Recombinant retroviruses encoding cell surface antigens as selectable markers.

Recombinant retroviruses are frequently used in the transfer and analysis of genes. This report describes new retrovirus vectors that incorporate a cDNA copy of a cell surface antigen to function as a selectable marker. By using techniques based on quantitative cell surface immunofluorescence, these vectors allow the rapid detection and isolation of infected cells. These vectors also allow the rapid detection of packaging cell lines producing large amounts of recombinant retroviruses. Potential applications of these vectors are demonstrated.     

On estimating the growth function of tumors

The growth rate of a cancerous tumor as a function of its age is a subject of intellectual and practical importance, as it influences both the effectiveness of proposed screening programs and the strategy of treatment. Obtaining direct evidence on the growth rate is quite difficult, owing to the ethical necessity to intervene when cancer is confirmed. The reasonable assumption that there is a common growth function of age and that probability of detection of a tumor in a short time period is proportional to its size allow the growth function to be inferred from data on sizes at detection. These results can be generalized to allow for individual variation in the rate of traversal of the common growth function. An estimator for the growth function from data on size at detection is obtained. Simulations indicate that it performs reasonably. Application of this estimator to data on a large series of cases of breast cancer at U.T. M. D. Anderson Hospital indicates that the growth function in the range of sizes seen at detection, can be adequately described by exponential growth, with rather large individual-to-individual variations in growth rate.     

Colour-dependent target detection by bees

The distance over which an object is detected by bees depends on the subtended visual angle and on spectral cues. At large angular subtenses detection is mediated only by chromatic cues. Achromatic targets, however, are also detectable. We investigated how chromatic and achromatic cues interact in detecting large-size targets. Coloured targets were used, with varied chromatic contrast that either did or did not present L-receptor contrast. Better detection correlated with higher chromatic contrast. Adding L-receptor contrast did not affect detection. It did allow the detection of achromatic targets, but at a lower level than most coloured ones, which indicates that the input from the achromatic system is negligible due to low sensitivity.     

A versatile fluorescence-based multiplexing assay for CAPS genotyping on capillary electrophoresis systems

Recent advances in next-generation sequencing techniques and the development of genomics resources for crop plants with large genomes allow the detection of a large number of single nucleotide polymorphisms (SNPs) and their use in a high-throughput manner. However, such large numbers of SNPs are on the one hand not needed in some plant breeding projects and on the other hand not affordable in some cases, raising the need for fast and low-cost innovative techniques for marker detection. In marker selection in plant breeding programs, cleaved amplified polymorphic sequence (CAPS) markers still play a significant role as a complement to other high-throughput methods for SNP genotyping. New methods focusing on the acceleration of CAPS-based genotyping are therefore highly desirable. The combination of the classical CAPS method and a M13-tailed primer multiplexing assay was used to develop an agarose-gel-free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence-labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected in a capillary electrophoresis system. This method allowed the cost-effective genotyping of several SNPs in barley in a multiplexed manner at an overall low cost in a short period of time. This new method was efficiently combined with the simultaneous detection of simple sequence repeats in the same electrophoresis run, resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.     

