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At least 1,099 records · Page 61Linked to original sources
PubMed2003

Nanodiagnostics: application of nanotechnology in molecular diagnostics.

Nanotechnology extends the limits of molecular diagnostics to the nanoscale. Nanotechnology-on-a-chip is one more dimension of microfluidic/lab-on-a-chip technology. Biological tests measuring the presence or activity of selected substances become quicker, more sensitive and more flexible when certain nanoscale particles are put to work as tags or labels. Magnetic nanoparticles, bound to a suitable antibody, are used to label specific molecules, structures or microorganisms. Magnetic immunoassay techniques have been developed in which the magnetic field generated by the magnetically labeled targets is detected directly with a sensitive magnetometer. Gold nanoparticles tagged with short segments of DNA can be used for detection of genetic sequence in a sample. Multicolor optical coding for biological assays has been achieved by embedding different-sized quantum dots into polymeric microbeads. Nanopore technology for analysis of nucleic acids converts strings of nucleotides directly into electronic signatures. DNA nanomachines can function as biomolecular detectors for homogeneous assays. Nanobarcodes, submicrometer metallic barcodes with striping patterns prepared by sequential electrochemical depositon of metal, show differential reflectivity of adjacent stripes enabling identification of the striping patterns by conventional light microscopy. All this has applications in population diagnostics and in point-of-care hand-held devices.

Animals↗
PubMedDate not supplied

Invasions by Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) in the Western Hemisphere: implications for South America.

The multicolored Asian lady beetle, Harmonia axyridis (Pallas), native to Asia, has recently been detected in South America after successfully invading North America and Europe. This coccinellid is a voracious predator; therefore, it is popular and effective in biological control. Unfortunately, H. axyridis also has associated adverse impacts (i.e., as a household pest, pest of fruit production, and threat to non-target organisms). To predict the potential geographic extent of impacts of H. axyridis in South America we review the history of its invasion in the Western Hemisphere and address various factors critical to the future invasion (i.e., arrival, establishment, and spread) of new areas of South America. The likelihood of continued introductions (i.e., arrival) of H. axyridis to South America seems high, due to its popularity as a biological control agent and through accidental introductions. Establishment also seems likely in broad regions of South America. Climate matching with the native range suggested that much of southern South America may be suitable for establishment. In contrast, habitat matching with the native range suggested that northern South America may be more suitable. In addition, prey availability should not limit establishment of this predator. Once established, H. axyridis seems likely to spread by flight and human-assisted means. Overall, the invasion of H. axyridis over broad areas in South America seems likely.

Animal Migration↗
PubMed2005

Seven novel and stable translocations associated with oncogenic gene expression in malignant melanoma.

Cytogenetics has not only precipitated the discovery of several oncogenes, but has also led to the molecular classification of numerous malignancies. The correct identification of aberrations in many tumors has, however, been hindered by extensive tumor complexity and the limitations of molecular cytogenetic techniques. In this study, we have investigated five malignant melanoma (MM) cell lines from at least three different passages using high-resolution R-banding and the recently developed methods of comparative genomic hybridization and multicolor or multiplex fluorescence in situ hybridization. We subsequently detected nine consistent translocations, seven of which were novel: dic(1;11)(p10;q14), der(9)t(3;9)(p12;p11), der(4)t(9;4;7)(q33:p15-q23:q21), der(14)t(5;14)(q12;q32), der(9)t(9;22)(p21;q11), der(19)t(19;20)(p13.3;p11), der(10)t(2;12;7;10)(q31:p12-->pter:q11.2-->q31:q21), der(19)t(10;19)(q23;q13), and der(20)t(Y;20)(q11.23;q13.3). Furthermore, using the human HG-U133A GeneChip, positive expression levels of oncogenes or tumor-related genes located at the regions of chromosomal breakpoints were identified, including AKT1, BMI1, CDK6, CTNNB1, E2F1, GPNMB, GPRK7, KBRAS2, LDB2, LIMK1, MAPK1, MEL, MP1, MUC18, NRCAM, PBX3, RAB22A, RAB38, SNK, and STK4, indicating an association between chromosomal breakpoints and altered gene expression. Moreover, we also show that growth of all five cell lines can be significantly reduced by downregulating CDK6 gene expression with small interfering RNA (siRNA). Because the majority of these breakpoints have been reported previously in MM, our results support the idea of common mechanisms in this disease.

Cell Line↗
PubMed2004

Influence of cover crops on insect pests and predators in conservation tillage cotton.

