Nonspecific PCR amplification by high-fidelity polymerases: implications for next-generation sequencing of AFLP markers.

High-fidelity 'proofreading' polymerases are often used in library construction for next-generation sequencing projects, in an effort to minimize errors in the resulting sequence data. The increased template fidelity of these polymerases can come at the cost of reduced template specificity, and library preparation methods based on the AFLP technique may be particularly susceptible. Here, we compare AFLP profiles generated with standard Taq and two versions of a high-fidelity polymerase. We find that Taq produces fewer and brighter peaks than high-fidelity polymerase, suggesting that Taq performs better at selectively amplifying templates that exactly match the primer sequences. Because the higher accuracy of proofreading polymerases remains important for sequencing applications, we suggest that it may be more effective to use alternative library preparation methods.

Probe-specific mixed-model approach to detect copy number differences using multiplex ligation-dependent probe amplification (MLPA)

Background: MLPA method is a potentially useful semi-quantitative method to detect copy number alterations in targeted regions. In this paper, we propose a method for the normalization procedure based on a non-linear mixed-model, as well as a new approach for determining the statistical significance of altered probes based on linear mixed-model. This method establishes a threshold by using different tolerance intervals that accommodates the specific random error variability observed in each test sample.Results: Through simulation studies we have shown that our proposed method outperforms two existing methods that are based on simple threshold rules or iterative regression. We have illustrated the method using a controlled MLPA assay in which targeted regions are variable in copy number in individuals suffering from different disorders such as Prader-Willi, DiGeorge or Autism showing the best performace.Conclusion: Using the proposed mixed-model, we are able to determine thresholds to decide whether a region is altered. These threholds are specific for each individual, incorporating experimental variability, resulting in improved sensitivity and specificity as the examples with real data have revealed.

Parametric Approach to Blind Deconvolution of Nonlinear Channels

A parametric procedure for the blind inversion of nonlinear channels is proposed, based on a recent method of blind source separation in nonlinear mixtures. Experiments show that the proposed algorithms perform efficiently, even in the presence of hard distortion. The method, based on the minimization of the output mutual information, needs the knowledge of log-derivative of input distribution (the so-called score function). Each algorithm consists of three adaptive blocks: one devoted to adaptive estimation of the score function, and two other blocks estimating the inverses of the linear and nonlinear parts of the channel, (quasi-)optimally adapted using the estimated score functions. This paper is mainly concerned by the nonlinear part, for which we propose two parametric models, the first based on a polynomial model and the second on a neural network, while [14, 15] proposed non-parametric approaches.

Amplification of 16S rRNA gene sequences to differentiate two highly related bradyrhizobia species

A 16S rRNA gene PCR-based assay was developed aiming at a fast molecular diagnostic method to differentiate the two phylogenetically closely related species Bradyrhizobium japonicum and B. elkanii, isolated from soybean nodules, in order to identify those more competitive and comprising greater nitrogen fixation ability for use in the formulation of commercial inoculants. The assay used was able to discriminate ten reference strains belonging to these two Bradyrhizobium species, as well as to efficiently identify 37 strains isolated from fields cultivated with soybean.

Robust parametric indirect estimates of the expected cost of a hospital stay with covariates and censored data.

We consider the problem of estimating the mean hospital cost of stays of a class of patients (e.g., a diagnosis-related group) as a function of patient characteristics. The statistical analysis is complicated by the asymmetry of the cost distribution, the possibility of censoring on the cost variable, and the occurrence of outliers. These problems have often been treated separately in the literature, and a method offering a joint solution to all of them is still missing. Indirect procedures have been proposed, combining an estimate of the duration distribution with an estimate of the conditional cost for a given duration. We propose a parametric version of this approach, allowing for asymmetry and censoring in the cost distribution and providing a mean cost estimator that is robust in the presence of extreme values. In addition, the new method takes covariate information into account.

Molecular investigation of lymph nodes in colon cancer patients using one-step nucleic acid amplification (OSNA): a new road to better staging?

BACKGROUND: A new diagnostic system, called one-step nucleic acid amplification (OSNA), has recently been designed to detect cytokeratin 19 mRNA as a surrogate for lymph node metastases. The objective of this prospective investigation was to compare the performance of OSNA with both standard hematoxylin and eosin (H&E) analysis and intensive histopathology in the detection of colon cancer lymph node metastases. METHODS: In total, 313 lymph nodes from 22 consecutive patients with stage I, II, and III colon cancer were assessed. Half of each lymph node was analyzed initially by H&E followed by an intensive histologic workup (5 levels of H&E and immunohistochemistry analyses, the gold standard for the assessment of sensitivity/specificity of OSNA), and the other half was analyzed using OSNA. RESULTS: OSNA was more sensitive in detecting small lymph node tumor infiltrates compared with H&E (11 results were OSNA positive/H&E negative). Compared with intensive histopathology, OSNA had 94.5% sensitivity, 97.6% specificity, and a concordance rate of 97.1%. OSNA resulted in an upstaging of 2 of 13 patients (15.3%) with lymph node-negative colon cancer after standard H&E examination. CONCLUSIONS: OSNA appeared to be a powerful and promising molecular tool for the detection of lymph node metastases in patients with colon cancer. OSNA had similar performance in the detection of lymph node metastases compared with intensive histopathologic investigations and appeared to be superior to standard histology with H&E. Most important, the authors concluded that OSNA may lead to a potential upstaging of >15% of patients with colon cancer.

One-step nucleic acid amplification (OSNA): a new road to better staging in colon cancer patients?

Introduction: Approximately one fifth of stage I and II colon cancer patients will suffer from recurrent disease. This is partly due to the presence of small nodal tumour infiltrates, which are undetected by standard histopathology using Haematoxylin & Eosin (H&E) staining on one slice and thus may not receive beneficial adjuvant therapy. A new diagnostic, semi-automatic system, called one-step nucleic acid amplification (OSNA), was recently designed for the detection of cytokeratin 19 (CK19) mRNA as a surrogate for lymph node metastases. The objective of the present investigation was to compare the performance of OSNA with both standard H&E as well as intensive histopathologic analyses in the detection of colon cancer lymph node micro- and macro-metastases.Methods: In this prospective study 313 lymph nodes from 22 consecutive stage I - III colon cancer patients were assessed. Half of each lymph node was analysed initially based on one slice of H&E followed by an intensive histologic work-up (5 levels of H&E and immuno-histochemistry staining for each slice), the other half was analysed using OSNA.Results: All OSNA results were available after less than 40 minutes. Fifty-one lymph nodes were positive and 246 lymph nodes negative with both OSNA and standard H&E. OSNA was more sensitive to detect small nodal tumor infiltrates compared to H&E (11 OSNA pos. /H&E neg.). Compared to intensive histopathologic analyses, OSNA had a sensitivity of 94.5% and a specificity of 97.6% to detect lymph node micro- and macro-metastases with a concordance rate of 97.1%. An upstaging due to OSNA was found in 2/13 (15.3%) initially node negative colon cancer patients.Conclusion: OSNA appears to be a powerful and promising molecular tool for the detection of lymph node macro- and micro-metastases in colon cancer patients. OSNA has a similar performance in the detection of micro- and macro-metastases compared to intensive histopathologic investigations and appears to be superior to standard histology with H&E. Since the use of OSNA allows the analysis of the whole lymph node, the problem of sampling bias and undetected tumor deposits due to uninvestigated material will be overcome in the future and OSNA may thus improve staging in colon cancer patients. It is hoped that this improved staging will lead to better patient selection for adjuvant therapy and consecutively improved local and distant control as well as better overall survival.

Pediatric reference intervals for plasma free and total metanephrines established with a parametric approach: Relevance to the diagnosis of neuroblastoma.

