Separation and detection of individual Aβ aggregates by capillary electrophoresis with laser-induced fluorescence detection

The separation and detection of individual amyloid beta (Aβ) aggregates by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was demonstrated. Samples were prepared with either Aβ (1-40) or Aβ (1-42) peptides and were characterized by CE with ultraviolet (UV) absorbance detection and transmission electron microscopy (TEM). Using thioflavin T (ThT) in the electrophoresis buffer, electrophoresis of aggregate-containing samples (5.0-s injection) produced up to several hundred narrow (< 20 ms FWHM [full width at half maximum]) fluorescence peaks. Injection of Aβ (1-40) monomer samples resulted in no additional peaks compared with controls. The CE-LIF results were validated by bulk ThT fluorescence measurements for the same samples. The potential of laser-induced fluorescence anisotropy (LIFA) with CE to characterize individual Aβ aggregates also was investigated.     

5′-Bispyrene molecular beacons for RNA detection

New fluorescent excimer-forming 5′-bispyrene molecular beacons for the detection of RNA were designed. The probes are 2′-O-methyl RNAs containing 5′-bispyrenylmethylphosphorodiamidate group (bispyrene group) at the 5′-end and a fluorescence quencher (BHQ1) at the 3′-end. A comparative study of the fluorescent properties of the probes having different distance between 5′-bispyrene group and target RNA upon the formation of hybridization complex was performed. The probes with bispyrene group located in the close proximity to the duplex exhibit the greatest excimer fluorescence upon binding to a complementary the 43-nt target RNA, in contrast to the probes with 5′-bispyrene group at dangling end. The feasibility of the new probes for visualization of intracellular RNA was demonstrated using 28S rRNA as a target. The results obtained confirm that the probes proposed in the study can be used as selective tools for RNA detection.     

Nanotechnologies for pathogen detection: Future alternatives?

The development of multiplex and flexible tests allowing the simultaneous analysis of pathogens presenting a transfusional risk is a real challenge. Current miniaturized platforms have been particularly marked by microarrays. These microsystems allow the optical detection of hundreds of individual targets simultaneously. However, they suffer from a low sensitivity and their combination with a preliminary target amplification step such as PCR is necessary. The variable level of expression of the infectious genomes of interest and their large diversity complicate multiplex amplification. Finally simultaneous analysis of multiple blood-transmitted agents poses numerous difficulties in diagnosis that remain unresolved by currently available technologies.Until recently, scientific and technological advances for pathogen detection have focused on target amplification and optical detection steps. Today, sample preparation is recognized as a critical area to improve. Nanotechnologies can reach the single-cell or molecular scale and consequently overcome several current technological obstacles. They offer new technological tools for improving sample preparation but also for avoiding target amplification and the current fluorescent labeling. The combination of nano-objects and nano-systems in current technologies offers new possibilities for potential applications in the detection of infectious agents.     

Cytochemical detection of catalase with 3,3′-diaminobenzidine

Summary The influence of various parameters of fixation and incubation upon the oxidation of DAB by catalase have been analyzed. Crystalline beef liver catalase was fixed with different concentrations of glutaraldehyde and peroxidatic activity was determined spectrophotometrically using DAB as hydrogen donor. Although aldehyde fixation appeared to be important in elicitation of the peroxidatic activity of catalase, the final pigment production after 60 min incubation was optimal with the lowest concentration of glutaraldehyde (1%), after the shortest fixation period (30 min), and at the lowest temperature (5° C) tested. Similarly cytochemical studies with rat kidney sections incubated for 10 min confirmed that the staining of peroxisomes in proximal tubules was strongest after the mildest fixation conditions. The pH and the temperature of incubation were closely interrelated, so that at room temperature (25° C) the maximal pigment production was obtained at pH 10.5 but incubation at 45° C gave the strongest staining at pH 8.5. The production of pigment increased with higher DAB concentrations which required larger amounts of H₂O₂ in the incubation medium. Cytochemical studies on renal peroxisomes were in agreement with these biochemical findings. The observations indicate that there are several options for the localization of catalase depending on the fixation and incubation conditions. Hence, these conditions should be selected according to the tissue and the purpose of the study. Examples for such selective applications are presented.     

PCR detection of cryptosporidium: the way forward?

In this article, Una Morgan and Andrew Thompson briefly review the latest information on polymerase chain reaction (PCR)detection of Cryptosporidium parvum in both clinical and environmental samples. Current detection methods for Cryptosporidium are cumbersome, time-consuming and lack sensitivity. A variety of PCR tests have been described recently in the literature and this article discusses the advantages and disadvantages of each new technique and their potential for future diagnosis of Cryptosporidium.     

A spelt-specific γ-gliadin gene: discovery and detection

Polymerase chain reaction (PCR) primers GAG5 and GAG6 were designed based on published γ-gliadin gene sequences and applied to 35 cultivars of closely related spelt (Triticum spelta L.) and hexaploid wheat (T. aestivum L.). Eight tetraploid durum wheat (T. durum Desf.) cultivars were included in the analysis. The obtained PCR products originated from two γ-gliadin genes which were mapped to homeologous chromosomes 1B and 1D and termed GAG56B and GAG56D, respectively. Two alleles of GAG56D differing in a 9-bp deletion/duplication and single nucleotide polymorphism were found. The 18 spelts tested and wheat cultivar ’Chinese Spring’ were discovered to carry a previously unknown γ-gliadin gene, while 16 wheat cultivars possessed its longer, already published allele. Two PCR-based detection systems for the diagnostic alleles were developed and applied. The occurrence of two alleles of GAG56B among the investigated durum wheats correlated with their expression of gluten quality markers γ-gliadins 42 or 45. Received: 10 March 1999 / Accepted: 17 March 1999     

Bioluminescence probe for γ-glutamyl transpeptidase detection in vivo

To detect γ-Glutamyl Transpeptidase (GGT) activity in vitro and in vivo, a bioluminescence probe with high sensitivity and specificity was well designed and synthesized. This probe can be recognized by GGT and release strong bioluminescence with its further reaction with luciferase. The performance of this probe was demonstrated in vitro and in cells. Finally, we applied the probe for detection of GGT activity in xenograft model.     

Does accounting for imperfect detection improve species distribution models?

Models of species distributions are increasingly being used to address a variety of problems in conservation biology. In many applications, perfect or constant detectability of species, given presence, is assumed. While this problem has been acknowledged and addressed through the development of occupancy models, we still know little regarding whether addressing the potential for imperfect detection improves the predictive performance of species distribution models in nature. Here, we contrast logistic regression models of species occurrence that do not correct for detectability to hierarchical occupancy models that explicitly estimate and adjust for detectability, and maximum entropy models that attempt to circumvent the detectability problem by using data from known presence locations only. We use a large‐scale, long‐term monitoring database across western Montana and northern Idaho to contrast these models for nine landbird species that cover a broad spectrum in detectability. Overall, occupancy models were similar to or better than other approaches in terms of predictive accuracy, as measured by the Area Under the ROC Curve (AUC) and Kappa, with maximum entropy tending to provide the lowest predictive accuracy. Models varied in the types of errors associated with predictions, such that some model approaches may be preferred over others in certain situations. As expected, predictive performance varied across a gradient in species detectability, with logistic regression providing lower relative performance for less detectable species and Maxent providing lower performance for highly detectable species. We conclude by discussing the advantages and limitations to each approach for developing large‐scale species distribution models.     

Is NMDA receptor-coincidence detection required for learning and memory?

The Mg2+ block of NMDA-type glutamate receptors (NMDARs) is crucial to their function as synaptic coincidence detectors. An analysis of Drosophila expressing a Mg2+-independent NMDAR by in this issue of Neuron concludes that the Mg2+ block is required primarily for long-term memory.     

Luminescence detection of SNARE–SNARE interaction in Arabidopsis protoplasts

Membrane associated proteins SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) provide the minimal fusion machinery necessary for cellular vesicles to fuse to target organelle membranes in eukaryotic cells. Despite the conserved nature of the fusion machinery in all eukaryotes, it still remains challenging to identify functional SNARE pairs in higher plants. We developed a method based on a split-luciferase complementation assay for detecting changes in SNARE–SNARE interaction by luminescence within Arabidopsis protoplasts that express recombinant proteins at physiological levels in 96-well plates. The reliability of the assay was confirmed by three experiments. First, reduction of the SNARE–SNARE interaction caused by a single amino acid substitution adjacent to the SNARE motif in endosome-localized AtVAM3/SYP22 (syntaxin of plant 22) was detected by a reduction of luminescence. Second, reduction of the interaction between plasma-membrane localized SYP121 and VAMP722 in response to sodium azide was detected in real-time. Third, the results of 21 SNARE pairs investigated by this method largely agreed with the results from previously reported co-immunoprecipitation assays. Using the method, we newly identified the interaction between SYP121 and VAMP722 was significantly increased when the protoplasts were incubated in the light. Microscopic observation of transgenic Arabidopsis expressing GFP–SYP121 (green fluorescent protein tagged SYP121) from its own promoter suggested that the plasma-membrane localization of GFP–SYP121 is maintained by light. These suggested that the vesicle trafficking pathway mediated by SYP121 might be regulated by light in Arabidopsis. In general, this article demonstrated the method that can generate new biological insight of the SNARE protein interactions in plant cells.     

Improved detection of β-thalassaemia carriers by a two-test method

Summary Seven red cell parameters, taken one at a time and in their 21 possible pairs, were investigated for their power to discriminate between adult carriers of the -thalassaemia allele and adult normal subjects. The red blood cell count (RBC), haemoglobin concentration (Hb), haematocrit (Hct), mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), and haemoglobin A₂(HbA₂) fraction were measured in 24 obligate heterozygotes and in 99 adult controls with comparable age and sex distributions. Quadratic discriminant functions were computed using Bayesian analysis of univariate and bivariate Gaussian density functions. Classification errors were then calculated by integrating the density function for one genotype over the region assigned to the other.In the univariate case, MCH led to the lowest cost of misclassification while MCV was the second best discriminant for all posterior probabilities considered. In the bivariate case, MCV combined with percentage Hb A₂ yielded the best discrimination and generated misclassification costs roughly 1/30 of those generated by the most efficient single parameter. When use of MCV alone cannot classify an individual reliably either as a heterozygote or as homozygous normal, combined use of MCV and percentage Hb A₂ is recommended for maximum accuracy.Application of this screening method to 260 adult subjects at risk for thalassaemia heterozygosity yielded an unbiased frequency of 0.067 for the adult carrier in the Montreal Greek community, a value similar to that reported in the source population in Greece. The improved discriminations thus achieved is particularly useful for sibs of affected subjects whose high prior probability of heterozygosity (0.67) impairs classification.     

Wiskott-Aldrich syndrome carrier detection with the hypervariable marker M27β

Summary Whole-blood cells of obligate carriers of the X-linked Wiskott-Aldrich syndrome (WAS) exhibit nonrandom inactivation of the X-chromosomes. However, because of the limited polymorphism of the probes available, the X-methylation pattern can only be determined in a restricted proportion of females. We thus analysed a large set of normal females and members of WAS families, using the recently described marker M27, which detects the hyperpolymorphic locus DXS255. The probe was used to detect differences in methylation between the active and inactive X-chromosome, and the findings were compared with the pattern obtained using the well-documented probes from the 5 end of the PGK and HPRT genes. All the normal females were found to use either X-chromosome randomly, and there was complete correlation between the three probes in the populations studied. Segregation analysis performed with M27 and other related markers in the WAS families was fully in accordance with the X-inactivation data. The use of M27, for both X-inactivation and segregation analysis of WAS kindreds, provides a basis for genetic counselling in the majority of families, including those with no surviving males.     

Is pulse oximetry a reliable tool for detection of aspiration?

This study was designed to determine whether significant differences in saturation levels existed among patients with aspiration and patients without and wether pulse oximetry can reliably detect aspiration in patients with dysphagia. We also examined the effects of gender and disease (neurologic versus non neurologic) on saturation levels. We studied 38 patients. They all underwent a videofluoroscopic study of swallowing (VFSS). Twenty patients aspirated on videofluoroscopic study of swallowing: ten patients were solid aspirators, ten patients were liquid aspirators. In each group (liquid aspirators, solid aspirators or non aspirators) we found no significant difference in saturation levels. We found however a significant difference in saturation levels between each group before, during and after videofluoroscopic study of swallowing. Both gender and disease had an effect on saturation levels. We conclude that pulse oximetry can not serve as a screening tool for detection of aspiration as saturation levels are dependent on many factors. Therefore one can not reliably predict aspiration with a single saturation screening.     

Systems for the detection and analysis of protein–protein interactions

The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review.K. Kuroda, M. Kato and J. Mima contributed equally to this work.     

Entropic Profiler – detection of conservation in genomes using information theory

Background

In the last decades, with the successive availability of whole genome sequences, many research efforts have been made to mathematically model DNA. Entropic Profiles (EP) were proposed recently as a new measure of continuous entropy of genome sequences. EP represent local information plots related to DNA randomness and are based on information theory and statistical concepts. They express the weighed relative abundance of motifs for each position in genomes. Their study is very relevant because under or over-representation segments are often associated with significant biological meaning.

Findings

The Entropic Profiler application here presented is a new tool designed to detect and extract under and over-represented DNA segments in genomes by using EP. It allows its computation in a very efficient way by recurring to improved algorithms and data structures, which include modified suffix trees. Available through a web interface http://kdbio.inesc-id.pt/software/ep/ and as downloadable source code, it allows to study positions and to search for motifs inside the whole sequence or within a specified range. DNA sequences can be entered from different sources, including FASTA files, pre-loaded examples or resuming a previously saved work. Besides the EP value plots, p-values and z-scores for each motif are also computed, along with the Chaos Game Representation of the sequence.

Conclusion

EP are directly related with the statistical significance of motifs and can be considered as a new method to extract and classify significant regions in genomes and estimate local scales in DNA. The present implementation establishes an efficient and useful tool for whole genome analysis.

     

FRET detection of lymphocyte function–associated antigen-1 conformational extension

Lymphocyte function–associated antigen 1 (LFA-1, CD11a/CD18, αLβ2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1–specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation.