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PubMed2006

Robustness of single-base extension against mismatches at the site of primer attachment in a clinical assay.

DNA genotyping is important for epidemiological and clinical studies and diagnosis for individuals. Genotyping error can strongly influence the outcome of such investigations. One possible reason for genotyping error is additional DNA sequence variation, which can lead to allelic dropout. Based on a published study where allelic dropout occurred in genotyping the cholesteryl ester transfer protein TaqIB polymorphism by a TaqMan-based method, we investigated the susceptibility of the single-base extension (SBE)-based GenoSNIP method to additional sequence variation at the primer attachment site. SBE genotyping was applied to 147 patient samples with known alleles and to synthetic SBE templates. Variables were positions of nucleotide mismatches, yield of SBE reactions, primer design, and ratio of alleles in the template. No allelic dropout occurred when genotyping the TaqIB polymorphism regardless of the reported nucleotide mismatch. Yields of SBE assays critical for allelic dropout were decreased in the presence of the reported nucleotide mismatch depending on SBE assay design. In a systematic mutation scan, only the position immediately adjacent to the polymorphism caused allelic dropout under standard conditions. Depending on SBE assay design, changes in allelic ratio due to a nucleotide mismatch were similar in appearance to changes due to sample mixture or copy number variation. In conclusion, we found the SBE genotyping assays to be relatively robust against interfering DNA variations. The importance of appropriate design and validation of assays, especially in regard to critical yields and potentially interfering nucleotide mismatches, should be emphasized particularly in clinical settings. Care should be taken when interpreting observed changes in the allelic ratio, which could be caused by nucleotide mismatches, sample mixtures, or copy number variation.

Alleles↗
PubMed2002

A new giant pterosaur with a robust skull from the latest cretaceous of Romania.

A new giant pterosaur, Hatzegopteryx thambema, nov.gen., nov.sp., from the Maastrichtian Densuş-Ciula Formation of Romania is remarkable for its very large size (estimated wing span > or = 12 m) and for the robustness of its large skull, which may have been nearly 3 m long. The stout skull bones contrast with the usually thin and slender skull elements of other pterosaurs, and raise the question of how the weight of the skull was reduced in order to make flight possible. The answer probably lies in the very peculiar internal structure of the bones, which consists of a dense network of very thin trabeculae enclosing small alveoli. This structure is reminiscent of expanded polystyrene and, like it, probably combined strength with lightness.

Animals↗
PubMed2003

Landmark memories are more robust when acquired at the nest site than en route: experiments in desert ants.

Foraging desert ants, Cataglyphis fortis, encounter different sequences of visual landmarks while navigating by path integration. This paper explores the question whether the storage of landmark information depends on the context in which the landmarks are learned during an ant's foraging journey. Two experimental set-ups were designed in which the ants experienced an artificial landmark panorama that was placed either around the nest entrance (nest marks) or along the vector route leading straight towards the feeder (route marks). The two training paradigms resulted in pronounced differences in the storage characteristics of the acquired landmark information: memory traces of nest marks were much more robust against extinction and/or suppression than those of route marks. In functional terms, this result is in accord with the observation that desert ants encounter new route marks during every foraging run but always pass the same landmarks when approaching the nest entrance.

Animals↗
PubMed2002

Identification of cut rose (Rosa hybrida) and rootstock varieties using robust sequence tagged microsatellite site markers.

In this study a DNA fingerprinting protocol was developed for the identification of rose varieties based on the variability of microsatellites. Microsatellites were isolated from Rosa hybrida L. using enriched small insert libraries. In total 24 polymorphic sequenced tagged microsatellite site (STMS) markers with easily scorable allele profiles, from six different linkage groups, were used to characterize 46 Hybrid Tea varieties and 30 rootstock varieties belonging to different species (Rosa canina L., Rosa indica Thory., Rosa chinensis Jacq., Rosa rubiginosa L., and Rosa rubrifolia glauca Pour.). Clones and known flower color mutants were identified as being identical, all other varieties were differentiated by a unique pattern with as few as three STMS markers. The high discriminating power of the loci suggests that a selection of the most-robust STMS markers may be able to differentiate any two varieties within rootstocks or Hybrid Teas except for mutants. The selected STMS markers will be useful as a tool for reference collection management, for assessing essential derivation of varieties and illegal propagation.