Semiconductor fluorescent nanocrystals (quantum dots) in biological biochips

Comprehension of biological processes in cells, tissues and organisms requires identification and analysis of numerous biological objects, mechanisms of their action and regulation. Microarray (biochips) technology is a rare tool to solve this problem. It is based on high-throughput recognition of a target to the probe and has the potential to measure simultaneously the presence of numerous molecules in multiplexed testes, all contained in a small drop of test fluid. Biochips allow the parallel analysis of genomic or proteomic content in healthy versus disease-affected or altered tissues or cells. The signals read-out from the biochips is done with organic dyes which often suffer from photobleaching, low brightness and background fluorescence. Recent data show that the use of fluorescent nanocrystals "quantum dots" (QDs) allows push away these restrictions. The QDs are sufficiently bright to be detected as individual particles, extremely resistant to photobleaching and provide unique possibilities for multiplexing thus supplying the microarray technology with the novel read-out option enabling the sensitivity of detection reaching the single molecule level. This paper is aimed at the development of the approaches to the QDs application in microarray-based detection. Possibilities of QDs application both in solid state (planar) biochips as well as intensively developing technique of suspension biochips (bead-based assays or liquid biochips) are demonstrated. The latter are more and more applied for simultaneous identification of very large numbers of molecules in proteomics, genomics, drug screening and clinical diagnostics. This assays base on spectral encoded elements (as a rule polymer microbeads). The benefits of using optically encoded microbeads (instead of the solid-state two-dimensional arrays) are derived from the freedom of bead to move in three dimensions. Polymeric beads optically encoded with organic dyes allow for a limited number of unique codes, whereas the use of semiconductor nanocrystals as fluorescent tags improves the beads multiplexed imaging capabilities, photostability and sensitivity of the biological objects detection. Additionally, an employment in suspension biochips of Frster resonance energy transfer (FRET) allows improving detection specificity. The absence of fluorescent background from non-interacting with the beads dye-labelled antibodies additionally increases the sensitivity of detection and further facilitates the multiplexing capabilities of nanocrystals-based detection and diagnostics. So the combination of the biochips and QDs techniques allow increasing detection sensitivity and significantly raising the number of detected objects (multiplexing capacities). Such combination should provide the breakthrough in proteomics, particularly in new drugs development, clinical diagnostics, new disease markers identification, better understanding of intracellular mechanisms.     

A medium designed for monitoring pitching yeast contamination in beer using a conductimetric technique

Nitrogen assimilation is the most readily utilized source of conductance changes when pitching yeast is grown in a glucose-based medium. A simple growth medium comprising yeast nitrogen base, in which nitrogen is supplied as ammonium sulphate, and glucose gave good growth but little change in conductance. Inclusion of a succinate buffer in the medium to remove protons liberated as a result of nitrogen uptake produced a large decrease in conductance and detection times that correlated well with enumeration of yeast by plate counting. The medium will allow more rapid and automated detection of pitching yeast survival in pasteurized beer although individual calibration for each beer type will be necessary.     

Highly efficient de novo mutant identification in a Sorghum bicolor TILLING population using the ComSeq approach

Screening large populations for carriers of known or de novo rare single nucleotide polymorphisms (SNPs) is required both in Targeting induced local lesions in genomes (TILLING) experiments in plants and in screening of human populations. We previously suggested an approach that combines the mathematical field of compressed sensing with next‐generation sequencing to allow such large‐scale screening. Based on pooled measurements, this method identifies multiple carriers of heterozygous or homozygous rare alleles while using only a small fraction of resources. Its rigorous mathematical foundations allow scalable and robust detection, and provide error correction and resilience to experimental noise. Here we present a large‐scale experimental demonstration of our computational approach, in which we targeted a TILLING population of 1024 Sorghum bicolor lines to detect carriers of de novo SNPs whose frequency was less than 0.1%, using only 48 pools. Subsequent validation confirmed that all detected lines were indeed carriers of the predicted mutations. This novel approach provides a highly cost‐effective and robust tool for biologists and breeders to allow identification of novel alleles and subsequent functional analysis.     

Genetic tests for the detection of chemically induced small deletions in Drosophila chromosomes

We have developed genetic tests for estimating the proportion of small deletions among chemically induced point mutations in Drosophila. The criteria used allow the detection of deletions that are large enough to include a viable visible mutation as well as a lethal, or a sex-linked lethal as well as a gene that is required for the development of a spermatogonium into a spermatozoon. On these criteria, we have concluded that DEB produces a high proportion of deletions among point mutations; that HA produces no deletions; and that DEN produces either no deletions or only very small ones that cannot be detected by our methods.     

Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC-MS using a QQLIT instrument

Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.     