BACKGROUND: Urine catecholamines, vanillylmandelic, and homovanillic acid are recognized biomarkers for the diagnosis and follow-up of neuroblastoma. Plasma free (f) and total (t) normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MT) could represent a convenient alternative to those urine markers. The primary objective of this study was to establish pediatric centile charts for plasma metanephrines. Secondarily, we explored their diagnostic performance in 10 patients with neuroblastoma. PROCEDURE: We recruited 191 children (69 females) free of neuroendocrine disease to establish reference intervals for plasma metanephrines, reported as centile curves for a given age and sex based on a parametric method using fractional polynomials models. Urine markers and plasma metanephrines were measured in 10 children with neuroblastoma at diagnosis. Plasma total metanephrines were measured by HPLC with coulometric detection and plasma free metanephrines by tandem LC-MS. RESULTS: We observed a significant age-dependence for tNMN, fNMN, and fMN, and a gender and age-dependence for tMN, fNMN, and fMN. Free MT was below the lower limit of quantification in 94% of the children. All patients with neuroblastoma at diagnosis were above the 97.5th percentile for tMT, tNMN, fNMN, and fMT, whereas their fMN and tMN were mostly within the normal range. As expected, urine assays were inconstantly predictive of the disease. CONCLUSIONS: A continuous model incorporating all data for a given analyte represents an appealing alternative to arbitrary partitioning of reference intervals across age categories. Plasma metanephrines are promising biomarkers for neuroblastoma, and their performances need to be confirmed in a prospective study on a large cohort of patients. Pediatr Blood Cancer 2015;62:587-593. © 2015 Wiley Periodicals, Inc.

Amplification and displacement of chaotic attractors by means of unidirectional chaotic driving

Chaotic systems, when used to drive copies of themselves (or parts of themselves) may induce interesting behaviors in the driven system. In case the later exhibits invariance under amplification or translation, they may show amplification (reduction), or displacement of the attractor. It is shown how the behavior to be obtained is implied by the symmetries involved. Two explicit examples are studied to show how these phenomena manifest themselves under perfect and imperfect coupling.

Identification of parasitoid species using PCR amplification and restriction enzyme digestion

Spodoptera frugiperda is a pest of great economic importance in the Americas. It is attacked by several species of parasitoids, which act as biological control agents. Parasitoids are morphologically identifiable as adults, but not as larvae. Laboratory rearing conditions are not always optimal to rear out parasitic wasps from S. frugiperda larvae collected from wild populations, and it frequently happens that parasitoids do not complete their life cycle and stop developing at the larval stage. Therefore, we explored ways to identify parasitoid larvae using molecular techniques. Sequencing is one possible technique, yet it is expensive. Here we present an alternate, cheaper way of identifying seven species of parasitoids (Cotesia marginiventris, Campoletis sonorensis, Pristomerus spinator, Chelonus insularis, Chelonus cautus, Eiphosoma vitticolle and Meteorus laphygmae) using PCR amplification of COI gene followed by a digestion with a combination of four restriction endonucleases. Each species was found to exhibit a specific pattern when the amplification product was run on an agarose gel. Identifying larvae revealed that conclusions on species composition of a population of parasitic wasps can be biased if only the emerging adults are taken into account.

Univariate Parametric Survival Analysis using GS-distributions

The GS-distribution is a family of distributions that provide an accurate representation of any unimodal univariate continuous distribution. In this contribution we explore the utility of this family as a general model in survival analysis. We show that the survival function based on the GS-distribution is able to provide a model for univariate survival data and that appropriate estimates can be obtained. We develop some hypotheses tests that can be used for checking the underlying survival model and for comparing the survival of different groups.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

Continuous quantification of HER2 expression by microfluidic precision immunofluorescence estimates HER2 gene amplification in breast cancer.

Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis. We show that the level of HER2 protein expression, as continuously quantified using microfluidic precision IF in 25 breast cancer cases, including several cases with equivocal IHC result, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization (FISH). Finally, we demonstrate that the working principle of this technology is not restricted to HER2 but can be extended to other biomarkers. We anticipate that our method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy.