Dynamics of breast-cancer relapse reveal late-recurring ER-positive genomic subgroups.

The rates and routes of lethal systemic spread in breast cancer are poorly understood owing to a lack of molecularly characterized patient cohorts with long-term, detailed follow-up data. Long-term follow-up is especially important for those with oestrogen-receptor (ER)-positive breast cancers, which can recur up to two decades after initial diagnosis1-6. It is therefore essential to identify patients who have a high risk of late relapse7-9. Here we present a statistical framework that models distinct disease stages (locoregional recurrence, distant recurrence, breast-cancer-related death and death from other causes) and competing risks of mortality from breast cancer, while yielding individual risk-of-recurrence predictions. We apply this model to 3,240 patients with breast cancer, including 1,980 for whom molecular data are available, and delineate spatiotemporal patterns of relapse across different categories of molecular information (namely immunohistochemical subtypes; PAM50 subtypes, which are based on gene-expression patterns10,11; and integrative or IntClust subtypes, which are based on patterns of genomic copy-number alterations and gene expression12,13). We identify four late-recurring integrative subtypes, comprising about one quarter (26%) of tumours that are both positive for ER and negative for human epidermal growth factor receptor 2, each with characteristic tumour-driving alterations in genomic copy number and a high risk of recurrence (mean 47-62%) up to 20 years after diagnosis. We also define a subgroup of triple-negative breast cancers in which cancer rarely recurs after five years, and a separate subgroup in which patients remain at risk. Use of the integrative subtypes improves the prediction of late, distant relapse beyond what is possible with clinical covariates (nodal status, tumour size, tumour grade and immunohistochemical subtype). These findings highlight opportunities for improved patient stratification and biomarker-driven clinical trials.

CIRCUST: A novel methodology for temporal order reconstruction of molecular rhythms; validation and application towards a daily rhythm gene expression atlas in humans.

The circadian system drives near-24-h oscillations in behaviors and biological processes. The underlying core molecular clock regulates the expression of other genes, and it has been shown that the expression of more than 50 percent of genes in mammals displays 24-h rhythmic patterns, with the specific genes that cycle varying from one tissue to another. Determining rhythmic gene expression patterns in human tissues sampled as single timepoints has several challenges, including the reconstruction of temporal order of highly noisy data. Previous methodologies have attempted to address these challenges in one or a small number of tissues for which rhythmic gene evolutionary conservation is assumed to be preserved. Here we introduce CIRCUST, a novel CIRCular-robUST methodology for analyzing molecular rhythms, that relies on circular statistics, is robust against noise, and requires fewer assumptions than existing methodologies. Next, we validated the method against four controlled experiments in which sampling times were known, and finally, CIRCUST was applied to 34 tissues from the Genotype-Tissue Expression (GTEx) dataset with the aim towards building a comprehensive daily rhythm gene expression atlas in humans. The validation and application shown here indicate that CIRCUST provides a flexible framework to formulate and solve the issues related to the analysis of molecular rhythms in human tissues. CIRCUST methodology is publicly available at https://github.com/yolandalago/CIRCUST/.

Dynamics of gliotoxin and bis(methylthio)gliotoxin production during the course of Aspergillus fumigatus infection.

As recently described, fungal secondary metabolism activates during infection in response to a hostile host environment. Gliotoxin and bis(methylthio)gliotoxin are two recognized secondary metabolites produced by Aspergillus fumigatus with differential cytotoxicity and involved in virulence. We sought to describe the temporal dynamics of gliotoxin and bis(methylthio)gliotoxin during A. fumigatus progression to further explore their role in the infection. First, we optimized the production of the mycotoxins under different in vitro growth conditions and then specifically measured them using an UHPLC/PDA method. The analytical conditions were selected after testing different parameters such as extraction procedures, column type, and mobile phase composition. We found that gliotoxin and bis(methylthio)gliotoxin are differentially excreted to the extracellular media during the course of A. fumigatus infection regardless of the growth format tested. Dynamic profiles show an early production of gliotoxin, which, after reaching a maximum, decreases coinciding with the increase in the production of the inactive derivative bis(methylthio)gliotoxin. Presence of gliotoxin may indicate an early phase of fungal development, whereas detection of bis(methylthio)gliotoxin may correspond to a more advanced stage of infection. Our chromatographic method successfully characterizes these secondary metabolites. Thus, it may potentially be used to further understand Aspergillus infection. LAY SUMMARY: Aspergillus fumigatus secondary metabolites may contribute to fungal survival. A new chromatographic method was applied to simultaneously characterize two relevant metabolites. Presence of toxic gliotoxin may indicate an early phase of development, whereas the detection of the inactive derivate may represent an advanced infection stage.

Order restricted inference in chronobiology.

This paper is motivated by applications in oscillatory systems where researchers are typically interested in discovering components of those systems that display rhythmic temporal patterns. The contributions of this paper are twofold. First, a methodology is developed based on a circular signal plus error model that is defined using order restrictions. This mathematical formulation of rhythmicity is simple, easily interpretable and very flexible, with the latter property derived from the nonparametric formulation of the signal. Second, we address various commonly encountered problems in the analysis of oscillatory systems data. Specifically, we propose a methodology for (a) detecting rhythmic signals in an oscillatory system and (b) estimating the unknown sampling time that occurs when tissues are obtained from subjects whose time of death is unknown. The proposed methodology is computationally efficient, outperforms the existing methods, and is broadly applicable to address a wide range of questions related to oscillatory systems.

Retrospective study on transmissible viral proventriculitis and chicken proventricular necrosis virus (CPNV) in the UK.

Chicken proventricular necrosis virus (CPNV) is a recently described birnavirus, which has been proposed to be the cause of transmissible viral proventriculitis (TVP). The understanding of the epidemiology of both the virus and the disease is very limited. A retrospective investigation on TVP and CPNV in broiler chicken submissions from the UK from between 1994 and 2015 was performed with the aims of assessing the longitudinal temporal evolution of TVP and CPNV, and to review the histological proventricular lesions in the studied chickens. Ninety-nine of the 135 included submissions (73.3%) fulfilled the TVP-diagnostic criteria, while the remaining 36 submissions (26.7%) displayed only lymphocytic proventriculitis (LP). The first detection of CPNV by PCR dated from 2009. Results showed a rise in the number of both TVP and positive CPNV RT-PCR submissions from 2009 with a peak in 2013, suggesting that they may be an emerging or re-emerging disease and pathogen, respectively. Twenty-two out of the 99 submissions displaying TVP lesions (22%) and four out of the 36 (11%) submissions with LP gave positive CPNV RT-PCR results, further supporting the association between CPNV and TVP and confirming that CPNV is present in a low proportion of proventriculi that do not fulfil the TVP-diagnostic criteria. In addition, intranuclear inclusion bodies were observed in 22 of the submissions with TVP. The vast majority of these cases (21 of 22, 96%) gave negative CPNV RT-PCR results, raising the question of whether a virus other than CPNV is responsible for some of these TVP-affected cases.RESEARCH HIGHLIGHTSTVP and CPNV have been present in British broilers since at least 1994 and 2009, respectively.TVP and CPNV seem to be an emerging and re-emerging disease and pathogen, respectively.CPNV was detected in proventriculi with both TVP and LP-lesions.Viruses other than CPNV may be responsible for some TVP-affected cases.

Fungal morphogenetic changes inside the mammalian host.

One of the main features of the majority of pathogenic fungi is the ability to switch between different types of morphological forms. These changes include the transition between cells of different shapes (such as the formation of pseudohyphae and hyphae), or the massive growth of the blastoconidia and formation of titan cells. Morphological changes occur during infection, and there is extensive evidence that they play a key role in processes required for disease, such as adhesion, invasion and dissemination, immune recognition evasion, and phagocytosis avoidance. In the present review, we will provide an overview of how morphological transitions contribute to the development of fungal disease, with special emphasis in two cases: Candida albicans as an example of yeast that switches between blastoconidia and filaments, and Cryptococcus neoformans as an example of a fungus that changes the size without modifying the shape of the cell.

Fowl aviadenovirus serotype 1 confirmed as the aetiological agent of gizzard erosions in replacement pullets and layer flocks in Great Britain by laboratory and in vivo studies.

An investigation into the aetiology and pathogenesis of adenoviral gizzard erosion has been conducted following three natural outbreaks affecting one flock of 6-week-old replacement pullets and two consecutive placements of free range layers at the age of 21 and 23 weeks. Affected flocks showed increased mortality (0.12-0.30% per week), and gizzard lesions were consistent with fowl aviadenovirus (FAdV) involvement. To substantiate the initial findings, a selection of archived formalin-fixed paraffin-embedded gizzard samples from another 12 pullet and layer flocks, for which macroscopic and histopathological diagnosis of the disease were recorded in Great Britain during the period 2009-2016, were also investigated. In situ hybridization (ISH), virology and/or PCR confirmed the presence of FAdV species-A, serotype-1 (FAdV-A, FAdV-1) DNA in gizzard samples of all 15 cases investigated. Co-infections with additional FAdV serotypes including FAdV-8a were detected by serology and/or virology in two of the pullet flocks. However, species-specific in situ hybridization revealed that pathological changes of affected gizzards were only associated with the detection of FAdV-A. A subsequent in vivo study infecting 21-day-old SPF pullets with FAdV-1 or FAdV-8a strains isolated from the 6-week-old replacement pullets revealed characteristic pathomorphological changes only in the gizzards from birds infected with FAdV-1. While infection with FAdV-8a was confirmed by virology and serology, infected SPF birds did not develop pathomorphological changes. Therefore, the aetiological involvement of the isolated FAdV-8a in the development of adenoviral gizzard erosion in commercial pullets has been ruled out.

Order restricted inference for oscillatory systems for detecting rhythmic signals.

MOTIVATION: Many biological processes, such as cell cycle, circadian clock, menstrual cycles, are governed by oscillatory systems consisting of numerous components that exhibit rhythmic patterns over time. It is not always easy to identify such rhythmic components. For example, it is a challenging problem to identify circadian genes in a given tissue using time-course gene expression data. There is a great potential for misclassifying non-rhythmic as rhythmic genes and vice versa. This has been a problem of considerable interest in recent years. In this article we develop a constrained inference based methodology called Order Restricted Inference for Oscillatory Systems (ORIOS) to detect rhythmic signals. Instead of using mathematical functions (e.g. sinusoidal) to describe shape of rhythmic signals, ORIOS uses mathematical inequalities. Consequently, it is robust and not limited by the biologist's choice of the mathematical model. We studied the performance of ORIOS using simulated as well as real data obtained from mouse liver, pituitary gland and data from NIH3T3, U2OS cell lines. Our results suggest that, for a broad collection of patterns of gene expression, ORIOS has substantially higher power to detect true rhythmic genes in comparison to some popular methods, while also declaring substantially fewer non-rhythmic genes as rhythmic. AVAILABILITY AND IMPLEMENTATION: A user friendly code implemented in R language can be downloaded from http://www.niehs.nih.gov/research/atniehs/labs/bb/staff/peddada/index.cfm CONTACT: peddada@niehs.nih.gov.

The formation of titan cells in Cryptococcus neoformans depends on the mouse strain and correlates with induction of Th2-type responses.

Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN-γ, TNF-α and IL17, while C57BL/BL mice had an increase in the anti-inflammatory cytokine IL-4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2-type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment.

Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host.

We have morphologically characterizedCandida tropicalisisolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of ß-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization ofGalleria mellonellalarvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.

Circular piecewise regression with applications to cell-cycle data.

Applications of circular regression models appear in many different fields such as evolutionary psychology, motor behavior, biology, and, in particular, in the analysis of gene expressions in oscillatory systems. Specifically, for the gene expression problem, a researcher may be interested in modeling the relationship among the phases of cell-cycle genes in two species with differing periods. This challenging problem reduces to the problem of constructing a piecewise circular regression model and, with this objective in mind, we propose a flexible circular regression model which allows different parameter values depending on sectors along the circle. We give a detailed interpretation of the parameters in the model and provide maximum likelihood estimators. We also provide a model selection procedure based on the concept of generalized degrees of freedom. The model is then applied to the analysis of two different cell-cycle data sets and through these examples we highlight the power of our new methodology.

Importance of Lid Hygiene Before Ocular Surgery: Qualitative and Quantitative Analysis of Eyelid and Conjunctiva Microbiota.

OBJECTIVE: To evaluate the efficacy of a cleansing eyelid wipe in reducing the microbiota present on the ocular surface before cataract surgery. METHODS: A single-center, prospective, single-blind phase IV study was conducted at the University Complutense of Madrid. Forty-five adult patients who were scheduled for ocular surgery after treatment with commercially available eyelid wipes were consecutively enrolled. The study lasted 5 days and the patients were examined at day 0 (D0), day 3 (D3), and day 5 (D5). They received instructions to apply the eyelid wipe only to the eye subject to surgery, using the other eye as a control with no treatment. Lid and conjunctival swabs were taken on each day and microbes identified. Ocular surface microbiota was estimated by measuring the area of the agar plate occupied by the grown colonies with respect to the total available area. RESULTS: Measurements at D3 and D5 showed a percent reduction of 58% and 63%, respectively, in the microbial load on the eyelid in the treated eyes (P=0.0011). There was also a reduction, although nonsignificant, in the microbiota of the conjunctiva of 72% and 69% on D3 and D5, respectively. CONCLUSIONS: The degree of microbiota reduction was comparable with that obtained after topical application of antibiotics in other studies. The results suggest the use of these eyelid wipes as a complementary prophylactic method before any ocular surgery.

Paradoxical growth of Candida albicans in the presence of caspofungin is associated with multiple cell wall rearrangements and decreased virulence.

In the last decade, echinocandins have emerged as an important family of antifungal drugs because of their fungicidal activity against Candida spp. Echinocandins inhibit the enzyme ß-1,3-d-glucan synthase, encoded by the FKS genes, and resistance to echinocandins is associated with mutations in this gene. In addition, echinocandin exposure can produce paradoxical growth, defined as the ability to grow at high antifungal concentrations but not at intermediate concentrations. In this work, we have demonstrated that paradoxical growth of Candida albicans in the presence of caspofungin is not due to antifungal degradation or instability. Media with high caspofungin concentrations recovered from wells where C. albicans showed paradoxical growth inhibited the growth of a Candida krusei reference strain. Cells exhibiting paradoxical growth at high caspofungin concentrations showed morphological changes such as enlarged size, abnormal septa, and absence of filamentation. Chitin content increased from the MIC to high caspofungin concentrations. Despite the high chitin levels, around 23% of cells died after treatment with caspofungin, indicating that chitin is required but not sufficient to protect the cells from the fungicidal effect of caspofungin. Moreover, we found that after paradoxical growth, ß-1,3-glucan was exposed at the cell wall surface. Cells grown at high caspofungin concentrations had decreased virulence in the invertebrate host Galleria mellonella. Cells grown at high caspofungin concentrations also induced a proinflammatory response in murine macrophages compared to control cells. Our work highlights important aspects about fungal adaptation to caspofungin, and although this adaptation is associated with reduced virulence, the clinical implications remain to be elucidated.

Performance and estimation of the true error rate of classification rules built with additional information. An application to a cancer trial.

Classification rules that incorporate additional information usually present in discrimination problems are receiving certain attention during the last years as they perform better than the usual rules. Fernández, M. A., C. Rueda and B. Salvador (2006): "Incorporating additional information to normal linear discriminant rules," J. Am. Stat. Assoc., 101, 569-577, proved that these rules have lower total misclassification probability than the usual Fisher's rule. In this paper we consider two issues; on the one hand, we compare these rules with those based on shrinkage estimators of the mean proposed by Tong, T., L. Chen and H. Zhao (2012): "Improved mean estimation and its application to diagonal discriminant analysis," Bioinformatics, 28(4): 531-537. with regard to four criteria: total misclassification probability, area under ROC curve, well-calibratedness and refinement; on the other hand, we consider the estimation of the true error rate, which is a very interesting parameter in applications. We prove results on the apparent error rate of the rules that expose the need of new estimators of their true error rate. We propose four such new estimators. Two of them are defined incorporating the additional information into the leave-one-out-bootstrap. The other two are the corresponding cross-validation after bootstrap versions. We compare these estimators with the usual ones in a simulation study and in a cancer trial application, showing the good behavior of the rules that incorporate additional information and of the new leave-one-out bootstrap estimators of their true error rate.

Identification of a core set of signature cell cycle genes whose relative order of time to peak expression is conserved across species.

A cell division cycle is a well-coordinated process in eukaryotes with cell cycle genes exhibiting a periodic expression over time. There is considerable interest among cell biologists to determine genes that are periodic in multiple organisms and whether such genes are also evolutionarily conserved in their relative order of time to peak expression. Interestingly, periodicity is not well-conserved evolutionarily. A conservative estimate of a number of periodic genes common to fission yeast (Schizosaccharomyces pombe) and budding yeast (Saccharomyces cerevisiae) ('core set FB') is 35, while those common to fission yeast and humans (Homo sapiens) ('core set FH') is 24. Using a novel statistical methodology, we discover that the relative order of peak expression is conserved in ∼80% of FB genes and in ∼40% of FH genes. We also discover that the order is evolutionarily conserved in six genes which are potentially the core set of signature cell cycle genes. These include ace2 (a transcription factor) and polo-kinase plo1, which are well-known hubs of early M-phase clusters, cdc18 a key component of pre-replication complexes, mik1 which is critical for the establishment and maintenance of DNA damage check point, and histones hhf1 and hta2.

ECGMiner: A flexible software for accurately digitizing ECG.

BACKGROUND AND OBJECTIVE: The electrocardiogram (ECG) is the most important non-invasive method for elucidating information about heart and cardiovascular disease diagnosis. Typically, the ECG system manufacturing companies provide ECG images, but store the numerical data in a proprietary format that is not interpretable and is not therefore useful for automatic diagnosis. There have been many efforts to digitize paper-based ECGs. The main limitations of previous works in ECG digitization are that they require manual selection of the regions of interest, only partly provide signal digitization, and offer limited accuracy. METHODS: We have developed the ECGMiner, an open-source software to digitize ECG images. It is precise, fast, and simple to use. This software digitizes ECGs in four steps: 1) recognizing the image composition; 2) removing the gridline; 3) extracting the signals; 4) post-processing and storing the data. RESULTS: We have evaluated the ECGMiner digitization capabilities using the Pearson Correlation Coefficient (PCC) and the Root Mean Square Error (RMSE) measures, and we consider ECG from two large, public, and widely used databases, LUDB and PTB-XL. The actual and digitized values of signals in both databases have been compared. The software's ability to correctly identify the location of characteristic waves has also been validated. Specifically, the PCC values are between 0.971 and 0.995, and the RMSE values are between 0.011 and 0.031 mV. CONCLUSIONS: The ECGMiner software presented in this paper is open access, easy to install, easy to use, and capable of precisely recovering the paper-based/digital ECG signal data, regardless of the input format and signal complexity. ECGMiner outperforms existing digitization algorithms in terms of PCC and RMSE values.

Comparison between the EUCAST procedure and the Etest for determination of the susceptibility of Candida species isolates to micafungin.

We compared the ability of the EUCAST EDef 7.2 and the Etest to detect the susceptibility to micafungin of 160 Candida and non-Candida clinical isolates. Agreement was higher when Etest MICs were obtained after 24 h of incubation; essential agreement was 90%, and categorical agreement was >90%. False susceptibility was seen only for Candida krusei (10%), and false resistance was observed in 6% of the isolates, ranging from 2.6% (C. glabrata) to 13% (C. albicans).

Molecular characterization of human-pathogenic microsporidia and Cyclospora cayetanensis isolated from various water sources in Spain: a year-long longitudinal study.

Recent studies suggest the involvement of water in the epidemiology of Cyclospora cayetanensis and some microsporidia. A total of 223 samples from four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) on four river basins from Madrid, Spain, were analyzed from spring 2008 to winter 2009. Microsporidia were detected in 49% of samples (109/223), Cyclospora spp. were detected in 9% (20/223), and both parasites were found in 5.4% (12/223) of samples. Human-pathogenic microsporidia were detected, including Enterocytozoon bieneusi (C, D, and D-like genotypes), Encephalitozoon intestinalis, Encephalitozoon cuniculi (genotypes I and III), and Anncaliia algerae. C. cayetanensis was identified in 17 of 20 samples. To our knowledge, this is the first study that shows a year-long longitudinal study of C. cayetanensis in drinking water treatment plants. Additionally, data about the presence and molecular characterization of the human-pathogenic microsporidia in drinking water, wastewater, and locations of influence during 1 year in Spain are shown. It is noteworthy that although the DWTPs and WWTPs studied meet European and national regulations on water sanitary quality, both parasites were found in water samples from these plants, supporting the idea that new and appropriate controls and regulations for drinking water, wastewater, and recreational waters should be proposed to avoid health risks from these pathogens.

isocir: An R Package for Constrained Inference using Isotonic Regression for Circular Data, with an Application to Cell Biology.

In many applications one may be interested in drawing inferences regarding the order of a collection of points on a unit circle. Due to the underlying geometry of the circle standard constrained inference procedures developed for Euclidean space data are not applicable. Recently, statistical inference for parameters under such order constraints on a unit circle was discussed in Rueda et al. (2009); Fernández et al. (2012). In this paper we introduce an R package called isocir which provides a set of functions that can be used for analyzing angular data subject to order constraints on a unit circle. Since this work is motivated by applications in cell biology, we illustrate the proposed package using a relevant cell cycle data.

Functional clustering of neuronal signals with FMM mixture models.

The identification of unlabeled neuronal electric signals is one of the most challenging open problems in neuroscience, widely known as Spike Sorting. Motivated to solve this problem, we propose a model-based approach within the mixture modeling framework for clustering oscillatory functional data called MixFMM. The core of the approach is the FMM (Frequency Modulated Möbius) waves, which are non-linear parametric time functions, flexible enough to describe different oscillatory patterns and simple enough to be estimated efficiently. In particular, specific model parameters describe the phase, amplitude and shape of the waveforms. A mixture model is defined using FMM waves as basic functions and gaussian errors, and an EM algorithm is proposed for estimating the parameters. Spike Sorting (SS) has received considerable attention in the literature, and different functional clustering approaches have been considered. We have conducted a fair comparative analysis of the MixFMM with three competitors. Two of them are traditional methods in functional clustering and widely used in Spike Sorting. The third is an approach that has proven superior to many others solving Spike Sorting problems. The datasets used for validation include benchmarking simulated and real cases. The internal and external validation indexes confirm a better performance of the MixFMM on real data sets against the three competitors and an outstanding performance in simulated data against traditional approaches.