conventional phenotypic methods: Topics by Science.gov

Sample records for conventional phenotypic methods

[A comparative study between the Vitek YBC and Microscan Walk Away RYID automated systems with conventional phenotypic methods for the identification of yeasts of clinical interest].

PubMed

Ferrara, Giuseppe; Mercedes Panizol, Maria; Mazzone, Marja; Delia Pequeneze, Maria; Reviakina, Vera

2014-12-01

The aim of this study was to compare the identification of clin- ically relevant yeasts by the Vitek YBC and Microscan Walk Away RYID automated methods with conventional phenotypic methods. One hundred and ninety three yeast strains isolated from clinical samples and five controls strains were used. All the yeasts were identified by the automated methods previously mentioned and conventional phenotypic methods such as carbohydrate assimilation, visualization of microscopic morphology on corn meal agar and the use of chromogenic agar. Variables were assessed by 2 x 2 contingency tables, McNemar's Chi square, the Kappa index, and concordance values were calculated, as well as major and minor errors for the automated methods. Yeasts were divided into two groups: (1) frequent isolation and (2) rare isolation. The Vitek YBC and Microscan Walk Away RYID systems were concordant in 88.4 and 85.9% respectively, when compared to conventional phenotypic methods. Although both automated systems can be used for yeasts identification, the presence of major and minor errors indicates the possibility of misidentifications; therefore, the operator of this equipment must use in parallel, phenotypic tests such as visualization of microscopic morphology on corn meal agar and chromogenic agar, especially against infrequently isolated yeasts. Automated systems are a valuable tool; however, the expertise and judgment of the microbiologist are an important strength to ensure the quality of the results.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid identification of fungal rhinosinusitis pathogens.

PubMed

Huang, Yanfei; Wang, Jinglin; Zhang, Mingxin; Zhu, Min; Wang, Mei; Sun, Yufeng; Gu, Haitong; Cao, Jingjing; Li, Xue; Zhang, Shaoya; Lu, Xinxin

2017-03-01

Filamentous fungi are among the most important pathogens, causing fungal rhinosinusitis (FRS). Current laboratory diagnosis of FRS pathogens mainly relies on phenotypic identification by culture and microscopic examination, which is time consuming and expertise dependent. Although matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS has been employed to identify various fungi, its efficacy in the identification of FRS fungi is less clear. A total of 153 FRS isolates obtained from patients were analysed at the Clinical Laboratory at the Beijing Tongren Hospital affiliated to the Capital Medical University, between January 2014 and December 2015. They were identified by traditional phenotypic methods and Bruker MALDI-TOF MS (Bruker, Biotyper version 3.1), respectively. Discrepancies between the two methods were further validated by sequencing. Among the 153 isolates, 151 had correct species identification using MALDI-TOF MS (Bruker, Biot 3.1, score ≥2.0 or 2.3). MALDI-TOF MS enabled identification of some very closely related species that were indistinguishable by conventional phenotypic methods, including 1/10 Aspergillus versicolor, 3/20 Aspergillus flavus, 2/30 Aspergillus fumigatus and 1/20 Aspergillus terreus, which were misidentified by conventional phenotypic methods as Aspergillus nidulans, Aspergillus oryzae, Aspergillus japonicus and Aspergillus nidulans, respectively. In addition, 2/2 Rhizopus oryzae and 1/1 Rhizopus stolonifer that were identified only to the genus level by the phenotypic method were correctly identified by MALDI-TOF MS. MALDI-TOF MS is a rapid and accurate technique, and could replace the conventional phenotypic method for routine identification of FRS fungi in clinical microbiology laboratories.
Comparative phenotypic and genotypic analysis of Edwardsiella spp. isolates from different hosts and geographic origins, with an emphasis on isolates formerly classified as E. tarda and an evaluation of diagnostic methods

USDA-ARS?s Scientific Manuscript database

Aims: Conventional phenotypic and genotypic analyses for the differentiation of phenotypically ambiguous Edwardsiella congeners was evaluated and historical E. tarda designations were linked to current taxonomic nomenclature. Methods and Results: Forty-seven Edwardsiella spp. isolates recovered over...
Comparative evaluation of matrix-assisted laser desorption ionisation-time of flight mass spectrometry and conventional phenotypic-based methods for identification of clinically important yeasts in a UK-based medical microbiology laboratory.

PubMed

Fatania, Nita; Fraser, Mark; Savage, Mike; Hart, Jason; Abdolrasouli, Alireza

2015-12-01

Performance of matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) was compared in a side-by side-analysis with conventional phenotypic methods currently in use in our laboratory for identification of yeasts in a routine diagnostic setting. A diverse collection of 200 clinically important yeasts (19 species, five genera) were identified by both methods using standard protocols. Discordant or unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene. MALDI-TOF and conventional methods were in agreement for 182 isolates (91%) with correct identification to species level. Eighteen discordant results (9%) were due to rarely encountered species, hence the difficulty in their identification using traditional phenotypic methods. MALDI-TOF MS enabled rapid, reliable and accurate identification of clinically important yeasts in a routine diagnostic microbiology laboratory. Isolates with rare, unusual or low probability identifications should be confirmed using robust molecular methods. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/
[Phenotypic and genotypic characteristics of Mycobacterium kansasii strains isolated in Spain (2000-2003)].

PubMed

Jiménez-Pajares, María Soledad; Herrera, Laura; Valverde, Azucena; Saiz, Pilar; Sáez-Nieto, Juan Antonio

2005-05-01

Mycobacterium kansasii is an opportunistic pathogen that mainly causes pulmonary infections. This species accounted for 9.7% of Mycobacteria other than tuberculosis complex identified in the reference laboratory of the Spanish Centro Nacional de Microbiologia during the period of 2000-2003. In this study we analyzed the phenotypic and genotypic characteristics of 298 M. kansasii strains isolated over this 4-year period. The phenotypic characteristics were determined by conventional methods: biochemical testing, culture and morphological study. Genotypic characteristics were studied using PCR restriction fragment analysis of a fragment of the hsp65 gene and digestion with BstEII and HaeIII, according to the method of Telenti. Among the total of tested strains, 57.4% had the typical phenotypic characteristics described for M. kansasii. The rest had atypical patterns that we grouped into 17 biotypes. Strains belonging to six of the seven described genotypes were identified, with 86.6% of the strains falling into genotype I. Analysis of the phenotypic characteristics of M. kansasii showed a higher discrimination index for intraspecific differentiation than genotypic methods. Nevertheless, the high variability of phenotypic characteristics, some of which were very specific for the species (e.g., photochromogenicity), could complicate their identification. Hence both conventional and molecular methods should be used to accurately identify the atypical isolates.
High-Throughput Microbore UPLC-MS Metabolic Phenotyping of Urine for Large-Scale Epidemiology Studies.

PubMed

Gray, Nicola; Lewis, Matthew R; Plumb, Robert S; Wilson, Ian D; Nicholson, Jeremy K

2015-06-05

A new generation of metabolic phenotyping centers are being created to meet the increasing demands of personalized healthcare, and this has resulted in a major requirement for economical, high-throughput metabonomic analysis by liquid chromatography-mass spectrometry (LC-MS). Meeting these new demands represents an emerging bioanalytical problem that must be solved if metabolic phenotyping is to be successfully applied to large clinical and epidemiological sample sets. Ultraperformance (UP)LC-MS-based metabolic phenotyping, based on 2.1 mm i.d. LC columns, enables comprehensive metabolic phenotyping but, when employed for the analysis of thousands of samples, results in high solvent usage. The use of UPLC-MS employing 1 mm i.d. columns for metabolic phenotyping rather than the conventional 2.1 mm i.d. methodology shows that the resulting optimized microbore method provided equivalent or superior performance in terms of peak capacity, sensitivity, and robustness. On average, we also observed, when using the microbore scale separation, an increase in response of 2-3 fold over that obtained with the standard 2.1 mm scale method. When applied to the analysis of human urine, the 1 mm scale method showed no decline in performance over the course of 1000 analyses, illustrating that microbore UPLC-MS represents a viable alternative to conventional 2.1 mm i.d. formats for routine large-scale metabolic profiling studies while also resulting in a 75% reduction in solvent usage. The modest increase in sensitivity provided by this methodology also offers the potential to either reduce sample consumption or increase the number of metabolite features detected with confidence due to the increased signal-to-noise ratios obtained. Implementation of this miniaturized UPLC-MS method of metabolic phenotyping results in clear analytical, economic, and environmental benefits for large-scale metabolic profiling studies with similar or improved analytical performance compared to conventional UPLC-MS.
Approximating genomic reliabilities for national genomic evaluation

USDA-ARS?s Scientific Manuscript database

With the introduction of standard methods for approximating effective daughter/data contribution by Interbull in 2001, conventional EDC or reliabilities contributed by daughter phenotypes are directly comparable across countries and used in routine conventional evaluations. In order to make publishe...
How to detect carbapenemase producers? A literature review of phenotypic and molecular methods.

PubMed

Hammoudi, D; Moubareck, C Ayoub; Sarkis, D Karam

2014-12-01

This review describes the current state-of-art of carbapenemase detection methods. Identification of carbapenemases is first based on conventional phenotypic tests including antimicrobial susceptibility testing, modified-Hodge test and carbapenemase-inhibitor culture tests. Second, molecular characterization of carbapenemase genes by PCR sequencing is essential. Third, innovative biochemical and spectrometric detection may be applied. Copyright © 2014 Elsevier B.V. All rights reserved.
Genomic assisted selection for enhancing line breeding: merging genomic and phenotypic selection in winter wheat breeding programs with preliminary yield trials.

PubMed

Michel, Sebastian; Ametz, Christian; Gungor, Huseyin; Akgöl, Batuhan; Epure, Doru; Grausgruber, Heinrich; Löschenberger, Franziska; Buerstmayr, Hermann

2017-02-01

Early generation genomic selection is superior to conventional phenotypic selection in line breeding and can be strongly improved by including additional information from preliminary yield trials. The selection of lines that enter resource-demanding multi-environment trials is a crucial decision in every line breeding program as a large amount of resources are allocated for thoroughly testing these potential varietal candidates. We compared conventional phenotypic selection with various genomic selection approaches across multiple years as well as the merit of integrating phenotypic information from preliminary yield trials into the genomic selection framework. The prediction accuracy using only phenotypic data was rather low (r = 0.21) for grain yield but could be improved by modeling genetic relationships in unreplicated preliminary yield trials (r = 0.33). Genomic selection models were nevertheless found to be superior to conventional phenotypic selection for predicting grain yield performance of lines across years (r = 0.39). We subsequently simplified the problem of predicting untested lines in untested years to predicting tested lines in untested years by combining breeding values from preliminary yield trials and predictions from genomic selection models by a heritability index. This genomic assisted selection led to a 20% increase in prediction accuracy, which could be further enhanced by an appropriate marker selection for both grain yield (r = 0.48) and protein content (r = 0.63). The easy to implement and robust genomic assisted selection gave thus a higher prediction accuracy than either conventional phenotypic or genomic selection alone. The proposed method took the complex inheritance of both low and high heritable traits into account and appears capable to support breeders in their selection decisions to develop enhanced varieties more efficiently.
The Role of 16S rRNA Gene Sequencing in Identification of Microorganisms Misidentified by Conventional Methods

PubMed Central

Petti, C. A.; Polage, C. R.; Schreckenberger, P.

2005-01-01

Traditional methods for microbial identification require the recognition of differences in morphology, growth, enzymatic activity, and metabolism to define genera and species. Full and partial 16S rRNA gene sequencing methods have emerged as useful tools for identifying phenotypically aberrant microorganisms. We report on three bacterial blood isolates from three different College of American Pathologists-certified laboratories that were referred to ARUP Laboratories for definitive identification. Because phenotypic identification suggested unusual organisms not typically associated with the submitted clinical diagnosis, consultation with the Medical Director was sought and further testing was performed including partial 16S rRNA gene sequencing. All three patients had endocarditis, and conventional methods identified isolates from patients A, B, and C as a Facklamia sp., Eubacterium tenue, and a Bifidobacterium sp. 16S rRNA gene sequencing identified the isolates as Enterococcus faecalis, Cardiobacterium valvarum, and Streptococcus mutans, respectively. We conclude that the initial identifications of these three isolates were erroneous, may have misled clinicians, and potentially impacted patient care. 16S rRNA gene sequencing is a more objective identification tool, unaffected by phenotypic variation or technologist bias, and has the potential to reduce laboratory errors. PMID:16333109
Design Space Toolbox V2: Automated Software Enabling a Novel Phenotype-Centric Modeling Strategy for Natural and Synthetic Biological Systems

PubMed Central

Lomnitz, Jason G.; Savageau, Michael A.

2016-01-01

Mathematical models of biochemical systems provide a means to elucidate the link between the genotype, environment, and phenotype. A subclass of mathematical models, known as mechanistic models, quantitatively describe the complex non-linear mechanisms that capture the intricate interactions between biochemical components. However, the study of mechanistic models is challenging because most are analytically intractable and involve large numbers of system parameters. Conventional methods to analyze them rely on local analyses about a nominal parameter set and they do not reveal the vast majority of potential phenotypes possible for a given system design. We have recently developed a new modeling approach that does not require estimated values for the parameters initially and inverts the typical steps of the conventional modeling strategy. Instead, this approach relies on architectural features of the model to identify the phenotypic repertoire and then predict values for the parameters that yield specific instances of the system that realize desired phenotypic characteristics. Here, we present a collection of software tools, the Design Space Toolbox V2 based on the System Design Space method, that automates (1) enumeration of the repertoire of model phenotypes, (2) prediction of values for the parameters for any model phenotype, and (3) analysis of model phenotypes through analytical and numerical methods. The result is an enabling technology that facilitates this radically new, phenotype-centric, modeling approach. We illustrate the power of these new tools by applying them to a synthetic gene circuit that can exhibit multi-stability. We then predict values for the system parameters such that the design exhibits 2, 3, and 4 stable steady states. In one example, inspection of the basins of attraction reveals that the circuit can count between three stable states by transient stimulation through one of two input channels: a positive channel that increases the count, and a negative channel that decreases the count. This example shows the power of these new automated methods to rapidly identify behaviors of interest and efficiently predict parameter values for their realization. These tools may be applied to understand complex natural circuitry and to aid in the rational design of synthetic circuits. PMID:27462346
Extreme-phenotype genome-wide association study (XP-GWAS): a method for identifying trait-associated variants by sequencing pools of individuals selected from a diversity panel.

PubMed

Yang, Jinliang; Jiang, Haiying; Yeh, Cheng-Ting; Yu, Jianming; Jeddeloh, Jeffrey A; Nettleton, Dan; Schnable, Patrick S

2015-11-01

Although approaches for performing genome-wide association studies (GWAS) are well developed, conventional GWAS requires high-density genotyping of large numbers of individuals from a diversity panel. Here we report a method for performing GWAS that does not require genotyping of large numbers of individuals. Instead XP-GWAS (extreme-phenotype GWAS) relies on genotyping pools of individuals from a diversity panel that have extreme phenotypes. This analysis measures allele frequencies in the extreme pools, enabling discovery of associations between genetic variants and traits of interest. This method was evaluated in maize (Zea mays) using the well-characterized kernel row number trait, which was selected to enable comparisons between the results of XP-GWAS and conventional GWAS. An exome-sequencing strategy was used to focus sequencing resources on genes and their flanking regions. A total of 0.94 million variants were identified and served as evaluation markers; comparisons among pools showed that 145 of these variants were statistically associated with the kernel row number phenotype. These trait-associated variants were significantly enriched in regions identified by conventional GWAS. XP-GWAS was able to resolve several linked QTL and detect trait-associated variants within a single gene under a QTL peak. XP-GWAS is expected to be particularly valuable for detecting genes or alleles responsible for quantitative variation in species for which extensive genotyping resources are not available, such as wild progenitors of crops, orphan crops, and other poorly characterized species such as those of ecological interest. © 2015 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.
Estimating causal effects with a non-paranormal method for the design of efficient intervention experiments

PubMed Central

2014-01-01

Background Knockdown or overexpression of genes is widely used to identify genes that play important roles in many aspects of cellular functions and phenotypes. Because next-generation sequencing generates high-throughput data that allow us to detect genes, it is important to identify genes that drive functional and phenotypic changes of cells. However, conventional methods rely heavily on the assumption of normality and they often give incorrect results when the assumption is not true. To relax the Gaussian assumption in causal inference, we introduce the non-paranormal method to test conditional independence in the PC-algorithm. Then, we present the non-paranormal intervention-calculus when the directed acyclic graph (DAG) is absent (NPN-IDA), which incorporates the cumulative nature of effects through a cascaded pathway via causal inference for ranking causal genes against a phenotype with the non-paranormal method for estimating DAGs. Results We demonstrate that causal inference with the non-paranormal method significantly improves the performance in estimating DAGs on synthetic data in comparison with the original PC-algorithm. Moreover, we show that NPN-IDA outperforms the conventional methods in exploring regulators of the flowering time in Arabidopsis thaliana and regulators that control the browning of white adipocytes in mice. Our results show that performance improvement in estimating DAGs contributes to an accurate estimation of causal effects. Conclusions Although the simplest alternative procedure was used, our proposed method enables us to design efficient intervention experiments and can be applied to a wide range of research purposes, including drug discovery, because of its generality. PMID:24980787
Estimating causal effects with a non-paranormal method for the design of efficient intervention experiments.

PubMed

Teramoto, Reiji; Saito, Chiaki; Funahashi, Shin-ichi

2014-06-30

Knockdown or overexpression of genes is widely used to identify genes that play important roles in many aspects of cellular functions and phenotypes. Because next-generation sequencing generates high-throughput data that allow us to detect genes, it is important to identify genes that drive functional and phenotypic changes of cells. However, conventional methods rely heavily on the assumption of normality and they often give incorrect results when the assumption is not true. To relax the Gaussian assumption in causal inference, we introduce the non-paranormal method to test conditional independence in the PC-algorithm. Then, we present the non-paranormal intervention-calculus when the directed acyclic graph (DAG) is absent (NPN-IDA), which incorporates the cumulative nature of effects through a cascaded pathway via causal inference for ranking causal genes against a phenotype with the non-paranormal method for estimating DAGs. We demonstrate that causal inference with the non-paranormal method significantly improves the performance in estimating DAGs on synthetic data in comparison with the original PC-algorithm. Moreover, we show that NPN-IDA outperforms the conventional methods in exploring regulators of the flowering time in Arabidopsis thaliana and regulators that control the browning of white adipocytes in mice. Our results show that performance improvement in estimating DAGs contributes to an accurate estimation of causal effects. Although the simplest alternative procedure was used, our proposed method enables us to design efficient intervention experiments and can be applied to a wide range of research purposes, including drug discovery, because of its generality.
Single nucleotide polymorphisms in the Mycobacterium bovis genome resolve phylogenetic relationships

USDA-ARS?s Scientific Manuscript database

Mycobacterium bovis isolates carry restricted allelic variation yet exhibit a range of disease phenotypes and host preferences. Conventional genotyping methods target small hyper-variable regions of their genome and provide anonymous biallelic information insufficient to develop phylogeny. To resolv...
Identifying Pleiotropic Genes in Genome-Wide Association Studies for Multivariate Phenotypes with Mixed Measurement Scales

PubMed Central

Williams, L. Keoki; Buu, Anne

2017-01-01

We propose a multivariate genome-wide association test for mixed continuous, binary, and ordinal phenotypes. A latent response model is used to estimate the correlation between phenotypes with different measurement scales so that the empirical distribution of the Fisher’s combination statistic under the null hypothesis is estimated efficiently. The simulation study shows that our proposed correlation estimation methods have high levels of accuracy. More importantly, our approach conservatively estimates the variance of the test statistic so that the type I error rate is controlled. The simulation also shows that the proposed test maintains the power at the level very close to that of the ideal analysis based on known latent phenotypes while controlling the type I error. In contrast, conventional approaches–dichotomizing all observed phenotypes or treating them as continuous variables–could either reduce the power or employ a linear regression model unfit for the data. Furthermore, the statistical analysis on the database of the Study of Addiction: Genetics and Environment (SAGE) demonstrates that conducting a multivariate test on multiple phenotypes can increase the power of identifying markers that may not be, otherwise, chosen using marginal tests. The proposed method also offers a new approach to analyzing the Fagerström Test for Nicotine Dependence as multivariate phenotypes in genome-wide association studies. PMID:28081206
In Amnio MRI of Mouse Embryos

PubMed Central

Roberts, Thomas A.; Norris, Francesca C.; Carnaghan, Helen; Savery, Dawn; Wells, Jack A.; Siow, Bernard; Scambler, Peter J.; Pierro, Agostino; De Coppi, Paolo; Eaton, Simon; Lythgoe, Mark F.

2014-01-01

Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px). To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community. PMID:25330230
Neuropsychological Criteria for Mild Cognitive Impairment Improves Diagnostic Precision, Biomarker Associations, and Progression Rates

PubMed Central

Bondi, Mark W.; Edmonds, Emily C.; Jak, Amy J.; Clark, Lindsay R.; Delano-Wood, Lisa; McDonald, Carrie R.; Nation, Daniel A.; Libon, David J.; Au, Rhoda; Galasko, Douglas; Salmon, David P.

2014-01-01

We compared two methods of diagnosing mild cognitive impairment (MCI): conventional Petersen/Winblad criteria as operationalized by the Alzheimer’s Disease Neuroimaging Initiative (ADNI) and an actuarial neuropsychological method put forward by Jak and Bondi designed to balance sensitivity and reliability. 1,150 ADNI participants were diagnosed at baseline as cognitively normal (CN) or MCI via ADNI criteria (MCI: n = 846; CN: n = 304) or Jak/Bondi criteria (MCI: n = 401; CN: n = 749), and the two MCI samples were submitted to cluster and discriminant function analyses. Resulting cluster groups were then compared and further examined for APOE allelic frequencies, cerebrospinal fluid (CSF) Alzheimer’s disease (AD) biomarker levels, and clinical outcomes. Results revealed that both criteria produced a mildly impaired Amnestic subtype and a more severely impaired Dysexecutive/Mixed subtype. The neuropsychological Jak/Bondi criteria uniquely yielded a third Impaired Language subtype, whereas conventional Petersen/Winblad ADNI criteria produced a third subtype comprising nearly one-third of the sample that performed within normal limits across the cognitive measures, suggesting this method’s susceptibility to false positive diagnoses. MCI participants diagnosed via neuropsychological criteria yielded dissociable cognitive phenotypes, significant CSF AD biomarker associations, more stable diagnoses, and identified greater percentages of participants who progressed to dementia than conventional MCI diagnostic criteria. Importantly, the actuarial neuropsychological method did not produce a subtype that performed within normal limits on the cognitive testing, unlike the conventional diagnostic method. Findings support the need for refinement of MCI diagnoses to incorporate more comprehensive neuropsychological methods, with resulting gains in empirical characterization of specific cognitive phenotypes, biomarker associations, stability of diagnoses, and prediction of progression. Refinement of MCI diagnostic methods may also yield gains in biomarker and clinical trial study findings because of improvements in sample compositions of ‘true positive’ cases and removal of ‘false positive’ cases. PMID:24844687
Development of Amplified 16S Ribosomal DNA Restriction Analysis for Identification of Actinomyces Species and Comparison with Pyrolysis-Mass Spectrometry and Conventional Biochemical Tests

PubMed Central

Hall, Val; O’Neill, G. L.; Magee, J. T.; Duerden, B. I.

1999-01-01

Identification of Actinomyces spp. by conventional phenotypic methods is notoriously difficult and unreliable. Recently, the application of chemotaxonomic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. A practical and discriminatory identification method is now needed for routine identification of clinical isolates. Amplified 16S ribosomal DNA restriction analysis (ARDRA) was applied to reference strains (n = 27) and clinical isolates (n = 36) of Actinomyces spp. and other gram-positive rods. Clinical strains were identified initially to the species level by conventional biochemical tests. However, given the low degree of confidence in conventional methods, the findings obtained by ARDRA were also compared with those obtained by pyrolysis-mass spectrometry. The ARDRA profiles generated by the combination of HaeIII and HpaII endonuclease digestion differentiated all reference strains to the species or subspecies level. The profiles correlated well with the findings obtained by pyrolysis-mass spectrometry and by conventional tests and enabled the identification of 31 of 36 clinical isolates to the species level. ARDRA was shown to be a simple, rapid, cost-effective, and highly discriminatory method for routine identification of Actinomyces spp. of clinical origin. PMID:10364594
Leaf epidermis images for robust identification of plants

PubMed Central

da Silva, Núbia Rosa; Oliveira, Marcos William da Silva; Filho, Humberto Antunes de Almeida; Pinheiro, Luiz Felipe Souza; Rossatto, Davi Rodrigo; Kolb, Rosana Marta; Bruno, Odemir Martinez

2016-01-01

This paper proposes a methodology for plant analysis and identification based on extracting texture features from microscopic images of leaf epidermis. All the experiments were carried out using 32 plant species with 309 epidermal samples captured by an optical microscope coupled to a digital camera. The results of the computational methods using texture features were compared to the conventional approach, where quantitative measurements of stomatal traits (density, length and width) were manually obtained. Epidermis image classification using texture has achieved a success rate of over 96%, while success rate was around 60% for quantitative measurements taken manually. Furthermore, we verified the robustness of our method accounting for natural phenotypic plasticity of stomata, analysing samples from the same species grown in different environments. Texture methods were robust even when considering phenotypic plasticity of stomatal traits with a decrease of 20% in the success rate, as quantitative measurements proved to be fully sensitive with a decrease of 77%. Results from the comparison between the computational approach and the conventional quantitative measurements lead us to discover how computational systems are advantageous and promising in terms of solving problems related to Botany, such as species identification. PMID:27217018

'Neisseria skkuensis' sp. nov., isolated from the blood of a diabetic patient with a foot ulcer.

PubMed

Lee, Mi Young; Park, Eu Gene; Choi, Ji Young; Cheong, Hae Suk; Chung, Doo Ryeon; Peck, Kyong Ran; Song, Jae-Hoon; Ko, Kwan Soo

2010-07-01

A Gram-negative bacterium was isolated from the blood of a patient with diabetes mellitus. However, it could not be identified by conventional microbiological methods, and so was characterized by phenotypic and genotypic analyses. 16S rRNA gene sequence analysis revealed that the strain belonged to the genus Neisseria. Based on the phenotypic and genotypic characteristics, we propose that strain SMC-A9199(T) (=KCTC 22696(T)=JCM 16127(T)) should be classified as a novel species, 'Neisseria skkuensis' sp. nov. The patient was further treated with amoxicillin-clavulanate and ciprofloxacin for 3 weeks.
Immune phenotype in children with therapy-naïve remitted and relapsed Crohn’s disease

PubMed Central

Cseh, Aron; Vasarhelyi, Barna; Molnar, Kriszta; Szalay, Balazs; Svec, Peter; Treszl, Andras; Dezsofi, Antal; Lakatos, Peter Laszlo; Arato, Andras; Tulassay, Tivadar; Veres, Gabor

2010-01-01

AIM: To characterize the prevalence of subpopulations of CD4+ cells along with that of major inhibitor or stimulator cell types in therapy-naïve childhood Crohn’s disease (CD) and to test whether abnormalities of immune phenotype are normalized with the improvement of clinical signs and symptoms of disease. METHODS: We enrolled 26 pediatric patients with CD. 14 therapy-naïve CD children; of those, 10 children remitted on conventional therapy and formed the remission group. We also tested another group of 12 children who relapsed with conventional therapy and were given infliximab; and 15 healthy children who served as controls. The prevalence of Th1 and Th2, naïve and memory, activated and regulatory T cells, along with the members of innate immunity such as natural killer (NK), NK-T, myeloid and plasmocytoid dendritic cells (DCs), monocytes and Toll-like receptor (TLR)-2 and TLR-4 expression were determined in peripheral blood samples. RESULTS: Children with therapy-naïve CD and those in relapse showed a decrease in Th1 cell prevalence. Simultaneously, an increased prevalence of memory and activated lymphocytes along with that of DCs and monocytes was observed. In addition, the ratio of myeloid /plasmocytoid DCs and the prevalence of TLR-2 or TLR-4 positive DCs and monocytes were also higher in therapy-naïve CD than in controls. The majority of alterations diminished in remitted CD irrespective of whether remission was obtained by conventional or biological therapy. CONCLUSION: The finding that immune phenotype is normalized in remission suggests a link between immune phenotype and disease activity in childhood CD. Our observations support the involvement of members of the adaptive and innate immune systems in childhood CD. PMID:21157977
Gene Variants Associated with Antisocial Behaviour: A Latent Variable Approach

ERIC Educational Resources Information Center

Bentley, Mary Jane; Lin, Haiqun; Fernandez, Thomas V.; Lee, Maria; Yrigollen, Carolyn M.; Pakstis, Andrew J.; Katsovich, Liliya; Olds, David L.; Grigorenko, Elena L.; Leckman, James F.

2013-01-01

Objective: The aim of this study was to determine if a latent variable approach might be useful in identifying shared variance across genetic risk alleles that is associated with antisocial behaviour at age 15 years. Methods: Using a conventional latent variable approach, we derived an antisocial phenotype in 328 adolescents utilizing data from a…
Comparative UAV and Field Phenotyping to Assess Yield and Nitrogen Use Efficiency in Hybrid and Conventional Barley.

PubMed

Kefauver, Shawn C; Vicente, Rubén; Vergara-Díaz, Omar; Fernandez-Gallego, Jose A; Kerfal, Samir; Lopez, Antonio; Melichar, James P E; Serret Molins, María D; Araus, José L

2017-01-01

With the commercialization and increasing availability of Unmanned Aerial Vehicles (UAVs) multiple rotor copters have expanded rapidly in plant phenotyping studies with their ability to provide clear, high resolution images. As such, the traditional bottleneck of plant phenotyping has shifted from data collection to data processing. Fortunately, the necessarily controlled and repetitive design of plant phenotyping allows for the development of semi-automatic computer processing tools that may sufficiently reduce the time spent in data extraction. Here we present a comparison of UAV and field based high throughput plant phenotyping (HTPP) using the free, open-source image analysis software FIJI (Fiji is just ImageJ) using RGB (conventional digital cameras), multispectral and thermal aerial imagery in combination with a matching suite of ground sensors in a study of two hybrids and one conventional barely variety with ten different nitrogen treatments, combining different fertilization levels and application schedules. A detailed correlation network for physiological traits and exploration of the data comparing between treatments and varieties provided insights into crop performance under different management scenarios. Multivariate regression models explained 77.8, 71.6, and 82.7% of the variance in yield from aerial, ground, and combined data sets, respectively.
Phenotypic landscape of non-conventional yeast species for different stress tolerance traits desirable in bioethanol fermentation.

PubMed

Mukherjee, Vaskar; Radecka, Dorota; Aerts, Guido; Verstrepen, Kevin J; Lievens, Bart; Thevelein, Johan M

2017-01-01

Non-conventional yeasts present a huge, yet barely exploited, resource of yeast biodiversity for industrial applications. This presents a great opportunity to explore alternative ethanol-fermenting yeasts that are more adapted to some of the stress factors present in the harsh environmental conditions in second-generation (2G) bioethanol fermentation. Extremely tolerant yeast species are interesting candidates to investigate the underlying tolerance mechanisms and to identify genes that when transferred to existing industrial strains could help to design more stress-tolerant cell factories. For this purpose, we performed a high-throughput phenotypic evaluation of a large collection of non-conventional yeast species to identify the tolerance limits of the different yeast species for desirable stress tolerance traits in 2G bioethanol production. Next, 12 multi-tolerant strains were selected and used in fermentations under different stressful conditions. Five strains out of which, showing desirable fermentation characteristics, were then evaluated in small-scale, semi-anaerobic fermentations with lignocellulose hydrolysates. Our results revealed the phenotypic landscape of many non-conventional yeast species which have not been previously characterized for tolerance to stress conditions relevant for bioethanol production. This has identified for each stress condition evaluated several extremely tolerant non- Saccharomyces yeasts. It also revealed multi-tolerance in several yeast species, which makes those species good candidates to investigate the molecular basis of a robust general stress tolerance. The results showed that some non-conventional yeast species have similar or even better fermentation efficiency compared to S. cerevisiae in the presence of certain stressful conditions. Prior to this study, our knowledge on extreme stress-tolerant phenotypes in non-conventional yeasts was limited to only few species. Our work has now revealed in a systematic way the potential of non- Saccharomyces species to emerge either as alternative host species or as a source of valuable genetic information for construction of more robust industrial S. serevisiae bioethanol production yeasts. Striking examples include yeast species like Pichia kudriavzevii and Wickerhamomyces anomalus that show very high tolerance to diverse stress factors. This large-scale phenotypic analysis has yielded a detailed database useful as a resource for future studies to understand and benefit from the molecular mechanisms underlying the extreme phenotypes of non-conventional yeast species.
Quantitative phenotyping of X-disease resistance in chokecherry using real-time PCR.

PubMed

Huang, Danqiong; Walla, James A; Dai, Wenhao

2014-03-01

A quantitative real-time SYBR Green PCR (qPCR) assay has been developed to detect and quantify X-disease phytoplasmas in chokecherry. An X-disease phytoplasma-specific and high sensitivity primer pair was designed based on the 16S rRNA gene sequence of X-disease phytoplasmas. This primer pair was specific to the 16SrIII group (X-disease) phytoplasmas. The qPCR method can quantify phytoplasmas from a DNA mix (a mix of both chokecherry and X-disease phytoplasma DNA) at as low as 0.001 ng, 10-fold lower than conventional PCR using the same primer pair. A significant correlation between the copy number of phytoplasmas and visual phenotypic rating scores of X-disease resistance in chokecherry plants was observed. Disease resistant chokecherries had a significantly lower titer of X-disease phytoplasmas than susceptible plants. This suggests that the qPCR assay provides a more objective tool to phenotype phytoplasma disease severity, particularly for early evaluation of host resistance; therefore, this method will facilitate quantitative phenotyping of disease resistance and has great potential in enhancing plant breeding. Copyright © 2013 Elsevier B.V. All rights reserved.
Engineering live cell surfaces with functional polymers via cytocompatible controlled radical polymerization

NASA Astrophysics Data System (ADS)

Niu, Jia; Lunn, David J.; Pusuluri, Anusha; Yoo, Justin I.; O'Malley, Michelle A.; Mitragotri, Samir; Soh, H. Tom; Hawker, Craig J.

2017-06-01

The capability to graft synthetic polymers onto the surfaces of live cells offers the potential to manipulate and control their phenotype and underlying cellular processes. Conventional grafting-to strategies for conjugating preformed polymers to cell surfaces are limited by low polymer grafting efficiency. Here we report an alternative grafting-from strategy for directly engineering the surfaces of live yeast and mammalian cells through cell surface-initiated controlled radical polymerization. By developing cytocompatible PET-RAFT (photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization), synthetic polymers with narrow polydispersity (Mw/Mn < 1.3) could be obtained at room temperature in 5 minutes. This polymerization strategy enables chain growth to be initiated directly from chain-transfer agents anchored on the surface of live cells using either covalent attachment or non-covalent insertion, while maintaining high cell viability. Compared with conventional grafting-to approaches, these methods significantly improve the efficiency of grafting polymer chains and enable the active manipulation of cellular phenotypes.
Identification of genus Acinetobacter: Standardization of in-house PCR and its comparison with conventional phenotypic methods.

PubMed

Kulkarni, Sughosh S; Madalgi, Radhika; Ajantha, Ganavalli S; Kulkarni, Raghavendra D

2017-01-01

Acinetobacter is grouped under nonfermenting Gram-negative bacilli. It is increasingly isolated from pathological samples. The ability of this genus to acquire drug resistance and spread in the hospital settings is posing a grave problem in healthcare. Specific treatment protocols are advocated for Acinetobacter infections. Hence, rapid identification and drug susceptibility profiling are critical in the management of these infections. To standardize an in-house polymerase chain reaction (PCR) for identification of genus Acinetobacter and to compare PCR with two protocols for its phenotypic identification. A total of 96 clinical isolates of Acinetobacter were included in the study. An in-house PCR for genus level identification of Acinetobacter was standardized. All the isolates were phenotypically identified by two protocols. The results of PCR and phenotypic identification protocols were compared. The in-house PCR standardized was highly sensitive and specific for the genus Acinetobacter . There was 100% agreement between the phenotypic and molecular identification of the genus. The preliminary identification tests routinely used in clinical laboratories were also in complete agreement with phenotypic and molecular identification. The in-house PCR for genus level identification is specific and sensitive. However, it may not be essential for routine identification as the preliminary phenotypic identification tests used in the clinical laboratory reliably identify the genus Acinetobacter .
Phenotypic mapping of metabolic profiles using self-organizing maps of high-dimensional mass spectrometry data.

PubMed

Goodwin, Cody R; Sherrod, Stacy D; Marasco, Christina C; Bachmann, Brian O; Schramm-Sapyta, Nicole; Wikswo, John P; McLean, John A

2014-07-01

A metabolic system is composed of inherently interconnected metabolic precursors, intermediates, and products. The analysis of untargeted metabolomics data has conventionally been performed through the use of comparative statistics or multivariate statistical analysis-based approaches; however, each falls short in representing the related nature of metabolic perturbations. Herein, we describe a complementary method for the analysis of large metabolite inventories using a data-driven approach based upon a self-organizing map algorithm. This workflow allows for the unsupervised clustering, and subsequent prioritization of, correlated features through Gestalt comparisons of metabolic heat maps. We describe this methodology in detail, including a comparison to conventional metabolomics approaches, and demonstrate the application of this method to the analysis of the metabolic repercussions of prolonged cocaine exposure in rat sera profiles.
Updated Histologic Classification of Adenomas and Carcinomas in the Colon of Carcinogen-treated Sprague-Dawley Rats.

PubMed

Rubio, Carlos A

2017-12-01

Recent studies have disclosed novel histological phenotypes of colon tumours in carcinogen-treated rats. The aim of this study was to update the current histological classification of colonic neoplasias in Sprague-Dawley (SD) rats. Archival sections from 398 SD rats having 408 neoplasias in previous experiments were re-evaluated. Of the 408 colonic neoplasias, 11% (44/408) were adenomas without invasive growth and 89% (364/408) invasive carcinomas. Out of the 44 adenomas, 82% were conventional (tubular or villous), 14% traditional serrated (TSA; with unlocked serrations or with closed microtubules) and 5% gut-associated lymphoid tissue (GALT)-associated adenomas. Out of 364 carcinomas, 57% were conventional carcinomas, 26% GALT carcinomas, 8% undifferentiated, 6% signet-ring cell carcinomas, and 4% traditional serrated carcinomas (TSC). Thus, conventional adenomas, conventional carcinomas and GALT-associated carcinomas predominated (p<0.05). The updated classification of colonic tumours in SD rats includes conventional adenomas, TSA, GALT-associated adenomas, conventional carcinomas, TSC, GALT-associated carcinomas, signet-ring cell carcinomas and undifferentiated carcinomas. Several of the histological phenotypes reported here are not included in any of the current classifications of colonic tumours in rodents. This updated classification fulfils the requirements for an animal model of human disease, inasmuch as similar histological phenotypes of colon neoplasias have been documented in humans. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
Revisiting the susceptibility testing of Mycobacterium tuberculosis to ethionamide in solid culture medium.

PubMed

Lakshmi, Rajagopalan; Ramachandran, Ranjani; Kumar, D Ravi; Sundar, A Syam; Radhika, G; Rahman, Fathima; Selvakumar, N; Kumar, Vanaja

2015-11-01

Increase in the isolation of drug resistant phenotypes of Mycobacterium tuberculosis necessitates accuracy in the testing methodology. Critical concentration defining resistance for ethionamide (ETO), needs re-evaluation in accordance with the current scenario. Thus, re-evaluation of conventional minimum inhibitory concentration (MIC) and proportion sensitivity testing (PST) methods for ETO was done to identify the ideal breakpoint concentration defining resistance. Isolates of M. tuberculosis (n=235) from new and treated patients were subjected to conventional MIC and PST methods for ETO following standard operating procedures. With breakpoint concentration set at 114 and 156 µg/ml, an increase in specificity was observed whereas sensitivity was high with 80 µg/ml as breakpoint concentration. Errors due to false resistant and susceptible isolates were least at 80 µg/ml concentration. Performance parameters at 80 µg/ml breakpoint concentration indicated significant association between PST and MIC methods.
Molecular and Phenotypic Identification and Speciation of Malassezia Yeasts Isolated from Egyptian Patients with Pityriasis Versicolor

PubMed Central

Elshabrawy, Walaa Othman; Sallam, Manar

2017-01-01

Introduction Pityriasis Versicolor (PV) is a common health problem caused by genus Malassezia, a lipophilic fungi found as a part of the normal flora of skin. Although PV is common in Egypt, there is little information regarding the Malassezia species distribution in PV patients to date. Aim To spot a light on the distribution and clinico-epidemiological features of the Malassezia species in PV patients and healthy individuals that were established by conventional phenotypic and molecular techniques. Materials and Methods A cross-sectional study including 167 individuals; 137 clinically suspected PV patients attending Mansoura University Hospitals, Egypt and 30 healthy control individuals, was carried out. Characterization of Malassezia species was performed phenotypically by conventional, culture-based methods and biochemical tests. Genomic DNA was extracted from isolated colonies for PCR amplification of the highly conserved 26S rDNA region with further species level identification by Restriction Fragment Length Polymorphism (RFLP) using Hha1 and BstC1 enzymes. The association of Malassezia species with epidemiological profile and clinical characteristics was studied. Results A 94.2% of PV samples and 13.3% of control samples were positive by Potassium Hydroxide (KOH) while 71.5% of PV samples and 16.7% of control samples yielded growth in culture with high statistically significant differences (p=0.0001, for both methods). By phenotypic methods, only 75.5% of isolates from patients were identified as: M. furfur (51.4%), M. globosa, (29.7%), M. restricta (13.5%) and M. pachydermatis (5.4%) while by RFLP technique, six species were revealed: M. furfur (44.9%), M. globosa (24.5%), M. sympodialis (12.2 %), M. restricta (10.2%), M. obtusa (4.1%) and M. pachydermatis (4.1%). Most species were isolated from hypopigmented lesions of PV patients aged between 20-29 years. Neck and back were the most common affected sites. Only M. furfur (10%) and M. globosa (6.7%) were identified in healthy controls. Conclusion M. furfur and M. globosa are the commonly encountered species in both healthy and diseased human skin although other species were identified in PV patients. PCR-RFLP method represents a considerably accurate technique in identification of different Malassezia species for better understanding of their effect on the clinico-epidemiological characterization of PV patients in Egypt. PMID:28969121
A two-step non-flowcytometry-based naïve B cell isolation method and its application in Staphylococcal enterotoxin B (SEB) presentation.

PubMed

Chokeshai-u-saha, Kaj; Buranapraditkun, Supranee; Jacquet, Alain; Nguyen, Catherine; Ruxrungtham, Kiat

2012-09-01

To study the role of human naïve B cells in antigen presentation and stimulation to naïve CD4+ T cell, a suitable method to reproducibly isolate sufficient naïve B cells is required. To improve the purity of isolated naive B cells obtained from a conventional one-step magnetic bead method, we added a rosetting step to enrich total B cell isolates from human whole blood samples prior to negative cell sorting by magnetic beads. The acquired naïve B cells were analyzed for phenotypes and for their role in Staphylococcal enterotoxin B (SEB) presentation to naïve CD4+ T cells. The mean (SD) naïve B cell (CD19+/CD27-) purity obtained from this two-step method compared with the one-step method was 97% (1.0) versus 90% (1.2), respectively. This two-step method can be used with a sample of whole blood as small as 10 ml. The isolated naive B cells were phenotypically at a resting state and were able to prime naïve CD4+ T cell activation by Staphylococcal enterotoxin B (SEB) presentation. This two-step non-flow cytometry-based approach improved the purity of isolated naïve B cells compared with conventional one-step magnetic bead method. It also worked well with a small blood volume. In addition, this study showed that the isolated naïve B cells can present a super-antigen "SEB" to activate naïve CD4 cells. These methods may thus be useful for further in vitro characterization of human naïve B cells and their roles as antigen presenting cells in various diseases.
A Field Evaluation of the Hardy TB MODS Kit™ for the Rapid Phenotypic Diagnosis of Tuberculosis and Multi-Drug Resistant Tuberculosis

PubMed Central

Martin, Laura; Coronel, Jorge; Faulx, Dunia; Valdez, Melissa; Metzler, Mutsumi; Crudder, Chris; Castillo, Edith; Caviedes, Luz; Grandjean, Louis; Rodriguez, Mitzi; Friedland, Jon S.; Gilman, Robert H.; Moore, David A. J.

2014-01-01

Background Even though the WHO-endorsed, non-commercial MODS assay offers rapid, reliable TB liquid culture and phenotypic drug susceptibility testing (DST) at lower cost than any other diagnostic, uptake has been patchy. In part this reflects misperceptions about in-house assay quality assurance, but user convenience of one-stop procurement is also important. A commercial MODS kit was developed by Hardy Diagnostics (Santa Maria, CA, USA) with PATH (Seattle, WA, USA) to facilitate procurement, simplify procedures through readymade media, and enhance safety with a sealing silicone plate lid. Here we report the results from a large-scale field evaluation of the MODS kit in a government service laboratory. Methods & Findings 2446 sputum samples were cultured in parallel in Lowenstein-Jensen (LJ), conventional MODS and in the MODS kit. MODS kit DST was compared with conventional MODS (direct) DST and proportion method (indirect) DST. 778 samples (31.8%) were Mycobacterium tuberculosis culture-positive. Compared to conventional MODS the sensitivity, specificity, positive, and negative predictive values (95% confidence intervals) of the MODS Kit were 99.3% (98.3–99.8%), 98.3% (97.5–98.8%), 95.8% (94.0–97.1%), and 99.7% (99.3–99.9%). Median (interquartile ranges) time to culture-positivity (and rifampicin and isoniazid DST) was 10 (9–13) days for conventional MODS and 8.5 (7–11) for MODS Kit (p<0.01). Direct rifampicin and isoniazid DST in MODS kit was almost universally concordant with conventional MODS (97.9% agreement, 665/679 evaluable samples) and reference indirect DST (97.9% agreement, 687/702 evaluable samples). Conclusions MODS kit delivers performance indistinguishable from conventional MODS and offers a convenient, affordable alternative with enhanced safety from the sealing silicone lid. The availability in the marketplace of this platform, which conforms to European standards (CE-marked), readily repurposed for second-line DST in the near future, provides a fresh opportunity for improving equity of access to TB diagnosis and first and second-line DST in settings where the need is greatest. PMID:25225802
Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children

DTIC Science & Technology

2010-09-01

Organization (WHO), ETEC is the second most common cause of diarrhea after rotavirus in children less than 5 years of age and is therefore an...pathogens (Shigella, Salmonella. Vibrio, Campylo/Jacter, Gi- ardia Iamblia, Cryptosporidium, and rotavirus ) by conventional methods. Five lactose...DAEC (2% and 3%, respec- tively), Campylobacter (20% and 6%, respectively), and Shi- gella (2% and 0%, respectively). Rotavirus was found in 10% ( 4
Using Machine Learning to Discover Latent Social Phenotypes in Free-Ranging Macaques

PubMed Central

Madlon-Kay, Seth; Brent, Lauren J. N.; Heller, Katherine A.; Platt, Michael L.

2017-01-01

Investigating the biological bases of social phenotypes is challenging because social behavior is both high-dimensional and richly structured, and biological factors are more likely to influence complex patterns of behavior rather than any single behavior in isolation. The space of all possible patterns of interactions among behaviors is too large to investigate using conventional statistical methods. In order to quantitatively define social phenotypes from natural behavior, we developed a machine learning model to identify and measure patterns of behavior in naturalistic observational data, as well as their relationships to biological, environmental, and demographic sources of variation. We applied this model to extensive observations of natural behavior in free-ranging rhesus macaques, and identified behavioral states that appeared to capture periods of social isolation, competition over food, conflicts among groups, and affiliative coexistence. Phenotypes, represented as the rate of being in each state for a particular animal, were strongly and broadly influenced by dominance rank, sex, and social group membership. We also identified two states for which variation in rates had a substantial genetic component. We discuss how this model can be extended to identify the contributions to social phenotypes of particular genetic pathways. PMID:28754001
Genome-wide association study identifies multiple loci influencing human serum metabolite levels

PubMed Central

Kettunen, Johannes; Tukiainen, Taru; Sarin, Antti-Pekka; Ortega-Alonso, Alfredo; Tikkanen, Emmi; Lyytikäinen, Leo-Pekka; Kangas, Antti J; Soininen, Pasi; Würtz, Peter; Silander, Kaisa; Dick, Danielle M; Rose, Richard J; Savolainen, Markku J; Viikari, Jorma; Kähönen, Mika; Lehtimäki, Terho; Pietiläinen, Kirsi H; Inouye, Michael; McCarthy, Mark I; Jula, Antti; Eriksson, Johan; Raitakari, Olli T; Salomaa, Veikko; Kaprio, Jaakko; Järvelin, Marjo-Riitta; Peltonen, Leena; Perola, Markus; Freimer, Nelson B; Ala-Korpela, Mika; Palotie, Aarno; Ripatti, Samuli

2013-01-01

Nuclear magnetic resonance assays allow for measurement of a wide range of metabolic phenotypes. We report here the results of a GWAS on 8,330 Finnish individuals genotyped and imputed at 7.7 million SNPs for a range of 216 serum metabolic phenotypes assessed by NMR of serum samples. We identified significant associations (P < 2.31 × 10−10) at 31 loci, including 11 for which there have not been previous reports of associations to a metabolic trait or disorder. Analyses of Finnish twin pairs suggested that the metabolic measures reported here show higher heritability than comparable conventional metabolic phenotypes. In accordance with our expectations, SNPs at the 31 loci associated with individual metabolites account for a greater proportion of the genetic component of trait variance (up to 40%) than is typically observed for conventional serum metabolic phenotypes. The identification of such associations may provide substantial insight into cardiometabolic disorders. PMID:22286219
Relationships between functional genes in Lactobacillus delbrueckii ssp. bulgaricus isolates and phenotypic characteristics associated with fermentation time and flavor production in yogurt elucidated using multilocus sequence typing.

PubMed

Liu, Wenjun; Yu, Jie; Sun, Zhihong; Song, Yuqin; Wang, Xueni; Wang, Hongmei; Wuren, Tuoya; Zha, Musu; Menghe, Bilige; Heping, Zhang

2016-01-01

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is well known for its worldwide application in yogurt production. Flavor production and acid producing are considered as the most important characteristics for starter culture screening. To our knowledge this is the first study applying functional gene sequence multilocus sequence typing technology to predict the fermentation and flavor-producing characteristics of yogurt-producing bacteria. In the present study, phenotypic characteristics of 35 L. bulgaricus strains were quantified during the fermentation of milk to yogurt and during its subsequent storage; these included fermentation time, acidification rate, pH, titratable acidity, and flavor characteristics (acetaldehyde concentration). Furthermore, multilocus sequence typing analysis of 7 functional genes associated with fermentation time, acid production, and flavor formation was done to elucidate the phylogeny and genetic evolution of the same L. bulgaricus isolates. The results showed that strains significantly differed in fermentation time, acidification rate, and acetaldehyde production. Combining functional gene sequence analysis with phenotypic characteristics demonstrated that groups of strains established using genotype data were consistent with groups identified based on their phenotypic traits. This study has established an efficient and rapid molecular genotyping method to identify strains with good fermentation traits; this has the potential to replace time-consuming conventional methods based on direct measurement of phenotypic traits. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
A new Microbacterium species isolated from the blood of a patient with fever: Microbacterium pyrexiae sp. nov.

PubMed

Ko, Kwan Soo; Oh, Won Sup; Lee, Mi Young; Peck, Kyong Ran; Lee, Nam Yong; Song, Jae-Hoon

2007-04-01

A Gram-positive bacterium, SMC-A8265(T), which was isolated from the blood of a patient with fever but could not be identified by a conventional microbiologic method, was finally characterized by performing phenotypic and genotypic analyses. 16S rRNA gene sequence analysis revealed that the strain SMC-A8265(T) belonged to the genus Microbacterium, but it did not correspond to any of the previously described Microbacterium spp. Biochemical tests and cellular fatty acid composition analysis also confirmed that this bacterium is distinct from other Microbacterium spp. Based on the phenotypic and genotypic characteristics, we propose that the strain SMC-A8265(T) should be classified as a new species, namely, Microbacterium pyrexiae sp. nov.
Comparison of phenotypic methods and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry for the identification of aero-tolerant Actinomyces spp. isolated from soft-tissue infections.

PubMed

Ng, L S Y; Sim, J H C; Eng, L C; Menon, S; Tan, T Y

2012-08-01

Aero-tolerant Actinomyces spp. are an under-recognised cause of cutaneous infections, in part because identification using conventional phenotypic methods is difficult and may be inaccurate. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a promising new technique for bacterial identification, but with limited data on the identification of aero-tolerant Actinomyces spp. This study evaluated the accuracy of a phenotypic biochemical kit, MALDI-TOF MS and genotypic identification methods for the identification of this problematic group of organisms. Thirty aero-tolerant Actinomyces spp. were isolated from soft-tissue infections over a 2-year period. Species identification was performed by 16 s rRNA sequencing and genotypic results were compared with results obtained by API Coryne and MALDI-TOF MS. There was poor agreement between API Coryne and genotypic identification, with only 33% of isolates correctly identified to the species level. MALDI-TOF MS correctly identified 97% of isolates to the species level, with 33% of identifications achieved with high confidence scores. MALDI-TOF MS is a promising new tool for the identification of aero-tolerant Actinomyces spp., but improvement of the database is required in order to increase the confidence level of identification.

Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for the identification of clinical filamentous fungi.

PubMed

Huang, Yanfei; Zhang, Mingxin; Zhu, Min; Wang, Mei; Sun, Yufeng; Gu, Haitong; Cao, Jingjing; Li, Xue; Zhang, Shaoya; Wang, Jinglin; Lu, Xinxin

2017-07-01

Infections caused by filamentous fungi have become a health concern, and require rapid and accurate identification in order for effective treatment of the pathogens. To compare the performance of two MALDI-TOF MS systems (Bruker Microflex LT and Xiamen Microtyper) in the identification of filamentous fungal species. A total of 374 clinical filamentous fungal isolates sequentially collected in the Clinical Laboratory at the Beijing Tongren Hospital between January 2014 and December 2015 were identified by traditional phenotypic methods, Bruker Microflex LT and Xiamen Microtyper MALDI-TOF MS, respectively. The discrepancy between these methods was resolved by sequencing for definitive identification. Bruker Microflex LT and Xiamen Microtyper had similar correct species ID (98.9 vs. 99.2%), genus ID (99.7 vs. 100%), mis-ID (0.3 vs. 0%) and no ID (0 vs. 0). The rate of correct species identification by both MALDI-TOF MS (98.9 and 99.2%, respectively) was much higher compared with phenotypic approach (91.9%). Both MALDI-TOF MS systems provide accurate identification of clinical filamentous fungi compared with conventional phenotypic method, and have the potential to replace identification for routine identification of these fungi in clinical mycology laboratories. Both systems have similar performance in the identification of clinical filamentous fungi.
A combined disc method with resazurin agar plate assay for early phenotypic screening of KPC, MBL and OXA-48 carbapenemases among Enterobacteriaceae.

PubMed

Teethaisong, Y; Eumkeb, G; Nakouti, I; Evans, K; Hobbs, G

2016-08-01

To validate a combined disc method along with resazurin chromogenic agar for early screening and differentiation of Klebsiella pneumoniae carbapenemase, metallo-β-lactamase and OXA-48 carbapenemase-producing Enterobacteriaceae. The combined disc test comprising of meropenem alone and with EDTA, phenylboronic acid or both EDTA and phenylboronic acid, and temocillin alone were evaluated with the resazurin chromogenic agar plate assay against a total of 86 molecularly confirmed Enterobacteriaceae clinical isolates (11 metallo-β-lactamases, eight Kl. pneumoniae carbapenemases, 11 OXA-48, 32 AmpC and 15 extended-spectrum-β-lactamase producers and nine co-producers of extended-spectrum-β-lactamase and AmpC). The inhibition zone diameters were measured and interpreted at 7 h for the presence of carbapenemase. All carbapenemase producers were phenotypically distinguished by this assay with 100% sensitivity and specificity. This early phenotypic method is very simple, inexpensive, and reliable in the detection and differentiation of carbapenemase-producing Enterobacteriaceae. It could be exploited in any microbiological laboratory for diagnosis of these recalcitrant bacteria. This assay poses excellent performance in discrimination of Kl. pneumoniae carbapenemase, metallo-β-lactamase and OXA-48 carbapenemases within 7 h, which is much faster than conventional disc diffusion methods. The rapid detection could help clinicians screen patients, control infection and provide epidemiological surveillance. © 2016 The Society for Applied Microbiology.
Phenotypic mutant library: potential for gene discovery

USDA-ARS?s Scientific Manuscript database

The rapid development of high throughput and affordable Next- Generation Sequencing (NGS) techniques has renewed interest in gene discovery using forward genetics. The conventional forward genetic approach starts with isolation of mutants with a phenotype of interest, mapping the mutation within a s...
An integrated one-step system to extract, analyze and annotate all relevant information from image-based cell screening of chemical libraries.

PubMed

Rabal, Obdulia; Link, Wolfgang; Serelde, Beatriz G; Bischoff, James R; Oyarzabal, Julen

2010-04-01

Here we report the development and validation of a complete solution to manage and analyze the data produced by image-based phenotypic screening campaigns of small-molecule libraries. In one step initial crude images are analyzed for multiple cytological features, statistical analysis is performed and molecules that produce the desired phenotypic profile are identified. A naïve Bayes classifier, integrating chemical and phenotypic spaces, is built and utilized during the process to assess those images initially classified as "fuzzy"-an automated iterative feedback tuning. Simultaneously, all this information is directly annotated in a relational database containing the chemical data. This novel fully automated method was validated by conducting a re-analysis of results from a high-content screening campaign involving 33 992 molecules used to identify inhibitors of the PI3K/Akt signaling pathway. Ninety-two percent of confirmed hits identified by the conventional multistep analysis method were identified using this integrated one-step system as well as 40 new hits, 14.9% of the total, originally false negatives. Ninety-six percent of true negatives were properly recognized too. A web-based access to the database, with customizable data retrieval and visualization tools, facilitates the posterior analysis of annotated cytological features which allows identification of additional phenotypic profiles; thus, further analysis of original crude images is not required.
Non-contact measurement of oxygen saturation with an RGB camera

PubMed Central

Guazzi, Alessandro R.; Villarroel, Mauricio; Jorge, João; Daly, Jonathan; Frise, Matthew C.; Robbins, Peter A.; Tarassenko, Lionel

2015-01-01

A novel method (Sophia) is presented to track oxygen saturation changes in a controlled environment using an RGB camera placed approximately 1.5 m away from the subject. The method is evaluated on five healthy volunteers (Fitzpatrick skin phenotypes II, III, and IV) whose oxygen saturations were varied between 80% and 100% in a purpose-built chamber over 40 minutes each. The method carefully selects regions of interest (ROI) in the camera image by calculating signal-to-noise ratios for each ROI. This allows it to track changes in oxygen saturation accurately with respect to a conventional pulse oximeter (median coefficient of determination, 0.85). PMID:26417504
Resolving the Effects of Maternal and Offspring Genotype on Dyadic Outcomes in Genome Wide Complex Trait Analysis (“M-GCTA”)

PubMed Central

Pourcain, Beate St.; Smith, George Davey; York, Timothy P.; Evans, David M.

2014-01-01

Genome wide complex trait analysis (GCTA) is extended to include environmental effects of the maternal genotype on offspring phenotype (“maternal effects”, M-GCTA). The model includes parameters for the direct effects of the offspring genotype, maternal effects and the covariance between direct and maternal effects. Analysis of simulated data, conducted in OpenMx, confirmed that model parameters could be recovered by full information maximum likelihood (FIML) and evaluated the biases that arise in conventional GCTA when indirect genetic effects are ignored. Estimates derived from FIML in OpenMx showed very close agreement to those obtained by restricted maximum likelihood using the published algorithm for GCTA. The method was also applied to illustrative perinatal phenotypes from ∼4,000 mother-offspring pairs from the Avon Longitudinal Study of Parents and Children. The relative merits of extended GCTA in contrast to quantitative genetic approaches based on analyzing the phenotypic covariance structure of kinships are considered. PMID:25060210
Towards precision medicine: from quantitative imaging to radiomics

PubMed Central

Acharya, U. Rajendra; Hagiwara, Yuki; Sudarshan, Vidya K.; Chan, Wai Yee; Ng, Kwan Hoong

2018-01-01

Radiology (imaging) and imaging-guided interventions, which provide multi-parametric morphologic and functional information, are playing an increasingly significant role in precision medicine. Radiologists are trained to understand the imaging phenotypes, transcribe those observations (phenotypes) to correlate with underlying diseases and to characterize the images. However, in order to understand and characterize the molecular phenotype (to obtain genomic information) of solid heterogeneous tumours, the advanced sequencing of those tissues using biopsy is required. Thus, radiologists image the tissues from various views and angles in order to have the complete image phenotypes, thereby acquiring a huge amount of data. Deriving meaningful details from all these radiological data becomes challenging and raises the big data issues. Therefore, interest in the application of radiomics has been growing in recent years as it has the potential to provide significant interpretive and predictive information for decision support. Radiomics is a combination of conventional computer-aided diagnosis, deep learning methods, and human skills, and thus can be used for quantitative characterization of tumour phenotypes. This paper discusses the overview of radiomics workflow, the results of various radiomics-based studies conducted using various radiological images such as computed tomography (CT), magnetic resonance imaging (MRI), and positron-emission tomography (PET), the challenges we are facing, and the potential contribution of radiomics towards precision medicine. PMID:29308604
The Expression of TALEN before Fertilization Provides a Rapid Knock-Out Phenotype in Xenopus laevis Founder Embryos.

PubMed

Miyamoto, Kei; Suzuki, Ken-Ichi T; Suzuki, Miyuki; Sakane, Yuto; Sakuma, Tetsushi; Herberg, Sarah; Simeone, Angela; Simpson, David; Jullien, Jerome; Yamamoto, Takashi; Gurdon, J B

2015-01-01

Recent advances in genome editing using programmable nucleases have revolutionized gene targeting in various organisms. Successful gene knock-out has been shown in Xenopus, a widely used model organism, although a system enabling less mosaic knock-out in founder embryos (F0) needs to be explored in order to judge phenotypes in the F0 generation. Here, we injected modified highly active transcription activator-like effector nuclease (TALEN) mRNA to oocytes at the germinal vesicle (GV) stage, followed by in vitro maturation and intracytoplasmic sperm injection, to achieve a full knock-out in F0 embryos. Unlike conventional injection methods to fertilized embryos, the injection of TALEN mRNA into GV oocytes allows expression of nucleases before fertilization, enabling them to work from an earlier stage. Using this procedure, most of developed embryos showed full knock-out phenotypes of the pigmentation gene tyrosinase and/or embryonic lethal gene pax6 in the founder generation. In addition, our method permitted a large 1 kb deletion. Thus, we describe nearly complete gene knock-out phenotypes in Xenopus laevis F0 embryos. The presented method will help to accelerate the production of knock-out frogs since we can bypass an extra generation of about 1 year in Xenopus laevis. Meantime, our method provides a unique opportunity to rapidly test the developmental effects of disrupting those genes that do not permit growth to an adult able to reproduce. In addition, the protocol shown here is considerably less invasive than the previously used host transfer since our protocol does not require surgery. The experimental scheme presented is potentially applicable to other organisms such as mammals and fish to resolve common issues of mosaicism in founders.
Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens.

PubMed

Minker, Katharine R; Biedrzycki, Meredith L; Kolagunda, Abhishek; Rhein, Stephen; Perina, Fabiano J; Jacobs, Samuel S; Moore, Michael; Jamann, Tiffany M; Yang, Qin; Nelson, Rebecca; Balint-Kurti, Peter; Kambhamettu, Chandra; Wisser, Randall J; Caplan, Jeffrey L

2018-02-01

The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms-from sample processing to image analysis-to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141-152, 2018. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Evaluation of two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems for the identification of Candida species.

PubMed

Lacroix, C; Gicquel, A; Sendid, B; Meyer, J; Accoceberry, I; François, N; Morio, F; Desoubeaux, G; Chandenier, J; Kauffmann-Lacroix, C; Hennequin, C; Guitard, J; Nassif, X; Bougnoux, M-E

2014-02-01

Candida spp. are responsible for severe infections in immunocompromised patients and those undergoing invasive procedures. The accurate identification of Candida species is important because emerging species can be associated with various antifungal susceptibility spectra. Conventional methods have been developed to identify the most common pathogens, but have often failed to identify uncommon species. Several studies have reported the efficiency of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of clinically relevant Candida species. In this study, we evaluated two commercially available MALDI-TOF systems, Andromas™ and Bruker Biotyper™, for Candida identification in routine diagnosis. For this purpose, we investigated 1383 Candida isolates prospectively collected in eight hospital laboratories during routine practice. MALDI-TOF MS results were compared with those obtained using conventional phenotypic methods. Analysis of rDNA gene sequences with internal transcribed regions or D1-D2 regions is considered the reference standard for identification. Both MALDI-TOF MS systems could accurately identify 98.3% of the isolates at the species level (1359/1383 for Andromas™; 1360/1383 for Bruker Biotyper™) vs. 96.5% for conventional techniques. Furthermore, whereas conventional methods failed to identify rare or emerging species, these were correctly identified by MALDI-TOF MS. Both MALDI-TOF MS systems are accurate and cost-effective alternatives to conventional methods for mycological identification of clinically relevant Candida species and should improve the diagnosis of fungal infections as well as patient management. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.
Genome-wide annotation of mutations in a phenotyped mutant library provides an efficient platform for discovery of casual gene mutations

USDA-ARS?s Scientific Manuscript database

Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. Conventionally, these mutations are identified by techniques that can detect single-nucleotide mismatches in heteroduplexes of individual PCR amplicons. We applied whole-genome sequencing to 256-phenotyped mutant l...
Unexpected Cartilage Phenotype in CD4-Cre-Conditional SOS-Deficient Mice.

