The Discrimination of Colored Acrylic, Cotton, and Wool Textile Fibers Using Micro-Raman Spectroscopy. Part 1 : In Situ Detection and Characterization of Dyes

Raman spectroscopy has been applied to characterize fiber dyes and determine the discriminating ability of the method. Black, blue, and red acrylic, cotton, and wool samples were analyzed. Four excitation sources were used to obtain complementary responses in the case of fluorescent samples. Fibers that did not provide informative spectra using a given laser were usually detected using another wavelength. For any colored acrylic, the 633-nm laser did not provide Raman information. The 514-nm laser provided the highest discrimination for blue and black cotton, but half of the blue cottons produced noninformative spectra. The 830-nm laser exhibited the highest discrimination for red cotton. Both visible lasers provided the highest discrimination for black and blue wool, and NIR lasers produced remarkable separation for red and black wool. This study shows that the discriminating ability of Raman spectroscopy depends on the fiber type, color, and the laser wavelength.

Modeling of in-situ crystallization processes in the Permian mafic layered intrusion of Mont Collon (Dent Blanche nappe, western Alps)

The Mont Collon mafic complex is one of the best preserved examples of the Early Permian magmatism in the Central Alps, related to the intra-continental collapse of the Variscan belt. It mostly consists (> 95 vol.%) of ol+hy-nonnative plagioclase-wehrlites, olivine- and cpx-gabbros with cumulitic structures, crosscut by acid dikes. Pegmatitic gabbros, troctolites and anorthosites outcrop locally. A well-preserved cumulative, sequence is exposed in the Dents de Bertol area (center of intrusion). PT-calculations indicate that this layered magma chamber emplaced at mid-crustal levels at about 0.5 GPa and 1100 degrees C. The Mont Collon cumulitic rocks record little magmatic differentiation, as illustrated by the restricted range of clinopyroxene mg-number (Mg#(cpx)=83-89). Whole-rock incompatible trace-element contents (e.g. Nb, Zr, Ba) vary largely and without correlation with major-element composition. These features are characteristic of an in-situ crystallization process with variable amounts of interstitial liquid L trapped between the cumulus mineral phases. LA-ICPMS measurements show that trace-element distribution in the latter is homogeneous, pointing to subsolidus re-equilibration between crystals and interstitial melts. A quantitative modeling based on Langmuir's in-situ crystallization equation successfully duplicated the REE concentrations in cumulitic minerals of all rock facies of the intrusion. The calculated amounts of interstitial liquid L vary between 0 and 35% for degrees of differentiation F of 0 to 20%, relative to the least evolved facies of the intrusion. L values are well correlated with the modal proportions of interstitial amphibole and whole-rock incompatible trace-element concentrations (e.g. Zr, Nb) of the tested samples. However, the in-situ crystallization model reaches its limitations with rock containing high modal content of REE-bearing minerals (i.e. zircon), such as pegmatitic gabbros. Dikes of anorthositic composition, locally crosscutting the layered lithologies, evidence that the Mont Collon rocks evolved in open system with mixing of intercumulus liquids of different origins and possibly contrasting compositions. The proposed model is not able to resolve these complex open systems, but migrating liquids could be partly responsible for the observed dispersion of points in some correlation diagrams. Absence of significant differentiation with recurrent lithologies in the cumulitic pile of Dents de Bertol points to an efficiently convective magma chamber, with possible periodic replenishment, (c) 2005 Elsevier B.V. All rights reserved.

In situ evaluation of single-cell lysis by cytosol extraction observation through fluorescence decay and dielectrophoretic trapping time

We present a new method for lysis of single cells in continuous flow, where cells are sequentially trapped, lysed and released in an automatic process. Using optimized frequencies, dielectrophoretic trapping allows exposing cells in a reproducible way to high electrical fields for long durations, thereby giving good control on the lysis parameters. In situ evaluation of cytosol extraction on single cells has been studied for Chinese hamster ovary (CHO) cells through out-diffusion of fluorescent molecules for different voltage amplitudes. A diffusion model is proposed to correlate this out-diffusion to the total area of the created pores, which is dependent on the potential drop across the cell membrane and enables evaluation of the total pore area in the membrane. The dielectrophoretic trapping is no longer effective after lysis because of the reduced conductivity inside the cells, leading to cell release. The trapping time is linked to the time required for cytosol extraction and can thus provide additional validation of the effective cytosol extraction for non-fluorescent cells. Furthermore, the application of one single voltage for both trapping and lysis provides a fully automatic process including cell trapping, lysis, and release, allowing operating the device in continuous flow without human intervention.

