Utility of sarcoma-specific fusion gene analysis in paraffin-embedded material for routine diagnosis at a specialist centre.

AIMS: Diagnosis of soft tissue sarcomas can be difficult. It can be aided by detection of specific genetic aberrations in many cases. This study assessed the utility of a molecular genetics/cytogenetics service as part of the routine diagnostic service at the Royal Marsden Hospital. METHODS: A retrospective audit was performed over a 15-month period to evaluate the diagnostic usefulness for soft tissue sarcomas with translocations of fluorescence in situ hybridisation (FISH) and reverse-transcriptase PCR (RT-PCR) in paraffin-embedded (PE) material. Results were compared with histology, and evaluated. RESULTS: Molecular investigations were performed on PE material in 158 samples (total 194 RT-PCR and 174 FISH tests), of which 85 were referral cases. Synovial sarcoma, Ewing sarcoma and low-grade fibromyxoid sarcoma were the most commonly tested tumours. Myxoid liposarcoma showed the best histological and molecular concordance, and alveolar rhabdomyosarcoma showed the best agreement between methods. FISH had a higher sensitivity for detecting tumours (73%, compared with 59% for RT-PCR) with a better success rate than RT-PCR, although the latter was specific in identifying the partner gene for each fusion. In particular, referral blocks in which methods of tissue fixation and processing were not certain resulted in higher RT-PCR failure rates. CONCLUSIONS: FISH and RT-PCR on PE tissue are practical and effective ancillary tools in the diagnosis of soft tissue sarcomas. They are useful in confirming doubtful histological diagnoses and excluding malignant diagnoses. PCR is less sensitive than FISH, and the use of both techniques is optimal for maximising the detection rate of translocation-positive sarcomas.

Low frequency of the TEL/AML1 fusion gene in acute lymphoblastic leukaemia in Spain.

We report on a series of Spanish patients with acute lymphoblastic leukaemia in whom the t(12;21) [TEL/AML1] translocation could not be identified with two sensitive techniques: reverse transcript-polymerase chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH). 101 cases were analysed: 38 children (29 B-cell precursor; nine T-cell precursor) and 63 adults (48 B-cell precursor; 15 T-cell precursor). Specific RT-PCR to amplify the TEL/AML1 fusion transcript was negative in all 101 cases. Moreover, all 38 paediatric samples were also negative by interphase FISH analysis for the presence of the TEL/AML1 fusion. These results suggest the existence of geographic/race variations in the genotype of acute lymphoblastic leukaemia (ALL).

Herceptest: HER2 expression and gene amplification in non-small cell lung cancer

HER2 is an erbB/HER type I tyrosine kinase receptor that is frequently over-expressed in malignant epithelial tumours. Herceptin, a humanised mouse monoclonal antibody to HER2, is proven therapeutically in the management of metastatic breast cancer, significantly prolonging survival when combined with cytotoxic chemotherapeutic agents. Immunohistochemical studies suggest that non-small-cell lung cancer (NSCLC) tumours may over-express HER2. Our aim was to evaluate HER2 gene amplification and semi-quantitative immuno-expression in NSCLC. A total of 344 NSCLC cases were immunostained for HER2 expression in 2 centres using the HercepTest. Fluorescence in situ hybridisation (FISH) analysis for HER2 gene amplification was performed on most positive cases and a subset of negative cases. Fifteen cases (4.3%) demonstrated 2+ or 3+ membranous HER2 immuno-expression. There was no correlation between immuno-expression and tumour histology or grade. Tumours from higher-stage disease were more often HercepTest-positive (p < 0.001). All 4 HercepTest 3 + cases demonstrated gene amplification. One of the 5 2+ cases tested for gene amplification showed areas of borderline amplification and areas of polyploidy. None of the 19 HercepTest-negative cases demonstrated gene amplification or polyploidy (p < 0.001). Gene amplification was demonstrated in all HercepTest 3+ scoring NSCLC cases. Unlike breast cancer, gene amplification and HER2 protein over-expression assessed by the HercepTest appeared to be uncommon in NSCLC. Herceptin may therefore target only a small proportion of NSCLC tumours and be of limited clinical value in this disease, particularly in the adjuvant setting. © 2001 Wiley-Liss, Inc.