PubMed

Guittard, Geoffrey; Gallardo, Devorah L; Li, Wenmei; Melis, Nicolas; Lui, Julian C; Kortum, Robert L; Shakarishvili, Nicholas G; Huh, Sunmee; Baron, Jeffrey; Weigert, Roberto; Kramer, Joshua A; Samelson, Lawrence E; Sommers, Connie L

2017-01-01

RAS signaling is central to many cellular processes and SOS proteins promote RAS activation. To investigate the role of SOS proteins in T cell biology, we crossed Sos1 f/f Sos2 -/- mice to CD4-Cre transgenic mice. We previously reported an effect of these mutations on T cell signaling and T cell migration. Unexpectedly, we observed nodules on the joints of greater than 90% of these mutant mice at 5 months of age, especially on the carpal joints. As the mice aged further, some also displayed joint stiffness, hind limb paralysis, and lameness. Histological analysis indicated that the abnormal growth in joints originated from dysplastic chondrocytes. Second harmonic generation imaging of the carpal nodules revealed that nodules were encased by rich collagen fibrous networks. Nodules formed in mice also deficient in RAG2, indicating that conventional T cells, which undergo rearrangement of the T cell antigen receptor, are not required for this phenotype. CD4-Cre expression in a subset of cells, either immune lineage cells (e.g., non-conventional T cells) or non-immune lineage cells (e.g., chondrocytes) likely mediates the dramatic phenotype observed in this study. Disruptions of genes in the RAS signaling pathway are especially likely to cause this phenotype. These results also serve as a cautionary tale to those intending to use CD4-Cre transgenic mice to specifically delete genes in conventional T cells.
Sjögren-Like Lacrimal Keratoconjunctivitis in Germ-Free Mice

PubMed Central

Wang, Changjun; Zaheer, Mahira; Bian, Fang; Quach, Darin; Swennes, Alton G.; Britton, Robert A.; Pflugfelder, Stephen C.

2018-01-01

Commensal bacteria play an important role in the formation of the immune system but their role in the maintenance of immune homeostasis at the ocular surface and lacrimal gland remains poorly understood. This study investigated the eye and lacrimal gland phenotype in germ-free and conventional C57BL/6J mice. Our results showed that germ-free mice had significantly greater corneal barrier disruption, greater goblet cell loss, and greater total inflammatory cell and CD4+ T cell infiltration within the lacrimal gland compared to the conventionally housed group. A greater frequency of CD4+IFN-γ+ cells was observed in germ-free lacrimal glands. Females exhibited a more severe phenotype compared to males. Adoptive transfer of CD4+ T cells isolated from female germ-free mice into RAG1KO mice transferred Sjögren-like lacrimal keratoconjunctivitis. Fecal microbiota transplant from conventional mice reverted dry eye phenotype in germ-free mice and decreased CD4+IFN-γ+ cells to levels similar to conventional C57BL/6J mice. These findings indicate that germ-free mice have a spontaneous lacrimal keratoconjunctivitis similar to that observed in Sjögren syndrome patients and demonstrate that commensal bacteria function in maintaining immune homeostasis on the ocular surface. Thus, manipulation of intestinal commensal bacteria has the potential to become a novel therapeutic approach to treat Sjögren Syndrome. PMID:29438346
Phenotypic and Genotypic Eligible Methods for Salmonella Typhimurium Source Tracking

PubMed Central

Ferrari, Rafaela G.; Panzenhagen, Pedro H. N.; Conte-Junior, Carlos A.

2017-01-01

Salmonellosis is one of the most common causes of foodborne infection and a leading cause of human gastroenteritis. Throughout the last decade, Salmonella enterica serotype Typhimurium (ST) has shown an increase report with the simultaneous emergence of multidrug-resistant isolates, as phage type DT104. Therefore, to successfully control this microorganism, it is important to attribute salmonellosis to the exact source. Studies of Salmonella source attribution have been performed to determine the main food/food-production animals involved, toward which, control efforts should be correctly directed. Hence, the election of a ST subtyping method depends on the particular problem that efforts must be directed, the resources and the data available. Generally, before choosing a molecular subtyping, phenotyping approaches such as serotyping, phage typing, and antimicrobial resistance profiling are implemented as a screening of an investigation, and the results are computed using frequency-matching models (i.e., Dutch, Hald and Asymmetric Island models). Actually, due to the advancement of molecular tools as PFGE, MLVA, MLST, CRISPR, and WGS more precise results have been obtained, but even with these technologies, there are still gaps to be elucidated. To address this issue, an important question needs to be answered: what are the currently suitable subtyping methods to source attribute ST. This review presents the most frequently applied subtyping methods used to characterize ST, analyses the major available microbial subtyping attribution models and ponders the use of conventional phenotyping methods, as well as, the most applied genotypic tools in the context of their potential applicability to investigates ST source tracking. PMID:29312260
Phenotypic and Genotypic Eligible Methods for Salmonella Typhimurium Source Tracking.

PubMed

Ferrari, Rafaela G; Panzenhagen, Pedro H N; Conte-Junior, Carlos A

2017-01-01

Salmonellosis is one of the most common causes of foodborne infection and a leading cause of human gastroenteritis. Throughout the last decade, Salmonella enterica serotype Typhimurium (ST) has shown an increase report with the simultaneous emergence of multidrug-resistant isolates, as phage type DT104. Therefore, to successfully control this microorganism, it is important to attribute salmonellosis to the exact source. Studies of Salmonella source attribution have been performed to determine the main food/food-production animals involved, toward which, control efforts should be correctly directed. Hence, the election of a ST subtyping method depends on the particular problem that efforts must be directed, the resources and the data available. Generally, before choosing a molecular subtyping, phenotyping approaches such as serotyping, phage typing, and antimicrobial resistance profiling are implemented as a screening of an investigation, and the results are computed using frequency-matching models (i.e., Dutch, Hald and Asymmetric Island models). Actually, due to the advancement of molecular tools as PFGE, MLVA, MLST, CRISPR, and WGS more precise results have been obtained, but even with these technologies, there are still gaps to be elucidated. To address this issue, an important question needs to be answered: what are the currently suitable subtyping methods to source attribute ST. This review presents the most frequently applied subtyping methods used to characterize ST, analyses the major available microbial subtyping attribution models and ponders the use of conventional phenotyping methods, as well as, the most applied genotypic tools in the context of their potential applicability to investigates ST source tracking.
Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis

PubMed Central

Dragovic, Rebecca A.; Gardiner, Christopher; Brooks, Alexandra S.; Tannetta, Dionne S.; Ferguson, David J.P.; Hole, Patrick; Carr, Bob; Redman, Christopher W.G.; Harris, Adrian L.; Dobson, Peter J.; Harrison, Paul; Sargent, Ian L.

2011-01-01

Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ∼50 nm and is far more sensitive than conventional flow cytometry (lower limit ∼300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined. From the Clinical Editor The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry. PMID:21601655
Self-reported pigmentary phenotypes and race are significant but incomplete predictors of Fitzpatrick skin phototype in an ethnically diverse population.

PubMed

He, Steven Y; McCulloch, Charles E; Boscardin, W John; Chren, Mary-Margaret; Linos, Eleni; Arron, Sarah T

2014-10-01

Fitzpatrick skin phototype (FSPT) is the most common method used to assess sunburn risk and is an independent predictor of skin cancer risk. Because of a conventional assumption that FSPT is predictable based on pigmentary phenotypes, physicians frequently estimate FSPT based on patient appearance. We sought to determine the degree to which self-reported race and pigmentary phenotypes are predictive of FSPT in a large, ethnically diverse population. A cross-sectional survey collected responses from 3386 individuals regarding self-reported FSPT, pigmentary phenotypes, race, age, and sex. Univariate and multivariate logistic regression analyses were performed to determine variables that significantly predict FSPT. Race, sex, skin color, eye color, and hair color are significant but weak independent predictors of FSPT (P<.0001). A multivariate model constructed using all independent predictors of FSPT only accurately predicted FSPT to within 1 point on the Fitzpatrick scale with 92% accuracy (weighted kappa statistic 0.53). Our study enriched for responses from ethnic minorities and does not fully represent the demographics of the US population. Patient self-reported race and pigmentary phenotypes are inaccurate predictors of sun sensitivity as defined by FSPT. There are limitations to using patient-reported race and appearance in predicting individual sunburn risk. Copyright © 2014 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.
Using tumor phenotype, histological tumor distribution, and mammographic appearance to explain the survival differences between screen-detected and clinically detected breast cancers.

PubMed

Chuang, Shu-Lin; Chen, Sam Li-Sheng; Yu, Cheng-Ping; Chang, King-Jen; Yen, Amy Ming-Fang; Chiu, Sherry Yueh-Hsia; Fann, Jean Ching-Yuan; Tabár, László; Stephen, Duffy W; Smith, Robert A; Chen, Hsiu-Hsi

2014-08-01

In the era of mass screening for breast cancer with mammography, it has been noted that conventional tumor attributes and mammographic appearance are insufficient to account for the better prognosis of screen-detected tumors. Such prognostication may require additional updated pathological information regarding tumor phenotype (e.g., basal status) and histological tumor distribution (focality). We investigated this hypothesis using a Bayesian approach to analyze breast cancer data from Dalarna County, Sweden. We used data for tumors diagnosed in the Swedish Two-County Trial and early service screening period, 1977-1995, and from the mature service screening period, 1996-1998. In the early period of mammographic screening (1977-1995), the crude hazard ratio (HR) of breast cancer death for screen-detected cases compared with symptomatic ones was 0.22 (95% CI: 0.17-0.29) compared with 0.53 (95% CI: 0.34-0.76) when adjusted for conventional tumor attributes only. Using the data from the mature service screening period, 1996-1998, the HR was 0.23 (95% CI: 0.08-0.44) unadjusted and 0.71 (95% CI: 0.26-1.47) after adjustment for tumor phenotype, mammographic appearance, histological tumor distribution, and conventional tumor attributes. The area under the ROC curve (AUC) for the prediction of breast cancer deaths using these variables without the detection mode was 0.82, only slightly less than that observed when additionally including the detection mode (AUC=0.83). Using Freedman statistics, conventional tumor attributes and mammographic appearances explained 58% (95% CI: 57.5-58.6%) of the difference of breast cancer survival between the screen-detected and the clinically detected breast cancers, whereas the corresponding figure was increased to 77% (95% CI: 75.6-77.6%) when adding the two information on tumor phenotype and histological tumor distribution. The results indicated that conventional tumor attributes and mammographic appearance are not sufficient to be interim markers for explaining the survival difference between screen-detected and clinically detected cancers in the era marked by the widespread use of mammography. Additional information on tumor phenotype and histological distribution may be added as effective interim markers for explaining the benefit of the early detection of breast cancer with mammography. © 2014 APMIS. Published by John Wiley & Sons Ltd.
Quality of the blood pressure phenotype in the GEnotipo, Fenotipo y Ambiente de la hipertensión arterial en UruguaY (GEFA-HT-UY) study.

PubMed

Luzardo, Leonella; Sottolano, Mariana; Lujambio, Inés; Robaina, Sebastián; Thijs, Lutgarde; da Rosa, Alicia; Krul, Nadia; Carusso, Florencia; Ríos, Ana C; Olascoaga, Alicia; Noboa, Oscar; Staessen, Jan A; Boggia, José

2014-12-01

In the ongoing GEnotipo, Fenotipo y Ambiente de la HiperTensión Arterial en UruguaY (GEFA-HT-UY) study, we applied standardized epidemiological methods to determine complex phenotypes including blood pressure (BP). In this report, we present the quality control of the conventionally measured BP. Three trained observers measured BP five times consecutively in the seated position at each of two home visits and one clinic visit according to the guidelines of the European Society of Hypertension. On 1 December 2013, 4379 single BP readings in 170 participants were available for analysis. Fewer BP readings than the five planned per contact occurred only at one home visit. Among observers, the frequency of identical consecutive readings for systolic or diastolic BP varied from 0 to 4.2%. The occurrence of odd readings ranged from 0.1 to 0.6%. Only 21.6% of the systolic and diastolic BP readings ended on zero (expected 20%). At home visits, there was a progressive decline in BP from the first to the fifth reading. The average of the five BP readings also decreased from the first to the second home visit (-5.63/-2.34 mmHg). Our study highlighted the necessity to implement a stringent quality control of the conventionally measured BP. The procedures set up in the GEFA-HT-UY study are resulting in a well-defined BP phenotype, which is consistent with that in other population studies.
Evaluations for service-sire conception rate for heifer and cow inseminations with conventional and sexed semen

USDA-ARS?s Scientific Manuscript database

Service-sire conception rate (SCR), a phenotypic fertility evaluation based on conventional (nonsexed) inseminations from parities 1 through 5, was implemented by USDA in August 2008. Using insemination data from 2005 through 2009, the SCR procedure was applied separately for nulliparous heifer inse...

Female-specific flightless (fsRIDL) phenotype for control of Aedes albopictus.

PubMed

Labbé, Geneviève M C; Scaife, Sarah; Morgan, Siân A; Curtis, Zoë H; Alphey, Luke

2012-01-01

Aedes albopictus, the Asian tiger mosquito, is a vector of several arboviruses including dengue and chikungunya, and is also a significant nuisance mosquito. It is one of the most invasive of mosquitoes with a relentlessly increasing geographic distribution. Conventional control methods have so far failed to control Ae. albopictus adequately. Novel genetics-based strategies offer a promising alternative or aid towards efficient control of this mosquito. We describe here the isolation, characterisation and use of the Ae. albopictus Actin-4 gene to drive a dominant lethal gene in the indirect flight muscles of Ae. albopictus, thus inducing a conditional female-specific late-acting flightless phenotype. We also show that in this context, the Actin-4 regulatory regions from both Ae. albopictus and Ae. aegypti can be used to provide conditional female-specific flightlessness in either species. With the disease-transmitting females incapacitated, the female flightless phenotype encompasses a genetic sexing mechanism and would be suitable for controlling Ae. albopictus using a male-only release approach as part of an integrated pest management strategy.
Whole genome sequencing in the prevention and control of Staphylococcus aureus infection.

PubMed

Price, J R; Didelot, X; Crook, D W; Llewelyn, M J; Paul, J

2013-01-01

Staphylococcus aureus remains a leading cause of hospital-acquired infection but weaknesses inherent in currently available typing methods impede effective infection prevention and control. The high resolution offered by whole genome sequencing has the potential to revolutionise our understanding and management of S. aureus infection. To outline the practicalities of whole genome sequencing and discuss how it might shape future infection control practice. We review conventional typing methods and compare these with the potential offered by whole genome sequencing. In contrast with conventional methods, whole genome sequencing discriminates down to single nucleotide differences and allows accurate characterisation of transmission events and outbreaks and additionally provides information about the genetic basis of phenotypic characteristics, including antibiotic susceptibility and virulence. However, translating its potential into routine practice will depend on affordability, acceptable turnaround times and on creating a reliable standardised bioinformatic infrastructure. Whole genome sequencing has the potential to provide a universal test that facilitates outbreak investigation, enables the detection of emerging strains and predicts their clinical importance. Copyright © 2012 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
A field evaluation of the Hardy TB MODS Kit™ for the rapid phenotypic diagnosis of tuberculosis and multi-drug resistant tuberculosis.

PubMed

Martin, Laura; Coronel, Jorge; Faulx, Dunia; Valdez, Melissa; Metzler, Mutsumi; Crudder, Chris; Castillo, Edith; Caviedes, Luz; Grandjean, Louis; Rodriguez, Mitzi; Friedland, Jon S; Gilman, Robert H; Moore, David A J

2014-01-01

Even though the WHO-endorsed, non-commercial MODS assay offers rapid, reliable TB liquid culture and phenotypic drug susceptibility testing (DST) at lower cost than any other diagnostic, uptake has been patchy. In part this reflects misperceptions about in-house assay quality assurance, but user convenience of one-stop procurement is also important. A commercial MODS kit was developed by Hardy Diagnostics (Santa Maria, CA, USA) with PATH (Seattle, WA, USA) to facilitate procurement, simplify procedures through readymade media, and enhance safety with a sealing silicone plate lid. Here we report the results from a large-scale field evaluation of the MODS kit in a government service laboratory. 2446 sputum samples were cultured in parallel in Lowenstein-Jensen (LJ), conventional MODS and in the MODS kit. MODS kit DST was compared with conventional MODS (direct) DST and proportion method (indirect) DST. 778 samples (31.8%) were Mycobacterium tuberculosis culture-positive. Compared to conventional MODS the sensitivity, specificity, positive, and negative predictive values (95% confidence intervals) of the MODS Kit were 99.3% (98.3-99.8%), 98.3% (97.5-98.8%), 95.8% (94.0-97.1%), and 99.7% (99.3-99.9%). Median (interquartile ranges) time to culture-positivity (and rifampicin and isoniazid DST) was 10 (9-13) days for conventional MODS and 8.5 (7-11) for MODS Kit (p<0.01). Direct rifampicin and isoniazid DST in MODS kit was almost universally concordant with conventional MODS (97.9% agreement, 665/679 evaluable samples) and reference indirect DST (97.9% agreement, 687/702 evaluable samples). MODS kit delivers performance indistinguishable from conventional MODS and offers a convenient, affordable alternative with enhanced safety from the sealing silicone lid. The availability in the marketplace of this platform, which conforms to European standards (CE-marked), readily repurposed for second-line DST in the near future, provides a fresh opportunity for improving equity of access to TB diagnosis and first and second-line DST in settings where the need is greatest.
Application of the stepwise focusing method to optimize the cost-effectiveness of genome-wide association studies with limited research budgets for genotyping and phenotyping.

PubMed

Ohashi, J; Clark, A G

2005-05-01

The recent cataloguing of a large number of SNPs enables us to perform genome-wide association studies for detecting common genetic variants associated with disease. Such studies, however, generally have limited research budgets for genotyping and phenotyping. It is therefore necessary to optimize the study design by determining the most cost-effective numbers of SNPs and individuals to analyze. In this report we applied the stepwise focusing method, with two-stage design, developed by Satagopan et al. (2002) and Saito & Kamatani (2002), to optimize the cost-effectiveness of a genome-wide direct association study using a transmission/disequilibrium test (TDT). The stepwise focusing method consists of two steps: a large number of SNPs are examined in the first focusing step, and then all the SNPs showing a significant P-value are tested again using a larger set of individuals in the second focusing step. In the framework of optimization, the numbers of SNPs and families and the significance levels in the first and second steps were regarded as variables to be considered. Our results showed that the stepwise focusing method achieves a distinct gain of power compared to a conventional method with the same research budget.
Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance

PubMed Central

Jeyapalan, J N; Noor, D A Mohamed; Lee, S-H; Tan, C L; Appleby, V A; Kilday, J P; Palmer, R D; Schwalbe, E C; Clifford, S C; Walker, D A; Murray, M J; Coleman, N; Nicholson, J C; Scotting, P J

2011-01-01

Background: Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. Methods: A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. Results: Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a ‘methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. Conclusion: Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy. PMID:21712824
Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test

PubMed Central

Lionel, Anath C; Costain, Gregory; Monfared, Nasim; Walker, Susan; Reuter, Miriam S; Hosseini, S Mohsen; Thiruvahindrapuram, Bhooma; Merico, Daniele; Jobling, Rebekah; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Sung, Wilson W L; Wang, Zhuozhi; Bikangaga, Peter; Boelman, Cyrus; Carter, Melissa T; Cordeiro, Dawn; Cytrynbaum, Cheryl; Dell, Sharon D; Dhir, Priya; Dowling, James J; Heon, Elise; Hewson, Stacy; Hiraki, Linda; Inbar-Feigenberg, Michal; Klatt, Regan; Kronick, Jonathan; Laxer, Ronald M; Licht, Christoph; MacDonald, Heather; Mercimek-Andrews, Saadet; Mendoza-Londono, Roberto; Piscione, Tino; Schneider, Rayfel; Schulze, Andreas; Silverman, Earl; Siriwardena, Komudi; Snead, O Carter; Sondheimer, Neal; Sutherland, Joanne; Vincent, Ajoy; Wasserman, Jonathan D; Weksberg, Rosanna; Shuman, Cheryl; Carew, Chris; Szego, Michael J; Hayeems, Robin Z; Basran, Raveen; Stavropoulos, Dimitri J; Ray, Peter N; Bowdin, Sarah; Meyn, M Stephen; Cohn, Ronald D; Scherer, Stephen W; Marshall, Christian R

2018-01-01

Purpose Genetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use. Methods We prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing. Results WGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24% P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A. Conclusion WGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort. PMID:28771251
Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test.

PubMed

Lionel, Anath C; Costain, Gregory; Monfared, Nasim; Walker, Susan; Reuter, Miriam S; Hosseini, S Mohsen; Thiruvahindrapuram, Bhooma; Merico, Daniele; Jobling, Rebekah; Nalpathamkalam, Thomas; Pellecchia, Giovanna; Sung, Wilson W L; Wang, Zhuozhi; Bikangaga, Peter; Boelman, Cyrus; Carter, Melissa T; Cordeiro, Dawn; Cytrynbaum, Cheryl; Dell, Sharon D; Dhir, Priya; Dowling, James J; Heon, Elise; Hewson, Stacy; Hiraki, Linda; Inbar-Feigenberg, Michal; Klatt, Regan; Kronick, Jonathan; Laxer, Ronald M; Licht, Christoph; MacDonald, Heather; Mercimek-Andrews, Saadet; Mendoza-Londono, Roberto; Piscione, Tino; Schneider, Rayfel; Schulze, Andreas; Silverman, Earl; Siriwardena, Komudi; Snead, O Carter; Sondheimer, Neal; Sutherland, Joanne; Vincent, Ajoy; Wasserman, Jonathan D; Weksberg, Rosanna; Shuman, Cheryl; Carew, Chris; Szego, Michael J; Hayeems, Robin Z; Basran, Raveen; Stavropoulos, Dimitri J; Ray, Peter N; Bowdin, Sarah; Meyn, M Stephen; Cohn, Ronald D; Scherer, Stephen W; Marshall, Christian R

2018-04-01

PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.
Super-Resolution of Plant Disease Images for the Acceleration of Image-based Phenotyping and Vigor Diagnosis in Agriculture.

PubMed

Yamamoto, Kyosuke; Togami, Takashi; Yamaguchi, Norio

2017-11-06

Unmanned aerial vehicles (UAVs or drones) are a very promising branch of technology, and they have been utilized in agriculture-in cooperation with image processing technologies-for phenotyping and vigor diagnosis. One of the problems in the utilization of UAVs for agricultural purposes is the limitation in flight time. It is necessary to fly at a high altitude to capture the maximum number of plants in the limited time available, but this reduces the spatial resolution of the captured images. In this study, we applied a super-resolution method to the low-resolution images of tomato diseases to recover detailed appearances, such as lesions on plant organs. We also conducted disease classification using high-resolution, low-resolution, and super-resolution images to evaluate the effectiveness of super-resolution methods in disease classification. Our results indicated that the super-resolution method outperformed conventional image scaling methods in spatial resolution enhancement of tomato disease images. The results of disease classification showed that the accuracy attained was also better by a large margin with super-resolution images than with low-resolution images. These results indicated that our approach not only recovered the information lost in low-resolution images, but also exerted a beneficial influence on further image analysis. The proposed approach will accelerate image-based phenotyping and vigor diagnosis in the field, because it not only saves time to capture images of a crop in a cultivation field but also secures the accuracy of these images for further analysis.
Super-Resolution of Plant Disease Images for the Acceleration of Image-based Phenotyping and Vigor Diagnosis in Agriculture

PubMed Central

Togami, Takashi; Yamaguchi, Norio

2017-01-01

Unmanned aerial vehicles (UAVs or drones) are a very promising branch of technology, and they have been utilized in agriculture—in cooperation with image processing technologies—for phenotyping and vigor diagnosis. One of the problems in the utilization of UAVs for agricultural purposes is the limitation in flight time. It is necessary to fly at a high altitude to capture the maximum number of plants in the limited time available, but this reduces the spatial resolution of the captured images. In this study, we applied a super-resolution method to the low-resolution images of tomato diseases to recover detailed appearances, such as lesions on plant organs. We also conducted disease classification using high-resolution, low-resolution, and super-resolution images to evaluate the effectiveness of super-resolution methods in disease classification. Our results indicated that the super-resolution method outperformed conventional image scaling methods in spatial resolution enhancement of tomato disease images. The results of disease classification showed that the accuracy attained was also better by a large margin with super-resolution images than with low-resolution images. These results indicated that our approach not only recovered the information lost in low-resolution images, but also exerted a beneficial influence on further image analysis. The proposed approach will accelerate image-based phenotyping and vigor diagnosis in the field, because it not only saves time to capture images of a crop in a cultivation field but also secures the accuracy of these images for further analysis. PMID:29113104
Phenotypic and Genotypic Characterisation of Inflammatory Bowel Disease Presenting Before the Age of 2 years

PubMed Central

Dziubak, Robert; Pescarin, Matilde; Drury, Suzanne; Godwin, Heather; Reeve, Kate; Chadokufa, Sibongile; Huggett, Bonita; Sider, Sara; James, Chela; Acton, Nikki; Cernat, Elena; Gasparetto, Marco; Noble-Jamieson, Gabi; Kiparissi, Fevronia; Elawad, Mamoun; Beales, Phil L.; Sebire, Neil J.; Gilmour, Kimberly; Uhlig, Holm H.; Bacchelli, Chiara; Shah, Neil

2017-01-01

Abstract Objectives: Inflammatory bowel disease [IBD] presenting in early childhood is extremely rare. More recently, progress has been made to identify children with monogenic forms of IBD predominantly presenting very early in life. In this study, we describe the heterogeneous phenotypes and genotypes of patients with IBD presenting before the age of 2 years and establish phenotypic features associated with underlying monogenicity. Methods: Phenotype data of 62 children with disease onset before the age of 2 years presenting over the past 20 years were reviewed. Children without previously established genetic diagnosis were prospectively recruited for next-generation sequencing. Results: In all, 62 patients [55% male] were identified. The median disease onset was 3 months of age (interquartile range [IQR]: 1 to 11). Conventional IBD classification only applied to 15 patients with Crohn’s disease [CD]-like [24%] and three with ulcerative colitis [UC]-like [5%] phenotype; 44 patients [71%] were diagnosed with otherwise unclassifiable IBD. Patients frequently required parenteral nutrition [40%], extensive immunosuppression [31%], haematopoietic stem-cell transplantation [29%], and abdominal surgery [19%]. In 31% of patients, underlying monogenic diseases were established [EPCAM, IL10, IL10RA, IL10RB, FOXP3, LRBA, SKIV2L, TTC37, TTC7A]. Phenotypic features significantly more prevalent in monogenic IBD were: consanguinity, disease onset before the 6th month of life, stunting, extensive intestinal disease and histological evidence of epithelial abnormalities. Conclusions: IBD in children with disease onset before the age of 2 years is frequently unclassifiable into Crohn’s disease and ulcerative colitis, particularly treatment resistant, and can be indistinguishable from monogenic diseases with IBD-like phenotype. PMID:27302973
An Image Analysis Method for the Precise Selection and Quantitation of Fluorescently Labeled Cellular Constituents

PubMed Central

Agley, Chibeza C.; Velloso, Cristiana P.; Lazarus, Norman R.

2012-01-01

The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture. Use of this method can be used to overcome many of the long-standing limitations of conventional approaches for quantifying muscle cell size in vitro. In addition, wider applications of Photoshop as a quantitative and semiquantitative tool in immunocytochemistry are explored. PMID:22511600
A multi-scale convolutional neural network for phenotyping high-content cellular images.

PubMed

Godinez, William J; Hossain, Imtiaz; Lazic, Stanley E; Davies, John W; Zhang, Xian

2017-07-01

Identifying phenotypes based on high-content cellular images is challenging. Conventional image analysis pipelines for phenotype identification comprise multiple independent steps, with each step requiring method customization and adjustment of multiple parameters. Here, we present an approach based on a multi-scale convolutional neural network (M-CNN) that classifies, in a single cohesive step, cellular images into phenotypes by using directly and solely the images' pixel intensity values. The only parameters in the approach are the weights of the neural network, which are automatically optimized based on training images. The approach requires no a priori knowledge or manual customization, and is applicable to single- or multi-channel images displaying single or multiple cells. We evaluated the classification performance of the approach on eight diverse benchmark datasets. The approach yielded overall a higher classification accuracy compared with state-of-the-art results, including those of other deep CNN architectures. In addition to using the network to simply obtain a yes-or-no prediction for a given phenotype, we use the probability outputs calculated by the network to quantitatively describe the phenotypes. This study shows that these probability values correlate with chemical treatment concentrations. This finding validates further our approach and enables chemical treatment potency estimation via CNNs. The network specifications and solver definitions are provided in Supplementary Software 1. william_jose.godinez_navarro@novartis.com or xian-1.zhang@novartis.com. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com
EHR-based phenotyping: Bulk learning and evaluation.

PubMed

Chiu, Po-Hsiang; Hripcsak, George

2017-06-01

In data-driven phenotyping, a core computational task is to identify medical concepts and their variations from sources of electronic health records (EHR) to stratify phenotypic cohorts. A conventional analytic framework for phenotyping largely uses a manual knowledge engineering approach or a supervised learning approach where clinical cases are represented by variables encompassing diagnoses, medicinal treatments and laboratory tests, among others. In such a framework, tasks associated with feature engineering and data annotation remain a tedious and expensive exercise, resulting in poor scalability. In addition, certain clinical conditions, such as those that are rare and acute in nature, may never accumulate sufficient data over time, which poses a challenge to establishing accurate and informative statistical models. In this paper, we use infectious diseases as the domain of study to demonstrate a hierarchical learning method based on ensemble learning that attempts to address these issues through feature abstraction. We use a sparse annotation set to train and evaluate many phenotypes at once, which we call bulk learning. In this batch-phenotyping framework, disease cohort definitions can be learned from within the abstract feature space established by using multiple diseases as a substrate and diagnostic codes as surrogates. In particular, using surrogate labels for model training renders possible its subsequent evaluation using only a sparse annotated sample. Moreover, statistical models can be trained and evaluated, using the same sparse annotation, from within the abstract feature space of low dimensionality that encapsulates the shared clinical traits of these target diseases, collectively referred to as the bulk learning set. Copyright © 2017 Elsevier Inc. All rights reserved.
New species of Bordetella, Bordetella ansorpii sp. nov., isolated from the purulent exudate of an epidermal cyst.

PubMed

Ko, Kwan Soo; Peck, Kyong Ran; Oh, Won Sup; Lee, Nam Yong; Lee, Jang Ho; Song, Jae-Hoon

2005-05-01

A gram-negative bacillus, SMC-8986(T), which was isolated from the purulent exudate of an epidermal cyst but could not be identified by a conventional microbiologic method, was characterized by a variety of phenotypic and genotypic analyses. Sequences of the 16S rRNA gene revealed that this bacterium belongs to the genus Bordetella but diverged distinctly from previously described Bordetella species. Analyses of cellular fatty acid composition and performance of biochemical tests confirmed that this bacterium is distinct from other Bordetella species. Furthermore, the results of comparative sequence analyses of two protein-coding genes (risA and ompA) also showed that this strain represents a new species within the genus Bordetella. Based on the evaluated phenotypic and genotypic characteristics, it is proposed that SMC-8986(T) should be classified as a new species, namely Bordetella ansorpii sp. nov.
Methicillin-Resistant Staphylococcus aureus: Comparison of Susceptibility Testing Methods and Analysis of mecA-Positive Susceptible Strains

PubMed Central

Sakoulas, George; Gold, Howard S.; Venkataraman, Lata; DeGirolami, Paola C.; Eliopoulos, George M.; Qian, Qinfang

2001-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections. Phenotypic heterogeneous drug resistance (heteroresistance) to antistaphylococcal beta-lactams affects the results of susceptibility testing. The present study compared the MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) for detection of PBP 2a with agar dilution, the VITEK-1 and VITEK-2 systems (bioMérieux, St. Louis, Mo.), and the oxacillin agar screen test for detection of MRSA, with PCR for the mecA gene used as the “gold standard” assay. Analysis of 107 methicillin-susceptible S. aureus (MSSA) isolates and 203 MRSA isolates revealed that the MRSA-Screen latex agglutination test is superior to any single phenotype-based susceptibility testing method, with a sensitivity of 100% and a specificity of 99.1%. Only one isolate that lacked mecA was weakly positive by the MRSA-Screen latex agglutination test. This isolate was phenotypically susceptible to oxacillin and did not contain the mecA gene by Southern blot hybridization. The oxacillin agar screen test, the VITEK-1 system, the VITEK-2 system, and agar dilution showed sensitivities of 99.0, 99.0, 99.5, and 99%, respectively, and specificities of 98.1, 100, 97.2, and 100%, respectively. The differences in sensitivity or specificity were not statistically significant. Oxacillin bactericidal assays showed that mecA- and PBP 2a-positive S. aureus isolates that are susceptible to antistaphylococcal beta-lactams by conventional methods are functionally resistant to oxacillin. We conclude that the accuracy of the MRSA-Screen latex agglutination method for detection of PBP 2a approaches the accuracy of PCR and is more accurate than any susceptibility testing method used alone for the detection of MRSA. PMID:11682512
Flow Cytometry Data Preparation Guidelines for Improved Automated Phenotypic Analysis.

PubMed

Jimenez-Carretero, Daniel; Ligos, José M; Martínez-López, María; Sancho, David; Montoya, María C

2018-05-15

Advances in flow cytometry (FCM) increasingly demand adoption of computational analysis tools to tackle the ever-growing data dimensionality. In this study, we tested different data input modes to evaluate how cytometry acquisition configuration and data compensation procedures affect the performance of unsupervised phenotyping tools. An analysis workflow was set up and tested for the detection of changes in reference bead subsets and in a rare subpopulation of murine lymph node CD103 + dendritic cells acquired by conventional or spectral cytometry. Raw spectral data or pseudospectral data acquired with the full set of available detectors by conventional cytometry consistently outperformed datasets acquired and compensated according to FCM standards. Our results thus challenge the paradigm of one-fluorochrome/one-parameter acquisition in FCM for unsupervised cluster-based analysis. Instead, we propose to configure instrument acquisition to use all available fluorescence detectors and to avoid integration and compensation procedures, thereby using raw spectral or pseudospectral data for improved automated phenotypic analysis. Copyright © 2018 by The American Association of Immunologists, Inc.
Assessment of real-time PCR based methods for quantification of pollen-mediated gene flow from GM to conventional maize in a field study.

PubMed

Pla, Maria; La Paz, José-Luis; Peñas, Gisela; García, Nora; Palaudelmàs, Montserrat; Esteve, Teresa; Messeguer, Joaquima; Melé, Enric

2006-04-01

Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1-10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions.
The phenotypic equilibrium of cancer cells: From average-level stability to path-wise convergence.

PubMed

Niu, Yuanling; Wang, Yue; Zhou, Da

2015-12-07

The phenotypic equilibrium, i.e. heterogeneous population of cancer cells tending to a fixed equilibrium of phenotypic proportions, has received much attention in cancer biology very recently. In the previous literature, some theoretical models were used to predict the experimental phenomena of the phenotypic equilibrium, which were often explained by different concepts of stabilities of the models. Here we present a stochastic multi-phenotype branching model by integrating conventional cellular hierarchy with phenotypic plasticity mechanisms of cancer cells. Based on our model, it is shown that: (i) our model can serve as a framework to unify the previous models for the phenotypic equilibrium, and then harmonizes the different kinds of average-level stabilities proposed in these models; and (ii) path-wise convergence of our model provides a deeper understanding to the phenotypic equilibrium from stochastic point of view. That is, the emergence of the phenotypic equilibrium is rooted in the stochastic nature of (almost) every sample path, the average-level stability just follows from it by averaging stochastic samples. Copyright © 2015 Elsevier Ltd. All rights reserved.
Digital Quantification of Goldmann Visual Fields (GVF) as a Means for Genotype-Phenotype Comparisons and Detection of Progression in Retinal Degenerations

PubMed Central

Zahid, Sarwar; Peeler, Crandall; Khan, Naheed; Davis, Joy; Mahmood, Mahdi; Heckenlively, John; Jayasundera, Thiran

2015-01-01

Purpose To develop a reliable and efficient digital method to quantify planimetric Goldmann visual field (GVF) data to monitor disease course and treatment responses in retinal degenerative diseases. Methods A novel method to digitally quantify GVF using Adobe Photoshop CS3 was developed for comparison to traditional digital planimetry (Placom 45C digital planimeter; EngineerSupply, Lynchburg, Virginia, USA). GVFs from 20 eyes from 10 patients with Stargardt disease were quantified to assess the difference between the two methods (a total of 230 measurements per method). This quantification approach was also applied to 13 patients with X-linked retinitis pigmentosa (XLRP) with mutations in RPGR. Results Overall, measurements using Adobe Photoshop were more rapidly performed than those using conventional planimetry. Photoshop measurements also exhibited less inter- and intra-observer variability. GVF areas for the I4e isopter in patients with the same mutation in RPGR who were nearby in age had similar qualitative and quantitative areas. Conclusions Quantification of GVF using Adobe Photoshop is quicker, more reliable, and less-user dependent than conventional digital planimetry. It will be a useful tool for both retrospective and prospective studies of disease course as well as for monitoring treatment response in clinical trials for retinal degenerative diseases. PMID:24664690
In vivo Phenotyping Methods: Cytochrome P450 Probes with Emphasis on the Cocktail Approach.

PubMed

Keller, Guillermo Alberto; Gago, María Laura Ferreirós; Diez, Roberto Alejandro; Di Girolamo, Guillermo

2017-01-01

Differences in drug response among patients are common. Most major drugs are effective in only 25 to 60 percent of the patients, in part due to the CYP enzymes, whose activity vary up to 50-fold between individuals for some index metabolic reactions. Several factors affect CYP activity, among which genetic polymorphisms have been studied as the major cause for long time. Age, gender, disease states, and environmental influences such as smoking, concomitant drug treatment or exposure to environmental chemicals are also important. This article reviews the available literature on multiple phenotypes assessment as an important tool to predict possible therapeutic failures or toxic reactions to conventional drug doses during patient evaluation. Probe drugs can be used in various combinations allowing for the in vivo assessment of multiple pathways of drug metabolism in a single experiment, configuring a new tool known as phenotyping "cocktails". There are several drug cocktails with different advantages and disadvantages. Most of them have sufficient clinical evidence and data validation to support their use in clinical setting as a surrogate for the risk of adverse reaction in the course of therapy, leading to a better balance between efficacy and safety. Probes characteristics and metabolic ratio measurements are important in the evaluation of phenotyping cocktails as near-future applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Random forests-based differential analysis of gene sets for gene expression data.

PubMed

Hsueh, Huey-Miin; Zhou, Da-Wei; Tsai, Chen-An

2013-04-10

In DNA microarray studies, gene-set analysis (GSA) has become the focus of gene expression data analysis. GSA utilizes the gene expression profiles of functionally related gene sets in Gene Ontology (GO) categories or priori-defined biological classes to assess the significance of gene sets associated with clinical outcomes or phenotypes. Many statistical approaches have been proposed to determine whether such functionally related gene sets express differentially (enrichment and/or deletion) in variations of phenotypes. However, little attention has been given to the discriminatory power of gene sets and classification of patients. In this study, we propose a method of gene set analysis, in which gene sets are used to develop classifications of patients based on the Random Forest (RF) algorithm. The corresponding empirical p-value of an observed out-of-bag (OOB) error rate of the classifier is introduced to identify differentially expressed gene sets using an adequate resampling method. In addition, we discuss the impacts and correlations of genes within each gene set based on the measures of variable importance in the RF algorithm. Significant classifications are reported and visualized together with the underlying gene sets and their contribution to the phenotypes of interest. Numerical studies using both synthesized data and a series of publicly available gene expression data sets are conducted to evaluate the performance of the proposed methods. Compared with other hypothesis testing approaches, our proposed methods are reliable and successful in identifying enriched gene sets and in discovering the contributions of genes within a gene set. The classification results of identified gene sets can provide an valuable alternative to gene set testing to reveal the unknown, biologically relevant classes of samples or patients. In summary, our proposed method allows one to simultaneously assess the discriminatory ability of gene sets and the importance of genes for interpretation of data in complex biological systems. The classifications of biologically defined gene sets can reveal the underlying interactions of gene sets associated with the phenotypes, and provide an insightful complement to conventional gene set analyses. Copyright © 2012 Elsevier B.V. All rights reserved.
Positive health effects of the natural outdoor environment in typical populations in different regions in Europe (PHENOTYPE): a study programme protocol

PubMed Central

Nieuwenhuijsen, Mark J; Kruize, Hanneke; Gidlow, Christopher; Andrusaityte, Sandra; Antó, Josep Maria; Basagaña, Xavier; Cirach, Marta; Dadvand, Payam; Danileviciute, Asta; Donaire-Gonzalez, David; Garcia, Judith; Jerrett, Michael; Jones, Marc; Julvez, Jordi; van Kempen, Elise; van Kamp, Irene; Maas, Jolanda; Seto, Edmund; Smith, Graham; Triguero, Margarita; Wendel-Vos, Wanda; Wright, John; Zufferey, Joris; van den Hazel, Peter Jan; Lawrence, Roderick; Grazuleviciene, Regina

2014-01-01

Introduction Growing evidence suggests that close contact with nature brings benefits to human health and well-being, but the proposed mechanisms are still not well understood and the associations with health remain uncertain. The Positive Health Effects of the Natural Outdoor environment in Typical Populations in different regions in Europe (PHENOTYPE) project investigates the interconnections between natural outdoor environments and better human health and well-being. Aims and methods The PHENOTYPE project explores the proposed underlying mechanisms at work (stress reduction/restorative function, physical activity, social interaction, exposure to environmental hazards) and examines the associations with health outcomes for different population groups. It implements conventional and new innovative high-tech methods to characterise the natural environment in terms of quality and quantity. Preventive as well as therapeutic effects of contact with the natural environment are being covered. PHENOTYPE further addresses implications for land-use planning and green space management. The main innovative part of the study is the evaluation of possible short-term and long-term associations of green space and health and the possible underlying mechanisms in four different countries (each with quite a different type of green space and a different use), using the same methodology, in one research programme. This type of holistic approach has not been undertaken before. Furthermore there are technological innovations such as the use of remote sensing and smartphones in the assessment of green space. Conclusions The project will produce a more robust evidence base on links between exposure to natural outdoor environment and human health and well-being, in addition to a better integration of human health needs into land-use planning and green space management in rural as well as urban areas. PMID:24740979
Predictive oncology: multidisciplinary, multi-scale in-silico modeling linking phenotype, morphology and growth

PubMed Central

Sanga, Sandeep; Frieboes, Hermann B.; Zheng, Xiaoming; Gatenby, Robert; Bearer, Elaine L.; Cristini, Vittorio

2007-01-01

Empirical evidence and theoretical studies suggest that the phenotype, i.e., cellular- and molecular-scale dynamics, including proliferation rate and adhesiveness due to microenvironmental factors and gene expression that govern tumor growth and invasiveness, also determine gross tumor-scale morphology. It has been difficult to quantify the relative effect of these links on disease progression and prognosis using conventional clinical and experimental methods and observables. As a result, successful individualized treatment of highly malignant and invasive cancers, such as glioblastoma, via surgical resection and chemotherapy cannot be offered and outcomes are generally poor. What is needed is a deterministic, quantifiable method to enable understanding of the connections between phenotype and tumor morphology. Here, we critically review advantages and disadvantages of recent computational modeling efforts (e.g., continuum, discrete, and cellular automata models) that have pursued this understanding. Based on this assessment, we propose and discuss a multi-scale, i.e., from the molecular to the gross tumor scale, mathematical and computational “first-principle” approach based on mass conservation and other physical laws, such as employed in reaction-diffusion systems. Model variables describe known characteristics of tumor behavior, and parameters and functional relationships across scales are informed from in vitro, in vivo and ex vivo biology. We demonstrate that this methodology, once coupled to tumor imaging and tumor biopsy or cell culture data, should enable prediction of tumor growth and therapy outcome through quantification of the relation between the underlying dynamics and morphological characteristics. In particular, morphologic stability analysis of this mathematical model reveals that tumor cell patterning at the tumor-host interface is regulated by cell proliferation, adhesion and other phenotypic characteristics: histopathology information of tumor boundary can be inputted to the mathematical model and used as phenotype-diagnostic tool and thus to predict collective and individual tumor cell invasion of surrounding host. This approach further provides a means to deterministically test effects of novel and hypothetical therapy strategies on tumor behavior. PMID:17629503
Phenotypic and genotypic analysis of anti-tuberculosis drug resistance in Mycobacterium tuberculosis isolates in Myanmar.

PubMed

Aung, Wah Wah; Ei, Phyu Win; Nyunt, Wint Wint; Swe, Thyn Lei; Lwin, Thandar; Htwe, Mi Mi; Kim, Kyung Jun; Lee, Jong Seok; Kim, Chang Ki; Cho, Sang Nae; Song, Sun Dae; Chang, Chulhun L

2015-09-01

Tuberculosis (TB) is one of the most serious health problems in Myanmar. Because TB drug resistance is associated with genetic mutation(s) relevant to responses to each drug, genotypic methods for detecting these mutations have been proposed to overcome the limitations of classic phenotypic drug susceptibility testing (DST). We explored the current estimates of drug-resistant TB and evaluated the usefulness of genotypic DST in Myanmar. We determined the drug susceptibility of Mycobacterium tuberculosis isolated from sputum smear-positive patients with newly diagnosed pulmonary TB at two main TB centers in Myanmar during 2013 by using conventional phenotypic DST and the GenoType MTBDRplus assay (Hain Lifescience, Germany). Discrepant results were confirmed by sequencing the genes relevant to each type of resistance (rpoB for rifampicin; katG and inhA for isoniazid). Of 191 isolates, phenotypic DST showed that 27.7% (n=53) were resistant to at least one first-line drug and 20.9% (n=40) were resistant to two or more, including 18.3% (n=35) multidrug-resistant TB (MDR-TB) strains. Monoresistant strains accounted for 6.8% (n=13) of the samples. Genotypic assay of 189 isolates showed 17.5% (n=33) MDR-TB and 5.3% (n=10) isoniazid-monoresistant strains. Genotypic susceptibility results were 99.5% (n=188) concordant and agreed almost perfectly with phenotypic DST (kappa=0.99; 95% confidence interval 0.96-1.01). The results highlight the burden of TB drug resistance and prove the usefulness of the genotypic DST in Myanmar.
GiNA, an Efficient and High-Throughput Software for Horticultural Phenotyping

PubMed Central

Diaz-Garcia, Luis; Covarrubias-Pazaran, Giovanny; Schlautman, Brandon; Zalapa, Juan

2016-01-01

Traditional methods for trait phenotyping have been a bottleneck for research in many crop species due to their intensive labor, high cost, complex implementation, lack of reproducibility and propensity to subjective bias. Recently, multiple high-throughput phenotyping platforms have been developed, but most of them are expensive, species-dependent, complex to use, and available only for major crops. To overcome such limitations, we present the open-source software GiNA, which is a simple and free tool for measuring horticultural traits such as shape- and color-related parameters of fruits, vegetables, and seeds. GiNA is multiplatform software available in both R and MATLAB® programming languages and uses conventional images from digital cameras with minimal requirements. It can process up to 11 different horticultural morphological traits such as length, width, two-dimensional area, volume, projected skin, surface area, RGB color, among other parameters. Different validation tests produced highly consistent results under different lighting conditions and camera setups making GiNA a very reliable platform for high-throughput phenotyping. In addition, five-fold cross validation between manually generated and GiNA measurements for length and width in cranberry fruits were 0.97 and 0.92. In addition, the same strategy yielded prediction accuracies above 0.83 for color estimates produced from images of cranberries analyzed with GiNA compared to total anthocyanin content (TAcy) of the same fruits measured with the standard methodology of the industry. Our platform provides a scalable, easy-to-use and affordable tool for massive acquisition of phenotypic data of fruits, seeds, and vegetables. PMID:27529547
GiNA, an Efficient and High-Throughput Software for Horticultural Phenotyping.

PubMed

Diaz-Garcia, Luis; Covarrubias-Pazaran, Giovanny; Schlautman, Brandon; Zalapa, Juan

2016-01-01

Traditional methods for trait phenotyping have been a bottleneck for research in many crop species due to their intensive labor, high cost, complex implementation, lack of reproducibility and propensity to subjective bias. Recently, multiple high-throughput phenotyping platforms have been developed, but most of them are expensive, species-dependent, complex to use, and available only for major crops. To overcome such limitations, we present the open-source software GiNA, which is a simple and free tool for measuring horticultural traits such as shape- and color-related parameters of fruits, vegetables, and seeds. GiNA is multiplatform software available in both R and MATLAB® programming languages and uses conventional images from digital cameras with minimal requirements. It can process up to 11 different horticultural morphological traits such as length, width, two-dimensional area, volume, projected skin, surface area, RGB color, among other parameters. Different validation tests produced highly consistent results under different lighting conditions and camera setups making GiNA a very reliable platform for high-throughput phenotyping. In addition, five-fold cross validation between manually generated and GiNA measurements for length and width in cranberry fruits were 0.97 and 0.92. In addition, the same strategy yielded prediction accuracies above 0.83 for color estimates produced from images of cranberries analyzed with GiNA compared to total anthocyanin content (TAcy) of the same fruits measured with the standard methodology of the industry. Our platform provides a scalable, easy-to-use and affordable tool for massive acquisition of phenotypic data of fruits, seeds, and vegetables.
Identification of novel drought-tolerant-associated SNPs in common bean (Phaseolus vulgaris)

PubMed Central

Villordo-Pineda, Emiliano; González-Chavira, Mario M.; Giraldo-Carbajo, Patricia; Acosta-Gallegos, Jorge A.; Caballero-Pérez, Juan

2015-01-01

Common bean (Phaseolus vulgaris L.) is a leguminous in high demand for human nutrition and a very important agricultural product. Production of common bean is constrained by environmental stresses such as drought. Although conventional plant selection has been used to increase production yield and stress tolerance, drought tolerance selection based on phenotype is complicated by associated physiological, anatomical, cellular, biochemical, and molecular changes. These changes are modulated by differential gene expression. A common method to identify genes associated with phenotypes of interest is the characterization of Single Nucleotide Polymorphims (SNPs) to link them to specific functions. In this work, we selected two drought-tolerant parental lines from Mesoamerica, Pinto Villa, and Pinto Saltillo. The parental lines were used to generate a population of 282 families (F3:5) and characterized by 169 SNPs. We associated the segregation of the molecular markers in our population with phenotypes including flowering time, physiological maturity, reproductive period, plant, seed and total biomass, reuse index, seed yield, weight of 100 seeds, and harvest index in three cultivation cycles. We observed 83 SNPs with significant association (p < 0.0003 after Bonferroni correction) with our quantified phenotypes. Phenotypes most associated were days to flowering and seed biomass with 58 and 44 associated SNPs, respectively. Thirty-seven out of the 83 SNPs were annotated to a gene with a potential function related to drought tolerance or relevant molecular/biochemical functions. Some SNPs such as SNP28 and SNP128 are related to starch biosynthesis, a common osmotic protector; and SNP18 is related to proline biosynthesis, another well-known osmotic protector. PMID:26257755
Identification of novel drought-tolerant-associated SNPs in common bean (Phaseolus vulgaris).

PubMed

Villordo-Pineda, Emiliano; González-Chavira, Mario M; Giraldo-Carbajo, Patricia; Acosta-Gallegos, Jorge A; Caballero-Pérez, Juan

2015-01-01

Common bean (Phaseolus vulgaris L.) is a leguminous in high demand for human nutrition and a very important agricultural product. Production of common bean is constrained by environmental stresses such as drought. Although conventional plant selection has been used to increase production yield and stress tolerance, drought tolerance selection based on phenotype is complicated by associated physiological, anatomical, cellular, biochemical, and molecular changes. These changes are modulated by differential gene expression. A common method to identify genes associated with phenotypes of interest is the characterization of Single Nucleotide Polymorphims (SNPs) to link them to specific functions. In this work, we selected two drought-tolerant parental lines from Mesoamerica, Pinto Villa, and Pinto Saltillo. The parental lines were used to generate a population of 282 families (F3:5) and characterized by 169 SNPs. We associated the segregation of the molecular markers in our population with phenotypes including flowering time, physiological maturity, reproductive period, plant, seed and total biomass, reuse index, seed yield, weight of 100 seeds, and harvest index in three cultivation cycles. We observed 83 SNPs with significant association (p < 0.0003 after Bonferroni correction) with our quantified phenotypes. Phenotypes most associated were days to flowering and seed biomass with 58 and 44 associated SNPs, respectively. Thirty-seven out of the 83 SNPs were annotated to a gene with a potential function related to drought tolerance or relevant molecular/biochemical functions. Some SNPs such as SNP28 and SNP128 are related to starch biosynthesis, a common osmotic protector; and SNP18 is related to proline biosynthesis, another well-known osmotic protector.
Enhancement of plant metabolite fingerprinting by machine learning.

PubMed

Scott, Ian M; Vermeer, Cornelia P; Liakata, Maria; Corol, Delia I; Ward, Jane L; Lin, Wanchang; Johnson, Helen E; Whitehead, Lynne; Kular, Baldeep; Baker, John M; Walsh, Sean; Dave, Anuja; Larson, Tony R; Graham, Ian A; Wang, Trevor L; King, Ross D; Draper, John; Beale, Michael H

2010-08-01

Metabolite fingerprinting of Arabidopsis (Arabidopsis thaliana) mutants with known or predicted metabolic lesions was performed by (1)H-nuclear magnetic resonance, Fourier transform infrared, and flow injection electrospray-mass spectrometry. Fingerprinting enabled processing of five times more plants than conventional chromatographic profiling and was competitive for discriminating mutants, other than those affected in only low-abundance metabolites. Despite their rapidity and complexity, fingerprints yielded metabolomic insights (e.g. that effects of single lesions were usually not confined to individual pathways). Among fingerprint techniques, (1)H-nuclear magnetic resonance discriminated the most mutant phenotypes from the wild type and Fourier transform infrared discriminated the fewest. To maximize information from fingerprints, data analysis was crucial. One-third of distinctive phenotypes might have been overlooked had data models been confined to principal component analysis score plots. Among several methods tested, machine learning (ML) algorithms, namely support vector machine or random forest (RF) classifiers, were unsurpassed for phenotype discrimination. Support vector machines were often the best performing classifiers, but RFs yielded some particularly informative measures. First, RFs estimated margins between mutant phenotypes, whose relations could then be visualized by Sammon mapping or hierarchical clustering. Second, RFs provided importance scores for the features within fingerprints that discriminated mutants. These scores correlated with analysis of variance F values (as did Kruskal-Wallis tests, true- and false-positive measures, mutual information, and the Relief feature selection algorithm). ML classifiers, as models trained on one data set to predict another, were ideal for focused metabolomic queries, such as the distinctiveness and consistency of mutant phenotypes. Accessible software for use of ML in plant physiology is highlighted.
Digital quantification of Goldmann visual fields (GVFs) as a means for genotype-phenotype comparisons and detection of progression in retinal degenerations.

PubMed

Zahid, Sarwar; Peeler, Crandall; Khan, Naheed; Davis, Joy; Mahmood, Mahdi; Heckenlively, John R; Jayasundera, Thiran

2014-01-01

To develop a reliable and efficient digital method to quantify planimetric Goldmann visual field (GVF) data to monitor disease course and treatment responses in retinal degenerative diseases. A novel method to digitally quantify GVFs using Adobe Photoshop CS3 was developed for comparison to traditional digital planimetry (Placom 45C digital planimeter; Engineer Supply, Lynchburg, Virginia, USA). GVFs from 20 eyes from 10 patients with Stargardt disease were quantified to assess the difference between the two methods (a total of 230 measurements per method). This quantification approach was also applied to 13 patients with X-linked retinitis pigmentosa (XLRP) with mutations in RPGR. Overall, measurements using Adobe Photoshop were more rapidly performed than those using conventional planimetry. Photoshop measurements also exhibited less inter- and intraobserver variability. GVF areas for the I4e isopter in patients with the same mutation in RPGR who were nearby in age had similar qualitative and quantitative areas. Quantification of GVFs using Adobe Photoshop is quicker, more reliable, and less user dependent than conventional digital planimetry. It will be a useful tool for both retrospective and prospective studies of disease course as well as for monitoring treatment response in clinical trials for retinal degenerative diseases.
Breeding to adapt agriculture to climate change: affordable phenotyping solutions.

PubMed

Araus, José L; Kefauver, Shawn C

2018-05-28

Breeding is one of the central pillars of adaptation of crops to climate change. However, phenotyping is a key bottleneck that is limiting breeding efficiency. The awareness of phenotyping as a breeding limitation is not only sustained by the lack of adequate approaches, but also by the perception that phenotyping is an expensive activity. Phenotyping is not just dependent on the choice of appropriate traits and tools (e.g. sensors) but relies on how these tools are deployed on their carrying platforms, the speed and volume of data extraction and analysis (throughput), the handling of spatial variability and characterization of environmental conditions, and finally how all the information is integrated and processed. Affordable high throughput phenotyping aims to achieve reasonably priced solutions for all the components comprising the phenotyping pipeline. This mini-review will cover current and imminent solutions for all these components, from the increasing use of conventional digital RGB cameras, within the category of sensors, to open-access cloud-structured data processing and the use of smartphones. Emphasis will be placed on field phenotyping, which is really the main application for day-to-day phenotyping. Copyright © 2018 Elsevier Ltd. All rights reserved.
Identification of anaerobic bacteria by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry with on-plate formic acid preparation.

PubMed

Schmitt, Bryan H; Cunningham, Scott A; Dailey, Aaron L; Gustafson, Daniel R; Patel, Robin

2013-03-01

Identification of anaerobic bacteria using phenotypic methods is often time-consuming; methods such as 16S rRNA gene sequencing are costly and may not be readily available. We evaluated 253 clinical isolates of anaerobic bacteria using the Bruker MALDI Biotyper (Bruker Daltonics, Billerica, MA) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system with a user-supplemented database and an on-plate formic acid-based preparation method and compared results to those of conventional identification using biochemical testing or 16S rRNA gene sequencing. A total of 179 (70.8%) and 232 (91.7%) isolates were correctly identified to the species and genus levels, respectively, using manufacturer-recommended score cutoffs. MALDI-TOF MS offers a rapid, inexpensive method for identification of anaerobic bacteria.
Distribution of Malassezia species on the skin of patients with atopic dermatitis, psoriasis, and healthy volunteers assessed by conventional and molecular identification methods

PubMed Central

2014-01-01

Background The Malassezia yeasts which belong to the physiological microflora of human skin have also been implicated in several dermatological disorders, including pityriasis versicolor (PV), atopic dermatitis (AD), and psoriasis (PS). The Malassezia genus has repeatedly been revised and it now accommodates 14 species, all but one being lipid-dependent species. The traditional, phenotype-based identification schemes of Malassezia species are fraught with interpretative ambiguities and inconsistencies, and are thus increasingly being supplemented or replaced by DNA typing methods. The aim of this study was to explore the species composition of Malassezia microflora on the skin of healthy volunteers and patients with AD and PS. Methods Species characterization was performed by conventional, culture-based methods and subsequently molecular techniques: PCR-RFLP and sequencing of the internal transcribed spacer (ITS) 1/2 regions and the D1/D2 domains of the 26S rRNA gene. The Chi-square test and Fisher’s exact test were used for statistical analysis. Results Malassezia sympodialis was the predominant species, having been cultured from 29 (82.9%) skin samples collected from 17 out of 18 subjects under the study. Whereas AD patients yielded exclusively M. sympodialis isolates, M. furfur isolates were observed only in PS patients. The isolation of M. sympodialis was statistically more frequent among AD patients and healthy volunteers than among PS patients (P < 0.03). Whether this mirrors any predilection of particular Malassezia species for certain clinical conditions needs to be further evaluated. The overall concordance between phenotypic and molecular methods was quite high (65%), with the discordant results being rather due to the presence of multiple species in a single culture (co-colonization) than true misidentification. All Malassezia isolates were susceptible to cyclopiroxolamine and azole drugs, with M. furfur isolates being somewhat more drug tolerant than other Malassezia species. Conclusions This study provides an important insight into the species composition of Malassezia microbiota in human skin. The predominance of M. sympodialis in both normal and pathologic skin, contrasts with other European countries, reporting M. globosa and M. restricta as the most frequently isolated Malassezia species. PMID:24602368
Rearing environment influences boldness and prey acquisition behavior, and brain and lens development of bull trout

USGS Publications Warehouse

Brignon, William R.; Pike, Martin M.; Ebbesson, Lars O.E.; Schaller, Howard A.; Peterson, James T.; Schreck, Carl B.

2018-01-01

Animals reared in barren captive environments exhibit different developmental trajectories and behaviors than wild counterparts. Hence, the captive phenotypes may influence the success of reintroduction and recovery programs for threatened and endangered species. We collected wild bull trout embryos from the Metolius River Basin, Oregon and reared them in differing environments to better understand how captivity affects the bull trout Salvelinus confluentusphenotype. We compared the boldness and prey acquisition behaviors and development of the brain and eye lens of bull trout reared in conventional barren and more structurally complex captive environments with that of wild fish. Wild fish and captive reared fish from complex habitats exhibited a greater level of boldness and prey acquisition ability, than fish reared in conventional captive environments. In addition, the eye lens of conventionally reared bull trout was larger than complex reared captive fish or same age wild fish. Interestingly, we detected wild fish had a smaller relative cerebellum than either captive reared treatment. Our results suggest that rearing fish in more complex captive environments can create a more wild-like phenotype than conventional rearing practices. A better understanding of the effects of captivity on the development and behavior of bull trout can inform rearing and reintroduction programs though prediction of the performance of released individuals.
Multi-modal classification of neurodegenerative disease by progressive graph-based transductive learning

PubMed Central

Wang, Zhengxia; Zhu, Xiaofeng; Adeli, Ehsan; Zhu, Yingying; Nie, Feiping; Munsell, Brent

2018-01-01

Graph-based transductive learning (GTL) is a powerful machine learning technique that is used when sufficient training data is not available. In particular, conventional GTL approaches first construct a fixed inter-subject relation graph that is based on similarities in voxel intensity values in the feature domain, which can then be used to propagate the known phenotype data (i.e., clinical scores and labels) from the training data to the testing data in the label domain. However, this type of graph is exclusively learned in the feature domain, and primarily due to outliers in the observed features, may not be optimal for label propagation in the label domain. To address this limitation, a progressive GTL (pGTL) method is proposed that gradually finds an intrinsic data representation that more accurately aligns imaging features with the phenotype data. In general, optimal feature-to-phenotype alignment is achieved using an iterative approach that: (1) refines inter-subject relationships observed in the feature domain by using the learned intrinsic data representation in the label domain, (2) updates the intrinsic data representation from the refined inter-subject relationships, and (3) verifies the intrinsic data representation on the training data to guarantee an optimal classification when applied to testing data. Additionally, the iterative approach is extended to multi-modal imaging data to further improve pGTL classification accuracy. Using Alzheimer’s disease and Parkinson’s disease study data, the classification accuracy of the proposed pGTL method is compared to several state-of-the-art classification methods, and the results show pGTL can more accurately identify subjects, even at different progression stages, in these two study data sets. PMID:28551556
[Use of multiple locus variable number tandem repeats analysis for the Brucella systematization].

PubMed

Kulakov, Iu K; Kovalev, D A; Misetova, E N; Golovneva, S I; Liapustina, L V; Zheludkov, M M

2012-01-01

The methods of molecular-genetic differentiation to strain level acquire increasing significance in the current system of struggle with brucellosis. MLVA (multiple locus variable number tandem repeats analysis) was selected for molecular-genetic differentiation to strain level and simultaneous establishment of the genetic relationship of investigated Brucella strains. The goal of this work was MLVA typing of three pathogenic Brucella species strains with the analysis of stability of chosen loci, discrimination power and concordance to conventional phenotypic methods of the Brucella differentiation for use in systematization of brucellosis causing agents. Twenty six Brucella strains representing reference (n = 15), vaccine (n = 2) and field strains of three pathogenic Brucella species were tested: B. melitensis (n = 3), B. abortus (n = 2), B. suis (n = 2), and isolates (n = 2) with unidentified taxonomic position using MLVA with 9 pairs primers on known variable loci of Brucella genome. The analysis of the stability of chosen loci, discrimination power on Hunter-Gaston discrimination index (HGDI) and consistency to phenotypic methods of identification was performed. MLVA was confirmed for the results of phenotypic methods of identification, stability of the chosen loci in majority reference, and vaccine strains with a high index of variability HGDI 0.9969 for all loci. A dendrogram was plotted on the basis of MLVA data on distributed Brucella strains in related clusters according to its taxonomic species and biovar positions and construction of 25 genotypes. B. melitensis strains formed cluster related to the reference strain of B. melitensis 63/9 biovar 2. Australian isolates of Brucella 83-4 and Brucella 83-6 isolated from rodents formed a cluster distant from other strains of Brucella. MLVA is a promising method for differentiation of Brucella strains with known and unresolved taxonomic status for their systematization and creation of MLVA genotype catalogue that will promote qualitative improvement of brucellosis surveillance system in Russia.
Phenotype frequencies of blood group systems (Rh, Kell, Kidd, Duffy, MNS, P, Lewis, and Lutheran) in blood donors of south Gujarat, India

PubMed Central

Kahar, Manoj A.; Patel, Rajnikant. D.

