Sensitivity Of Such Methods Research Articles

Relaxation-Assisted Magnetization Transfer Phenomena for a Sensitivity-Enhanced 2D NMR.

2D NOESY and TOCSY play central roles in contemporary NMR. We have recently discussed how solvent-driven exchanges can significantly enhance the sensitivity of such methods when attempting correlations between labile and nonlabile protons. This study explores two scenarios where similar sensitivity enhancements can be achieved in the absence of solvent exchange: the first one involves biomolecular paramagnetic systems, while the other involves small organic molecules in natural abundance. It is shown that, in both cases, the effects introduced by either differential paramagnetic shift and relaxation or by polarization sharing among networks of protons can provide a similar sensitivity boost, as previously discussed for solvent exchange. The origin and potential of the resulting enhancements are analyzed, and experiments that demonstrate them in protein and natural products are exemplified. Limitations and future improvements of these approaches are also briefly discussed.

Sensitivity estimation for dark matter subhalos in synthetic Gaia DR2 using deep learning

The abundance of dark matter subhalos orbiting a host galaxy is a generic prediction of the cosmological framework, and is a promising way to constrain the nature of dark matter. In this paper, we investigate the use of machine learning-based tools to quantify the magnitude of phase-space perturbations caused by the passage of dark matter subhalos. A simple binary classifier and an anomaly detection model are proposed to estimate if stars or star particles close to dark matter subhalos are statistically detectable in simulations. The simulated datasets are three Milky Way-like galaxies and nine synthetic Gaia DR2 surveys derived from these. Firstly, we find that the anomaly detection algorithm, trained on a simulated galaxy with full 6D kinematic observables and applied on another galaxy, is nontrivially sensitive to the dark matter subhalo population. On the other hand, the classification-based approach is not sufficiently sensitive due to the extremely low statistics of signal stars for supervised training. Finally, the sensitivity of both algorithms in the Gaia-like surveys is negligible. The enormous size of the Gaia dataset motivates the further development of scalable and accurate data analysis methods that could be used to select potential regions of interest for dark matter searches to ultimately constrain the Milky Way’s subhalo mass function, as well as simulations where to study the sensitivity of such methods under different signal hypotheses.

CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry.

The quantification of the specific disease-associated populations of circulating extracellular membrane nanovesicles (ENVs) has opened up new opportunities for liquid biopsy in cancer and other chronic diseases. However, the sensitivity of such methods is mediated by an optimal combination of the isolation and labeling approaches, and is not yet sufficient for routine clinical application. The presented study aimed to develop, characterize, and explore a new approach to non-specific ENV staining, followed by size-exclusive chromatography (SEC), which allows us to increase the sensitivity of bead-assisted flow cytometry. Plasma from healthy donors was purified from large components, stained with lipophilic CM-Dil dye, and fractionated by means of SEC. The obtained fractions were analyzed in terms of particle size and concentration using NTA, as well as vesicular markers and plasma protein content via dot-blotting. We characterized the process of CM-Dil-stained plasma fractionation in detail and indicated the fractions with optimal characteristics. Finally, we explored the sensitivity of on-bead flow cytometry for the analysis of specific populations of plasma ENVs and demonstrated the advantages and limitations of the proposed technique.

SeqURE \u2013 a new copy-capture based method for sequencing of unknown Retroposition events

BackgroundRetroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated.ResultsHere we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions.ConclusionsThis study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.

Aptamers: Promising Tools for the Detection of Circulating Tumor Cells

Circulating tumor cells (CTCs) are cells that shed from a primary tumor and freely circulate in the blood, retaining the ability to initiate metastasis and form a secondary tumor in distant organs in the body. CTCs reflect the molecular profile of the primary tumor, therefore studying CTCs can allow for an understanding of the mechanism of metastasis, and an opportunity to monitor the prognosis of cancer. Unfortunately, the detection of CTCs is a considerable challenge due to their low abundance in the bloodstream and the lack of consistent markers present to recognize these cells. The aim of this review is to summarize some of the aptamer-based affinity methods for the detection of CTCs. The basic biological concept of how metastasis occurs and the role of CTCs in this process are presented. Some methods of CTC detection employing antibodies or peptides are mentioned here for comparison. The review of present literature suggests that aptamers are emerging as competitive technology in the detection of CTCs, especially due to their unique properties, but there still remain several challenges to be met, including the need to improve the throughput and sensitivity of such methods.

Current methods of the analysis of immunosuppressive agents in clinical materials: A review

More than 100000 solid organ transplantations are performed every year worldwide. Calcineurin (cyclosporine A, tacrolimus), serine/threonine kinase (sirolimus, everolimus) and inosine monophosphate dehydrogenase inhibitor (mycophenolate mofetil), are the most common drugs used as immunosuppressive agents after solid organ transplantation. Immunosuppressive therapy, although necessary after transplantation, is associated with many adverse consequences, including the formation of secondary metabolites of drugs and the induction of their side effects. Calcineurin inhibitors are associated with nephrotoxicity, cardiotoxicity and neurotoxicity; moreover, they increase the risk of many diseases after transplantation. The review presents a study of the movement of drugs in the body, including the processes of absorption, distribution, localisation in tissues, biotransformation and excretion, and also their accompanying side effects. Therefore, there is a necessity to monitor immunosuppressants, especially because these drugs are characterised by narrow therapeutic ranges. Their incorrect concentrations in a patient’s blood could result in transplant rejection or in the accumulation of toxic effects. Immunosuppressive pharmaceuticals are macrolide lactones, peptides, and high molecular weight molecules that can be metabolised to several metabolites. Therefore the two main analytical methods used for their determination are high performance liquid chromatography with various detection methods and immunoassay methods. Despite the rapid development of new analytical methods of analysing immunosuppressive agents, the application of the latest generation of detectors and increasing sensitivity of such methods, there is still a great demand for the development of highly selective, sensitive, specific, rapid and relatively simple methods of immunosuppressive drugs analysis.

Substitution Model Adequacy and Assessing the Reliability of Estimates of Virus Evolutionary Rates and Time Scales.

Determining the time scale of virus evolution is central to understanding their origins and emergence. The phylogenetic methods commonly used for this purpose can be misleading if the substitution model makes incorrect assumptions about the data. Empirical studies consider a pool of models and select that with the highest statistical fit. However, this does not allow the rejection of all models, even if they poorly describe the data. An alternative is to use model adequacy methods that evaluate the ability of a model to predict hypothetical future observations. This can be done by comparing the empirical data with data generated under the model in question. We conducted simulations to evaluate the sensitivity of such methods with nucleotide, amino acid, and codon data. These effectively detected underparameterized models, but failed to detect mutational saturation and some instances of nonstationary base composition, which can lead to biases in estimates of tree topology and length. To test the applicability of these methods with real data, we analyzed nucleotide and amino acid data sets from the genus Flavivirus of RNA viruses. In most cases these models were inadequate, with the exception of a data set of relatively closely related sequences of Dengue virus, for which the GTR+Γ nucleotide and LG+Γ amino acid substitution models were adequate. Our results partly explain the lack of consensus over estimates of the long-term evolutionary time scale of these viruses, and indicate that assessing the adequacy of substitution models should be routinely used to determine whether estimates are reliable.

Target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery.

Viral infections cause many different diseases stemming both from well-characterized viral pathogens but also from emerging viruses, and the search for novel viruses continues to be of great importance. High-throughput sequencing is an important technology for this purpose. However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue. Techniques to enrich viral targets in high-throughput sequencing have been reported, but the sensitivity of such methods is not well established. This study compares different library preparation techniques targeting both DNA and RNA with and without virion enrichment. By optimizing the selection of intact virus particles, both by physical and enzymatic approaches, we assessed the effectiveness of the specific enrichment of viral sequences as compared to non-enriched sample preparations by selectively looking for and counting read sequences obtained from shotgun sequencing. Using shotgun sequencing of total DNA or RNA, viral targets were detected at concentrations corresponding to the predicted level, providing a foundation for estimating the effectiveness of virion enrichment. Virion enrichment typically produced a 1000-fold increase in the proportion of DNA virus sequences. For RNA virions the gain was less pronounced with a maximum 13-fold increase. This enrichment varied between the different sample concentrations, with no clear trend. Despite that less sequencing was required to identify target sequences, it was not evident from our data that a lower detection level was achieved by virion enrichment compared to shotgun sequencing.