Alleles↗
PubMed2003

A simple and robust set-up for on-column sample preconcentration--nano-liquid chromatography--electrospray ionization mass spectrometry for the analysis of N-acylhomoserine lactones.

A simple method for the simultaneous, rapid and sensitive determination of N-acylhomoserine lactone signaling molecules in bacterial isolates, without prior sample preconcentration and with minimal sample cleanup, is presented. The analysis relies on the combination of analyte preconcentration and separation on a single device: a relatively large sample volume (1-5 microL) is directly loaded onto a laboratory-made, miniaturized (75 microm i. d.) reverse phase nano-liquid chromatography column, connected on-line to a microelectrospray-ionization ion trap mass spectrometer. In a first step the analyte is adsorbed (and so concentrated) at the beginning of the column, and is eluted and selectively separated in a second step by the organic mobile phase. Sample preconcentration follows the mechanisms of solid phase extraction on a nano-scale, while separation takes place according to classical liquid chromatography separation principles. The columns can be manufactured easily, are simply connected, and used with minimal solvent amounts; this makes this method extremely robust and cost-effective. The analytical setup was found to be routinely quantitative down to a concentration of 10 ng/mL (corresponding to a total analyte amount of 10 pg or ca. 50 fmol). The limit of detection was reached at 1 ng/mL (1 pg, ca. 5 fmol). Compared to the classical AHL analysis of bacterial cultures with biosensors, where selectivity and sensitivity is often limited, this rapid analytical technique is a substantial qualitative and quantitative improvement. Two unsubstituted N-acylhomoserine lactones could be identified and quantified from a Burkholderia cepacia culture supernatant in a chloroform extract.

4-Butyrolactone↗
PubMed2005

Development and validation of a selective and robust LC-MS/MS method for quantifying amlodipine in human plasma.

A liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) for quantifying amlodipine in human plasma was developed and validated. Sample preparation was based on liquid-liquid extraction using NaOH and a mixture of ethyl acetate/hexane (80/20; v/v). Chromatography was performed on a C-18 analytical column and the retention times were 1.9 and 3.0 min for amlodipine and nimodipine (internal standard), respectively. The ionization was optimized using ESI(+) and enhanced selectivity was achieved using tandem mass spectrometric analysis via two MRM functions, 409 --> 238 and 418 --> 343 for amlodipine and nimodipine. The calibration curve ranged from 0.2 to 20.0 ng/mL. The inter-day precision and accuracy and the relative standard deviation (RSD) were <15%. The analyte was shown to be stable over the time-scale of the whole procedure. The robustness of the method was demonstrated by the good reproducibility of the results obtained during the analysis of clinical samples.

Amlodipine↗
PubMed2005

Surviving high-intensity field pulses: strategies for improving robustness and performance of electrotransfection and electrofusion.

Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10-20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.

Animals↗
PubMed2004

Recent mammalian gene duplications: robust search for functionally divergent gene pairs.

Comparison of 317 gene pairs in human and mouse that were duplicated after the most recent common ancestor of the two species was used to search for candidates that may have undergone functional differentiation. Even when corrected for multiple tests, Tajima's relative rate test showed significant rate differences in 36% of cases for which the test was applicable. However, a significant result in this case was increasingly likely as the sequence length increased; thus, a statistically significant result of a relative rate test may not be biologically meaningful. We used regression methods to provide more robust methods of testing for functionally differentiated gene pairs, which take into account the variation in the entire data set by examination of residuals from regression-identified gene pairs with unusually high nonsynonymous divergence from a reference sequence and from each other. This approach identified six duplicate gene pairs that appeared to be candidates for functional differentiation as a result of positive Darwinian selection.

Amino Acid Sequence↗
PubMed2005

Expanding metabolic networks: scopes of compounds, robustness, and evolution.

A new method for the mathematical analysis of large metabolic networks is presented. Based on the fact that the occurrence of a metabolic reaction generally requires the existence of other reactions providing its substrates, series of metabolic networks are constructed. In each step of the corresponding expansion process those reactions are incorporated whose substrates are made available by the networks of the previous generations. The method is applied to the set of all metabolic reactions included in the KEGG database. Starting with one or more seed compounds, the expansion results in a final network whose compounds define the scope of the seed. Scopes of all metabolic compounds are calculated and it is shown that large parts of cellular metabolism can be considered as the combined scope of simple building blocks. Analyses of various expansion processes reveal crucial metabolites whose incorporation allows for the increase in network complexity. Among these metabolites are common cofactors such as NAD+, ATP, and coenzyme A. We demonstrate that the outcome of network expansion is in general very robust against elimination of single or few reactions. There exist, however, crucial reactions whose elimination results in a dramatic reduction of scope sizes. It is hypothesized that the expansion process displays characteristics of the evolution of metabolism such as the temporal order of the emergence of metabolic pathways.

Adenosine Triphosphate↗
PubMed2006

Using confidence set heuristics during topology search improves the robustness of phylogenetic inference.

We examine the impact of likelihood surface characteristics on phylogenetic inference. Amino acid data sets simulated from topologies with branch length features chosen to represent varying degrees of difficulty for likelihood maximization are analyzed. We present situations where the tree found to achieve the global maximum in likelihood is often not equal to the true tree. We use the program covSEARCH to demonstrate how the use of adaptively sized pools of candidate trees that are updated using confidence tests results in solution sets that are highly likely to contain the true tree. This approach requires more computation than traditional maximum likelihood methods, hence covSEARCH is best suited to small to medium-sized alignments or large alignments with some constrained nodes. The majority rule consensus tree computed from the confidence sets also proves to be different from the generating topology. Although low phylogenetic signal in the input alignment can result in large confidence sets of trees, some biological information can still be obtained based on nodes that exhibit high support within the confidence set. Two real data examples are analyzed: mammal mitochondrial proteins and a small tubulin alignment. We conclude that the technique of confidence set optimization can significantly improve the robustness of phylogenetic inference at a reasonable computational cost. Additionally, when either very short internal branches or very long terminal branches are present, confident resolution of specific bipartitions or subtrees, rather than whole-tree phylogenies, may be the most realistic goal for phylogenetic methods.

Algorithms↗
PubMed2000

The robust statistical bases of the coevolution theory of genetic code origin.

A paper (Amirnovin R, J Mol Evol 44:473-476, 1997) seems to undermine the validity of the coevolution theory of genetic code origin by shedding doubt on the connection between the biosynthetic relationships between amino acids and the organization of the genetic code, at a time when the literature on the topic takes this for granted. However, as a few papers cite this paper as evidence against the coevolution theory, and to cast aside all doubt on the subject, we have decided to reanalyze the statistical bases on which this theory is founded. We come to the following conclusions: (1) the methods used in the above referred paper contain certain mistakes, and (2) the statistical foundations on which the coevolution theory is based are extremely robust. We have done this by critically appraising Amirnovin's paper and suggesting an alternative method based on the generation of random codes which, along with the method reported in the literature, allows us to evaluate the significance, in the genetic code, of different sets of amino acid pairs in biosynthetic relationships. In particular, by using this method and after building up a certain set of amino acid pairs reflecting the expectations of the coevolution theory, we show that the presence of this set in the genetic code would be obtained, purely by chance, with a probability of 6x10(-5). This observation seems to provide particularly strong support to the coevolution theory.

Amino Acids↗
PubMed2001

Robust processing of intracranial CT angiograms for 3D volume rendering.

The goal of this study was to develop a robust and simple technique for processing of cranial CT angiograms (CTA) in the clinical setting. The method described in this paper involves segmentation of the bone, then dilation of the skull by adding three or four layers of voxels. This dilated skull is subtracted from the vessels object on a voxel-by-voxel basis, allowing segmentation and subsequent display of the vessels only. For evaluation of the technique, three groups of operators processed one CTA, and the quality of the 3D views obtained and the times taken were compared. One group was given training by an expert and a "recipe" for guidance, the second was given only the "recipe," and the third group consisted of expert operators. All operators were able to produce good or acceptable shaded-surface displays when compared with digital subtraction angiography, within 10 min for experienced users, an average of 17 min for trained operators and 26 min for those using only the recipe sheet. Using a simple scoring system for the appearance of feeding vessels and draining veins, no significant differences were found between the three levels of training and experience. This technique simplifies the processing of CTAs and is quick enough to make such examinations part of a routine clinical service.

Cerebral Angiography↗
PubMed2003

Robustness of neuroprosthetic decoding algorithms.