2014-01-01

Background: This is the first study on phenotype frequencies of various blood group systems in blood donors of south Gujarat, India using conventional tube technique. Material and Methods: A total of 115 “O” blood group donors from three different blood banks of south Gujarat were typed for D, C, c, E, e, K, Jka, Lea, Leb, P1, M, and N antigens using monoclonal antisera and k, Kpa, Kpb, Fya,Fyb, Jkb, S,s, Lua, and Lub antigens were typed using polyclonal antisera employing Indirect Antiglobulin Test. Antigens and phenotype frequencies were expressed as percentages. Results: From the 115 blood donor samples used for extended antigen typing in the Rh system, e antigen was found in 100% donors, followed by D [84.35%], C [81.74%], c [56.32%], and E [21.74%] with DCe/DCe (R1 R1, 40.87%) as the most common phenotype. k was found to be positive in 100% of donors and no K+k- phenotype was found in Kell system. For Kidd and Duffy blood group system, Jk(a+b+) and Fy(a-b-) were the most common phenotypes with frequency of 52.17% and 48.69%, respectively. In the MNS system, 39.13% donors were typed as M+N+, 37.39% as M+N-, and 23.48% as M-N+. S+s+ was found in 24.35% of donors, S+s- in 8.69%, and S-s+ as the commonest amongst donors with 66.96%. No Lu(a+b+) or Lu(a+b-) phenotypes were detected in 115 donors typed for Lutheran antigens. A rare Lu(a-b-) phenotype was found in 2.61% donors. Conclusion: Data base for antigen frequency of various blood group systems in local donors help provide antigen negative compatible blood units to patients with multiple antibodies in order to formulate in-house red cells for antibody detection and identification and for preparing donor registry for rare blood groups. PMID:24678176
Faster and economical screening for vancomycin-resistant enterococci by sequential use of chromogenic agar and real-time polymerase chain reaction.

PubMed

Tan, Thean Yen; Jiang, Boran; Ng, Lily Siew Yong

2017-08-01

Screening for vancomycin-resistant enterococci (VRE) by culture takes days to generate results, while polymerase chain reaction (PCR) testing directly from clinical specimens lacks specificity. The aims of this study were to develop a real-time PCR to detect and identify Enterococcus faecium, Enterococcus faecalis, and vanA and vanB genes, and to evaluate the impact of this PCR on test-reporting times when performing it directly from suspect VRE isolates present on screening chromogenic media. The tetraplex PCR primers were designed to amplify E. faecium, E. faecalis, and vanA and vanB genes, with melt-curve analysis of PCR products. Following analytical and clinical validation of the molecular assay, PCR testing was performed for target colonies present on VRE chromogenic media. PCR results were evaluated against conventional phenotypic identification and susceptibility testing, with the time to result being monitored for both modalities. A total of 519 colonies from clinical specimens were tested concurrently by real-time PCR and phenotypic methods. In all, 223 isolates were identified with phenotypic vancomycin resistance (vanA, n = 108; vanB, n = 105; non-vanA/vanB = 10), with complete agreement between PCR and phenotypic testing for vancomycin-resistant E. faecium and E. faecalis. The majority (88.6%) of PCR results were reported, on average, 24.8 hours earlier than those of phenotypic testing, with 68% reduction in total costs. The use of culture on selective media, followed by direct colony PCR confirmation allows faster and economical VRE screening. Copyright © 2015. Published by Elsevier B.V.
Enhancement of Plant Metabolite Fingerprinting by Machine Learning1[W

PubMed Central

Scott, Ian M.; Vermeer, Cornelia P.; Liakata, Maria; Corol, Delia I.; Ward, Jane L.; Lin, Wanchang; Johnson, Helen E.; Whitehead, Lynne; Kular, Baldeep; Baker, John M.; Walsh, Sean; Dave, Anuja; Larson, Tony R.; Graham, Ian A.; Wang, Trevor L.; King, Ross D.; Draper, John; Beale, Michael H.

2010-01-01

Metabolite fingerprinting of Arabidopsis (Arabidopsis thaliana) mutants with known or predicted metabolic lesions was performed by 1H-nuclear magnetic resonance, Fourier transform infrared, and flow injection electrospray-mass spectrometry. Fingerprinting enabled processing of five times more plants than conventional chromatographic profiling and was competitive for discriminating mutants, other than those affected in only low-abundance metabolites. Despite their rapidity and complexity, fingerprints yielded metabolomic insights (e.g. that effects of single lesions were usually not confined to individual pathways). Among fingerprint techniques, 1H-nuclear magnetic resonance discriminated the most mutant phenotypes from the wild type and Fourier transform infrared discriminated the fewest. To maximize information from fingerprints, data analysis was crucial. One-third of distinctive phenotypes might have been overlooked had data models been confined to principal component analysis score plots. Among several methods tested, machine learning (ML) algorithms, namely support vector machine or random forest (RF) classifiers, were unsurpassed for phenotype discrimination. Support vector machines were often the best performing classifiers, but RFs yielded some particularly informative measures. First, RFs estimated margins between mutant phenotypes, whose relations could then be visualized by Sammon mapping or hierarchical clustering. Second, RFs provided importance scores for the features within fingerprints that discriminated mutants. These scores correlated with analysis of variance F values (as did Kruskal-Wallis tests, true- and false-positive measures, mutual information, and the Relief feature selection algorithm). ML classifiers, as models trained on one data set to predict another, were ideal for focused metabolomic queries, such as the distinctiveness and consistency of mutant phenotypes. Accessible software for use of ML in plant physiology is highlighted. PMID:20566707
Phenotypic Identification of Actinomyces and Related Species Isolated from Human Sources

PubMed Central

Sarkonen, Nanna; Könönen, Eija; Summanen, Paula; Könönen, Mauno; Jousimies-Somer, Hannele

2001-01-01

Recent advancements in chemotaxonomic and molecular biology-based identification methods have clarified the taxonomy of the genus Actinomyces and have led to the recognition of several new Actinomyces and related species. Actinomyces-like gram-positive rods have increasingly been isolated from various clinical specimens. Thus, an easily accessible scheme for reliable differentiation at the species level is needed in clinical and oral microbiology laboratories, where bacterial identification is mainly based on conventional biochemical methods. In the present study we designed a two-step protocol that consists of a flowchart that describes rapid, cost-efficient tests for preliminary identification of Actinomyces and closely related species and an updated more comprehensive scheme that also uses fermentation reactions for accurate differentiation of Actinomyces and closely related species. PMID:11682514

Rapid phenotypic antimicrobial susceptibility testing using nanoliter arrays.

PubMed

Avesar, Jonathan; Rosenfeld, Dekel; Truman-Rosentsvit, Marianna; Ben-Arye, Tom; Geffen, Yuval; Bercovici, Moran; Levenberg, Shulamit

2017-07-18

Antibiotic resistance is a major global health concern that requires action across all sectors of society. In particular, to allow conservative and effective use of antibiotics clinical settings require better diagnostic tools that provide rapid determination of antimicrobial susceptibility. We present a method for rapid and scalable antimicrobial susceptibility testing using stationary nanoliter droplet arrays that is capable of delivering results in approximately half the time of conventional methods, allowing its results to be used the same working day. In addition, we present an algorithm for automated data analysis and a multiplexing system promoting practicality and translatability for clinical settings. We test the efficacy of our approach on numerous clinical isolates and demonstrate a 2-d reduction in diagnostic time when testing bacteria isolated directly from urine samples.
Comparison of the genomic sequence of the microminipig, a novel breed of swine, with the genomic database for conventional pig.

PubMed

Miura, Naoki; Kucho, Ken-Ichi; Noguchi, Michiko; Miyoshi, Noriaki; Uchiumi, Toshiki; Kawaguchi, Hiroaki; Tanimoto, Akihide

2014-01-01

The microminipig, which weighs less than 10 kg at an early stage of maturity, has been reported as a potential experimental model animal. Its extremely small size and other distinct characteristics suggest the possibility of a number of differences between the genome of the microminipig and that of conventional pigs. In this study, we analyzed the genomes of two healthy microminipigs using a next-generation sequencer SOLiD™ system. We then compared the obtained genomic sequences with a genomic database for the domestic pig (Sus scrofa). The mapping coverage of sequenced tag from the microminipig to conventional pig genomic sequences was greater than 96% and we detected no clear, substantial genomic variance from these data. The results may indicate that the distinct characteristics of the microminipig derive from small-scale alterations in the genome, such as Single Nucleotide Polymorphisms or translational modifications, rather than large-scale deletion or insertion polymorphisms. Further investigation of the entire genomic sequence of the microminipig with methods enabling deeper coverage is required to elucidate the genetic basis of its distinct phenotypic traits. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
Ultra-high frequency ultrasound biomicroscopy and high throughput cardiovascular phenotyping in a large scale mouse mutagenesis screen

NASA Astrophysics Data System (ADS)

Liu, Xiaoqin; Francis, Richard; Tobita, Kimimasa; Kim, Andy; Leatherbury, Linda; Lo, Cecilia W.

2013-02-01

Ultrasound biomicroscopy (UBM) is ideally suited for phenotyping fetal mice for congenital heart disease (CHD), as imaging can be carried out noninvasively to provide both hemodynamic and structural information essential for CHD diagnosis. Using the UBM (Vevo 2100; 40Hz) in conjunction with the clinical ultrasound system (Acuson Sequioa C512; 15Hz), we developed a two-step screening protocol to scan thousands fetuses derived from ENU mutagenized pedigrees. A wide spectrum of CHD was detected by the UBM, which were subsequently confirmed with follow-up necropsy and histopathology examination with episcopic fluorescence image capture. CHD observed included outflow anomalies, left/right heart obstructive lesions, septal/valvular defects and cardiac situs anomalies. Meanwhile, various extracardiac defects were found, such as polydactyly, craniofacial defects, exencephaly, omphalocele-cleft palate, most of which were associated with cardiac defects. Our analyses showed the UBM was better at assessing cardiac structure and blood flow profiles, while conventional ultrasound allowed higher throughput low-resolution screening. Our study showed the integration of conventional clinical ultrasound imaging with the UBM for fetal mouse cardiovascular phenotyping can maximize the detection and recovery of CHD mutants.
Early seedling vigour, an imperative trait for direct-seeded rice: an overview on physio-morphological parameters and molecular markers.

PubMed

Mahender, A; Anandan, A; Pradhan, S K

2015-05-01

Rapid uniform germination and accumulation of biomass during initial phase of seedling establishment is an essential phenotypic trait considered as early seedling vigour for direct seeded situation in rice irrespective of environment. Enhanced role of carbohydrate, amylase, growth hormones, antioxidant enzymes and ascorbic acid brings changes in vigour and phenotype of seedling. Early establishment and demanding life form dominate the surroundings. Crop plant that has better growth overdrives the weed plant and suppresses its growth. Seedling early vigour is the characteristic of seed quality and describes the rapid, uniform germination and the establishment of strong seedlings in any environmental condition. The phenotype of modern rice varieties has been changed into adaptable for transplanted rice with thirst toward water and selection pressure for semi-dwarf architecture resulting in reduced early vigour. Decreasing freshwater availability and rising labour cost drives the search for a suitable alternative management system to enhance grain yield productivity for the burgeoning world population. In view of these issues, much attention has been focused on dry direct-seeded rice, because it demands low input. A rice cultivar with a strong seedling vigour trait is desirable in case of direct seeding. However, seedling vigour has not been selected in crop improvement programmes in conventional breeding due to its complex nature and quantitative inheritance. Molecular markers have been proven effective in increasing selection efficiency, particularly for quantitative traits that are simply inherited. Marker-assisted selection approach has facilitated efficient and precise transfer of genes/QTL(s) into many crop species and suggests a speedy and efficient technique over conventional breeding and selection methods. In this review, we present the findings and investigations in the field of seedling vigour in rice that includes the nature of inheritance of physio-morphological and biochemical traits and QTLs to assist plant breeders who work for direct-seeded rice.
Enhancing the reliability and throughput of neurosphere culture on hydrogel microwell arrays.

PubMed

Cordey, Myriam; Limacher, Monika; Kobel, Stefan; Taylor, Verdon; Lutolf, Matthias P

2008-10-01

The neurosphere assay is the standard retrospective assay to test the self-renewal capability and multipotency of neural stem cells (NSCs) in vitro. However, it has recently become clear that not all neurospheres are derived from a NSC and that on conventional cell culture substrates, neurosphere motility may cause frequent neurosphere "merging" [Nat Methods 2006;3:801-806; Stem Cells 2007;25:871-874]. Combining biomimetic hydrogel matrix technology with microengineering, we developed a microwell array platform on which NSC fate and neurosphere formation can be unequivocally attributed to a single founding cell. Using time-lapse microscopy and retrospective immunostaining, the fate of several hundred single NSCs was quantified. Compared with conventional neurosphere culture methods on plastic dishes, we detected a more than 100% increase in single NSC viability on soft hydrogels. Effective confinement of single proliferating cells to microwells led to neurosphere formation of vastly different sizes, a high percentage of which showed stem cell phenotypes after one week in culture. The reliability and increased throughput of this platform should help to better elucidate the function of sphere-forming stem/progenitor cells independent of their proliferation dynamics. Disclosure of potential conflicts of interest is found at the end of this article.
Comparison of methods for the identification of microorganisms isolated from blood cultures.

PubMed

Monteiro, Aydir Cecília Marinho; Fortaleza, Carlos Magno Castelo Branco; Ferreira, Adriano Martison; Cavalcante, Ricardo de Souza; Mondelli, Alessandro Lia; Bagagli, Eduardo; da Cunha, Maria de Lourdes Ribeiro de Souza

2016-08-05

Bloodstream infections are responsible for thousands of deaths each year. The rapid identification of the microorganisms causing these infections permits correct therapeutic management that will improve the prognosis of the patient. In an attempt to reduce the time spent on this step, microorganism identification devices have been developed, including the VITEK(®) 2 system, which is currently used in routine clinical microbiology laboratories. This study evaluated the accuracy of the VITEK(®) 2 system in the identification of 400 microorganisms isolated from blood cultures and compared the results to those obtained with conventional phenotypic and genotypic methods. In parallel to the phenotypic identification methods, the DNA of these microorganisms was extracted directly from the blood culture bottles for genotypic identification by the polymerase chain reaction (PCR) and DNA sequencing. The automated VITEK(®) 2 system correctly identified 94.7 % (379/400) of the isolates. The YST and GN cards resulted in 100 % correct identifications of yeasts (15/15) and Gram-negative bacilli (165/165), respectively. The GP card correctly identified 92.6 % (199/215) of Gram-positive cocci, while the ANC card was unable to correctly identify any Gram-positive bacilli (0/5). The performance of the VITEK(®) 2 system was considered acceptable and statistical analysis showed that the system is a suitable option for routine clinical microbiology laboratories to identify different microorganisms.
Heritability Analyses of IQ Scores: Science or Numerology?

ERIC Educational Resources Information Center

Layzer, David

1974-01-01

Examines limitations of the heritability concept and heritability analysis, and discusses a conventional application of heritability analysis, IQ scores as measurements of a phenotypic character, the heritability of IQ, and the relationship of IQ and race. (JR)
Distribution of Kell phenotype among pregnant women in Sokoto, North Western Nigeria.

PubMed

Osaro, Erhabor; Ladan, Malami Aisha; Zama, Isaac; Ahmed, Yakubu; Mairo, Hassan

2015-01-01

Kell antigen is highly immunogenic and is the common cause of antibody production in mismatched blood transfusions, haemolytic transfusion reaction (HTR) and maternal alloimmunization, which causes severe anaemia in neonates. The aim of this study is to determine the prevalence and ethnic variation of the Kell phenotype among pregnant women in Sokoto, Nigeria. Kell antigen status of 150 pregnant women aged 18-45 years and mean age 27.19 ±4.69 years attending antenatal clinic in UDUTH Sokoto Nigeria was determined using the conventional tube method and anti-Kell reagents (Lorne Laboratories, UK). Among the 150 subjects studied, 3 (2.0%) of subjects were positive and 147 (98.0) were negative for K antigen. Of the 150 pregnant subjects; 32 (21.3%) were primigravidae while 118 (78.7%) were multigravidae. Kell phenotype was more prevalent among primigravidae (3.1%) compared to multigravidae (1.7%) women. The distribution of Kell phenotype among the pregnant subjects was compared based on ethnicity. The prevalence of Kell antigen was significantly higher among the Hausa ethnic group (3.2%) compared to other ethnic groups which indicated zero prevalence (p = 0.001). Kell negative phenotype was ≥ 96.8% among all the ethnic groups. Our observed prevalence of Kell phenotype is consistent with previous studies among Blacks and Asians but significantly lower than values observed in previous studies among Caucasians. We recommend that all pregnant women should be screened for the presence clinically significant red cell antigens including Kell antigen on their first antenatal visit. Kell negative red cell should be routinely provided for all pregnant women and women with child bearing potential to reduce the risk of Kell-associated HDFN. There is need to introduce routine screening of pregnant women for clinically significant red cell antibodies to facilitate the effective management of HDFN as well as prevent HTR. There is also need for sustained health education of pregnant women in the area to encourage early booking for antenatal care.
Distribution of Malassezia species on the skin of patients with atopic dermatitis, psoriasis, and healthy volunteers assessed by conventional and molecular identification methods.

PubMed

Jagielski, Tomasz; Rup, Elżbieta; Ziółkowska, Aleksandra; Roeske, Katarzyna; Macura, Anna B; Bielecki, Jacek

2014-03-07

The Malassezia yeasts which belong to the physiological microflora of human skin have also been implicated in several dermatological disorders, including pityriasis versicolor (PV), atopic dermatitis (AD), and psoriasis (PS). The Malassezia genus has repeatedly been revised and it now accommodates 14 species, all but one being lipid-dependent species. The traditional, phenotype-based identification schemes of Malassezia species are fraught with interpretative ambiguities and inconsistencies, and are thus increasingly being supplemented or replaced by DNA typing methods. The aim of this study was to explore the species composition of Malassezia microflora on the skin of healthy volunteers and patients with AD and PS. Species characterization was performed by conventional, culture-based methods and subsequently molecular techniques: PCR-RFLP and sequencing of the internal transcribed spacer (ITS) 1/2 regions and the D1/D2 domains of the 26S rRNA gene. The Chi-square test and Fisher's exact test were used for statistical analysis. Malassezia sympodialis was the predominant species, having been cultured from 29 (82.9%) skin samples collected from 17 out of 18 subjects under the study. Whereas AD patients yielded exclusively M. sympodialis isolates, M. furfur isolates were observed only in PS patients. The isolation of M. sympodialis was statistically more frequent among AD patients and healthy volunteers than among PS patients (P < 0.03). Whether this mirrors any predilection of particular Malassezia species for certain clinical conditions needs to be further evaluated. The overall concordance between phenotypic and molecular methods was quite high (65%), with the discordant results being rather due to the presence of multiple species in a single culture (co-colonization) than true misidentification. All Malassezia isolates were susceptible to cyclopiroxolamine and azole drugs, with M. furfur isolates being somewhat more drug tolerant than other Malassezia species. This study provides an important insight into the species composition of Malassezia microbiota in human skin. The predominance of M. sympodialis in both normal and pathologic skin, contrasts with other European countries, reporting M. globosa and M. restricta as the most frequently isolated Malassezia species.
Accuracy of different bioinformatics methods in detecting antibiotic resistance and virulence factors from Staphylococcus aureus whole genome sequences.

PubMed

Mason, Amy; Foster, Dona; Bradley, Phelim; Golubchik, Tanya; Doumith, Michel; Gordon, N Claire; Pichon, Bruno; Iqbal, Zamin; Staves, Peter; Crook, Derrick; Walker, A Sarah; Kearns, Angela; Peto, Tim

2018-06-20

Background : In principle, whole genome sequencing (WGS) can predict phenotypic resistance directly from genotype, replacing laboratory-based tests. However, the contribution of different bioinformatics methods to genotype-phenotype discrepancies has not been systematically explored to date. Methods : We compared three WGS-based bioinformatics methods (Genefinder (read-based), Mykrobe (de Bruijn graph-based) and Typewriter (BLAST-based)) for predicting presence/absence of 83 different resistance determinants and virulence genes, and overall antimicrobial susceptibility, in 1379 Staphylococcus aureus isolates previously characterised by standard laboratory methods (disc diffusion, broth and/or agar dilution and PCR). Results : 99.5% (113830/114457) of individual resistance-determinant/virulence gene predictions were identical between all three methods, with only 627 (0.5%) discordant predictions, demonstrating high overall agreement (Fliess-Kappa=0.98, p<0.0001). Discrepancies when identified were in only one of the three methods for all genes except the cassette recombinase, ccrC(b ). Genotypic antimicrobial susceptibility prediction matched laboratory phenotype in 98.3% (14224/14464) cases (2720 (18.8%) resistant, 11504 (79.5%) susceptible). There was greater disagreement between the laboratory phenotypes and the combined genotypic predictions (97 (0.7%) phenotypically-susceptible but all bioinformatic methods reported resistance; 89 (0.6%) phenotypically-resistant, but all bioinformatics methods reported susceptible) than within the three bioinformatics methods (54 (0.4%) cases, 16 phenotypically-resistant, 38 phenotypically-susceptible). However, in 36/54 (67%), the consensus genotype matched the laboratory phenotype. Conclusions : In this study, the choice between these three specific bioinformatic methods to identify resistance-determinants or other genes in S. aureus did not prove critical, with all demonstrating high concordance with each other and phenotypic/molecular methods. However, each has some limitations and therefore consensus methods provide some assurance. Copyright © 2018 American Society for Microbiology.
Effect of L-thyroxine treatment on peripheral blood dendritic cell subpopulations in patients with Hashimoto's thyroiditis.

PubMed

Stasiolek, Mariusz; Dedecjus, Marek; Adamczewski, Zbigniew; Sliwka, Przemyslaw Wiktor; Brzezinski, Jan; Lewinski, Andrzej

2014-01-01

Recent reports suggested dendritic cells (DCs) to be important players in the pathogenesis of autoimmune thyroid processes in humans. However, there are virtually no data addressing the influence of thyroid autoaggression-associated disturbances of thyrometabolic conditions on DCs biology. The aim of the study was to evaluate the influence of L-thyroxine supplementation on conventional and plasmacytoid peripheral blood DCs subtypes in patients with hypothyroidism due to Hashimoto's thyroiditis (HT). Eighteen patients with newly diagnosed hypothyroidism due to HT were included into the study. All patients received L-thyroxine treatment with doses adjusted to reach euthyroidism. Peripheral blood DC subtypes structure and immunoregulatory phenotype were analyzed by flow cytometry in the same patient prospectively at two time points: (i) before and (ii) 3 months after beginning of L-thyroxine treatment (hypothyroidism vs. euthyroidism, respectively). Percentage of plasmacytoid DCs in peripheral blood mononuclear cells fraction was significantly decreased in the course of L-thyroxine treatment (0.27 ± 0.19 vs. 0.11 ± 0.08; p < 0.05), whereas we did not observe any changes in the number of conventional DCs. However, the phenotypic analysis showed a significant increase of conventional DCs expressing CD86 and CD91 (64.25 ± 21.6% vs. 86.3 ± 11%; p < 0.05 and 30.75 ± 11.66% vs. 44.5 ± 13.3%; p < 0.05; respectively) in euthyroid patients. Standard L-thyroxine supplementation in HT patients exerted significant immunoregulatory effects, associated with quantitative and phenotypic changes of peripheral blood DC subpopulations.
Measurement of Lp(a) with a two-step monoclonal competitive sandwich ELISA method.

PubMed

Morikawa, W; Iki, R; Terano, T; Funatsu, A; Sugiuchi, H; Uji, Y; Okabe, H

1995-06-01

To evaluate the results of Lipoprotein (a)[Lp(a)] measurements by a competitive two-step monoclonal enzyme-linked immuno sorbent assay method comparing them with those by a conventional ELISA. Serum having various isoforms of Lp(a) and purified Lp(a) were assayed using the method described here and commercially available kits. The reference range was determined with the use of 324 normal subjects by means of calculation from Lp(a) results of logarithmic transformation. Our method takes advantage of a competitive reaction between fixed antibody and free antibody to Lp(a), having the detection range up to 1000 mg/L with the lowest detection limit of 2 mg/L. The anti-Lp(a) monoclonal antibody employed in the assay system reacts uniformly with all phenotypes of Lp(a) but showing very low cross-reactivity for plasminogen and LDL. Within-run and between-run precisions were excellent, giving CVs of 2.9 and 4.0% with mean values of 145 and 635 mg/L, respectively. In comparison of the results by our method with those by a polyclonal method (Biopool) or a monoclonal antibody method (Terumo), they correlated well; Y (our method) = 0.99 x (polyclonal method, Biopool) - 1.9, r = 0.994 (n = 60), and Y = 0.94 X(monoclonal method, Terumo) -9.8, r = 0.97 (n = 60), respectively. The reference range was 105.9 +/- 25.4 mg/L, the difference between the sexes was not significant. Our method has proven highly accurate and specific. It is applicable with auto analyzer because it does not require such a pre-dilution step as is necessary for Lp(a) determination by conventional ELISA assay. Accordingly, we can conclude that our test method is workable for both clinical laboratories and mass screening.
Identification of rare pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

PubMed

Seng, Piseth; Abat, Cedric; Rolain, Jean Marc; Colson, Philippe; Lagier, Jean-Christophe; Gouriet, Frédérique; Fournier, Pierre Edouard; Drancourt, Michel; La Scola, Bernard; Raoult, Didier

2013-07-01

During the past 5 years, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.
Identification of Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

PubMed Central

Seng, Piseth; Abat, Cedric; Rolain, Jean Marc; Colson, Philippe; Lagier, Jean-Christophe; Gouriet, Frédérique; Fournier, Pierre Edouard; Drancourt, Michel; La Scola, Bernard

2013-01-01

During the past 5 years, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories. PMID:23637301
Genome and metabolic engineering in non-conventional yeasts: Current advances and applications.

PubMed

Löbs, Ann-Kathrin; Schwartz, Cory; Wheeldon, Ian

2017-09-01

Microbial production of chemicals and proteins from biomass-derived and waste sugar streams is a rapidly growing area of research and development. While the model yeast Saccharomyces cerevisia e is an excellent host for the conversion of glucose to ethanol, production of other chemicals from alternative substrates often requires extensive strain engineering. To avoid complex and intensive engineering of S. cerevisiae, other yeasts are often selected as hosts for bioprocessing based on their natural capacity to produce a desired product: for example, the efficient production and secretion of proteins, lipids, and primary metabolites that have value as commodity chemicals. Even when using yeasts with beneficial native phenotypes, metabolic engineering to increase yield, titer, and production rate is essential. The non-conventional yeasts Kluyveromyces lactis, K. marxianus, Scheffersomyces stipitis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris have been developed as eukaryotic hosts because of their desirable phenotypes, including thermotolerance, assimilation of diverse carbon sources, and high protein secretion. However, advanced metabolic engineering in these yeasts has been limited. This review outlines the challenges of using non-conventional yeasts for strain and pathway engineering, and discusses the developed solutions to these problems and the resulting applications in industrial biotechnology.
Improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of difficult-to-identify bacteria and its impact in the workflow of a clinical microbiology laboratory.

PubMed

Rodríguez-Sánchez, Belén; Marín, Mercedes; Sánchez-Carrillo, Carlos; Cercenado, Emilia; Ruiz, Adrián; Rodríguez-Créixems, Marta; Bouza, Emilio

2014-05-01

This study evaluates matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) capability for the identification of difficult-to-identify microorganisms. A total of 150 bacterial isolates inconclusively identified with conventional phenotypic tests were further assessed by 16S rRNA sequencing and by MALDI-TOF MS following 2 methods: a) a simplified formic acid-based, on-plate extraction and b) performing a tube-based extraction step. Using the simplified method, 29 isolates could not be identified. For the remaining 121 isolates (80.7%), we obtained a reliable identification by MALDI-TOF: in 103 isolates, the identification by 16S rRNA sequencing and MALDI TOF coincided at the species level (68.7% from the total 150 analyzed isolates and 85.1% from the samples with MALDI-TOF result), and in 18 isolates, the identification by both methods coincided at the genus level (12% from the total and 14.9% from the samples with MALDI-TOF results). No discordant results were observed. The performance of the tube-based extraction step allowed the identification at the species level of 6 of the 29 unidentified isolates by the simplified method. In summary, MALDI-TOF can be used for the rapid identification of many bacterial isolates inconclusively identified by conventional methods. Copyright © 2014 Elsevier Inc. All rights reserved.
Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

PubMed Central

Gallerani, Giulia; Cocchi, Claudia; Bocchini, Martine; Piccinini, Filippo; Fabbri, Francesco

2017-01-01

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments. PMID:29286485
FOXP3: required but not sufficient. the role of GARP (LRRC32) as a safeguard of the regulatory phenotype.

PubMed

Probst-Kepper, M; Balling, R; Buer, J

2010-08-01

FOXP3 is essential for the development and function of regulatory CD4(+)CD25(hi) T (T(reg)) cells. However, recent evidence suggests that FOXP3 alone is not sufficient to completely explain the regulatory phenotype of these key players in autoimmunity and inflammation: after being activated, conventional human CD4(+) T cells transiently up-regulate FOXP3 without acquiring a regulatory function. Researchers have recently found that glycoprotein A repetitions predominant (GARP, or LRRC32) is a T(reg)-specific receptor that binds latent TGF-beta and dominantly controls FOXP3 and the regulatory phenotype via a positive feedback loop. This finding provides a missing link in our molecular understanding of FOXP3 in T(reg) cells. This viewpoint focuses on GARP as safeguard of FOXP3 and the regulatory phenotype.
The role of ecological opportunity in shaping disparate diversification trajectories in a bicontinental primate radiation.

PubMed

Tran, Lucy A P

2014-04-22

Exceptional species and phenotypic diversity commonly are attributed to ecological opportunity (EO). The conventional EO model predicts that rates of lineage diversification and phenotypic evolution are elevated early in a radiation only to decline later in response to niche availability. Foregut fermentation is hypothesized to be a key innovation that allowed colobine monkeys (subfamily Colobinae), the only primates with this trait, to successfully colonize folivore adaptive zones unavailable to other herbivorous species. Therefore, diversification rates also are expected to be strongly linked with the evolution of traits related to folivory in these monkeys. Using dated molecular phylogenies and a dataset of feeding morphology, I test predictions of the EO model to evaluate the role of EO conferred by foregut fermentation in shaping the African and Asian colobine radiations. Findings from diversification methods coupled with colobine biogeographic history provide compelling evidence that decreasing availability of new adaptive zones during colonization of Asia together with constraints presented by dietary specialization underlie temporal changes in diversification in the Asian but not African clade. Additionally, departures from the EO model likely reflect iterative diversification events in Asia.
Identification of Candida lusitaniae as an opportunistic yeast in humans.

PubMed

Holzschu, D L; Presley, H L; Miranda, M; Phaff, H J

1979-08-01

Four yeast strains, causally associated with infection in a patient with acute myelogenous leukemia, were identified by standard methods currently used in yeast taxonomy as representatives of Candida lusitania van Uden et do Carmo-Sousa. Because this species has not been recognized previously as an opportunistic yeast in humans, molecular taxonomic methods were applied to confirm its identity. The nuclear deoxyribonucleic acid (DNA) base composition of two clinical isolates was shown to be 45.1 mol% guanine plus cytosine as compared to 44.7 mol% guanine plus cytosine for the type strain of this species. DNA/DNA reassociation experiments revealed more than 95% complementarity between the DNAs from the clinical isolates and that of the type strain of C. lusitaniae, thus confirming their classification by conventional taxonomy. A key is provided to differentiate C. lusitaniae from two phenotypically similar Candida species.

Introduction: Food Allergy - A Toxicologists Point Of View

EPA Science Inventory

Incorporation of new proteins into crops to promote resistance to pests and other stressors, improve nutrition, or otherwise modify phenotype is a technology that has many advantages over conventional approaches. There is concern however, that introduction of a novel protein into...
Appraisal of Biofilm Formation in Diabetic Foot Infections by Comparing Phenotypic Methods With the Ultrastructural Analysis.

PubMed

Pugazhendhi, Sugandhi; Dorairaj, Arvind Prasanth

Diabetic patients are more prone to the development of foot ulcers, because their underlying tissues are exposed to colonization by various pathogenic organisms. Hence, biofilm formation plays a vital role in disease progression by antibiotic resistance to the pathogen found in foot infections. The present study has demonstrated the correlation of biofilm assay with the clinical characteristics of diabetic foot infection. The clinical characteristics such as the ulcer duration, size, nature, and grade were associated with biofilm production. Our results suggest that as the size of the ulcer with poor glycemic control increased, the organism was more likely to be positive for biofilm formation. A high-degree of antibiotic resistance was exhibited by the biofilm-producing gram-positive isolates for erythromycin and gram-negative isolates for cefpodoxime. Comparisons of biofilm production using 3 different conventional methods were performed. The strong producers with the tube adherence method were able to produce biofilm using the cover slip assay method, and the weak producers in tube adherence method had difficulty in producing biofilm using the other 2 methods, indicating that the tube adherence method is the best method for assessing biofilm formation. The strong production of biofilm with the conventional method was further confirmed by scanning electron microscopy analysis, because bacteria attached as a distinct layer of biofilm. Thus, the high degree of antibiotic resistance was exhibited by biofilm producers compared with nonbiofilm producers. The tube adherence and cover slip assay were found to be the better method for biofilm evaluation. Copyright © 2018 The American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.
Adult onset Niemann-Pick type C disease: A clinical, neuroimaging and molecular genetic study.

PubMed

Battisti, Carla; Tarugi, Patrizla; Dotti, Maria Teresa; De Stefano, Nicola; Vattimo, Angelo; Chierichetti, Francesea; Calandra, Sebastiano; Federico, Antonio

2003-11-01

We report on a patient with adult-onset Niemann-Pick type C (NPC) disease, carrying the mutations P1007 and I1061T in the NPC1 gene, presenting with marked psychiatric changes followed by dystonia and cognitive impairment. Filipin staining, single photon emission computed tomography perfusional, positron emission tomography metabolic, conventional magnetic resonance imaging, and magnetic resonance spectroscopy findings suggested a pathophysiological correlation with phenotype expression. This case expands the clinical and genetic spectrum of the rare adult-onset NPC disease phenotype.
How genetic errors in GPCRs affect their function: Possible therapeutic strategies

PubMed Central

Stoy, Henriette; Gurevich, Vsevolod V.

2015-01-01

Activating and inactivating mutations in numerous human G protein-coupled receptors (GPCRs) are associated with a wide range of disease phenotypes. Here we use several class A GPCRs with a particularly large set of identified disease-associated mutations, many of which were biochemically characterized, along with known GPCR structures and current models of GPCR activation, to understand the molecular mechanisms yielding pathological phenotypes. Based on this mechanistic understanding we also propose different therapeutic approaches, both conventional, using small molecule ligands, and novel, involving gene therapy. PMID:26229975
Genetic diversity in soybean genotypes using phenotypic characters and enzymatic markers.

PubMed

Zambiazzi, E V; Bruzi, A T; Sales, A P; Borges, I M M; Guilherme, S R; Zuffo, A M; Lima, J G; Ribeiro, F O; Mendes, A E S; Godinho, S H M; Carvalho, M L M

2017-09-21

The objective of this study was to evaluate the genetic diversity of soybean cultivars by adopting phenotypic traits and enzymatic markers, the relative contribution of agronomic traits to diversity, as well as diversity between the level of technology used in soybean cultivars and genetic breeding programs in which cultivars were inserted. The experiments were conducted on the field at the Center for Scientific and Technological Development in crop-livestock production and the Electrophoresis Laboratory of Lavras Federal University. The agronomic traits adopted were grain yield, plant height, first legume insertion, plant lodging, the mass of one thousand seeds, and days for complete maturation, in which the Euclidean distance, grouped by Tocher and UPGMA criteria, was obtained. After electrophorese gels for enzymatic systems, dehydrogenase alcohol, esterase, superoxide dismutase, and peroxidase were performed. The genetic similarity estimative was also obtained between genotypes by the Jaccard coefficient with subsequent grouping by the UPGMA method. The formation of two groups was shown using phenotypic characters in the genetic diversity study and individually discriminating the cultivar 97R73 RR. The character with the greatest contribution to the genetic divergence was grain yield with contribution higher than 90.0%. To obtain six different groups, individually discriminating the cultivars CG 8166 RR, FPS Jupiter RR, and BRS MG 780 RR, enzymatic markers were used. Cultivars carrying the RR technology presented more divergence than conventional cultivars and IPRO cultivars.
Noninvasive assessment of altered activity following restraint in mice using an automated physiological monitoring system.

PubMed

Spiers, Jereme G; Chen, Hsiao-Jou Cortina; Steyn, Frederik J; Lavidis, Nickolas A; Woodruff, Trent M; Lee, John D

2017-01-01

In the laboratory setting, typical endocrine and targeted behavioral tests are limited in their ability to provide a direct assessment of stress in animals housed in undisturbed conditions. We hypothesized that an automated phenotyping system would allow the detection of subtle stress-related behavioral changes well beyond the time-frames examined using conventional methods. In this study, we have utilized the TSE PhenoMaster system to continuously record basal behaviors and physiological parameters including activity, body weight, food intake and oxygen consumption in undisturbed and stressed C57Bl/6J male mice (n = 12/group), with a pharmacological intervention using the conventional anxiolytic, diazepam (5 mg kg -1 i.p.; n = 8/group). We observed significant 20-30% reductions in locomotor activity in the dark phase, with subtle reductions in light phase activity for up to 96 h following a single 2 h episode of restraint stress. A single administration of diazepam reduced plasma corticosterone concentrations by 30-35% during stress exposure when compared to mice treated with vehicle. This treatment did not result in significantly different locomotor activity compared to vehicle within the first 48 h following restraint stress. However, diazepam treatment facilitated restoration of locomotor activity at 72 and 96 h after restraint stress exposure in comparison to vehicle-treated mice. Hence, the use of an automated phenotyping system allows a real time assessment of basal behaviors and empirical metabolism following exposure to restraint stress and demonstrates major and subtle changes in activity persist for several days after stress exposure.
Discovery of a drug targeting microenvironmental support for lymphoma cells by screening using patient-derived xenograft cells

PubMed Central

Sugimoto, Keiki; Hayakawa, Fumihiko; Shimada, Satoko; Morishita, Takanobu; Shimada, Kazuyuki; Katakai, Tomoya; Tomita, Akihiro; Kiyoi, Hitoshi; Naoe, Tomoki

2015-01-01

Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, especially in the points of rapid growth rate and microenvironment independency. Consequently, the majority of conventional anti-cancer drugs are less sensitive to slow growing cells and do not target microenvironmental support, although most primary cancer cells grow slower than cell lines and depend on microenvironmental support. Here, we developed a novel high throughput drug screening system using patient-derived xenograft (PDX) cells of lymphoma that maintained primary cancer cell phenotype more than cell lines. The library containing 2613 known pharmacologically active substance and off-patent drugs were screened by this system. We could find many compounds showing higher cytotoxicity than conventional anti-tumor drugs. Especially, pyruvinium pamoate showed the highest activity and its strong anti-tumor effect was confirmed also in vivo. We extensively investigated its mechanism of action and found that it inhibited glutathione supply from stromal cells to lymphoma cells, implying the importance of the stromal protection from oxidative stress for lymphoma cell survival and a new therapeutic strategy for lymphoma. Our system introduces a primary cancer cell phenotype into cell-based phenotype screening and sheds new light on anti-cancer drug development. PMID:26278963
Using iterative cluster merging with improved gap statistics to perform online phenotype discovery in the context of high-throughput RNAi screens

PubMed Central

Yin, Zheng; Zhou, Xiaobo; Bakal, Chris; Li, Fuhai; Sun, Youxian; Perrimon, Norbert; Wong, Stephen TC

2008-01-01

Background The recent emergence of high-throughput automated image acquisition technologies has forever changed how cell biologists collect and analyze data. Historically, the interpretation of cellular phenotypes in different experimental conditions has been dependent upon the expert opinions of well-trained biologists. Such qualitative analysis is particularly effective in detecting subtle, but important, deviations in phenotypes. However, while the rapid and continuing development of automated microscope-based technologies now facilitates the acquisition of trillions of cells in thousands of diverse experimental conditions, such as in the context of RNA interference (RNAi) or small-molecule screens, the massive size of these datasets precludes human analysis. Thus, the development of automated methods which aim to identify novel and biological relevant phenotypes online is one of the major challenges in high-throughput image-based screening. Ideally, phenotype discovery methods should be designed to utilize prior/existing information and tackle three challenging tasks, i.e. restoring pre-defined biological meaningful phenotypes, differentiating novel phenotypes from known ones and clarifying novel phenotypes from each other. Arbitrarily extracted information causes biased analysis, while combining the complete existing datasets with each new image is intractable in high-throughput screens. Results Here we present the design and implementation of a novel and robust online phenotype discovery method with broad applicability that can be used in diverse experimental contexts, especially high-throughput RNAi screens. This method features phenotype modelling and iterative cluster merging using improved gap statistics. A Gaussian Mixture Model (GMM) is employed to estimate the distribution of each existing phenotype, and then used as reference distribution in gap statistics. This method is broadly applicable to a number of different types of image-based datasets derived from a wide spectrum of experimental conditions and is suitable to adaptively process new images which are continuously added to existing datasets. Validations were carried out on different dataset, including published RNAi screening using Drosophila embryos [Additional files 1, 2], dataset for cell cycle phase identification using HeLa cells [Additional files 1, 3, 4] and synthetic dataset using polygons, our methods tackled three aforementioned tasks effectively with an accuracy range of 85%–90%. When our method is implemented in the context of a Drosophila genome-scale RNAi image-based screening of cultured cells aimed to identifying the contribution of individual genes towards the regulation of cell-shape, it efficiently discovers meaningful new phenotypes and provides novel biological insight. We also propose a two-step procedure to modify the novelty detection method based on one-class SVM, so that it can be used to online phenotype discovery. In different conditions, we compared the SVM based method with our method using various datasets and our methods consistently outperformed SVM based method in at least two of three tasks by 2% to 5%. These results demonstrate that our methods can be used to better identify novel phenotypes in image-based datasets from a wide range of conditions and organisms. Conclusion We demonstrate that our method can detect various novel phenotypes effectively in complex datasets. Experiment results also validate that our method performs consistently under different order of image input, variation of starting conditions including the number and composition of existing phenotypes, and dataset from different screens. In our findings, the proposed method is suitable for online phenotype discovery in diverse high-throughput image-based genetic and chemical screens. PMID:18534020
Prevalence and molecular characterization of methicillin resistance among Coagulase-negative Staphylococci at a tertiary care center.

PubMed

Bhatt, Puneet; Tandel, Kundan; Singh, Alina; Kumar, M; Grover, Naveen; Sahni, A K

2016-12-01

Methicillin-resistant Coagulase-negative Staphylococci (MR-CoNS) have emerged as an important cause of nosocomial infections especially in patients with prosthetic devices and implants. This study was conducted with an aim to determine the prevalence of methicillin resistance among CoNS isolates at a tertiary care center by both phenotypic and genotypic methods. This cross sectional study was carried out from September 2011 to February 2014 in which 150 non-repetitive clinical isolates of CoNS were identified at the species level by conventional phenotypic methods. Cefoxitin disk (30 μg) diffusion testing was used to determine methicillin resistance and confirmed by detection of mec A gene by polymerase chain reaction (PCR). Out of 150 CoNS isolates, 51 were methicillin resistant by cefoxitin disk diffusion method. Out of these 51 isolates, mec A gene was detected only in 45 isolates. Moreover, mec A gene was also detected in 4 isolates, which were cefoxitin sensitive. Thus, the prevalence of methicillin resistance among CoNS was found to be 32.7% by PCR. The prevalence of methicillin resistance among Coagulase-negative Staphylococci (CoNS) was 32.7% by PCR detection of mec A gene. The sensitivity and specificity of cefoxitin disk diffusion method against mec A gene detection by PCR were found to be more than 90%. It can be concluded from this study that cefoxitin disk diffusion test can be used as a useful screening method to detect methicillin resistance among CoNS isolates. However, detection of mec A gene by PCR remains a more accurate method of detecting methicillin resistance among CoNS.
The Use of Duplication-Generating Rearrangements for Studying Heterokaryon Incompatibility Genes in Neurospora

PubMed Central

Perkins, David D.

1975-01-01

Heterokaryon (vegetative) incompatibility, governing the fusion of somatic hyphal filaments to form stable heterokaryons, is of interest because of its widespread occurrence in fungi and its bearing on cellular recognition. Conventional investigations of the genetic basis of heterokaryon incompatibility in N. crassa are difficult because in commonly used stocks differences are present at several het loci, all with similar incompatibility phenotypes. This difficulty is overcome by using duplications (partial diploids) that are unlikely to contain more than one het locus. A phenotypically expressed incompatibility reaction occurs when unlike het alleles are present within the same somatic nucleus, and this parallels the heterokaryon incompatibility reaction that occurs when unlike alleles in different haploid nuclei are introduced into the same somatic hypha by mycelial fusion.—Nontandem duplications were used to confirm that the incompatibility reactions in heterokaryons and in duplications are alternate expressions of the same genes. This was demonstrated for three loci which had previously been established by conventional heterokaryon tests—het-e, het-c and mt. These were each obtained in duplications as recombinant meiotic segregants from crosses heterozygous for duplication-generating chromosome rearrangements. The particular method of producing the duplications is irrelevant so long as the incompatibility alleles are heterozygous.—The duplication technique has made it possible to determine easily the het-e and het-c genotypes of numerous laboratory and wild strains of unknown constitution. In laboratory strains both loci are represented simply by two alleles. Analysis of het-c is more complicated in some wild strains, where differences have been demonstrated at one or more additional het loci within the duplication used and multiple allelism is also possible.—The results show that the duplication method can be used to identify and map additional vegetative incompatibility loci, without the necessity of heterokaryon tests. PMID:124288
Individuality, phenotypic differentiation, dormancy and ‘persistence’ in culturable bacterial systems: commonalities shared by environmental, laboratory, and clinical microbiology

PubMed Central

Kell, Douglas; Potgieter, Marnie; Pretorius, Etheresia

2015-01-01

For bacteria, replication mainly involves growth by binary fission. However, in a very great many natural environments there are examples of phenotypically dormant, non-growing cells that do not replicate immediately and that are phenotypically ‘nonculturable’ on media that normally admit their growth. They thereby evade detection by conventional culture-based methods. Such dormant cells may also be observed in laboratory cultures and in clinical microbiology. They are usually more tolerant to stresses such as antibiotics, and in clinical microbiology they are typically referred to as ‘persisters’. Bacterial cultures necessarily share a great deal of relatedness, and inclusive fitness theory implies that there are conceptual evolutionary advantages in trading a variation in growth rate against its mean, equivalent to hedging one’s bets. There is much evidence that bacteria exploit this strategy widely. We here bring together data that show the commonality of these phenomena across environmental, laboratory and clinical microbiology. Considerable evidence, using methods similar to those common in environmental microbiology, now suggests that many supposedly non-communicable, chronic and inflammatory diseases are exacerbated (if not indeed largely caused) by the presence of dormant or persistent bacteria (the ability of whose components to cause inflammation is well known). This dormancy (and resuscitation therefrom) often reflects the extent of the availability of free iron. Together, these phenomena can provide a ready explanation for the continuing inflammation common to such chronic diseases and its correlation with iron dysregulation. This implies that measures designed to assess and to inhibit or remove such organisms (or their access to iron) might be of much therapeutic benefit. PMID:26629334
Characterization of bacterial coliform occurrences in different zones of a drinking water distribution system.

PubMed

Blanch, A R; Galofré, B; Lucena, F; Terradillos, A; Vilanova, X; Ribas, F

2007-03-01

To compare the bacterial coliforms detected from occurrences in three zones of a water distribution system supplied by two separate water sources. Conventional and standardized protocols for identifying enterobacterial populations were applied. Additional tests to confirm isolates were included. Analyses of diversity and population similarity were performed using the Phene Plate System, a miniaturized biochemical phenotyping method. Isolates were identified by the API 20E system in tandem with biochemical phenotyping. A total of 16 576 samples were taken from the water distribution system, with 1416 isolates analysed. A low number of coliform occurrences were observed (2%). Escherichia coli was not detected in either water origin or in Zone 2 samples; however, in Zones 1 and 3 a low number of cases of E. coli were recorded. The percentages of E. coli depended on the identification criteria. Eight biochemical profiles for coliform populations were defined according to the results of the confirmative tests. There was a high diversity among these populations in the three zones studied, although no significant variations in their composition (associated with occurrences in the different zones) were observed. Klebsiella oxytoca was the most commonly detected species irrespective of zone, although seven other enterobacterial genera were also found. Analysis of the enzymatic activity of beta-glucuronidase or application of the criteria established in the norm ISO 9308-1, in tandem with thermotolerance was needed to evaluate the occurrence of E. coli in the distribution systems. Detected occurrences of bacterial coliforms could be associated with re-growth patterns for specific sampling points in the distribution system. Seasonal differences, independent of the studied zones, were observed. Biochemical phenotyping of bacterial coliforms was shown to be a useful method on the characterization of occurrences in water distribution systems.
Causal inference in biology networks with integrated belief propagation.

PubMed

Chang, Rui; Karr, Jonathan R; Schadt, Eric E

2015-01-01

Inferring causal relationships among molecular and higher order phenotypes is a critical step in elucidating the complexity of living systems. Here we propose a novel method for inferring causality that is no longer constrained by the conditional dependency arguments that limit the ability of statistical causal inference methods to resolve causal relationships within sets of graphical models that are Markov equivalent. Our method utilizes Bayesian belief propagation to infer the responses of perturbation events on molecular traits given a hypothesized graph structure. A distance measure between the inferred response distribution and the observed data is defined to assess the 'fitness' of the hypothesized causal relationships. To test our algorithm, we infer causal relationships within equivalence classes of gene networks in which the form of the functional interactions that are possible are assumed to be nonlinear, given synthetic microarray and RNA sequencing data. We also apply our method to infer causality in real metabolic network with v-structure and feedback loop. We show that our method can recapitulate the causal structure and recover the feedback loop only from steady-state data which conventional method cannot.
Molecular cytogenetics characterization of seven small supernumerary marker chromosomes derived from chromosome 19: Genotype-phenotype correlation and review of the literature.

PubMed

Recalcati, Maria Paola; Bonati, Maria Teresa; Beltrami, Nicola; Cardarelli, Laura; Catusi, Ilaria; Costa, Asia; Garzo, Maria; Mammi, Isabella; Mattina, Teresa; Nalesso, Elisa; Nardone, Anna Maria; Postorivo, Diana; Sajeva, Anna; Varricchio, Aminta; Verri, Annapia; Villa, Nicoletta; Larizza, Lidia; Giardino, Daniela

2018-03-01

Only a few subjects carrying supernumerary marker chromosomes derived from 19 chromosome (sSMC(19)) have been described to date and for a small portion of them the genic content has been defined at the molecular level. We present seven new different sSMCs(19) identified in eight individuals, seven of whom unrelated. The presence of the sSMC is associated with a clinical phenotype in five subjects, while the other three carriers, two of whom related, are normal. All sSMCs(19) have been characterized by means of conventional and molecular cytogenetics. We compare the sSMCs(19) carriers with a clinical phenotype to already described patients with gains (sSMCs or microduplications) of overlapping genomic regions with the aim to deepen the pathogenicity of the encountered imbalances and to assess the role of the involved genes on the phenotype. The present work supports the correlation between the gain of some chromosome 19 critical regions and specific phenotypes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
Recognition of unusual presentation of natural killer cell leukemia.

PubMed

Gardiner, C M; Reen, D J; O'Meara, A

1995-10-01

Expansion of the natural killer (NK) subset of lymphocytes represents a rare leukemia phenotype with variations in clinical presentation, morphology, surface phenotype, and effector function. This paper reports on a 5-year-old male patient who had an unusual presentation of an NK cell leukemia that was initially diagnosed as neuroblastoma. A bone marrow (BM) aspirate showed clumps of undifferentiated cells with the following phenotype: CD56bright+, CD33dim+, CD45-, CD2-, CD19-, CD16-, and CD57-. Cytochemistry was noncontributory. The patient, having failed to respond to conventional neuroblastoma chemotherapy, was subsequently diagnosed as having NK cell leukemia based on functional in vitro assays. The patient responded to acute lymphoblastic leukemia (ALL) chemotherapy but relapsed 4 weeks into treatment and eventually died 25 weeks after initial presentation. The cell surface phenotype observed is consistent with a rare NK cell subset, the biology of which has not been well defined. Freshly isolated BM cells killed K562 cells in a conventional 51Cr-release assay. Both interleukin-2 (IL-2) and interferon-alpha (IFN-alpha) induced LAK activity against the Daudi cell line. IL-2 induced proliferation of the leukemic cells. TNF-alpha, IFN-gamma, IL-6, IL-1ra, and TGF-beta levels were assessed and found to be concentrated in BM, in contrast to plasma samples. TNF-alpha was present at a high concentration in BM (150.9 pg/ml), probably a reflection of the associated disease pathology of severe bone pain and pyrexia. In summary, this paper details clinical and laboratory investigations of a leukemia of a rare NK cell subset.
Usefulness of multiple chalk-based food colorings for inducing better gene silencing by feeding RNA interference in planarians.

PubMed

Hattori, Miki; Miyamoto, Mai; Hosoda, Kazutaka; Umesono, Yoshihiko

2018-01-01

Planarians have become widely recognized as one of the major animal models for regeneration studies in invertebrates. To induce RNA interference (RNAi) by feeding in planarians, the widely accepted protocol is one in which animals undergo two or three feedings of food containing double-stranded RNA (dsRNA) plus visible food coloring (e.g., blood) for confirmation of feeding by individual animals. However, one possible problem is that incorporated food coloring is often retained within the gut for several days, which makes it difficult to confirm the success of each round of dsRNA feeding based on the difference of the color density within the gut before and after feeding. As a consequence, the difference of appetite levels among individuals undergoing dsRNA feeding leads to phenotypic variability among them due to insufficient knockdown. In our attempts to overcome this problem, we have developed a novel method for achieving robust confirmation of the success of dsRNA feeding in individuals fed multiple times by means of including a combination of three different colored chalks (pink, yellow and blue) as food coloring. Notably, we found that this method is superior to the conventional method for positively marking individuals that actively consumed the dsRNA-containing food during four times of once-daily feeding. Using these selected animals, we obtained stable and sufficiently strong RNAi-induced phenotypes. We termed this improved multi-colored chalk-spiked method of feeding RNAi "Candi" and propose its benefits for gene function analysis in planarians. © 2017 Japanese Society of Developmental Biologists.
Quantification and clustering of phenotypic screening data using time-series analysis for chemotherapy of schistosomiasis

PubMed Central

2012-01-01

Background Neglected tropical diseases, especially those caused by helminths, constitute some of the most common infections of the world's poorest people. Development of techniques for automated, high-throughput drug screening against these diseases, especially in whole-organism settings, constitutes one of the great challenges of modern drug discovery. Method We present a method for enabling high-throughput phenotypic drug screening against diseases caused by helminths with a focus on schistosomiasis. The proposed method allows for a quantitative analysis of the systemic impact of a drug molecule on the pathogen as exhibited by the complex continuum of its phenotypic responses. This method consists of two key parts: first, biological image analysis is employed to automatically monitor and quantify shape-, appearance-, and motion-based phenotypes of the parasites. Next, we represent these phenotypes as time-series and show how to compare, cluster, and quantitatively reason about them using techniques of time-series analysis. Results We present results on a number of algorithmic issues pertinent to the time-series representation of phenotypes. These include results on appropriate representation of phenotypic time-series, analysis of different time-series similarity measures for comparing phenotypic responses over time, and techniques for clustering such responses by similarity. Finally, we show how these algorithmic techniques can be used for quantifying the complex continuum of phenotypic responses of parasites. An important corollary is the ability of our method to recognize and rigorously group parasites based on the variability of their phenotypic response to different drugs. Conclusions The methods and results presented in this paper enable automatic and quantitative scoring of high-throughput phenotypic screens focused on helmintic diseases. Furthermore, these methods allow us to analyze and stratify parasites based on their phenotypic response to drugs. Together, these advancements represent a significant breakthrough for the process of drug discovery against schistosomiasis in particular and can be extended to other helmintic diseases which together afflict a large part of humankind. PMID:22369037
Genetic modification of Arachis hypogaea for quality traits

USDA-ARS?s Scientific Manuscript database

TILLING, targeting induced local lesions in genomes, combines conventional mutagenesis with targeted screening of known genes. Advantages are that a series of alleles can be recovered to assist with functional analysis, and mutations can be identified in polyploids where a phenotype is likely to be...
Evidence for mesenchymal-like sub-populations within squamous cell carcinomas possessing chemoresistance and phenotypic plasticity.

PubMed

Basu, D; Nguyen, T-T K; Montone, K T; Zhang, G; Wang, L-P; Diehl, J A; Rustgi, A K; Lee, J T; Weinstein, G S; Herlyn, M

2010-07-22

Variable drug responses among malignant cells within individual tumors may represent a barrier to their eradication using chemotherapy. Carcinoma cells expressing mesenchymal markers resist conventional and epidermal growth factor receptor (EGFR)-targeted chemotherapy. In this study, we evaluated whether mesenchymal-like sub-populations within human squamous cell carcinomas (SCCs) with predominantly epithelial features contribute to overall therapy resistance. We identified a mesenchymal-like subset expressing low E-cadherin (Ecad-lo) and high vimentin within the upper aerodigestive tract SCCs. This subset was both isolated from the cell lines and was identified in xenografts and primary clinical specimens. The Ecad-lo subset contained more low-turnover cells, correlating with resistance to the conventional chemotherapeutic paclitaxel in vitro. Epidermal growth factor induced less stimulation of the mitogen-activated protein kinase and phosphatidylinositol-3-kinase pathways in Ecad-lo cells, which was likely due to lower EGFR expression in this subset and correlated with in vivo resistance to the EGFR-targeted antibody, cetuximab. The Ecad-lo and high E-cadherin subsets were dynamic in phenotype, showing the capacity to repopulate each other from single-cell clones. Taken together, these results provide evidence for a low-turnover, mesenchymal-like sub-population in SCCs with diminished EGFR pathway function and intrinsic resistance to conventional and EGFR-targeted chemotherapies.
[Applications of MALDI-TOF-MS in clinical microbiology laboratory].

PubMed

Carbonnelle, Etienne; Nassif, Xavier

2011-10-01

For twenty years, mass spectrometry (MS) has emerged as a particularly powerful tool for analysis and characterization of proteins in research. It is only recently that this technology, especially MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization Time-Of-Flight) has entered the field of routine microbiology. This method has proven to be reliable and safe for the identification of bacteria, yeasts, filamentous fungi and dermatophytes. MALDI-TOF-MS is a rapid, precise and cost-effective method for identification, compared to conventional phenotypic techniques or molecular biology. Its ability to analyse whole microorganisms with few sample preparation has greatly reduced the time to identification (1-2 min). Furthermore, this technology can be used to identify bacteria directly from clinical samples as blood culture bottles or urines. Future applications will be developed in order to provide direct information concerning virulence or resistance protein markers. © 2011 médecine/sciences – Inserm / SRMS.

Identification of Candida lusitaniae as an opportunistic yeast in humans.

PubMed Central

Holzschu, D L; Presley, H L; Miranda, M; Phaff, H J

1979-01-01

Four yeast strains, causally associated with infection in a patient with acute myelogenous leukemia, were identified by standard methods currently used in yeast taxonomy as representatives of Candida lusitania van Uden et do Carmo-Sousa. Because this species has not been recognized previously as an opportunistic yeast in humans, molecular taxonomic methods were applied to confirm its identity. The nuclear deoxyribonucleic acid (DNA) base composition of two clinical isolates was shown to be 45.1 mol% guanine plus cytosine as compared to 44.7 mol% guanine plus cytosine for the type strain of this species. DNA/DNA reassociation experiments revealed more than 95% complementarity between the DNAs from the clinical isolates and that of the type strain of C. lusitaniae, thus confirming their classification by conventional taxonomy. A key is provided to differentiate C. lusitaniae from two phenotypically similar Candida species. PMID:292646
Positive health effects of the natural outdoor environment in typical populations in different regions in Europe (PHENOTYPE): a study programme protocol.

PubMed

Nieuwenhuijsen, Mark J; Kruize, Hanneke; Gidlow, Christopher; Andrusaityte, Sandra; Antó, Josep Maria; Basagaña, Xavier; Cirach, Marta; Dadvand, Payam; Danileviciute, Asta; Donaire-Gonzalez, David; Garcia, Judith; Jerrett, Michael; Jones, Marc; Julvez, Jordi; van Kempen, Elise; van Kamp, Irene; Maas, Jolanda; Seto, Edmund; Smith, Graham; Triguero, Margarita; Wendel-Vos, Wanda; Wright, John; Zufferey, Joris; van den Hazel, Peter Jan; Lawrence, Roderick; Grazuleviciene, Regina

2014-04-16

Growing evidence suggests that close contact with nature brings benefits to human health and well-being, but the proposed mechanisms are still not well understood and the associations with health remain uncertain. The Positive Health Effects of the Natural Outdoor environment in Typical Populations in different regions in Europe (PHENOTYPE) project investigates the interconnections between natural outdoor environments and better human health and well-being. The PHENOTYPE project explores the proposed underlying mechanisms at work (stress reduction/restorative function, physical activity, social interaction, exposure to environmental hazards) and examines the associations with health outcomes for different population groups. It implements conventional and new innovative high-tech methods to characterise the natural environment in terms of quality and quantity. Preventive as well as therapeutic effects of contact with the natural environment are being covered. PHENOTYPE further addresses implications for land-use planning and green space management. The main innovative part of the study is the evaluation of possible short-term and long-term associations of green space and health and the possible underlying mechanisms in four different countries (each with quite a different type of green space and a different use), using the same methodology, in one research programme. This type of holistic approach has not been undertaken before. Furthermore there are technological innovations such as the use of remote sensing and smartphones in the assessment of green space. The project will produce a more robust evidence base on links between exposure to natural outdoor environment and human health and well-being, in addition to a better integration of human health needs into land-use planning and green space management in rural as well as urban areas.
Direct identification of pathogens from positive blood cultures using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

PubMed

Rodríguez-Sánchez, B; Sánchez-Carrillo, C; Ruiz, A; Marín, M; Cercenado, E; Rodríguez-Créixems, M; Bouza, E

2014-07-01

In recent years, matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has proved a rapid and reliable method for the identification of bacteria and yeasts that have already been isolated. The objective of this study was to evaluate this technology as a routine method for the identification of microorganisms directly from blood culture bottles (BCBs), before isolation, in a large collection of samples. For this purpose, 1000 positive BCBs containing 1085 microorganisms have been analysed by conventional phenotypic methods and by MALDI-TOF MS. Discrepancies have been resolved using molecular methods: the amplification and sequencing of the 16S rRNA gene or the Superoxide Dismutase gene (sodA) for streptococcal isolates. MALDI-TOF predicted a species- or genus-level identification of 81.4% of the analysed microorganisms. The analysis by episode yielded a complete identification of 814 out of 1000 analysed episodes (81.4%). MALDI-TOF identification is available for clinicians within hours of a working shift, as oppose to 18 h later when conventional identification methods are performed. Moreover, although further improvement of sample preparation for polymicrobial BCBs is required, the identification of more than one pathogen in the same BCB provides a valuable indication of unexpected pathogens when their presence may remain undetected in Gram staining. Implementation of MALDI-TOF identification directly from the BCB provides a rapid and reliable identification of the causal pathogen within hours. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.
MALDI-TOF-mass spectrometry applications in clinical microbiology.

PubMed

Seng, Piseth; Rolain, Jean-Marc; Fournier, Pierre Edouard; La Scola, Bernard; Drancourt, Michel; Raoult, Didier

2010-11-01

MALDI-TOF-mass spectrometry (MS) has been successfully adapted for the routine identification of microorganisms in clinical microbiology laboratories in the past 10 years. This revolutionary technique allows for easier and faster diagnosis of human pathogens than conventional phenotypic and molecular identification methods, with unquestionable reliability and cost-effectiveness. This article will review the application of MALDI-TOF-MS tools in routine clinical diagnosis, including the identification of bacteria at the species, subspecies, strain and lineage levels, and the identification of bacterial toxins and antibiotic-resistance type. We will also discuss the application of MALDI-TOF-MS tools in the identification of Archaea, eukaryotes and viruses. Pathogenic identification from colony-cultured, blood-cultured, urine and environmental samples is also reviewed.
Application of MALDI-TOF MS for the Identification of Food Borne Bacteria

PubMed Central

Pavlovic, Melanie; Huber, Ingrid; Konrad, Regina; Busch, Ulrich

2013-01-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful tool for the routine identification of clinical isolates. MALDI-TOF MS based identification of bacteria has been shown to be more rapid, accurate and cost-efficient than conventional phenotypic techniques or molecular methods. Rapid and reliable identification of food-associated bacteria is also of crucial importance for food processing and product quality. This review is concerned with the applicability of MALDI-TOF MS for routine identification of foodborne bacteria taking the specific requirements of food microbiological laboratories and the food industry into account. The current state of knowledge including recent findings and new approaches are discussed. PMID:24358065
Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

PubMed

Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

2015-04-01

Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Genetic fingerprinting of potato varieties from the Northwest Potato Variety Development Program

USDA-ARS?s Scientific Manuscript database

The Northwest Potato Variety Development Program using conventional breeding has successfully released more than 40 improved varieties of potato since its inception in 1983. Potato breeders rely primarily on morphological and phenotypic data for selection and breeding of potato cultivars. With the a...
Integrated rare variant-based risk gene prioritization in disease case-control sequencing studies.

PubMed

Lin, Jhih-Rong; Zhang, Quanwei; Cai, Ying; Morrow, Bernice E; Zhang, Zhengdong D

2017-12-01

Rare variants of major effect play an important role in human complex diseases and can be discovered by sequencing-based genome-wide association studies. Here, we introduce an integrated approach that combines the rare variant association test with gene network and phenotype information to identify risk genes implicated by rare variants for human complex diseases. Our data integration method follows a 'discovery-driven' strategy without relying on prior knowledge about the disease and thus maintains the unbiased character of genome-wide association studies. Simulations reveal that our method can outperform a widely-used rare variant association test method by 2 to 3 times. In a case study of a small disease cohort, we uncovered putative risk genes and the corresponding rare variants that may act as genetic modifiers of congenital heart disease in 22q11.2 deletion syndrome patients. These variants were missed by a conventional approach that relied on the rare variant association test alone.
Quantification and clustering of phenotypic screening data using time-series analysis for chemotherapy of schistosomiasis.

PubMed

Lee, Hyokyeong; Moody-Davis, Asher; Saha, Utsab; Suzuki, Brian M; Asarnow, Daniel; Chen, Steven; Arkin, Michelle; Caffrey, Conor R; Singh, Rahul

2012-01-01

Neglected tropical diseases, especially those caused by helminths, constitute some of the most common infections of the world's poorest people. Development of techniques for automated, high-throughput drug screening against these diseases, especially in whole-organism settings, constitutes one of the great challenges of modern drug discovery. We present a method for enabling high-throughput phenotypic drug screening against diseases caused by helminths with a focus on schistosomiasis. The proposed method allows for a quantitative analysis of the systemic impact of a drug molecule on the pathogen as exhibited by the complex continuum of its phenotypic responses. This method consists of two key parts: first, biological image analysis is employed to automatically monitor and quantify shape-, appearance-, and motion-based phenotypes of the parasites. Next, we represent these phenotypes as time-series and show how to compare, cluster, and quantitatively reason about them using techniques of time-series analysis. We present results on a number of algorithmic issues pertinent to the time-series representation of phenotypes. These include results on appropriate representation of phenotypic time-series, analysis of different time-series similarity measures for comparing phenotypic responses over time, and techniques for clustering such responses by similarity. Finally, we show how these algorithmic techniques can be used for quantifying the complex continuum of phenotypic responses of parasites. An important corollary is the ability of our method to recognize and rigorously group parasites based on the variability of their phenotypic response to different drugs. The methods and results presented in this paper enable automatic and quantitative scoring of high-throughput phenotypic screens focused on helmintic diseases. Furthermore, these methods allow us to analyze and stratify parasites based on their phenotypic response to drugs. Together, these advancements represent a significant breakthrough for the process of drug discovery against schistosomiasis in particular and can be extended to other helmintic diseases which together afflict a large part of humankind.
Assessing the Efficiency of Phenotyping Early Traits in a Greenhouse Automated Platform for Predicting Drought Tolerance of Soybean in the Field.

PubMed

Peirone, Laura S; Pereyra Irujo, Gustavo A; Bolton, Alejandro; Erreguerena, Ignacio; Aguirrezábal, Luis A N

2018-01-01

Conventional field phenotyping for drought tolerance, the most important factor limiting yield at a global scale, is labor-intensive and time-consuming. Automated greenhouse platforms can increase the precision and throughput of plant phenotyping and contribute to a faster release of drought tolerant varieties. The aim of this work was to establish a framework of analysis to identify early traits which could be efficiently measured in a greenhouse automated phenotyping platform, for predicting the drought tolerance of field grown soybean genotypes. A group of genotypes was evaluated, which showed variation in their drought susceptibility index (DSI) for final biomass and leaf area. A large number of traits were measured before and after the onset of a water deficit treatment, which were analyzed under several criteria: the significance of the regression with the DSI, phenotyping cost, earliness, and repeatability. The most efficient trait was found to be transpiration efficiency measured at 13 days after emergence. This trait was further tested in a second experiment with different water deficit intensities, and validated using a different set of genotypes against field data from a trial network in a third experiment. The framework applied in this work for assessing traits under different criteria could be helpful for selecting those most efficient for automated phenotyping.
An efficient genome-wide association test for multivariate phenotypes based on the Fisher combination function.

PubMed

Yang, James J; Li, Jia; Williams, L Keoki; Buu, Anne

2016-01-05

In genome-wide association studies (GWAS) for complex diseases, the association between a SNP and each phenotype is usually weak. Combining multiple related phenotypic traits can increase the power of gene search and thus is a practically important area that requires methodology work. This study provides a comprehensive review of existing methods for conducting GWAS on complex diseases with multiple phenotypes including the multivariate analysis of variance (MANOVA), the principal component analysis (PCA), the generalizing estimating equations (GEE), the trait-based association test involving the extended Simes procedure (TATES), and the classical Fisher combination test. We propose a new method that relaxes the unrealistic independence assumption of the classical Fisher combination test and is computationally efficient. To demonstrate applications of the proposed method, we also present the results of statistical analysis on the Study of Addiction: Genetics and Environment (SAGE) data. Our simulation study shows that the proposed method has higher power than existing methods while controlling for the type I error rate. The GEE and the classical Fisher combination test, on the other hand, do not control the type I error rate and thus are not recommended. In general, the power of the competing methods decreases as the correlation between phenotypes increases. All the methods tend to have lower power when the multivariate phenotypes come from long tailed distributions. The real data analysis also demonstrates that the proposed method allows us to compare the marginal results with the multivariate results and specify which SNPs are specific to a particular phenotype or contribute to the common construct. The proposed method outperforms existing methods in most settings and also has great applications in GWAS on complex diseases with multiple phenotypes such as the substance abuse disorders.
Generation of computationally predicted Adverse Outcome Pathway networks through integration of publicly available in vivo, in vitro, phenotype, and biological pathway data.

EPA Science Inventory

The Adverse Outcome Pathway (AOP) framework is becoming a widely used tool for organizing and summarizing the mechanistic information connecting molecular perturbations by environmental stressors with adverse ecological and human health outcomes. However, the conventional process...
Recent advances in phytoplasma research: from genetic diversity and genome evolution to pathogenic redirection of plant stem cell fate

USDA-ARS?s Scientific Manuscript database

Parasitizing phloem sieve cells and being transmitted by insects, phytoplasmas are a unique group of cell wall-less bacteria responsible for numerous plant diseases worldwide. Due to difficulties in establishing axenic culture of phytoplasmas, phenotypic characters suitable for conventional microbia...
SEE locomotor behavior test discriminates C57BL/6J and DBA/2J mouse inbred strains across laboratories and protocol conditions.

PubMed

Kafkafi, Neri; Lipkind, Dina; Benjamini, Yoav; Mayo, Cheryl L; Elmer, Gregory I; Golani, Ilan

2003-06-01

Conventional tests of behavioral phenotyping frequently have difficulties differentiating certain genotypes and replicating these differences across laboratories and protocol conditions. This study explores the hypothesis that automated tests can be designed to quantify ethologically relevant behavior patterns that more readily characterize heritable and replicable phenotypes. It used SEE (Strategy for the Exploration of Exploration) to phenotype the locomotor behavior of the C57BL/6 and DBA/2 mouse inbred strains across 3 laboratories. The 2 genotypes differed in 15 different measures of behavior, none of which had a significant genotype-laboratory interaction. Within the same laboratory, most of these differences were replicated in additional experiments despite the test photoperiod phase being changed and saline being injected. Results suggest that well-designed tests may considerably enhance replicability across laboratories.
Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory.

PubMed

Jia, Dongya; Jolly, Mohit Kumar; Kulkarni, Prakash; Levine, Herbert

2017-06-22

Waddington's epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of "landscape" in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an "attractor" that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of "cancer attractors"-hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes "recanalization", i.e., the exit from "cancer attractors" and re-entry into "normal attractors", is more likely to succeed rather than a conventional approach that targets individual molecules/pathways.
In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timraz, Sara B.H., E-mail: sara.timraz@kustar.ac.ae; Farhat, Ilyas A.H., E-mail: ilyas.farhat@outlook.com; Alhussein, Ghada, E-mail: ghada.alhussein@kustar.ac.ae

In vitro research on vascular tissue engineering has extensively used isolated primary human or animal smooth muscle cells (SMC). Research programs that lack such facilities tend towards commercially available primary cells sources. Here, we aim to evaluate the capacity of commercially available human SMC to maintain their contractile phenotype, and determine if dedifferentiation towards the synthetic phenotype occurs in response to conventional cell culture and passaging without any external biochemical or mechanical stimuli. Lower passage SMC adopted a contractile phenotype marked by a relatively slower proliferation rate, higher expression of proteins of the contractile apparatus and smoothelin, elongated morphology, andmore » reduced deposition of collagen types I and III. As the passage number increased, migratory capacity was enhanced, average cell speed, total distance and net distance travelled increased up to passage 8. Through the various assays, corroborative evidence pinpoints SMC at passage 7 as the transition point between the contractile and synthetic phenotypes, while passage 8 distinctly and consistently exhibited characteristics of synthetic phenotype. This knowledge is particularly useful in selecting SMC of appropriate passage number for the target vascular tissue engineering application, for example, a homeostatic vascular graft for blood vessel replacement versus recreating atherosclerotic blood vessel model in vitro. - Highlights: • Ability of human smooth muscle cells to alter phenotype in culture is evaluated. • Examined the effect of passaging human smooth muscle cells on phenotype. • Phenotype is assessed based on morphology, proliferation, markers, and migration. • Multi-resolution assessment methodology, single-cell and cell-population. • Lower and higher passages than P7 adopted a contractile and synthetic phenotype respectively.« less
Physiologically based pharmacokinetic model for 6-mercpatopurine: exploring the role of genetic polymorphism in TPMT enzyme activity

PubMed Central

Ogungbenro, Kayode; Aarons, Leon

2015-01-01

Aims To extend the physiologically based pharmacokinetic (PBPK) model developed for 6-mercaptopurine to account for intracellular metabolism and to explore the role of genetic polymorphism in the TPMT enzyme on the pharmacokinetics of 6-mercaptopurine. Methods The developed PBPK model was extended for 6-mercaptopurine to account for intracellular metabolism and genetic polymorphism in TPMT activity. System and drug specific parameters were obtained from the literature or estimated using plasma or intracellular red blood cell concentrations of 6-mercaptopurine and its metabolites. Age-dependent changes in parameters were implemented for scaling, and variability was also introduced for simulation. The model was validated using published data. Results The model was extended successfully. Parameter estimation and model predictions were satisfactory. Prediction of intracellular red blood cell concentrations of 6-thioguanine nucleotide for different TPMT phenotypes (in a clinical study that compared conventional and individualized dosing) showed results that were consistent with observed values and reported incidence of haematopoietic toxicity. Following conventional dosing, the predicted mean concentrations for homozygous and heterozygous variants, respectively, were about 10 times and two times the levels for wild-type. However, following individualized dosing, the mean concentration was around the same level for the three phenotypes despite different doses. Conclusions The developed PBPK model has been extended for 6-mercaptopurine and can be used to predict plasma 6-mercaptopurine and tissue concentration of 6-mercaptopurine, 6-thioguanine nucleotide and 6-methylmercaptopurine ribonucleotide in adults and children. Predictions of reported data from clinical studies showed satisfactory results. The model may help to improve 6-mercaptopurine dosing, achieve better clinical outcome and reduce toxicity. PMID:25614061
‘N-of-1-pathways’ unveils personal deregulated mechanisms from a single pair of RNA-Seq samples: towards precision medicine

PubMed Central

Gardeux, Vincent; Achour, Ikbel; Li, Jianrong; Maienschein-Cline, Mark; Li, Haiquan; Pesce, Lorenzo; Parinandi, Gurunadh; Bahroos, Neil; Winn, Robert; Foster, Ian; Garcia, Joe G N; Lussier, Yves A

2014-01-01

Background The emergence of precision medicine allowed the incorporation of individual molecular data into patient care. Indeed, DNA sequencing predicts somatic mutations in individual patients. However, these genetic features overlook dynamic epigenetic and phenotypic response to therapy. Meanwhile, accurate personal transcriptome interpretation remains an unmet challenge. Further, N-of-1 (single-subject) efficacy trials are increasingly pursued, but are underpowered for molecular marker discovery. Method ‘N-of-1-pathways’ is a global framework relying on three principles: (i) the statistical universe is a single patient; (ii) significance is derived from geneset/biomodules powered by paired samples from the same patient; and (iii) similarity between genesets/biomodules assesses commonality and differences, within-study and cross-studies. Thus, patient gene-level profiles are transformed into deregulated pathways. From RNA-Seq of 55 lung adenocarcinoma patients, N-of-1-pathways predicts the deregulated pathways of each patient. Results Cross-patient N-of-1-pathways obtains comparable results with conventional genesets enrichment analysis (GSEA) and differentially expressed gene (DEG) enrichment, validated in three external evaluations. Moreover, heatmap and star plots highlight both individual and shared mechanisms ranging from molecular to organ-systems levels (eg, DNA repair, signaling, immune response). Patients were ranked based on the similarity of their deregulated mechanisms to those of an independent gold standard, generating unsupervised clusters of diametric extreme survival phenotypes (p=0.03). Conclusions The N-of-1-pathways framework provides a robust statistical and relevant biological interpretation of individual disease-free survival that is often overlooked in conventional cross-patient studies. It enables mechanism-level classifiers with smaller cohorts as well as N-of-1 studies. Software http://lussierlab.org/publications/N-of-1-pathways PMID:25301808
‘N-of-1- pathways ’ unveils personal deregulated mechanisms from a single pair of RNA-Seq samples: Towards precision medicine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardeux, Vincent; Achour, Ikbel; Li, Jianrong

Background: The emergence of precision medicine allowed the incorporation of individual molecular data into patient care. This research entails, DNA sequencing predicts somatic mutations in individual patients. However, these genetic features overlook dynamic epigenetic and phenotypic response to therapy. Meanwhile, accurate personal transcriptome interpretation remains an unmet challenge. Further, N-of-1 (single-subject) efficacy trials are increasingly pursued, but are underpowered for molecular marker discovery. Method: ‘N-of-1- pathways’ is a global framework relying on three principles: (i) the statistical universe is a single patient; (ii) significance is derived from geneset/biomodules powered by paired samples from the same patient; and (iii) similarity betweenmore » genesets/biomodules assesses commonality and differences, within-study and cross-studies. Thus, patient gene-level profiles are transformed into deregulated pathways. From RNA-Seq of 55 lung adenocarcinoma patients, N-of-1- pathways predicts the deregulated pathways of each patient. Results: Cross-patient N-of-1- pathways obtains comparable results with conventional genesets enrichment analysis (GSEA) and differentially expressed gene (DEG) enrichment, validated in three external evaluations. Moreover, heatmap and star plots highlight both individual and shared mechanisms ranging from molecular to organ-systems levels (eg, DNA repair, signaling, immune response). Patients were ranked based on the similarity of their deregulated mechanisms to those of an independent gold standard, generating unsupervised clusters of diametric extreme survival phenotypes (p=0.03). Conclusions: The N-of-1- pathways framework provides a robust statistical and relevant biological interpretation of individual disease-free survival that is often overlooked in conventional cross-patient studies. It enables mechanism-level classifiers with smaller cohorts as well as N-of-1 studies.« less
‘N-of-1- pathways ’ unveils personal deregulated mechanisms from a single pair of RNA-Seq samples: Towards precision medicine

DOE PAGES

Gardeux, Vincent; Achour, Ikbel; Li, Jianrong; ...

2014-11-01

Background: The emergence of precision medicine allowed the incorporation of individual molecular data into patient care. This research entails, DNA sequencing predicts somatic mutations in individual patients. However, these genetic features overlook dynamic epigenetic and phenotypic response to therapy. Meanwhile, accurate personal transcriptome interpretation remains an unmet challenge. Further, N-of-1 (single-subject) efficacy trials are increasingly pursued, but are underpowered for molecular marker discovery. Method: ‘N-of-1- pathways’ is a global framework relying on three principles: (i) the statistical universe is a single patient; (ii) significance is derived from geneset/biomodules powered by paired samples from the same patient; and (iii) similarity betweenmore » genesets/biomodules assesses commonality and differences, within-study and cross-studies. Thus, patient gene-level profiles are transformed into deregulated pathways. From RNA-Seq of 55 lung adenocarcinoma patients, N-of-1- pathways predicts the deregulated pathways of each patient. Results: Cross-patient N-of-1- pathways obtains comparable results with conventional genesets enrichment analysis (GSEA) and differentially expressed gene (DEG) enrichment, validated in three external evaluations. Moreover, heatmap and star plots highlight both individual and shared mechanisms ranging from molecular to organ-systems levels (eg, DNA repair, signaling, immune response). Patients were ranked based on the similarity of their deregulated mechanisms to those of an independent gold standard, generating unsupervised clusters of diametric extreme survival phenotypes (p=0.03). Conclusions: The N-of-1- pathways framework provides a robust statistical and relevant biological interpretation of individual disease-free survival that is often overlooked in conventional cross-patient studies. It enables mechanism-level classifiers with smaller cohorts as well as N-of-1 studies.« less

Comparative analysis of Edwardsiella isolates from fish in the eastern United States identifies two distinct genetic taxa amongst organisms phenotypically classified as E. tarda

USGS Publications Warehouse

Griffin, Matt J.; Quiniou, Sylvie M.; Cody, Theresa; Tabuchi, Maki; Ware, Cynthia; Cipriano, Rocco C.; Mauel, Michael J.; Soto, Esteban

2013-01-01

Edwardsiella tarda, a Gram-negative member of the family Enterobacteriaceae, has been implicated in significant losses in aquaculture facilities worldwide. Here, we assessed the intra-specific variability of E. tarda isolates from 4 different fish species in the eastern United States. Repetitive sequence mediated PCR (rep-PCR) using 4 different primer sets (ERIC I & II, ERIC II, BOX, and GTG5) and multi-locus sequence analysis of 16S SSU rDNA, groEl, gyrA, gyrB, pho, pgi, pgm, and rpoA gene fragments identified two distinct genotypes of E. tarda (DNA group I; DNA group II). Isolates that fell into DNA group II demonstrated more similarity to E. ictaluri than DNA group I, which contained the reference E. tarda strain (ATCC #15947). Conventional PCR analysis using published E. tarda-specific primer sets yielded variable results, with several primer sets producing no observable amplification of target DNA from some isolates. Fluorometric determination of G + C content demonstrated 56.4% G + C content for DNA group I, 60.2% for DNA group II, and 58.4% for E. ictaluri. Surprisingly, these isolates were indistinguishable using conventional biochemical techniques, with all isolates demonstrating phenotypic characteristics consistent with E. tarda. Analysis using two commercial test kits identified multiple phenotypes, although no single metabolic characteristic could reliably discriminate between genetic groups. Additionally, anti-microbial susceptibility and fatty acid profiles did not demonstrate remarkable differences between groups. The significant genetic variation (<90% similarity at gyrA, gyrB, pho, phi and pgm; <40% similarity by rep-PCR) between these groups suggests organisms from DNA group II may represent an unrecognized, genetically distinct taxa of Edwardsiella that is phenotypically indistinguishable from E. tarda.
The role of ecological opportunity in shaping disparate diversification trajectories in a bicontinental primate radiation

PubMed Central

Tran, Lucy A. P.

2014-01-01

Exceptional species and phenotypic diversity commonly are attributed to ecological opportunity (EO). The conventional EO model predicts that rates of lineage diversification and phenotypic evolution are elevated early in a radiation only to decline later in response to niche availability. Foregut fermentation is hypothesized to be a key innovation that allowed colobine monkeys (subfamily Colobinae), the only primates with this trait, to successfully colonize folivore adaptive zones unavailable to other herbivorous species. Therefore, diversification rates also are expected to be strongly linked with the evolution of traits related to folivory in these monkeys. Using dated molecular phylogenies and a dataset of feeding morphology, I test predictions of the EO model to evaluate the role of EO conferred by foregut fermentation in shaping the African and Asian colobine radiations. Findings from diversification methods coupled with colobine biogeographic history provide compelling evidence that decreasing availability of new adaptive zones during colonization of Asia together with constraints presented by dietary specialization underlie temporal changes in diversification in the Asian but not African clade. Additionally, departures from the EO model likely reflect iterative diversification events in Asia. PMID:24598417
Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationships of sarcomere components in Caenorhabditis elegans.

PubMed

Hwang, Hyundoo; Barnes, Dawn E; Matsunaga, Yohei; Benian, Guy M; Ono, Shoichiro; Lu, Hang

2016-01-29

The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Despite decades of genetics work, the functional relationships and the roles of those sarcomeric proteins in animal behaviors remain unclear. In this paper, we demonstrate that optogenetic activation of the motor neurons that induce muscle contraction can facilitate quantitative studies of muscle kinetics in C. elegans. To increase the throughput of the study, we trapped multiple worms in parallel in a microfluidic device and illuminated for photoactivation of channelrhodopsin-2 to induce contractions in body wall muscles. Using image processing, the change in body size was quantified over time. A total of five parameters including rate constants for contraction and relaxation were extracted from the optogenetic assay as descriptors of sarcomere functions. To potentially relate the genes encoding the sarcomeric proteins functionally, a hierarchical clustering analysis was conducted on the basis of those parameters. Because it assesses physiological output different from conventional assays, this method provides a complement to the phenotypic analysis of C. elegans muscle mutants currently performed in many labs; the clusters may provide new insights and drive new hypotheses for functional relationships among the many sarcomere components.
Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationships of sarcomere components in Caenorhabditis elegans

NASA Astrophysics Data System (ADS)

Hwang, Hyundoo; Barnes, Dawn E.; Matsunaga, Yohei; Benian, Guy M.; Ono, Shoichiro; Lu, Hang

2016-01-01

The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Despite decades of genetics work, the functional relationships and the roles of those sarcomeric proteins in animal behaviors remain unclear. In this paper, we demonstrate that optogenetic activation of the motor neurons that induce muscle contraction can facilitate quantitative studies of muscle kinetics in C. elegans. To increase the throughput of the study, we trapped multiple worms in parallel in a microfluidic device and illuminated for photoactivation of channelrhodopsin-2 to induce contractions in body wall muscles. Using image processing, the change in body size was quantified over time. A total of five parameters including rate constants for contraction and relaxation were extracted from the optogenetic assay as descriptors of sarcomere functions. To potentially relate the genes encoding the sarcomeric proteins functionally, a hierarchical clustering analysis was conducted on the basis of those parameters. Because it assesses physiological output different from conventional assays, this method provides a complement to the phenotypic analysis of C. elegans muscle mutants currently performed in many labs; the clusters may provide new insights and drive new hypotheses for functional relationships among the many sarcomere components.
Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

PubMed Central

Fish, Margaret B.; Cho, Ken W. Y.

2016-01-01

CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce ‘leapfrogging’, a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species. PMID:27385011
Adaptability and phenotypic stability of soybean cultivars for grain yield and oil content.

PubMed

Silva, K B; Bruzi, A T; Zuffo, A M; Zambiazzi, E V; Soares, I O; de Rezende, P M; Fronza, V; Vilela, G D L; Botelho, F B S; Teixeira, C M; de O Coelho, M A

2016-04-25

The aim of this study was to verify the adaptability and stability of soybean cultivars with regards to yield and oil content. Data of soybean yield and oil content were used from experiments set up in six environments in the 2011/12 and 2012/13 crop seasons in the municipalities of Patos de Minas, Uberaba, Lavras, and São Gotardo, Minas Gerais, Brazil, testing 36 commercial soybean cultivars of both conventional and transgenic varieties. The Wricke method and GGE biplot analysis were used to evaluate adaptability and stability of these cultivars. Large variations were observed in grain yield in relation to the different environments studied, showing that these materials are adaptable. The cultivars exhibited significant differences in oil content. The cultivars BRSGO204 (Goiânia) and BRSMG (Garantia) exhibited the greatest average grain yield in the different environments studied, and the cultivar BRSMG 760 SRR had the greatest oil content among the cultivars evaluated. Ecovalence was adopted to identify the most stable cultivars, and the estimates were nearly uniform both for grain yield and oil content, showing a variation of 0.07 and 0.01%, respectively. The GGE biplot was efficient at identifying cultivars with high adaptability and phenotype stability.
Evaluation of phage assay for rapid phenotypic detection of rifampicin resistance in Mycobacterium tuberculosis

PubMed Central

Yzquierdo, Sergio Luis; Lemus, Dihadenys; Echemendia, Miguel; Montoro, Ernesto; McNerney, Ruth; Martin, Anandi; Palomino, Juan Carlos

2006-01-01

Background Conventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba. Methods Phage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay. Results Phage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97.8 % and 100% respectively. Conclusion Phage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem. PMID:16630356
Indirect antiglobulin test-crossmatch using low-ionic-strength saline-albumin enhancement medium and reduced incubation time: effectiveness in the detection of most clinically significant antibodies and impact on blood utilization.

PubMed

Dinardo, C L; Bonifácio, S L; Mendrone, A

2014-01-01

Indirect antiglobulin test-crossmatch (IAT-XM) using enhancement media such as low-ionic-strength saline (LISS) and polyethylene glycol (PEG) usually requires 15 minutes of incubation. These methods are necessary when testing samples from blood recipients who have a higher risk of alloimmunization. In emergency situations, IAT-XM can be time-consuming and can influence presurgery routine, resulting in more red blood cell (RBC) units being tested and stored to avoid the transfusion of uncrossmatched ones. The objective of this study was to evaluate the performance of a LISS-albumin enhancer to intensify antigen-antibody reaction after 5 minutes of 37oC incubation and compare this performance with that of other enhancers, gel, and conventional tube testing. Second, the study evaluated the impact of this method's implementation in the C:T ratio (crossmatched to transfused RBC units) of a transfusion laboratory. Ninety serum samples containing alloantibodies of potential clinical significance were tested against phenotyped RBCs using four different methods: (1) tube with LISS-albumin enhancer (5 minutes of incubation), (2) tube with LISS-albumin and PEG (15 minutes of incubation), (3) gel, and (4) conventional tube method (60 minutes of incubation). In parallel, the study compared the C:T ratio of a tertiary-hospital transfusion laboratory in two different periods: 3 months before and 3 months after the implementation of the 5-minute IAT-XM protocol. The use of LISS-albumin with 5 minutes of incubation exhibited the same performance as LISS-albumin, PEG, and gel with 15 minutes of incubation. Conventional tube method results were equally comparable, but reactions were significantly less intense, except for anti-c (p = 0.406). Accuracy was 100 percent for all selected methods. After the implementation of the 5-minute IAT-XM protocol, the C:T ratio fell from 2.74 to 1.29 (p < 0.001). IAT-XM can have its incubation time reduced to 5 minutes with the use of LISS-albumin enhancement. We suggest this strategy should be used to quickly prepare RBC units for surgical patients, keeping transfusion safety without compromising blood supplies.
A Statistical Approach for Testing Cross-Phenotype Effects of Rare Variants

PubMed Central

Broadaway, K. Alaine; Cutler, David J.; Duncan, Richard; Moore, Jacob L.; Ware, Erin B.; Jhun, Min A.; Bielak, Lawrence F.; Zhao, Wei; Smith, Jennifer A.; Peyser, Patricia A.; Kardia, Sharon L.R.; Ghosh, Debashis; Epstein, Michael P.

2016-01-01

Increasing empirical evidence suggests that many genetic variants influence multiple distinct phenotypes. When cross-phenotype effects exist, multivariate association methods that consider pleiotropy are often more powerful than univariate methods that model each phenotype separately. Although several statistical approaches exist for testing cross-phenotype effects for common variants, there is a lack of similar tests for gene-based analysis of rare variants. In order to fill this important gap, we introduce a statistical method for cross-phenotype analysis of rare variants using a nonparametric distance-covariance approach that compares similarity in multivariate phenotypes to similarity in rare-variant genotypes across a gene. The approach can accommodate both binary and continuous phenotypes and further can adjust for covariates. Our approach yields a closed-form test whose significance can be evaluated analytically, thereby improving computational efficiency and permitting application on a genome-wide scale. We use simulated data to demonstrate that our method, which we refer to as the Gene Association with Multiple Traits (GAMuT) test, provides increased power over competing approaches. We also illustrate our approach using exome-chip data from the Genetic Epidemiology Network of Arteriopathy. PMID:26942286
Evaluation of chromogenic media and seminested PCR in the identification of Candida species

PubMed Central

Daef, Enas; Moharram, Ahmed; Eldin, Salwa Seif; Elsherbiny, Nahla; Mohammed, Mona

2014-01-01

Identification of Candida cultured from various clinical specimens to the species level is increasingly necessary for clinical laboratories. Although sn PCR identifies the species within hours but its cost-effectiveness is to be considered. So there is always a need for media which help in the isolation and identification at the species level. The study aimed to evaluate the performance of different chromogenic media and to compare the effectiveness of the traditional phenotypic methods vs. seminested polymerase chain reaction (sn PCR) for identification of Candida species. One hundred and twenty seven Candida strains isolated from various clinical specimens were identified by conventional methods, four different chromogenic media and sn PCR. HiCrome Candida Differential and CHROMagar Candida media showed comparably high sensitivities and specificities in the identification of C. albicans, C. tropicalis, C. glabrata and C. krusei. CHROMagar Candida had an extra advantage of identifying all C. parapsilosis isolates. CHROMagar-Pal’s medium identified C. albicans, C. tropicalis and C. krusei with high sensitivities and specificities, but couldn’t identify C. glabrata or C. parapsilosis. It was the only medium that identified C. dubliniensis with a sensitivity and specificity of 100%. Biggy agar showed the least sensitivities and specificities. The overall concordance of the snPCR compared to the conventional tests including CHROMAgar Candida in the identification of Candida species was 97.5%. The use of CHROMAgar Candida medium is an easy and accurate method for presumptive identification of the most commonly encountered Candida spp. PMID:24948942
Text-based phenotypic profiles incorporating biochemical phenotypes of inborn errors of metabolism improve phenomics-based diagnosis.

PubMed

Lee, Jessica J Y; Gottlieb, Michael M; Lever, Jake; Jones, Steven J M; Blau, Nenad; van Karnebeek, Clara D M; Wasserman, Wyeth W

2018-05-01

Phenomics is the comprehensive study of phenotypes at every level of biology: from metabolites to organisms. With high throughput technologies increasing the scope of biological discoveries, the field of phenomics has been developing rapid and precise methods to collect, catalog, and analyze phenotypes. Such methods have allowed phenotypic data to be widely used in medical applications, from assisting clinical diagnoses to prioritizing genomic diagnoses. To channel the benefits of phenomics into the field of inborn errors of metabolism (IEM), we have recently launched IEMbase, an expert-curated knowledgebase of IEM and their disease-characterizing phenotypes. While our efforts with IEMbase have realized benefits, taking full advantage of phenomics requires a comprehensive curation of IEM phenotypes in core phenomics projects, which is dependent upon contributions from the IEM clinical and research community. Here, we assess the inclusion of IEM biochemical phenotypes in a core phenomics project, the Human Phenotype Ontology. We then demonstrate the utility of biochemical phenotypes using a text-based phenomics method to predict gene-disease relationships, showing that the prediction of IEM genes is significantly better using biochemical rather than clinical profiles. The findings herein provide a motivating goal for the IEM community to expand the computationally accessible descriptions of biochemical phenotypes associated with IEM in phenomics resources.
Harnessing Diversity in Wheat to Enhance Grain Yield, Climate Resilience, Disease and Insect Pest Resistance and Nutrition Through Conventional and Modern Breeding Approaches

PubMed Central

Mondal, Suchismita; Rutkoski, Jessica E.; Velu, Govindan; Singh, Pawan K.; Crespo-Herrera, Leonardo A.; Guzmán, Carlos; Bhavani, Sridhar; Lan, Caixia; He, Xinyao; Singh, Ravi P.

2016-01-01

Current trends in population growth and consumption patterns continue to increase the demand for wheat, a key cereal for global food security. Further, multiple abiotic challenges due to climate change and evolving pathogen and pests pose a major concern for increasing wheat production globally. Triticeae species comprising of primary, secondary, and tertiary gene pools represent a rich source of genetic diversity in wheat. The conventional breeding strategies of direct hybridization, backcrossing and selection have successfully introgressed a number of desirable traits associated with grain yield, adaptation to abiotic stresses, disease resistance, and bio-fortification of wheat varieties. However, it is time consuming to incorporate genes conferring tolerance/resistance to multiple stresses in a single wheat variety by conventional approaches due to limitations in screening methods and the lower probabilities of combining desirable alleles. Efforts on developing innovative breeding strategies, novel tools and utilizing genetic diversity for new genes/alleles are essential to improve productivity, reduce vulnerability to diseases and pests and enhance nutritional quality. New technologies of high-throughput phenotyping, genome sequencing and genomic selection are promising approaches to maximize progeny screening and selection to accelerate the genetic gains in breeding more productive varieties. Use of cisgenic techniques to transfer beneficial alleles and their combinations within related species also offer great promise especially to achieve durable rust resistance. PMID:27458472
Electrocardiographic Characterization of Cardiac Hypertrophy in Mice that Overexpress the ErbB2 Receptor Tyrosine Kinase

PubMed Central

Sysa-Shah, Polina; Sørensen, Lars L; Abraham, M Roselle; Gabrielson, Kathleen L

2015-01-01

Electrocardiography is an important method for evaluation and risk stratification of patients with cardiac hypertrophy. We hypothesized that the recently developed transgenic mouse model of cardiac hypertrophy (ErbB2tg) will display distinct ECG features, enabling WT (wild type) mice to be distinguished from transgenic mice without using conventional PCR genotyping. We evaluated more than 2000 mice and developed specific criteria for genotype determination by using cageside ECG, during which unanesthetized mice were manually restrained for less than 1 min. Compared with those from WT counterparts, the ECG recordings of ErbB2tg mice were characterized by higher P- and R-wave amplitudes, broader QRS complexes, inverted T waves, and ST interval depression. Pearson's correlation matrix analysis of combined WT and ErbB2tg data revealed significant correlation between heart weight and the ECG parameters of QT interval (corrected for heart rate), QRS interval, ST height, R amplitude, P amplitude, and PR interval. In addition, the left ventricular posterior wall thickness as determined by echocardiography correlated with ECG-determined ST height, R amplitude, QRS interval; echocardiographic left ventricular mass correlated with ECG-determined ST height and PR interval. In summary, we have determined phenotypic ECG criteria to differentiate ErbB2tg from WT genotypes in 98.8% of mice. This inexpensive and time-efficient ECG-based phenotypic method might be applied to differentiate between genotypes in other rodent models of cardiac hypertrophy. Furthermore, with appropriate modifications, this method might be translated for use in other species. PMID:26310459
A strategy to apply quantitative epistasis analysis on developmental traits.

PubMed

Labocha, Marta K; Yuan, Wang; Aleman-Meza, Boanerges; Zhong, Weiwei

2017-05-15

Genetic interactions are keys to understand complex traits and evolution. Epistasis analysis is an effective method to map genetic interactions. Large-scale quantitative epistasis analysis has been well established for single cells. However, there is a substantial lack of such studies in multicellular organisms and their complex phenotypes such as development. Here we present a method to extend quantitative epistasis analysis to developmental traits. In the nematode Caenorhabditis elegans, we applied RNA interference on mutants to inactivate two genes, used an imaging system to quantitatively measure phenotypes, and developed a set of statistical methods to extract genetic interactions from phenotypic measurement. Using two different C. elegans developmental phenotypes, body length and sex ratio, as examples, we showed that this method could accommodate various metazoan phenotypes with performances comparable to those methods in single cell growth studies. Comparing with qualitative observations, this method of quantitative epistasis enabled detection of new interactions involving subtle phenotypes. For example, several sex-ratio genes were found to interact with brc-1 and brd-1, the orthologs of the human breast cancer genes BRCA1 and BARD1, respectively. We confirmed the brc-1 interactions with the following genes in DNA damage response: C34F6.1, him-3 (ortholog of HORMAD1, HORMAD2), sdc-1, and set-2 (ortholog of SETD1A, SETD1B, KMT2C, KMT2D), validating the effectiveness of our method in detecting genetic interactions. We developed a reliable, high-throughput method for quantitative epistasis analysis of developmental phenotypes.
Bull fertility evaluations for Angus service sires bred to Holstein cows

USDA-ARS?s Scientific Manuscript database

Sire conception rate (SCR), a phenotypic evaluation of service-sire fertility implemented in August 2008, is based on data from the most recent 4 years, conventional-semen breedings up to 7 services, and cow parities 1 through 5. Many US dairy cows are now being bred to Angus sires because beef pric...
Combinatorial genetic perturbation to refine metabolic circuits for producing biofuels and biochemicals.

PubMed

Kim, Hyo Jin; Turner, Timothy Lee; Jin, Yong-Su

2013-11-01

Recent advances in metabolic engineering have enabled microbial factories to compete with conventional processes for producing fuels and chemicals. Both rational and combinatorial approaches coupled with synthetic and systematic tools play central roles in metabolic engineering to create and improve a selected microbial phenotype. Compared to knowledge-based rational approaches, combinatorial approaches exploiting biological diversity and high-throughput screening have been demonstrated as more effective tools for improving various phenotypes of interest. In particular, identification of unprecedented targets to rewire metabolic circuits for maximizing yield and productivity of a target chemical has been made possible. This review highlights general principles and the features of the combinatorial approaches using various libraries to implement desired phenotypes for strain improvement. In addition, recent applications that harnessed the combinatorial approaches to produce biofuels and biochemicals will be discussed. Copyright © 2013 Elsevier Inc. All rights reserved.
Shadows of complexity: what biological networks reveal about epistasis and pleiotropy

PubMed Central

Tyler, Anna L.; Asselbergs, Folkert W.; Williams, Scott M.; Moore, Jason H.

2011-01-01

Pleiotropy, in which one mutation causes multiple phenotypes, has traditionally been seen as a deviation from the conventional observation in which one gene affects one phenotype. Epistasis, or gene-gene interaction, has also been treated as an exception to the Mendelian one gene-one phenotype paradigm. This simplified perspective belies the pervasive complexity of biology and hinders progress toward a deeper understanding of biological systems. We assert that epistasis and pleiotropy are not isolated occurrences, but ubiquitous and inherent properties of biomolecular networks. These phenomena should not be treated as exceptions, but rather as fundamental components of genetic analyses. A systems level understanding of epistasis and pleiotropy is, therefore, critical to furthering our understanding of human genetics and its contribution to common human disease. Finally, graph theory offers an intuitive and powerful set of tools with which to study the network bases of these important genetic phenomena. PMID:19204994
A multiplex real-time PCR assay for the identification and differentiation of Salmonella enterica serovar Typhimurium and monophasic serovar 4,[5],12:i:-.

PubMed

Prendergast, Deirdre M; Hand, Darren; Nί Ghallchóir, Eadaoin; McCabe, Evonne; Fanning, Seamus; Griffin, Margaret; Egan, John; Gutierrez, Montserrat

2013-08-16

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is considered to be a monophasic variant of Salmonella Typhimurium and is increasingly associated with human infections. The use of PCR for the unequivocal identification of strains identified by conventional serotyping as 4,[5],12:i:- has been recommended by the European Food Safety Authority (EFSA), in particular the conventional multiplex PCR developed by Tennant et al. (2010). An alternative protocol for the identification and differentiation of S. Typhimurium and S. Typhimurium-like strains, including its monophasic variants, based on a multiplex real-time PCR assay was developed in our laboratory. A panel of 206 Salmonella strains was used to validate our multiplex real-time PCR against the conventional multiplex PCR recommended by EFSA, i.e. 43 Salmonella strains of serovars other than Typhimurium and 163 routine isolates determined by slide agglutination serotyping to have an incomplete antigenic formula compatible with the S. Typhimurium formula 4,[5],12:i:1,2. Both methods correctly identified the 43 Salmonella strains as non S. Typhimurium. Among the 163 isolates of undetermined serovar by conventional serotyping, both PCR protocols identified 54 isolates as S. Typhimurium, 101 as monophasic S. Typhimurium and 8 as non-S. Typhimurium. Twenty isolates phenotypically lacking the phase-2 H antigen were positive for the fljB.1,2 gene. These strains have been recently described in the literature by other workers and have been referred to as "inconsistent" variants of S. Typhimurium. Antimicrobial resistance and phage typing were also performed on the S. Typhimurium isolates, including monophasic variants, and approximately half of the isolates identified as monophasic S. Typhimurium by our multiplex real-time PCR protocol were DT193 with the resistance pattern ASSuT. There was 100% concordance between the conventional PCR and the multiplex real-time PCR method developed in this study which proved that our protocol is equivalent to the one recommended by EFSA. In comparison to the conventional PCR, this new protocol is faster and is currently being applied routinely in our laboratory to all isolates that could potentially be S. Typhimurium. Copyright © 2013 Elsevier B.V. All rights reserved.
Parametric and Nonparametric Statistical Methods for Genomic Selection of Traits with Additive and Epistatic Genetic Architectures

PubMed Central

Howard, Réka; Carriquiry, Alicia L.; Beavis, William D.

2014-01-01

Parametric and nonparametric methods have been developed for purposes of predicting phenotypes. These methods are based on retrospective analyses of empirical data consisting of genotypic and phenotypic scores. Recent reports have indicated that parametric methods are unable to predict phenotypes of traits with known epistatic genetic architectures. Herein, we review parametric methods including least squares regression, ridge regression, Bayesian ridge regression, least absolute shrinkage and selection operator (LASSO), Bayesian LASSO, best linear unbiased prediction (BLUP), Bayes A, Bayes B, Bayes C, and Bayes Cπ. We also review nonparametric methods including Nadaraya-Watson estimator, reproducing kernel Hilbert space, support vector machine regression, and neural networks. We assess the relative merits of these 14 methods in terms of accuracy and mean squared error (MSE) using simulated genetic architectures consisting of completely additive or two-way epistatic interactions in an F2 population derived from crosses of inbred lines. Each simulated genetic architecture explained either 30% or 70% of the phenotypic variability. The greatest impact on estimates of accuracy and MSE was due to genetic architecture. Parametric methods were unable to predict phenotypic values when the underlying genetic architecture was based entirely on epistasis. Parametric methods were slightly better than nonparametric methods for additive genetic architectures. Distinctions among parametric methods for additive genetic architectures were incremental. Heritability, i.e., proportion of phenotypic variability, had the second greatest impact on estimates of accuracy and MSE. PMID:24727289
Assessing the Efficiency of Phenotyping Early Traits in a Greenhouse Automated Platform for Predicting Drought Tolerance of Soybean in the Field

PubMed Central

Peirone, Laura S.; Pereyra Irujo, Gustavo A.; Bolton, Alejandro; Erreguerena, Ignacio; Aguirrezábal, Luis A. N.

2018-01-01

Conventional field phenotyping for drought tolerance, the most important factor limiting yield at a global scale, is labor-intensive and time-consuming. Automated greenhouse platforms can increase the precision and throughput of plant phenotyping and contribute to a faster release of drought tolerant varieties. The aim of this work was to establish a framework of analysis to identify early traits which could be efficiently measured in a greenhouse automated phenotyping platform, for predicting the drought tolerance of field grown soybean genotypes. A group of genotypes was evaluated, which showed variation in their drought susceptibility index (DSI) for final biomass and leaf area. A large number of traits were measured before and after the onset of a water deficit treatment, which were analyzed under several criteria: the significance of the regression with the DSI, phenotyping cost, earliness, and repeatability. The most efficient trait was found to be transpiration efficiency measured at 13 days after emergence. This trait was further tested in a second experiment with different water deficit intensities, and validated using a different set of genotypes against field data from a trial network in a third experiment. The framework applied in this work for assessing traits under different criteria could be helpful for selecting those most efficient for automated phenotyping. PMID:29774042

QTLs associated with agronomic traits in the Attila × CDC Go spring wheat population evaluated under conventional management

PubMed Central

Zou, Jun; Iqbal, Muhammad; Chen, Hua; Asif, Mohammad; N’Diaye, Amidou; Navabi, Alireza; Perez-Lara, Enid; Pozniak, Curtis; Yang, Rong-Cai; Randhawa, Harpinder; Spaner, Dean

2017-01-01

Recently, we investigated the effect of the wheat 90K single nucleotide polymorphic (SNP) array and three gene-specific (Ppd-D1, Vrn-A1 and Rht-B1) markers on quantitative trait loci (QTL) detection in a recombinant inbred lines (RILs) population derived from a cross between two spring wheat (Triticum aestivum L.) cultivars, ‘Attila’ and ‘CDC Go’, and evaluated for eight agronomic traits at three environments under organic management. The objectives of the present study were to investigate the effect of conventional management on QTL detection in the same mapping population using the same set of markers as the organic management and compare the results with organic management. Here, we evaluated 167 RILs for number of tillers (tillering), flowering time, maturity, plant height, test weight (grain volume weight), 1000 kernel weight, grain yield, and grain protein content at seven conventionally managed environments from 2008 to 2014. Using inclusive composite interval mapping (ICIM) on phenotypic data averaged across seven environments and a subset of 1203 informative markers (1200 SNPs and 3 gene specific markers), we identified a total of 14 QTLs associated with flowering time (1), maturity (2), plant height (1), grain yield (1), test weight (2), kernel weight (4), tillering (1) and grain protein content (2). Each QTL individually explained from 6.1 to 18.4% of the phenotypic variance. Overall, the QTLs associated with each trait explained from 9.7 to 35.4% of the phenotypic and from 22.1 to 90.8% of the genetic variance. Three chromosomal regions on chromosomes 2D (61–66 cM), 4B (80–82 cM) and 5A (296–297 cM) harbored clusters of QTLs associated with two to three traits. The coincidental region on chromosome 5A harbored QTL clusters for both flowering and maturity time, and mapped about 2 cM proximal to the Vrn-A1 gene, which was in high linkage disequilibrium (0.70 ≤ r2 ≤ 0.75) with SNP markers that mapped within the QTL confidence interval. Six of the 14 QTLs (one for flowering time and plant height each, and two for maturity and kernel weight each) were common between the conventional and organic management systems, which suggests issues in directly utilizing gene discovery results based on conventional management to make in detail selection (decision) for organic management. PMID:28158253
Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates.

PubMed

Cheng, Keding; Chui, Huixia; Domish, Larissa; Sloan, Angela; Hernandez, Drexler; McCorrister, Stuart; Robinson, Alyssia; Walker, Matthew; Peterson, Lorea A M; Majcher, Miles; Ratnam, Sam; Haldane, David J M; Bekal, Sadjia; Wylie, John; Chui, Linda; Tyler, Shaun; Xu, Bianli; Reimer, Aleisha; Nadon, Celine; Knox, J David; Wang, Gehua

2016-08-01

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing. Copyright © 2016 Cheng et al.
Genotypic and phenotypic virulence characteristics and antimicrobial resistance of Yersinia spp. isolated from meat and milk products.

PubMed

Özdemir, Fatma; Arslan, Seza

2015-06-01

A total of 300 food samples including 180 milk and 120 meat products have been examined for the presence of Yersinia spp. using the ISO 10273 and the cold enrichment method. The overall prevalence of Yersinia spp. was 84 (28%). Yersinia enterocolitica was isolated from 18 (6%) of the 300 samples. The other Yersinia species were detected in the samples Yersinia rohdei 15 (5%), Yersinia intermedia 14 (4.7%), Yersinia pseudotuberculosis 12 (4%), Yersinia ruckeri 12 (4%), Yersinia mollaretii 5 (1.7%), Yersinia bercovieri 4 (1.3%), and atypical Yersinia spp. 4 (1.3%). The conventionally identified Y. enterocolitica strains were also confirmed by the 16S rRNA gene sequencing. All Y. enterocolitica strains biotyped as 1A had negative results in the phenotypic virulence tests. The 84 Yersinia strains were also examined genotypically for the presence of virulence genes. None of the Y. enterocolitica and other Yersinia strains contained the ail, ystA, yadA, and virF except only 1 Y. intermedia and 2 Y. enterocolitica strains that were found to be positive for ystB. Antimicrobial resistance of 84 Yersinia to 16 antimicrobial agents was determined by the disk diffusion method. All strains were sensitive to tobramycine and imipenem while resistant to clindamycin. Although 84.5% of the strains were resistant to at least 3 or more antimicrobial agents, 64.3% of them were resistant to 4 or more antimicrobial agents. © 2015 Institute of Food Technologists®
Federated Tensor Factorization for Computational Phenotyping

PubMed Central

Kim, Yejin; Sun, Jimeng; Yu, Hwanjo; Jiang, Xiaoqian

2017-01-01

Tensor factorization models offer an effective approach to convert massive electronic health records into meaningful clinical concepts (phenotypes) for data analysis. These models need a large amount of diverse samples to avoid population bias. An open challenge is how to derive phenotypes jointly across multiple hospitals, in which direct patient-level data sharing is not possible (e.g., due to institutional policies). In this paper, we developed a novel solution to enable federated tensor factorization for computational phenotyping without sharing patient-level data. We developed secure data harmonization and federated computation procedures based on alternating direction method of multipliers (ADMM). Using this method, the multiple hospitals iteratively update tensors and transfer secure summarized information to a central server, and the server aggregates the information to generate phenotypes. We demonstrated with real medical datasets that our method resembles the centralized training model (based on combined datasets) in terms of accuracy and phenotypes discovery while respecting privacy. PMID:29071165
[Differentiation and characterization of yeasts pathogenic for humans (Candida albicans, Exophiala dermatitidis) and algae pathogenic for animals (Prototheca spp.) using Fourier transform infrared spectroscopy (FTIR) in comparison with conventional methods].

PubMed

Schmalreck, A F; Tränkle, P; Vanca, E; Blaschke-Hellmessen, R

1998-01-01

Due to the Fourier-Transform Infrared Spectroscopy (FT-IR) of strain specific traits demonstrated to be a suitable and efficient method for diagnostic and epidemiological determinations for the yeasts Candida albicans, Exophiala dermatitidis and the chlorophylless algae of the genus Prototheca. FT-IR leads in a rapid and economical way to reproducible results according to the spectral differences of intact cells (IR-fingerprints). Different genera, species and sub-species respectively, different strains can be recognized and grouped into different clusters and subclusters. The FT-IR analysis of Candida albicans isolates (n = 150) of 22 newborns-at-risk of an intensive care unit showed, that 86% of the children were colonised with several (2-4) different strains in the oral cavities and faeces. Stationary cross-infections could definitely be determined. Exophiala dermatitidis isolates (n = 31), mostly isolated repetitively within a period of 3 years from sputa of patients suffering from cystic fibrosis could be characterized and grouped patient-specifically over the total sampling period. Of 6 from 8 patients (75%) their individual strains remain the same and could be tracked over the three years. Cross-infections during the stationary treatment could be clearly identified by FT-IR. The Prototheca isolate (n = 43) from live-stock and farm environment showed clear distinguishable clusters differentiating the species P. wickerhamii, P. zopfii and P. stagnora. In addition, the biotypes of P. zopfii could be distinguished, especially the subclusters of variants II and III. It could be demonstrated, that FT-IR is suitable for the routine identification and differentiation of yeasts and algae. However, in spite of the gain of knowledge by using FT-IR for the characterization of microorganisms, the conventional phenotyping and/or genetic analysis of yeast or algae strains cannot be replaced completely. For a final taxonomic classification a combination of conventional methods on FT-IR together with more sophisticated molecular genetic procedures is necessary.
Revealing plant cryptotypes: defining meaningful phenotypes among infinite traits.

PubMed

Chitwood, Daniel H; Topp, Christopher N

2015-04-01

The plant phenotype is infinite. Plants vary morphologically and molecularly over developmental time, in response to the environment, and genetically. Exhaustive phenotyping remains not only out of reach, but is also the limiting factor to interpreting the wealth of genetic information currently available. Although phenotyping methods are always improving, an impasse remains: even if we could measure the entirety of phenotype, how would we interpret it? We propose the concept of cryptotype to describe latent, multivariate phenotypes that maximize the separation of a priori classes. Whether the infinite points comprising a leaf outline or shape descriptors defining root architecture, statistical methods to discern the quantitative essence of an organism will be required as we approach measuring the totality of phenotype. Copyright © 2015 Elsevier Ltd. All rights reserved.
Genetic validation of bipolar disorder identified by automated phenotyping using electronic health records.

PubMed

Chen, Chia-Yen; Lee, Phil H; Castro, Victor M; Minnier, Jessica; Charney, Alexander W; Stahl, Eli A; Ruderfer, Douglas M; Murphy, Shawn N; Gainer, Vivian; Cai, Tianxi; Jones, Ian; Pato, Carlos N; Pato, Michele T; Landén, Mikael; Sklar, Pamela; Perlis, Roy H; Smoller, Jordan W

2018-04-18

Bipolar disorder (BD) is a heritable mood disorder characterized by episodes of mania and depression. Although genomewide association studies (GWAS) have successfully identified genetic loci contributing to BD risk, sample size has become a rate-limiting obstacle to genetic discovery. Electronic health records (EHRs) represent a vast but relatively untapped resource for high-throughput phenotyping. As part of the International Cohort Collection for Bipolar Disorder (ICCBD), we previously validated automated EHR-based phenotyping algorithms for BD against in-person diagnostic interviews (Castro et al. Am J Psychiatry 172:363-372, 2015). Here, we establish the genetic validity of these phenotypes by determining their genetic correlation with traditionally ascertained samples. Case and control algorithms were derived from structured and narrative text in the Partners Healthcare system comprising more than 4.6 million patients over 20 years. Genomewide genotype data for 3330 BD cases and 3952 controls of European ancestry were used to estimate SNP-based heritability (h 2 g ) and genetic correlation (r g ) between EHR-based phenotype definitions and traditionally ascertained BD cases in GWAS by the ICCBD and Psychiatric Genomics Consortium (PGC) using LD score regression. We evaluated BD cases identified using 4 EHR-based algorithms: an NLP-based algorithm (95-NLP) and three rule-based algorithms using codified EHR with decreasing levels of stringency-"coded-strict", "coded-broad", and "coded-broad based on a single clinical encounter" (coded-broad-SV). The analytic sample comprised 862 95-NLP, 1968 coded-strict, 2581 coded-broad, 408 coded-broad-SV BD cases, and 3 952 controls. The estimated h 2 g were 0.24 (p = 0.015), 0.09 (p = 0.064), 0.13 (p = 0.003), 0.00 (p = 0.591) for 95-NLP, coded-strict, coded-broad and coded-broad-SV BD, respectively. The h 2 g for all EHR-based cases combined except coded-broad-SV (excluded due to 0 h 2 g ) was 0.12 (p = 0.004). These h 2 g were lower or similar to the h 2 g observed by the ICCBD + PGCBD (0.23, p = 3.17E-80, total N = 33,181). However, the r g between ICCBD + PGCBD and the EHR-based cases were high for 95-NLP (0.66, p = 3.69 × 10 -5 ), coded-strict (1.00, p = 2.40 × 10 -4 ), and coded-broad (0.74, p = 8.11 × 10 -7 ). The r g between EHR-based BD definitions ranged from 0.90 to 0.98. These results provide the first genetic validation of automated EHR-based phenotyping for BD and suggest that this approach identifies cases that are highly genetically correlated with those ascertained through conventional methods. High throughput phenotyping using the large data resources available in EHRs represents a viable method for accelerating psychiatric genetic research.
7 Tesla proton magnetic resonance spectroscopic imaging in adult X-linked adrenoleukodystrophy

PubMed Central

Ratai, Eva; Kok, Trina; Wiggins, Christopher; Wiggins, Graham; Grant, Ellen; Gagoski, Borjan; O'Neill, Gilmore; Adalsteinsson, Elfar; Eichler, Florian

2010-01-01

Background Adult patients with X-linked adrenoleukodystrophy (X-ALD) remain at risk for progressive neurological deterioration. Phenotypes vary in their pathology, ranging from axonal degeneration to inflammatory demyelination. The severity of symptoms is poorly explained by conventional imaging. Objective To test the hypothesis that neurochemistry in normal appearing brain differs among adult phenotypes of X-ALD, and that neurochemical changes correlate with the severity of symptoms. Patients and Methods Using a 7 Tesla scanner we performed structural and proton MRSI in 13 adult patients with X-ALD, including 4 patients with adult cerebral ALD (ACALD), 5 with adrenomyeloneuropathy (AMN) and 4 female heterozygotes. Studies were also performed in nine healthy controls. Results Among adult X-ALD phenotypes, MI/Cr was 46% higher and Cho/Cr 21% higher in normal appearing white matter of ACALD compared to AMN (p < 0.05). Both NAA/Cr and Glu/Cr ratios were lower in AMN patients (p = 0.028 and p = 0.036, respectively) than in controls. There were no significant differences between AMN and female heterozygotes. In cortex, ACALD patients had lower values of NAA/Cr compared to female heterozygotes and controls (p = 0.022). The global MI/Cr ratio demonstrated a significant association with the EDSS (Spearman ρ = 0.66, p = 0.039). Conclusion 7 Tesla proton MRSI reveals differences in the neurochemistry of ACALD but is unable to distinguish AMN from female heterozygotes. MI/Cr correlates with the severity of the symptoms and may be a meaningful biomarker in adult X-ALD. PMID:19001168
Face shape differs in phylogenetically related populations.

PubMed

Hopman, Saskia M J; Merks, Johannes H M; Suttie, Michael; Hennekam, Raoul C M; Hammond, Peter

2014-11-01

3D analysis of facial morphology has delineated facial phenotypes in many medical conditions and detected fine grained differences between typical and atypical patients to inform genotype-phenotype studies. Next-generation sequencing techniques have enabled extremely detailed genotype-phenotype correlative analysis. Such comparisons typically employ control groups matched for age, sex and ethnicity and the distinction between ethnic categories in genotype-phenotype studies has been widely debated. The phylogenetic tree based on genetic polymorphism studies divides the world population into nine subpopulations. Here we show statistically significant face shape differences between two European Caucasian populations of close phylogenetic and geographic proximity from the UK and The Netherlands. The average face shape differences between the Dutch and UK cohorts were visualised in dynamic morphs and signature heat maps, and quantified for their statistical significance using both conventional anthropometry and state of the art dense surface modelling techniques. Our results demonstrate significant differences between Dutch and UK face shape. Other studies have shown that genetic variants influence normal facial variation. Thus, face shape difference between populations could reflect underlying genetic difference. This should be taken into account in genotype-phenotype studies and we recommend that in those studies reference groups be established in the same population as the individuals who form the subject of the study.
Spiroplasma infection causes either early or late male killing in Drosophila, depending on maternal host age

NASA Astrophysics Data System (ADS)

Kageyama, Daisuke; Anbutsu, Hisashi; Shimada, Masakazu; Fukatsu, Takema

2007-04-01

Symbiont-induced male-killing phenotypes have been found in a variety of insects. Conventionally, these phenotypes have been divided into two categories according to the timing of action: early male killing at embryonic stages and late male killing at late larval stages. In Drosophila species, endosymbiotic bacteria of the genus Spiroplasma have been known to cause early male killing. Here, we report that a spiroplasma strain normally causing early male killing also induces late male killing depending on the maternal host age: male-specific mortality of larvae and pupae was more frequently observed in the offspring of young females. As the lowest spiroplasma density and occasional male production were also associated with newly emerged females, we proposed the density-dependent hypothesis for the expression of early and late male-killing phenotypes. Our finding suggested that (1) early and late male-killing phenotypes can be caused by the same symbiont and probably by the same mechanism; (2) late male killing may occur as an attenuated expression of early male killing; (3) expression of early and late male-killing phenotypes may be dependent on the symbiont density, and thus, could potentially be affected by the host immunity and regulation; and (4) early male killing and late male killing could be alternative strategies adopted by microbial reproductive manipulators.
Migration ability and Toll-like receptor expression of human mesenchymal stem cells improves significantly after three-dimensional culture.

PubMed

Zhou, Panpan; Liu, Zilin; Li, Xue; Zhang, Bing; Wang, Xiaoyuan; Lan, Jing; Shi, Qing; Li, Dong; Ju, Xiuli

2017-09-16

While the conventional two-dimensional (2D) culture protocol is well accepted for the culture of mesenchymal stem cells (MSCs), this method fails to recapitulate the in vivo native three-dimensional (3D) cellular microenvironment, and may result in phenotypic changes, and homing and migration capacity impairments. MSC preparation in 3D culture systems has been considered an attractive preparatory and delivery method recently. We seeded human umbilical cord-derived MSCs (hUCMSCs) in a 3D culture system with porcine acellular dermal matrix (PADM), and investigated the phenotypic changes, the expression changes of some important receptors, including Toll-like receptors (TLRs) and C-X-C chemokine receptor type 4 (CXCR4) when hUCMSCs were transferred from 2D to 3D systems, as well as the alterations in in vivo homing and migration potential. It was found that the percentage of CD105-positive cells decreased significantly, whereas that of CD34- and CD271-positive cells increased significantly in 3D culture, compared to that in 2D culture. The mRNA and protein expression levels of TLR2, TLR3, TLR4, TLR6, and CXCR4 in hUCMSCs were increased significantly upon culturing with PADM for 3 days, compared to the levels in 2D culture. The numbers of migratory 3D hUCMSCs in the heart, liver, spleen, and bone marrow were significantly greater than the numbers of 2D hUCMSCs, and the worst migration occurred in 3D + AMD3100 (CXCR4 antagonist) hUCMSCs. These results suggested that 3D culture of hUCMSCs with PADM could alter the phenotypic characteristics of hUCMSCs, increase their TLR and CXCR4 expression levels, and promote their migratory and homing capacity in which CXCR4 plays an important role. Copyright © 2017 Elsevier Inc. All rights reserved.
Advantage of MALDI-TOF-MS over biochemical-based phenotyping for microbial identification illustrated on industrial applications.

PubMed

Urwyler, S K; Glaubitz, J

2016-02-01

Fast microbial identification is becoming increasingly necessary in industry to improve microbial control and reduce biocide consumption. We compared the performances of two systems based on MALDI-TOF MS (VITEK MS and BIOTYPER) and two based on biochemical testing (BIOLOG, VITEK 2 Compact) with genetic methods for the identification of environmental bacteria. At genus level both MALDI-TOF MS-based systems showed the lowest number of false (4%) and approx. 60% correct identifications. In contrast, the biochemical-based systems assigned 25% of the genera incorrectly. The differences were even more apparent at the species level. The BIOTYPER was most conservative, where assigning a species led to the lowest percentage of species identifications (54%) but also to the least wrong assignments (4%). The other three systems showed higher levels of false assignments: 8·7, 40 and 46% respectively. The genus identification performance on four industrial products of the BIOTYPER could be increased up to 94·3% (average 88% of 167 isolates) by evolving the database in a product specific manner. Comparison of the bacterial population in the example of paints, and raw materials used therein, at different production steps demonstrated unequivocally that the contamination of the final paint product originated not from the main raw material. MALDI-TOF-MS has revolutionized speed and precision of microbial identification for clinical isolates outperforming conventional methods. In contrast, few performance studies have been published so far focusing on suitability for particularly industrial applications, geomicrobiology and environmental analytics. This study evaluates the performance of this proteomic phenotyping on such industrial isolates in comparison with biochemical-based phenotyping and genotyping. Further the study exemplifies the power of MALDI-TOF-MS to trace cost-efficiently the dominating cultivable bacterial species throughout an industrial paint production process. Vital information can be retrieved to identify the most crucial contaminating source for the final product. © 2015 The Authors published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.
[Frequency of enzymes associated with reduced sensitivity to beta-lactam antibiotics in enterobacteria isolates, Caracas, Venezuela].

PubMed

Marcano, Daniel; De Jesús, Andreína; Hernández, Luis; Torres, Luis

2011-12-01

To determine the frequency of enzymatic mechanisms associated with reduced sensitivity to broad-spectrum beta-lactam antibiotics in enterobacteria isolates obtained at hospital centers in Caracas, Venezuela. A cross-sectional study was conducted on enterobacteria isolated from patients at eight hospital centers in Caracas, Venezuela, from 15 October 2009 to 15 January 2010. The species were identified using conventional biochemical tests, and their susceptibility to antimicrobial drugs was assessed by antibiogram (Kirby-Bauer method), using the 2010 performance standards published by the Clinical and Laboratory Standards Institute. Beta-lactam-resistant genes were detected using an enhanced polymerase chain reaction assay. Of 1 235 isolates, 207 (16.8%) exhibited resistance to third- and fourth-generation cephalosporins, carbapenems, or both. They presented the following phenotypes: extended-spectrum beta-lactamase (ESBL), 93.8%; depressed AmpC, 4.3%; and carbapenemase, 1.9%. Further characterization of the first two phenotypes yielded the following breakdown of types: SHV, 36.7%; CTX-M-1 group, 22.3%; TEM, 21.7%; CTX-M-1 group with impermeability, 5.2%; two-enzyme combinations, 4.5%; CTX-M-2 group, 4.3%; PER, 3.4%; and KPC, 1.9%. The SHV type was predominant in the public hospital strains, whereas the CTX-M-1 group was most common in the strains from the private hospitals. Of the enzymatic mechanisms investigated, the SHV type was the most frequent, followed by the CTX-M-1 group and the TEM type. Also, a high percentage of type KPC was found. The research reported here is one of only a few multicenter studies that have been conducted in Venezuela to evaluate the frequency of this type of antimicrobial resistance mechanism, including phenotypical and molecular characterization. It was shown that the detection methods require proper interpretation of sensitivity profiles and molecular confirmation of the mechanism present.
Three cases of Moraxella osloensis meningitis: a difficult experience in species identification and determination of clinical significance.

PubMed

Roh, Kyoung Ho; Kim, Chang Ki; Koh, Eunmi; Kim, Myung Sook; Yong, Dongeun; Park, Soo Chul; Lee, Kyungwon; Chong, Yunsop

2010-03-01

We had three cases of Moraxella osloensis meningitis. The species identification was impossible by conventional and commercial phenotypic tests. However, we could identify the species using the 16S rRNA gene sequencing. Determination of clinical significance was difficult in one patient. All three patients recovered by appropriate antimicrobial therapy.
Effect of personalized nutrition on health-related behaviour change: evidence from the Food4Me European randomized controlled trial.

PubMed

Celis-Morales, Carlos; Livingstone, Katherine M; Marsaux, Cyril Fm; Macready, Anna L; Fallaize, Rosalind; O'Donovan, Clare B; Woolhead, Clara; Forster, Hannah; Walsh, Marianne C; Navas-Carretero, Santiago; San-Cristobal, Rodrigo; Tsirigoti, Lydia; Lambrinou, Christina P; Mavrogianni, Christina; Moschonis, George; Kolossa, Silvia; Hallmann, Jacqueline; Godlewska, Magdalena; Surwillo, Agnieszka; Traczyk, Iwona; Drevon, Christian A; Bouwman, Jildau; van Ommen, Ben; Grimaldi, Keith; Parnell, Laurence D; Matthews, John Ns; Manios, Yannis; Daniel, Hannelore; Martinez, J Alfredo; Lovegrove, Julie A; Gibney, Eileen R; Brennan, Lorraine; Saris, Wim Hm; Gibney, Mike; Mathers, John C

2017-04-01

Optimal nutritional choices are linked with better health, but many current interventions to improve diet have limited effect. We tested the hypothesis that providing personalized nutrition (PN) advice based on information on individual diet and lifestyle, phenotype and/or genotype would promote larger, more appropriate, and sustained changes in dietary behaviour. : Adults from seven European countries were recruited to an internet-delivered intervention (Food4Me) and randomized to: (i) conventional dietary advice (control) or to PN advice based on: (ii) individual baseline diet; (iii) individual baseline diet plus phenotype (anthropometry and blood biomarkers); or (iv) individual baseline diet plus phenotype plus genotype (five diet-responsive genetic variants). Outcomes were dietary intake, anthropometry and blood biomarkers measured at baseline and after 3 and 6 months' intervention. At baseline, mean age of participants was 39.8 years (range 18-79), 59% of participants were female and mean body mass index (BMI) was 25.5 kg/m 2 . From the enrolled participants, 1269 completed the study. Following a 6-month intervention, participants randomized to PN consumed less red meat [-5.48 g, (95% confidence interval:-10.8,-0.09), P = 0.046], salt [-0.65 g, (-1.1,-0.25), P = 0.002] and saturated fat [-1.14 % of energy, (-1.6,-0.67), P < 0.0001], increased folate [29.6 µg, (0.21,59.0), P = 0.048] intake and had higher Healthy Eating Index scores [1.27, (0.30, 2.25), P = 0.010) than those randomized to the control arm. There was no evidence that including phenotypic and phenotypic plus genotypic information enhanced the effectiveness of the PN advice. Among European adults, PN advice via internet-delivered intervention produced larger and more appropriate changes in dietary behaviour than a conventional approach. © The Author 2016; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association
Utility of 16S rDNA Sequencing for Identification of Rare Pathogenic Bacteria.

PubMed

Loong, Shih Keng; Khor, Chee Sieng; Jafar, Faizatul Lela; AbuBakar, Sazaly

2016-11-01

Phenotypic identification systems are established methods for laboratory identification of bacteria causing human infections. Here, the utility of phenotypic identification systems was compared against 16S rDNA identification method on clinical isolates obtained during a 5-year study period, with special emphasis on isolates that gave unsatisfactory identification. One hundred and eighty-seven clinical bacteria isolates were tested with commercial phenotypic identification systems and 16S rDNA sequencing. Isolate identities determined using phenotypic identification systems and 16S rDNA sequencing were compared for similarity at genus and species level, with 16S rDNA sequencing as the reference method. Phenotypic identification systems identified ~46% (86/187) of the isolates with identity similar to that identified using 16S rDNA sequencing. Approximately 39% (73/187) and ~15% (28/187) of the isolates showed different genus identity and could not be identified using the phenotypic identification systems, respectively. Both methods succeeded in determining the species identities of 55 isolates; however, only ~69% (38/55) of the isolates matched at species level. 16S rDNA sequencing could not determine the species of ~20% (37/187) of the isolates. The 16S rDNA sequencing is a useful method over the phenotypic identification systems for the identification of rare and difficult to identify bacteria species. The 16S rDNA sequencing method, however, does have limitation for species-level identification of some bacteria highlighting the need for better bacterial pathogen identification tools. © 2016 Wiley Periodicals, Inc.
Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells

NASA Astrophysics Data System (ADS)

Okumus, Burak; Landgraf, Dirk; Lai, Ghee Chuan; Bakhsi, Somenath; Arias-Castro, Juan Carlos; Yildiz, Sadik; Huh, Dann; Fernandez-Lopez, Raul; Peterson, Celeste N.; Toprak, Erdal; El Karoui, Meriem; Paulsson, Johan

2016-05-01

Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3-4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting.
[Mass spectrometry in the clinical microbiology laboratory].

PubMed

Jordana-Lluch, Elena; Martró Català, Elisa; Ausina Ruiz, Vicente

2012-12-01

Infectious diseases are still a cause of high mortality and morbidity rates. Current microbiological diagnostic methods are based on culture and phenotypic identification of isolated microorganisms, which can be obtained in about 24-48 h. Given that the microbiological identification is of major importance for patient management, new diagnostic methods are needed in order to detect and identify microorganisms in a timely and accurate manner. Over the last few years, several molecular techniques based on the amplification of microbial nucleic acids have been developed with the aim of reducing the time needed for the identification of the microorganisms involved in different infectious processes. On the other hand, mass spectrometry has emerged as a rapid and consistent alternative to conventional methods for microorganism identification. This review describes the most widely used mass spectrometry technologies -matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization time-of-flight (ESI-TOF)-, both for protein and nucleic acid analysis, as well as the commercial platforms available. Related publications of most interest in clinical microbiology are also reviewed. Copyright © 2011 Elsevier España, S.L. All rights reserved.
Identification of clinical yeasts by Vitek MS system compared with API ID 32 C.

PubMed

Durán-Valle, M Teresa; Sanz-Rodríguez, Nuria; Muñoz-Paraíso, Carmen; Almagro-Moltó, María; Gómez-Garcés, José Luis

2014-05-01

We performed a clinical evaluation of the Vitek MS matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) system with the commercial database version 2.0 for rapid identification of medically important yeasts as compared with the conventional phenotypic method API ID 32 C. We tested 161 clinical isolates, nine isolates from culture collections and five reference strains. In case of discrepant results or no identification with one or both methods, molecular identification techniques were employed. Concordance between both methods was observed with 160/175 isolates (91.42%) and misidentifications by both systems occurred only when taxa were not included in the respective databases, i.e., one isolate of Candida etchellsii was identified as C. globosa by Vitek MS and two isolates of C. orthopsilosis were identified as C. parapsilosis by API ID 32 C. Vitek MS could not identify nine strains (5.14%) and API ID 32 C did not identify 13 (7.42%). Vitek MS was more reliable than API ID 32 C and reduced the time required for the identification of clinical isolates to only a few minutes.
Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory

PubMed Central

Kulkarni, Prakash; Levine, Herbert

2017-01-01

Waddington’s epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of “landscape” in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an “attractor” that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of “cancer attractors”—hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes “recanalization”, i.e., the exit from “cancer attractors” and re-entry into “normal attractors”, is more likely to succeed rather than a conventional approach that targets individual molecules/pathways. PMID:28640191

SAP is required for the development of innate phenotype in H2-M3-restricted CD8+ T cells1

PubMed Central

Bediako, Yaw; Bian, Yao; Zhang, Hong; Cho, Hoonsik; Stein, Paul L.; Wang, Chyung-Ru

2012-01-01

H2-M3-restricted T cells have a pre-activated surface phenotype, rapidly expand and produce cytokines upon stimulation and as such, are classified as innate T cells. Unlike most innate T cells, M3-restricted T cells also express CD8αβ co-receptors and a diverse TCR repertoire: hallmarks of conventional MHC Ia-restricted CD8+ T cells. Although iNKT cells are also innate lymphocytes, they are selected exclusively on hematopoietic cells (HC), while M3-restricted T cells can be selected on either hematopoietic or thymic epithelial cells (TEC). Moreover, their phenotypes differ depending on what cells mediate their selection. Though there is a clear correlation between selection on HC and development of innate phenotype, the underlying mechanism remains unclear. SAP is required for the development of iNKT cells and mediates signals from SLAM receptors that are exclusively expressed on HC. Based on their dual selection pathway, M3-restricted T cells present a unique model for studying the development of innate T cell phenotype. Using both polyclonal and transgenic mouse models we demonstrate that while M3-restricted T cells are capable of developing in the absence of SAP, SAP is required for HC-mediated selection, development of pre-activated phenotype and heightened effector functions of M3-restricted T cells. These findings are significant because they directly demonstrate the need for SAP in HC-mediated acquisition of innate T cell phenotype and suggest that due to their SAP-dependent HC-mediated selection, M3-restricted T cells develop a pre-activated phenotype and an intrinsic ability to proliferate faster upon stimulation, allowing for an important role in the early response to infection. PMID:23041566
Fold or hold: experimental evolution in vitro

PubMed Central

Collins, S; Rambaut, A; Bridgett, S J

2013-01-01

We introduce a system for experimental evolution consisting of populations of short oligonucleotides (Oli populations) evolving in a modified quantitative polymerase chain reaction (qPCR). It is tractable at the genetic, genomic, phenotypic and fitness levels. The Oli system uses DNA hairpins designed to form structures that self-prime under defined conditions. Selection acts on the phenotype of self-priming, after which differences in fitness are amplified and quantified using qPCR. We outline the methodological and bioinformatics tools for the Oli system here and demonstrate that it can be used as a conventional experimental evolution model system by test-driving it in an experiment investigating adaptive evolution under different rates of environmental change. PMID:24003997
Phenotypic, Genotypic, and Antimicrobial Characteristics of Streptococcus halichoeri Isolates from Humans, Proposal To Rename Streptococcus halichoeri as Streptococcus halichoeri subsp. halichoeri, and Description of Streptococcus halichoeri subsp. hominis subsp. nov., a Bacterium Associated with Human Clinical Infections.

PubMed

Shewmaker, P L; Whitney, A M; Humrighouse, B W

2016-03-01

Phenotypic, genotypic, and antimicrobial characteristics of six phenotypically distinct human clinical isolates that most closely resembled the type strain of Streptococcus halichoeri isolated from a seal are presented. Sequencing of the 16S rRNA, rpoB, sodA, and recN genes; comparative whole-genome analysis; conventional biochemical and Rapid ID 32 Strep identification methods; and antimicrobial susceptibility testing were performed on the human isolates, the type strain of S. halichoeri, and type strains of closely related species. The six human clinical isolates were biochemically indistinguishable from each other and showed 100% 16S rRNA, rpoB, sodA, and recN gene sequence similarity. Comparative 16S rRNA gene sequencing analysis revealed 98.6% similarity to S. halichoeri CCUG 48324(T), 97.9% similarity to S. canis ATCC 43496(T), and 97.8% similarity to S. ictaluri ATCC BAA-1300(T). A 3,530-bp fragment of the rpoB gene was 98.8% similar to the S. halichoeri type strain, 84.6% to the S. canis type strain, and 83.8% to the S. ictaluri type strain. The S. halichoeri type strain and the human clinical isolates were susceptible to the antimicrobials tested based on CLSI guidelines for Streptococcus species viridans group with the exception of tetracycline and erythromycin. The human isolates were phenotypically distinct from the type strain isolated from a seal; comparative whole-genome sequence analysis confirmed that the human isolates were S. halichoeri. On the basis of these results, a novel subspecies, Streptococcus halichoeri subsp. hominis, is proposed for the human isolates and Streptococcus halichoeri subsp. halichoeri is proposed for the gray seal isolates. The type strain of the novel subspecies is SS1844(T) = CCUG 67100(T) = LMG 28801(T). Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry.

PubMed

Arraud, Nicolas; Gounou, Céline; Turpin, Delphine; Brisson, Alain R

2016-02-01

Plasma contains cell-derived extracellular vesicles (EVs) which participate in various physiopathological processes and have potential biomedical applications. Despite intense research activity, knowledge on EVs is limited mainly due to the difficulty of isolating and characterizing sub-micrometer particles like EVs. We have recently reported that a simple flow cytometry (FCM) approach based on triggering the detection on a fluorescence signal enabled the detection of 50× more Annexin-A5 binding EVs (Anx5+ EVs) in plasma than the conventional FCM approach based on light scattering triggering. Here, we present the application of the fluorescence triggering approach to the enumeration and phenotyping of EVs from platelet free plasma (PFP), focusing on CD41+ and CD235a+ EVs, as well as their sub-populations which bind or do not bind Anx5. Higher EV concentrations were detected by fluorescence triggering as compared to light scattering triggering, namely 40× for Anx5+ EVs, 75× for CD41+ EVs, and 15× for CD235a+ EVs. We found that about 30% of Anx5+ EVs were of platelet origin while only 3% of them were of erythrocyte origin. In addition, a majority of EVs from platelet and erythrocyte origin do not expose PS, in contrast to the classical theory of EV formation. Furthermore, the same PFP samples were analyzed fresh and after freeze-thawing, showing that freeze-thawing processes induce an increase, of about 35%, in the amount of Anx5+ EVs, while the other EV phenotypes remain unchanged. The method of EV detection and phenotyping by fluorescence triggering is simple, sensitive and reliable. We foresee that its application to EV studies will improve our understanding on the formation mechanisms and functions of EVs in health and disease and help the development of EV-based biomarkers. © 2015 International Society for Advancement of Cytometry.
Colorimetric Method of Loop-Mediated Isothermal Amplification with the Pre-Addition of Calcein for Detecting Flavobacterium columnare and its Assessment in Tilapia Farms.

PubMed

Suebsing, Rungkarn; Kampeera, Jantana; Sirithammajak, Sarawut; Withyachumnarnkul, Boonsirm; Turner, Warren; Kiatpathomchai, Wansika

2015-03-01

Flavobacterium columnare, the causative agent of columnaris disease in fish, affects many economically important freshwater fish species. A colorimetric method of loop-mediated isothermal amplification with the pre-addition of calcein (LAMP-calcein) was developed and used to detect the presence of F. columnare in farmed tilapia (Nile Tilapia Oreochromis niloticus and red tilapia [Nile Tilapia × Mozambique Tilapia O. mossambicus]) and rearing water. The detection method, based on a change in color from orange to green, could be performed within 45 min at 63°C. The method was highly specific, as it had no cross-detections with 14 other bacterial species, including other fish pathogens and two Flavobacterium species. The method has a minimum detection limit of 2.2 × 10(2) F. columnare CFU; thus, it is about 10 times more sensitive than conventional PCR. With this method, F. columnare was detected in gonad, gill, and blood samples from apparently healthy tilapia broodstock as well as in samples of fertilized eggs, newly hatched fry, and rearing water. The bacteria isolated from the blood were further characterized biochemically and found to be phenotypically identical to F. columnare. The amplified products from the LAMP-calcein method had 97% homology with the DNA sequence of F. columnare.
PhenomeExpress: a refined network analysis of expression datasets by inclusion of known disease phenotypes.

PubMed

Soul, Jamie; Hardingham, Timothy E; Boot-Handford, Raymond P; Schwartz, Jean-Marc

2015-01-29

We describe a new method, PhenomeExpress, for the analysis of transcriptomic datasets to identify pathogenic disease mechanisms. Our analysis method includes input from both protein-protein interaction and phenotype similarity networks. This introduces valuable information from disease relevant phenotypes, which aids the identification of sub-networks that are significantly enriched in differentially expressed genes and are related to the disease relevant phenotypes. This contrasts with many active sub-network detection methods, which rely solely on protein-protein interaction networks derived from compounded data of many unrelated biological conditions and which are therefore not specific to the context of the experiment. PhenomeExpress thus exploits readily available animal model and human disease phenotype information. It combines this prior evidence of disease phenotypes with the experimentally derived disease data sets to provide a more targeted analysis. Two case studies, in subchondral bone in osteoarthritis and in Pax5 in acute lymphoblastic leukaemia, demonstrate that PhenomeExpress identifies core disease pathways in both mouse and human disease expression datasets derived from different technologies. We also validate the approach by comparison to state-of-the-art active sub-network detection methods, which reveals how it may enhance the detection of molecular phenotypes and provide a more detailed context to those previously identified as possible candidates.
Digital Assays Part II: Digital Protein and Cell Assays.

PubMed

Basu, Amar S

2017-08-01

A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.
Rodent motor and neuropsychological behaviour measured in home cages using the integrated modular platform SmartCage™

PubMed Central

Khroyan, Taline V; Zhang, Jingxi; Yang, Liya; Zou, Bende; Xie, James; Pascual, Conrado; Malik, Adam; Xie, Julian; Zaveri, Nurulain T; Vazquez, Jacqueline; Polgar, Willma; Toll, Lawrence; Fang, Jidong; Xie, Xinmin

2017-01-01

SUMMARY To facilitate investigation of diverse rodent behaviours in rodents’ home cages, we have developed an integrated modular platform, the SmartCage™ system (AfaSci, Inc. Burlingame, CA, USA), which enables automated neurobehavioural phenotypic analysis and in vivo drug screening in a relatively higher-throughput and more objective manner.The individual platform consists of an infrared array, a vibration floor sensor and a variety of modular devices. One computer can simultaneously operate up to 16 platforms via USB cables.The SmartCage™ detects drug-induced increases and decreases in activity levels, as well as changes in movement patterns. Wake and sleep states of mice can be detected using the vibration floor sensor. The arousal state classification achieved up to 98% accuracy compared with results obtained by electroencephalography and electromyography. More complex behaviours, including motor coordination, anxiety-related behaviours and social approach behaviour, can be assessed using appropriate modular devices and the results obtained are comparable with results obtained using conventional methods.In conclusion, the SmartCage™ system provides an automated and accurate tool to quantify various rodent behaviours in a ‘stress-free’ environment. This system, combined with the validated testing protocols, offers powerful a tool kit for transgenic phenotyping and in vivo drug screening. PMID:22540540
A CpG island methylator phenotype of colorectal cancer that is contiguous with conventional adenomas, but not serrated polyps

PubMed Central

HOKAZONO, KOJI; UEKI, TAKASHI; NAGAYOSHI, KINUKO; NISHIOKA, YASUNOBU; HATAE, TATSUNOBU; KOGA, YUTAKA; HIRAHASHI, MINAKO; ODA, YOSHINAO; TANAKA, MASAO

2014-01-01

A subset of colorectal cancers (CRCs) harbor the CpG island methylator phenotype (CIMP), with concurrent multiple promoter hypermethylation of tumor-related genes. A serrated pathway in which CIMP is developed from serrated polyps is proposed. The present study characterized CIMP and morphologically examined precursor lesions of CIMP. In total, 104 CRCs treated between January 1996 and December 2004 were examined. Aberrant promoter methylation of 15 cancer-related genes was analyzed. CIMP status was classified according to the number of methylated genes and was correlated with the clinicopathological features, including the concomitant polyps in and around the tumors. The frequency of aberrant methylation in each CRC showed a bimodal pattern, and the CRCs were classified as CIMP-high (CIMP-H), CIMP-low (CIMP-L) and CIMP-negative (CIMP-N). CIMP-H was associated with aberrant methylation of MLH1 (P=0.005) and with an improved recurrence-free survival (RFS) rate following curative resection compared with CIMP-L/N (five-year RFS rate, 93.8 vs. 67.1%; P=0.044), while CIMP-N tumors were associated with frequent distant metastases at diagnosis (P=0.023). No concomitant serrated lesions were present in the tumors, whereas conventional adenoma was contiguous with 11 (10.6%) of 104 CRCs, including four CIMP-H CRCs. CIMP-H was classified in CRCs by a novel CIMP marker panel and the presence of concomitant tumors revealed that certain CIMP-H CRCs may have arisen from conventional adenomas. PMID:25289081
A CpG island methylator phenotype of colorectal cancer that is contiguous with conventional adenomas, but not serrated polyps.

PubMed

Hokazono, Koji; Ueki, Takashi; Nagayoshi, Kinuko; Nishioka, Yasunobu; Hatae, Tatsunobu; Koga, Yutaka; Hirahashi, Minako; Oda, Yoshinao; Tanaka, Masao

2014-11-01

A subset of colorectal cancers (CRCs) harbor the CpG island methylator phenotype (CIMP), with concurrent multiple promoter hypermethylation of tumor-related genes. A serrated pathway in which CIMP is developed from serrated polyps is proposed. The present study characterized CIMP and morphologically examined precursor lesions of CIMP. In total, 104 CRCs treated between January 1996 and December 2004 were examined. Aberrant promoter methylation of 15 cancer-related genes was analyzed. CIMP status was classified according to the number of methylated genes and was correlated with the clinicopathological features, including the concomitant polyps in and around the tumors. The frequency of aberrant methylation in each CRC showed a bimodal pattern, and the CRCs were classified as CIMP-high (CIMP-H), CIMP-low (CIMP-L) and CIMP-negative (CIMP-N). CIMP-H was associated with aberrant methylation of MLH1 (P=0.005) and with an improved recurrence-free survival (RFS) rate following curative resection compared with CIMP-L/N (five-year RFS rate, 93.8 vs. 67.1%; P=0.044), while CIMP-N tumors were associated with frequent distant metastases at diagnosis (P=0.023). No concomitant serrated lesions were present in the tumors, whereas conventional adenoma was contiguous with 11 (10.6%) of 104 CRCs, including four CIMP-H CRCs. CIMP-H was classified in CRCs by a novel CIMP marker panel and the presence of concomitant tumors revealed that certain CIMP-H CRCs may have arisen from conventional adenomas.
Testing cross-phenotype effects of rare variants in longitudinal studies of complex traits.

PubMed

Rudra, Pratyaydipta; Broadaway, K Alaine; Ware, Erin B; Jhun, Min A; Bielak, Lawrence F; Zhao, Wei; Smith, Jennifer A; Peyser, Patricia A; Kardia, Sharon L R; Epstein, Michael P; Ghosh, Debashis

2018-06-01

Many gene mapping studies of complex traits have identified genes or variants that influence multiple phenotypes. With the advent of next-generation sequencing technology, there has been substantial interest in identifying rare variants in genes that possess cross-phenotype effects. In the presence of such effects, modeling both the phenotypes and rare variants collectively using multivariate models can achieve higher statistical power compared to univariate methods that either model each phenotype separately or perform separate tests for each variant. Several studies collect phenotypic data over time and using such longitudinal data can further increase the power to detect genetic associations. Although rare-variant approaches exist for testing cross-phenotype effects at a single time point, there is no analogous method for performing such analyses using longitudinal outcomes. In order to fill this important gap, we propose an extension of Gene Association with Multiple Traits (GAMuT) test, a method for cross-phenotype analysis of rare variants using a framework based on the distance covariance. The approach allows for both binary and continuous phenotypes and can also adjust for covariates. Our simple adjustment to the GAMuT test allows it to handle longitudinal data and to gain power by exploiting temporal correlation. The approach is computationally efficient and applicable on a genome-wide scale due to the use of a closed-form test whose significance can be evaluated analytically. We use simulated data to demonstrate that our method has favorable power over competing approaches and also apply our approach to exome chip data from the Genetic Epidemiology Network of Arteriopathy. © 2018 WILEY PERIODICALS, INC.
Scaling by shrinking: empowering single-cell ‘omics’ with microfluidic devices

PubMed Central

Prakadan, Sanjay M.; Shalek, Alex K.; Weitz, David A.

2017-01-01

Recent advances in cellular profiling have demonstrated substantial heterogeneity in the behaviour of cells once deemed ‘identical’, challenging fundamental notions of cell ‘type’ and ‘state’. Not surprisingly, these findings have elicited substantial interest in deeply characterizing the diversity, interrelationships and plasticity among cellular phenotypes. To explore these questions, experimental platforms are needed that can extensively and controllably profile many individual cells. Here, microfluidic structures—whether valve-, droplet- or nanowell-based—have an important role because they can facilitate easy capture and processing of single cells and their components, reducing labour and costs relative to conventional plate-based methods while also improving consistency. In this article, we review the current state-of-the-art methodologies with respect to microfluidics for mammalian single-cell ‘omics’ and discuss challenges and future opportunities. PMID:28392571
Genetic and Computational Approaches for Studying Plant Development and Abiotic Stress Responses Using Image-Based Phenotyping

NASA Astrophysics Data System (ADS)

Campbell, M. T.; Walia, H.; Grondin, A.; Knecht, A.

2017-12-01

The development of abiotic stress tolerant crops (i.e. drought, salinity, or heat stress) requires the discovery of DNA sequence variants associated with stress tolerance-related traits. However, many traits underlying adaptation to abiotic stress involve a suite of physiological pathways that may be induced at different times throughout the duration of stress. Conventional single-point phenotyping approaches fail to fully capture these temporal responses, and thus downstream genetic analysis may only identify a subset of the genetic variants that are important for adaptation to sub-optimal environments. Although genomic resources for crops have advanced tremendously, the collection of phenotypic data for morphological and physiological traits is laborious and remains a significant bottleneck in bridging the phenotype-genotype gap. In recent years, the availability of automated, image-based phenotyping platforms has provided researchers with an opportunity to collect morphological and physiological traits non-destructively in a highly controlled environment. Moreover, these platforms allow abiotic stress responses to be recorded throughout the duration of the experiment, and have facilitated the use of function-valued traits for genetic analyses in major crops. We will present our approaches for addressing abiotic stress tolerance in cereals. This talk will focus on novel open-source software to process and extract biological meaningful data from images generated from these phenomics platforms. In addition, we will discuss the statistical approaches to model longitudinal phenotypes and dissect the genetic basis of dynamic responses to these abiotic stresses throughout development.
Unmanned Aerial Vehicle Remote Sensing for Field-Based Crop Phenotyping: Current Status and Perspectives.

PubMed

Yang, Guijun; Liu, Jiangang; Zhao, Chunjiang; Li, Zhenhong; Huang, Yanbo; Yu, Haiyang; Xu, Bo; Yang, Xiaodong; Zhu, Dongmei; Zhang, Xiaoyan; Zhang, Ruyang; Feng, Haikuan; Zhao, Xiaoqing; Li, Zhenhai; Li, Heli; Yang, Hao

2017-01-01

Phenotyping plays an important role in crop science research; the accurate and rapid acquisition of phenotypic information of plants or cells in different environments is helpful for exploring the inheritance and expression patterns of the genome to determine the association of genomic and phenotypic information to increase the crop yield. Traditional methods for acquiring crop traits, such as plant height, leaf color, leaf area index (LAI), chlorophyll content, biomass and yield, rely on manual sampling, which is time-consuming and laborious. Unmanned aerial vehicle remote sensing platforms (UAV-RSPs) equipped with different sensors have recently become an important approach for fast and non-destructive high throughput phenotyping and have the advantage of flexible and convenient operation, on-demand access to data and high spatial resolution. UAV-RSPs are a powerful tool for studying phenomics and genomics. As the methods and applications for field phenotyping using UAVs to users who willing to derive phenotypic parameters from large fields and tests with the minimum effort on field work and getting highly reliable results are necessary, the current status and perspectives on the topic of UAV-RSPs for field-based phenotyping were reviewed based on the literature survey of crop phenotyping using UAV-RSPs in the Web of Science™ Core Collection database and cases study by NERCITA. The reference for the selection of UAV platforms and remote sensing sensors, the commonly adopted methods and typical applications for analyzing phenotypic traits by UAV-RSPs, and the challenge for crop phenotyping by UAV-RSPs were considered. The review can provide theoretical and technical support to promote the applications of UAV-RSPs for crop phenotyping.
Unmanned Aerial Vehicle Remote Sensing for Field-Based Crop Phenotyping: Current Status and Perspectives

PubMed Central

Yang, Guijun; Liu, Jiangang; Zhao, Chunjiang; Li, Zhenhong; Huang, Yanbo; Yu, Haiyang; Xu, Bo; Yang, Xiaodong; Zhu, Dongmei; Zhang, Xiaoyan; Zhang, Ruyang; Feng, Haikuan; Zhao, Xiaoqing; Li, Zhenhai; Li, Heli; Yang, Hao

2017-01-01

Phenotyping plays an important role in crop science research; the accurate and rapid acquisition of phenotypic information of plants or cells in different environments is helpful for exploring the inheritance and expression patterns of the genome to determine the association of genomic and phenotypic information to increase the crop yield. Traditional methods for acquiring crop traits, such as plant height, leaf color, leaf area index (LAI), chlorophyll content, biomass and yield, rely on manual sampling, which is time-consuming and laborious. Unmanned aerial vehicle remote sensing platforms (UAV-RSPs) equipped with different sensors have recently become an important approach for fast and non-destructive high throughput phenotyping and have the advantage of flexible and convenient operation, on-demand access to data and high spatial resolution. UAV-RSPs are a powerful tool for studying phenomics and genomics. As the methods and applications for field phenotyping using UAVs to users who willing to derive phenotypic parameters from large fields and tests with the minimum effort on field work and getting highly reliable results are necessary, the current status and perspectives on the topic of UAV-RSPs for field-based phenotyping were reviewed based on the literature survey of crop phenotyping using UAV-RSPs in the Web of Science™ Core Collection database and cases study by NERCITA. The reference for the selection of UAV platforms and remote sensing sensors, the commonly adopted methods and typical applications for analyzing phenotypic traits by UAV-RSPs, and the challenge for crop phenotyping by UAV-RSPs were considered. The review can provide theoretical and technical support to promote the applications of UAV-RSPs for crop phenotyping. PMID:28713402
A functional U-statistic method for association analysis of sequencing data.

PubMed

Jadhav, Sneha; Tong, Xiaoran; Lu, Qing

2017-11-01

Although sequencing studies hold great promise for uncovering novel variants predisposing to human diseases, the high dimensionality of the sequencing data brings tremendous challenges to data analysis. Moreover, for many complex diseases (e.g., psychiatric disorders) multiple related phenotypes are collected. These phenotypes can be different measurements of an underlying disease, or measurements characterizing multiple related diseases for studying common genetic mechanism. Although jointly analyzing these phenotypes could potentially increase the power of identifying disease-associated genes, the different types of phenotypes pose challenges for association analysis. To address these challenges, we propose a nonparametric method, functional U-statistic method (FU), for multivariate analysis of sequencing data. It first constructs smooth functions from individuals' sequencing data, and then tests the association of these functions with multiple phenotypes by using a U-statistic. The method provides a general framework for analyzing various types of phenotypes (e.g., binary and continuous phenotypes) with unknown distributions. Fitting the genetic variants within a gene using a smoothing function also allows us to capture complexities of gene structure (e.g., linkage disequilibrium, LD), which could potentially increase the power of association analysis. Through simulations, we compared our method to the multivariate outcome score test (MOST), and found that our test attained better performance than MOST. In a real data application, we apply our method to the sequencing data from Minnesota Twin Study (MTS) and found potential associations of several nicotine receptor subunit (CHRN) genes, including CHRNB3, associated with nicotine dependence and/or alcohol dependence. © 2017 WILEY PERIODICALS, INC.
Expression of conventional and novel glucose transporters, GLUT1, -9, -10, and -12, in vascular smooth muscle cells

PubMed Central

Pyla, Rajkumar; Poulose, Ninu; Jun, John Y.

2013-01-01

Intimal hyperplasia is characterized by exaggerated proliferation of vascular smooth muscle cells (VSMCs). Enhanced VSMC growth is dependent on increased glucose uptake and metabolism. Facilitative glucose transporters (GLUTs) are comprised of conventional GLUT isoforms (GLUT1–5) and novel GLUT isoforms (GLUT6–14). Previous studies demonstrate that GLUT1 overexpression or GLUT10 downregulation contribute to phenotypic changes in VSMCs. To date, the expression profile of all 14 GLUT isoforms has not been fully examined in VSMCs. Using the proliferative and differentiated phenotypes of human aortic VSMCs, the present study has determined the relative abundance of GLUT1–14 mRNAs by quantitative real-time PCR analysis. Twelve GLUT mRNAs excluding GLUT7 and GLUT14 were detectable in VSMCs. In the proliferative phenotype, the relative abundance of key GLUT mRNAs was GLUT1 (∼43%) > GLUT10 (∼26%) > GLUT9 (∼13%) > GLUT12 (∼4%), whereas in the differentiated phenotype the relative abundance was GLUT10 (∼28%) > GLUT1 (∼25%) > GLUT12 (∼20%) > GLUT9 (∼14%), together constituting 86–87% of total GLUT transcripts. To confirm the expression of key GLUT proteins, immunoblot and immunocytochemical analyses were performed using GLUT isoform-specific primary antibodies. The protein bands characteristic of GLUT1, -9, -10, and -12 were detected in VSMCs in parallel with respective positive controls. In particular, GLUT1 protein expression showed different molecular forms representative of altered glycosylation. While GLUT1 protein displayed a predominant distribution in the plasma membrane, GLUT9, -10, and -12 proteins were mostly distributed in the intracellular compartments. The present study provides the first direct evidence for GLUT9 and GLUT12 expression in VSMCs in conjunction with the previously identified GLUT1 and GLUT10. PMID:23302780
Literature-based prediction of novel drug indications considering relationships between entities.

PubMed

Jang, Giup; Lee, Taekeon; Lee, Byung Mun; Yoon, Youngmi

2017-06-27

There have been many attempts to identify and develop new uses for existing drugs, which is known as drug repositioning. Among these efforts, text mining is an effective means of discovering novel knowledge from a large amount of literature data. We identify a gene regulation by a drug and a phenotype based on the biomedical literature. Drugs or phenotypes can activate or inhibit gene regulation. We calculate the therapeutic possibility that a drug acts on a phenotype by means of these two types of regulation. We assume that a drug treats a phenotype if the genes regulated by the phenotype are inversely correlated with the genes regulated by the drug. Based on this hypothesis, we identify drug-phenotype associations with therapeutic possibility. To validate the drug-phenotype associations predicted by our method, we make an enrichment comparison with known drug-phenotype associations. We also identify candidate drugs for drug repositioning from novel associations and thus reveal that our method is a novel approach to drug repositioning.
An Assessment of Phylogenetic Tools for Analyzing the Interplay Between Interspecific Interactions and Phenotypic Evolution.

PubMed

Drury, J P; Grether, G F; Garland, T; Morlon, H

2018-05-01

Much ecological and evolutionary theory predicts that interspecific interactions often drive phenotypic diversification and that species phenotypes in turn influence species interactions. Several phylogenetic comparative methods have been developed to assess the importance of such processes in nature; however, the statistical properties of these methods have gone largely untested. Focusing mainly on scenarios of competition between closely-related species, we assess the performance of available comparative approaches for analyzing the interplay between interspecific interactions and species phenotypes. We find that many currently used statistical methods often fail to detect the impact of interspecific interactions on trait evolution, that sister-taxa analyses are particularly unreliable in general, and that recently developed process-based models have more satisfactory statistical properties. Methods for detecting predictors of species interactions are generally more reliable than methods for detecting character displacement. In weighing the strengths and weaknesses of different approaches, we hope to provide a clear guide for empiricists testing hypotheses about the reciprocal effect of interspecific interactions and species phenotypes and to inspire further development of process-based models.
Three Cases of Moraxella osloensis Meningitis: A Difficult Experience in Species Identification and Determination of Clinical Significance

PubMed Central

Roh, Kyoung Ho; Kim, Chang Ki; Koh, Eunmi; Kim, Myung Sook; Yong, Dongeun; Park, Soo Chul; Chong, Yunsop

2010-01-01

We had three cases of Moraxella osloensis meningitis. The species identification was impossible by conventional and commercial phenotypic tests. However, we could identify the species using the 16S rRNA gene sequencing. Determination of clinical significance was difficult in one patient. All three patients recovered by appropriate antimicrobial therapy. PMID:20191057

CHEMICAL EFFECTS IN BIOLOGICAL SYSTEMS – DATA DICTIONARY (CEBS-DD): A COMPENDIUM OF TERMS FOR THE CAPTURE AND INTEGRATION OF BIOLOGICAL STUDY DESIGN DESCRIPTION, CONVENTIONAL PHENOTYPES AND ‘OMICS’ DATA

EPA Science Inventory

A critical component in the design of the Chemical Effects in Biological Systems (CEBS) Knowledgebase is a strategy to capture toxicogenomics study protocols and the toxicity endpoint data (clinical pathology and histopathology). A Study is generally an experiment carried out du...
Williams-Beuren syndrome: phenotypic variability and deletions of chromosomes 7, 11, and 22 in a series of 52 patients.

PubMed Central

Joyce, C A; Zorich, B; Pike, S J; Barber, J C; Dennis, N R

1996-01-01

Fluorescence in situ hybridisation (FISH) and conventional chromosome analysis were performed on a series of 52 patients with classical Williams-Beuren syndrome (WBS), suspected WBS, or supravalvular aortic stenosis (SVAS). In the classical WBS group, 22/23 (96%) had a submicroscopic deletion of the elastin locus on chromosome 7, but the remaining patient had a unique interstitial deletion of chromosome 11 (del(11)(q13.5q14.2)). In the suspected WBS group 2/22 (9%) patients had elastin deletions but a third patient had a complex karyotype including a ring chromosome 22 with a deletion of the long arm (r(22)(p11-->q13)). In the SVAS group, 1/7 (14%) had an elastin gene deletion, despite having normal development and minimal signs of WBS. Overall, some patients with submicroscopic elastin deletions have fewer features of Williams-Beuren syndrome than those with other cytogenetic abnormalities. These results, therefore, emphasise the importance of a combined conventional and molecular cytogenetic approach to diagnosis and suggest that the degree to which submicroscopic deletions of chromosome 7 extend beyond the elastin locus may explain some of the phenotypic variability found in Williams-Beuren syndrome. Images PMID:9004128
Unsupervised automated high throughput phenotyping of RNAi time-lapse movies.

PubMed

Failmezger, Henrik; Fröhlich, Holger; Tresch, Achim

2013-10-04

Gene perturbation experiments in combination with fluorescence time-lapse cell imaging are a powerful tool in reverse genetics. High content applications require tools for the automated processing of the large amounts of data. These tools include in general several image processing steps, the extraction of morphological descriptors, and the grouping of cells into phenotype classes according to their descriptors. This phenotyping can be applied in a supervised or an unsupervised manner. Unsupervised methods are suitable for the discovery of formerly unknown phenotypes, which are expected to occur in high-throughput RNAi time-lapse screens. We developed an unsupervised phenotyping approach based on Hidden Markov Models (HMMs) with multivariate Gaussian emissions for the detection of knockdown-specific phenotypes in RNAi time-lapse movies. The automated detection of abnormal cell morphologies allows us to assign a phenotypic fingerprint to each gene knockdown. By applying our method to the Mitocheck database, we show that a phenotypic fingerprint is indicative of a gene's function. Our fully unsupervised HMM-based phenotyping is able to automatically identify cell morphologies that are specific for a certain knockdown. Beyond the identification of genes whose knockdown affects cell morphology, phenotypic fingerprints can be used to find modules of functionally related genes.
Multiple Phenotype Association Tests Using Summary Statistics in Genome-Wide Association Studies

PubMed Central

Liu, Zhonghua; Lin, Xihong

2017-01-01

Summary We study in this paper jointly testing the associations of a genetic variant with correlated multiple phenotypes using the summary statistics of individual phenotype analysis from Genome-Wide Association Studies (GWASs). We estimated the between-phenotype correlation matrix using the summary statistics of individual phenotype GWAS analyses, and developed genetic association tests for multiple phenotypes by accounting for between-phenotype correlation without the need to access individual-level data. Since genetic variants often affect multiple phenotypes differently across the genome and the between-phenotype correlation can be arbitrary, we proposed robust and powerful multiple phenotype testing procedures by jointly testing a common mean and a variance component in linear mixed models for summary statistics. We computed the p-values of the proposed tests analytically. This computational advantage makes our methods practically appealing in large-scale GWASs. We performed simulation studies to show that the proposed tests maintained correct type I error rates, and to compare their powers in various settings with the existing methods. We applied the proposed tests to a GWAS Global Lipids Genetics Consortium summary statistics data set and identified additional genetic variants that were missed by the original single-trait analysis. PMID:28653391
Multiple phenotype association tests using summary statistics in genome-wide association studies.

PubMed

Liu, Zhonghua; Lin, Xihong

2018-03-01

We study in this article jointly testing the associations of a genetic variant with correlated multiple phenotypes using the summary statistics of individual phenotype analysis from Genome-Wide Association Studies (GWASs). We estimated the between-phenotype correlation matrix using the summary statistics of individual phenotype GWAS analyses, and developed genetic association tests for multiple phenotypes by accounting for between-phenotype correlation without the need to access individual-level data. Since genetic variants often affect multiple phenotypes differently across the genome and the between-phenotype correlation can be arbitrary, we proposed robust and powerful multiple phenotype testing procedures by jointly testing a common mean and a variance component in linear mixed models for summary statistics. We computed the p-values of the proposed tests analytically. This computational advantage makes our methods practically appealing in large-scale GWASs. We performed simulation studies to show that the proposed tests maintained correct type I error rates, and to compare their powers in various settings with the existing methods. We applied the proposed tests to a GWAS Global Lipids Genetics Consortium summary statistics data set and identified additional genetic variants that were missed by the original single-trait analysis. © 2017, The International Biometric Society.
Method for restoration of normal phenotype in cancer cells

DOEpatents

Bissell, Mina J.; Weaver, Valerie M.

2000-01-01

A method for reversing expression of malignant phenotype in cancer cells is described. The method comprises applying .beta..sub.1 integrin function-blocking antibody to the cells. The method can be used to assess the progress of cancer therapy. Human breast epithelial cells were shown to be particularly responsive.
Phenotypic and genetic relations between the HEXACO dimensions and trait emotional intelligence.

PubMed

Veselka, Livia; Petrides, K V; Schermer, Julie Aitken; Cherkas, Lynn F; Spector, Tim D; Vernon, Philip A

2010-02-01

The present study investigated the location of trait emotional intelligence (trait EI or trait emotional self-efficacy) within the context of the HEXACO model - a more comprehensive personality framework than the conventional Big Five structure. A total of 666 MZ and 526 DZ adult twin pairs from the United Kingdom completed the short form of the Trait Emotional Intelligence Questionnaire (TEIQue-SF) and the short form of the HEXACO Personality Inventory (HEXACO-60). Many significant phenotypic correlations between the TEIQue-SF and the HEXACO-60 were obtained, which were strongest for HEXACO Extraversion, and weakest for HEXACO Honesty-Humility. As was expected, Emotionality was the only HEXACO dimension to correlate negatively with TEIQue-SF scores. Bivariate behavioral genetic analyses revealed that all phenotypic correlations were attributable to common genetic and common nonshared environmental factors. The study confirms the validity of trait EI as a constellation of emotional self-perceptions located at the lower levels of personality.
The usefulness of DNA sequencing after extraction by Whatman FTA filter matrix technology and phenotypic tests for differentiation of Candida albicans and Candida dubliniensis.

PubMed

Kiraz, Nuri; Oz, Yasemin; Aslan, Huseyin; Muslumanoglu, Hamza

2014-02-01

Since C. dubliniensis is similar to C. albicans phenotypically, it can be misidentified as C. albicans. We aimed to investigate the prevalence of C. dubliniensis among isolates previously identified as C. albicans in our stocks and to compare the phenotypic methods and DNA sequencing of D1/D2 region on the ribosomal large subunit (rLSU) gene. A total of 850 isolates included in this study. Phenotypic identification was performed based on germ tube formation, chlamydospore production, colony colors on chromogenic agar, inability of growth at 45 °C and growth on hypertonic Sabouraud dextrose agar. Eighty isolates compatible with C. dubliniensis by at least one phenotypic test were included in the sequence analysis. Nested PCR amplification of D1/D2 region of the rLSU gene was performed after the fungal DNA extraction by Whatman FTA filter paper technology. The sequencing analysis of PCR products carried out by an automated capillary gel electrophoresis device. The rate of C. dubliniensis was 2.35 % (n = 20) among isolates previously described as C. albicans. Consequently, none of the phenotypic tests provided satisfactory performance alone in our study, and molecular methods required special equipment and high cost. Thus, at least two phenotypic methods can be used for identification of C. dubliniensis, and molecular methods can be used for confirmation.
Next generation phenotyping using narrative reports in a rare disease clinical data warehouse.

PubMed

Garcelon, Nicolas; Neuraz, Antoine; Salomon, Rémi; Bahi-Buisson, Nadia; Amiel, Jeanne; Picard, Capucine; Mahlaoui, Nizar; Benoit, Vincent; Burgun, Anita; Rance, Bastien

2018-05-31

Secondary use of data collected in Electronic Health Records opens perspectives for increasing our knowledge of rare diseases. The clinical data warehouse (named Dr. Warehouse) at the Necker-Enfants Malades Children's Hospital contains data collected during normal care for thousands of patients. Dr. Warehouse is oriented toward the exploration of clinical narratives. In this study, we present our method to find phenotypes associated with diseases of interest. We leveraged the frequency and TF-IDF to explore the association between clinical phenotypes and rare diseases. We applied our method in six use cases: phenotypes associated with the Rett, Lowe, Silver Russell, Bardet-Biedl syndromes, DOCK8 deficiency and Activated PI3-kinase Delta Syndrome (APDS). We asked domain experts to evaluate the relevance of the top-50 (for frequency and TF-IDF) phenotypes identified by Dr. Warehouse and computed the average precision and mean average precision. Experts concluded that between 16 and 39 phenotypes could be considered as relevant in the top-50 phenotypes ranked by descending frequency discovered by Dr. Warehouse (resp. between 11 and 41 for TF-IDF). Average precision ranges from 0.55 to 0.91 for frequency and 0.52 to 0.95 for TF-IDF. Mean average precision was 0.79. Our study suggests that phenotypes identified in clinical narratives stored in Electronic Health Record can provide rare disease specialists with candidate phenotypes that can be used in addition to the literature. Clinical Data Warehouses can be used to perform Next Generation Phenotyping, especially in the context of rare diseases. We have developed a method to detect phenotypes associated with a group of patients using medical concepts extracted from free-text clinical narratives.
Integrating Multiple Correlated Phenotypes for Genetic Association Analysis by Maximizing Heritability

PubMed Central

Zhou, Jin J.; Cho, Michael H.; Lange, Christoph; Lutz, Sharon; Silverman, Edwin K.; Laird, Nan M.

2015-01-01

Many correlated disease variables are analyzed jointly in genetic studies in the hope of increasing power to detect causal genetic variants. One approach involves assessing the relationship between each phenotype and each single nucleotide polymorphism (SNP) individually and using a Bonferroni correction for the effective number of tests conducted. Alternatively, one can apply a multivariate regression or a dimension reduction technique, such as principal component analysis (PCA), and test for the association with the principal components (PC) of the phenotypes rather than the individual phenotypes. Inspired by the previous approaches of combining phenotypes to maximize heritability at individual SNPs, in this paper, we propose to construct a maximally heritable phenotype (MaxH) by taking advantage of the estimated total heritability and co-heritability. The heritability and co-heritability only need to be estimated once, therefore our method is applicable to genome-wide scans. MaxH phenotype is a linear combination of the individual phenotypes with increased heritability and power over the phenotypes being combined. Simulations show that the heritability and power achieved agree well with the theory for large samples and two phenotypes. We compare our approach with commonly used methods and assess both the heritability and the power of the MaxH phenotype. Moreover we provide suggestions for how to choose the phenotypes for combination. An application of our approach to a COPD genome-wide association study shows the practical relevance. PMID:26111731
The MAM rodent model of schizophrenia

PubMed Central

Lodge, Daniel J.

2013-01-01

Rodent models of human disease are essential to obtain a better understanding of disease pathology, the mechanism of action underlying conventional treatments, as well as for the generation of novel therapeutic approaches. There are a number of rodent models of schizophrenia based on either genetic manipulations, acute or sub-chronic drug administration, or developmental disturbances. The prenatal methylazoxymethanol acetate (MAM) rodent model is a developmental disruption model gaining increased attention because it displays a number of histological, neurophysiological and behavioral deficits analogous to those observed in schizophrenia patients. This unit describes the procedures required to safely induce the MAM phenotype in rats. In addition, we describe a simple behavioral procedure, amphetamine-induced hyper-locomotion, which can be utilized to verify the MAM phenotype. PMID:23559309
Phenotypic approaches to drought in cassava: review

PubMed Central

Okogbenin, Emmanuel; Setter, Tim L.; Ferguson, Morag; Mutegi, Rose; Ceballos, Hernan; Olasanmi, Bunmi; Fregene, Martin

2012-01-01

Cassava is an important crop in Africa, Asia, Latin America, and the Caribbean. Cassava can be produced adequately in drought conditions making it the ideal food security crop in marginal environments. Although cassava can tolerate drought stress, it can be genetically improved to enhance productivity in such environments. Drought adaptation studies in over three decades in cassava have identified relevant mechanisms which have been explored in conventional breeding. Drought is a quantitative trait and its multigenic nature makes it very challenging to effectively manipulate and combine genes in breeding for rapid genetic gain and selection process. Cassava has a long growth cycle of 12–18 months which invariably contributes to a long breeding scheme for the crop. Modern breeding using advances in genomics and improved genotyping, is facilitating the dissection and genetic analysis of complex traits including drought tolerance, thus helping to better elucidate and understand the genetic basis of such traits. A beneficial goal of new innovative breeding strategies is to shorten the breeding cycle using minimized, efficient or fast phenotyping protocols. While high throughput genotyping have been achieved, this is rarely the case for phenotyping for drought adaptation. Some of the storage root phenotyping in cassava are often done very late in the evaluation cycle making selection process very slow. This paper highlights some modified traits suitable for early-growth phase phenotyping that may be used to reduce drought phenotyping cycle in cassava. Such modified traits can significantly complement the high throughput genotyping procedures to fast track breeding of improved drought tolerant varieties. The need for metabolite profiling, improved phenomics to take advantage of next generation sequencing technologies and high throughput phenotyping are basic steps for future direction to improve genetic gain and maximize speed for drought tolerance breeding. PMID:23717282
Phenotypic and Genotypic Characterisation of Inflammatory Bowel Disease Presenting Before the Age of 2 years.

PubMed

Kammermeier, Jochen; Dziubak, Robert; Pescarin, Matilde; Drury, Suzanne; Godwin, Heather; Reeve, Kate; Chadokufa, Sibongile; Huggett, Bonita; Sider, Sara; James, Chela; Acton, Nikki; Cernat, Elena; Gasparetto, Marco; Noble-Jamieson, Gabi; Kiparissi, Fevronia; Elawad, Mamoun; Beales, Phil L; Sebire, Neil J; Gilmour, Kimberly; Uhlig, Holm H; Bacchelli, Chiara; Shah, Neil

2017-01-01

Inflammatory bowel disease [IBD] presenting in early childhood is extremely rare. More recently, progress has been made to identify children with monogenic forms of IBD predominantly presenting very early in life. In this study, we describe the heterogeneous phenotypes and genotypes of patients with IBD presenting before the age of 2 years and establish phenotypic features associated with underlying monogenicity. Phenotype data of 62 children with disease onset before the age of 2 years presenting over the past 20 years were reviewed. Children without previously established genetic diagnosis were prospectively recruited for next-generation sequencing. In all, 62 patients [55% male] were identified. The median disease onset was 3 months of age (interquartile range [IQR]: 1 to 11). Conventional IBD classification only applied to 15 patients with Crohn's disease [CD]-like [24%] and three with ulcerative colitis [UC]-like [5%] phenotype; 44 patients [71%] were diagnosed with otherwise unclassifiable IBD. Patients frequently required parenteral nutrition [40%], extensive immunosuppression [31%], haematopoietic stem-cell transplantation [29%], and abdominal surgery [19%]. In 31% of patients, underlying monogenic diseases were established [EPCAM, IL10, IL10RA, IL10RB, FOXP3, LRBA, SKIV2L, TTC37, TTC7A]. Phenotypic features significantly more prevalent in monogenic IBD were: consanguinity, disease onset before the 6th month of life, stunting, extensive intestinal disease and histological evidence of epithelial abnormalities. IBD in children with disease onset before the age of 2 years is frequently unclassifiable into Crohn's disease and ulcerative colitis, particularly treatment resistant, and can be indistinguishable from monogenic diseases with IBD-like phenotype. Copyright © 2016 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Acquisition of Epithelial-Mesenchymal Transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of Notch signaling pathway

PubMed Central

Wang, Zhiwei; Li, Yiwei; Kong, Dejuan; Banerjee, Sanjeev; Ahmad, Aamir; Azmi, Asfar Sohail; Ali, Shadan; Abbruzzese, James L.; Gallick, Gary E.; Sarkar, Fazlul H

2009-01-01

Despite rapid advances in many fronts, pancreatic cancer (PC) remains one of the most difficult human malignancies to treat, in part due to de novo and acquired chemo- and radio-resistance. Gemcitabine alone or in combination with other conventional therapeutics is the standard of care for the treatment of advanced PC without any significant improvement in the overall survival of patients diagnosed with this deadly disease. Previous studies have shown that PC cells that are gemcitabine-resistant (GR) acquired epithelial-mesenchymal transition (EMT) phenotype which is reminiscent of “cancer stem-like cells (CSC)”; however the molecular mechanism that led to EMT phenotype has not been fully investigated. The present study demonstrates that Notch-2 and its ligand Jagged-1 are highly up-regulated in GR cells, which is consistent with the role of Notch signaling pathway in the acquisition of EMT and CSC phenotype. We also found that the down-regulation of Notch signaling was associated with decreased invasive behavior of GR cells. Moreover, down-regulation of Notch signaling by siRNA approach led to partial reversal of the EMT phenotype, resulting in the mesenchymal-epithelial transition (MET), which was associated with decreased expression of vimentin, ZEB1, Slug, Snail and NF-κB. These results provide molecular evidence showing that the activation of Notch signaling is mechanistically linked with chemo-resistance phenotype (EMT phenotype) of PC cells, suggesting that the inactivation of Notch signaling by novel strategies could be a potential targeted therapeutic approach for overcoming chemo-resistance toward the prevention of tumor progression and/or treatment of metastatic PC. PMID:19276344
Molecular Surveillance of True Nontypeable Haemophilus influenzae: An Evaluation of PCR Screening Assays

PubMed Central

Binks, Michael J.; Temple, Beth; Kirkham, Lea-Ann; Wiertsema, Selma P.; Dunne, Eileen M.; Richmond, Peter C.; Marsh, Robyn L.; Leach, Amanda J.; Smith-Vaughan, Heidi C.

2012-01-01

Background Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Methodology/Principal Findings Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Conclusions/Significance Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease. PMID:22470516
Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays.

PubMed

Binks, Michael J; Temple, Beth; Kirkham, Lea-Ann; Wiertsema, Selma P; Dunne, Eileen M; Richmond, Peter C; Marsh, Robyn L; Leach, Amanda J; Smith-Vaughan, Heidi C

2012-01-01

Unambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination. Here we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction. Our data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease.
Development of a Melanoma Risk Prediction Model Incorporating MC1R Genotype and Indoor Tanning Exposure: Impact of Mole Phenotype on Model Performance

PubMed Central

Penn, Lauren A.; Qian, Meng; Zhang, Enhan; Ng, Elise; Shao, Yongzhao; Berwick, Marianne; Lazovich, DeAnn; Polsky, David

2014-01-01

Background Identifying individuals at increased risk for melanoma could potentially improve public health through targeted surveillance and early detection. Studies have separately demonstrated significant associations between melanoma risk, melanocortin receptor (MC1R) polymorphisms, and indoor ultraviolet light (UV) exposure. Existing melanoma risk prediction models do not include these factors; therefore, we investigated their potential to improve the performance of a risk model. Methods Using 875 melanoma cases and 765 controls from the population-based Minnesota Skin Health Study we compared the predictive ability of a clinical melanoma risk model (Model A) to an enhanced model (Model F) using receiver operating characteristic (ROC) curves. Model A used self-reported conventional risk factors including mole phenotype categorized as “none”, “few”, “some” or “many” moles. Model F added MC1R genotype and measures of indoor and outdoor UV exposure to Model A. We also assessed the predictive ability of these models in subgroups stratified by mole phenotype (e.g. nevus-resistant (“none” and “few” moles) and nevus-prone (“some” and “many” moles)). Results Model A (the reference model) yielded an area under the ROC curve (AUC) of 0.72 (95% CI = 0.69, 0.74). Model F was improved with an AUC = 0.74 (95% CI = 0.71–0.76, p<0.01). We also observed substantial variations in the AUCs of Models A & F when examined in the nevus-prone and nevus-resistant subgroups. Conclusions These results demonstrate that adding genotypic information and environmental exposure data can increase the predictive ability of a clinical melanoma risk model, especially among nevus-prone individuals. PMID:25003831
Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients.

PubMed

Hoshino, Tomonori; Fujiwara, Taku; Kilian, Mogens

2005-12-01

The aim of this study was to evaluate molecular and phenotypic methods for the identification of nonhemolytic streptococci. A collection of 148 strains consisting of 115 clinical isolates from cases of infective endocarditis, septicemia, and meningitis and 33 reference strains, including type strains of all relevant Streptococcus species, were examined. Identification was performed by phylogenetic analysis of nucleotide sequences of four housekeeping genes, ddl, gdh, rpoB, and sodA; by PCR analysis of the glucosyltransferase (gtf) gene; and by conventional phenotypic characterization and identification using two commercial kits, Rapid ID 32 STREP and STREPTOGRAM and the associated databases. A phylogenetic tree based on concatenated sequences of the four housekeeping genes allowed unequivocal differentiation of recognized species and was used as the reference. Analysis of single gene sequences revealed deviation clustering in eight strains (5.4%) due to homologous recombination with other species. This was particularly evident in S. sanguinis and in members of the anginosus group of streptococci. The rate of correct identification of the strains by both commercial identification kits was below 50% but varied significantly between species. The most significant problems were observed with S. mitis and S. oralis and 11 Streptococcus species described since 1991. Our data indicate that identification based on multilocus sequence analysis is optimal. As a more practical alternative we recommend identification based on sodA sequences with reference to a comprehensive set of sequences that is available for downloading from our server. An analysis of the species distribution of 107 nonhemolytic streptococci from bacteremic patients showed a predominance of S. oralis and S. anginosus with various underlying infections.
PCAN: phenotype consensus analysis to support disease-gene association.

PubMed

Godard, Patrice; Page, Matthew

2016-12-07

Bridging genotype and phenotype is a fundamental biomedical challenge that underlies more effective target discovery and patient-tailored therapy. Approaches that can flexibly and intuitively, integrate known gene-phenotype associations in the context of molecular signaling networks are vital to effectively prioritize and biologically interpret genes underlying disease traits of interest. We describe Phenotype Consensus Analysis (PCAN); a method to assess the consensus semantic similarity of phenotypes in a candidate gene's signaling neighborhood. We demonstrate that significant phenotype consensus (p < 0.05) is observable for ~67% of 4,549 OMIM disease-gene associations, using a combination of high quality String interactions + Metabase pathways and use Joubert Syndrome to demonstrate the ease with which a significant result can be interrogated to highlight discriminatory traits linked to mechanistically related genes. We advocate phenotype consensus as an intuitive and versatile method to aid disease-gene association, which naturally lends itself to the mechanistic deconvolution of diverse phenotypes. We provide PCAN to the community as an R package ( http://bioconductor.org/packages/PCAN/ ) to allow flexible configuration, extension and standalone use or integration to supplement existing gene prioritization workflows.
Identification by 16S rRNA gene sequencing of an Actinomyces hongkongensis isolate recovered from a patient with pelvic actinomycosis.

PubMed

Flynn, A N; Lyndon, C A; Church, D L

2013-08-01

A case of Actinomyces hongkongensis pelvic actinomycosis in an adult woman is described. Conventional phenotypic tests failed to identify the Gram-positive bacillus isolated from a fluid aspirate of a pelvic abscess. The bacterium was identified by 16S rRNA gene sequencing and analysis using the SmartGene Integrated Database Network System software.

Heterogeneity in the distribution of genetically modified and conventional oilseed rape within fields and seed lots.

PubMed

Begg, Graham S; Elliott, Martin J; Cullen, Danny W; Iannetta, Pietro P M; Squire, Geoff R

2008-10-01

The implementation of co-existence in the commercialisation of GM crops requires GM and non-GM products to be segregated in production and supply. However, maintaining segregation in oilseed rape will be made difficult by the highly persistent nature of this species. An understanding of its population dynamics is needed to predict persistence and develop potential strategies for control, while to ensure segregation is being achieved, the production of GM oilseed rape must be accompanied by the monitoring of GM levels in crop or seed populations. Heterogeneity in the spatial distribution of oilseed rape has the potential to affect both control and monitoring and, although a universal phenomenon in arable weeds and harvested seed lots, spatial heterogeneity in oilseed rape populations remains to be demonstrated and quantified. Here we investigate the distribution of crop and volunteer populations in a commercial field before and during the cultivation of the first conventional oilseed rape (winter) crop since the cultivation of a GM glufosinate-tolerant oilseed rape crop (spring) three years previously. GM presence was detected by ELISA for the PAT protein in each of three morphologically distinguishable phenotypes: autumn germinating crop-type plants (3% GM), autumn-germinating 'regrowths' (72% GM) and spring germinating 'small-type' plants (17% GM). Statistical models (Poisson log-normal and binomial logit-normal) were used to describe the spatial distribution of these populations at multiple spatial scales in the field and of GM presence in the harvested seed lot. Heterogeneity was a consistent feature in the distribution of GM and conventional oilseed rape. Large trends across the field (50 x 400 m) and seed lot (4 x 1.5 x 1.5 m) were observed in addition to small-scale heterogeneity, less than 20 m in the field and 20 cm in the seed lot. The heterogeneity was greater for the 'regrowth' and 'small' phenotypes, which were likely to be volunteers and included most of the GM plants detected, than for the largely non-GM 'crop' phenotype. The implications of the volunteer heterogeneity for field management and GM-sampling are discussed.
PhenStat | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

PhenStat is a freely available R package that provides a variety of statistical methods for the identification of phenotypic associations from model organisms developed for the International Mouse Phenotyping Consortium (IMPC at www.mousephenotype.org ). The methods have been developed for high throughput phenotyping pipelines implemented across various experimental designs with an emphasis on managing temporal variation and is being adapted for analysis with PDX mouse strains.
The Second Report on the State of the World's Animal Genetic Resources for Food and Agriculture, Part 4, The State of the Art: Box 4A4: A digital enumeration method for collecting phenotypic data for genome association

USDA-ARS?s Scientific Manuscript database

Consistent data across animal populations are required to inform genomic science aimed at finding important adaptive genetic variations. The ADAPTMap Digital Phenotype Collection- Prototype Method will yield a new procedure to provide consistent phenotypic data by digital enumeration of categorical ...
Conventional and hyperspectral time-series imaging of maize lines widely used in field trials

PubMed Central

Liang, Zhikai; Pandey, Piyush; Stoerger, Vincent; Xu, Yuhang; Qiu, Yumou; Ge, Yufeng

2018-01-01

Abstract Background Maize (Zea mays ssp. mays) is 1 of 3 crops, along with rice and wheat, responsible for more than one-half of all calories consumed around the world. Increasing the yield and stress tolerance of these crops is essential to meet the growing need for food. The cost and speed of plant phenotyping are currently the largest constraints on plant breeding efforts. Datasets linking new types of high-throughput phenotyping data collected from plants to the performance of the same genotypes under agronomic conditions across a wide range of environments are essential for developing new statistical approaches and computer vision–based tools. Findings A set of maize inbreds—primarily recently off patent lines—were phenotyped using a high-throughput platform at University of Nebraska-Lincoln. These lines have been previously subjected to high-density genotyping and scored for a core set of 13 phenotypes in field trials across 13 North American states in 2 years by the Genomes 2 Fields Consortium. A total of 485 GB of image data including RGB, hyperspectral, fluorescence, and thermal infrared photos has been released. Conclusions Correlations between image-based measurements and manual measurements demonstrated the feasibility of quantifying variation in plant architecture using image data. However, naive approaches to measuring traits such as biomass can introduce nonrandom measurement errors confounded with genotype variation. Analysis of hyperspectral image data demonstrated unique signatures from stem tissue. Integrating heritable phenotypes from high-throughput phenotyping data with field data from different environments can reveal previously unknown factors that influence yield plasticity. PMID:29186425
Cumulative live birth rates after IVF in patients with polycystic ovaries: phenotype matters.

PubMed

De Vos, Michel; Pareyn, Stéphanie; Drakopoulos, Panagiotis; Raimundo, José M; Anckaert, Ellen; Santos-Ribeiro, Samuel; Polyzos, Nikolaos P; Tournaye, Herman; Blockeel, Christophe

2018-05-07

Do cumulative live birth rates (CLBR) vary among women with different polycystic ovary syndrome (PCOS) phenotypes who undergo IVF/intracytoplasmic sperm injection (ICSI) treatment? In this retrospective cohort study, data from 567 patients undergoing an assisted reproductive technology (ART) cycle between January 2010 and December 2015 were collected. Demographical traits, cycle characteristics and clinical and laboratory data were analysed. After conventional ovarian stimulation using a gonadotrophin-releasing hormone antagonist protocol, the median number of oocytes retrieved ranged between 11 and 13.5 and did not differ significantly among the studied groups. Live birth rate (LBR) after fresh embryo transfer and CLBR after transfer of all fresh and vitrified embryos were significantly lower in women with hyperandrogenic PCOS phenotypes A (LBR 16.7%, CLBR 25.8%) and C (LBR 18.5%, CLBR 27.8%) compared with women with normoandrogenic PCOS phenotype D (LBR 33.7%, CLBR 48%) (P-value for LBR 0.01 and 0.03, respectively; P-value for CLBR 0.002 and 0.01, respectively) and controls with a polycystic ovarian morphology (LBR 37.1%, CLBR 53.3%) (P-value for LBR 0.002 and 0.01, respectively; P-value for CLBR <0.001 and 0.001, respectively). Multivariate regression analysis indicated that after adjustment for relevant confounders, PCOS phenotype was an independent predictor for CLBR. Hyperandrogenic PCOS phenotypes confer significantly lower CLBR compared with their normoandrogenic counterparts. These findings may imply the need for adapted counselling and tailored approaches when treating PCOS patients with hyperandrogenism who require ART. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
Conventional and hyperspectral time-series imaging of maize lines widely used in field trials.