Adaptive Filtering for Robust Proprioceptive Robot Impact Detection Under Model Uncertainties

In the context of safe human–robot physical interaction, this paper introduces a new method for the detection of dynamic impacts of flexible-joint robot manipulators with their environment. The objective is to detect external impacts applied to the robot using only proprioceptive information with maximal sensitivity. Several model-based detection methods in robotics are based on the difference, called residual, between the estimated and the actual applied torques. Sensitivity of such methods can be limited by model uncertainties that originate either from errors on experimentally identified model parameters, possibly varying with the operating conditions, or the use of simplified models, which results in a residual dependence on the robot’s state. The main contribution of this paper consists of a new adaptive residual evaluation method that takes into account this dependence, which otherwise can lead to a tradeoff between sensitivity and false alarm rate. The proposed approach uses only proprioceptive motor-side measurements and does not require any additional joint position sensors or force/torque sensors. Dynamic effects of a collision on the residual are isolated using bandpass filtering and comparison with a state-dependent dynamic threshold. Adaptive online estimation of filter coefficients avoids the need for extensive experiments for parametric model identification. Experimental evaluation on the CEA backdrivable ASSIST robot arm illustrates the enhancement of the detection sensitivity.

Testing systemic fishing responses with ecosystem indicators

Successful implementation of Ecosystem Based Fisheries Management (EBFM) requires practical methods of translating information on system status into management actions. Threshold values in ecosystem indicators have been demonstrated to provide insight for characterizing change points in marine ecosystems and suggested as reference points for EBFM. We used a guild based multispecies simulation model of the Georges Bank finfish community to quantify tradeoffs and changes among values for proposed ecological indicators given alternative fishing scenarios, and tested the performance of indicator-based approaches for setting system ceilings on annual catches.Values for ecosystem indicators were sensitive to the exploitation rates on guilds, with total biomass of the community being most sensitive to groundfish exploitation rate. Setting ceilings on system-wide annual catches was successful in constraining values for indicators and revealed levels of system catch associated with indicator change. Community composition indicators showed catch thresholds lower than provided by the total biomass indicator. Ceilings based on community composition indicators more frequently resulted in higher yields and fewer species being overfished than when ceilings were set using total biomass or when no ceiling was in place.Simulations demonstrated that threshold values in ecosystem indicators could be used to determine reference points in an EBFM context. The broad ranges for threshold values obtained demonstrates the sensitivity of such methods to exploitation history, underscoring the need to both incorporate expert knowledge and relate reference point determination to management objectives.

Evaluation of buffer-radius modelling approaches used in forest conservation and planning

Summary Spatial modelling approaches are increasingly being used to direct forest management and conservation planning at the landscape scale. A popular approach is the use of buffer-radius methods, which create buffers around distinct forest habitat patches to assess habitat connectivity within anthropogenic landscapes. However, the effectiveness and sensitivity of such methods have rarely been evaluated. In this study, Euclidean and least-cost buffer-radius approaches were used to predict functional ecological networks within the wooded landscape of the Isle of Wight (UK). To parameterize the models, a combination of empirical evidence and expert knowledge was used relating to the dispersal ability of a model species, the wood cricket (Nemobius sylvestris Bosc.). Three scenarios were developed to assess the influence of increasing the amount of spatial and species-specific input data on the model outcomes. This revealed that the level of habitat fragmentation for the model species is likely to be underestimated when few empirical data are available. Furthermore, the least-cost buffer approach outperformed simple Euclidean buffer in predicting presence and absence for the model species. Sensitivity analyses on model performance revealed high sensitivity of the models to variation in buffer distance (i.e. maximum dispersal distance) and permeability of common landscape features such as roads, watercourses, grassland and semi-natural habitat. This indicates that when data are lacking with which to parameterize buffer-radius models, the model outcomes need to be interpreted with caution. This study also showed that if sufficient empirical data are available, least-cost buffer approaches have the potential to be a valuable tool to assist forest managers in making informed decisions. However, least-cost approaches should always be used as an indicative rather than prescriptive management tool to support forest landscape conservation and planning.