We assessed the ability of two algorithms to predict hand kinematics from neural activity as a function of the amount of data used to determine the algorithm parameters. Using chronically implanted intracortical arrays, single- and multineuron discharge was recorded during trained step tracking and slow continuous tracking tasks in macaque monkeys. The effect of increasing the amount of data used to build a neural decoding model on the ability of that model to predict hand kinematics accurately was examined. We evaluated how well a maximum-likelihood model classified discrete reaching directions and how well a linear filter model reconstructed continuous hand positions over time within and across days. For each of these two models we asked two questions: (1) How does classification performance change as the amount of data the model is built upon increases? (2) How does varying the time interval between the data used to build the model and the data used to test the model affect reconstruction? Less than 1 min of data for the discrete task (8 to 13 neurons) and less than 3 min (8 to 18 neurons) for the continuous task were required to build optimal models. Optimal performance was defined by a cost function we derived that reflects both the ability of the model to predict kinematics accurately and the cost of taking more time to build such models. For both the maximum-likelihood classifier and the linear filter model, increasing the duration between the time of building and testing the model within a day did not cause any significant trend of degradation or improvement in performance. Linear filters built on one day and tested on neural data on a subsequent day generated error-measure distributions that were not significantly different from those generated when the linear filters were tested on neural data from the initial day (p<0.05, Kolmogorov-Smirnov test). These data show that only a small amount of data from a limited number of cortical neurons appears to be necessary to construct robust models to predict kinematic parameters for the subsequent hours. Motor-control signals derived from neurons in motor cortex can be reliably acquired for use in neural prosthetic devices. Adequate decoding models can be built rapidly from small numbers of cells and maintained with daily calibration sessions.

Action Potentials↗
PubMed2002

Universal, robust, highly quantitative SNP allele frequency measurement in DNA pools.

Detecting alleles that confer small increments in susceptibility to disease will require large-scale allelic association studies of single-nucleotide polymorphisms (SNPs) in candidate, or positional candidate, genes. However, current genotyping technologies are one to two orders of magnitude too expensive to permit the analysis of thousands of SNPs in large samples. We have developed and thoroughly validated a highly accurate protocol for SNP allele frequency estimation in DNA pools based upon the SNaPshot (Applied Biosystems) chemistry adaptation of primer extension. Using this assay, we were able to estimate the difference in allele frequencies between pooled cases and controls (Delta) with a mean error of 0.01. Moreover, when we genotyped seven different SNPs in a single multiplex reaction, the results were similar, with a mean error for Delta of 0.008. The assay performed well for alleles of low frequency alleles (f approximately 0.05) and was accurate even with relatively poor quality DNA template extracted from mouthwashes. Our assay conditions are generalisable, universal, robust and, therefore, for the first time, permit high-throughput association analysis at a realistic cost.

DNA↗
PubMed2004

Origin and spread of the 1278insTATC mutation causing Tay-Sachs disease in Ashkenazi Jews: genetic drift as a robust and parsimonious hypothesis.

The 1278insTATC is the most prevalent beta-hexosaminidase A ( HEXA) gene mutation causing Tay-Sachs disease (TSD), one of the four lysosomal storage diseases (LSDs) occurring at elevated frequencies among Ashkenazi Jews (AJs). To investigate the genetic history of this mutation in the AJ population, a conserved haplotype (D15S981:175-D15S131:240-D15S1050:284-D15S197:144-D15S188:418) was identified in 1278insTATC chromosomes from 55 unrelated AJ individuals (15 homozygotes and 40 heterozygotes for the TSD mutation), suggesting the occurrence of a common founder. When two methods were used for analysis of linkage disequilibrium (LD) between flanking polymorphic markers and the disease locus and for the study of the decay of LD over time, the estimated age of the insertion was found to be 40+/-12 generations (95% confidence interval: 30-50 generations), so that the most recent common ancestor of the mutation-bearing chromosomes would date to the 8th-9th century. This corresponds with the demographic expansion of AJs in central Europe, following the founding of the Ashkenaz settlement in the early Middle Ages. The results are consistent with the geographic distribution of the main TSD mutation, 1278insTATC being more common in central Europe, and with the coalescent times of mutations causing two other LSDs, Gaucher disease and mucolipidosis type IV. Evidence for the absence of a determinant positive selection (heterozygote advantage) over the mutation is provided by a comparison between the estimated age of 1278insTATC and the probability of the current AJ frequency of the mutant allele as a function of its age, calculated by use of a branching-process model. Therefore, the founder effect in a rapidly expanding population arising from a bottleneck provides a robust parsimonious hypothesis explaining the spread of 1278insTATC-linked TSD in AJ individuals.