Visualization of in situ intracellular aggregation of two cataract-associated human gamma-crystallin mutants: Lose a tail, lose transparency

PURPOSE. To understand the molecular features underlying autosomal dominant congenital cataracts caused by the deletion mutations W156X in human gamma D-crystallin and W157X in human gamma C-crystallin. METHODS. Normal and mutant cDNAs (with the enhanced green fluorescent protein [EGFP] tag in the front) were cloned into the pEGFP-C1 vector, transfected into various cell lines, and observed under a confocal microscope for EGFP fluorescence. Normal and W156X gamma D cDNAs were also cloned into the pET21a(+) vector, and the recombinant proteins were overexpressed in the BL-21(DE3) pLysS strain of Escherichia coli, purified, and isolated. The conformational features, structural stability, and solubility in aqueous solution of the mutant protein were compared with those of the wild type using spectroscopic methods. Comparative molecular modeling was performed to provide additional structural information. RESULTS. Transfection of the EGFP-tagged mutant cDNAs into several cell lines led to the visualization of aggregates, whereas that of wild-type cDNAs did not. Turning to the properties of the expressed proteins, the mutant molecules show remarkable reduction in solubility. They also seem to have a greater degree of surface hydrophobicity than the wild-type molecules, most likely accounting for self-aggregation. Molecular modeling studies support these features. CONCLUSIONS. The deletion of C-terminal 18 residues of human gamma C-and gamma D-crystallins exposes the side chains of several hydrophobic residues in the sequence to the solvent, causing the molecule to self-aggregate. This feature appears to be reflected in situ on the introduction of the mutants in human lens epithelial cells.

Directing role of functional groups in selective generation of C-H center dot center dot center dot pi interactions: In situ cryo-crystallographic studies on benzyl derivatives

The in situ cryo-crystallization study of benzyl derivatives reveals that the molecular packing in these compounds is either through methylene (sp(3)) C-H center dot center dot center dot pi or aromatic (sp(2)) C-H center dot center dot center dot pi interactions depending on the level of acidity of the benzyl proton. These studies of low melting compounds bring out the subtle features of such weak interactions and point to the directional preferences depending on the nature (electron withdrawing, polarizability) of the neighbouring functional group.

An electron spectroscopic study of the adsorption of nitrogen on a Ni/TiO2 catalyst prepared in situ in the spectrometer: evidence for dissociative adsorption in the strong-metal-support-interaction (SMSI) state

Nitrogen is dissociatively adsorbed on an annealed Ni/TiO2 surface just as on a Ti–Ni alloy surface while it is molecularly adsorbed on a Ni/Al2O3 surface.

Susceptibility of sperm chromatin to acid denaturation in situ: A study in endogenous FSH-deprived adult male bonnet monkeys (Macaca radiata)

Acid denaturation of calf thymus DNA in vitro followed by acridine orange (AO) binding induced a 112% increase in the emission of red, a 58% decrease in green, and a consequential decrease in the ratio of green:red fluorescences from 1.7 to 0.9. This metachromatic property of AO on binding to DNA following acid denaturation was utilized to study the susceptibility of normal and ovine follicle-stimulating hormone (oFSH) actively immunized bonnet monkey spermatozoa voided throughout the year. For analyses, the scattergram generated by the emission of red and green fluorescences by 10,000 AO-bound sperm from each semen sample was divided into 4 quadrant zones representing percentage cells fluorescing high green-low red (Q1), high green-high red (Q2), low green-low red (Q3) and low green-high red. (Q4). Normal monkey sperm obtained during the months of July-December exhibited 76, 13, and 11% cells in Q2, Q3, and Q4 quadrants, respectively. However, during January-June, when the females of the species are markedly subfertile, noncycling, and amenorrhoeic, the spermatozoa ejaculated by the male monkeys exhibited 38, 39, and 23% sperm in Q2, Q3, and Q4, respectively, the differences being highly significant (p < .01-.001). FSH deprivation induced significant shifts in fluorescence emissions, from respective controls, with 39, 33, and 28% cells in Q2, Q3, and Q4, respectively, during July-December, and 15, 48, and 37% sperm in Q2, Q3, and Q4 quadrants, respectively, during January-June. It is postulated that the altered kinetics of germ cell transformations and the deficient spermiogenesis observed earlier following FSH deprivation in these monkeys may have induced the enhanced susceptibility to acid denaturation in sperm.

Role of in situ generated tribofilm on the tribological characteristics of monolith and TiB2 reinforced MoSi2 intermetallic

Wear experiments performed on steel disc with increasing load for monolithic MoSi2 of different densities and its composite with TiB2 showed three distinct wear regimes. The specimens exhibited severe wear rate below the lower and above the upper critical loads and mild wear in between the two critical loads. The increase in density of the monolith and the reinforcement of TiB2 were effective in reducing the coefficient of friction and the specific wear rate. The wear experiments have been performed in these three regimes (15, 50 and 75 N). The tribofilm formed on the pin surface was found to contain both pin and disc materials. The temperature of the pins during the sliding against EN-24 disc was calculated using one dimensional heat transfer equation at different loads for each composition. The composite experiences lower temperatures compared to the monoliths. (C) 2002 Elsevier Science B.V. All rights reserved.

Crystallization of SrCO3 on a self-assembled monolayer substrate: an in-situ synchrotron X-ray study

Self-assembled monolayers (SAMs) of alkanethiols on gold surfaces show great promise in controlling the nucleation and growth of inorganic minerals from solution. In doing so, they mimic the role of some biogenic macromolecules in natural biomineralisation processes. Crystallization on SAM surfaces is usually monitored ex-situ; by allowing the process to commence and to evolve for some time, removing the substrate from the mother solution, and then examining it using microscopy, diffraction etc. We present here for the first time, the use of high energy monochromatic synchrotron X-radiation in conjunction with a two dimensional detector to monitor in situ, in a time resolved fashion, the growth of SrCO3 (strontianite) crystals on a SAM substrate.

Optically transparent polymer devices for in situ assessment of cell electroporation

In order to study cell electroporation in situ, polymer devices have been fabricated from poly-dimethyl siloxane with transparent indium tin oxide parallel plate electrodes in horizontal geometry. This geometry with cells located on a single focal plane at the interface of the bottom electrode allows a longer observation time in both transmitted bright-field and reflected fluorescence microscopy modes. Using propidium iodide (PI) as a marker dye, the number of electroporated cells in a typical culture volume of 10-100 mu l was quantified in situ as a function of applied voltage from 10 to 90 V in a series of 2-ms pulses across 0.5-mm electrode spacing. The electric field at the interface and device current was calculated using a model that takes into account bulk screening of the transient pulse. The voltage dependence of the number of electroporated cells could be explained using a stochastic model for the electroporation kinetics, and the free energy for pore formation was found to be kT at room temperature. With this device, the optimum electroporation conditions can be quickly determined by monitoring the uptake of PI marker dye in situ under the application of millisecond voltage pulses. The electroporation efficiency was also quantified using an ex situ fluorescence-assisted cell sorter, and the morphology of cultured cells was evaluated after the pulsing experiment. Importantly, the efficacy of the developed device was tested independently using two cell lines (C2C12 mouse myoblast cells and yeast cells) as well as in three different electroporation buffers (phosphate buffer saline, electroporation buffer and 10 % glycerol).

Composición quimica y digestibilidad in situ de los pastos angleton (Dichantium aristatum, poir.), Colonial (Panicum maximum, Jacq.) y Taiwan (Pennisetum purpureum, Schum)

Se determinó la digestibilidad de los pastos Angleton, Colonial y Taiwan mediante el método in situ, disponiendo para ello de tres novillos de la raza criolla Reyna cuya edad oscilaba entre 12 y 18 meses y con un peso promedio de 261 kg, los cuales estuvieron provistos de una fístula ruminal. El objetivo propuesto fue obtener y comparar los estimados cuantitativos relativos a la degradación ruminal a diferentes tiempos de incubación (24,48 y 72 horas} tanto de materia seca como de proteína bruta. Los pastos fueron cosechados a los 35 días después del rebrote y se analizaron químicamente según procedimientos de la A.O.A.C (1984) para materia seca (MS), proteína bruta (PB), extracto etéreo (EE), extracto libre de nitrógeno (ELN), fibra bruta (FB) y cenizas (C) (Weende), y según el método de Van Soest (CATIE, 1987) para fibra neutro detergente (FND)y fibra ácido detergente (FAD), así como Hemicelulosa (HC). Se incubaron 10 gr de las muestras de cada uno de los pastos en bolsas de nylon. Para analizar estadísticamente los valores de degradación obtenidos, se utilizaron análisis de varianza dentro de un DCA para determinar la significancia entre pastos en los tiempos medidos y prueba de rango múltiple de Duncan para comparar medias de los pastos dentro de cada tiempo, obteniéndose diferencias altamente significativas entre ellos (P <0.01), y al observar la separación de medias se manifestó la superioridad del Taiwan en todos los tiempos de incubación, sin embargo el Colonial, no presentó diferencias significativas con el Taiwan y el Angleton en el tiempo de 72 horas. Se concluye como resultado de este estudio, que a una edad de rebrote de 35 días, el. Taiwán es superior al Angleton y al Colonial en lo que respecta a solubilidad de materia seca v proteína bruta al mismo tiempo el Colonial. mostró superioridad ante el Angl.eton debido a su mayor solubilidad de materia seca. Las mayores degradaciones de materia seca se presentaron en el. período de 0 a 24 horas de fermentación para los tres pastos; en cambio para proteína bruta ocurrieron para el. Angleton y el Taiwán entra 1as 24 y 48 horas y para e1 Colonia1 entre O y 24 horas. En qenera1, a través de la dinámica de digestión de 1os pastos se observó la influencia negativa que ejerce proporcionalmente a su contenido, la fracción de fibra (fibra neutro detergente y fibra acido detergente).