In fall 2000, an on-farm sustainable agricultural research project was established for cotton, Gossypium hirsutum L., in Tift County, Georgia. The objective of our 2-yr research project was to determine the impact of several cover crops on pest and predator insects in cotton. The five cover crop treatments included 1) cereal rye, Secale cereale L., a standard grass cover crop; 2) crimson clover, Trifolium incarnatum L., a standard legume cover crop; 3) a legume mixture of balansa clover, Trifolium michelianum Savi; crimson clover; and hairy vetch, Vicia villosa Roth; 4) a legume mixture + rye combination; and 5) no cover crop in conventionally tilled fields. Three main groups or species of pests were collected in cover crops and cotton: 1) the heliothines Heliothis virescens (F.) and Helicoverpa zea (Boddie); 2) the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois); and 3) stink bugs. The main stink bugs collected were the southern green stink bug, Nezara viridula (L.); the brown stink bug, Euschistus servus (Say); and the green stink bug, Acrosternum hilare (Say). Cotton aphids, Aphis gossypii Glover, were collected only on cotton. For both years of the study, the heliothines were the only pests that exceeded their economic threshold in cotton, and the number of times this threshold was exceeded in cotton was higher in control cotton than in crimson clover and rye cotton. Heliothine predators and aphidophagous lady beetles occurred in cover crops and cotton during both years of the experiment. Geocoris punctipes (Say), Orius insidiosus (Say), and red imported fire ant, Solenopsis invicta Buren were relatively the most abundant heliothine predators observed. Lady beetles included the convergent lady beetle, Hippodamia convergens Guérin-Méneville; the sevenspotted lady beetle, Coccinella septempunctata L.; spotted lady beetle, Coleomegilla maculata (DeGeer); and the multicolored Asian lady beetle, Harmonia axyridis (Pallas). Density of G. punctipes was higher in cotton fields previously planted in crimson clover compared with control cotton fields for all combined sampling dates in 2001. Intercropping cotton in live strips of cover crop was probably responsible for the relay of G. punctipes onto cotton in these crimson clover fields. Density of O. insidiosus was not significantly different between cover crop and control cotton fields. Lady beetles seemed to relay from cover crops into cotton. Conservation of the habitat of fire ants during planting probably was responsible for the higher density of red imported fire ants observed in all conservation tillage cotton fields relative to control cotton fields. Reduction in the number of times in which economic thresholds for heliothines were exceeded in crimson clover and rye compared with control fields indicated that the buildup of predaceous fire ants and G. punctipes in these cover crops subsequently resulted in reduction in the level of heliothines in conservation tillage cotton with these cover crops compared with conventional tillage cotton without cover crops.

Agriculture↗
PubMedDate not supplied

Characterization of cells with a high aldehyde dehydrogenase activity from cord blood and acute myeloid leukemia samples.

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme that is responsible for the oxidation of intracellular aldehydes. Elevated levels of ALDH have been demonstrated in murine and human progenitor cells compared with other hematopoietic cells, and this is thought to be important in chemoresistance. A method for the assessment of ALDH activity in viable cells recently has been developed and made commercially available in a kit format. In this study, we confirmed the use of the ALDH substrate kit to identify cord blood stem/progenitor cells. Via multicolor flow cytometry of cord blood ALDH+ cells, we have expanded on their phenotypic analysis. We then assessed the incidence, morphology, phenotype, and nonobese diabetic/ severe combined immunodeficiency engraftment ability of ALDH+ cells from acute myeloid leukemia (AML) samples. AML samples had no ALDH+ cells at all, an extremely rare nonmalignant stem/progenitor cell population, or a less rare, leukemic stem cell population. Hence, in addition to identifying nonmalignant stem cells within some AML samples, a high ALDH activity also identifies some patients' CD34+/ CD38- leukemic stem cells. The incidence of normal or leukemic stem cells with an extremely high ALDH activity may have important implications for resistance to chemotherapy. Identification and isolation of leukemic cells on the basis of ALDH activity provides a tool for their isolation and further analysis.

ADP-ribosyl Cyclase↗
PubMed2005

Simple and efficient isolation of hematopoietic stem cells from H2K-zFP transgenic mice.

We have generated a transgenic mouse line that allows for simple and highly efficient enrichment for mouse hematopoietic stem cells (HSCs). The transgene expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs express exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2K-zFP mice. Up to 90% of zFP(bright) cells are also c-kit(high), Sca-1(high), Lin(neg), Flk-2(neg), which is a bona fide phenotype for long-term HSCs. Double-sorted zFP(bright) HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2K-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs without using fluorescence-conjugated monoclonal antibodies. This approach will greatly facilitate gene transfer, including short interfering RNA for gene knockdown, into HSCs and, consequently, into all other hematopoietic lineages.

Animals↗
PubMed2002

Karyotypes of human lymphocytes exposed to high-energy iron ions.

Chromosomal aberrations were analyzed using multicolor fluorescence in situ hybridization (mFISH) in human peripheral blood lymphocytes after in vitro exposure to gamma rays or accelerated (56)Fe ions (1 GeV/nucleon, 145 keV/microm) at Brookhaven National Laboratory (Upton, NY). Doses of 0.3 and 3 Gy were used for both radiation types. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid the population selection bias observed at metaphase as a result of the severe cell cycle delays induced by heavy ions. A total of 1053 karyotypes (G(2) and M phases) were analyzed in irradiated lymphocytes. Results revealed different distribution patterns for chromosomal aberrations after low- and high-LET radiation exposures: Heavy ions induced a much higher fraction of cells with multiple aberrations, while the majority of the aberrant cells induced by low doses of gamma rays contained a single aberration. The high fraction of complex-type exchanges after heavy ions leads to an overestimation of simple-type asymmetrical interchanges (dicentrics) from analysis of Giemsa-stained samples. However, even after a dose of 3 Gy iron ions, about 30% of the cells presented no complex-type exchanges. The involvement of individual chromosomes in exchanges was similar for densely and sparsely ionizing radiation, and no statistically significant evidence of a nonrandom involvement of specific chromosomes was detected.

Chromosome Aberrations↗
PubMed2003

Truly incomplete and complex exchanges in prematurely condensed chromosomes of human fibroblasts exposed in vitro to energetic heavy ions.

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 degrees C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/microm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy gamma rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.

Cell Cycle↗
PubMed2004

Stable intrachromosomal biomarkers of past exposure to densely ionizing radiation in several chromosomes of exposed individuals.

A multicolor banding (mBAND) fluorescence in situ hybridization technique was used to investigate the presence inhuman populations of a stable biomarker-intrachromosomal chromosome aberrations-of past exposure to high-LET radiation. Peripheral blood lymphocytes were taken from healthy Russian nuclear workers occupationally exposed from 1949 onward to either plutonium, gamma rays or both. Metaphase spreads were produced and chromosomes 1 and 2 were hybridized with mBAND FISH probes and scored for intra-chromosomal aberrations. A large yield of intrachromosomal aberrations was observed in both chromosomes of the individuals exposed to high doses of plutonium, whereas there was no significant increase over the (low) background control rate in the population who were exposed to high doses of gamma rays. Interchromosome aberration yields were similar in both the high plutonium and the high gamma-ray groups. These results for chromosome 1 and 2 confirm and extend data published previously for chromosome 5. Intrachromosomal aberrations thus represent a potential biomarker for past exposure to high-LET radiations such as alpha particles and neutrons and could possibly be used as a biodosimeter to estimate both the dose and type of radiation exposure in previously exposed populations.

Aged↗
PubMed2005

Clonally expanded T-cell populations in atomic bomb survivors do not show excess levels of chromosome instability.

Radiation-induced genomic instability has been studied primarily in cultured cells, while in vivo studies have been limited. One major obstacle for in vivo studies is the lack of reliable biomarkers that are capable of distinguishing genetic alterations induced by delayed radiation effects from those that are induced immediately after a radiation exposure. Here we describe a method to estimate cytogenetic instability in vivo using chromosomally marked clonal T-cell populations in atomic bomb survivors. The basic idea is that clonal translocations are derived from single progenitor cells that acquired an aberration, most likely after a radiation exposure, and then multiplied extensively in vivo, resulting in a large number of progeny cells that eventually comprise several percent of the total lymphocyte population. Therefore, if chromosome instability began to operate soon after a radiation exposure, an elevated frequency of additional but solitary chromosome aberrations in clonal cell populations would be expected. In the present study, six additional translocations were found among 936 clonal cells examined with the G-band method (0.6%); the corresponding value with multicolor FISH analysis was 1.2% (4/333). Since these frequencies were no higher than 1.2% (219/17,878 cells), the mean translocation frequency observed in control subjects using the G-band method, it is concluded that chromosome instabilities that could give rise to an increased frequency of persisting, exchange-type aberrations were not commonly generated by radiation exposure.

