Fluorescence in situ hybridization of potato somatohaploids and their somatic hybrid donors using two Solanum brevidens specific sequences

Selostus: Perunan somaattisten hybridien ja niiden somatohaploidien fluoresenssi in situ -hybridisaatio Solanum brevidens -lajin spesifisten sekvenssien avulla

Clinical assessment of fluorescence cytoscopy during transurethral bladder resection in superficial bladder cancer.

The prognosis of superficial bladder cancer in terms of recurrence and disease progression is related to bladder tumor multiplicity and the presence of concomitant "plane" tumors such as high-grade dysplasia and carcinoma in situ. This study in 33 patients aimed to demonstrate the role of fluorescence cystoscopy in transurethral resection of superficial bladder cancer. The method is based on the detection of protoporphyrin-IX-induced fluorescence in urothelial cancer cells by topical administration of 5-aminolevulinic acid. The sensitivity and the specificity of this procedure on apparently normal mucosa in superficial bladder cancer are estimated to be 82.9% and 81.3%, respectively. Thus, fluorescence cytoscopy is a simple and reliable method for mapping the bladder mucosa, especially in the case of multifocal bladder disease, and it facilitates the screening of occult dysplasia.

Concrete Pavement Mixture Design and Analysis (MDA): Application of a Portable X-Ray Fluorescence Technique to Assess Concrete Mix Proportions , TPF-5(205), 2012

Any transportation infrastructure system is inherently concerned with durability and performance issues. The proportioning and uniformity control of concrete mixtures are critical factors that directly affect the longevity and performance of the portland cement concrete pavement systems. At present, the only means available to monitor mix proportions of any given batch are to track batch tickets created at the batch plant. However, this does not take into account potential errors in loading materials into storage silos, calibration errors, and addition of water after dispatch. Therefore, there is a need for a rapid, cost-effective, and reliable field test that estimates the proportions of as-delivered concrete mixtures. In addition, performance based specifications will be more easily implemented if there is a way to readily demonstrate whether any given batch is similar to the proportions already accepted based on laboratory performance testing. The goal of the present research project is to investigate the potential use of a portable x-ray fluorescence (XRF) technique to assess the proportions of concrete mixtures as they are delivered. Tests were conducted on the raw materials, paste and mortar samples using a portable XRF device. There is a reasonable correlation between the actual and calculated mix proportions of the paste samples, but data on mortar samples was less reliable.

Thrombin-sensitive dual fluorescence imaging and therapeutic agent for detection and treatment of synovial inflammation in murine rheumatoid arthritis.

We have developed a thrombin-sensitive polymeric photosensitizer prodrug (T-PS) to selectively image and eradicate inflammatory lesions in rheumatoid arthritis (RA). Thrombin is a serine protease up-regulated in synovial tissues of rheumatoid arthritis (RA) patients. T-PS consists of a polymeric backbone, to which multiple photosensitizer (PS) units are tethered via short thrombin-cleavable peptide linkers. Fluorescence emission and phototoxicity of the prodrug are efficiently quenched due to the interaction of neighboring photosensitizer units. The prodrug is passively delivered to the inflammation site via the enhanced permeability and retention (EPR) effect. Subsequent site-selective proteolytic cleavage of the peptide linkers restores its photoactivity by increasing the mutual distance between PS. Whole animal imaging in murine collagen-induced arthritis, an experimental model of RA revealed a dose-dependent fluorescence increase in arthritic paws after systemic prodrug injection. In addition, administration of T-PS resulted in much higher fluorescence selectivity for arthritic joints as compared to the free PS. Irradiation of the arthritic joints induced light dose dependent phototoxic effects such as apoptosis, vascular damage and local hemorrhage. Long-term observations showed complete regression of the latter. Irradiated non-arthritic tissues or non-irradiated arthritic tissues showed no histological effects after photodynamic therapy with T-PS. This illustrates that T-PS can localize inflammatory lesions with excellent selectivity and induce apoptosis and vascular shut down after irradiation.

Correlation of photodynamic activity and fluorescence signaling for free and pegylated mTHPC in mesothelioma xenografts

Rapport de synthèse : Introduction : Les premières applications cliniques de la thérapie photodynamique (PDT) remontent à plus d'une vingtaine d'années. Basée sur l'activation d'un médicament photosensibilisateur par une source lumineuse à une longueur d'onde spécifique, la PDT permet la destruction sélective de tissus contenant le produit actif. Ce procédé a été expérimenté dans le traitement de cancers en raison de la propriété du médicament à se concentrer dans les tumeurs tout en épargnant les structures normales contigües. Cependant, les photosensibilisateurs utilisés jusqu'à ce jour n'ont pas démontré une accumulation exclusive dans les tissus néoplasiques mais également dans les structures saines avoisinantes induisant une destruction tissulaire non sélective. Notamment, d'importantes complications ont été rapportées suite à l'utilisation de la PDT dans la cavité thoracique après la résection de mésothéliomes pleuraux, et ce malgré l'arrivée de photosensibilisateurs de secondes générations. De ce fait, plusieurs études expérimentales ont été menées afin d'améliorer la sélectivité tumorale du médicament en modulant différentes conditions de traitement et en modifiant la structure du photosensibilisateur par pégylation. Le but de cette étude expérimentale est de corréler l'activité photodynamique, la phototoxicité et la distribution du m-tetrahydroxyphenylchlorin (mTHPC) et de sa forme pégylée, le PEG-mTHPC. De ce fait, un modèle de souris nues porteur de xenogreffes de mésothéliome humain a été utilisé pour étudier les deux photosensibilisateurs. De récents travaux avec ce modèle ont montré que la mesure de la concentration tissulaire du mTHPC et de sa forme pégylée par HPLC restait limitée afin de prédire l'activité photodynamique. De ce fait, nous pensons que les mesures de fluorescence peuvent être plus appropriée. Le signalement fluorescent est mesuré dans le tissu tumoral et dans une région contrôle de la peau afin d'étudier la distribution et l'intensité des deux sensibilisateurs. Méthode : Des souris nues (cd1nu/nu mice) de 8 semaines ont été transplantées avec des fragments de mésothéliome malin humain (H-meso-1). Ces derniers ont été obtenus à partir d'une suspension cellulaire. Au moins trois passages ont été faits dans les animaux, avant que le traitement soit initié. Deux groupes de 6 souris chacun ont été utilisés pour l'injection intraveineuse par la queue du mTHPC à 0.15 mg/kg et du PEG-mTHPC à dose équimolaire. Après trois jour, la tumeur ainsi qu'une région contrôle de la cuisse ont été illuminées sur une surface d'un diamètre de 1.2 cm et pendant 133 secondes avec un laser à une longueur d'onde à 652 nm (fluence 20 J/cm2, fluence rate 150 mW/cm2). Les animaux ont été ensuite sacrifiés 72 heures après l'illumination. L'étendue de la nécrose tumorale et de la région contrôle ont été déterminées en aveugle par histomorphometrie par un pathologue (HJA). La fluorescence microscopique a été évaluée dans 12 souris à une concentration de 0.15 et 0.5 mg/kg pour le mTHPC, et à doses équimolaires pour le PEG-mTHPC. Trois animaux ont été injectés avec le mTHPC à 0.15 mg/kg, 3 autres à dose équimolaire avec la forme pégylée et 6 souris avec le mTHPC à 0.5 mg/kg et à dose équimolaire. Les animaux ont été sacrifiés 72 heures après injection. L'intensité fluorescente des sensibilisateurs a été mesurée dans la tumeur et la région contrôle. Suite à cela, les coupes ont été fixées par H&E et superposées aux images fluorescentes, afin de localiser la distribution des deux photosensibilisateurs dans les différents compartiments tissulaires. Six souris transplantées n'ayant ni été injectées avec les sensibilisateurs ou illuminées ont servi de groupe contrôle. Résultats : Trois jours après l'illumination, la PDT provoque une nécrose tumorale de 10 ±5.4 mm2 pour le mTHPC à 0.15mg/kg et 5.2 ± 4.6 mm2 pour sa forme pégylée à dose équimolaire. Cependant, la nécrose tumorale induite par les deux formulations du sensibilisateur est significativement plus élevée que dans le groupe contrôle (0.33 ± 0.58 mm2) (P=0.02). Toutefois, le mTHPC pégylé provoque une photosensibilité cutanée moins importante que la forme non-pegylée. Dans les deux groupes, aucune nécrose n'a été observée dans la cuisse des animaux. Trois jours après l'injection du mTHPC et de la forme pégylée à 0.15 mg/kg, aucune activité fluorescente n'a été détectée. Cependant, à 0.5 mg/kg, la fluorescence microscopique révèle une distribution hétérogène des deux photo-sensibilisateurs dans le tissu tumoral avec une accumulation prédominante dans les régions peri-vasculaires. Les deux médicaments montrent une distribution intracellulaire homogène dans le cytoplasme et une absence de signalement dans le nucleus. La mesure de l'intensité fluorescente du mTHPC à 0.5mg/kg ne montre pas de différence significative entre le tissu tumoral et la région contrôle. Par contre, le PEG-mTHPC montre une intensité fluorescente supérieure dans le tissu tumoral que dans la peau (ratio tumeur- peau 0.94 pour le mTHPC et 1.73 pour le PEG-mTHPC). Conclusion : L'utilisation du mTHPC à 0.15mg/kg induit une nécrose tumorale similaire à celle du PEG-mTHPC à dose équimolaire. Cependant, ce dernier démontre une photo-toxicité plus atténuée de la peau. La fluorescence microscopique permet de localiser les deux sensibilisateurs dans les différents compartiments tissulaires à partir d'une dose de 0.5 mg/kg. Le PEG-mTHPC induit un signalement fluorescent supérieur dans le tissu tumoral par rapport à la peau. La mesure du signalement fluorescent a le potentiel de prédire l'activité photodynamique du mTHPC et de sa forme pégylée dans les xénogreffes de mésothéliome humain dans un modèle de souris nue.