Alleles↗
PubMed2005

Identification and characterisation of soluble epoxide hydrolase in mouse brain by a robust protein biochemical method.

The central nervous system is an important potential target for certain environmental prototoxins, but relatively little is known regarding brain-specific expression of biotransformation enzyme systems. On the other hand, developments in the field of molecular biology and advances in high-throughput screening methods continue to increase the number and amounts of available proteins. We used thus a robust and reliable technique, two-dimensional gel electrophoresis coupled to matrix assisted laser desorption/ionisation mass spectroscopy followed by tandem mass spectrometry and identified for the first time soluble epoxide hydrolase and added other biotransformation enzymes in the hippocampal region of mouse brain. Soluble epoxide hydrolase has an Mr of 61.5 kDa, pI of 5.9, twenty-six matching peptides and sequence coverage of 56% and was unambiguously identified by MS/MS. Since localised biotransformation events in regions of the central nervous system may account for pathologies and/or toxicities initiated by exposure to certain endogenous and/or environmental chemicals, identification of these enzymes would present an opportunity for developing novel therapeutic targets or would have critical toxicologic significance.

Amino Acid Sequence↗
PubMed2003

Robust microsatellite instability (MSI) analysis by denaturing high-performance liquid chromatography (DHPLC).

Microsatellite instability (MSI) plays an important biological role in various types of cancers, and especially in colorectal cancers. This study aimed to develop a simple, efficient, new method for robust MSI analysis. DNA was extracted from 175 (105 proximal colon and 70 distal colorectal) cancer samples and matched normal tissues, and five Bethesda microsatellite markers (BAT-25, BAT-26, D5S346, D2S123, and D17S250) were examined for MSI by denaturing high-performance liquid chromatography (DHPLC) analysis at a temperature of 50 degrees C and a flow rate of 0.9 ml/min. It took just 9 min per PCR product to determine MSI or microsatellite stability (MSS) using the new protocol. The DHPLC results were confirmed with conventional gel-based electrophoresis and capillary-based sequencing method. Of 175 samples, 45 (26%) showed high microsatellite instability (MSI-H), 12 (7%) showed low microsatellite instability (MSI-L), and 118 (67%) showed MSS. All MSI samples were deletion mutants and all 12 MSI-L cases had MSI in dinucleotide markers (D5S346, D2S123, and D17S250). MSI was significantly associated with proximal colon cancers ( p<0.0001), as previously reported. The MSI-H tumors were also associated with tumor node metastasis (TNM) I/II stages ( p=0.05) and high-grade tumors ( p<0.01). Here, we propose a DHPLC-based method as an alternative for MSI analysis.

Chemistry Techniques, Analytical↗
PubMed2005

Robust micromechanical neurite elicitation in synapse-competent neurons via magnetic bead force application.

The ability to engineer living networks of interconnected neurons with specified connectivity would facilitate the study of synaptogenesis and information processing in the nervous system. Previously, we found that a neurite can be elicited from embryonic chick forebrain neurons by direct mechanical means using magnetic bead force application (MBFA); however, our previous studies and others focused on young, synapse-incompetent neurons. To address this issue, we tested cultures of embryonic chick forebrain neurons of varying age and found that neurites could be micromechanically elicited via MBFA at all ages tested, which ranged between 7 and 22 embryonic equivalent (EE) days (days in ovo plus days in vitro). The probability of neurite initiation was at least 40% for all ages, with a maximum of approximately 80% after 2-4 days in vitro, and a decrease to approximately 60% by day 10 in vitro. The force required to elicit a neurite was approximately 1500 pN with a minimum of approximately 700 pN at embryonic equivalent day 14. The probability of success was similar for two rates of force application (10 and 500 pN/s). Neurite initiation via micromechanical force is robust with respect to cell age, and micromechanical force can induce neurites in synapse-competent neurons.

Animals↗