimation to canopy density.

The regulation of photosynthetic acclimation to canopy density was investigated in tobacco canopies and in tobacco and Arabidopsis plants with part of their foliage experimentally shaded. Both species acclimated to canopy light gradients and partial shading by allocating photosynthetic capacity to leaves in high light and adjusting chloroplast organization to the local light conditions. An investigation was carried out to determine whether signalling mediated by photoreceptors, sugars, cytokinin, and nitrate is involved in and necessary for proper photosynthetic acclimation. No evidence was found for a role for sugars, or for nitrate. The distribution of cytokinins in tobacco stands of contrasting density could be explained in part by irradiance-dependent delivery of cytokinins through the transpiration stream. Functional studies using a comprehensive selection of Arabidopsis mutants and transgenics showed that normal wild-type responses to partial shading were retained when signalling mediated by photoreceptors or cytokinins was disrupted. This indicates that these pathways probably operate in a redundant manner. However, the reduction of the chlorophyll a/b ratio in response to local shade was completely absent in the Arabidopsis Ws-2 accession mutated in PHYTOCHROME D and in the triple phyAphyCphyD mutant. Moreover, cytokinin receptor mutants also showed a reduced response, suggesting a previously unrecognized function of phyD and cytokinins.

[Quantum dots in oncological surgery: the future for surgical margin status]. / Quantum dots en chirurgie oncologique : un rôle d'avenir pour le marquage des berges d'exérèse?

Quantum dots (QDs) are semi-conductor nanocrystals that emit fluorescence on excitation with a light source. They have excellent optical properties, including high brightness and resistance to photobleaching. Their spectroscopic properties can be modulated by many factors. Recent progress in developing QDs enable us to control the size, shape and surface functionality of nanoparticles for potential application in cancer imaging. QDs with near-infrared emission could be applied to identify sentinel lymph-node. Conjugation of QDs with biomolecules could be used to target tumors in vivo. This article reviewed recent developments and issues in nanotechnology with a particular focus on applications to the surgery.

Respiration as a percentage of daily photosynthesis in whole plants is homeostatic at moderate, but not high, growth temperatures.

Here, we investigated the impact of temperature on the carbon economy of two Plantago species from contrasting habitats. The lowland Plantago major and the alpine Plantago euryphylla were grown hydroponically at three constant temperatures: 13, 20 and 27 degrees C. Rates of photosynthetic CO(2) uptake (P) and respiratory CO(2) release (R) in shoots and R in roots were measured at the growth temperature using intact plants. At each growth temperature, air temperatures were changed to establish short-term temperature effects on the ratio of R to P (R/P). In both species, R/P was essentially constant in plants grown at 13 and 20 degrees C. However, R/P was substantially greater in 27 degrees C-grown plants, particularly in P. euryphylla. The increase in R/P at 27 degrees C would have been even greater had biomass allocation to roots not decreased with increasing growth temperature. Short-term increases in air temperature increased R/P in both species, with the effects of air temperature being most pronounced in 13 degrees C-grown plants. We conclude that temperature-mediated changes in biomass allocation play an important role in determining whole-plant R/P values, and, while homeostasis of R/P is achieved across moderate growth temperatures, homeostasis is not maintained when plants are exposed to growth temperatures higher than usually experienced in the natural habitat.

FlgM anti-sigma factors: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure-function relationships.

FlgM proteins, also known as Anti-sigma-28 factor (sigma28), are negative regulators of flagellin synthesis. Recently, a three-dimensional structure of the Aquifex aeolicus sigma28/FlgM complex (PDB code: 1rp3) was determined by X-ray crystallography at 2.3 A resolution. Furthermore, experimental data on bacterial FlgM, including site-directed mutagenesis and structural characterization by NMR are also available. However, an interpretation of the sequence-structure-function relationships combining X-ray and NMR data with the evolutionary information extracted from the increasing number of FlgM-related sequences annotated in databases is not available. In the present study, we combined database sequence searches and sequence-analysis tools to update the multiple sequence alignment of a previously characterized cluster of orthologs (COG2747) and the PFAM classification of protein domains (PF04316) for the FlgM family. A phylogenetic analysis of 77 protein sequences revealed the presence of at least three major sequence clades within the FlgM family. Besides, we predicted functional residues using a SequenceSpace method. We also generated homology models for Bacillus subtilis and Salmonella typhimurium FlgM proteins, for which sequence-structure-function relationship data are available, and used the docking program ClusPro to hypothesize about the dimer association between FlgM proteins. In conclusion, the analysis presented in this work will be useful in designing new experiments to understand better protein-protein interactions between FglM, sigma factors, and putative molecules from the flagellar export apparatus. Electronic Supplementary Material is available in the online version of this article at http://link.springer.de/

Phenotypic plasticity and growth temperature: understanding interspecific variability.

The subject of this review is the impact of long-term changes in temperature on plant growth and its underlying components. The discussion highlights the extent to which thermal acclimation of metabolism is intrinsically linked to the plasticity of a range of biochemical and morphological traits. The fact that there is often a trade-off between temperature-mediated changes in net assimilation rates (NAR) and biomass allocation [in particular the specific leaf area (SLA)] when plants are grown at different temperatures is also highlighted. Also discussed is the role of temperature-mediated changes in photosynthesis and respiration in determining NAR values. It is shown that in comparisons that do not take phylogeny into account, fast-growing species exhibit greater temperature-dependent changes in RGR, SLA, and NAR than slow-growing plants. For RGR and NAR, such trends are maintained within phylogenetically independent contrasts (i.e. species adapted to more-favourable habitats consistently exhibit greater temperature-mediated changes than their congeneric counterparts adapted to less-favourable habitats). By contrast, SLA was not consistently more thermally plastic in species from favourable habitats. Interestingly, biomass allocation between leaves and roots was consistently more plastic in slow-growing species within individual phylogenetically independent contrasts, when plants were grown under contrasting temperatures. Finally, how interspecific variations in NAR account for an increasing proportion of variability in RGR as growth temperatures decrease is highlighted. Conversely, SLA played a more dominant role in determining interspecific variability in RGR at higher growth temperatures; thus, the importance of SLA in determining interspecific variation in RGR could potentially increase if annual mean temperatures increase in the future.

Performance of trees in forest canopies: explorations with a bottom-up functional-structural plant growth model.

Here we present a functional-structural plant model that integrates the growth of metamers into a growing, three-dimensional tree structure, and study the effects of different constraints and strategies on tree performance in different canopies. The tree is a three-dimensional system of connected metamers, and growth is defined by the flush probability of metamers. Tree growth was simulated for different canopy light environments. The result suggest that: the constraints result in an exponential, logistic and decay phase; a mono-layered-leaf crown results from self-shading in a closed canopy; a strong apical control results in slender trees like tall stature species; the interaction between weak apical control and light response results in a crown architecture and performance known from short stature species in closed forest; correlated leaf traits explain interspecific differences in growth, survival and adult stature. The model successfully unravels the interaction effects of different constraints and strategies on tree growth in different canopy light environments.

Electro-optic response of second-harmonic generation membrane potential sensors.

We quantify and ascertain the nature of the second-harmonic generation (SHG) response of amphiphilic push-pull chromophores to a transmembrane electric field. Our technique is based on the application of an alternating field across labeled giant unilamelar vesicles. For chromophore responses that are purely electro-optic, our technique provides an estimate of photoinduced charge shifts based on the observed dispersion of the field response, in accord with a two-level perturbation theory. These results are applicable to the optimization of membrane potential sensors for SHG microscopy.

Emergencia hipertensiva secundaria a terapia electroconvulsiva en un anciano / Hypertensive emenrgency secondary to electroconvulsive therapy in an elderly person

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Acclimation of plants to light gradients in leaf canopies: evidence for a possible role for cytokinins transported in the transpiration stream.

The mechanism of response of plants to vertical light intensity gradients in leaf canopies was investigated. Since shaded leaves transpire less than leaves in high light, it was hypothesized that cytokinins (CKs) carried by mass transport in the transpiration stream would be distributed over the leaf area of partially shaded plants parallel to the gradient in light intensity. It was also hypothesized that this causes the distribution of leaf growth, leaf N and photosynthetic capacity, and possibly chloroplast acclimation as observed in plants growing in leaf canopies. In a field experiment, the distribution of Ca, N and CKs in a bean leaf canopy of a dense and an open stand supported the concept of a role for CKs in the response of N allocation to the light gradient when a decreasing sensitivity for CKs with increasing leaf age is assumed. Both shading of one leaf of the pair of primary bean leaves and independent reduction of its transpiration rate in a growth cabinet experiment caused lower dry mass, N and Ca per unit leaf area in comparison to the opposite not treated leaf. Shading caused a parallel reduction in CK concentration, which supports the hypothesis, but independent reduction of transpiration rate failed to do the same. Application of benzylaminopurine (BA) counteracted the reduction caused by shade of leaf N, photosynthetic capacity and leaf area growth. The experiments show an important role for the transpiration stream in the response of plants to light gradients. Evidence is presented here that CKs carried in the transpiration stream may be important mediators for the acclimation of plants to leaf canopy density.

Molecular cloning of an alpha-glucosidase-like gene from Penicillium minioluteum and structure prediction of its gene product.

The dexC cDNA, which is expressed in dextran-containing medium by the filamentous fungus Penicillium minioluteum, was cloned and sequence characterized. The cDNA sequence comprises 1859 bp plus a poly (A) tail, coding for a predicted protein of 597 amino acids. The genomic counterpart was isolated by PCR, finding three introns in its sequence. The dexC gene was located by Southern blot in the same 9-kb fragment that the previously isolated dextranase-encoding gene (dexA). Sequence analysis revealed that the deduced DexC protein belongs to glycosyl hydrolase family 13, showing a high sequence identity (58%) with Aspergillus parasiticus alpha-1,6-glucosidase. In addition, the high sequence identity (51%) between DexC protein and oligo-1,6-glucosidase of Bacillus cereus, with three-dimensional (3D) structure determined, leads us to proposed a 3D model for the structural core of DexC protein.

Prevalencia de patología disfuncional masticatoria en una muestra de pacientes de la clínica odontológica universitaria / Prevalence of disfuncional masticatorry pathology in a patients sample of the university dental clinic

El objetivo de este trabajo es valorar los signos y síntomas más frecuentes relativos a la patología temporomandibular en una muestra de pacientes. Para ello se estudian las respuestas a un cuestionario y los resultados obtenidos en un protocolo de exploración aplicado a 63 pacientes visitados en la clínica odontológica universitaria de la Facultad de Odontología de la Universidad de Barcelona. La semiología más prevalente Dallada incluye la que se asocia al apretamiento diurno de los dientes, seguida de clics, apretamiento nocturno de los dientes los desgastes oclusales cuya repercusión física parece afectar con mayor or frecuencia al disco u menisco articular (AU)

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Progressive transneuronal changes in the brainstem and thalamus after long-term dorsal rhizotomies in adult macaque monkeys.

This study deals with a potential brainstem and thalamic substrate for the extensive reorganization of somatosensory cortical maps that occurs after chronic, large-scale loss of peripheral input. Transneuronal atrophy occurred in neurons of the dorsal column (DCN) and ventral posterior lateral thalamic (VPL) nuclei in monkeys subjected to cervical and upper thoracic dorsal rhizotomies for 13-21 years and that had shown extensive representational plasticity in somatosensory cortex and thalamus in other experiments. Volumes of DCN and VPL, number and sizes of neurons, and neuronal packing density were measured by unbiased stereological techniques. When compared with the opposite, unaffected, side, the ipsilateral cuneate nucleus (CN), external cuneate nucleus (ECN), and contralateral VPL showed reductions in volume: 44-51% in CN, 37-48% in ECN, and 32-38% in VPL. In the affected nuclei, neurons were progressively shrunken with increasing survival time, and their packing density increased, but there was relatively little loss of neurons (10-16%). There was evidence for loss of axons of atrophic CN cells in the medial lemniscus and in the thalamus, with accompanying severe disorganization of the parts of the ventral posterior nuclei representing the normally innervated face and the deafferented upper limb. Secondary transneuronal atrophy in VPL, associated with retraction of axons of CN neurons undergoing primary transneuronal atrophy, is likely to be associated with similar withdrawal of axons from the cerebral cortex and should be a powerful influence on reorganization of somatotopic maps in the somatosensory cortex.

Leaf respiration of snow gum in the light and dark. Interactions between temperature and irradiance.

We investigated the effect of temperature and irradiance on leaf respiration (R, non-photorespiratory mitochondrial CO(2) release) of snow gum (Eucalyptus pauciflora Sieb. ex Spreng). Seedlings were hydroponically grown under constant 20 degrees C, controlled-environment conditions. Measurements of R (using the Laisk method) and photosynthesis (at 37 Pa CO(2)) were made at several irradiances (0-2,000 micromol photons m(-2) s(-1)) and temperatures (6 degrees C-30 degrees C). At 15 degrees C to 30 degrees C, substantial inhibition of R occurred at 12 micromol photons m(-2) s(-1), with maximum inhibition occurring at 100 to 200 micromol photons m(-2) s(-1). Higher irradiance had little additional effect on R at these moderate temperatures. The irradiance necessary to maximally inhibit R at 6 degrees C to 10 degrees C was lower than that at 15 degrees C to 30 degrees C. Moreover, although R was inhibited by low irradiance at 6 degrees C to 10 degrees C, it recovered with progressive increases in irradiance. The temperature sensitivity of R was greater in darkness than under bright light. At 30 degrees C and high irradiance, light-inhibited rates of R represented 2% of gross CO(2) uptake (v(c)), whereas photorespiratory CO(2) release was approximately 20% of v(c). If light had not inhibited leaf respiration at 30 degrees C and high irradiance, R would have represented 11% of v(c). Variations in light inhibition of R can therefore have a substantial impact on the proportion of photosynthesis that is respired. We conclude that the rate of R in the light is highly variable, being dependent on irradiance and temperature.

Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity.

The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a beta-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern H-x (2)-[PTV]-x (4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC][GA]3 for this family.

Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity.

beta-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from alpha-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in beta-fructofuranosidases.