The influence of meal size on prey DNA detectability in piscivorous birds

Molecular methods allow noninvasive assessment of vertebrate predator–prey systems at high taxonomic resolution by examining dietary samples such as faeces and pellets. To facilitate the interpretation of field‐derived data, feeding trials, investigating the impacts of biological, methodological and environmental factors on prey DNA detection, have been conducted. The effect of meal size, however, has not yet been explicitly considered for vertebrate consumers. Moreover, different noninvasively obtained sample types remain to be compared in such experiments. Here, we present a feeding trial on abundant piscivorous birds, Great Cormorants (Phalacrocorax carbo), to assess meal size effects on postfeeding prey DNA detection success. Faeces and pellets were sampled twice a day after the feed of large (350–540 g), medium (190–345 g) and small (15–170 g) fish meals contributing either a large (>79%) or small (<38%) share to the daily consumption. Samples were examined for prey DNA and fish hard parts. Molecular analysis of faeces revealed that both large meal size and share had a significantly positive effect on prey DNA detection rate postfeeding. Furthermore, large meals were detectable for a significantly longer time span with a detection limit at ~76 hr and a 50% detection probability at ~32 hr postfeeding. In pellets, molecular methods reliably identified the meal consumed the previous day, which was not possible via morphological analysis or when examining individual faeces. The less reliable prey DNA detection of small meals or meal shares in faeces signifies the importance of large numbers of dietary samples to obtain reliable trophic data.     

A Comparison of Methods for Estimating Northern Bobwhite Covey Detection Probabilities

Abstract: We compared the time-of-detection and logistic regression methods of estimating probability of detection for northern bobwhite (Colinus virginianus) coveys. Both methods are unusual in that they allow estimation of the total probability of detection (i.e., the product of the probability that a covey is available for detection [i.e., that a covey vocalizes] and detection given availability). The logistic regression method produced an average detection probability of 0.596 (SE = 0.020) and the time-of-detection method produced a detection probability estimate of 0.540 (SE = 0.086), and the 2 estimates were not significantly different. This is the first evaluation of the time-of-detection method with empirical field data. Although the time-of-detection and logistic regression method each have advantages, both can be used under appropriate conditions to improve estimates of bobwhite abundance by allowing for the estimation of detection probabilities. Improved estimates of bobwhite abundance will allow land managers to make more informed management decisions.     

Detection of Iberian terrestrial mammals employing olfactory,visual and auditory attractants

Whereas high lure specificity for mammal monitoring is often sought, little effort has been put into identifying general-purpose attractants for multispecies mammal surveys. We examined whether the olfactory lures ‘fatty acid scent’ (FAS) and catnip oil differ in their ability to detect Iberian larger mammals (body mass >?800 g, i.e. lagomorphs, ungulates and most carnivores); whether the addition of visual and acoustic stimuli increases detection efficiency; and whether environmental variables interact with lures to influence detection. Three treatments were randomly assigned to 192 detection plots: olfactory lures only, visual lures (a combination of olfactory and visual stimuli) and auditory lures (all three types of stimuli). The design was balanced across two detection methods and three landscapes. Out of 13 target species, two were absent in the study plots and six (three common and three rare species) were detected with low frequency. The two olfactory attractants produced similar detection rates for species allowing analysis. The addition of visual and auditory stimuli did not increase detection and apparently induced repulsion for some species. Environmental variables associated with scent diffusion and lure visibility did not significantly influence mammal detection. Monitoring of mammal communities over large areas may gain efficiency by selecting lures that are simple, have a constant chemical composition, require short handling times and allow detection of all occurring species. A single olfactory attractant (FAS) could be suitable for designing large-scale monitoring programmes at least for five common mammal species, provided that passive detection methods and lure use are chosen.     

A unique advantage for giant eyes in giant squid

Giant and colossal deep-sea squid (Architeuthis and Mesonychoteuthis) have the largest eyes in the animal kingdom [1, 2], but there is no explanation for why they would need eyes that are nearly three times the diameter of those of any other extant animal. Here we develop a theory for visual detection in pelagic habitats, which predicts that such giant eyes are unlikely to evolve for detecting mates or prey at long distance but are instead uniquely suited for detecting very large predators, such as sperm whales. We also provide photographic documentation of an eyeball of about 27 cm with a 9 cm pupil in a giant squid, and we predict that, below 600 m depth, it would allow detection of sperm whales at distances exceeding 120 m. With this long range of vision, giant squid get an early warning of approaching sperm whales. Because the sonar range of sperm whales exceeds 120 m [3-5], we hypothesize that a well-prepared and powerful evasive response to hunting sperm whales may have driven the evolution of huge dimensions in both eyes and bodies of giant and colossal squid. Our theory also provides insights into the vision of Mesozoic ichthyosaurs with unusually large eyes.     

Development of a fluorescence in situ hybridization method for cheese using a 16S rRNA probe

A 16S rRNA fluorescence in situ hybridization (FISH) method for cheese was developed to allow detection in situ of microorganisms within the dairy matrix. An embedding procedure using a plastic resin was applied to Stilton cheese, providing intact embedded cheese sections withstanding the hybridization reaction. The use of a fluorescein-labelled 16S rRNA Domain Bacteria probe allowed observation of large colonies of microbial cells homogeneously distributed in the cheese matrix. FISH experiments performed on cheese suspensions provided images of the different microbial morphotypes occurring. The technique has great potential to study the spatial distribution of microbial populations in situ in foods, especially where the matrix is too fragile to allow manipulation of cryosections.     

PBXL-1: a new fluorochrome applied to detection of proteins on membranes

An easy, sensitive and direct fluorescent immunodetection method for proteins is described using the new fluorochrome PBXL-1 imaged with the FMBIO II Laser Scanning Imaging System. PBXL-1 is derived from a protein supra-molecular complex that contains a large number of chromophores. This complex, the phycobilisome, is extracted from a red alga then chemically stabilized to allow its use in specific binding assays. PBXL-1 was cross-linked to goat anti-rabbit IgG or streptavidin with heterobifunctional cross-linkers. The detection limit of PBXL-1 was determined by applying it on nitrocellulose membranes then imaging the membrane using an ytterbium aluminum garnet (YAG) laser. Evaluation of PBXL-1 sensitivity in a specific binding assay was tested on streptavidin/biotin and an antibody system. PBXL-1 provides high sensitivity in direct fluorescent applications due to a physical amplification of signal (i.e., a large number of fluorophores per binding event). PBXL-1 provides a linear response over two orders of magnitude while providing sub-amol sensitivity, indicating broad applicability for detection of a variety of targets. To our knowledge, this is the most sensitive direct fluorescent detection method available.     

A robust in vivo positive-readout system for monitoring siRNA delivery to xenograft tumors

Delivering small interfering RNA (siRNA) to tumors is the major technical hurdle that prevents the advancement of siRNA-based cancer therapy. One of the difficulties associated with the development of clinically relevant delivery systems is the lack of reliable tools for monitoring siRNA delivery to tumors in vivo. We describe here a novel, positive-readout system where siRNA-mediated target knockdown elicits a rapid and robust increase of reporter activity. Using the positive-readout system, we created (1) β-galactosidase-based tumor models that allow the detection of target knockdown in 1%-2% of tumor cells and can distinguish between tumor areas where effective target knockdown occurs versus tumor areas that are not accessible to delivery, and (2) luciferase-based tumor models that allow the quantitative assessment of a large number of delivery systems. Using these positive-readout models, we screened a number of literature-described siRNA delivery systems and identified lipid nanoparticles as a promising delivery platform for siRNA-based cancer therapy.     

Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay

The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens.     

Functional assays for screening GPCR targets

G-protein-coupled receptors (GPCRs) are valuable molecular targets for drug discovery. An important aspect of the early drug discovery process is the design and implementation of high-throughput GPCR functional assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates. Several functional assay kits based on fluorescence and/or chemiluminescence detection are commercially available for convenient screen development, each having advantages and disadvantages. In addition, new GPCR biosensors and high-content imaging technologies have recently been developed that hold promise for the development of functional GPCR screens in living cells.