Breast Neoplasms↗
PubMed2006

Chromosomes lacking telomeres are present in the progeny of human lymphocytes exposed to heavy ions.

High-charge and energy (HZE) nuclei represent one of the main health risks for human space exploration, yet little is known about the mechanisms responsible for the high biological effectiveness of these particles. We have used in situ hybridization probes for cross-species multicolor banding (RxFISH) in combination with telomere detection to compare yields of different types of chromosomal aberrations in the progeny of human peripheral blood lymphocytes exposed to either high-energy iron ions or gamma rays. Terminal deletions showed the greatest relative variation, with many more of these types of aberrations induced after exposure to accelerated iron ions (energy 1 GeV/nucleon) compared with the same dose of gamma rays. We found that truncated chromosomes without telomeres could be transmitted for at least three cell cycles after exposure and represented about 10% of all aberrations observed in the progeny of cells exposed to iron ions. On the other hand, the fraction of cells carrying stable, transmissible chromosomal aberrations was similar in the progeny of cells exposed to the same dose of densely or sparsely ionizing radiation. The results demonstrate that unrejoined chromosome breaks are an important component of aberration spectra produced by the exposure to HZE nuclei. This finding may well be related to the ability of such energetic particles to produce untoward late effects in irradiated organisms.

Cell Division↗
PubMed2006

Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum.

O6-alkylguanine-DNA alkyltransferase (AGT) fusion proteins can be specifically and covalently labeled with fluorescent O6-benzylguanine (O6-BG) derivatives for multicolor live cell imaging approaches. Here, we characterize several new BG fluorophores suitable for in vivo AGT labeling that display fluorescence emission maxima covering the visible spectrum from 472 to 673 nm, thereby extending the spectral limits set by fluorescent proteins. We show that the photostability of the cell-permeable dyes BG Rhodamine Green (BG505) and CP tetramethylrhodamine (CP-TMR) is in the range of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP), and that BG diethylaminomethyl coumarin (BGDEAC), a derivative of coumarin, is even more stable than enhanced cyan fluorescent protein (ECFP). Due to the increasing number of new BG derivatives with interesting fluorescence properties, such as far-red emission, fluorescence labeling of AGT fusion proteins is becoming a versatile alternative to existing live cell imaging approaches.

Animals↗
PubMed2006

Multiframe particle tracking in intravital imaging: defining Lagrangian coordinates in the microcirculation.

The cellular composition of the microcirculation creates blood flow that can be unsteady and nonuniform. To obtain information about nonuniform cellular trajectories, we describe in vivo imaging techniques that provide both detailed tracking of individual particles as well as an approach to simultaneous multicolor particle tracking. Particularly relevant to biologic systems, Lagrangian methods provide information about the fate of individual particles and flow in the system.

Animals↗
PubMed2000

Microsatellite genotyping of post-PCR fluorescently labeled markers.

We show that a post-PCR multicolor fluorescence-labeling technique is applicable to multiplex microsatellite genotyping. Forty-three dinucleotide microsatellite markers, which are located on 11q13-23, a candidate region for dominant familial exudative vitreoretinopathy (FEVR), were used to evaluate the quality of the marker profile produced by this technique. Thirty-eight people from six families with this disease were subjects for genotyping. The samples revealed clearly separated fragment peaks with signal intensities sufficient for analysis. All genotypes were consistent with Mendelian inheritance within each family. This method is cost effective because it does not use expensive fluorescently labeled primers. It also has the advantage of avoiding ambiguity in the analysis, which may arise from the addition of non-template nucleotides by Taq DNA polymerase.

Fluorescence↗
PubMed2002

Red fluorescent protein DsRed from Discosoma sp. as a reporter protein in higher plants.

GFP from Aequorea victoria is a standard genetic marker widely used to visualize cellular events in a noninvasive manner. For simultaneous imaging of different processes, in vivo mutants of GFP with shifted wavelength spectra (e.g., blue fluorescent protein) are conventionally used. The recently reported red fluorescent protein from Discosoma sp., DsRed, represents a new marker that can be used together with GFP variants for multicolor imaging. DsRed is an interesting marker protein for use in plants because of its red-shifted wavelength spectrum that will avoid damaging cells and tissues by excitation light. In this report, we show that DsRed is an excellent marker in higher plants in spite of the interfering red autofluorescence of chlorophyll, which can be eliminated by using the appropriate filter sets. Transient expression of DsRed1-C1 and a soluble-modified, red-shifted GFP variant has been carried out both individually and jointly in the epidermal cells of three different Nicotiana species and Chenopodium quinoa, which gives rise to dual labeling in plants. For this purpose, a human codon-optimized variant of DsRed has been adopted for expression in plants. Moreover, the DsRed reporter gene was expressed by using a labeled plant viral vector derived from an infectious full-length clone of potato virus X.

Animals↗
PubMed2002

Software for machine-independent quantitative interpretation of SSCP in capillary array electrophoresis (QUISCA).

PCR single-stranded conformational polymorphism (SSCP) analysis is a simple and rapid electrophoretic technique for the sensitive detection of sequence variants of PCR products. Here we describe a cross-platform program package, quantitative interpretation of SSCP in capillary array (QUISCA), which allows semi-automated quantitative detection of sequence variants separated by multicolor fluorescence-based SSCP electrophoresis using various capillary array apparatus. The program, together with the QUISCAview as a graphical user interface, takes trace data in ASCII format and processes them with three modules: signal denoising/baseline subtraction, color-matrix construction/application, and calibration of peak positions between multiple capillary runs using internal standard peaks. QUISCA is compatible with data from various widely used capillary array sequencers and is suitable not only for finding or typing SNPs in individual DNAs but also for the accurate estimation of the allele frequencies of many SNPs using a pooled DNA strategy. QUISCA can also serve as a versatile core program for various fragment analyses.

Algorithms↗
PubMed1997

Generation of multiple mRNA fingerprints using fluorescence-based differential display and an automated DNA sequencer.

Differential display is a method for the survey, analysis and comparison of gene expression in eukaryotic cells and tissues. Differential display involves isolation of high-quality nondegraded RNA, selective reverse transcription of polyadenylated mRNA using specific anchored oligopolydeoxythymidine [oligo(dT)] primers, and the subsequent PCR amplification of the cDNA with the same oligo(dT), an arbitrary upstream primer and radioisotopes for labeling the PCR products. The radioisotopically labeled products are then separated on a sequencing gel. In this report, we describe a rapid, specific, nonradioactive fluorescent differential display methodology in which fluorescently differentially labeled anchored oligo(dT) downstream primers are used in the reaction, with subsequent analysis of fluorescently labeled PCR products on an automated sequencer. Complete gene expression profiles, containing multiple mRNA fingerprints are possible by the simultaneous comparison of the multicolored banding patterns of the fluorescently differentially labeled products from several primer combinations. This modification of the differential display technique simplifies the assay and increases the throughput of high sample volumes required for comparative gene expression studies in various clinical applications.

Antibodies↗
PubMed2004

Time-resolved confocal fluorescence imaging and spectrocopy system with single molecule sensitivity and sub-micrometer resolution.

We present novel technical features and results from a two channel confocal fluorescence lifetime microscope, which allows to efficiently investigate fluorescence dynamics down to the single molecule level. The MicroTime 200 time-resolved fluorescence microscope offers a multicolor excitation where different picosecond diode lasers are used. For imaging and positioning purposes we utilize a compact Piezo scanner which allows, due to a novel scanning algorithm and synchronisation technique, a superior movement and positioning accuracy. The data acquisition is completely based on time-correlated single photon counting, where every photon is detected and stored individually with its specific timing information (Time-Tagged Time-Resolved mode). This multiparameter data acquisition scheme offers the opportunity to analyse the parameter dependencies in a multitude of different ways. Standard intensity analysis can be used to reconstruct 2D-images or the temporal evolution (time trace) of the fluorescence of a single spot. The information from the two distinct detector channels additionally allows to investigate the polarisation of the emitted light or its spectral composition, for example for analysis of Fluorescence Resonance Energy Transfer (FRET). The timing information down to a picosecond scale offers the possibility not only to reconstruct fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function of any single spot of interest. The flexible multichannel detector scheme enables in this case also a cross-correlation between spectrally separated parts of the emission light, or even identical parts of the fluorescence to eliminate detector artifacts. The photon arrival coincidence analysis can also be expanded in the sub-ns range to study fluorescence antibunching in the fluorescence emission of single molecules. The ability of combining these different pieces of temporal information allows the construction of extremely powerful analysis methods and assays. We demonstrate a variety of these capabilities with results obtained from fluorescently labeled latex beads, biological samples, and single molecules excited in the blue or red wavelength region.

Equipment Design↗