PubMed

Liang, Zhikai; Pandey, Piyush; Stoerger, Vincent; Xu, Yuhang; Qiu, Yumou; Ge, Yufeng; Schnable, James C

2018-02-01

Maize (Zea mays ssp. mays) is 1 of 3 crops, along with rice and wheat, responsible for more than one-half of all calories consumed around the world. Increasing the yield and stress tolerance of these crops is essential to meet the growing need for food. The cost and speed of plant phenotyping are currently the largest constraints on plant breeding efforts. Datasets linking new types of high-throughput phenotyping data collected from plants to the performance of the same genotypes under agronomic conditions across a wide range of environments are essential for developing new statistical approaches and computer vision-based tools. A set of maize inbreds-primarily recently off patent lines-were phenotyped using a high-throughput platform at University of Nebraska-Lincoln. These lines have been previously subjected to high-density genotyping and scored for a core set of 13 phenotypes in field trials across 13 North American states in 2 years by the Genomes 2 Fields Consortium. A total of 485 GB of image data including RGB, hyperspectral, fluorescence, and thermal infrared photos has been released. Correlations between image-based measurements and manual measurements demonstrated the feasibility of quantifying variation in plant architecture using image data. However, naive approaches to measuring traits such as biomass can introduce nonrandom measurement errors confounded with genotype variation. Analysis of hyperspectral image data demonstrated unique signatures from stem tissue. Integrating heritable phenotypes from high-throughput phenotyping data with field data from different environments can reveal previously unknown factors that influence yield plasticity. © The Authors 2017. Published by Oxford University Press.
Overcoming the anaerobic hurdle in phenotypic microarrays: Generation andvisualization of growth curve data for Desulfovibrio vulgaris Hildenborough

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borglin, Sharon E; Joyner, Dominique; Jacobsen, Janet

2008-10-04

Growing anaerobic microorganisms in phenotypic microarrays (PM) and 96-well microtiter plates is an emerging technology that allows high throughput survey of the growth and physiology and/or phenotype of cultivable microorganisms. For non-model bacteria, a method for phenotypic analysis is invaluable, not only to serve as a starting point for further evaluation, but also to provide a broad understanding of the physiology of an uncharacterized wild-type organism or the physiology/phenotype of a newly created mutant of that organism. Given recent advances in genetic characterization and targeted mutations to elucidate genetic networks and metabolic pathways, high-throughput methods for determining phenotypic differences aremore » essential. Here we outline challenges presented in studying the physiology and phenotype of a sulfate reducing anaerobic delta proteobacterium, Desulfovibrio vulgaris Hildenborough. Modifications of the commercially available OmniLog(TM) system (Hayward, CA) for experimental setup, and configuration, as well as considerations in PM data analysis are presented. Also highlighted here is data viewing software that enables users to view and compare multiple PM data sets. The PM method promises to be a valuable strategy in our systems biology approach to D. vulgaris studies and is readily applicable to other anaerobic and aerobic bacteria.« less
Restoration of normal phenotype in cancer cells

DOEpatents

Bissell, M.J.; Weaver, V.M.

1998-12-08

A method for reversing expression of malignant phenotype in cancer cells is described. The method comprises applying {beta}{sub 1} integrin function-blocking antibody to the cells. The method can be used to assess the progress of cancer therapy. Human breast epithelial cells were shown to be particularly responsive. 14 figs.
Restoration of normal phenotype in cancer cells

DOEpatents

Bissell, Mina J.; Weaver, Valerie M.

1998-01-01

A method for reversing expression of malignant phenotype in cancer cells is described. The method comprises applying .beta..sub.1 integrin function-blocking antibody to the cells. The method can be used to assess the progress of cancer therapy. Human breast epithelial cells were shown to be particularly responsive.
Characteristics of Fluency and Speech in Two Families with High Incidences of Stuttering

ERIC Educational Resources Information Center

Stager, Sheila V.; Freeman, Frances J.; Braun, Allen

2015-01-01

Purpose: This study presents data from 2 families with high incidence of stuttering, comparing methods of phenotype assignment and exploring the presence of other fluency disorders and corresponding speech characteristics. Method: Three methods for assigning phenotype of stuttering were used: self-identification, family identification, and expert…
Conceptual dissonance: evaluating the efficacy of natural language processing techniques for validating translational knowledge constructs.

PubMed

Payne, Philip R O; Kwok, Alan; Dhaval, Rakesh; Borlawsky, Tara B

2009-03-01

The conduct of large-scale translational studies presents significant challenges related to the storage, management and analysis of integrative data sets. Ideally, the application of methodologies such as conceptual knowledge discovery in databases (CKDD) provides a means for moving beyond intuitive hypothesis discovery and testing in such data sets, and towards the high-throughput generation and evaluation of knowledge-anchored relationships between complex bio-molecular and phenotypic variables. However, the induction of such high-throughput hypotheses is non-trivial, and requires correspondingly high-throughput validation methodologies. In this manuscript, we describe an evaluation of the efficacy of a natural language processing-based approach to validating such hypotheses. As part of this evaluation, we will examine a phenomenon that we have labeled as "Conceptual Dissonance" in which conceptual knowledge derived from two or more sources of comparable scope and granularity cannot be readily integrated or compared using conventional methods and automated tools.
A commentary on the role of molecular technology and automation in clinical diagnostics

PubMed Central

O’Connor, Ciara; Fitzgibbon, Marie; Powell, James; Barron, Denis; O’Mahony, Jim; Power, Lorraine; O’Connell, Nuala H; Dunne, Colum

2014-01-01

Historically, the identification of bacterial or yeast isolates has been based on phenotypic characteristics such as growth on defined media, colony morphology, Gram stain, and various biochemical reactions, with significant delay in diagnosis. Clinical microbiology as a medical specialty has embraced advances in molecular technology for rapid species identification with broad-range 16S rDNA polymerase chain reaction (PCR) and matrix-assisted laser desorption and/or ionization time of flight (MALDI-TOF) mass spectrometry demonstrated as accurate, rapid, and cost-effective methods for the identification of most, but not all, bacteria and yeasts. Protracted conventional incubation times previously necessary to identify certain species have been mitigated, affording patients quicker diagnosis with associated reduction in exposure to empiric broad-spectrum antimicrobial therapy and shortened hospital stay. This short commentary details such molecular advances and their implications in the clinical microbiology setting. PMID:24658184
Media Ion Composition Controls Regulatory and Virulence Response of Salmonella in Spaceflight

PubMed Central

Wilson, James W.; Ott, C. Mark; Quick, Laura; Davis, Richard; zu Bentrup, Kerstin Höner; Crabbé, Aurélie; Richter, Emily; Sarker, Shameema; Barrila, Jennifer; Porwollik, Steffen; Cheng, Pui; McClelland, Michael; Tsaprailis, George; Radabaugh, Timothy; Hunt, Andrea; Shah, Miti; Nelman-Gonzalez, Mayra; Hing, Steve; Parra, Macarena; Dumars, Paula; Norwood, Kelly; Bober, Ramona; Devich, Jennifer; Ruggles, Ashleigh; CdeBaca, Autumn; Narayan, Satro; Benjamin, Joseph; Goulart, Carla; Rupert, Mark; Catella, Luke; Schurr, Michael J.; Buchanan, Kent; Morici, Lisa; McCracken, James; Porter, Marc D.; Pierson, Duane L.; Smith, Scott M.; Mergeay, Max; Leys, Natalie; Stefanyshyn-Piper, Heidemarie M.; Gorie, Dominic; Nickerson, Cheryl A.

2008-01-01

The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth. PMID:19079590
Regulating transgenic crops sensibly: lessons from plant breeding, biotechnology and genomics.

PubMed

Bradford, Kent J; Van Deynze, Allen; Gutterson, Neal; Parrott, Wayne; Strauss, Steven H

2005-04-01

The costs of meeting regulatory requirements and market restrictions guided by regulatory criteria are substantial impediments to the commercialization of transgenic crops. Although a cautious approach may have been prudent initially, we argue that some regulatory requirements can now be modified to reduce costs and uncertainty without compromising safety. Long-accepted plant breeding methods for incorporating new diversity into crop varieties, experience from two decades of research on and commercialization of transgenic crops, and expanding knowledge of plant genome structure and dynamics all indicate that if a gene or trait is safe, the genetic engineering process itself presents little potential for unexpected consequences that would not be identified or eliminated in the variety development process before commercialization. We propose that as in conventional breeding, regulatory emphasis should be on phenotypic rather than genomic characteristics once a gene or trait has been shown to be safe.
A commentary on the role of molecular technology and automation in clinical diagnostics.

PubMed

O'Connor, Ciara; Fitzgibbon, Marie; Powell, James; Barron, Denis; O'Mahony, Jim; Power, Lorraine; O'Connell, Nuala H; Dunne, Colum

2014-01-01

Historically, the identification of bacterial or yeast isolates has been based on phenotypic characteristics such as growth on defined media, colony morphology, Gram stain, and various biochemical reactions, with significant delay in diagnosis. Clinical microbiology as a medical specialty has embraced advances in molecular technology for rapid species identification with broad-range 16S rDNA polymerase chain reaction (PCR) and matrix-assisted laser desorption and/or ionization time of flight (MALDI-TOF) mass spectrometry demonstrated as accurate, rapid, and cost-effective methods for the identification of most, but not all, bacteria and yeasts. Protracted conventional incubation times previously necessary to identify certain species have been mitigated, affording patients quicker diagnosis with associated reduction in exposure to empiric broad-spectrum antimicrobial therapy and shortened hospital stay. This short commentary details such molecular advances and their implications in the clinical microbiology setting.
Analysis of Exhaled Breath for Disease Detection

NASA Astrophysics Data System (ADS)

Amann, Anton; Miekisch, Wolfram; Schubert, Jochen; Buszewski, Bogusław; Ligor, Tomasz; Jezierski, Tadeusz; Pleil, Joachim; Risby, Terence

2014-06-01

Breath analysis is a young field of research with great clinical potential. As a result of this interest, researchers have developed new analytical techniques that permit real-time analysis of exhaled breath with breath-to-breath resolution in addition to the conventional central laboratory methods using gas chromatography-mass spectrometry. Breath tests are based on endogenously produced volatiles, metabolites of ingested precursors, metabolites produced by bacteria in the gut or the airways, or volatiles appearing after environmental exposure. The composition of exhaled breath may contain valuable information for patients presenting with asthma, renal and liver diseases, lung cancer, chronic obstructive pulmonary disease, inflammatory lung disease, or metabolic disorders. In addition, oxidative stress status may be monitored via volatile products of lipid peroxidation. Measurement of enzyme activity provides phenotypic information important in personalized medicine, whereas breath measurements provide insight into perturbations of the human exposome and can be interpreted as preclinical signals of adverse outcome pathways.
Tepidimonas arfidensis Sp. Nov., a Novel Gram-negative and thermophilic bacterium isolated from the bone marrow of a patient with leukemia in Korea.

PubMed

Ko, Kwan Soo; Lee, Nam Yong; Oh, Won Sup; Lee, Jang Ho; Ki, Hyun Kyun; Peck, Kyong Ran; Song, Jae-Hoon

2005-01-01

A Gram-negative bacillus, SMC-6271(T), which was isolated from the bone marrow of a patient with leukemia but could not be identified by a conventional microbiologic method, was characterized by a genotypic analysis of 16S rRNA gene. Sequences of the 16S rRNA gene revealed that this bacterium was closely related to Tepidimonas ignava and other slightly thermophilic isolates but diverged distinctly from them. Analyses of cellular fatty acid composition and performance of biochemical tests confirmed that this bacterium is a distinct species from the other Tepidimonas species. Based on the evaluated phenotypic and genotypic characteristics, it is proposed that SMC-6271T (=ABB 0301T =KCTC 12412T =JCM 13232T) should be classified as a new species, namely Tepidimonas arfidensis sp. nov.
Application of Digital Anthropometry for Craniofacial Assessment

PubMed Central

Jayaratne, Yasas S. N.; Zwahlen, Roger A.

2014-01-01

Craniofacial anthropometry is an objective technique based on a series of measurements and proportions, which facilitate the characterization of phenotypic variation and quantification of dysmorphology. With the introduction of stereophotography, it is possible to acquire a lifelike three-dimensional (3D) image of the face with natural color and texture. Most of the traditional anthropometric landmarks can be identified on these 3D photographs using specialized software. Therefore, it has become possible to compute new digital measurements, which were not feasible with traditional instruments. The term “digital anthropometry” has been used by researchers based on such systems to separate their methods from conventional manual measurements. Anthropometry has been traditionally used as a research tool. With the advent of digital anthropometry, this technique can be employed in several disciplines as a noninvasive tool for quantifying facial morphology. The aim of this review is to provide a broad overview of digital anthropometry and discuss its clinical applications. PMID:25050146
Use of NAP gene to manipulate leaf senescence in plants

DOEpatents

Gan, Susheng; Guo, Yongfeng

2013-04-16

The present invention discloses transgenic plants having an altered level of NAP protein compared to that of a non-transgenic plant, where the transgenic plants display an altered leaf senescence phenotype relative to a non-transgenic plant, as well as mutant plants comprising an inactivated NAP gene, where mutant plants display a delayed leaf senescence phenotype compared to that of a non-mutant plant. The present invention also discloses methods for delaying leaf senescence in a plant, as well as methods of making a mutant plant having a decreased level of NAP protein compared to that of a non-mutant plant, where the mutant plant displays a delayed leaf senescence phenotype relative to a non-mutant plant. Methods for causing precocious leaf senescence or promoting leaf senescence in a plant are also disclosed. Also disclosed are methods of identifying a candidate plant suitable for breeding that displays a delayed leaf senescence and/or enhanced yield phenotype.
GC[Formula: see text]NMF: A Novel Matrix Factorization Framework for Gene-Phenotype Association Prediction.

PubMed

Zhang, Yaogong; Liu, Jiahui; Liu, Xiaohu; Hong, Yuxiang; Fan, Xin; Huang, Yalou; Wang, Yuan; Xie, Maoqiang

2018-04-24

Gene-phenotype association prediction can be applied to reveal the inherited basis of human diseases and facilitate drug development. Gene-phenotype associations are related to complex biological processes and influenced by various factors, such as relationship between phenotypes and that among genes. While due to sparseness of curated gene-phenotype associations and lack of integrated analysis of the joint effect of multiple factors, existing applications are limited to prediction accuracy and potential gene-phenotype association detection. In this paper, we propose a novel method by exploiting weighted graph constraint learned from hierarchical structures of phenotype data and group prior information among genes by inheriting advantages of Non-negative Matrix Factorization (NMF), called Weighted Graph Constraint and Group Centric Non-negative Matrix Factorization (GC[Formula: see text]NMF). Specifically, first we introduce the depth of parent-child relationships between two adjacent phenotypes in hierarchical phenotypic data as weighted graph constraint for a better phenotype understanding. Second, we utilize intra-group correlation among genes in a gene group as group constraint for gene understanding. Such information provides us with the intuition that genes in a group probably result in similar phenotypes. The model not only allows us to achieve a high-grade prediction performance, but also helps us to learn interpretable representation of genes and phenotypes simultaneously to facilitate future biological analysis. Experimental results on biological gene-phenotype association datasets of mouse and human demonstrate that GC[Formula: see text]NMF can obtain superior prediction accuracy and good understandability for biological explanation over other state-of-the-arts methods.

A Brief Critique of the TATES Procedure.

PubMed

Aliev, Fazil; Salvatore, Jessica E; Agrawal, Arpana; Almasy, Laura; Chan, Grace; Edenberg, Howard J; Hesselbrock, Victor; Kuperman, Samuel; Meyers, Jacquelyn; Dick, Danielle M

2018-03-01

The Trait-based test that uses the Extended Simes procedure (TATES) was developed as a method for conducting multivariate GWAS for correlated phenotypes whose underlying genetic architecture is complex. In this paper, we provide a brief methodological critique of the TATES method using simulated examples and a mathematical proof. Our simulated examples using correlated phenotypes show that the Type I error rate is higher than expected, and that more TATES p values fall outside of the confidence interval relative to expectation. Thus the method may result in systematic inflation when used with correlated phenotypes. In a mathematical proof we further demonstrate that the distribution of TATES p values deviates from expectation in a manner indicative of inflation. Our findings indicate the need for caution when using TATES for multivariate GWAS of correlated phenotypes.
A nondestructive method to estimate the chlorophyll content of Arabidopsis seedlings

DOE PAGES

Liang, Ying; Urano, Daisuke; Liao, Kang-Ling; ...

2017-04-14

Chlorophyll content decreases in plants under stress conditions, therefore it is used commonly as an indicator of plant health. Arabidopsis thaliana offers a convenient and fast way to test physiological phenotypes of mutations and treatments. But, chlorophyll measurements with conventional solvent extraction are not applicable to Arabidopsis leaves due to their small size, especially when grown on culture dishes. We provide a nondestructive method for chlorophyll measurement whereby the red, green and blue (RGB) values of a color leaf image is used to estimate the chlorophyll content from Arabidopsis leaves. The method accommodates different profiles of digital cameras by incorporatingmore » the ColorChecker chart to make the digital negative profiles, to adjust the white balance, and to calibrate the exposure rate differences caused by the environment so that this method is applicable in any environment. We chose an exponential function model to estimate chlorophyll content from the RGB values, and fitted the model parameters with physical measurements of chlorophyll contents. As further proof of utility, this method was used to estimate chlorophyll content of G protein mutants grown on different sugar to nitrogen ratios. Our method is a simple, fast, inexpensive, and nondestructive estimation of chlorophyll content of Arabidopsis seedlings. This method lead to the discovery that G proteins are important in sensing the C/N balance to control chlorophyll content in Arabidopsis.« less
Development of a new culture system for human lymphokine-activated killer cells: comparison with a conventional static culture method.

PubMed

Murata, M; Yano, T; Yoshino, I; Togami, M; Sogabe, M; Yasumoto, K; Sugimachi, K; Kimura, G; Nomoto, K

1991-10-01

We recently developed a new culture system based on dialysis perfusion (designated JCC-device) for the generation and expansion of human lymphokine-activated killer (LAK) cells (Murata et al., 1990). More recently we have scaled up the volume of the culture vessel of the JCC-device from 100 ml to 400 ml for clinical use. In the present study, using this new 400 ml JCC-device, we cultured human lymph node lymphocytes (LNL) obtained from 8 surgical patients with primary lung cancer, and investigated the cellular characteristics in comparison with a conventional batchwise culture system using tissue culture dishes. With the JCC-device, the cell density reached a maximum 2.7 x 10(7) cells/ml with greater than 90% viability by the appropriate exchange of perfusion medium and by making additions at the appropriate intervals for recombinant interleukin-2 (rIL-2). The expansion fold of LNL with the JCC-device, ranging 6.6- to 19.2-fold (mean 13.8-fold), was not significantly different from that in dish cultures. There was no marked difference in cell surface phenotypes between the two culture systems in 7 out of 8 cases. As for LAK activity of LNL, the JCC culture was either superior or equal in 4 out of 8 cases, but inferior in the other 4 cases to the conventional dish cultures. In the latter cases, the usage of serum for the JCC culture was limited, which might have resulted in the low LAK activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Detecting Genetic Interactions for Quantitative Traits Using m-Spacing Entropy Measure

PubMed Central

Yee, Jaeyong; Kwon, Min-Seok; Park, Taesung; Park, Mira

2015-01-01

A number of statistical methods for detecting gene-gene interactions have been developed in genetic association studies with binary traits. However, many phenotype measures are intrinsically quantitative and categorizing continuous traits may not always be straightforward and meaningful. Association of gene-gene interactions with an observed distribution of such phenotypes needs to be investigated directly without categorization. Information gain based on entropy measure has previously been successful in identifying genetic associations with binary traits. We extend the usefulness of this information gain by proposing a nonparametric evaluation method of conditional entropy of a quantitative phenotype associated with a given genotype. Hence, the information gain can be obtained for any phenotype distribution. Because any functional form, such as Gaussian, is not assumed for the entire distribution of a trait or a given genotype, this method is expected to be robust enough to be applied to any phenotypic association data. Here, we show its use to successfully identify the main effect, as well as the genetic interactions, associated with a quantitative trait. PMID:26339620
Hybrid & El Tor variant biotypes of Vibrio cholerae O1 in Thailand

PubMed Central

Na-Ubol, M.; Srimanote, P.; Chongsa-nguan, M.; Indrawattana, N.; Sookrung, N.; Tapchaisri, P.; Yamazaki, S.; Bodhidatta, L.; Eampokalap, B.; Kurazono, H.; Hayashi, H.; Nair, G.B.; Takeda, Y.; Chaicumpa, W.

2011-01-01

Background & objectives: El Tor Vibrio cholerae O1 carrying ctxBC trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand. Methods: A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpAC, tcpAE, hlyAC and hlyAE were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles. Results: All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid. Interpretation & conclusions: In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxBC). PMID:21537091
Text-mined phenotype annotation and vector-based similarity to improve identification of similar phenotypes and causative genes in monogenic disease patients.

PubMed

Saklatvala, Jake R; Dand, Nick; Simpson, Michael A

2018-05-01

The genetic diagnosis of rare monogenic diseases using exome/genome sequencing requires the true causal variant(s) to be identified from tens of thousands of observed variants. Typically a virtual gene panel approach is taken whereby only variants in genes known to cause phenotypes resembling the patient under investigation are considered. With the number of known monogenic gene-disease pairs exceeding 5,000, manual curation of personalized virtual panels using exhaustive knowledge of the genetic basis of the human monogenic phenotypic spectrum is challenging. We present improved probabilistic methods for estimating phenotypic similarity based on Human Phenotype Ontology annotation. A limitation of existing methods for evaluating a disease's similarity to a reference set is that reference diseases are typically represented as a series of binary (present/absent) observations of phenotypic terms. We evaluate a quantified disease reference set, using term frequency in phenotypic text descriptions to approximate term relevance. We demonstrate an improved ability to identify related diseases through the use of a quantified reference set, and that vector space similarity measures perform better than established information content-based measures. These improvements enable the generation of bespoke virtual gene panels, facilitating more accurate and efficient interpretation of genomic variant profiles from individuals with rare Mendelian disorders. These methods are available online at https://atlas.genetics.kcl.ac.uk/~jake/cgi-bin/patient_sim.py. © 2018 Wiley Periodicals, Inc.
Rodent motor and neuropsychological behaviour measured in home cages using the integrated modular platform SmartCage™.

PubMed

Khroyan, Taline V; Zhang, Jingxi; Yang, Liya; Zou, Bende; Xie, James; Pascual, Conrado; Malik, Adam; Xie, Julian; Zaveri, Nurulain T; Vazquez, Jacqueline; Polgar, Willma; Toll, Lawrence; Fang, Jidong; Xie, Xinmin

2012-07-01

1. To facilitate investigation of diverse rodent behaviours in rodents' home cages, we have developed an integrated modular platform, the SmartCage(™) system (AfaSci, Inc. Burlingame, CA, USA), which enables automated neurobehavioural phenotypic analysis and in vivo drug screening in a relatively higher-throughput and more objective manner. 2, The individual platform consists of an infrared array, a vibration floor sensor and a variety of modular devices. One computer can simultaneously operate up to 16 platforms via USB cables. 3. The SmartCage(™) detects drug-induced increases and decreases in activity levels, as well as changes in movement patterns. Wake and sleep states of mice can be detected using the vibration floor sensor. The arousal state classification achieved up to 98% accuracy compared with results obtained by electroencephalography and electromyography. More complex behaviours, including motor coordination, anxiety-related behaviours and social approach behaviour, can be assessed using appropriate modular devices and the results obtained are comparable with results obtained using conventional methods. 4. In conclusion, the SmartCage(™) system provides an automated and accurate tool to quantify various rodent behaviours in a 'stress-free' environment. This system, combined with the validated testing protocols, offers powerful a tool kit for transgenic phenotyping and in vivo drug screening. © 2012 The Authors. Clinical and Experimental Pharmacology and Physiology © 2012 Blackwell Publishing Asia Pty Ltd.
Psoriasiform Eruptions During Kawasaki Disease: A Distinct Phenotype

PubMed Central

Haddock, Ellen S.; Calame, Antoanella; Shimizu, Chisato; Tremoulet, Adriana H.; Burns, Jane C.; Tom, Wynnis L.

2016-01-01

Background A psoriasis-like eruption develops in a subset of patients with Kawasaki disease (KD). Objective To systematically compare KD-associated psoriasiform eruptions with classic psoriasis and the outcomes of KD in children with and without this rash. Methods This was a retrospective study of 11 KD cases with a psoriasiform eruption matched 1:2 by age, gender, and ethnicity with psoriasis-only and KD-only controls. Genotyping was performed in 10 cases for a deletion of two late cornified envelope (LCE) genes, LCE3C_LCE3B-del, associated with increased risk for pediatric-onset psoriasis. Results Similar to classic psoriasis, KD-associated eruptions were characterized clinically by well-demarcated, scaly pink plaques and histopathologically by intraepidermal neutrophils, suprabasilar keratin 16 expression, and increased Ki-67 expression. They showed less frequent diaper area involvement, more crust and serous exudate, and an enduring remission (91% vs. 23% with confirmed resolution; p < 0.001). Frequency of LCE3C_LCE3B-del and major KD outcomes were similar between cases and controls. Limitations The study was limited by the small number of cases, treatment variation, and availability of skin biopsy specimens. Conclusions Although the overall clinical and histopathologic findings were similar to conventional psoriasis, this appears to be a distinct phenotype with significantly greater propensity for remission. No adverse effect on KD outcomes was noted. PMID:26946987
Collective Functionality through Bacterial Individuality

NASA Astrophysics Data System (ADS)

Ackermann, Martin

According to the conventional view, the properties of an organism are a product of nature and nurture - of its genes and the environment it lives in. Recent experiments with unicellular organisms have challenged this view: several molecular mechanisms generate phenotypic variation independently of environmental signals, leading to variation in clonal groups. My presentation will focus on the causes and consequences of this microbial individuality. Using examples from bacterial genetic model systems, I will first discuss different molecular and cellular mechanisms that give rise to bacterial individuality. Then, I will discuss the consequences of individuality, and focus on how phenotypic variation in clonal populations of bacteria can promote interactions between individuals, lead to the division of labor, and allow clonal groups of bacteria to cope with environmental uncertainty. Variation between individuals thus provides clonal groups with collective functionality.
Red fluorescent proteins: advanced imaging applications and future design.

PubMed

Shcherbakova, Daria M; Subach, Oksana M; Verkhusha, Vladislav V

2012-10-22

In the past few years a large series of the advanced red-shifted fluorescent proteins (RFPs) has been developed. These enhanced RFPs provide new possibilities to study biological processes at the levels ranging from single molecules to whole organisms. Herein the relationship between the properties of the RFPs of different phenotypes and their applications to various imaging techniques are described. Existing and emerging imaging approaches are discussed for conventional RFPs, far-red FPs, RFPs with a large Stokes shift, fluorescent timers, irreversibly photoactivatable and reversibly photoswitchable RFPs. Advantages and limitations of specific RFPs for each technique are presented. Recent progress in understanding the chemical transformations of red chromophores allows the future RFP phenotypes and their respective novel imaging applications to be foreseen. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Genome‐wide association study of facial emotion recognition in children and association with polygenic risk for mental health disorders

PubMed Central

Coleman, Jonathan R.I.; Lester, Kathryn J.; Keers, Robert; Munafò, Marcus R.; Breen, Gerome

2017-01-01

Emotion recognition is disrupted in many mental health disorders, which may reflect shared genetic aetiology between this trait and these disorders. We explored genetic influences on emotion recognition and the relationship between these influences and mental health phenotypes. Eight‐year‐old participants (n = 4,097) from the Avon Longitudinal Study of Parents and Children (ALSPAC) completed the Diagnostic Analysis of Non‐Verbal Accuracy (DANVA) faces test. Genome‐wide genotype data was available from the Illumina HumanHap550 Quad microarray. Genome‐wide association studies were performed to assess associations with recognition of individual emotions and emotion in general. Exploratory polygenic risk scoring was performed using published genomic data for schizophrenia, bipolar disorder, depression, autism spectrum disorder, anorexia, and anxiety disorders. No individual genetic variants were identified at conventional levels of significance in any analysis although several loci were associated at a level suggestive of significance. SNP‐chip heritability analyses did not identify a heritable component of variance for any phenotype. Polygenic scores were not associated with any phenotype. The effect sizes of variants influencing emotion recognition are likely to be small. Previous studies of emotion identification have yielded non‐zero estimates of SNP‐heritability. This discrepancy is likely due to differences in the measurement and analysis of the phenotype. PMID:28608620
Vertically transmitted fecal IgA levels distinguish extra-chromosomal phenotypic variation

PubMed Central

Wallace, Meghan A.; D, Carey-Ann; Burnham; Virgin, Herbert W.; Stappenbeck, Thaddeus S.

2014-01-01

Summary The proliferation of genetically modified mouse models has exposed phenotypic variation between investigators and institutions that has been challenging to control1-5. In many cases, the microbiota is the presumed culprit of the variation. Current solutions to account for phenotypic variability include littermate and maternal controls or defined microbial consortia in gnotobiotic mice6,7. In conventionally raised mice, the microbiome is transmitted from the dam2,8,9. Here we show that microbially–driven dichotomous fecal IgA levels in WT mice within the same facility mimic the effects of chromosomal mutations. We observed in multiple facilities that vertically-transmissible bacteria in IgA-Low mice dominantly lowered fecal IgA levels in IgA-High mice after cohousing or fecal transplantation. In response to injury, IgA-Low mice showed increased damage that was transferable by fecal transplantation and driven by fecal IgA differences. We found that bacteria from IgA-Low mice degraded the secretory component (SC) of SIgA as well as IgA itself. These data indicate that phenotypic comparisons between mice must take into account the non-chromosomal hereditary variation between different breeders. We propose fecal IgA as one marker of microbial variability and conclude that cohousing and/or fecal transplantation enables analysis of progeny from different dams. PMID:25686606
Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE).

PubMed

Shekhar, Karthik; Brodin, Petter; Davis, Mark M; Chakraborty, Arup K

2014-01-07

Mass cytometry enables an unprecedented number of parameters to be measured in individual cells at a high throughput, but the large dimensionality of the resulting data severely limits approaches relying on manual "gating." Clustering cells based on phenotypic similarity comes at a loss of single-cell resolution and often the number of subpopulations is unknown a priori. Here we describe ACCENSE, a tool that combines nonlinear dimensionality reduction with density-based partitioning, and displays multivariate cellular phenotypes on a 2D plot. We apply ACCENSE to 35-parameter mass cytometry data from CD8(+) T cells derived from specific pathogen-free and germ-free mice, and stratify cells into phenotypic subpopulations. Our results show significant heterogeneity within the known CD8(+) T-cell subpopulations, and of particular note is that we find a large novel subpopulation in both specific pathogen-free and germ-free mice that has not been described previously. This subpopulation possesses a phenotypic signature that is distinct from conventional naive and memory subpopulations when analyzed by ACCENSE, but is not distinguishable on a biaxial plot of standard markers. We are able to automatically identify cellular subpopulations based on all proteins analyzed, thus aiding the full utilization of powerful new single-cell technologies such as mass cytometry.
Forensic DNA Phenotyping: Predicting human appearance from crime scene material for investigative purposes.

PubMed

Kayser, Manfred

2015-09-01

Forensic DNA Phenotyping refers to the prediction of appearance traits of unknown sample donors, or unknown deceased (missing) persons, directly from biological materials found at the scene. "Biological witness" outcomes of Forensic DNA Phenotyping can provide investigative leads to trace unknown persons, who are unidentifiable with current comparative DNA profiling. This intelligence application of DNA marks a substantially different forensic use of genetic material rather than that of current DNA profiling presented in the courtroom. Currently, group-specific pigmentation traits are already predictable from DNA with reasonably high accuracies, while several other externally visible characteristics are under genetic investigation. Until individual-specific appearance becomes accurately predictable from DNA, conventional DNA profiling needs to be performed subsequent to appearance DNA prediction. Notably, and where Forensic DNA Phenotyping shows great promise, this is on a (much) smaller group of potential suspects, who match the appearance characteristics DNA-predicted from the crime scene stain or from the deceased person's remains. Provided sufficient funding being made available, future research to better understand the genetic basis of human appearance will expectedly lead to a substantially more detailed description of an unknown person's appearance from DNA, delivering increased value for police investigations in criminal and missing person cases involving unknowns. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance.

PubMed

Jeyapalan, J N; Noor, D A Mohamed; Lee, S-H; Tan, C L; Appleby, V A; Kilday, J P; Palmer, R D; Schwalbe, E C; Clifford, S C; Walker, D A; Murray, M J; Coleman, N; Nicholson, J C; Scotting, P J

2011-08-09

Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.
Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy.

PubMed

Nakano, Sachie; Tsukimura, Takahiro; Togawa, Tadayasu; Ohashi, Toya; Kobayashi, Masahisa; Takayama, Katsuyoshi; Kobayashi, Yukuharu; Abiko, Hiroshi; Satou, Masatsugu; Nakahata, Tohru; Warnock, David G; Sakuraba, Hitoshi; Shibasaki, Futoshi

2015-01-01

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy.
Mechanical slowing-down of cytoplasmic diffusion allows in vivo counting of proteins in individual cells

PubMed Central

Okumus, Burak; Landgraf, Dirk; Lai, Ghee Chuan; Bakhsi, Somenath; Arias-Castro, Juan Carlos; Yildiz, Sadik; Huh, Dann; Fernandez-Lopez, Raul; Peterson, Celeste N.; Toprak, Erdal; El Karoui, Meriem; Paulsson, Johan

2016-01-01

Many key regulatory proteins in bacteria are present in too low numbers to be detected with conventional methods, which poses a particular challenge for single-cell analyses because such proteins can contribute greatly to phenotypic heterogeneity. Here we develop a microfluidics-based platform that enables single-molecule counting of low-abundance proteins by mechanically slowing-down their diffusion within the cytoplasm of live Escherichia coli (E. coli) cells. Our technique also allows for automated microscopy at high throughput with minimal perturbation to native physiology, as well as viable enrichment/retrieval. We illustrate the method by analysing the control of the master regulator of the E. coli stress response, RpoS, by its adapter protein, SprE (RssB). Quantification of SprE numbers shows that though SprE is necessary for RpoS degradation, it is expressed at levels as low as 3–4 molecules per average cell cycle, and fluctuations in SprE are approximately Poisson distributed during exponential phase with no sign of bursting. PMID:27189321
Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems.

PubMed

Zhang, Hongkai; Torkamani, Ali; Jones, Teresa M; Ruiz, Diana I; Pons, Jaume; Lerner, Richard A

2011-08-16

Use of large combinatorial antibody libraries and next-generation sequencing of nucleic acids are two of the most powerful methods in modern molecular biology. The libraries are screened using the principles of evolutionary selection, albeit in real time, to enrich for members with a particular phenotype. This selective process necessarily results in the loss of information about less-fit molecules. On the other hand, sequencing of the library, by itself, gives information that is mostly unrelated to phenotype. If the two methods could be combined, the full potential of very large molecular libraries could be realized. Here we report the implementation of a phenotype-information-phenotype cycle that integrates information and gene recovery. After selection for phage-encoded antibodies that bind to targets expressed on the surface of Escherichia coli, the information content of the selected pool is obtained by pyrosequencing. Sequences that encode specific antibodies are identified by a bioinformatic analysis and recovered by a stringent affinity method that is uniquely suited for gene isolation from a highly degenerate collection of nucleic acids. This approach can be generalized for selection of antibodies against targets that are present as minor components of complex systems.
EHR Big Data Deep Phenotyping

PubMed Central

Lenert, L.; Lopez-Campos, G.

2014-01-01

Summary Objectives Given the quickening speed of discovery of variant disease drivers from combined patient genotype and phenotype data, the objective is to provide methodology using big data technology to support the definition of deep phenotypes in medical records. Methods As the vast stores of genomic information increase with next generation sequencing, the importance of deep phenotyping increases. The growth of genomic data and adoption of Electronic Health Records (EHR) in medicine provides a unique opportunity to integrate phenotype and genotype data into medical records. The method by which collections of clinical findings and other health related data are leveraged to form meaningful phenotypes is an active area of research. Longitudinal data stored in EHRs provide a wealth of information that can be used to construct phenotypes of patients. We focus on a practical problem around data integration for deep phenotype identification within EHR data. The use of big data approaches are described that enable scalable markup of EHR events that can be used for semantic and temporal similarity analysis to support the identification of phenotype and genotype relationships. Conclusions Stead and colleagues’ 2005 concept of using light standards to increase the productivity of software systems by riding on the wave of hardware/processing power is described as a harbinger for designing future healthcare systems. The big data solution, using flexible markup, provides a route to improved utilization of processing power for organizing patient records in genotype and phenotype research. PMID:25123744
NCI Workshop Report: Clinical and Computational Requirements for Correlating Imaging Phenotypes with Genomics Signatures.

PubMed

Colen, Rivka; Foster, Ian; Gatenby, Robert; Giger, Mary Ellen; Gillies, Robert; Gutman, David; Heller, Matthew; Jain, Rajan; Madabhushi, Anant; Madhavan, Subha; Napel, Sandy; Rao, Arvind; Saltz, Joel; Tatum, James; Verhaak, Roeland; Whitman, Gary

2014-10-01

The National Cancer Institute (NCI) Cancer Imaging Program organized two related workshops on June 26-27, 2013, entitled "Correlating Imaging Phenotypes with Genomics Signatures Research" and "Scalable Computational Resources as Required for Imaging-Genomics Decision Support Systems." The first workshop focused on clinical and scientific requirements, exploring our knowledge of phenotypic characteristics of cancer biological properties to determine whether the field is sufficiently advanced to correlate with imaging phenotypes that underpin genomics and clinical outcomes, and exploring new scientific methods to extract phenotypic features from medical images and relate them to genomics analyses. The second workshop focused on computational methods that explore informatics and computational requirements to extract phenotypic features from medical images and relate them to genomics analyses and improve the accessibility and speed of dissemination of existing NIH resources. These workshops linked clinical and scientific requirements of currently known phenotypic and genotypic cancer biology characteristics with imaging phenotypes that underpin genomics and clinical outcomes. The group generated a set of recommendations to NCI leadership and the research community that encourage and support development of the emerging radiogenomics research field to address short-and longer-term goals in cancer research.

Identification by 16S rRNA Gene Sequencing of an Actinomyces hongkongensis Isolate Recovered from a Patient with Pelvic Actinomycosis

PubMed Central

Flynn, A. N.; Lyndon, C. A.

2013-01-01

A case of Actinomyces hongkongensis pelvic actinomycosis in an adult woman is described. Conventional phenotypic tests failed to identify the Gram-positive bacillus isolated from a fluid aspirate of a pelvic abscess. The bacterium was identified by 16S rRNA gene sequencing and analysis using the SmartGene Integrated Database Network System software. PMID:23698532
FAVR (Filtering and Annotation of Variants that are Rare): methods to facilitate the analysis of rare germline genetic variants from massively parallel sequencing datasets

PubMed Central

2013-01-01

Background Characterising genetic diversity through the analysis of massively parallel sequencing (MPS) data offers enormous potential to significantly improve our understanding of the genetic basis for observed phenotypes, including predisposition to and progression of complex human disease. Great challenges remain in resolving genetic variants that are genuine from the millions of artefactual signals. Results FAVR is a suite of new methods designed to work with commonly used MPS analysis pipelines to assist in the resolution of some of the issues related to the analysis of the vast amount of resulting data, with a focus on relatively rare genetic variants. To the best of our knowledge, no equivalent method has previously been described. The most important and novel aspect of FAVR is the use of signatures in comparator sequence alignment files during variant filtering, and annotation of variants potentially shared between individuals. The FAVR methods use these signatures to facilitate filtering of (i) platform and/or mapping-specific artefacts, (ii) common genetic variants, and, where relevant, (iii) artefacts derived from imbalanced paired-end sequencing, as well as annotation of genetic variants based on evidence of co-occurrence in individuals. We applied conventional variant calling applied to whole-exome sequencing datasets, produced using both SOLiD and TruSeq chemistries, with or without downstream processing by FAVR methods. We demonstrate a 3-fold smaller rare single nucleotide variant shortlist with no detected reduction in sensitivity. This analysis included Sanger sequencing of rare variant signals not evident in dbSNP131, assessment of known variant signal preservation, and comparison of observed and expected rare variant numbers across a range of first cousin pairs. The principles described herein were applied in our recent publication identifying XRCC2 as a new breast cancer risk gene and have been made publically available as a suite of software tools. Conclusions FAVR is a platform-agnostic suite of methods that significantly enhances the analysis of large volumes of sequencing data for the study of rare genetic variants and their influence on phenotypes. PMID:23441864
Molecular basis of differentiation therapy for soft tissue sarcomas

PubMed Central

Luther, Gaurav; Rames, Richard; Wagner, Eric R.; Zhu, Gaohui; Luo, Qing; Bi, Yang; Kim, Stephanie H.; Gao, Jian-Li; Huang, Enyi; Yang, Ke; Wang, Linyuan; Liu, Xing; Li, Mi; Hu, Ning; Su, Yuxi; Luo, Xiaoji; Chen, Liang; Luo, Jinyong; Haydon, Rex C.; Luu, Hue H.; Zhou, Lan; He, Tong-Chuan

2015-01-01

Stem cells are undifferentiated precursor cells with the capacity for proliferation or terminal differentiation. Progression down the differentiation cascade results in a loss of proliferative potential in exchange for the differentiated phenotype. This balance is tightly regulated in the physiologic state. Recent studies, however, have demonstrated that during tumorigenesis, disruptions preventing terminal differentiation allow cancer cells to maintain a proliferative, precursor cell phenotype. Current therapies (i.e., chemotherapy and radiation therapy) target the actively proliferating cells in tumor masses, which in many cases inevitably induce therapy-resistant cancer cells. It is conceivable that promising therapy regimens can be developed by treating human cancers by inducing terminal differentiation, thereby restoring the interrupted pathway and shifting the balance from proliferation to differentiation. For example, osteosarcoma (OS) is a primary bone cancer caused by differentiation defects in mesenchymal stem cells (MSCs) for which several differentiation therapies have shown great promise. In this review, we discuss the various differentiation therapies in the treatment of human sarcomas with a focus on OS. Such therapies hold great promise as they not only inhibit tumorigenesis, but also avoid the adverse effects associated with conventional chemotherapy regimens. Furthermore, it is conceivable that a combination of conventional therapies with differentiation therapy should significantly improve anticancer efficacy and reduce drug-resistance in the clinical management of human cancers, including sarcomas. PMID:26912947
Uniparental disomy and prenatal phenotype

PubMed Central

Li, Xiaofei; Liu, Yan; Yue, Song; Wang, Li; Zhang, Tiejuan; Guo, Cuixia; Hu, Wenjie; Kagan, Karl-Oliver; Wu, Qingqing

2017-01-01

Abstract Rationale: Uniparental disomy (UPD) gives a description of the inheritance of both homologues of a chromosome pair from the same parent. The consequences of UPD depend on the specific chromosome/segment involved and its parental origin. Patient concerns: We report prenatal phenotypes of 2 rare cases of UPD. Diagnoses: The prenatal phenotype of case 1 included sonographic markers such as enlarged nuchal translucency (NT), absent nasal bone, short femur and humerus length, and several structural malformations involving Dandy–Walker malformation and congenital heart defects. The prenatal phenotype of Case 2 are sonographic markers, including enlarged NT, thickened nuchal fold, ascites, and polyhydramnios without apparent structural malformations. Interventions: Conventional G-band karyotype appears normal in case 1, while it shows normal chromosomes with a small supernumerary marker chromosome (sSMC) in case 2. Genetic etiology was left unknown until single-nucleotide polymorphism-based array (SNP-array) was performed, and segmental paternal UPD 22 was identified in case 1 and segmental paternal UPD 14 was found in case 2. Outcomes: The parents of case 1 chose termination of pregnancy. The neonate of case 2 was born prematurely with a bellshaped small thorax and died within a day. Lessons: UPD cases are rare and the phenotypes are different, which depend on the origin and affected chromosomal part. If a fetus shows multiple anomalies that cannot be attributed to a common aneuploidy or a genetic syndrome, or manifests some features possibly related to an UPD syndrome, such as detection of sSMC, SNP-array should be considered. PMID:29137034
Uniparental disomy and prenatal phenotype: Two case reports and review.

PubMed

Li, Xiaofei; Liu, Yan; Yue, Song; Wang, Li; Zhang, Tiejuan; Guo, Cuixia; Hu, Wenjie; Kagan, Karl-Oliver; Wu, Qingqing

2017-11-01

Uniparental disomy (UPD) gives a description of the inheritance of both homologues of a chromosome pair from the same parent. The consequences of UPD depend on the specific chromosome/segment involved and its parental origin. We report prenatal phenotypes of 2 rare cases of UPD. The prenatal phenotype of case 1 included sonographic markers such as enlarged nuchal translucency (NT), absent nasal bone, short femur and humerus length, and several structural malformations involving Dandy-Walker malformation and congenital heart defects. The prenatal phenotype of Case 2 are sonographic markers, including enlarged NT, thickened nuchal fold, ascites, and polyhydramnios without apparent structural malformations. Conventional G-band karyotype appears normal in case 1, while it shows normal chromosomes with a small supernumerary marker chromosome (sSMC) in case 2. Genetic etiology was left unknown until single-nucleotide polymorphism-based array (SNP-array) was performed, and segmental paternal UPD 22 was identified in case 1 and segmental paternal UPD 14 was found in case 2. The parents of case 1 chose termination of pregnancy. The neonate of case 2 was born prematurely with a bellshaped small thorax and died within a day. UPD cases are rare and the phenotypes are different, which depend on the origin and affected chromosomal part. If a fetus shows multiple anomalies that cannot be attributed to a common aneuploidy or a genetic syndrome, or manifests some features possibly related to an UPD syndrome, such as detection of sSMC, SNP-array should be considered.
Estrogen receptor-α, progesterone receptor, and c-erbB/HER-family receptor mRNA detection and phenotype analysis in spontaneous canine models of breast cancer

PubMed Central

Kabir, Farruk M. Lutful; DeInnocentes, Patricia; Agarwal, Payal; Mill, Christopher P.; Riese, David J.

2017-01-01

Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer. PMID:27515268
Using text mining techniques to extract phenotypic information from the PhenoCHF corpus

PubMed Central

2015-01-01

Background Phenotypic information locked away in unstructured narrative text presents significant barriers to information accessibility, both for clinical practitioners and for computerised applications used for clinical research purposes. Text mining (TM) techniques have previously been applied successfully to extract different types of information from text in the biomedical domain. They have the potential to be extended to allow the extraction of information relating to phenotypes from free text. Methods To stimulate the development of TM systems that are able to extract phenotypic information from text, we have created a new corpus (PhenoCHF) that is annotated by domain experts with several types of phenotypic information relating to congestive heart failure. To ensure that systems developed using the corpus are robust to multiple text types, it integrates text from heterogeneous sources, i.e., electronic health records (EHRs) and scientific articles from the literature. We have developed several different phenotype extraction methods to demonstrate the utility of the corpus, and tested these methods on a further corpus, i.e., ShARe/CLEF 2013. Results Evaluation of our automated methods showed that PhenoCHF can facilitate the training of reliable phenotype extraction systems, which are robust to variations in text type. These results have been reinforced by evaluating our trained systems on the ShARe/CLEF corpus, which contains clinical records of various types. Like other studies within the biomedical domain, we found that solutions based on conditional random fields produced the best results, when coupled with a rich feature set. Conclusions PhenoCHF is the first annotated corpus aimed at encoding detailed phenotypic information. The unique heterogeneous composition of the corpus has been shown to be advantageous in the training of systems that can accurately extract phenotypic information from a range of different text types. Although the scope of our annotation is currently limited to a single disease, the promising results achieved can stimulate further work into the extraction of phenotypic information for other diseases. The PhenoCHF annotation guidelines and annotations are publicly available at https://code.google.com/p/phenochf-corpus. PMID:26099853
Genomic scan as a tool for assessing the genetic component of phenotypic variance in wild populations.

PubMed

Herrera, Carlos M

2012-01-01

Methods for estimating quantitative trait heritability in wild populations have been developed in recent years which take advantage of the increased availability of genetic markers to reconstruct pedigrees or estimate relatedness between individuals, but their application to real-world data is not exempt from difficulties. This chapter describes a recent marker-based technique which, by adopting a genomic scan approach and focusing on the relationship between phenotypes and genotypes at the individual level, avoids the problems inherent to marker-based estimators of relatedness. This method allows the quantification of the genetic component of phenotypic variance ("degree of genetic determination" or "heritability in the broad sense") in wild populations and is applicable whenever phenotypic trait values and multilocus data for a large number of genetic markers (e.g., amplified fragment length polymorphisms, AFLPs) are simultaneously available for a sample of individuals from the same population. The method proceeds by first identifying those markers whose variation across individuals is significantly correlated with individual phenotypic differences ("adaptive loci"). The proportion of phenotypic variance in the sample that is statistically accounted for by individual differences in adaptive loci is then estimated by fitting a linear model to the data, with trait value as the dependent variable and scores of adaptive loci as independent ones. The method can be easily extended to accommodate quantitative or qualitative information on biologically relevant features of the environment experienced by each sampled individual, in which case estimates of the environmental and genotype × environment components of phenotypic variance can also be obtained.
Prediction of gene-phenotype associations in humans, mice, and plants using phenologs.

PubMed

Woods, John O; Singh-Blom, Ulf Martin; Laurent, Jon M; McGary, Kriston L; Marcotte, Edward M

2013-06-21

Phenotypes and diseases may be related to seemingly dissimilar phenotypes in other species by means of the orthology of underlying genes. Such "orthologous phenotypes," or "phenologs," are examples of deep homology, and may be used to predict additional candidate disease genes. In this work, we develop an unsupervised algorithm for ranking phenolog-based candidate disease genes through the integration of predictions from the k nearest neighbor phenologs, comparing classifiers and weighting functions by cross-validation. We also improve upon the original method by extending the theory to paralogous phenotypes. Our algorithm makes use of additional phenotype data--from chicken, zebrafish, and E. coli, as well as new datasets for C. elegans--establishing that several types of annotations may be treated as phenotypes. We demonstrate the use of our algorithm to predict novel candidate genes for human atrial fibrillation (such as HRH2, ATP4A, ATP4B, and HOPX) and epilepsy (e.g., PAX6 and NKX2-1). We suggest gene candidates for pharmacologically-induced seizures in mouse, solely based on orthologous phenotypes from E. coli. We also explore the prediction of plant gene-phenotype associations, as for the Arabidopsis response to vernalization phenotype. We are able to rank gene predictions for a significant portion of the diseases in the Online Mendelian Inheritance in Man database. Additionally, our method suggests candidate genes for mammalian seizures based only on bacterial phenotypes and gene orthology. We demonstrate that phenotype information may come from diverse sources, including drug sensitivities, gene ontology biological processes, and in situ hybridization annotations. Finally, we offer testable candidates for a variety of human diseases, plant traits, and other classes of phenotypes across a wide array of species.
Supervised segmentation of phenotype descriptions for the human skeletal phenome using hybrid methods.

PubMed

Groza, Tudor; Hunter, Jane; Zankl, Andreas

2012-10-15

Over the course of the last few years there has been a significant amount of research performed on ontology-based formalization of phenotype descriptions. In order to fully capture the intrinsic value and knowledge expressed within them, we need to take advantage of their inner structure, which implicitly combines qualities and anatomical entities. The first step in this process is the segmentation of the phenotype descriptions into their atomic elements. We present a two-phase hybrid segmentation method that combines a series individual classifiers using different aggregation schemes (set operations and simple majority voting). The approach is tested on a corpus comprised of skeletal phenotype descriptions emerged from the Human Phenotype Ontology. Experimental results show that the best hybrid method achieves an F-Score of 97.05% in the first phase and F-Scores of 97.16% / 94.50% in the second phase. The performance of the initial segmentation of anatomical entities and qualities (phase I) is not affected by the presence / absence of external resources, such as domain dictionaries. From a generic perspective, hybrid methods may not always improve the segmentation accuracy as they are heavily dependent on the goal and data characteristics.
[Strategies to identify supernumerary chromosomal markers in constitutional cytogenetics].

PubMed

Douet-Guilbert, N; Basinko, A; Le Bris, M-J; Herry, A; Morel, F; De Braekeleer, M

2008-09-01

Supernumerary marker chromosomes (SMCs) are defined as extrastructurally abnormal chromosomes which origin and composition cannot be determined by conventional cytogenetics. SMCs are an heterogeneous group of abnormalities concerning all chromosomes with variable structure and size and are associated with phenotypic heterogeneity. The characterisation of SMCs is of utmost importance for genetic counselling. Different molecular techniques are used to identify chromosomal material present in markers such as 24-colour FISH (MFISH, SKY), centromere specific multicolour FISH (cenMFISH) and derivatives (acroMFISH, subcenMFISH), comparative genomic hybridisation (CGH), arrayCGH, and targeted FISH techniques (banding techniques, whole chromosome painting...). Based on the morphology of SMC with conventional cytogenetic and clinical data, we tried to set up different molecular strategies with all available techniques.
Inferring fitness landscapes and selection on phenotypic states from single-cell genealogical data

PubMed Central

Kussell, Edo

2017-01-01

Recent advances in single-cell time-lapse microscopy have revealed non-genetic heterogeneity and temporal fluctuations of cellular phenotypes. While different phenotypic traits such as abundance of growth-related proteins in single cells may have differential effects on the reproductive success of cells, rigorous experimental quantification of this process has remained elusive due to the complexity of single cell physiology within the context of a proliferating population. We introduce and apply a practical empirical method to quantify the fitness landscapes of arbitrary phenotypic traits, using genealogical data in the form of population lineage trees which can include phenotypic data of various kinds. Our inference methodology for fitness landscapes determines how reproductivity is correlated to cellular phenotypes, and provides a natural generalization of bulk growth rate measures for single-cell histories. Using this technique, we quantify the strength of selection acting on different cellular phenotypic traits within populations, which allows us to determine whether a change in population growth is caused by individual cells’ response, selection within a population, or by a mixture of these two processes. By applying these methods to single-cell time-lapse data of growing bacterial populations that express a resistance-conferring protein under antibiotic stress, we show how the distributions, fitness landscapes, and selection strength of single-cell phenotypes are affected by the drug. Our work provides a unified and practical framework for quantitative measurements of fitness landscapes and selection strength for any statistical quantities definable on lineages, and thus elucidates the adaptive significance of phenotypic states in time series data. The method is applicable in diverse fields, from single cell biology to stem cell differentiation and viral evolution. PMID:28267748
TATES: Efficient Multivariate Genotype-Phenotype Analysis for Genome-Wide Association Studies

PubMed Central

van der Sluis, Sophie; Posthuma, Danielle; Dolan, Conor V.

2013-01-01

To date, the genome-wide association study (GWAS) is the primary tool to identify genetic variants that cause phenotypic variation. As GWAS analyses are generally univariate in nature, multivariate phenotypic information is usually reduced to a single composite score. This practice often results in loss of statistical power to detect causal variants. Multivariate genotype–phenotype methods do exist but attain maximal power only in special circumstances. Here, we present a new multivariate method that we refer to as TATES (Trait-based Association Test that uses Extended Simes procedure), inspired by the GATES procedure proposed by Li et al (2011). For each component of a multivariate trait, TATES combines p-values obtained in standard univariate GWAS to acquire one trait-based p-value, while correcting for correlations between components. Extensive simulations, probing a wide variety of genotype–phenotype models, show that TATES's false positive rate is correct, and that TATES's statistical power to detect causal variants explaining 0.5% of the variance can be 2.5–9 times higher than the power of univariate tests based on composite scores and 1.5–2 times higher than the power of the standard MANOVA. Unlike other multivariate methods, TATES detects both genetic variants that are common to multiple phenotypes and genetic variants that are specific to a single phenotype, i.e. TATES provides a more complete view of the genetic architecture of complex traits. As the actual causal genotype–phenotype model is usually unknown and probably phenotypically and genetically complex, TATES, available as an open source program, constitutes a powerful new multivariate strategy that allows researchers to identify novel causal variants, while the complexity of traits is no longer a limiting factor. PMID:23359524
Carbapenem-resistant Gram-negative bacteria associated with catheter-related bloodstream infections in three intensive care units in Egypt.

PubMed

Abdulall, Abeer K; Tawfick, Mahmoud M; El Manakhly, Arwa R; El Kholy, Amani

2018-06-23

We aimed to identify the carbapenem-resistant Gram-negative bacteria (GNB) causing catheter-related bloodstream infections (CRBSI) in intensive care units (ICU) in a tertiary care Egyptian hospital, to study their resistance mechanisms by phenotypic and genetic tests, and to use ERIC-PCR for assessing their relatedness. The study was conducted over 2 years in three ICUs in a tertiary care hospital in Egypt during 2015-2016. We identified 194 bloodstream infections (BSIs); 130 (67.01%) were caused by GNB, of which 57 were isolated from CRBSI patients (73.84%). Identification of isolates was performed using conventional methods and MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was done by disc diffusion following CLSI guidelines. Phenotypic detection of carbapenemases enzymes activity was by modified Hodge test and the Carba-NP method. Isolates were investigated for the most common carbapenemases encoding genes bla KPC , bla NDM , and bla OXA-48 using multiplex PCR. Molecular typing of carbapenem-resistant isolates was done by ERIC-PCR followed by sequencing of common resistance genes. The overall rate of CRBSI in our study was 3.6 per 1000 central venous catheter (CVC) days. Among 57 Gram-negative CRBSI isolates, Klebsiella pneumoniae (K. pneumoniae) was the most frequently isolated (27/57; 47.4%), of which more than 70% were resistant to Meropenem. Phenotypic tests for carbapenemases showed that 37.9% of isolates were positive by modified Hodge test and 63.8% by Carba-NP detection. Multiplex PCR assay detected the bla NDM in 28.6% of the isolates and bla KPC in 26.8%, bla NDM and bla KPC were detected together in the same isolate in 5.6%, while bla OXA-48 -like were not detected. ERIC-PCR detected limited genetic relatedness between K. pneumoniae isolates. Elevated resistance rates were observed to all antibiotics including carbapenems among K. pneumoniae isolates causing CRBSI. ERIC-PCR showed that the resistant isolates were mainly polyclonal. Our results call for reinforcement of antimicrobial stewardship and measures to prevent CRBSI.
Measuring nanoscale viscoelastic parameters of cells directly from AFM force-displacement curves.

PubMed

Efremov, Yuri M; Wang, Wen-Horng; Hardy, Shana D; Geahlen, Robert L; Raman, Arvind

2017-05-08

Force-displacement (F-Z) curves are the most commonly used Atomic Force Microscopy (AFM) mode to measure the local, nanoscale elastic properties of soft materials like living cells. Yet a theoretical framework has been lacking that allows the post-processing of F-Z data to extract their viscoelastic constitutive parameters. Here, we propose a new method to extract nanoscale viscoelastic properties of soft samples like living cells and hydrogels directly from conventional AFM F-Z experiments, thereby creating a common platform for the analysis of cell elastic and viscoelastic properties with arbitrary linear constitutive relations. The method based on the elastic-viscoelastic correspondence principle was validated using finite element (FE) simulations and by comparison with the existed AFM techniques on living cells and hydrogels. The method also allows a discrimination of which viscoelastic relaxation model, for example, standard linear solid (SLS) or power-law rheology (PLR), best suits the experimental data. The method was used to extract the viscoelastic properties of benign and cancerous cell lines (NIH 3T3 fibroblasts, NMuMG epithelial, MDA-MB-231 and MCF-7 breast cancer cells). Finally, we studied the changes in viscoelastic properties related to tumorigenesis including TGF-β induced epithelial-to-mesenchymal transition on NMuMG cells and Syk expression induced phenotype changes in MDA-MB-231 cells.
Liquid Biopsy in Metastasized Breast Cancer as Basis for Treatment Decisions.

PubMed

Krawczyk, Natalia; Fehm, Tanja; Banys-Paluchowski, Malgorzata; Janni, Wolfgang; Schramm, Amelie

2016-01-01

According to current guidelines, the additional biopsy of breast cancer metastases to analyze the receptor status for phenotype assessment is recommended. However, due to clinical difficulties in performing biopsies of metastatic lesions, the phenotype of the primary tumor most often determines the treatment decisions in metastatic breast cancer. Liquid biopsy allows the analysis of several circulating biomarkers like circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) in peripheral blood samples of cancer patients. Thus, it is an elegant and easily practicable technique that delivers information on the current disease status. Determination of the CTC phenotype regarding the hormone receptor and human epidermal growth factor receptor 2 (HER2) status might replace additional tissue biopsy for planning further therapy strategies. Liquid biopsy is a crucial step towards a more individualized cancer therapy. In contrast to the conventional concept of tissue biopsy, it offers an easy, less invasive acquisition of biomaterial. In addition, it allows multiple repetitions and real-time monitoring of metastasized disease in the clinical routine. However, the clinical utility of liquid biopsy still needs to be evaluated. © 2016 S. Karger GmbH, Freiburg.
Ethical, legal and social implications of forensic molecular phenotyping in South Africa.

PubMed

Slabbert, Nandi; Heathfield, Laura Jane

2018-06-01

Conventional forensic DNA analysis involves a matching principle, which compares DNA profiles from evidential samples to those from reference samples of known origin. In casework, however, the accessibility to a reference sample is not guaranteed which limits the use of DNA as an investigative tool. This has led to the development of phenotype prediction, which uses SNP analysis to estimate the physical appearance of the sample donor. Physical traits, such as eye, hair and skin colour, have been associated with certain alleles within specific genes involved in the melanogenesis pathways. These genetic markers are also associated with ancestry and their trait prediction ability has mainly been assessed in European and North American populations. This has prompted research investigating the discriminatory power of these markers in other populations, especially those exhibiting admixture. South Africa is well known for its diversity, and the viability of these particular SNPs still needs to be assessed within this population. South African law currently restricts the use of DNA for molecular phenotyping, and there are also numerous ethical and social considerations, all of which are discussed. © 2018 John Wiley & Sons Ltd.
Prolonged partial upper airway obstruction during sleep – an underdiagnosed phenotype of sleep-disordered breathing

PubMed Central

Anttalainen, Ulla; Tenhunen, Mirja; Rimpilä, Ville; Polo, Olli; Rauhala, Esa; Himanen, Sari-Leena; Saaresranta, Tarja

2016-01-01

Obstructive sleep apnea syndrome (OSAS) is a well-recognized disorder conventionally diagnosed with an elevated apnea–hypopnea index. Prolonged partial upper airway obstruction is a common phenotype of sleep-disordered breathing (SDB), which however is still largely underreported. The major reasons for this are that cyclic breathing pattern coupled with arousals and arterial oxyhemoglobin saturation are easy to detect and considered more important than prolonged episodes of increased respiratory effort with increased levels of carbon dioxide in the absence of cycling breathing pattern and repetitive arousals. There is also a growing body of evidence that prolonged partial obstruction is a clinically significant form of SDB, which is associated with symptoms and co-morbidities which may partially differ from those associated with OSAS. Partial upper airway obstruction is most prevalent in women, and it is treatable with the nasal continuous positive pressure device with good adherence to therapy. This review describes the characteristics of prolonged partial upper airway obstruction during sleep in terms of diagnostics, pathophysiology, clinical presentation, and comorbidity to improve recognition of this phenotype and its timely and appropriate treatment. PMID:27608271
Modeling thrombin generation: plasma composition based approach.

PubMed

Brummel-Ziedins, Kathleen E; Everse, Stephen J; Mann, Kenneth G; Orfeo, Thomas

2014-01-01

Thrombin has multiple functions in blood coagulation and its regulation is central to maintaining the balance between hemorrhage and thrombosis. Empirical and computational methods that capture thrombin generation can provide advancements to current clinical screening of the hemostatic balance at the level of the individual. In any individual, procoagulant and anticoagulant factor levels together act to generate a unique coagulation phenotype (net balance) that is reflective of the sum of its developmental, environmental, genetic, nutritional and pharmacological influences. Defining such thrombin phenotypes may provide a means to track disease progression pre-crisis. In this review we briefly describe thrombin function, methods for assessing thrombin dynamics as a phenotypic marker, computationally derived thrombin phenotypes versus determined clinical phenotypes, the boundaries of normal range thrombin generation using plasma composition based approaches and the feasibility of these approaches for predicting risk.
[Methods of high-throughput plant phenotyping for large-scale breeding and genetic experiments].