Reducing the false positive rate in the non-parametric analysis of molecular coevolution

BackgroundThe strength of selective constraints operating on amino acid sites of proteins has a multifactorial nature. In fact, amino acid sites within proteins coevolve due to their functional and/or structural relationships. Different methods have been developed that attempt to account for the evolutionary dependencies between amino acid sites. Researchers have invested a significant effort to increase the sensitivity of such methods. However, the difficulty in disentangling functional co-dependencies from historical covariation has fuelled the scepticism over their power to detect biologically meaningful results. In addition, the biological parameters connecting linear sequence evolution to structure evolution remain elusive. For these reasons, most of the evolutionary studies aimed at identifying functional dependencies among protein domains have focused on the structural properties of proteins rather than on the information extracted from linear multiple sequence alignments (MSA). Non-parametric methods to detect coevolution have been reported to be especially susceptible to produce false positive results based on the properties of MSAs. However, no formal statistical analysis has been performed to definitively test the differential effects of these properties on the sensitivity of such methods.ResultsHere we test the effect that variations on the MSA properties have over the sensitivity of non-parametric methods to detect coevolution. We test the effect that the size of the MSA (number of sequences), mean pairwise amino acid distance per site and the strength of the coevolution signal have on the ability of non-parametric methods to detect coevolution. Our results indicate that all three factors have significant effects on the accuracy of non-parametric methods. Further, introducing statistical filters improves the sensitivity and increases the statistical power of the methods to detect functional coevolution. Statistical analysis of the physico-chemical properties of amino acid sites in the context of the protein structure reveals striking dependencies among amino acid sites. Results indicate a covariation trend in the hydrophobicities and molecular weight characteristics of amino acid sites when analysing a non-redundant set of 8000 protein structures. Using this biological information as filter in coevolutionary analyses minimises the false positive rate of these methods. Application of these filters to three different proteins with known functional domains supports the importance of using biological filters to detect coevolution.ConclusionCoevolutionary analyses using non-parametric methods have proved difficult and highly prone to provide spurious results depending on the properties of MSAs and on the strength of coevolution between amino acid sites. The application of statistical filters to the number of pairs detected as coevolving reduces significantly the number of artifactual results. Analysis of the physico-chemical properties of amino acid sites in the protein structure context reveals their structure-dependent covariation. The application of this known biological information to the analysis of covariation greatly enhances the functional coevolutionary signal and removes historical covariation. Simultaneous use of statistical and biological data is instrumental in the detection of functional amino acid sites dependencies and compensatory changes at the protein level.

Real-time nucleic acid amplification in clinical microbiology

The use of molecular methods in clinical microbiology has increased exponentially over the past two decades. The main reason for this influx of molecular-based methods has been the continued development and improvement of PCR and the introduction of other nucleic acid amplification formats. The exquisite sensitivity of such methods has surpassed even the traditional “gold standards” for diagnostic testing. However, it has also brought an additional awareness of sample contamination containment to laboratories. The implementation of these methods suffered initially because of longer turnaround time than routine methods, even though they provided a sensitivity and specificity not achievable by classic microbiological methods. The recent development of real-time nucleic acid amplification methods has elevated clinical molecular nucleic acid amplification testing to a new level. Assay turnaround time can now be as short as 1 to 2 h because of the simultaneous product amplification and detection step. Additionally, the closed system has essentially reduced the risk of contamination to negligible levels. This article provides a brief review of the principles of real-time PCR and also provides a discussion of various clinical applications of real-time nucleic acid amplification technology.

How is the Drosophila research community making use of the genome sequence?

A report on the 44th Annual Drosophila Research Conference, Chicago, USA, 5-9 March, 2003.

Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part two: colorigenic methods and comparison with chemiluminescent methods

The diagnosis of genetic infections and cancerous diseases is carried out more and more often at a molecular level using Southern's technique which is based on the use of 32P-labelled DNA. In order to circumvent the risks and rapid decrease in radioactivity associated with these latter techniques, new colorigenic methods have been developed. In this work, we describe the use of dTTP analogues (digoxigenin-dUTP and biotin-dUTP) for the labelling of probes and detection of target DNA. Using digoxigenin-11-dUTP, 0.1 aM of a 561 bp target DNA was detected by using a modified Southern procedure. The reliability and the high sensitivity of such methods make them a good tool for DNA investigation in research as well as in testing laboratories.

Viscoelastic and dielectric study of thermally aged ethylene—propylene diene monomer (EPDM) compounds

Three ethylene—propylene—hexadiene-1,4 terpolymer-based rubbers were submitted to thermal ageing in the temperature range 110–135°C. Degradation effects on the rubbers were studied using a direct method, FTIR spectroscopy, and two indirect methods, dynamic mechanical analysis (DMA) and dielectrical analysis (DEA). The necessary sensitivity of such methods for the determination of the molecular effects of thermal ageing has been clearly illustrated. Changes in the main relaxation peak height allow the thermooxidation to be quantified.

Detection of aberrations in the occurrence of notifiable diseases surveillance data

The detection of unusual patterns in the occurrence of diseases and other health events presents an important challenge to public health surveillance. This paper discusses three analytic methods for identifying aberrations in underlying distributions. The methods are illustrated on selected infectious diseases included in the National Notifiable Diseases Surveillance System of the Centers for Disease Control. Results suggest the utility of such an analytic approach. Further work will determine the sensitivity of such methods to variations in the occurrence of disease. These methods are useful for evaluating and monitoring public health surveillance data.

Influence of glutathione on the formation of cysteine alkylation products in human hemoglobin

Human blood samples were treated in vitro with iodoacetamide. At low concentrations - less than 1 mM - only a low fraction of the β93 cysteine in hemoglobin was alkylated, whereas the alkylating reaction with glutathione was extensive. At higher iodoacetamide concentrations the glutathione pool became exhausted leading to more than proportional increases in the alkylation of the sulfhydryl group in hemoglobin. When diethyl maleate was used as a glutathione depletor prior to incubation with iodoacetamide, low concentrations of iodoacetamide were sufficient to obtain high degrees of hemoglobin sulfhydryl alkylation. N-Ethylmaleimide could not be used as glutathione depletor because the reaction with glutathione appeared to be reversible. The lower reactivity of the thiol group in hemoglobin in comparison with that of glutathione was also found for the isolated biomolecules. The protection of hemoglobin by glutathione present in the human erythrocyte renders the measurement of hemoglobin alkylation less attractive for biological effect monitoring. The sensitivity of such methods is lowered, while the important relation between the alkylation of hemoglobin and that of DNA in the target tissues is affected by interindividual differences in the ratios of the effectiveness of glutathione protection between erythrocytes and target cells.

The Role of Viruses in Systemic Lupus Erythematosus

SUMMARY AND CONCLUSIONS The hypothesis that a chronic virus infection causes human SLE is based largely on the model of New Zealand mouse disease, where type C viruses are involved. Directevidence islacking, butrecent studies suggest there may be enhancedexpression oftypeC virus antigens in human SLE. This is compatible with the hypothesis that a vertically transmitted type C virus causes defective cell-mediated immunity in genetically predisposed individuals, the result ofwhich isenhancedproductionofviral antigens, a hyper-reactive antibody response and immunologically-mediated disease. Future studies will utilise newly developed and increasingly sensitive methods for isolation and detection ofvirus and viral components. The isolation of a true human type C virus(es)would lead to an increase in the sensitivity of such methods. This shouldallow the hypothesisthata virus causes SLEto beprovenor notwithin five years.