Treatment and detection of ALK-rearranged NSCLC.

The recent approval of crizotinib for the treatment of anaplastic lymphoma kinase (ALK)-rearranged advanced non-small cell lung cancer (NSCLC) in the US and other countries has provoked intense interest in ALK rearrangements as oncogenic drivers, and promises to revolutionise the way in which NSCLC is diagnosed and treated. Here, we review clinical data to date for the use of crizotinib to treat patients with advanced, ALK-positive NSCLC and consider issues surrounding the detection of ALK-positivity including the use of fluorescence in situ hybridisation and the other potential techniques available, and their suitability for ALK screening. We also discuss the emergence of resistance to crizotinib therapy and the range of other ALK inhibitors currently in development.

Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon.

We present a silicon chip-based approach for the enhanced sensitivity detection of surface-immobilized fluorescent molecules. Green fluorescent protein (GFP) is bound to the silicon substrate by a disuccinimidyl terephtalate-aminosilane immobilization procedure. The immobilized organic layers are characterized by surface analysis techniques, like ellipsometry, atomic force microscopy (AFM) and X-ray induced photoelectron spectroscopy. We obtain a 20-fold enhancement of the fluorescent signal, using constructive interference effects in a fused silica dielectric layer, deposited before immobilization onto the silicon. Our method opens perspectives to increase by an order of magnitude the fluorescent response of surface immobilized DNA- or protein-based layers for a variety of biosensor applications.

Simple method for detection of MYH11 DNA rearrangements in patients with inv(16)(p13q22) and acute myeloid leukemia.

The pericentric inversion on chromosome 16 [inv(16)(p13q22)] and related t(16;16)(p13;q22) are recurrent aberrations associated with acute myeloid leukemia (AML) M4 Eo. Both abberations result in a fusion of the core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11). A selected genomic 6.9-kb BamHl probe detects MYH11 DNA rearrangements in 18 of 19 inv(16)/t(16;16) patients tested using HindIII digested DNA. The rearranged fragments were not detectable after remission in two cases tested, while they were present after relapse in one of these two cases tested.

The application of NMR and MS methods for detection of adulteration of wine, fruit juices, and olive oil. A review

This review covers two important techniques, high resolution nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), used to characterize food products and detect possible adulteration of wine, fruit juices, and olive oil, all important products of the Mediterranean Basin. Emphasis is placed on the complementary use of SNIF-NMR (site-specific natural isotopic fractionation nuclear magnetic resonance) and IRMS (isotope-ratio mass spectrometry) in association with chemometric methods for detecting the adulteration.

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.

Contribution of ultrasonographic examination to the prenatal detection of chromosomal abnormalities in 19 centres across Europe.

The objective of this study was to evaluate the prenatal detection of chromosomal abnormalities by fetal ultrasonographic examination in a large database provided by 19 Registries of Congenital Anomalies from 11 European countries. This study included 1738 cases of chromosomal abnormalities, liveborn, stillborn or termination of pregnancy regardless of maternal age from a population of 664,340 births during the period 1996 - 1998. The most frequent chromosomal anomalies were Down syndrome (n=1050), trisomy 18 (n=191), Turner syndrome (n=125), trisomy 13 (n=86), and triploidy (n=56). Fetal ultrasonographic examination resulted in the prenatal detection of 37.7% of the chromosomal abnormalities, thereby resulting in a reduction of 28.6% in their prevalence at birth due to terminations of pregnancy. The detection rate by ultrasound examination varied according to local policies of prenatal diagnosis : it was lower in countries where routine scan were not performed and higher in countries in which at least one routine anomaly scan during the second trimester of pregnancy was performed. The ultrasound detection varied according to the specific chromosomal anomaly and was lowest for Klinefelter syndrome (5.7%) and highest for triploidy (78.6%). For Down syndrome it was 26.4%. Termination of pregnancy was performed in 75.9% of the cases. Among the 655 cases detected by ultrasound, the most frequent ultrasound signs by category of chromosomal abnormalities were analysed. This study shows that ultrasound screening is an important tool in the prenatal detection of chromosomal abnormalities in Europe, leading to a significant reduction in the prevalence of livebirth children with chromosomal anomalies.

Reference Cluster Normalization Improves Detection of Frontotemporal Lobar Degeneration by Means of FDG-PET.