PubMed

Afonnikov, D A; Genaev, M A; Doroshkov, A V; Komyshev, E G; Pshenichnikova, T A

2016-07-01

Phenomics is a field of science at the junction of biology and informatics which solves the problems of rapid, accurate estimation of the plant phenotype; it was rapidly developed because of the need to analyze phenotypic characteristics in large scale genetic and breeding experiments in plants. It is based on using the methods of computer image analysis and integration of biological data. Owing to automation, new approaches make it possible to considerably accelerate the process of estimating the characteristics of a phenotype, to increase its accuracy, and to remove a subjectivism (inherent to humans). The main technologies of high-throughput plant phenotyping in both controlled and field conditions, their advantages and disadvantages, and also the prospects of their use for the efficient solution of problems of plant genetics and breeding are presented in the review.

GiNA, an efficient and high-throughput software for horticultural phenotyping

USDA-ARS?s Scientific Manuscript database

Traditional methods for trait phenotyping have been a bottleneck for research in many crop species due to their intensive labor, high cost, complex implementation, lack of reproducibility and propensity to subjective bias. Recently, multiple high-throughput phenotyping platforms have been developed,...
Quantifying male attractiveness.

PubMed Central

McNamara, John M; Houston, Alasdair I; Marques Dos Santos, Miguel; Kokko, Hanna; Brooks, Rob

2003-01-01

Genetic models of sexual selection are concerned with a dynamic process in which female preference and male trait values coevolve. We present a rigorous method for characterizing evolutionary endpoints of this process in phenotypic terms. In our phenotypic characterization the mate-choice strategy of female population members determines how attractive females should find each male, and a population is evolutionarily stable if population members are actually behaving in this way. This provides a justification of phenotypic explanations of sexual selection and the insights into sexual selection that they provide. Furthermore, the phenotypic approach also has enormous advantages over a genetic approach when computing evolutionarily stable mate-choice strategies, especially when strategies are allowed to be complex time-dependent preference rules. For simplicity and clarity our analysis deals with haploid mate-choice genetics and a male trait that is inherited phenotypically, for example by vertical cultural transmission. The method is, however, easily extendible to other cases. An example illustrates that the sexy son phenomenon can occur when there is phenotypic inheritance of the male trait. PMID:14561306
The urinary microbiome and its contribution to lower urinary tract symptoms; ICI-RS 2015.

PubMed

Drake, Marcus J; Morris, Nicola; Apostolidis, Apostolos; Rahnama'i, Mohammad S; Marchesi, Julian R

2017-04-01

The microbiome is the term used for the symbiotic microbial colonisation of healthy organs. Studies have found bacterial identifiers within voided urine which is apparently sterile on conventional laboratory culture, and accordingly there may be health and disease implications. The International Consultation on Incontinence Research Society (ICI-RS) established a literature review and expert consensus discussion focussed on the increasing awareness of the urinary microbiome, and potential research priorities. The consensus considered the discrepancy between findings of conventional clinical microbiology methods, which generally rely on culture parameters predisposed towards certain "expected" organisms. Discrepancy between selective culture and RNA sequencing to study species-specific 16S ribosomal RNA is increasingly clear, and highlights the possibility that protective or harmful bacteria may be overlooked where microbiological methods are selective. There are now strong signals of the existence of a "core" urinary microbiome for the human urinary tract, particularly emerging with ageing. The consensus reviewed the potential relationship between a patient's microbiome and lower urinary tract dysfunction, whether low-count bacteriuria may be clinically significant and mechanisms which could associate micro-organisms with lower urinary tract symptoms. Key research priorities identified include the need to establish the scope of microbiome across the range of normality and clinical presentations, and gain consensus on testing protocols. Proteomics to study enzymatic and other functions may be necessary, since different bacteria may have overlapping phenotype. Longitudinal studies into risk factors for exposure, cumulative risk, and emergence of disease need to undertaken. Neurourol. Urodynam. 36:850-853, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
Interoperability between phenotype and anatomy ontologies.

PubMed

Hoehndorf, Robert; Oellrich, Anika; Rebholz-Schuhmann, Dietrich

2010-12-15

Phenotypic information is important for the analysis of the molecular mechanisms underlying disease. A formal ontological representation of phenotypic information can help to identify, interpret and infer phenotypic traits based on experimental findings. The methods that are currently used to represent data and information about phenotypes fail to make the semantics of the phenotypic trait explicit and do not interoperate with ontologies of anatomy and other domains. Therefore, valuable resources for the analysis of phenotype studies remain unconnected and inaccessible to automated analysis and reasoning. We provide a framework to formalize phenotypic descriptions and make their semantics explicit. Based on this formalization, we provide the means to integrate phenotypic descriptions with ontologies of other domains, in particular anatomy and physiology. We demonstrate how our framework leads to the capability to represent disease phenotypes, perform powerful queries that were not possible before and infer additional knowledge. http://bioonto.de/pmwiki.php/Main/PheneOntology.
Somatic NLRP3 mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes.

PubMed

Nakagawa, Kenji; Gonzalez-Roca, Eva; Souto, Alejandro; Kawai, Toshinao; Umebayashi, Hiroaki; Campistol, Josep María; Cañellas, Jeronima; Takei, Syuji; Kobayashi, Norimoto; Callejas-Rubio, Jose Luis; Ortego-Centeno, Norberto; Ruiz-Ortiz, Estíbaliz; Rius, Fina; Anton, Jordi; Iglesias, Estibaliz; Jimenez-Treviño, Santiago; Vargas, Carmen; Fernandez-Martin, Julian; Calvo, Inmaculada; Hernández-Rodríguez, José; Mendez, María; Dordal, María Teresa; Basagaña, Maria; Bujan, Segundo; Yashiro, Masato; Kubota, Tetsuo; Koike, Ryuji; Akuta, Naoko; Shimoyama, Kumiko; Iwata, Naomi; Saito, Megumu K; Ohara, Osamu; Kambe, Naotomo; Yasumi, Takahiro; Izawa, Kazushi; Kawai, Tomoki; Heike, Toshio; Yagüe, Jordi; Nishikomori, Ryuta; Aróstegui, Juan I

2015-03-01

: Familial cold autoinflammatory syndrome, Muckle-Wells syndrome (MWS), and chronic, infantile, neurological, cutaneous and articular (CINCA) syndrome are dominantly inherited autoinflammatory diseases associated to gain-of-function NLRP3 mutations and included in the cryopyrin-associated periodic syndromes (CAPS). A variable degree of somatic NLRP3 mosaicism has been detected in ≈35% of patients with CINCA. However, no data are currently available regarding the relevance of this mechanism in other CAPS phenotypes. To evaluate somatic NLRP3 mosaicism as the disease-causing mechanism in patients with clinical CAPS phenotypes other than CINCA and NLRP3 mutation-negative. NLRP3 analyses were performed by Sanger sequencing and by massively parallel sequencing. Apoptosis-associated Speck-like protein containing a CARD (ASC)-dependent nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) activation and transfection-induced THP-1 cell death assays determined the functional consequences of the detected variants. A variable degree (5.5-34.9%) of somatic NLRP3 mosaicism was detected in 12.5% of enrolled patients, all of them with a MWS phenotype. Six different missense variants, three novel (p.D303A, p.K355T and p.L411F), were identified. Bioinformatics and functional analyses confirmed that they were disease-causing, gain-of-function NLRP3 mutations. All patients treated with anti-interleukin1 drugs showed long-lasting positive responses. We herein show somatic NLRP3 mosaicism underlying MWS, probably representing a shared genetic mechanism in CAPS not restricted to CINCA syndrome. The data here described allowed definitive diagnoses of these patients, which had serious implications for gaining access to anti-interleukin 1 treatments under legal indication and for genetic counselling. The detection of somatic mosaicism is difficult when using conventional methods. Potential candidates should benefit from the use of modern genetic tools. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
A collaborative approach to developing an electronic health record phenotyping algorithm for drug-induced liver injury

PubMed Central

Overby, Casey Lynnette; Pathak, Jyotishman; Gottesman, Omri; Haerian, Krystl; Perotte, Adler; Murphy, Sean; Bruce, Kevin; Johnson, Stephanie; Talwalkar, Jayant; Shen, Yufeng; Ellis, Steve; Kullo, Iftikhar; Chute, Christopher; Friedman, Carol; Bottinger, Erwin; Hripcsak, George; Weng, Chunhua

2013-01-01

Objective To describe a collaborative approach for developing an electronic health record (EHR) phenotyping algorithm for drug-induced liver injury (DILI). Methods We analyzed types and causes of differences in DILI case definitions provided by two institutions—Columbia University and Mayo Clinic; harmonized two EHR phenotyping algorithms; and assessed the performance, measured by sensitivity, specificity, positive predictive value, and negative predictive value, of the resulting algorithm at three institutions except that sensitivity was measured only at Columbia University. Results Although these sites had the same case definition, their phenotyping methods differed by selection of liver injury diagnoses, inclusion of drugs cited in DILI cases, laboratory tests assessed, laboratory thresholds for liver injury, exclusion criteria, and approaches to validating phenotypes. We reached consensus on a DILI phenotyping algorithm and implemented it at three institutions. The algorithm was adapted locally to account for differences in populations and data access. Implementations collectively yielded 117 algorithm-selected cases and 23 confirmed true positive cases. Discussion Phenotyping for rare conditions benefits significantly from pooling data across institutions. Despite the heterogeneity of EHRs and varied algorithm implementations, we demonstrated the portability of this algorithm across three institutions. The performance of this algorithm for identifying DILI was comparable with other computerized approaches to identify adverse drug events. Conclusions Phenotyping algorithms developed for rare and complex conditions are likely to require adaptive implementation at multiple institutions. Better approaches are also needed to share algorithms. Early agreement on goals, data sources, and validation methods may improve the portability of the algorithms. PMID:23837993
Whole genome prediction and heritability of childhood asthma phenotypes.

PubMed

McGeachie, Michael J; Clemmer, George L; Croteau-Chonka, Damien C; Castaldi, Peter J; Cho, Michael H; Sordillo, Joanne E; Lasky-Su, Jessica A; Raby, Benjamin A; Tantisira, Kelan G; Weiss, Scott T

2016-12-01

While whole genome prediction (WGP) methods have recently demonstrated successes in the prediction of complex genetic diseases, they have not yet been applied to asthma and related phenotypes. Longitudinal patterns of lung function differ between asthmatics, but these phenotypes have not been assessed for heritability or predictive ability. Herein, we assess the heritability and genetic predictability of asthma-related phenotypes. We applied several WGP methods to a well-phenotyped cohort of 832 children with mild-to-moderate asthma from CAMP. We assessed narrow-sense heritability and predictability for airway hyperresponsiveness, serum immunoglobulin E, blood eosinophil count, pre- and post-bronchodilator forced expiratory volume in 1 sec (FEV 1 ), bronchodilator response, steroid responsiveness, and longitudinal patterns of lung function (normal growth, reduced growth, early decline, and their combinations). Prediction accuracy was evaluated using a training/testing set split of the cohort. We found that longitudinal lung function phenotypes demonstrated significant narrow-sense heritability (reduced growth, 95%; normal growth with early decline, 55%). These same phenotypes also showed significant polygenic prediction (areas under the curve [AUCs] 56% to 62%). Including additional demographic covariates in the models increased prediction 4-8%, with reduced growth increasing from 62% to 66% AUC. We found that prediction with a genomic relatedness matrix was improved by filtering available SNPs based on chromatin evidence, and this result extended across cohorts. Longitudinal reduced lung function growth displayed extremely high heritability. All phenotypes with significant heritability showed significant polygenic prediction. Using SNP-prioritization increased prediction across cohorts. WGP methods show promise in predicting asthma-related heritable traits.
Using leaf optical properties to detect ozone effects on foliar biochemistry

USDA-ARS?s Scientific Manuscript database

Efficient methods for accurate and meaningful high-throughput plant phenotyping are limiting the development and breeding of stress-tolerant crops. A number of emerging techniques, specifically remote sensing methods, have been identified as promising tools for plant phenotyping. These remote-sensin...
Methods and compositions to modulate ethylene sensitivity

DOEpatents

Stepanova, Anna N.; Ecker, Joseph R.

2007-01-30

The field of the invention relates to plants and plant genes, including both plant mutants and transgenic plants containing a gene that confers an ethylene insensitive phenotype. Also encompassed by the invention are methods of using the disclosed plant gene to confer an ethylene insensitive phenotype.
Development of Poly(Ethylene Glycol) Hydrogels for Salivary Gland Tissue Engineering Applications

PubMed Central

Shubin, Andrew D.; Felong, Timothy J.; Graunke, Dean; Ovitt, Catherine E.

2015-01-01

More than 40,000 patients are diagnosed with head and neck cancers annually in the United States with the vast majority receiving radiation therapy. Salivary glands are irreparably damaged by radiation therapy resulting in xerostomia, which severely affects patient quality of life. Cell-based therapies have shown some promise in mouse models of radiation-induced xerostomia, but they suffer from insufficient and inconsistent gland regeneration and accompanying secretory function. To aid in the development of regenerative therapies, poly(ethylene glycol) hydrogels were investigated for the encapsulation of primary submandibular gland (SMG) cells for tissue engineering applications. Different methods of hydrogel formation and cell preparation were examined to identify cytocompatible encapsulation conditions for SMG cells. Cell viability was much higher after thiol-ene polymerizations compared with conventional methacrylate polymerizations due to reduced membrane peroxidation and intracellular reactive oxygen species formation. In addition, the formation of multicellular microspheres before encapsulation maximized cell–cell contacts and increased viability of SMG cells over 14-day culture periods. Thiol-ene hydrogel-encapsulated microspheres also promoted SMG proliferation. Lineage tracing was employed to determine the cellular composition of hydrogel-encapsulated microspheres using markers for acinar (Mist1) and duct (Keratin5) cells. Our findings indicate that both acinar and duct cell phenotypes are present throughout the 14 day culture period. However, the acinar:duct cell ratios are reduced over time, likely due to duct cell proliferation. Altogether, permissive encapsulation methods for primary SMG cells have been identified that promote cell viability, proliferation, and maintenance of differentiated salivary gland cell phenotypes, which allows for translation of this approach for salivary gland tissue engineering applications. PMID:25762214
Mechanism-based facilitated maturation of human pluripotent stem cell-derived cardiomyocytes

PubMed Central

Lieu, Deborah K.; Fu, Ji-Dong; Chiamvimonvat, Nipavan; Chan Tung, Kelvin W.; McNerney, Gregory P.; Huser, Thomas; Keller, Gordon; Kong, Chi-Wing

2013-01-01

Background Human embryonic stem cells (hESCs) can be efficiently and reproducibly directed into cardiomyocytes (CMs) using stage-specific induction protocols. However, their functional properties and suitability for clinical and other applications have not been evaluated. Methods and Results Here we showed that CMs derived from multiple pluripotent human stem cell lines (hESC: H1, HES2) and types (induced pluripotent stem cell or iPSC) using different in vitro differentiation protocols (embryoid body formation, endodermal induction, directed differentiation) commonly displayed immature, pro-arrhythmic action potential (AP) properties such as high-degree of automaticity, depolarized resting membrane potential (RMP), Phase 4- depolarization and delayed after-depolarization (DAD). Among the panoply of sarcolemmal ionic currents investigated (INa+/ICaL2+/IKr+/INCX+/If+/Ito+/IK1-/IKs-), we pinpointed the lack of the Kir2.1-encoded inwardly rectifying K+ current (IK1) as the single mechanistic contributor to the observed immature electrophysiological properties in hESC-CMs. Forced expression of Kir2.1 in hESC-CMs led to robust expression of Ba2+-sensitive IK1 and more importantly, completely ablated all the pro-arrhythmic AP traits, rendering the electrophysiological phenotype indistinguishable from the adult counterparts. These results provided the first link of a complex developmentally arrested phenotype to a major effector gene, and importantly, further led us to develop a biomimetic culturing strategy for enhancing maturation. Conclusions By providing the environmental cues that are missing in conventional culturing method, this approach did not require any genetic or pharmacological interventions. Our findings can facilitate clinical applications, drug discovery and cardiotoxicity screening by improving the yield, safety and efficacy of derived CMs. PMID:23392582
Feasibility for improving phytonutrient content in vegetable crops using conventional breeding strategies: case study with carotenoids and tocopherols in sweet corn and broccoli.

PubMed

Ibrahim, Khalid E; Juvik, John A

2009-06-10

Among vegetables, sweet corn ( Zea mays L.) and broccoli ( Brassica oleracea L. ssp. italica) are important sources of dietary carotenoids and tocopherols. Because medical evidence suggests that carotenoid and tocopherol health-promoting activity acts in a dose-dependent manner, conventional breeding to develop elite sweet corn and broccoli germplasm with enhanced levels of these phytochemicals will potentially promote health among the consuming public. This investigation includes the quantitative analysis of carotenoid and tocopherol contents of 41 corn and 24 broccoli genotypes grown in multiple environments (years and seasons in one location) to partition the variation into genetic, environment, and genotype by environment interaction (GxE) components and measure the phenotypic stability of genotypes for these phytochemicals. The primary carotenoids and tocopherols in corn were lutein and gamma-tocopherol (65 and 73% of total carotenoid and tocopherol, respectively), whereas beta-carotene and alpha-tocopherol were dominant in broccoli (65 and 79% of total carotenoid and tocopherol, respectively). Partitioning of the variance indicated that genetic differences among the genotypes averaged for the primary compounds in corn (lutein, zeaxanthin, and alpha- and gamma-tocopherol) and broccoli (beta-carotene, lutein, and alpha- and gamma-tocopherol) accounted for the largest proportion of the variation (67 and 55% of total phenotypic variation averaged across the phytochemicals in sweet corn and broccoli, respectively). Stability analysis identified several corn (IL451b sh2 and IL2027-8 sh2) and broccoli ('Pirate' and 'Baccus') genotypes with relatively high mean concentrations for the various carotenoids and tocopherols that were comparatively stable across seasons and years. The results of this investigation suggest that sweet corn and broccoli germplasm with enhanced concentrations of carotenoids and tocopherols can be developed using conventional breeding protocols.
Design and baseline characteristics of the Food4Me study: a web-based randomised controlled trial of personalised nutrition in seven European countries.

PubMed

Celis-Morales, Carlos; Livingstone, Katherine M; Marsaux, Cyril F M; Forster, Hannah; O'Donovan, Clare B; Woolhead, Clara; Macready, Anna L; Fallaize, Rosalind; Navas-Carretero, Santiago; San-Cristobal, Rodrigo; Kolossa, Silvia; Hartwig, Kai; Tsirigoti, Lydia; Lambrinou, Christina P; Moschonis, George; Godlewska, Magdalena; Surwiłło, Agnieszka; Grimaldi, Keith; Bouwman, Jildau; Daly, E J; Akujobi, Victor; O'Riordan, Rick; Hoonhout, Jettie; Claassen, Arjan; Hoeller, Ulrich; Gundersen, Thomas E; Kaland, Siv E; Matthews, John N S; Manios, Yannis; Traczyk, Iwona; Drevon, Christian A; Gibney, Eileen R; Brennan, Lorraine; Walsh, Marianne C; Lovegrove, Julie A; Alfredo Martinez, J; Saris, Wim H M; Daniel, Hannelore; Gibney, Mike; Mathers, John C

2015-01-01

Improving lifestyle behaviours has considerable potential for reducing the global burden of non-communicable diseases, promoting better health across the life-course and increasing well-being. However, realising this potential will require the development, testing and implementation of much more effective behaviour change interventions than are used conventionally. Therefore, the aim of this study was to conduct a multi-centre, web-based, proof-of-principle study of personalised nutrition (PN) to determine whether providing more personalised dietary advice leads to greater improvements in eating patterns and health outcomes compared to conventional population-based advice. A total of 5,562 volunteers were screened across seven European countries; the first 1,607 participants who fulfilled the inclusion criteria were recruited into the trial. Participants were randomly assigned to one of the following intervention groups for a 6-month period: Level 0-control group-receiving conventional, non-PN advice; Level 1-receiving PN advice based on dietary intake data alone; Level 2-receiving PN advice based on dietary intake and phenotypic data; and Level 3-receiving PN advice based on dietary intake, phenotypic and genotypic data. A total of 1,607 participants had a mean age of 39.8 years (ranging from 18 to 79 years). Of these participants, 60.9 % were women and 96.7 % were from white-European background. The mean BMI for all randomised participants was 25.5 kg m(-2), and 44.8 % of the participants had a BMI ≥ 25.0 kg m(-2). Food4Me is the first large multi-centre RCT of web-based PN. The main outcomes from the Food4Me study will be submitted for publication during 2015.
Behavioural consequences of IVC cages on male and female C57BL/6J mice.

PubMed

Logge, W; Kingham, J; Karl, T

2013-05-01

Recent developments in the technology to breed and house laboratory rodents for medical research has produced individually ventilated cage (IVC) systems. These IVC systems produce a cage environment significantly different to conventional cages. As it is not known in detail whether housing mice in IVCs impacts on their baseline and drug-induced behaviours compared to mice of conventional filter-top cages a comprehensive multi-tiered phenotyping strategy was used to test the behavioural consequences of IVC housing in male and female C57BL/6JArc mice. IVC had anxiety-like effects in the elevated plus maze, which were more pronounced in female mice whereas cognition and locomotion of all test mice were not modified by IVC housing. Mice raised in IVC cage systems were socially more active than mice of filter-top systems. Furthermore, males raised in IVC exhibited an increased sensitivity to the locomotor-stimulating effects of acute MK-801 treatment compared to males in conventional cages. In summary, this is the first study revealing the longer-term effects of IVC housing on social behaviours and the locomotor response to an acute MK-801 challenge. In conclusion, researchers upgrading their holding facilities to IVC housing may encounter a shift in experimental outcomes (e.g. post pharmacological challenges) and the behavioural phenotype of test mice. Furthermore, differences between the housing conditions of breeding facilities and test facilities must carefully be considered. Finally, researchers should clarify in detail the type of housing test animals have been exposed to when publishing experimental animal research data. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.
A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

PubMed Central

Martins, Marta; McCusker, Matthew P; Viveiros, Miguel; Couto, Isabel; Fanning, Séamus; Pagès, Jean-Marie; Amaral, Leonard

2013-01-01

It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors. PMID:23589748
Phenotype Instance Verification and Evaluation Tool (PIVET): A Scaled Phenotype Evidence Generation Framework Using Web-Based Medical Literature

PubMed Central

Ke, Junyuan; Ho, Joyce C; Ghosh, Joydeep; Wallace, Byron C

2018-01-01

Background Researchers are developing methods to automatically extract clinically relevant and useful patient characteristics from raw healthcare datasets. These characteristics, often capturing essential properties of patients with common medical conditions, are called computational phenotypes. Being generated by automated or semiautomated, data-driven methods, such potential phenotypes need to be validated as clinically meaningful (or not) before they are acceptable for use in decision making. Objective The objective of this study was to present Phenotype Instance Verification and Evaluation Tool (PIVET), a framework that uses co-occurrence analysis on an online corpus of publically available medical journal articles to build clinical relevance evidence sets for user-supplied phenotypes. PIVET adopts a conceptual framework similar to the pioneering prototype tool PheKnow-Cloud that was developed for the phenotype validation task. PIVET completely refactors each part of the PheKnow-Cloud pipeline to deliver vast improvements in speed without sacrificing the quality of the insights PheKnow-Cloud achieved. Methods PIVET leverages indexing in NoSQL databases to efficiently generate evidence sets. Specifically, PIVET uses a succinct representation of the phenotypes that corresponds to the index on the corpus database and an optimized co-occurrence algorithm inspired by the Aho-Corasick algorithm. We compare PIVET’s phenotype representation with PheKnow-Cloud’s by using PheKnow-Cloud’s experimental setup. In PIVET’s framework, we also introduce a statistical model trained on domain expert–verified phenotypes to automatically classify phenotypes as clinically relevant or not. Additionally, we show how the classification model can be used to examine user-supplied phenotypes in an online, rather than batch, manner. Results PIVET maintains the discriminative power of PheKnow-Cloud in terms of identifying clinically relevant phenotypes for the same corpus with which PheKnow-Cloud was originally developed, but PIVET’s analysis is an order of magnitude faster than that of PheKnow-Cloud. Not only is PIVET much faster, it can be scaled to a larger corpus and still retain speed. We evaluated multiple classification models on top of the PIVET framework and found ridge regression to perform best, realizing an average F1 score of 0.91 when predicting clinically relevant phenotypes. Conclusions Our study shows that PIVET improves on the most notable existing computational tool for phenotype validation in terms of speed and automation and is comparable in terms of accuracy. PMID:29728351
Active Learning Strategies for Phenotypic Profiling of High-Content Screens.

PubMed

Smith, Kevin; Horvath, Peter

2014-06-01

High-content screening is a powerful method to discover new drugs and carry out basic biological research. Increasingly, high-content screens have come to rely on supervised machine learning (SML) to perform automatic phenotypic classification as an essential step of the analysis. However, this comes at a cost, namely, the labeled examples required to train the predictive model. Classification performance increases with the number of labeled examples, and because labeling examples demands time from an expert, the training process represents a significant time investment. Active learning strategies attempt to overcome this bottleneck by presenting the most relevant examples to the annotator, thereby achieving high accuracy while minimizing the cost of obtaining labeled data. In this article, we investigate the impact of active learning on single-cell-based phenotype recognition, using data from three large-scale RNA interference high-content screens representing diverse phenotypic profiling problems. We consider several combinations of active learning strategies and popular SML methods. Our results show that active learning significantly reduces the time cost and can be used to reveal the same phenotypic targets identified using SML. We also identify combinations of active learning strategies and SML methods which perform better than others on the phenotypic profiling problems we studied. © 2014 Society for Laboratory Automation and Screening.
A Comprehensive Evaluation of Xpert MTB/RIF Assay With Bronchoalveolar Lavage Fluid as a Single Test or Combined With Conventional Assays for Diagnosis of Pulmonary Tuberculosis in China: A Two-Center Prospective Study

PubMed Central

Pan, Xiaofu; Yang, Shoufeng; Deighton, Margaret A.; Qu, Yue; Hong, Liang; Su, Feifei

2018-01-01

Introduction: The Xpert MTB/RIF is recommended by the World Health Organization as a first line rapid test for the diagnosis of pulmonary tuberculosis (TB); however, China does not routinely use this test, partially due to the lack of a sufficient number of systematic evaluations of this assay in local patients. The aims of this study were to comprehensively assess the diagnostic performance of Xpert MTB/RIF, either alone or in combination with conventional assays for the diagnosis of pulmonary TB in adult Chinese patients. Methods: Xpert MTB/RIF tests were performed in 190 adult patients with suspected pulmonary TB, using bronchoalveolar lavage fluid (BALF) as test specimens. In parallel, conventional tests were carried out using the same BALF samples. Using two different reference standards, the performance of Xpert MTB/RIF, conventional assays and their combinations were evaluated. Results: Using mycobacterial culture as the reference comparator, Xpert MTB/RIF was found to be superior to smear-microscopy in detecting Mycobacterium tuberculosis. When final diagnosis, based on clinical criteria, was employed as the reference standard, Xpert MTB/RIF showed an even higher accuracy of 72.1%, supported by a sensitivity of 61.1% and specificity of 96.6%. Xpert MTB/RIF also demonstrated a powerful capability to identify pulmonary TB cases undetected by culture or smear-microscopy. Combining smear-microscopy and Xpert MTB/RIF was found to be the most accurate early predictor for pulmonary TB. Rifampicin resistance reported by Xpert MTB/RIF slightly deviated from that by phenotypic antibiotic susceptibility testing and requires further study with a larger sample size. Conclusion: This two-center prospective study highlights the value of Xpert MTB/RIF with BALF in diagnosing pulmonary TB in adult Chinese patients. These findings might contribute to the optimization of current diagnostic algorithms for pulmonary TB in China. PMID:29593688
A Comprehensive Evaluation of Xpert MTB/RIF Assay With Bronchoalveolar Lavage Fluid as a Single Test or Combined With Conventional Assays for Diagnosis of Pulmonary Tuberculosis in China: A Two-Center Prospective Study.

PubMed

Pan, Xiaofu; Yang, Shoufeng; Deighton, Margaret A; Qu, Yue; Hong, Liang; Su, Feifei

2018-01-01

Introduction: The Xpert MTB/RIF is recommended by the World Health Organization as a first line rapid test for the diagnosis of pulmonary tuberculosis (TB); however, China does not routinely use this test, partially due to the lack of a sufficient number of systematic evaluations of this assay in local patients. The aims of this study were to comprehensively assess the diagnostic performance of Xpert MTB/RIF, either alone or in combination with conventional assays for the diagnosis of pulmonary TB in adult Chinese patients. Methods: Xpert MTB/RIF tests were performed in 190 adult patients with suspected pulmonary TB, using bronchoalveolar lavage fluid (BALF) as test specimens. In parallel, conventional tests were carried out using the same BALF samples. Using two different reference standards, the performance of Xpert MTB/RIF, conventional assays and their combinations were evaluated. Results: Using mycobacterial culture as the reference comparator, Xpert MTB/RIF was found to be superior to smear-microscopy in detecting Mycobacterium tuberculosis . When final diagnosis, based on clinical criteria, was employed as the reference standard, Xpert MTB/RIF showed an even higher accuracy of 72.1%, supported by a sensitivity of 61.1% and specificity of 96.6%. Xpert MTB/RIF also demonstrated a powerful capability to identify pulmonary TB cases undetected by culture or smear-microscopy. Combining smear-microscopy and Xpert MTB/RIF was found to be the most accurate early predictor for pulmonary TB. Rifampicin resistance reported by Xpert MTB/RIF slightly deviated from that by phenotypic antibiotic susceptibility testing and requires further study with a larger sample size. Conclusion: This two-center prospective study highlights the value of Xpert MTB/RIF with BALF in diagnosing pulmonary TB in adult Chinese patients. These findings might contribute to the optimization of current diagnostic algorithms for pulmonary TB in China.
Molecular diagnosis of cystic fibrosis.

PubMed

Shrimpton, Antony E

2002-05-01

A review of the current molecular diagnosis of cystic fibrosis including an introduction to cystic fibrosis, the gene function, the phenotypic variation, who should be screened for which mutation, newborn and couple screening, quality assurance, phenotype-genotype correlation, methods and method limitations, options, statements, recommendations, useful Websites and treatments.

Prediction of Human Phenotype Ontology terms by means of hierarchical ensemble methods.

PubMed

Notaro, Marco; Schubach, Max; Robinson, Peter N; Valentini, Giorgio

2017-10-12

The prediction of human gene-abnormal phenotype associations is a fundamental step toward the discovery of novel genes associated with human disorders, especially when no genes are known to be associated with a specific disease. In this context the Human Phenotype Ontology (HPO) provides a standard categorization of the abnormalities associated with human diseases. While the problem of the prediction of gene-disease associations has been widely investigated, the related problem of gene-phenotypic feature (i.e., HPO term) associations has been largely overlooked, even if for most human genes no HPO term associations are known and despite the increasing application of the HPO to relevant medical problems. Moreover most of the methods proposed in literature are not able to capture the hierarchical relationships between HPO terms, thus resulting in inconsistent and relatively inaccurate predictions. We present two hierarchical ensemble methods that we formally prove to provide biologically consistent predictions according to the hierarchical structure of the HPO. The modular structure of the proposed methods, that consists in a "flat" learning first step and a hierarchical combination of the predictions in the second step, allows the predictions of virtually any flat learning method to be enhanced. The experimental results show that hierarchical ensemble methods are able to predict novel associations between genes and abnormal phenotypes with results that are competitive with state-of-the-art algorithms and with a significant reduction of the computational complexity. Hierarchical ensembles are efficient computational methods that guarantee biologically meaningful predictions that obey the true path rule, and can be used as a tool to improve and make consistent the HPO terms predictions starting from virtually any flat learning method. The implementation of the proposed methods is available as an R package from the CRAN repository.
Multiple testing and power calculations in genetic association studies.

PubMed

So, Hon-Cheong; Sham, Pak C

2011-01-01

Modern genetic association studies typically involve multiple single-nucleotide polymorphisms (SNPs) and/or multiple genes. With the development of high-throughput genotyping technologies and the reduction in genotyping cost, investigators can now assay up to a million SNPs for direct or indirect association with disease phenotypes. In addition, some studies involve multiple disease or related phenotypes and use multiple methods of statistical analysis. The combination of multiple genetic loci, multiple phenotypes, and multiple methods of evaluating associations between genotype and phenotype means that modern genetic studies often involve the testing of an enormous number of hypotheses. When multiple hypothesis tests are performed in a study, there is a risk of inflation of the type I error rate (i.e., the chance of falsely claiming an association when there is none). Several methods for multiple-testing correction are in popular use, and they all have strengths and weaknesses. Because no single method is universally adopted or always appropriate, it is important to understand the principles, strengths, and weaknesses of the methods so that they can be applied appropriately in practice. In this article, we review the three principle methods for multiple-testing correction and provide guidance for calculating statistical power.
Neuroanatomical phenotyping of the mouse brain with three-dimensional autofluorescence imaging

PubMed Central

Wong, Michael D.; Dazai, Jun; Altaf, Maliha; Mark Henkelman, R.; Lerch, Jason P.; Nieman, Brian J.

2012-01-01

The structural organization of the brain is important for normal brain function and is critical to understand in order to evaluate changes that occur during disease processes. Three-dimensional (3D) imaging of the mouse brain is necessary to appreciate the spatial context of structures within the brain. In addition, the small scale of many brain structures necessitates resolution at the ∼10 μm scale. 3D optical imaging techniques, such as optical projection tomography (OPT), have the ability to image intact large specimens (1 cm3) with ∼5 μm resolution. In this work we assessed the potential of autofluorescence optical imaging methods, and specifically OPT, for phenotyping the mouse brain. We found that both specimen size and fixation methods affected the quality of the OPT image. Based on these findings we developed a specimen preparation method to improve the images. Using this method we assessed the potential of optical imaging for phenotyping. Phenotypic differences between wild-type male and female mice were quantified using computer-automated methods. We found that optical imaging of the endogenous autofluorescence in the mouse brain allows for 3D characterization of neuroanatomy and detailed analysis of brain phenotypes. This will be a powerful tool for understanding mouse models of disease and development and is a technology that fits easily within the workflow of biology and neuroscience labs. PMID:22718750
SU-F-R-53: CT-Based Radiomics Analysis of Non-Small Cell Lung Cancer Patients Treated with Stereotactic Body Radiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huynh, E; Coroller, T; Narayan, V

Purpose: Stereotactic body radiation therapy (SBRT) is the standard of care for medically inoperable non-small cell lung cancer (NSCLC) patients and has demonstrated excellent local control and survival. However, some patients still develop distant metastases and local recurrence, and therefore, there is a clinical need to identify patients at high-risk of disease recurrence. The aim of the current study is to use a radiomics approach to identify imaging biomarkers, based on tumor phenotype, for clinical outcomes in SBRT patients. Methods: Radiomic features were extracted from free breathing computed tomography (CT) images of 113 Stage I-II NSCLC patients treated with SBRT.more » Their association to and prognostic performance for distant metastasis (DM), locoregional recurrence (LRR) and survival was assessed and compared with conventional features (tumor volume and diameter) and clinical parameters (e.g. performance status, overall stage). The prognostic performance was evaluated using the concordance index (CI). Multivariate model performance was evaluated using cross validation. All p-values were corrected for multiple testing using the false discovery rate. Results: Radiomic features were associated with DM (one feature), LRR (one feature) and survival (four features). Conventional features were only associated with survival and one clinical parameter was associated with LRR and survival. One radiomic feature was significantly prognostic for DM (CI=0.670, p<0.1 from random), while none of the conventional and clinical parameters were significant for DM. The multivariate radiomic model had a higher median CI (0.671) for DM than the conventional (0.618) and clinical models (0.617). Conclusion: Radiomic features have potential to be imaging biomarkers for clinical outcomes that conventional imaging metrics and clinical parameters cannot predict in SBRT patients, such as distant metastasis. Development of a radiomics biomarker that can identify patients at high-risk of recurrence could facilitate personalization of their treatment regimen for an optimized clinical outcome. R.M. had consulting interest with Amgen (ended in 2015).« less
Behavioural Phenotypes and Special Educational Needs: Is Aetiology Important in the Classroom?

ERIC Educational Resources Information Center

Reilly, C.

2012-01-01

Background: A number of genetic conditions with associated intellectual disability and/or special educational needs have increasingly well-defined behavioural phenotypes. Thus, the concept of "behavioural phenotype" and aetiology of intellectual disability may be important with regard to school-based interventions. Method: The evidence for…
Auditory Phenotype of Smith-Magenis Syndrome

ERIC Educational Resources Information Center

Brendal, Megan A.; King, Kelly A.; Zalewski, Christopher K.; Finucane, Brenda M.; Introne, Wendy; Brewer, Carmen C.; Smith, Ann C. M.

2017-01-01

Purpose: The purpose of this study was to describe the auditory phenotype of a large cohort with Smith-Magenis syndrome (SMS), a rare disorder including physical anomalies, cognitive deficits, sleep disturbances, and a distinct behavioral phenotype. Method: Hearing-related data were collected for 133 individuals with SMS aged 1-49 years. Audiogram…
Adjusting data to body size: a comparison of methods as applied to quantitative trait loci analysis of musculoskeletal phenotypes.

PubMed

Lang, Dean H; Sharkey, Neil A; Lionikas, Arimantas; Mack, Holly A; Larsson, Lars; Vogler, George P; Vandenbergh, David J; Blizard, David A; Stout, Joseph T; Stitt, Joseph P; McClearn, Gerald E

2005-05-01

The aim of this study was to compare three methods of adjusting skeletal data for body size and examine their use in QTL analyses. It was found that dividing skeletal phenotypes by body mass index induced erroneous QTL results. The preferred method of body size adjustment was multiple regression. Many skeletal studies have reported strong correlations between phenotypes for muscle, bone, and body size, and these correlations add to the difficulty in identifying genetic influence on skeletal traits that are not mediated through overall body size. Quantitative trait loci (QTL) identified for skeletal phenotypes often map to the same chromosome regions as QTLs for body size. The actions of a QTL identified as influencing BMD could therefore be mediated through the generalized actions of growth on body size or muscle mass. Three methods of adjusting skeletal phenotypes to body size were performed on morphologic, structural, and compositional measurements of the femur and tibia in 200-day-old C57BL/6J x DBA/2 (BXD) second generation (F(2)) mice (n = 400). A common method of removing the size effect has been through the use of ratios. This technique and two alternative techniques using simple and multiple regression were performed on muscle and skeletal data before QTL analyses, and the differences in QTL results were examined. The use of ratios to remove the size effect was shown to increase the size effect by inducing spurious correlations, thereby leading to inaccurate QTL results. Adjustments for body size using multiple regression eliminated these problems. Multiple regression should be used to remove the variance of co-factors related to skeletal phenotypes to allow for the study of genetic influence independent of correlated phenotypes. However, to better understand the genetic influence, adjusted and unadjusted skeletal QTL results should be compared. Additional insight can be gained by observing the difference in LOD score between the adjusted and nonadjusted phenotypes. Identifying QTLs that exert their effects on skeletal phenotypes through body size-related pathways as well as those having a more direct and independent influence on bone are equally important in deciphering the complex physiologic pathways responsible for the maintenance of bone health.
Autoimmune-autoinflammatory rheumatoid arthritis overlaps: a rare but potentially important subgroup of diseases

PubMed Central

Savic, Sinisa; Mistry, Anoop; Wilson, Anthony G; Barcenas-Morales, Gabriela; Doffinger, Rainer; Emery, Paul; McGonagle, Dennis

2017-01-01

At the population level, rheumatoid arthritis (RA) is generally viewed as autoimmune in nature with a small subgroup of cases having a palindromic form or systemic autoinflammatory disorder (SAID) phenotype. Herein, we describe resistant cases of classical autoantibody associated RA that had clinical, genetic and therapeutic responses indicative of coexistent autoinflammatory disease. Five patients with clinically overlapping features between RA and SAID including polysynovitis and autoantibody/shared epitope positivity, and who had abrupt severe self-limiting attacks including fevers and serositis, are described. Mutations or single nucleotide polymorphisms in recognised autoinflammatory pathways were evident. Generally, these cases responded poorly to conventional Disease-modifying anti-rheumatic drugs (DMARD) treatment with some excellent responses to colchicine or interleukin 1 pathway blockade. A subgroup of RA cases have a mixed autoimmune-autoinflammatory phenotype and genotype with therapeutic implications. PMID:29177082
Autoimmune-autoinflammatory rheumatoid arthritis overlaps: a rare but potentially important subgroup of diseases.

PubMed

Savic, Sinisa; Mistry, Anoop; Wilson, Anthony G; Barcenas-Morales, Gabriela; Doffinger, Rainer; Emery, Paul; McGonagle, Dennis

2017-01-01

At the population level, rheumatoid arthritis (RA) is generally viewed as autoimmune in nature with a small subgroup of cases having a palindromic form or systemic autoinflammatory disorder (SAID) phenotype. Herein, we describe resistant cases of classical autoantibody associated RA that had clinical, genetic and therapeutic responses indicative of coexistent autoinflammatory disease. Five patients with clinically overlapping features between RA and SAID including polysynovitis and autoantibody/shared epitope positivity, and who had abrupt severe self-limiting attacks including fevers and serositis, are described. Mutations or single nucleotide polymorphisms in recognised autoinflammatory pathways were evident. Generally, these cases responded poorly to conventional Disease-modifying anti-rheumatic drugs (DMARD) treatment with some excellent responses to colchicine or interleukin 1 pathway blockade. A subgroup of RA cases have a mixed autoimmune-autoinflammatory phenotype and genotype with therapeutic implications.
SigniSite: Identification of residue-level genotype-phenotype correlations in protein multiple sequence alignments.

PubMed

Jessen, Leon Eyrich; Hoof, Ilka; Lund, Ole; Nielsen, Morten

2013-07-01

Identifying which mutation(s) within a given genotype is responsible for an observable phenotype is important in many aspects of molecular biology. Here, we present SigniSite, an online application for subgroup-free residue-level genotype-phenotype correlation. In contrast to similar methods, SigniSite does not require any pre-definition of subgroups or binary classification. Input is a set of protein sequences where each sequence has an associated real number, quantifying a given phenotype. SigniSite will then identify which amino acid residues are significantly associated with the data set phenotype. As output, SigniSite displays a sequence logo, depicting the strength of the phenotype association of each residue and a heat-map identifying 'hot' or 'cold' regions. SigniSite was benchmarked against SPEER, a state-of-the-art method for the prediction of specificity determining positions (SDP) using a set of human immunodeficiency virus protease-inhibitor genotype-phenotype data and corresponding resistance mutation scores from the Stanford University HIV Drug Resistance Database, and a data set of protein families with experimentally annotated SDPs. For both data sets, SigniSite was found to outperform SPEER. SigniSite is available at: http://www.cbs.dtu.dk/services/SigniSite/.
Molecular epidemiology of Pseudomonas aeruginosa.

PubMed

Speert, David P

2002-10-01

Pseudomonas aeruginosa is a serious opportunistic pathogen in certain compromised hosts, such as those with cystic fibrosis, thermal burns and cancer. It also causes less severe noninvasive disease, such as otitis externa and hot tub folliculitis, in normal hosts. P. aeruginosa is phenotypically very unstable, particularly in patients with chronic infection. Phenotypic typing techniques are useful for understanding the epidemiology of acute infections, but they are limited by their discriminatory power and by their inability to group isolates that are phenotypically unrelated but genetically homologous. Molecular typing techniques, developed over the past decade, are highly discriminatory and are useful for typing strains from patients with chronic infection where the bacterial phenotype is unstable; this is particularly true in cystic fibrosis, where patients often are infected with the same strain for several decades, but the bacteria undergo phenotypic alteration. Molecular typing techniques, which have proven useful in typing P. aeruginosa for epidemiological purposes, include pulsed field gel electrophoresis, restriction fragment length polymorphic DNA analysis, random amplified polymorphic DNA analysis, repetitive extrapalindromic PCR analysis, and multilocus sequence typing. These methods are generally only available in specialized laboratories, but they should be used when data from phenotypic typing analysis are ambiguous or when phenotypic methods are unreliable, such as in cystic fibrosis.
HIV coreceptor phenotyping in the clinical setting.

PubMed

Low, Andrew J; Swenson, Luke C; Harrigan, P Richard

2008-01-01

The introduction of CCR5 antagonists increases the options available for constructing antiretroviral regimens. However, this option is coupled with the caveat that patients should be tested for HIV coreceptor tropism prior to initiating CCR5 antagonist-based therapy. Failure to screen for CXCR4 usage increases the risk of using an ineffective drug, thus reducing the likelihood of viral suppression and increasing their risk for developing antiretroviral resistance. This review discusses current and future methods of determining HIV tropism, with a focus on their utility in the clinical setting for screening purposes. Some of these methods include recombinant phenotypic tests, such as the Monogram Trofile assay, as well as genotype-based predictors, heteroduplex tracking assays, and flow cytometry based methods. Currently, the best evidence supports the use of phenotypic methods, although other methods of screening for HIV coreceptor usage prior to the administration of CCR5 antagonists may reduce costs and increase turnaround time over phenotypic methods. The presence of low levels of X4 virus is a challenge to all assay methods, resulting in reduced sensitivity in clinical, patient-derived samples when compared to clonally derived samples. Gaining a better understanding of the output of these assays and correlating them with clinical progression and therapy response will provide some indication on how both genotype-based, and phenotypic assays for determining HIV coreceptor usage can be improved. In addition, leveraging new technologies capable of detecting low-level minority species may provide the most significant advances in ensuring that individuals with low levels of dual/mixed tropic virus are not inadvertently prescribed CCR5 antagonists.
Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

PubMed

Krämer, Christina E M; Wiechert, Wolfgang; Kohlheyer, Dietrich

2016-09-01

Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.
Multivariate generalized multifactor dimensionality reduction to detect gene-gene interactions

PubMed Central

2013-01-01

Background Recently, one of the greatest challenges in genome-wide association studies is to detect gene-gene and/or gene-environment interactions for common complex human diseases. Ritchie et al. (2001) proposed multifactor dimensionality reduction (MDR) method for interaction analysis. MDR is a combinatorial approach to reduce multi-locus genotypes into high-risk and low-risk groups. Although MDR has been widely used for case-control studies with binary phenotypes, several extensions have been proposed. One of these methods, a generalized MDR (GMDR) proposed by Lou et al. (2007), allows adjusting for covariates and applying to both dichotomous and continuous phenotypes. GMDR uses the residual score of a generalized linear model of phenotypes to assign either high-risk or low-risk group, while MDR uses the ratio of cases to controls. Methods In this study, we propose multivariate GMDR, an extension of GMDR for multivariate phenotypes. Jointly analysing correlated multivariate phenotypes may have more power to detect susceptible genes and gene-gene interactions. We construct generalized estimating equations (GEE) with multivariate phenotypes to extend generalized linear models. Using the score vectors from GEE we discriminate high-risk from low-risk groups. We applied the multivariate GMDR method to the blood pressure data of the 7,546 subjects from the Korean Association Resource study: systolic blood pressure (SBP) and diastolic blood pressure (DBP). We compare the results of multivariate GMDR for SBP and DBP to the results from separate univariate GMDR for SBP and DBP, respectively. We also applied the multivariate GMDR method to the repeatedly measured hypertension status from 5,466 subjects and compared its result with those of univariate GMDR at each time point. Results Results from the univariate GMDR and multivariate GMDR in two-locus model with both blood pressures and hypertension phenotypes indicate best combinations of SNPs whose interaction has significant association with risk for high blood pressures or hypertension. Although the test balanced accuracy (BA) of multivariate analysis was not always greater than that of univariate analysis, the multivariate BAs were more stable with smaller standard deviations. Conclusions In this study, we have developed multivariate GMDR method using GEE approach. It is useful to use multivariate GMDR with correlated multiple phenotypes of interests. PMID:24565370
An efficient genome-wide association test for mixed binary and continuous phenotypes with applications to substance abuse research.

PubMed

Buu, Anne; Williams, L Keoki; Yang, James J

2018-03-01

We propose a new genome-wide association test for mixed binary and continuous phenotypes that uses an efficient numerical method to estimate the empirical distribution of the Fisher's combination statistic under the null hypothesis. Our simulation study shows that the proposed method controls the type I error rate and also maintains its power at the level of the permutation method. More importantly, the computational efficiency of the proposed method is much higher than the one of the permutation method. The simulation results also indicate that the power of the test increases when the genetic effect increases, the minor allele frequency increases, and the correlation between responses decreases. The statistical analysis on the database of the Study of Addiction: Genetics and Environment demonstrates that the proposed method combining multiple phenotypes can increase the power of identifying markers that may not be, otherwise, chosen using marginal tests.
Toward high-throughput phenotyping: unbiased automated feature extraction and selection from knowledge sources.

PubMed

Yu, Sheng; Liao, Katherine P; Shaw, Stanley Y; Gainer, Vivian S; Churchill, Susanne E; Szolovits, Peter; Murphy, Shawn N; Kohane, Isaac S; Cai, Tianxi

2015-09-01

Analysis of narrative (text) data from electronic health records (EHRs) can improve population-scale phenotyping for clinical and genetic research. Currently, selection of text features for phenotyping algorithms is slow and laborious, requiring extensive and iterative involvement by domain experts. This paper introduces a method to develop phenotyping algorithms in an unbiased manner by automatically extracting and selecting informative features, which can be comparable to expert-curated ones in classification accuracy. Comprehensive medical concepts were collected from publicly available knowledge sources in an automated, unbiased fashion. Natural language processing (NLP) revealed the occurrence patterns of these concepts in EHR narrative notes, which enabled selection of informative features for phenotype classification. When combined with additional codified features, a penalized logistic regression model was trained to classify the target phenotype. The authors applied our method to develop algorithms to identify patients with rheumatoid arthritis and coronary artery disease cases among those with rheumatoid arthritis from a large multi-institutional EHR. The area under the receiver operating characteristic curves (AUC) for classifying RA and CAD using models trained with automated features were 0.951 and 0.929, respectively, compared to the AUCs of 0.938 and 0.929 by models trained with expert-curated features. Models trained with NLP text features selected through an unbiased, automated procedure achieved comparable or slightly higher accuracy than those trained with expert-curated features. The majority of the selected model features were interpretable. The proposed automated feature extraction method, generating highly accurate phenotyping algorithms with improved efficiency, is a significant step toward high-throughput phenotyping. © The Author 2015. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Anti-atherosclerotic plants which modulate the phenotype of vascular smooth muscle cells.

PubMed

Saleh Al-Shehabi, Tuqa; Iratni, Rabah; Eid, Ali H

2016-10-15

Cardiovascular disease (CVD) remains the leading cause of global death, with atherosclerosis being a major contributor to this mortality. Several mechanisms are implicated in the pathogenesis of this disease. A key element in the development and progression of atherosclerotic lesions is the phenotype of vascular smooth muscle cells. Under pathophysiologic conditions such as injury, these cells switch from a contractile to a synthetic phenotype that often possesses high proliferative and migratory capacities. Despite major advances made in the management and treatment of atherosclerosis, mortality associated with this disease remains high. This mandates that other approaches be sought. Herbal medicine, especially for the treatment of CVD, has been gaining more attention in recent years. This is in no small part due to the evidence-based values associated with the consumption of many plants as well as the relatively cheaper prices, easier access and conventional folk medicine "inherited" over generations. Sections: In this review, we provide a brief introduction about the pathogenesis of atherosclerosis then we highlight the role of vascular smooth muscle cells in this disease, especially when a phenotypic switch of these cells arises. We then thoroughly discuss the various plants that show potentially beneficial effects as anti-atherosclerotic, with prime attention given to herbs and plants that inhibit the phenotypic switch of vascular smooth muscle cells. Accumulating evidence provides the justification for the use of botanicals in the treatment or prevention of atherosclerosis. However, further studies, especially clinical ones, are warranted to better define several pharmacological parameters of these herbs, such as toxicity, tolerability, and efficacy. Copyright © 2015 Elsevier Ltd. All rights reserved.
Female patient with autistic disorder, intellectual disability, and co-morbid anxiety disorder: Expanding the phenotype associated with the recurrent 3q13.2-q13.31 microdeletion.

PubMed

Quintela, Ines; Gomez-Guerrero, Lorena; Fernandez-Prieto, Montse; Resches, Mariela; Barros, Francisco; Carracedo, Angel

2015-12-01

In recent years, the advent of comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) arrays and its use as a first genetic test for the diagnosis of patients with neurodevelopmental phenotypes has allowed the identification of novel submicroscopic chromosomal abnormalities (namely, copy number variants or CNVs), imperceptible by conventional cytogenetic techniques. The 3q13.31 microdeletion syndrome (OMIM #615433) has been defined as a genomic disorder mainly characterized by developmental delay, postnatal overgrowth, hypotonia, genital abnormalities in males, and characteristic craniofacial features. Although the 3q13.31 CNVs are variable in size, a 3.4 Mb recurrently altered region at 3q13.2-q13.31 has been recently described and non-allelic homologous recombination (NAHR) mediated by flanking human endogenous retrovirus (HERV-H) elements has been suggested as the mechanism of deletion formation. We expand the phenotypic spectrum associated with this recurrent deletion performing the clinical description of a 9-year-old female patient with autistic disorder, total absence of language, intellectual disability, anxiety disorder and disruptive, and compulsive eating behaviors. The array-based molecular karyotyping allowed the identification of a de novo recurrent 3q13.2-q13.31 deletion encompassing 25 genes. In addition, we compare her clinical phenotype with previous reports of patients with neurodevelopmental and behavioral disorders and proximal 3q microdeletions. Finally, we also review the candidate genes proposed so far for these phenotypes. © 2015 Wiley Periodicals, Inc.
Comment on ''Effects of long-term high CO2 exposure on two species of coccolithophore'' by Müller et al. (2010)

NASA Astrophysics Data System (ADS)

Collins, S.

2010-07-01

Populations can respond to environmental change over tens or hundreds of generations by shifts in phenotype that can be the result of a sustained physiological response, evolutionary (genetic) change, shifts in community composition, or some combination of these factors. Microbes evolve on human timescales, and evolution may contribute to marine phytoplankton responses to global change over the coming decades. However, it is still unknown whether evolutionary responses are likely to contribute significantly to phenotypic change in marine microbial communities under high pCO2 regimes or other aspects of global change. Recent work by Müller et al. (2010) highlights that long-term responses of marine microbes to global change must be empirically measured and the underlying cause of changes in phenotype explained. Here, I briefly discuss how tools from experimental microbial evolution may be used to detect and measure evolutionary responses in marine phytoplankton grown in high CO2 environments and other environments of interest. I outline why the particular biology of marine microbes makes conventional experimental evolution challenging right now and make a case that marine microbes are good candidates for the development of new model systems in experimental evolution. I suggest that "black box" frameworks that focus on partitioning phenotypic change, such as the Price equation, may be useful in cases where direct measurements of evolutionary responses alone are difficult, and that such approaches could be used to test hypotheses about the underlying causes of phenotypic shifts in marine microbe communities responding to global change.
A homozygous PMS2 founder mutation with an attenuated constitutional mismatch repair deficiency phenotype.

PubMed

Li, Lili; Hamel, Nancy; Baker, Kristi; McGuffin, Michael J; Couillard, Martin; Gologan, Adrian; Marcus, Victoria A; Chodirker, Bernard; Chudley, Albert; Stefanovici, Camelia; Durandy, Anne; Hegele, Robert A; Feng, Bing-Jian; Goldgar, David E; Zhu, Jun; De Rosa, Marina; Gruber, Stephen B; Wimmer, Katharina; Young, Barbara; Chong, George; Tischkowitz, Marc D; Foulkes, William D

2015-05-01

Inherited mutations in DNA mismatch repair genes predispose to different cancer syndromes depending on whether they are mono-allelic or bi-allelic. This supports a causal relationship between expression level in the germline and phenotype variation. As a model to study this relationship, our study aimed to define the pathogenic characteristics of a recurrent homozygous coding variant in PMS2 displaying an attenuated phenotype identified by clinical genetic testing in seven Inuit families from Northern Quebec. Pathogenic characteristics of the PMS2 mutation NM_000535.5:c.2002A>G were studied using genotype-phenotype correlation, single-molecule expression detection and single genome microsatellite instability analysis. This PMS2 mutation generates a de novo splice site that competes with the authentic site. In homozygotes, expression of the full-length protein is reduced to a level barely detectable by conventional diagnostics. Median age at primary cancer diagnosis is 22 years among 13 NM_000535.5:c.2002A>G homozygotes, versus 8 years in individuals carrying bi-allelic truncating mutations. Residual expression of full-length PMS2 transcript was detected in normal tissues from homozygotes with cancers in their 20s. Our genotype-phenotype study of c.2002A>G illustrates that an extremely low level of PMS2 expression likely delays cancer onset, a feature that could be exploited in cancer preventive intervention. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

A novel Markov Blanket-based repeated-fishing strategy for capturing phenotype-related biomarkers in big omics data.

PubMed

Li, Hongkai; Yuan, Zhongshang; Ji, Jiadong; Xu, Jing; Zhang, Tao; Zhang, Xiaoshuai; Xue, Fuzhong

2016-03-09

We propose a novel Markov Blanket-based repeated-fishing strategy (MBRFS) in attempt to increase the power of existing Markov Blanket method (DASSO-MB) and maintain its advantages in omic data analysis. Both simulation and real data analysis were conducted to assess its performances by comparing with other methods including χ(2) test with Bonferroni and B-H adjustment, least absolute shrinkage and selection operator (LASSO) and DASSO-MB. A serious of simulation studies showed that the true discovery rate (TDR) of proposed MBRFS was always close to zero under null hypothesis (odds ratio = 1 for each SNPs) with excellent stability in all three scenarios of independent phenotype-related SNPs without linkage disequilibrium (LD) around them, correlated phenotype-related SNPs without LD around them, and phenotype-related SNPs with strong LD around them. As expected, under different odds ratio and minor allel frequency (MAFs), MBRFS always had the best performances in capturing the true phenotype-related biomarkers with higher matthews correlation coefficience (MCC) for all three scenarios above. More importantly, since proposed MBRFS using the repeated fishing strategy, it still captures more phenotype-related SNPs with minor effects when non-significant phenotype-related SNPs emerged under χ(2) test after Bonferroni multiple correction. The various real omics data analysis, including GWAS data, DNA methylation data, gene expression data and metabolites data, indicated that the proposed MBRFS always detected relatively reasonable biomarkers. Our proposed MBRFS can exactly capture the true phenotype-related biomarkers with the reduction of false negative rate when the phenotype-related biomarkers are independent or correlated, as well as the circumstance that phenotype-related biomarkers are associated with non-phenotype-related ones.
Macrophage polarization at the crossroad between HIV-1 infection and cancer development.

PubMed

Alfano, Massimo; Graziano, Francesca; Genovese, Luca; Poli, Guido

2013-06-01

Mononuclear phagocytes play a fundamental role in the tissue homeostasis and innate defenses against viruses and other microbial pathogens. In addition, they are likely involved in several steps of cancer development. Circulating monocytes and tissue macrophages are target cells of viral infections, including human cytomegalovirus, human herpes virus 8, and the HIV, and alterations of their functional and phenotypic properties are likely involved in many tissue-degenerative diseases, including atherosclerosis and cancer. Different tissue microenvironments as well as their pathological alterations can profoundly affect the polarization state of macrophages toward the extreme phenotypes conventionally termed M1 and M2. Thus, targeting disease-associated macrophages is considered a potential approach particularly in the context of cancer-associated tumor-associated macrophages, supporting malignant cell growth and progression toward a metastatic phenotype. Of note is the fact that tumor-associated macrophages isolated from established tumors display phenotypic and functional features similar to those of in vitro-derived M2-polarized cells. Concerning HIV-1 infection, viral eradication strategies in the context of combination antiretroviral therapy should also consider the possibility to deplete, at least transiently, certain mononuclear phagocytes subsets, although the possibility of distinguishing those that are either infected or pathogenically altered remains a goal of future research. In the present review, we will focus on the recent literature concerning the role of human macrophage polarization in viral infections and cancer.
Characterization of an Enterococcus faecium small-colony variant isolated from blood culture.

PubMed

Gröbner, Sabine; Beck, Julia; Schaller, Martin; Autenrieth, Ingo B; Schulte, Berit

2012-01-01

Small-colony variants (SCVs) of bacteria are slow-growing subpopulations which can cause latent or recurrent infections due to better intracellular survival compared to their wild-type counterparts. Atypical colony morphology and altered biochemical profile may lead to failure in identification of SCV strains. We here report for the first time the isolation of an Enterococcus faecium SCV phenotype. The case of a 65-year-old woman with acute myeloid leukaemia who developed symptoms of sepsis during induction chemotherapy is presented. E. faecium with normal and SCV phenotype was isolated from blood cultures. At the same time urine culture was positive with E. faecium suggesting that bacteraemia originated from the urinary tract. The SCV phenotype was characterized by atypical growth behaviour. Electron microscopic analyses revealed perturbation of the separation of daughter cells and the accumulation of cell wall material. Accordingly, the SCV variant showed a dysfunction or lack of spontaneous autolysis whereas the normal phenotype did not. In contrast to conventional identification systems based on biochemical characteristics, the E. faecium SCV was precisely identified by MALDI-TOF MS analysis implemented in our laboratory. Hence, the increasing use of MALDI-TOF MS analysis for the identification of bacteria might be an appropriate tool for the detection of SCV variants, the diagnosis of which is of importance for the clinical outcome and the antibiotic treatment. Copyright © 2011. Published by Elsevier GmbH.
Effect of chorioamnionitis on regulatory T cells in moderate/late preterm neonates☆

PubMed Central

Rueda, Cesar M.; Wells, Casey B.; Gisslen, Tate; Jobe, Alan H.; Kallapur, Suhas G.; Chougnet, Claire A.

2014-01-01

Regulatory T-cells (Treg) have a protective role for the control of immune activation and tissue damage. The effects of chorioamnionitis (chorio) on Treg in moderate/late preterm newborns are not known. We hypothesized that infants exposed to chorio would have decreased Treg frequency and/or function. We isolated mononuclear cells from adult peripheral blood and cord blood from term and moderate/late preterm infants who were classified for severity of chorio exposure. Mononuclear cells were analyzed by flow cytometry for Treg frequency and phenotype. Treg suppression of activation of conventional T-cells (Tcon) was also quantified. Treg frequencies were similar in all groups of neonates, but lower than that found in adults. Newborn Treg had a naïve phenotype, with decreased levels of CD45RO, HLA-DR, CD39 and TIGIT compared to adult Treg and chorio did not affect the phenotype. Treg from preterm newborns exposed to severe chorio had higher expression of Ki67 compared to the other groups. Treg from preterm newborns were less suppressive than Treg from adults or term, and the level of suppression was reduced with severe chorio. Relative to term, Treg frequency and phenotype were not affected by prematurity and chorio but their functionality was decreased. Lower Treg activity may contribute to inflammation in newborns that is often associated with chorioamnionitis. PMID:25451985
Analysis of ABCA4 in mixed Spanish families segregating different retinal dystrophies.

PubMed

Paloma, Eva; Coco, Rosa; Martínez-Mir, Amalia; Vilageliu, Lluïsa; Balcells, Susana; Gonzàlez-Duarte, Roser

2002-12-01

Genotype-phenotype correlations highlighted the function of ABCA4 in retinitis pigmentosa (RP),cone-rod dystrophy (CRD) and Stargardt/Fundus Flavimaculatus disease (STGD/FFM). Initial screening of ABCA4 variants showed a correlation between the type of mutation and the severity of the disease. In the present study we have undertaken mutational and haplotype analysis of ABCA4 in three mixed pedigrees segregating different retinal dystrophies. In family I, we have shown cosegregation of different ABCA4 alleles with CRD (homozygosity for L1940P) and three subtypes of STGD/FFM. The first, a mild form, consisting on fundus flavimaculatus-like distribution of flecks, but good visual acuity and absence of dark choroid, was found to cosegregate with alleles R1097C and F553L; the second, a conventional Stargardt phenotype was associated to alleles L1940P/R1097C and the third, displaying severely reduced visual acuity and dark choroid (named FFM), was associated to L1940P/F553L. In family II, segregating STGD and RP phenotypes, while the involvement of ABCA4 in STGD seems clear this is not the case for RP. Finally, in family III, also segregating STGD and RP, ABCA4 fails to explain either phenotype. Our data highlight the wide allelic heterogeneity involving this gene and support the genetic variability (beyond ABCA4) of mixed STGD/RP pedigrees. Copyright 2002 Wiley-Liss, Inc.
Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line

PubMed Central

2012-01-01

Background Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs) have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies. Methods CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis. Results The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs. Conclusions MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma. PMID:22475227
The Phantom Menace for Patients with Hepatobiliary Diseases: Shewanella haliotis, Often Misidentified as Shewanella algae in Biochemical Tests and MALDI-TOF Analysis.

PubMed

Byun, Jung-Hyun; Park, Hyunwoong; Kim, Sunjoo

2017-03-24

Although Shewanella algae has been known to have weak pathogenicity, case reports on infections with this species have been steadily increasing. S. algae and S. haliotis are difficult to distinguish from each other with conventional phenotypic methods. We reviewed the microbiological and clinical features of S. algae and S. haliotis infections at our institute. Bacterial culture and identification reports from patient samples from 2010 to 2014 were reviewed to screen the cases of Shewanella infections. In addition to conventional biochemical tests, 16S rRNA gene sequence analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were performed for 19 stored bacterial isolates. Medical records were reviewed for clinical characteristics and laboratory findings. All isolates were identified as S. algae by using VITEK 2. MALDI-TOF also identified all isolates as S. algae with a 99.9 confidence value. In contrast, 16S rRNA analysis identified 10 isolates as S. algae and 9 isolates as S. haliotis. Both S. algae (60%) and S. haliotis (77%) infections were strongly associated with diseases of the hepatobiliary tract and pancreas. To distinguish between S. algae and S. haliotis, 16S rRNA gene sequence analysis seems more accurate than biochemical tests or MALDI-TOF. Patients with underlying diseases in the hepatobiliary tract and pancreas seem to be susceptible to these marine pathogens.
Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level.