High-magnification vascular imaging to reject false-positive sites in situ during Hexvix® fluorescence cystoscopy.

Fluorescence imaging for detection of non-muscle-invasive bladder cancer is based on the selective production and accumulation of fluorescing porphyrins-mainly, protoporphyrin IX-in cancerous tissues after the instillation of Hexvix®. Although the sensitivity of this procedure is very good, its specificity is somewhat limited due to fluorescence false-positive sites. Consequently, magnification cystoscopy has been investigated in order to discriminate false from true fluorescence positive findings. Both white-light and fluorescence modes are possible with the magnification cystoscope, allowing observation of the bladder wall with magnification ranging between 30× for standard observation and 650×. The optical zooming setup allows adjusting the magnification continuously in situ. In the high-magnification (HM) regime, the smallest diameter of the field of view is 600 microns and the resolution is 2.5 microns when in contact with the bladder wall. With this cystoscope, we characterized the superficial vascularization of the fluorescing sites in order to discriminate cancerous from noncancerous tissues. This procedure allowed us to establish a classification based on observed vascular patterns. Seventy-two patients subject to Hexvix® fluorescence cystoscopy were included in the study. Comparison of HM cystoscopy classification with histopathology results confirmed 32?33 (97%) cancerous biopsies and rejected 17?20 (85%) noncancerous lesions.

Fas ligand expression is restricted to nonlymphoid thymic components in situ.

The cell surface receptor Fas (Apo-1/CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in activation-induced death of mature T cells and in killing mediated by cytolytic T cells. The role of the Fas pathway in apoptosis associated with thymic selection events is, however, controversial. Although Fas and FasL are known to be expressed in the thymus, the nature and in vivo localization of FasL-expressing cells have not been determined. Using recently developed anti-FasL Abs in combination with in situ hybridization on tissue sections, we show in this work that FasL-expressing cells are present in the thymus, particularly within the medulla. FasL mRNA was detected readily in thymic stromal cell extracts, but not in isolated thymocytes. Moreover, immunohistochemical analysis of serial tissue sections stained with Abs against FasL in conjunction with epithelial and dendritic cell markers indicated that both thymic epithelial and dendritic cells express FasL in situ. The coexistence of FasL-expressing stromal cells and Fas-expressing thymocytes may have important implications for the role of the Fas pathway in apoptosis associated with thymic selection events.

The in vivo performance of magnetic particle-loaded injectable, in situ gelling, carriers for the delivery of local hyperthermia.

We investigated the use of in situ implant formation that incorporates superparamagnetic iron oxide nanoparticles (SPIONs) as a form of minimally invasive treatment of cancer lesions by magnetically induced local hyperthermia. We developed injectable formulations that form gels entrapping magnetic particles into a tumor. We used SPIONs embedded in silica microparticles to favor syringeability and incorporated the highest proportion possible to allow large heating capacities. Hydrogel, single-solvent organogel and cosolvent (low-toxicity hydrophilic solvent) organogel formulations were injected into human cancer tumors xenografted in mice. The thermoreversible hydrogels (poloxamer, chitosan), which accommodated 20% w/v of the magnetic microparticles, proved to be inadequate. Alginate hydrogels, however, incorporated 10% w/v of the magnetic microparticles, and the external gelation led to strong implants localizing to the tumor periphery, whereas internal gelation failed in situ. The organogel formulations, which consisted of precipitating polymers dissolved in single organic solvents, displayed various microstructures. A 8% poly(ethylene-vinyl alcohol) in DMSO containing 40% w/v of magnetic microparticles formed the most suitable implants in terms of tumor casting and heat delivery. Importantly, it is of great clinical interest to develop cosolvent formulations with up to 20% w/v of magnetic microparticles that show reduced toxicity and centered tumor implantation.

In-situ revascularisation for secondary aorto-enteric fistulae: the success of silver-coated Dacron is closely linked to a suitable bowel repair.

OBJECTIVES: The purpose of this study was to assess short- and mid-term results of in-situ revascularisation (ISR) using silver-coated Dacron prostheses and bowel repair for management of secondary aorto-enteric fistulae (SAEF). DESIGN: Single-centre retrospective chart review. MATERIAL AND METHODS: This study includes all the patients treated by ISR using silver-coated Dacron for SAEF between 2006 and 2010. Primary end points were mortality and survival rates. Secondary end points were reinfection-free survival and secondary patency rates. RESULTS: Eighteen male patients with SAEF with a median age of 64 years were operated by ISR using silver-coated Dacron during the study period without operative death. The 30-day mortality was 22% and the in-hospital mortality rate was 39%. Indeed, during hospitalisation, a duodenal leak was observed in four patients including three who died. Four others patients died due to multi-system organ failure. Median follow-up was 16 months (range 1-66). The survival rate at 12 months was 55%. One duodenal leak was observed leading to death. The reinfection-free survival and the secondary patency rates at 12 months were 60% and 89%, respectively. CONCLUSION: In-situ revascularisation with silver-coated Dacron provides acceptable results in terms of mortality. This treatment may be useful for simple vascular reconstruction and allow greater attention to bowel repair that is a determinant in short- and mid-term survival.

Carcinomes canalaires in situ: données anatomo-pathologiques actuelles [Ductal carcinoma in situ: current anatomo-pathologic data]

Ductal carcinoma in situ (DCIS), accounting for 15-25% of all breast cancers, is frequently diagnosed by mammographic examination. This heterogeneous disease requires a rigorous local treatment based, in about two-third of cases, on conservative surgery and radiotherapy. DCIS are currently classified on the basis of nuclear grade. Most lesions, and especially high nuclear grade DCIS, are limited to one quadrant. Micropapillary DCIS are likely to be of larger size/extent and thus a conservative approach is often difficult. A careful pathological examination of an oriented excisional biopsy is a pre-requisite for optimal therapy.

Differential distribution of two microtubule-associated proteins, MAP2 and MAP5, during chick dorsal root ganglion development in situ and in culture.

Microtubule-associated proteins (MAPs) are essential components necessary for the early growth process of axons and dendrites, and for the structural organization within cells. Both MAP2 and MAP5 are involved in these events, MAP2 occupying a role predominantly in dendrites, and MAP5 being involved in both axonal and dendritic growth. In the chick dorsal root ganglia, pseudo-unipolar sensory neurons have a T-shaped axon and are devoid of any dendrites. Therefore, they offer an ideal model to study the differential expression of MAPs during DRG development, specifically during axonal growth. In this study we have analyzed the expression and localization of MAP2 and MAP5 isoforms during chick dorsal root ganglia development in vivo, and in cell culture. In DRG, both MAPs appeared as early as E5. MAP2 consists of the 3 isoforms MAP2a, b and c. On blots, no MAP2a could be found at any stage. MAP2b increased between E6 and E10 and thereafter diminished slowly in concentration, while MAP2c was found between stages E6 and E10 in DRG. By immunocytochemistry, MAP2 isoforms were mainly located in the neuronal perikarya and in the proximal portion of axons, but could not be localized to distal axonal segments, nor in sciatic nerve at any developmental stage. On blots, MAP5 was present in two isoforms, MAP5a and MAP5b. The concentration of MAP5a was highest at E6 and then decreased to a low level at E18. In contrast, MAP5b increased between E6 and E10, and rapidly decreased after E14. Only MAP5a was present in sciatic nerve up to E14. Immunocytochemistry revealed that MAP5 was localized mainly in axons, although neuronal perikarya exhibited a faint immunostaining. Strong staining of axons was observed between E10 and E14, at a time coincidental to a period of intense axonal outgrowth. After E14 immunolabeling of MAP5 decreased abruptly. In DRG culture, MAP2 was found exclusively in the neuronal perikarya and the most proximal neurite segment. In contrast, MAP5 was detected in the neuronal cell bodies and all along their neurites. In conclusion, MAP2 seems involved in the early establishment of the cytoarchitecture of cell bodies and the proximal axon segment of somatosensory neurons, while MAP5 is clearly related to axonal growth.

In situ stromal expression of the urokinase/plasmin system correlates with epithelial dysplasia in colorectal adenomas.

An increase of urokinase-type plasminogen activator (uPA) and a decrease of tissue-type PA (tPA) have been associated with the transition from normal to adenomatous colorectal mucosa. Serial sections from 25 adenomas were used to identify PA-related caseinolytic activities by in situ zymography, blocking selectively uPA or tPA. The distribution of uPA, tPA, and type 1 PA inhibitor mRNAs was investigated by nonradioactive in situ hybridization, and the receptor for uPA was detected by immunostaining. Low- and high-grade epithelial cell dysplasia was mapped histologically. Results show that 23 of 25 adenomas expressed uPA-related lytic activity located predominantly in the periphery whereas tPA-related activity was mainly in central areas of adenomas. In 15 of 25 adenomas, uPA mRNA was expressed in stromal cells clustered in foci that coincided with areas of uPA lytic activity. The probability of finding uPA mRNA-reactive cells was significantly higher in areas with high-grade epithelial dysplasia. uPA receptor was mainly stromal and expressed at the periphery. Type 1 PA inhibitor mRNA cellular expression was diffuse in the stroma, in endothelial cells, and in a subpopulation of alpha-smooth muscle cell actin-reactive cells. These results show that a stromal up-regulation of the uPA/plasmin system is associated with foci of severe dysplasia in a subset of colorectal adenomas.

Local moderate magnetically induced hyperthermia using an implant formed in situ in a mouse tumor model.

Purpose: We investigate a new heat delivery technique for the local treatment of solid tumors. The technique involves injecting a formulation that solidifies to form an implant in situ. This implant entraps superparamagnetic iron oxide nanoparticles (SPIONs) embedded in silica microbeads for magnetically induced moderate hyperthermia. Particle entrapment prevents phagocytosis and distant migration of SPIONs. The implant can be repeatedly heated by magnetic induction. Methods: We evaluated heating and treatment efficacies by means of thermometry and survival studies in nude mice carrying subcutaneous human colocarcinomas. At day 1, we injected the formulation into the tumor. At day 2, a single 20-min hyperthermia treatment was delivered by 141-kHz magnetic induction using field strengths of 9 to 12 mT under thermometry. Results: SPIONs embedded in silica microbeads were effectively confined within the implant at the injection site. Heat-induced necro-apoptosis was assessed by histology on day 3. On average, 12 mT resulted in tumor temperature of 47.8 degrees C, and over 70% tumor necrosis that correlated to the heat dose (AUC = 282 degrees C.min). In contrast, a 9-mT field strength induced tumoral temperature of 40 degrees C (AUC = 131 degrees C.min) without morphologically identifiable necrosis. Survival after treatment with 10.5 or 12 mT fields was significantly improved compared to non-implanted and implanted controls. Median survival times were 27 and 37 days versus 12 and 21 days respectively. Conclusion: Five of eleven mice (45%) of the 12 mT group survived one year without any tumor recurrence, holding promise for tumor therapy using magnetically induced moderate hyperthermia through injectable implants.

Evaluation of a PCR-RFLP assay for the identification of dermatophytes in situ

Dermatophytes are the main cause of superficial mycoses. These fungi have the capacity to invade keratinized tissue of humans or animals to produce infections that are generally restricted to the corneocytes of the skin, hair, and nails. Nevertheless, it is common to obtain negative results from fungal cultures of dermatological specimens where direct mycological examination showed fungal elements (30-40%). However, correct identification of the isolated dermatophytes from Tinea is important to choose the appropriate treatment. Therefore, we aim to develop a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on 28S rDNA that is able to identify dermatophytes species in positive dermatological samples. PCR-RFLP identification of dermatophytes in skin or hair allowed validation of the results obtained in culture. It was also possible to identify the infectious dermatophytes when direct hair/ skin mycological examination showed fungal elements, but negative results were obtained from fungal culture. As a conclusion, PCR methods may provide significant benefits in the rapid diagnosis of Tinea. First, there is an increase in sensitivity of dermatophytes identification when enough material is available. Secondly, identification of the infecting agent can be obtained in 24 hours with PCR-RFLP or sequencing, whereas results from fungal cultures can take 2-3 weeks.

MOLECULAR REORGANIZATION FOLLOWING SENSORY STIMULATION IN THE ADULT MOUSE BARREL CORTEX

Sensory information is an important factor in shaping neuronal circuits during development and adulthood. In the barrel cortex of adult rodents, cells from layer IV are able to adapt their functional state to an increased flow of sensory information from the mystacial whisker follicles. Previous studies in our group have shown that whisker stimulation induces the formation of inhibitory synapses in the corresponding barrel (Knott et al., 2002) and decreases neuronal responses toward the deflection of the stimulated whisker (Quairiaux et al., 2007). Together these observations have turned the barrel cortex into a model to study homeostatic plasticity. At the cellular level, neuronal activity triggers intracellular signaling cascades leading to a transcriptional response. To further characterize the molecular pathways involved in the synaptic changes after whisker stimulation in the adult mouse, a previous doctoral student in our group performed a microarray analysis on laser-dissected barrels in sections through layer IV. This study identified the regulation (up and down) of a series of genes in the stimulated barrels (thesis of Johnston-Wenger, 2010). We here focused on ten genes that presented the highest fold change according to the microarray analysis. Out of these genes, 7 are known as neuronal activity-dependent genes (Tnncl, Nptx2, Sorcs3, Ptgs2, Nr4a2, Npas4 and Adcyapl) whereas three have so far not been related to neuronal plasticity (Scn7a, Pcdhl5 and Cede3). The study aimed at confirming the results of the microarray analysis and localizing molecular modifications in the stimulated barrel column at the cellular level. In situ hybridization for Pcdhl5 after different periods of whisker stimulation (3, 6, 9, 15, 24 hrs) allowed us to confirm that the 1.25 fold change used for the microarray analysis is an appropriate threshold for considering a regulation significant after sensory-stimulation. Moreover, we confirmed with in situ hybridization a significant upregulation of the genes of interest in the stimulated barrels. In situ hybridization and immunohistochemistry allowed us to observe the distribution of the genes of interest and the corresponding protein products at the cellular level. Three observations were made: 1) alterations of the expression was restricted to the stimulated barrels for all genes tested; 2) within a barrel column not all cells responded to whisker stimulation with an altered gene expression; 3) in the stimulated barrels, two different patterns of mRNA and protein expression can be distinguished. We hypothesize that this segregation of the activity-induced gene expression reflects the segregation of the two principal thalamocortical pathways conveying the sensory information to the barrel cortex. Moreover, only neurons reaching the critical threshold will modify their gene expression program resulting in structural as well as physiological modifications that prevent the subsequent propagation of the excess of excitation to the postsynaptic targets. The activity-induced gene expression is therefore adapted in a cell-type-specific manner to induce a homeostatic response to the entire neuronal network involved in the integration of the sensory information. This to our knowledge the first study showing the distinct, but complementary contribution of the two thalamocortical pathways in experience-dependent plasticity in the adult mouse barrel cortex. -- L'information sensorielle nous permet de continuellement façonner nos circuits neuronaux autant durant le développement qu'à l'âge adulte. Chez le rongeur l'information sensorielle perçue par les vibrisses est intégrée au niveau du cortex somatosensoriel primaire (appelé en anglais « barrel cortex ») dont les cellules de la couche IV sont capables d'adapter leur état fonctionnel en réponse à une augmentation d'activité neuronale. Ce modèle expérimental a permis à notre groupe de recherche d'observer des changements rapides du circuit neuronal en fonction de l'activité sensorielle. En effet, la stimulation continue d'une vibrisse d'une souris adulte pendant 24 heures induit non seulement un remaniement synaptique (Knott et al., 2002), mais également des changements physiologiques au niveau des neurones du tonneau correspondant (Quairiaux et al., 2007). Ces observations nous permettent d'affirmer que le « barrel cortex » est un modèle approprié pour y étudier la plasticité synaptique. Au niveau cellulaire, l'activité neuronale déclenche des cascades de signalisation intracellulaire résultant en une réponse transcriptionnelle. Afin de caractériser les voies moléculaires impliquées dans la plasticité synaptique, une puce à ARN nous a permis de comparer l'expression de gènes entre un tonneau correspondant à une vibrisse stimulée et un tonneau d'une vibrisse non-stimulée (Nathalie). Cette analyse a révélé un certain nombre de gènes régulés de manière positive ou négative par l'augmentation de l'activité neuronale. Nous nous sommes concentrés sur 10 gènes dont l'expression est fortement régulée. L'expression de sept d'entre eux a déjà été démontrée comme dépendante de l'activité neuronale (Tnncl, Nptx2, Sorcs3, Ptgs2, Nr4a2, Npas4 otAdcyapl) alors que l'expression des trois autres (Scn7a, Pcdhl5 et Cedei) n'a pour le moment pas encore été liée à la plasticité neuronale. Le but de cette thèse est de confirmer les résultats de la puce à ARN et de déterminer dans quel type cellulaire ces gènes sont exprimés. L'hybridation in situ pour le gène Pcdhl5, après différentes périodes de stimulation des vibrisses (3, 6, 9, 15 et 24 heures), nous a permis de confirmer que le seuil de 1.25x utilisé dans l'analyse de la puce à ARN est approprié pour considérer qu'un gène est régulé de manière significative par la stimulation sensorielle. Nous avons également pu confirmer à l'aide de cette technique que la stimulation sensorielle augmente significativement l'expression de ces dix gènes. L'expression de ces gènes au niveau cellulaire a été observée à l'aide des techniques d'hybridation in situ et d'immunohistochimie. Trois observations ont été faites : 1) la régulation de ces gènes est restreinte aux tonneaux correspondants aux vibrisses stimulées ; 2) au niveau d'une colonne corticale correspondant aux vibrisses stimulées, seules certaines cellules présentent une altération de leur expression génique ; 3) au niveau des tonneaux stimulés, deux profils d'expression d'ARNm et de protéines sont observés. Notre hypothèse est que cette distribution pourrait correspondre à la terminaison ségrégée des deux voies thalamocortical qui amènent l'information sensorielle dans le cortex cérébral. De plus, seul les neurones atteignant le seuil critique d'activation modifient leur expression génique en réponse à la stimulation sensorielle. Ces changements d'expression géniques vont permettre à la cellule de modifier ses propriétés structurales et physiologiques de manière a prevenir la propagation d'un excès d'activité neuronale au niveau de ses cibles postsynaptics. L'activité neuronale agit donc spécifiquement sur certains types cellulaires de maniere a induire une réponse homéostatique au niveau du réseau neuronal impliqué dans l'integration de l'information sensorielle. Nos travaux démontrent pour une première fois que les deux voies sensorielles contribuent d'une manière distincte et complémentaire à la plasticité corticale induite par un changement de l'activité sensorielle chez la souris adulte.

Using flow cytometry for in situ monitoring of antimicrobial compound production in the biocontrol bacterium Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens strain CHA0 is able to protect plants against a variety of pathogens, notably by producing the two antimicrobial compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the expression of these compounds is affected by many biotic factors, such as fungal pathogens, rhizosphere bacteria as well as plant species. Therefore, the influence of some plant phenolic compounds on the expression of DAPG and PLT biosynthetic genes has been tested using GFP-based reporter, monitored by standard fluometry and flow cytometry. In situ experiments were also performed with cucumber plants. We found that several plant metabolites such as IAA and umbelliferone are able to modify significantly the expression of DAPG and PLT. The use of flow cytometry with autofluorescents proteins seems to be a promising method to study rhizobacteria-plant interactions.