Positron emission tomography with [18F] fluorodeoxyglucose (FDG-PET) plays a well-established role in assisting early detection of frontotemporal lobar degeneration (FTLD). Here, we examined the impact of intensity normalization to different reference areas on accuracy of FDG-PET to discriminate between patients with mild FTLD and healthy elderly subjects. FDG-PET was conducted at two centers using different acquisition protocols: 41 FTLD patients and 42 controls were studied at center 1, 11 FTLD patients and 13 controls were studied at center 2. All PET images were intensity normalized to the cerebellum, primary sensorimotor cortex (SMC), cerebral global mean (CGM), and a reference cluster with most preserved FDG uptake in the aforementioned patients group of center 1. Metabolic deficits in the patient group at center 1 appeared 1.5, 3.6, and 4.6 times greater in spatial extent, when tracer uptake was normalized to the reference cluster rather than to the cerebellum, SMC, and CGM, respectively. Logistic regression analyses based on normalized values from FTLD-typical regions showed that at center 1, cerebellar, SMC, CGM, and cluster normalizations differentiated patients from controls with accuracies of 86%, 76%, 75% and 90%, respectively. A similar order of effects was found at center 2. Cluster normalization leads to a significant increase of statistical power in detecting early FTLD-associated metabolic deficits. The established FTLD-specific cluster can be used to improve detection of FTLD on a single case basis at independent centers - a decisive step towards early diagnosis and prediction of FTLD syndromes enabling specific therapies in the future.

Nanopore detection of single molecule RNAP-DNA transcription complex.

In the past decade, a number of single-molecule methods have been developed with the aim of investigating single protein and nucleic acid interactions. For the first time we use solid-state nanopore sensing to detect a single E. coli RNAP-DNA transcription complex and single E. coli RNAP enzyme. On the basis of their specific conductance translocation signature, we can discriminate and identify between those two types of molecular translocations and translocations of bare DNA. This opens up a new perspectives for investigating transcription processes at the single-molecule level.

Influence of neuroblastoma stage on serum-based detection of MYCN amplification.

BACKGROUND: MYCN oncogene amplification has been defined as the most important prognostic factor for neuroblastoma (NB), the most common solid extracranial neoplasm in children. High copy numbers are strongly associated with rapid tumor progression and poor outcome, independently of tumor stage or patient age, and this has become an important factor in treatment stratification. PROCEDURE: By real-time quantitative PCR analysis, we evaluated the clinical relevance of circulating MYCN DNA of 267 patients with locoregional or metastatic NB in children less than 18 months of age. RESULTS: For patients in this age group with INSS stage 4 or 4S NB and stage 3 patients, serum-based determination of MYCN DNA sequences had good sensitivity (85%, 83%, and 75% respectively) and high specificity (100%) when compared to direct tumor gene determination. In contrast, the approach showed low sensitivity patients with stages 1 and 2 disease. CONCLUSION: Our results show that the sensitivity of the serum-based MYCN DNA sequence determination depends on the stage of the disease. However, this simple, reproducible assay may represent a reasonably sensitive and very specific tool to assess tumor MYCN status in cases with stage 3 and metastatic disease for whom a wait and see strategy is often recommended.

Short-term moderate hypocapnia augments detection of optimal cerebral perfusion pressure.

An autoregulation-oriented strategy has been proposed to guide neurocritical therapy toward the optimal cerebral perfusion pressure (CPPOPT). The influence of ventilation changes is, however, unclear. We sought to find out whether short-term moderate hypocapnia (HC) shifts the CPPOPT or affects its detection. Thirty patients with traumatic brain injury (TBI), who required sedation and mechanical ventilation, were studied during 20 min of normocapnia (5.1±0.4 kPa) and 30 min of moderate HC (4.4±3.0 kPa). Monitoring included bilateral transcranial Doppler of the middle cerebral arteries (MCA), invasive arterial blood pressure (ABP), and intracranial pressure (ICP). Mx -autoregulatory index provided a measure for the CPP responsiveness of MCA flow velocity. CPPOPT was assessed as the CPP at which autoregulation (Mx) was working with the maximal efficiency. During normocapnia, CPPOPT (left: 80.65±6.18; right: 79.11±5.84 mm Hg) was detectable in 12 of 30 patients. Moderate HC did not shift this CPPOPT but enabled its detection in another 17 patients (CPPOPT left: 83.94±14.82; right: 85.28±14.73 mm Hg). The detection of CPPOPT was achieved via significantly improved Mx-autoregulatory index and an increase of CPP mean. It appeared that short-term moderate HC augmented the detection of an optimum CPP, and may therefore usefully support CPP-guided therapy in patients with TBI.

Globally aligned photomosaic of the Lucky Strike hydrothermal vent field (Mid-Atlantic Ridge, 37°18.5’N): Release of georeferenced data, mosaic construction, and viewing software

We present a georeferenced photomosaic of the Lucky Strike hydrothermal vent field (Mid-Atlantic Ridge, 37°18’N). The photomosaic was generated from digital photographs acquired using the ARGO II seafloor imaging system during the 1996 LUSTRE cruise, which surveyed a ~1 km2 zone and provided a coverage of ~20% of the seafloor. The photomosaic has a pixel resolution of 15 mm and encloses the areas with known active hydrothermal venting. The final mosaic is generated after an optimization that includes the automatic detection of the same benthic features across different images (feature-matching), followed by a global alignment of images based on the vehicle navigation. We also provide software to construct mosaics from large sets of images for which georeferencing information exists (location, attitude, and altitude per image), to visualize them, and to extract data. Georeferencing information can be provided by the raw navigation data (collected during the survey) or result from the optimization obtained from imatge matching. Mosaics based solely on navigation can be readily generated by any user but the optimization and global alignment of the mosaic requires a case-by-case approach for which no universally software is available. The Lucky Strike photomosaics (optimized and navigated-only) are publicly available through the Marine Geoscience Data System (MGDS, http://www.marine-geo.org). The mosaic-generating and viewing software is available through the Computer Vision and Robotics Group Web page at the University of Girona (http://eia.udg.es/_rafa/mosaicviewer.html)

Development of bioreporter assays for the detection of bioavailability of long-chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2.

Long-chain alkanes are a major component of crude oil and therefore potentially good indicators of hydrocarbon spills. Here we present a set of new bacterial bioreporters and assays that allow to detect long-chain alkanes. These reporters are based on the regulatory protein AlkS and the alkB1 promoter from Alcanivorax borkumensis SK2, a widespread alkane degrader in marine habitats. Escherichia coli cells with the reporter construct reacted strongly to octane in short-term (6 h) aqueous suspension assays but very slightly only to tetradecane, in line with what is expected from its low water solubility. In contrast, long-term assays (up to 5 days) with A. borkumensis bioreporters showed strong induction with tetradecane and crude oil. Gel-immobilized A. borkumensis reporter cells were used to demonstrate tetradecane and crude oil bioavailability at a distance from a source. Alcanivorax borkumensis bioreporters induced fivefold more rapid and more strongly when allowed physical contact with the oil phase in standing flask assays, suggesting a major contribution of adhered cells to the overall reporter signal. Using the flask assays we further demonstrated the effect of oleophilic nutrients and biosurfactants on oil availability and degradation by A. borkumensis. The fluorescence signal from flask assays could easily be captured with a normal digital camera, making such tests feasible to be carried out on, e.g. marine oil responder vessels in case of oil accidents.

Detection of bovine foetal DNA from amniotic fluid using the polymerase chain reaction

Selostus: Sikiön DNA:n tunnistaminen naudan sikiövedestä polymeraasiketjureaktion avulla

Detection of moving groups among early type stars

Our procedure to detect moving groups in the solar neighbourhood (Chen et al., 1997) in the four-dimensional space of the stellar velocity components and age has been improved. The method, which takes advantadge of non-parametric estimators of density distribution to avoid any a priori knowledge of the kinematic properties of these stellar groups, now includes the effect of observational errors on the process to select moving group stars, uses a better estimation of the density distribution of the total sample and field stars, and classifies moving group stars using all the available information. It is applied here to an accurately selected sample of early-type stars with known radial velocities and Strömgren photometry. Astrometric data are taken from the HIPPARCOS catalogue (ESA, 1997), which results in an important decrease in the observational errors with respect to ground-based data, and ensures the uniformity of the observed data. Both the improvement of our method and the use of precise astrometric data have allowed us not only to confirm the existence of classical moving groups, but also to detect finer structures that in several cases can be related to kinematic properties of nearby open clusters or associations.