Determinación de la digestibilidad "in situ" del afrecho de cerveza

El presente trabajo se realizó en la Hda. Las Mercedes, perteneciente a la Universidad Nacional Agraria, Situada en el Kilómetro once y medio carretera Norte, Managua, con el objetivo de determinar el valor nutritivo del afrecho de cerveza y la degradación de la MS y PB del mismo, por un tiempo determinado. Se empleó una vaca adulta, con previa fistulación, el manejo de la vaca fue similar al que se utiliza en la Hacienda. El afrecho de cerveza fue secado y molido y se efectúo análisis bromatológico según metodología de la A.O.A.C. (1984). Se incubaron en el rumen de la vaca fistulada ocho bolsas con 10 grs. de afrecho de cerveza cada una, para ser sacadas las bolsas de dos en dos en loa intervalos de tiempo de 6, 12, 24 y 48 hrs., se cálculo el porcentaje de MS y PB según metodología A.O.A.C. (1984). Además se utilizó una bolsa testigo, conteniendo también 10 grs. de afrecho de cerveza, la cual sirvió para determinar las pérdidas de MS y PB que se dieron por el lavado o por rápida solubilización, conocido como tiempo cero. Calculándose el porcentaje de degradación, de la MS (44.9, 60.2, 61.2 y 73.0) y la PB (60.6, 79.5, 83.8 y 93.3); se hicieron curvas de degradación según ecuación; D = A+B(l-e-ct)de Orskov y McDonald (1979). Donde los porcentajes de la ecuación para la MS fueron de 44.9, 63.0, 77.3, 82.2 y para la PB de 60.2, 82.3, 93.9 y 95.9. Se efectuó la prueba de t (student) para datos pareados, para comparar, la degradación de la MS y PB de la parte experimental vs la degradación de la ecuación, para lo cual se encontró, que no existen diferencias significativas (P<0.05), tanto para la MS experimental vs MS ecuación, como para la PB experimental vs PB ecuación. Lo cual indica que la estimación es eficiente. Se concluyó, que el afrecho de cerveza según su valor nutritivo y tasa de degradación, puede ser considerado un suplemento proteico de lenta degradación. La máxima degradación de la MS y PB, fue a las 48 horas, presentando porcentajes altos de degradación 73% y 93.3% respectivamente.

Estudio agromorfológico de quince poblaciones de frijol común (Phaseolus vulgaris L.) conservadas in situ y ex situ

Los objetivos del trabajo fueron describir la variabilidad fenotípica presente tanto entre como dentro de las poblaciones evaluadas, y a su vez determinar los efectos de color de semilla y tipo de conservación (ex situ e in situ ) sobre dicha diversidad, El estudio consistió en un experimento trifactorial, des balanceado y parcialmente anidado en un diseño de bloques completo al azar con tres repeticiones, cuyos factores de estudio fueron: Color de semilla, tipo de conservación y poblaciones anidadas dentro de cada uno de los factores. Se sometieron a análisis variables cualitativas de la flor, fenológicas y de producción (Rendimiento y sus componentes). Los resultados indicaron que el color de la semilla y el tipo de conservación resultaron ser los criterios principales de diferenciación de las poblaciones bajo estudiado destacándose el grupo de semilla de color café, la interacción de los efectos color de semilla y tipo de conservación resultaron significativas para el rendimiento, cierto de sus componentes e índice de cosecha, mostrando los materiales genéticos de color de grano crema y conservadas actualmente por los agricultores los mayores valores para la variable antes mencionadas. Las poblaciones conservadas in situ comparadas a las conservadas ex situ, presentaron valores diferentes para las variables bajo estudio