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.

Fluorescence-activated cell sorting and cloning of bona fide CD8+ CTL with reversible MHC-peptide and antibody Fab' conjugates.

The isolation of subsets of Ag-specific T cells for in vitro and in vivo studies by FACS is compromised by the fact that the soluble MHC-peptide complexes and Abs used for staining, especially when combined, induce unwanted T cell activation and eventually apoptosis. This is especially a problem for CD8+ CTL, which are susceptible to activation-dependent cell death. In this study, we show that reversible MHC-peptide complexes (tetramers) can be prepared by conjugating MHC-peptide monomers with desthiobiotin (DTB; also called dethiobiotin) and multimerization by reaction with fluorescent streptavidin. While in the cold these reagents are stable and allow good staining, they rapidly dissociate in monomers at elevated temperatures, especially in the presence of free biotin. FACS cloning of Melan-A (MART-1)-specific CTL from a melanoma-infiltrated lymph node with reversible HLA-A2 Melan-A26-35 multimers yielded over two times more clones than when using the conventional biotin-containing multimers. CTL clones obtained by means of reversible multimers killed Melan-A-positive tumor cells more efficiently as compared with clones obtained with the stable multimers. Among the CTL obtained with the reversible multimers, but much less among those obtained with the stable multimers, a high proportion of clones exhibited high functional and physical avidity and died upon incubation with soluble MHC-peptide complexes. Finally, we show that Fab' of an anti-CD8 Ab can be converted in reversible DTB streptavidin conjugates the same way. These DTB reagents efficiently and reversibly stained murine and human CTL without affecting their viability.

Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations.

BACKGROUND: This study validates the use of phycoerythrin (PE) and allophycocyanin (APC) for fluorescence energy transfer (FRET) analyzed by flow cytometry. METHODS: FRET was detected when a pair of antibody conjugates directed against two noncompetitive epitopes on the same CD8alpha chain was used. FRET was also detected between antibody conjugate pairs specific for the two chains of the heterodimeric alpha (4)beta(1) integrin. Similarly, the association of T-cell receptor (TCR) with a soluble antigen ligand was detected by FRET when anti-TCR antibody and MHC class I/peptide complexes (<<tetramers>>) were used. RESULTS: FRET efficiency was always less than 10%, probably because of steric effects associated with the size and structure of PE and APC. Some suggestions are given to take into account this and other effects (e.g., donor and acceptor concentrations) for a better interpretation of FRET results obtained with this pair of fluorochromes. CONCLUSIONS: We conclude that FRET assays can be carried out easily with commercially available antibodies and flow cytometers to study arrays of multimolecular complexes.

Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood.

To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.

In situ fluorescence imaging of glutamate-evoked mitochondrial Na+ responses in astrocytes.

Astrocytes can experience large intracellular Na+ changes following the activation of the Na+-coupled glutamate transport. The present study investigated whether cytosolic Na+ changes are transmitted to mitochondria, which could therefore influence their function and contribute to the overall intracellular Na+ regulation. Mitochondrial Na+ (Na+(mit)) changes were monitored using the Na+-sensitive fluorescent probe CoroNa Red (CR) in intact primary cortical astrocytes, as opposed to the classical isolated mitochondria preparation. The mitochondrial localization and Na+ sensitivity of the dye were first verified and indicated that it can be safely used as a selective Na+(mit) indicator. We found by simultaneously monitoring cytosolic and mitochondrial Na+ using sodium-binding benzofuran isophthalate and CR, respectively, that glutamate-evoked cytosolic Na+ elevations are transmitted to mitochondria. The resting Na+(mit) concentration was estimated at 19.0 +/- 0.8 mM, reaching 30.1 +/- 1.2 mM during 200 microM glutamate application. Blockers of conductances potentially mediating Na+ entry (calcium uniporter, monovalent cation conductances, K+(ATP) channels) were not able to prevent the Na+(mit) response to glutamate. However, Ca2+ and its exchange with Na+ appear to play an important role in mediating mitochondrial Na+ entry as chelating intracellular Ca2+ with BAPTA or inhibiting Na+/Ca2+ exchanger with CGP-37157 diminished the Na+(mit) response. Moreover, intracellular Ca2+ increase achieved by photoactivation of caged Ca2+ also induced a Na+(mit) elevation. Inhibition of mitochondrial Na/H antiporter using ethylisopropyl-amiloride caused a steady increase in Na+(mit) without increasing cytosolic Na+, indicating that Na+ extrusion from mitochondria is mediated by these exchangers. Thus, mitochondria in intact astrocytes are equipped to efficiently sense cellular Na+ signals and to dynamically regulate their Na+ content.

Enhanced-reality video fluorescence: a real-time assessment of intestinal viability.

OBJECTIVE: Our aim was to evaluate a fluorescence-based enhanced-reality system to assess intestinal viability in a laparoscopic mesenteric ischemia model. MATERIALS AND METHODS: A small bowel loop was exposed, and 3 to 4 mesenteric vessels were clipped in 6 pigs. Indocyanine green (ICG) was administered intravenously 15 minutes later. The bowel was illuminated with an incoherent light source laparoscope (D-light-P, KarlStorz). The ICG fluorescence signal was analyzed with Ad Hoc imaging software (VR-RENDER), which provides a digital perfusion cartography that was superimposed to the intraoperative laparoscopic image [augmented reality (AR) synthesis]. Five regions of interest (ROIs) were marked under AR guidance (1, 2a-2b, 3a-3b corresponding to the ischemic, marginal, and vascularized zones, respectively). One hour later, capillary blood samples were obtained by puncturing the bowel serosa at the identified ROIs and lactates were measured using the EDGE analyzer. A surgical biopsy of each intestinal ROI was sent for mitochondrial respiratory rate assessment and for metabolites quantification. RESULTS: Mean capillary lactate levels were 3.98 (SD = 1.91) versus 1.05 (SD = 0.46) versus 0.74 (SD = 0.34) mmol/L at ROI 1 versus 2a-2b (P = 0.0001) versus 3a-3b (P = 0.0001), respectively. Mean maximal mitochondrial respiratory rate was 104.4 (±21.58) pmolO2/second/mg at the ROI 1 versus 191.1 ± 14.48 (2b, P = 0.03) versus 180.4 ± 16.71 (3a, P = 0.02) versus 199.2 ± 25.21 (3b, P = 0.02). Alanine, choline, ethanolamine, glucose, lactate, myoinositol, phosphocholine, sylloinositol, and valine showed statistically significant different concentrations between ischemic and nonischemic segments. CONCLUSIONS: Fluorescence-based AR may effectively detect the boundary between the ischemic and the vascularized zones in this experimental model.

Feasibility of direct mapping of cerebral fluorodeoxy-D-glucose metabolism in situ at subcellular resolution using soft X-ray fluorescence.

Glucose metabolism is difficult to image with cellular resolution in mammalian brain tissue, particularly with (18) fluorodeoxy-D-glucose (FDG) positron emission tomography (PET). To this end, we explored the potential of synchrotron-based low-energy X-ray fluorescence (LEXRF) to image the stable isotope of fluorine (F) in phosphorylated FDG (DG-6P) at 1 μm(2) spatial resolution in 3-μm-thick brain slices. The excitation-dependent fluorescence F signal at 676 eV varied linearly with FDG concentration between 0.5 and 10 mM, whereas the endogenous background F signal was undetectable in brain. To validate LEXRF mapping of fluorine, FDG was administered in vitro and in vivo, and the fluorine LEXRF signal from intracellular trapped FDG-6P over selected brain areas rich in radial glia was spectrally quantitated at 1 μm(2) resolution. The subsequent generation of spatial LEXRF maps of F reproduced the expected localization and gradients of glucose metabolism in retinal Müller glia. In addition, FDG uptake was localized to periventricular hypothalamic tanycytes, whose morphological features were imaged simultaneously by X-ray absorption. We conclude that the high specificity of photon emission from F and its spatial mapping at ≤1 μm resolution demonstrates the ability to identify glucose uptake at subcellular resolution and holds remarkable potential for imaging glucose metabolism in biological tissue. © 2012 Wiley Periodicals, Inc.