PubMed

Cherkaoui, Abdessalam; Hibbs, Jonathan; Emonet, Stéphane; Tangomo, Manuela; Girard, Myriam; Francois, Patrice; Schrenzel, Jacques

2010-04-01

Bacterial identification relies primarily on culture-based methodologies requiring 24 h for isolation and an additional 24 to 48 h for species identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to the problem of bacterial species identification. We evaluated two MALDI-TOF MS systems with 720 consecutively isolated bacterial colonies under routine clinical laboratory conditions. Isolates were analyzed in parallel on both devices, using the manufacturers' default recommendations. We compared MS with conventional biochemical test system identifications. Discordant results were resolved with "gold standard" 16S rRNA gene sequencing. The first MS system (Bruker) gave high-confidence identifications for 680 isolates, of which 674 (99.1%) were correct; the second MS system (Shimadzu) gave high-confidence identifications for 639 isolates, of which 635 (99.4%) were correct. Had MS been used for initial testing and biochemical identification used only in the absence of high-confidence MS identifications, the laboratory would have saved approximately US$5 per isolate in marginal costs and reduced average turnaround time by more than an 8-h shift, with no loss in accuracy. Our data suggest that implementation of MS as a first test strategy for one-step species identification would improve timeliness and reduce isolate identification costs in clinical bacteriology laboratories now.
Fine phenotyping of pod and seed traits in Arachis germplasm accessions using digital image analysis

USDA-ARS?s Scientific Manuscript database

Reliable and objective phenotyping of peanut pod and seed traits is important for cultivar selection and genetic mapping of yield components. To develop useful and efficient methods to quantitatively define peanut pod and seed traits, a group of peanut germplasm with high levels of phenotypic varia...
Comparison of the lysis centrifugation method with the conventional blood culture method in cases of sepsis in a tertiary care hospital.

PubMed

Parikh, Harshal R; De, Anuradha S; Baveja, Sujata M

2012-07-01

Physicians and microbiologists have long recognized that the presence of living microorganisms in the blood of a patient carries with it considerable morbidity and mortality. Hence, blood cultures have become critically important and frequently performed test in clinical microbiology laboratories for diagnosis of sepsis. To compare the conventional blood culture method with the lysis centrifugation method in cases of sepsis. Two hundred nonduplicate blood cultures from cases of sepsis were analyzed using two blood culture methods concurrently for recovery of bacteria from patients diagnosed clinically with sepsis - the conventional blood culture method using trypticase soy broth and the lysis centrifugation method using saponin by centrifuging at 3000 g for 30 minutes. Overall bacteria recovered from 200 blood cultures were 17.5%. The conventional blood culture method had a higher yield of organisms, especially Gram positive cocci. The lysis centrifugation method was comparable with the former method with respect to Gram negative bacilli. The sensitivity of lysis centrifugation method in comparison to conventional blood culture method was 49.75% in this study, specificity was 98.21% and diagnostic accuracy was 89.5%. In almost every instance, the time required for detection of the growth was earlier by lysis centrifugation method, which was statistically significant. Contamination by lysis centrifugation was minimal, while that by conventional method was high. Time to growth by the lysis centrifugation method was highly significant (P value 0.000) as compared to time to growth by the conventional blood culture method. For the diagnosis of sepsis, combination of the lysis centrifugation method and the conventional blood culture method with trypticase soy broth or biphasic media is advocable, in order to achieve faster recovery and a better yield of microorganisms.
A work study of the CAD/CAM method and conventional manual method in the fabrication of spinal orthoses for patients with adolescent idiopathic scoliosis.

PubMed

Wong, M S; Cheng, J C Y; Wong, M W; So, S F

2005-04-01

A study was conducted to compare the CAD/CAM method with the conventional manual method in fabrication of spinal orthoses for patients with adolescent idiopathic scoliosis. Ten subjects were recruited for this study. Efficiency analyses of the two methods were performed from cast filling/ digitization process to completion of cast/image rectification. The dimensional changes of the casts/ models rectified by the two cast rectification methods were also investigated. The results demonstrated that the CAD/CAM method was faster than the conventional manual method in the studied processes. The mean rectification time of the CAD/CAM method was shorter than that of the conventional manual method by 108.3 min (63.5%). This indicated that the CAD/CAM method took about 1/3 of the time of the conventional manual to finish cast rectification. In the comparison of cast/image dimensional differences between the conventional manual method and the CAD/CAM method, five major dimensions in each of the five rectified regions namely the axilla, thoracic, lumbar, abdominal and pelvic regions were involved. There were no significant dimensional differences (p < 0.05) in 19 out of the 25 studied dimensions. This study demonstrated that the CAD/CAM system could save the time in the rectification process and offer a relatively high resemblance in cast rectification as compared with the conventional manual method.
Quantitative modeling of the molecular steps underlying shut-off of rhodopsin activity in rod phototransduction

PubMed Central

Kraft, Timothy W.

2016-01-01

Purpose To examine the predictions of alternative models for the stochastic shut-off of activated rhodopsin (R*) and their implications for the interpretation of experimentally recorded single-photon responses (SPRs) in mammalian rods. Theory We analyze the transitions that an activated R* molecule undergoes as a result of successive phosphorylation steps and arrestin binding. We consider certain simplifying cases for the relative magnitudes of the reaction rate constants and derive the probability distributions for the time to arrestin binding. In addition to the conventional model in which R* catalytic activity declines in a graded manner with successive phosphorylations, we analyze two cases in which the activity is assumed to occur not via multiple small steps upon each phosphorylation but via a single large step. We refer to these latter two cases as the binary R* shut-off and three-state R* shut-off models. Methods We simulate R*’s stochastic reactions numerically for the three models. In the simplifying cases for the ratio of rate constants in the binary and three-state models, we show that the probability distribution of the time to arrestin binding is accurately predicted. To simulate SPRs, we then integrate the differential equations for the downstream reactions using a standard model of the rod outer segment that includes longitudinal diffusion of cGMP and Ca2+. Results Our simulations of SPRs in the conventional model of graded shut-off of R* conform closely to the simulations in a recent study. However, the gain factor required to account for the observed mean SPR amplitude is higher than can be accounted for from biochemical experiments. In addition, a substantial minority of the simulated SPRs exhibit features that have not been reported in published experiments. Our simulations of SPRs using the model of binary R* shut-off appear to conform closely to experimental results for wild type (WT) mouse rods, and the required gain factor conforms to biochemical expectations. However, for the arrestin knockout (Arr−/−) phenotype, the predictions deviated from experimental findings and led us to invoke a low-activity state that R* enters before arrestin binding. Our simulations of this three-state R* shut-off model are very similar to those of the binary model in the WT case but are preferred because they appear to accurately predict the mean SPRs for four mutant phenotypes, Arr+/−, Arr−/−, GRK1+/−, and GRK1−/−, in addition to the WT phenotype. When we additionally treated the formation and shut-off of activated phosphodiesterase (E*) as stochastic, the simulated SPRs appeared even more similar to real SPRs, and there was very little change in the ensemble mean and standard deviation or in the amplitude distribution. Conclusions We conclude that the conventional model of graded reduction in R* activity through successive phosphorylation steps appears to be inconsistent with experimental results. Instead, we find that two variants of a model in which R* activity initially remains high and then declines abruptly after several phosphorylation steps appears capable of providing a better description of experimentally measured SPRs. PMID:27375353
Methodology for the inference of gene function from phenotype data.

PubMed

Ascensao, Joao A; Dolan, Mary E; Hill, David P; Blake, Judith A

2014-12-12

Biomedical ontologies are increasingly instrumental in the advancement of biological research primarily through their use to efficiently consolidate large amounts of data into structured, accessible sets. However, ontology development and usage can be hampered by the segregation of knowledge by domain that occurs due to independent development and use of the ontologies. The ability to infer data associated with one ontology to data associated with another ontology would prove useful in expanding information content and scope. We here focus on relating two ontologies: the Gene Ontology (GO), which encodes canonical gene function, and the Mammalian Phenotype Ontology (MP), which describes non-canonical phenotypes, using statistical methods to suggest GO functional annotations from existing MP phenotype annotations. This work is in contrast to previous studies that have focused on inferring gene function from phenotype primarily through lexical or semantic similarity measures. We have designed and tested a set of algorithms that represents a novel methodology to define rules for predicting gene function by examining the emergent structure and relationships between the gene functions and phenotypes rather than inspecting the terms semantically. The algorithms inspect relationships among multiple phenotype terms to deduce if there are cases where they all arise from a single gene function. We apply this methodology to data about genes in the laboratory mouse that are formally represented in the Mouse Genome Informatics (MGI) resource. From the data, 7444 rule instances were generated from five generalized rules, resulting in 4818 unique GO functional predictions for 1796 genes. We show that our method is capable of inferring high-quality functional annotations from curated phenotype data. As well as creating inferred annotations, our method has the potential to allow for the elucidation of unforeseen, biologically significant associations between gene function and phenotypes that would be overlooked by a semantics-based approach. Future work will include the implementation of the described algorithms for a variety of other model organism databases, taking full advantage of the abundance of available high quality curated data.
[Agrobacterium rubi strains from blueberry plants are highly diverse].

PubMed

Abrahamovich, Eliana; López, Ana C; Alippi, Adriana M

2014-01-01

The diversity of a collection of Agrobacterium rubi strains isolated from blueberries from different regions of Argentina was studied by conventional microbiological tests and molecular techniques. Results from biochemical and physiological reactions, as well as from rep-PCR and RFLP analysis of PCR-amplified 23S rDNA showed high phenotypic and genotypic intraspecific variation. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.
Pathway-based factor analysis of gene expression data produces highly heritable phenotypes that associate with age.

PubMed

Anand Brown, Andrew; Ding, Zhihao; Viñuela, Ana; Glass, Dan; Parts, Leopold; Spector, Tim; Winn, John; Durbin, Richard

2015-03-09

Statistical factor analysis methods have previously been used to remove noise components from high-dimensional data prior to genetic association mapping and, in a guided fashion, to summarize biologically relevant sources of variation. Here, we show how the derived factors summarizing pathway expression can be used to analyze the relationships between expression, heritability, and aging. We used skin gene expression data from 647 twins from the MuTHER Consortium and applied factor analysis to concisely summarize patterns of gene expression to remove broad confounding influences and to produce concise pathway-level phenotypes. We derived 930 "pathway phenotypes" that summarized patterns of variation across 186 KEGG pathways (five phenotypes per pathway). We identified 69 significant associations of age with phenotype from 57 distinct KEGG pathways at a stringent Bonferroni threshold ([Formula: see text]). These phenotypes are more heritable ([Formula: see text]) than gene expression levels. On average, expression levels of 16% of genes within these pathways are associated with age. Several significant pathways relate to metabolizing sugars and fatty acids; others relate to insulin signaling. We have demonstrated that factor analysis methods combined with biological knowledge can produce more reliable phenotypes with less stochastic noise than the individual gene expression levels, which increases our power to discover biologically relevant associations. These phenotypes could also be applied to discover associations with other environmental factors. Copyright © 2015 Brown et al.
The CIMP Phenotype in BRAF Mutant Serrated Polyps from a Prospective Colonoscopy Patient Cohort

PubMed Central

Fernando, Winnie C.; Miranda, Mariska S.; Worthley, Daniel L.; Togashi, Kazutomo; Watters, Dianne J.; Leggett, Barbara A.; Spring, Kevin J.

2014-01-01

Colorectal cancers arising via the serrated pathway are often associated with BRAF V600E mutation, CpG island methylator phenotype (CIMP), and microsatellite instability. Previous studies have shown a strong association between BRAF V600E mutation and serrated polyps. This study aims to evaluate CIMP status of all the serrated polyp subtypes and its association with functionally important genes such as MLH1, p16, and IGFBP7. CIMP status and methylation were evaluated using the real-time based MethyLight assay in 154 serrated polyps and 63 conventional adenomas. Results showed that CIMP-high serrated polyps were strongly associated with BRAF mutation and proximal colon. CIMP-high was uncommon in conventional adenomas (1.59%), occurred in 8.25% of hyperplastic polyps (HPs), and became common in sessile serrated adenomas (SSAs) (51.43%). MLH1 methylation was mainly observed in the proximal colon and was significantly associated with BRAF mutation and CIMP-high. The number of samples methylated for p16 and IGFBP7 was the highest in SSAs. The methylation panel we used to detect CIMP is highly specific for CIMP-high cancers. With this panel, we demonstrate that CIMP-high is much more common in SSAs than HPs. This suggests that CIMP-high correlates with increased risk of malignant transformation which was also observed in methylation of functionally important genes. PMID:24812557
The CIMP Phenotype in BRAF Mutant Serrated Polyps from a Prospective Colonoscopy Patient Cohort.

PubMed

Fernando, Winnie C; Miranda, Mariska S; Worthley, Daniel L; Togashi, Kazutomo; Watters, Dianne J; Leggett, Barbara A; Spring, Kevin J

2014-01-01

Colorectal cancers arising via the serrated pathway are often associated with BRAF V600E mutation, CpG island methylator phenotype (CIMP), and microsatellite instability. Previous studies have shown a strong association between BRAF V600E mutation and serrated polyps. This study aims to evaluate CIMP status of all the serrated polyp subtypes and its association with functionally important genes such as MLH1, p16, and IGFBP7. CIMP status and methylation were evaluated using the real-time based MethyLight assay in 154 serrated polyps and 63 conventional adenomas. Results showed that CIMP-high serrated polyps were strongly associated with BRAF mutation and proximal colon. CIMP-high was uncommon in conventional adenomas (1.59%), occurred in 8.25% of hyperplastic polyps (HPs), and became common in sessile serrated adenomas (SSAs) (51.43%). MLH1 methylation was mainly observed in the proximal colon and was significantly associated with BRAF mutation and CIMP-high. The number of samples methylated for p16 and IGFBP7 was the highest in SSAs. The methylation panel we used to detect CIMP is highly specific for CIMP-high cancers. With this panel, we demonstrate that CIMP-high is much more common in SSAs than HPs. This suggests that CIMP-high correlates with increased risk of malignant transformation which was also observed in methylation of functionally important genes.
Comparison among conventional and advanced MRI, 18F-FDG PET/CT, phenotype and genotype in glioblastoma.

PubMed

Valentini, Maria Consuelo; Mellai, Marta; Annovazzi, Laura; Melcarne, Antonio; Denysenko, Tetyana; Cassoni, Paola; Casalone, Cristina; Maurella, Cristiana; Grifoni, Silvia; Fania, Piercarlo; Cistaro, Angelina; Schiffer, Davide

2017-10-31

Glioblastoma (GB) is a highly heterogeneous tumor. In order to identify in vivo the most malignant tumor areas, the extent of tumor infiltration and the sites giving origin to GB stem cells (GSCs), we combined positron emission tomography/computed tomography (PET/CT) and conventional and advanced magnetic resonance imaging (MRI) with histology, immunohistochemistry and molecular genetics. Prior to dura opening and tumor resection, forty-eight biopsy specimens [23 of contrast-enhancing (CE) and 25 of non-contrast enhancing (NE) regions] from 12 GB patients were obtained by a frameless image-guided stereotactic biopsy technique. The highest values of 2-[18F]-fluoro-2-deoxy-D-glucose maximum standardized uptake value ( 18 F-FDG SUV max ), relative cerebral blood volume (rCBV), Choline/Creatine (Cho/Cr), Choline/N-acetylaspartate (Cho/NAA) and Lipids/Lactate (LL) ratio have been observed in the CE region. They corresponded to the most malignant tumor phenotype, to the greatest molecular spectrum and stem cell potential. On the contrary, apparent diffusion coefficient (ADC) and fractional anisotropy (FA) in the CE region were very variable. 18 F-FDG SUV max , Cho/Cr and Cho/NAA ratio resulted the most suitable parameters to detect tumor infiltration. In edematous areas, reactive astrocytes and microglia/macrophages were influencing variables. Combined MRI and 18 F-FDG PET/CT allowed to recognize the specific biological significance of the different identified areas of GB.
Comparison among conventional and advanced MRI, 18F-FDG PET/CT, phenotype and genotype in glioblastoma

PubMed Central

Valentini, Maria Consuelo; Mellai, Marta; Annovazzi, Laura; Melcarne, Antonio; Denysenko, Tetyana; Cassoni, Paola; Casalone, Cristina; Maurella, Cristiana; Grifoni, Silvia; Fania, Piercarlo; Cistaro, Angelina; Schiffer, Davide

2017-01-01

Glioblastoma (GB) is a highly heterogeneous tumor. In order to identify in vivo the most malignant tumor areas, the extent of tumor infiltration and the sites giving origin to GB stem cells (GSCs), we combined positron emission tomography/computed tomography (PET/CT) and conventional and advanced magnetic resonance imaging (MRI) with histology, immunohistochemistry and molecular genetics. Prior to dura opening and tumor resection, forty-eight biopsy specimens [23 of contrast-enhancing (CE) and 25 of non-contrast enhancing (NE) regions] from 12 GB patients were obtained by a frameless image-guided stereotactic biopsy technique. The highest values of 2-[18F]-fluoro-2-deoxy-D-glucose maximum standardized uptake value (18F-FDG SUVmax), relative cerebral blood volume (rCBV), Choline/Creatine (Cho/Cr), Choline/N-acetylaspartate (Cho/NAA) and Lipids/Lactate (LL) ratio have been observed in the CE region. They corresponded to the most malignant tumor phenotype, to the greatest molecular spectrum and stem cell potential. On the contrary, apparent diffusion coefficient (ADC) and fractional anisotropy (FA) in the CE region were very variable. 18F-FDG SUVmax, Cho/Cr and Cho/NAA ratio resulted the most suitable parameters to detect tumor infiltration. In edematous areas, reactive astrocytes and microglia/macrophages were influencing variables. Combined MRI and 18F-FDG PET/CT allowed to recognize the specific biological significance of the different identified areas of GB. PMID:29207673
Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients

PubMed Central

Livério, Harisson Oliveira; Ruiz, Luciana da Silva; de Freitas, Roseli Santos; Nishikaku, Angela; de Souza, Ana Clara; Paula, Claudete Rodrigues; Domaneschi, Carina

2017-01-01

ABSTRACT The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species. PMID:28423089

Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients.

PubMed

Livério, Harisson Oliveira; Ruiz, Luciana da Silva; Freitas, Roseli Santos de; Nishikaku, Angela; Souza, Ana Clara de; Paula, Claudete Rodrigues; Domaneschi, Carina

2017-04-13

The aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species.
Determining which phenotypes underlie a pleiotropic signal

PubMed Central

Majumdar, Arunabha; Haldar, Tanushree; Witte, John S.

2016-01-01

Discovering pleiotropic loci is important to understand the biological basis of seemingly distinct phenotypes. Most methods for assessing pleiotropy only test for the overall association between genetic variants and multiple phenotypes. To determine which specific traits are pleiotropic, we evaluate via simulation and application three different strategies. The first is model selection techniques based on the inverse regression of genotype on phenotypes. The second is a subset-based meta-analysis ASSET [Bhattacharjee et al., 2012], which provides an optimal subset of non-null traits. And the third is a modified Benjamini-Hochberg (B-H) procedure of controlling the expected false discovery rate [Benjamini and Hochberg, 1995] in the framework of phenome-wide association study. From our simulations we see that an inverse regression based approach MultiPhen [O’Reilly et al., 2012] is more powerful than ASSET for detecting overall pleiotropic association, except for when all the phenotypes are associated and have genetic effects in the same direction. For determining which specific traits are pleiotropic, the modified B-H procedure performs consistently better than the other two methods. The inverse regression based selection methods perform competitively with the modified B-H procedure only when the phenotypes are weakly correlated. The efficiency of ASSET is observed to lie below and in between the efficiency of the other two methods when the traits are weakly and strongly correlated, respectively. In our application to a large GWAS, we find that the modified B-H procedure also performs well, indicating that this may be an optimal approach for determining the traits underlying a pleiotropic signal. PMID:27238845
Markov Logic Networks in the Analysis of Genetic Data

PubMed Central

Sakhanenko, Nikita A.

2010-01-01

Abstract Complex, non-additive genetic interactions are common and can be critical in determining phenotypes. Genome-wide association studies (GWAS) and similar statistical studies of linkage data, however, assume additive models of gene interactions in looking for genotype-phenotype associations. These statistical methods view the compound effects of multiple genes on a phenotype as a sum of influences of each gene and often miss a substantial part of the heritable effect. Such methods do not use any biological knowledge about underlying mechanisms. Modeling approaches from the artificial intelligence (AI) field that incorporate deterministic knowledge into models to perform statistical analysis can be applied to include prior knowledge in genetic analysis. We chose to use the most general such approach, Markov Logic Networks (MLNs), for combining deterministic knowledge with statistical analysis. Using simple, logistic regression-type MLNs we can replicate the results of traditional statistical methods, but we also show that we are able to go beyond finding independent markers linked to a phenotype by using joint inference without an independence assumption. The method is applied to genetic data on yeast sporulation, a complex phenotype with gene interactions. In addition to detecting all of the previously identified loci associated with sporulation, our method identifies four loci with smaller effects. Since their effect on sporulation is small, these four loci were not detected with methods that do not account for dependence between markers due to gene interactions. We show how gene interactions can be detected using more complex models, which can be used as a general framework for incorporating systems biology with genetics. PMID:20958249
Characterization of Lactobacillus from Algerian Goat’S Milk Based on Phenotypic, 16S rDNA Sequencing and their Technological Properties

PubMed Central

Marroki, Ahmed; Zúñiga, Manuel; Kihal, Mabrouk; Pérez- Martínez, Gaspar

2011-01-01

Nineteen strains of Lactobacillus isolated from goat’s milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility) was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21) and one strain of L. rhamnosus (LbMF25) have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria. PMID:24031617
Slump sitting X-ray of the lumbar spine is superior to the conventional flexion view in assessing lumbar spine instability.

PubMed

Hey, Hwee Weng Dennis; Lau, Eugene Tze-Chun; Lim, Joel-Louis; Choong, Denise Ai-Wen; Tan, Chuen-Seng; Liu, Gabriel Ka-Po; Wong, Hee-Kit

2017-03-01

Flexion radiographs have been used to identify cases of spinal instability. However, current methods are not standardized and are not sufficiently sensitive or specific to identify instability. This study aimed to introduce a new slump sitting method for performing lumbar spine flexion radiographs and comparison of the angular range of motions (ROMs) and displacements between the conventional method and this new method. This study used is a prospective study on radiological evaluation of the lumbar spine flexion ROMs and displacements using dynamic radiographs. Sixty patients were recruited from a single spine tertiary center. Angular and displacement measurements of lumbar spine flexion were carried out. Participants were randomly allocated into two groups: those who did the new method first, followed by the conventional method versus those who did the conventional method first, followed by the new method. A comparison of the angular and displacement measurements of lumbar spine flexion between the conventional method and the new method was performed and tested for superiority and non-inferiority. The measurements of global lumbar angular ROM were, on average, 17.3° larger (p<.0001) using the new slump sitting method compared with the conventional method. They were most significant at the levels of L3-L4, L4-L5, and L5-S1 (p<.0001, p<.0001 and p=.001, respectively). There was no significant difference between both methods when measuring lumbar displacements (p=.814). The new method of slump sitting dynamic radiograph was shown to be superior to the conventional method in measuring the angular ROM and non-inferior to the conventional method in the measurement of displacement. Copyright © 2016 Elsevier Inc. All rights reserved.
High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation.

PubMed

Vega, Sebastián L; Liu, Er; Arvind, Varun; Bushman, Jared; Sung, Hak-Joon; Becker, Matthew L; Lelièvre, Sophie; Kohn, Joachim; Vidi, Pierre-Alexandre; Moghe, Prabhas V

2017-02-01

Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative "imaging-derived" parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions. Copyright © 2017 Elsevier Inc. All rights reserved.
Strategies for Advancing Disease Definition Using Biomarkers and Genetics: The Bipolar and Schizophrenia Network for Intermediate Phenotypes.

PubMed

Tamminga, Carol A; Pearlson, Godfrey D; Stan, Ana D; Gibbons, Robert D; Padmanabhan, Jaya; Keshavan, Matcheri; Clementz, Brett A

2017-01-01

It is critical for psychiatry as a field to develop approaches to define the molecular, cellular, and circuit basis of its brain diseases, especially for serious mental illnesses, and then to use these definitions to generate biologically based disease categories, as well as to explore disease mechanisms and illness etiologies. Our current reliance on phenomenology is inadequate to support exploration of molecular treatment targets and disease formulations, and the leap directly from phenomenology to disease biology has been limiting because of broad heterogeneity within conventional diagnoses. The questions addressed in this review are formulated around how we can use brain biomarkers to achieve disease categories that are biologically based. We have grouped together a series of vignettes as examples of early approaches, all using the Bipolar and Schizophrenia Network on Intermediate Phenotypes (BSNIP) biomarker database and collaborators, starting off with describing the foundational statistical methods for these goals. We use primarily criterion-free statistics to identify pertinent groups of involved genes related to psychosis as well as symptoms, and finally, to create new biologically based disease cohorts within the psychopathological dimension of psychosis. Although we do not put these results forward as final formulations, they represent a novel effort to rely minimally on phenomenology as a diagnostic tool and to fully embrace brain characteristics of structure, as well as molecular and cellular characteristics and function, to support disease definition in psychosis. Copyright © 2016. Published by Elsevier Inc.
De Novo Assembly of the Donkey White Blood Cell Transcriptome and a Comparative Analysis of Phenotype-Associated Genes between Donkeys and Horses

PubMed Central

Xie, Feng-Yun; Feng, Yu-Long; Wang, Hong-Hui; Ma, Yun-Feng; Yang, Yang; Wang, Yin-Chao; Shen, Wei; Pan, Qing-Jie; Yin, Shen; Sun, Yu-Jiang; Ma, Jun-Yu

2015-01-01

Prior to the mechanization of agriculture and labor-intensive tasks, humans used donkeys (Equus africanus asinus) for farm work and packing. However, as mechanization increased, donkeys have been increasingly raised for meat, milk, and fur in China. To maintain the development of the donkey industry, breeding programs should focus on traits related to these new uses. Compared to conventional marker-assisted breeding plans, genome- and transcriptome-based selection methods are more efficient and effective. To analyze the coding genes of the donkey genome, we assembled the transcriptome of donkey white blood cells de novo. Using transcriptomic deep-sequencing data, we identified 264,714 distinct donkey unigenes and predicted 38,949 protein fragments. We annotated the donkey unigenes by BLAST searches against the non-redundant (NR) protein database. We also compared the donkey protein sequences with those of the horse (E. caballus) and wild horse (E. przewalskii), and linked the donkey protein fragments with mammalian phenotypes. As the outer ear size of donkeys and horses are obviously different, we compared the outer ear size-associated proteins in donkeys and horses. We identified three ear size-associated proteins, HIC1, PRKRA, and KMT2A, with sequence differences among the donkey, horse, and wild horse loci. Since the donkey genome sequence has not been released, the de novo assembled donkey transcriptome is helpful for preliminary investigations of donkey cultivars and for genetic improvement. PMID:26208029
De Novo Assembly of the Donkey White Blood Cell Transcriptome and a Comparative Analysis of Phenotype-Associated Genes between Donkeys and Horses.

PubMed

Xie, Feng-Yun; Feng, Yu-Long; Wang, Hong-Hui; Ma, Yun-Feng; Yang, Yang; Wang, Yin-Chao; Shen, Wei; Pan, Qing-Jie; Yin, Shen; Sun, Yu-Jiang; Ma, Jun-Yu

2015-01-01

Prior to the mechanization of agriculture and labor-intensive tasks, humans used donkeys (Equus africanus asinus) for farm work and packing. However, as mechanization increased, donkeys have been increasingly raised for meat, milk, and fur in China. To maintain the development of the donkey industry, breeding programs should focus on traits related to these new uses. Compared to conventional marker-assisted breeding plans, genome- and transcriptome-based selection methods are more efficient and effective. To analyze the coding genes of the donkey genome, we assembled the transcriptome of donkey white blood cells de novo. Using transcriptomic deep-sequencing data, we identified 264,714 distinct donkey unigenes and predicted 38,949 protein fragments. We annotated the donkey unigenes by BLAST searches against the non-redundant (NR) protein database. We also compared the donkey protein sequences with those of the horse (E. caballus) and wild horse (E. przewalskii), and linked the donkey protein fragments with mammalian phenotypes. As the outer ear size of donkeys and horses are obviously different, we compared the outer ear size-associated proteins in donkeys and horses. We identified three ear size-associated proteins, HIC1, PRKRA, and KMT2A, with sequence differences among the donkey, horse, and wild horse loci. Since the donkey genome sequence has not been released, the de novo assembled donkey transcriptome is helpful for preliminary investigations of donkey cultivars and for genetic improvement.
Isolation and characterization of progenitor cells from surgically created - early healing alveolar defects in humans. A preliminary study.

PubMed

Sant'Ana, Adriana Campos Passanezi; Damante, Carla Andreotti; Martinez, Maria Alejandra Frias; Valdivia, Maria Alejandra Medina; Karam, Paula Stefânia Hage; de Oliveira, Flavia Amadeu; de Oliveira, Rodrigo Cardoso; Gasparoto, Thais Helena; Campanelli, Ana Paula; Zangrando, Mariana Schutzer Ragghianti; de Rezende, Maria Lúcia Rubo; Greghi, Sebastião Luiz Aguiar; Passanezi, Euloir

2018-05-30

The granulation tissue (GT) present in surgically-created early healing sockets has been considered as a possible source of osteoprogenitor cells for periodontal regeneration, as demonstrated in animal studies. However, the in vitro osteogenic properties of tissue removed from human surgically-created early healing alveolar defects (SC-EHAD) remains to be established, being that the aim of this study. Surgical defects were created in the edentulous ridge of two systemically healthy adults. The healing tissue present in these defects was removed 21 days later for the establishment of primary culture. The in vitro characteristics of the cultured cells were determined by Armelin method, MTT assay, immunohistochemistry, alkaline phosphatase (ALP) activity, mineralization assay and flow cytometry for detection of stem cells/osteoprogenitor cell markers. Cells were able to adhere to the plastic and assumed spindle-shaped morphology at earlier passages, changing to a cuboidal one with increasing passages. Differences in the proliferation rate were observed with increasing passages, suggesting osteogenic differentiation. ALP and mineralization activities were detected in conventional and osteogenic medium. Fresh samples of SC-EHAD tissue exhibited CD34 - and CD45 - phenotypes. Cells at later passages (14 th ) exhibited CD34 - , CD45 - , CD105 - , CD166 - and collagen type I + phenotype. Tissue removed from SC-EHAD is a possible source of progenitor cells. This article is protected by copyright. All rights reserved. © 2018 American Academy of Periodontology.
Detection of Extended-Spectrum β-Lactamases in Clinical Isolates of Enterobacter cloacae and Enterobacter aerogenes

PubMed Central

Tzelepi, Eva; Giakkoupi, Panagiota; Sofianou, Danai; Loukova, Veneta; Kemeroglou, Anastassia; Tsakris, Athanassios

2000-01-01

The aim of the present study was to investigate the frequency of extended-spectrum β-lactamases (ESBLs) in a consecutive collection of clinical isolates of Enterobacter spp. The abilities of various screening methods to detect ESBLs in enterobacters were simultaneously tested. Among the 68 consecutive isolates (56 Enterobacter cloacae and 12 Enterobacter aerogenes isolates) that were analyzed for β-lactamase content, 21 (25 and 58%, respectively) possessed transferable ESBLs with pIs of 8.2 and phenotypic characteristics of SHV-type enzymes, 8 (14.3%) of the E. cloacae isolates produced a previously nondescribed, clavulanate-susceptible ESBL that exhibited a pI of 6.9 and that conferred a ceftazidime resistance phenotype on Escherichia coli transconjugants, and 2 E. cloacae isolates produced both of these enzymes. Among the total of 31 isolates that were considered ESBL producers, the Vitek ESBL detection test was positive for 2 (6.5%) strains, and the conventional double-disk synergy test (DDST) with amoxicillin-clavulanate and with expanded-spectrum cephalosporins and aztreonam was positive for 5 (16%) strains. Modifications of the DDST consisting of closer application of the disks (at 20 instead of 30 mm), the use of cefepime, and the use of both modifications increased the sensitivity of this test to 71, 61, and 90%, respectively. Of the 37 isolates for which isoelectric focusing failed to determine ESBLs, the Vitek test was false positive for 1 isolate and the various forms of DDSTs were false-positive for 3 isolates. PMID:10655342
Comparison of Molecular and Phenotypic Methods for the Detection and Characterization of Carbapenem Resistant Enterobacteriaceae.

PubMed

Somily, Ali M; Garaween, Ghada A; Abukhalid, Norah; Absar, Muhammad M; Senok, Abiola C

2016-03-01

In recent years, there has been a rapid dissemination of carbapenem resistant Enterobacteriaceae (CRE). This study aimed to compare phenotypic and molecular methods for detection and characterization of CRE isolates at a large tertiary care hospital in Saudi Arabia. This study was carried out between January 2011 and November 2013 at the King Khalid University Hospital (KKUH) in Saudi Arabia. Determination of presence of extended-spectrum beta-lactamases (ESBL) and carbapenem resistance was in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Phenotypic classification was done by the MASTDISCS(TM) ID inhibitor combination disk method. Genotypic characterization of ESBL and carbapenemase genes was performed by the Check-MDR CT102. Diversilab rep-PCR was used for the determination of clonal relationship. Of the 883 ESBL-positive Enterobacteriaceae detected during the study period, 14 (1.6%) isolates were carbapenem resistant. Both the molecular genotypic characterization and phenotypic testing were in agreement in the detection of all 8 metalo-beta-lactamases (MBL) producing isolates. Of these 8 MBL-producers, 5 were positive for blaNDM gene and 3 were positive for blaVIM gene. Molecular method identified additional blaOXA gene isolates while MASTDISCS(TM) ID detected one AmpC producer isolate. Both methods agreed in identifying 2 carbapenem resistant isolates which were negative for carbapenemase genes. Diversilab rep-PCR analysis of the 9 Klebsiella pneumoniae isolates revealed polyclonal distribution into eight clusters. MASTDISCS(TM) ID is a reliable simple cheap phenotypic method for detection of majority of carbapenemase genes with the exception of the blaOXA gene. We recommend to use such method in the clinical laboratory.
Evaluation of VITEK mass spectrometry (MS), a matrix-assisted laser desorption ionization time-of-flight MS system for identification of anaerobic bacteria.

PubMed

Lee, Wonmok; Kim, Myungsook; Yong, Dongeun; Jeong, Seok Hoon; Lee, Kyungwon; Chong, Yunsop

2015-01-01

By conventional methods, the identification of anaerobic bacteria is more time consuming and requires more expertise than the identification of aerobic bacteria. Although the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems are relatively less studied, they have been reported to be a promising method for the identification of anaerobes. We evaluated the performance of the VITEK MS in vitro diagnostic (IVD; 1.1 database; bioMérieux, France) in the identification of anaerobes. We used 274 anaerobic bacteria isolated from various clinical specimens. The results for the identification of the bacteria by VITEK MS were compared to those obtained by phenotypic methods and 16S rRNA gene sequencing. Among the 249 isolates included in the IVD database, the VITEK MS correctly identified 209 (83.9%) isolates to the species level and an additional 18 (7.2%) at the genus level. In particular, the VITEK MS correctly identified clinically relevant and frequently isolated anaerobic bacteria to the species level. The remaining 22 isolates (8.8%) were either not identified or misidentified. The VITEK MS could not identify the 25 isolates absent from the IVD database to the species level. The VITEK MS showed reliable identifications for clinically relevant anaerobic bacteria.
[Group B Streptococcus carriers among pregnant women].

PubMed

García, S D; Eliseth, M Cora; Lazzo, M J; Copolillo, E; Barata, A D; de Torres, R; Vay, C A; Famiglietti, A M

2003-01-01

Streptococcus agalactiae--group B streptococci (GBS)--is a main cause of severe neonatal infections with a high mortality rate. The detection of pregnant GBS carriers (5-35%) allows intrapartum administration of antibiotic prophylaxis to these women and prevents perinatal infection. We studied the prevalence of GBS in 259 patients between 28 and 37 weeks gestation from April 2000 to March 2002. The anorectum (AR) and vaginal introitus swabs (VI) were cultured in selective Todd-Hewitt broth containing colistin (10 micrograms/ml) and nalidixic acid (15 micrograms/ml) while vaginal swabs (VFS) were cultured following conventional methods. A total of 47 strains of EGB were isolated from 259 patients (18.15%). The prevalence in different samples were: 5.40% in VFS, 13.51% in VI, 11.58% in AR and 17.76% in VI + AR (reference method). The isolates were tested against penicillin, ceftriaxone, erythromycin, clindamycin, vancomycin, gentamicin and streptomycin to determine the minimum inhibitory concentration. The resistance phenotypes of erythromycin-resistant GBS were determined by the double-disk test. All strains were susceptible to penicillin, ceftriaxone and vancomycin, only one strain was erythromycin and clindamycin resistant by IMLSB mechanism. None of the isolated strains had a high resistant level to aminoglycosides. The sensitivity of cultures increased when selective broths were used as the primary detection method.
Methodological and Clinical Aspects of the Molecular Epidemiology of Mycobacterium tuberculosis and Other Mycobacteria

PubMed Central

Minias, Alina; van Ingen, Jakko; Rastogi, Nalin; Brzostek, Anna; Żaczek, Anna; Dziadek, Jarosław

2016-01-01

SUMMARY Molecular typing has revolutionized epidemiological studies of infectious diseases, including those of a mycobacterial etiology. With the advent of fingerprinting techniques, many traditional concepts regarding transmission, infectivity, or pathogenicity of mycobacterial bacilli have been revisited, and their conventional interpretations have been challenged. Since the mid-1990s, when the first typing methods were introduced, a plethora of other modalities have been proposed. So-called molecular epidemiology has become an essential subdiscipline of modern mycobacteriology. It serves as a resource for understanding the key issues in the epidemiology of tuberculosis and other mycobacterial diseases. Among these issues are disclosing sources of infection, quantifying recent transmission, identifying transmission links, discerning reinfection from relapse, tracking the geographic distribution and clonal expansion of specific strains, and exploring the genetic mechanisms underlying specific phenotypic traits, including virulence, organ tropism, transmissibility, or drug resistance. Since genotyping continues to unravel the biology of mycobacteria, it offers enormous promise in the fight against and prevention of the diseases caused by these pathogens. In this review, molecular typing methods for Mycobacterium tuberculosis and nontuberculous mycobacteria elaborated over the last 2 decades are summarized. The relevance of these methods to the epidemiological investigation, diagnosis, evolution, and control of mycobacterial diseases is discussed. PMID:26912567
Temporal abstraction-based clinical phenotyping with Eureka!

PubMed

Post, Andrew R; Kurc, Tahsin; Willard, Richie; Rathod, Himanshu; Mansour, Michel; Pai, Akshatha Kalsanka; Torian, William M; Agravat, Sanjay; Sturm, Suzanne; Saltz, Joel H

2013-01-01

Temporal abstraction, a method for specifying and detecting temporal patterns in clinical databases, is very expressive and performs well, but it is difficult for clinical investigators and data analysts to understand. Such patterns are critical in phenotyping patients using their medical records in research and quality improvement. We have previously developed the Analytic Information Warehouse (AIW), which computes such phenotypes using temporal abstraction but requires software engineers to use. We have extended the AIW's web user interface, Eureka! Clinical Analytics, to support specifying phenotypes using an alternative model that we developed with clinical stakeholders. The software converts phenotypes from this model to that of temporal abstraction prior to data processing. The model can represent all phenotypes in a quality improvement project and a growing set of phenotypes in a multi-site research study. Phenotyping that is accessible to investigators and IT personnel may enable its broader adoption.
PHENOstruct: Prediction of human phenotype ontology terms using heterogeneous data sources.

PubMed

Kahanda, Indika; Funk, Christopher; Verspoor, Karin; Ben-Hur, Asa

2015-01-01

The human phenotype ontology (HPO) was recently developed as a standardized vocabulary for describing the phenotype abnormalities associated with human diseases. At present, only a small fraction of human protein coding genes have HPO annotations. But, researchers believe that a large portion of currently unannotated genes are related to disease phenotypes. Therefore, it is important to predict gene-HPO term associations using accurate computational methods. In this work we demonstrate the performance advantage of the structured SVM approach which was shown to be highly effective for Gene Ontology term prediction in comparison to several baseline methods. Furthermore, we highlight a collection of informative data sources suitable for the problem of predicting gene-HPO associations, including large scale literature mining data.
Early Learning and Adaptive Behaviour in Toddlers with Down Syndrome: Evidence for an Emerging Behavioural Phenotype?

ERIC Educational Resources Information Center

Fidler, Deborah; Hepburn, Susan; Rogers, Sally

2006-01-01

Background: Though the Down syndrome behavioural phenotype has been described as involving relative strengths in visuo-spatial processing and sociability, and relative weaknesses in verbal skills and motor planning, the early emergence of this phenotypic pattern of strengths and weaknesses has not yet been fully explored. Method: In this study, we…
Using text mining techniques to extract phenotypic information from the PhenoCHF corpus.

PubMed

Alnazzawi, Noha; Thompson, Paul; Batista-Navarro, Riza; Ananiadou, Sophia

2015-01-01

Phenotypic information locked away in unstructured narrative text presents significant barriers to information accessibility, both for clinical practitioners and for computerised applications used for clinical research purposes. Text mining (TM) techniques have previously been applied successfully to extract different types of information from text in the biomedical domain. They have the potential to be extended to allow the extraction of information relating to phenotypes from free text. To stimulate the development of TM systems that are able to extract phenotypic information from text, we have created a new corpus (PhenoCHF) that is annotated by domain experts with several types of phenotypic information relating to congestive heart failure. To ensure that systems developed using the corpus are robust to multiple text types, it integrates text from heterogeneous sources, i.e., electronic health records (EHRs) and scientific articles from the literature. We have developed several different phenotype extraction methods to demonstrate the utility of the corpus, and tested these methods on a further corpus, i.e., ShARe/CLEF 2013. Evaluation of our automated methods showed that PhenoCHF can facilitate the training of reliable phenotype extraction systems, which are robust to variations in text type. These results have been reinforced by evaluating our trained systems on the ShARe/CLEF corpus, which contains clinical records of various types. Like other studies within the biomedical domain, we found that solutions based on conditional random fields produced the best results, when coupled with a rich feature set. PhenoCHF is the first annotated corpus aimed at encoding detailed phenotypic information. The unique heterogeneous composition of the corpus has been shown to be advantageous in the training of systems that can accurately extract phenotypic information from a range of different text types. Although the scope of our annotation is currently limited to a single disease, the promising results achieved can stimulate further work into the extraction of phenotypic information for other diseases. The PhenoCHF annotation guidelines and annotations are publicly available at https://code.google.com/p/phenochf-corpus.
Advanced Cell Classifier: User-Friendly Machine-Learning-Based Software for Discovering Phenotypes in High-Content Imaging Data.

PubMed

Piccinini, Filippo; Balassa, Tamas; Szkalisity, Abel; Molnar, Csaba; Paavolainen, Lassi; Kujala, Kaisa; Buzas, Krisztina; Sarazova, Marie; Pietiainen, Vilja; Kutay, Ulrike; Smith, Kevin; Horvath, Peter

2017-06-28

High-content, imaging-based screens now routinely generate data on a scale that precludes manual verification and interrogation. Software applying machine learning has become an essential tool to automate analysis, but these methods require annotated examples to learn from. Efficiently exploring large datasets to find relevant examples remains a challenging bottleneck. Here, we present Advanced Cell Classifier (ACC), a graphical software package for phenotypic analysis that addresses these difficulties. ACC applies machine-learning and image-analysis methods to high-content data generated by large-scale, cell-based experiments. It features methods to mine microscopic image data, discover new phenotypes, and improve recognition performance. We demonstrate that these features substantially expedite the training process, successfully uncover rare phenotypes, and improve the accuracy of the analysis. ACC is extensively documented, designed to be user-friendly for researchers without machine-learning expertise, and distributed as a free open-source tool at www.cellclassifier.org. Copyright © 2017 Elsevier Inc. All rights reserved.

Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

PubMed

Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A; Bathe, Mark

2016-01-01

Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.
Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector.

PubMed

Chakkaramakkil Verghese, Santhosh; Goloviznina, Natalya A; Kurre, Peter

2016-11-19

Fanconi anemia (FA) is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC)-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV) particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.
Habituation of medaka (Oryzias latipes) demonstrated by open-field testing.

PubMed

Matsunaga, Wataru; Watanabe, Eiji

2010-10-01

Habituation to novel environments is frequently studied to analyze cognitive phenotypes in animals, and an open-field test is generally conducted to investigate the changes that occur in animals during habituation. The test has not been used in behavioral studies of medaka (Oryzias latipes), which is recently being used in behavioral research. Therefore, we examined the open-field behavior of medaka on the basis of temporal changes in 2 conventional indexes of locomotion and position. The findings of our study clearly showed that medaka changed its behavior through multiple temporal phases as it became more familiar with new surroundings; this finding is consistent with those of other ethological studies in animals. During repeated open-field testing on 2 consecutive days, we observed that horizontal locomotion on the second day was less than that on the first day, which suggested that habituation is retained in fish for days. This temporal habituation was critically affected by water factors or visual cues of the tank, thereby suggesting that fish have spatial memory of their surroundings. Thus, the data from this study will afford useful fundamental information for behavioral phenotyping of medaka and for elucidating cognitive phenotypes in animals. Copyright (c) 2010 Elsevier B.V. All rights reserved.
A unique mosaic Turner syndrome patient with androgen receptor gene derived marker chromosome.

PubMed

Kalkan, Rasime; Özdağ, Nermin; Bundak, Rüveyde; Çirakoğlu, Ayşe; Serakinci, Nedime

2016-01-01

Patients with Turner syndrome are generally characterized by having short stature with no secondary sexual characteristics. Some abnormalities, such as webbed neck, renal malformations (>50%) and cardiac defects (10%) are less common. The intelligence of these patients is considered normal. Non-mosaic monosomy X is observed in approximately 45% of postnatal patients with Turner syndrome and the rest of the patients have structural abnormalities or mosaicism involving 46,X,i(Xq), 45,X/46,XX, 45,X and other variants. The phenotype of 45,X/46,X,+mar individuals varies by the genetic continent and degree of the mosaicism. The gene content of the marker chromosome is the most important when correlating the phenotype with the genotype. Here we present an 11-year-old female who was referred for evaluation of her short stature and learning disabilities. Conventional cytogenetic investigation showed a mosaic 45,X/46,X,+mar karyotype. Fluorescence in situ hybridization showed that the marker chromosome originated from the X chromosome within the androgen receptor (AR) and X-inactive specific transcript (XIST) genes. Therefore, it is possible that aberrant activation of the marker chromosome, compromising the AR and XIST genes, may modify the Turner syndrome phenotype.
RNAi trigger fragment truncation attenuates soybean FAD2-1 transcript suppression and yields intermediate oil phenotypes.

PubMed

Wagner, Nicholas; Mroczka, Andrew; Roberts, Peter D; Schreckengost, William; Voelker, Toni

2011-09-01

Suppression of the microsomal ω6 oleate desaturase during the seed development of soybean (Glycine max) with the 420-bp soybean FAD2-1A intron as RNAi trigger shifts the conventional fatty acid composition of soybean oil from 20% oleic and 60% polyunsaturates to one containing greater than 80% oleic acid and less than 10% polyunsaturates. To determine whether RNAi could be attenuated by reducing the trigger fragment length, transgenic plants were generated to express successively shorter 5' or 3' deletion derivatives of the FAD2-1A intron. We observed a gradual reduction in transcript suppression with shorter trigger fragments. Fatty acid composition was less affected with shorter triggers, and triggers less than 60 bp had no phenotypic effect. No trigger sequences conferring significantly higher or lower suppression efficiencies were found, and the primary determinant of suppression effect was sequence length. The observed relationship of transcript suppression with the induced fatty acid phenotype indicates that RNAi is a saturation process and not a step change between suppressed and nonsuppressed states and intermediate suppression states can be achieved. © 2010 Monsanto. Plant Biotechnology Journal © 2010 Society for Experimental Biology and Blackwell Publishing Ltd.
Gene variants associated with antisocial behaviour: A latent variable approach

PubMed Central

Bentley, Mary Jane; Lin, Haiqun; Fernandez, Thomas V.; Lee, Maria; Yrigollen, Carolyn M.; Pakstis, Andrew J.; Katsovich, Liliya; Olds, David L.; Grigorenko, Elena L.; Leckman, James F.

2013-01-01

Objective The aim of this study was to determine if a latent variable approach might be useful in identifying shared variance across genetic risk alleles that is associated with antisocial behaviour at age 15 years. Methods Using a conventional latent variable approach, we derived an antisocial phenotype in 328 adolescents utilizing data from a 15-year follow-up of a randomized trial of a prenatal and infancy nurse-home visitation program in Elmira, New York. We then investigated, via a novel latent variable approach, 450 informative genetic polymorphisms in 71 genes previously associated with antisocial behaviour, drug use, affiliative behaviours, and stress response in 241 consenting individuals for whom DNA was available. Haplotype and Pathway analyses were also performed. Results Eight single-nucleotide polymorphisms (SNPs) from 8 genes contributed to the latent genetic variable that in turn accounted for 16.0% of the variance within the latent antisocial phenotype. The number of risk alleles was linearly related to the latent antisocial variable scores. Haplotypes that included the putative risk alleles for all 8 genes were also associated with higher latent antisocial variable scores. In addition, 33 SNPs from 63 of the remaining genes were also significant when added to the final model. Many of these genes interact on a molecular level, forming molecular networks. The results support a role for genes related to dopamine, norepinephrine, serotonin, glutamate, opioid, and cholinergic signaling as well as stress response pathways in mediating susceptibility to antisocial behaviour. Conclusions This preliminary study supports use of relevant behavioural indicators and latent variable approaches to study the potential “co-action” of gene variants associated with antisocial behaviour. It also underscores the cumulative relevance of common genetic variants for understanding the etiology of complex behaviour. If replicated in future studies, this approach may allow the identification of a ‘shared’ variance across genetic risk alleles associated with complex neuropsychiatric dimensional phenotypes using relatively small numbers of well-characterized research participants. PMID:23822756
High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vega, Sebastián L.; Liu, Er; Arvind, Varun

Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regionsmore » of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative “imaging-derived” parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions. - Highlights: • High-content analysis of nuclear shape and organization classify stem and progenitor cells poised for distinct lineages. • Early oncogenic changes in mesenchymal stem cells (MSCs) are also detected with nuclear descriptors. • A new class of cancer-mitigating biomaterials was identified based on image informatics. • Textural metrics of the nuclear structural protein NuMA are sufficient to parse emergent cell phenotypes.« less
Assessing CpG island methylator phenotype, 1p/19q codeletion, and MGMT promoter methylation from epigenome-wide data in the biomarker cohort of the NOA-04 trial

PubMed Central

Wiestler, Benedikt; Capper, David; Hovestadt, Volker; Sill, Martin; Jones, David T.W.; Hartmann, Christian; Felsberg, Joerg; Platten, Michael; Feiden, Wolfgang; Keyvani, Kathy; Pfister, Stefan M.; Wiestler, Otmar D.; Meyermann, Richard; Reifenberger, Guido; Pietsch, Thorsten; von Deimling, Andreas; Weller, Michael; Wick, Wolfgang

2014-01-01

Background Molecular biomarkers including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q codeletion, and O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation may improve prognostication and guide treatment decisions for patients with World Health Organization (WHO) anaplastic gliomas. At present, each marker is individually tested by distinct assays. Illumina Infinium HumanMethylation450 BeadChip arrays (HM450) enable the determination of large-scale methylation profiles and genome-wide DNA copy number changes. Algorithms have been developed to detect the glioma CpG island methylator phenotype (G-CIMP) associated with IDH1/2 mutation, 1p/19q codeletion, and MGMT promoter methylation using a single assay. Methods Here, we retrospectively investigated the diagnostic and prognostic performance of these algorithms in comparison to individual marker testing and patient outcome in the biomarker cohort (n = 115 patients) of the NOA-04 trial. Results Concordance for IDH and 1p/19q status was very high: In 92% of samples, the HM450 and reference data agreed. In discordant samples, survival analysis by Kaplan-Meier and Cox regression analyses suggested a more accurate assessment of biological phenotype by the HM450 analysis. The HM450-derived MGMT-STP27 model to calculate MGMT promoter methylation probability revealed this aberration in a significantly higher fraction of samples than conventional methylation-specific PCR, with 87 of 91 G-CIMP tumors predicted as MGMT promoter-methylated. Pyrosequencing of discordant samples confirmed the HM450 assessment in 14 of 17 cases. Conclusions G-CIMP and 1p/19q codeletion are reliably detectable by HM450 analysis and are associated with prognosis in the NOA-04 trial. For MGMT, HM450 suggests promoter methylation in the vast majority of G-CIMP tumors, which is supported by pyrosequencing. PMID:25028501
Towards improving phenotype representation in OWL

PubMed Central

2012-01-01

Background Phenotype ontologies are used in species-specific databases for the annotation of mutagenesis experiments and to characterize human diseases. The Entity-Quality (EQ) formalism is a means to describe complex phenotypes based on one or more affected entities and a quality. EQ-based definitions have been developed for many phenotype ontologies, including the Human and Mammalian Phenotype ontologies. Methods We analyze formalizations of complex phenotype descriptions in the Web Ontology Language (OWL) that are based on the EQ model, identify several representational challenges and analyze potential solutions to address these challenges. Results In particular, we suggest a novel, role-based approach to represent relational qualities such as concentration of iron in spleen, discuss its ontological foundation in the General Formal Ontology (GFO) and evaluate its representation in OWL and the benefits it can bring to the representation of phenotype annotations. Conclusion Our analysis of OWL-based representations of phenotypes can contribute to improving consistency and expressiveness of formal phenotype descriptions. PMID:23046625
Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution

PubMed Central

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D.; Rainey, Paul B.; de Visser, J. Arjan G. M.; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes–via growth–over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology. PMID:27077662
Lineage Tracking for Probing Heritable Phenotypes at Single-Cell Resolution.

PubMed

Cottinet, Denis; Condamine, Florence; Bremond, Nicolas; Griffiths, Andrew D; Rainey, Paul B; de Visser, J Arjan G M; Baudry, Jean; Bibette, Jérôme

2016-01-01

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes-via growth-over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.
A novel 3D imaging system for strawberry phenotyping.

PubMed

He, Joe Q; Harrison, Richard J; Li, Bo

2017-01-01

Accurate and quantitative phenotypic data in plant breeding programmes is vital in breeding to assess the performance of genotypes and to make selections. Traditional strawberry phenotyping relies on the human eye to assess most external fruit quality attributes, which is time-consuming and subjective. 3D imaging is a promising high-throughput technique that allows multiple external fruit quality attributes to be measured simultaneously. A low cost multi-view stereo (MVS) imaging system was developed, which captured data from 360° around a target strawberry fruit. A 3D point cloud of the sample was derived and analysed with custom-developed software to estimate berry height, length, width, volume, calyx size, colour and achene number. Analysis of these traits in 100 fruits showed good concordance with manual assessment methods. This study demonstrates the feasibility of an MVS based 3D imaging system for the rapid and quantitative phenotyping of seven agronomically important external strawberry traits. With further improvement, this method could be applied in strawberry breeding programmes as a cost effective phenotyping technique.
Multiplex ligation-dependant probe amplification study of children with idiopathic mental retardation in South India

PubMed Central

John, Neetha; Rajasekhar, Moka; Girisha, Katta Mohan; Sharma, Podila Satya Venkata Narasimha; Gopinath, Puthiya Mundyat

2013-01-01

BACKGROUND: Mental retardation (MR) is a heterogeneous dysfunction of the central nervous system exhibiting complex phenotypes and has an estimated prevalence of 1-3% in the general population. However, in about 50% of the children diagnosed with any form of intellectual disability or developmental delay the cause goes undetected contributing to idiopathic intellectual disability. MATERIALS AND METHODS: A total of 122 children with developmental delay/MR were studied to identify the microscopic and submicroscopic chromosome rearrangements by using the conventional cytogenetics and multiplex ligation dependent probe amplification (MLPA) analysis using SALSA MLPA kits from Microbiology Research Centre Holland [MRC] Holland. RESULTS: All the recruited children were selected for this study, after thorough clinical assessment and metaphases prepared were analyzed by using automated karyotyping system. None was found to have chromosomal abnormality; MLPA analysis was carried out in all subjects and identified in 11 (9%) patients. CONCLUSION: Karyotype analysis in combination with MLPA assays for submicroscopic micro-deletions may be recommended for children with idiopathic MR. PMID:24019617
Signatures of negative selection in the genetic architecture of human complex traits.

PubMed

Zeng, Jian; de Vlaming, Ronald; Wu, Yang; Robinson, Matthew R; Lloyd-Jones, Luke R; Yengo, Loic; Yap, Chloe X; Xue, Angli; Sidorenko, Julia; McRae, Allan F; Powell, Joseph E; Montgomery, Grant W; Metspalu, Andres; Esko, Tonu; Gibson, Greg; Wray, Naomi R; Visscher, Peter M; Yang, Jian

2018-05-01

We develop a Bayesian mixed linear model that simultaneously estimates single-nucleotide polymorphism (SNP)-based heritability, polygenicity (proportion of SNPs with nonzero effects), and the relationship between SNP effect size and minor allele frequency for complex traits in conventionally unrelated individuals using genome-wide SNP data. We apply the method to 28 complex traits in the UK Biobank data (N = 126,752) and show that on average, 6% of SNPs have nonzero effects, which in total explain 22% of phenotypic variance. We detect significant (P < 0.05/28) signatures of natural selection in the genetic architecture of 23 traits, including reproductive, cardiovascular, and anthropometric traits, as well as educational attainment. The significant estimates of the relationship between effect size and minor allele frequency in complex traits are consistent with a model of negative (or purifying) selection, as confirmed by forward simulation. We conclude that negative selection acts pervasively on the genetic variants associated with human complex traits.
Survival strategies of Bacillus spores in food.

PubMed

Stecchini, Mara Lucia; Del Torre, Manuela; Polese, Pierluigi

2013-11-01

Control of bacterial spores is one of the major problem in the food preservation. Spores of Bacillus genus are commonly present in different environments, including soil and the gut of insects and animals and, as a result, they can be spread to all kind of foods. Due to their high resistance properties, their complete inactivation in food is often impossible without changing the product characteristics. Surviving spores can germinate and grow out to vegetative cells, with the consequent great risk of food spoilage and food poisoning after consumption. Spores have evolved various mechanisms, including phenotypic variability, to protect themselves from a wide range of damage resulting from food preservation treatments. Even if the phenotypic heterogeneity contributes to increase the chances of survival of Bacillus spore to conventional preservation treatments, in some specific instances, an homogeneous response could be the result of a strategy adopted by the spores to increase resistance to those treatments.
Liver-resident NK cells and their potential functions.

PubMed

Peng, Hui; Sun, Rui

2017-09-18

Natural killer (NK) cells represent a heterogeneous population of innate lymphocytes with phenotypically and functionally distinct subsets. In particular, recent studies have identified a unique subset of NK cells residing within the liver that are maintained as tissue-resident cells, confer antigen-specific memory responses and exhibit different phenotypical and developmental characteristics compared with conventional NK (cNK) cells. These findings have encouraged researchers to uncover tissue-resident NK cells at other sites, and detailed analyses have revealed that these tissue-resident NK cells share many similarities with liver-resident NK cells and tissue-resident memory T cells. Here, we present a brief historical perspective on the discovery of liver-resident NK cells and discuss their relationship to cNK cells and other emerging NK cell subsets and their potential functions.Cellular &Molecular Immunology advance online publication, 18 September 2017; doi:10.1038/cmi.2017.72.
Raffinose, a plant galactoside, inhibits Pseudomonas aeruginosa biofilm formation via binding to LecA and decreasing cellular cyclic diguanylate levels

NASA Astrophysics Data System (ADS)

Kim, Han-Shin; Cha, Eunji; Kim, Yunhye; Jeon, Young Ho; Olson, Betty H.; Byun, Youngjoo; Park, Hee-Deung

2016-05-01

Biofilm formation on biotic or abiotic surfaces has unwanted consequences in medical, clinical, and industrial settings. Treatments with antibiotics or biocides are often ineffective in eradicating biofilms. Promising alternatives to conventional agents are biofilm-inhibiting compounds regulating biofilm development without toxicity to growth. Here, we screened a biofilm inhibitor, raffinose, derived from ginger. Raffinose, a galactotrisaccharide, showed efficient biofilm inhibition of Pseudomonas aeruginosa without impairing its growth. Raffinose also affected various phenotypes such as colony morphology, matrix formation, and swarming motility. Binding of raffinose to a carbohydrate-binding protein called LecA was the cause of biofilm inhibition and altered phenotypes. Furthermore, raffinose reduced the concentration of the second messenger, cyclic diguanylate (c-di-GMP), by increased activity of a c-di-GMP specific phosphodiesterase. The ability of raffinose to inhibit P. aeruginosa biofilm formation and its molecular mechanism opens new possibilities for pharmacological and industrial applications.
3D imaging of optically cleared tissue using a simplified CLARITY method and on-chip microscopy

PubMed Central

Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

2017-01-01

High-throughput sectioning and optical imaging of tissue samples using traditional immunohistochemical techniques can be costly and inaccessible in resource-limited areas. We demonstrate three-dimensional (3D) imaging and phenotyping in optically transparent tissue using lens-free holographic on-chip microscopy as a low-cost, simple, and high-throughput alternative to conventional approaches. The tissue sample is passively cleared using a simplified CLARITY method and stained using 3,3′-diaminobenzidine to target cells of interest, enabling bright-field optical imaging and 3D sectioning of thick samples. The lens-free computational microscope uses pixel super-resolution and multi-height phase recovery algorithms to digitally refocus throughout the cleared tissue and obtain a 3D stack of complex-valued images of the sample, containing both phase and amplitude information. We optimized the tissue-clearing and imaging system by finding the optimal illumination wavelength, tissue thickness, sample preparation parameters, and the number of heights of the lens-free image acquisition and implemented a sparsity-based denoising algorithm to maximize the imaging volume and minimize the amount of the acquired data while also preserving the contrast-to-noise ratio of the reconstructed images. As a proof of concept, we achieved 3D imaging of neurons in a 200-μm-thick cleared mouse brain tissue over a wide field of view of 20.5 mm2. The lens-free microscope also achieved more than an order-of-magnitude reduction in raw data compared to a conventional scanning optical microscope imaging the same sample volume. Being low cost, simple, high-throughput, and data-efficient, we believe that this CLARITY-enabled computational tissue imaging technique could find numerous applications in biomedical diagnosis and research in low-resource settings. PMID:28819645
CD4 T-helper cell cytokine phenotypes and antibody response following tetanus toxoid booster immunization

USDA-ARS?s Scientific Manuscript database

Routine methods for enumerating antigen-specific T-helper cells may not identify low-frequency phenotypes such as Th2 cells. We compared methods of evaluating such responses to identify tetanus toxoid- (TT) specific Th1, Th2, Th17 and IL10+ cells. Eight healthy subjects were given a TT booster vacci...
Linkage analysis of alternative anxiety phenotypes in multiply affected panic disorder families

PubMed Central

Fyer, Abby J.; Costa, Ramiro; Haghighi, Fatemeh; Logue, Mark W.; Knowles, James A.; Weissman, Myrna M.; Hodge, Susan E.; Hamilton, Steven P.

2013-01-01

Background The choice of phenotype definitions for genetic studies of panic and phobic disorders is complicated by family, twin and neurobiological data indicating both distinct and shared risk factors as well as heterogeneity within categories. We previously reported a genome scan in 120 multiplex panic disorder (PD) families using a phenotype that closely adhered to the DSM IV PD definition. Here we extend this work by conducting exploratory linkage analyses in this same pedigree set using ten additional literature- based panic and phobia-related phenotypes that take into account aspects of these hypothesized complexities. Methods Multiply affected families (> 2 individuals with PD) were recruited from clinical and non-clinical sources, evaluated by clinician administered semi-structured interview and subsequent blind consensus best estimate procedure. Each phenotype was analyzed under dominant and recessive models using parametric 2-point (homogeneity and heterogeneity), multipoint, and non-parametric methods. Empirically based permutations were used to estimate model specific and global (across all phenotypes) p-values. Results The highest score was a 2-point lod (4.27, global p < 0.08) on chromosome 13 (D13S793, 76cM) for the phenotype “specific or social phobia” under a recessive model and conditions of homogeneity. There was minimal support for linkage to any of the remaining nine phenotypes. Conclusions Though interpretation of findings is limited by sample size and the large number of phenotypes and models analyzed these data suggest a region on chromosome 13 as a potential site for further exploration in relation to risk for specific and social phobias. PMID:22525237

Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

PubMed Central

Li, Yiyan; Yang, Xing; Zhao, Weian

2018-01-01

Rapid bacterial identification (ID) and antibiotic susceptibility testing (AST) are in great demand due to the rise of drug-resistant bacteria. Conventional culture-based AST methods suffer from a long turnaround time. By necessity, physicians often have to treat patients empirically with antibiotics, which has led to an inappropriate use of antibiotics, an elevated mortality rate and healthcare costs, and antibiotic resistance. Recent advances in miniaturization and automation provide promising solutions for rapid bacterial ID/AST profiling, which will potentially make a significant impact in the clinical management of infectious diseases and antibiotic stewardship in the coming years. In this review, we summarize and analyze representative emerging micro- and nanotechnologies, as well as automated systems for bacterial ID/AST, including both phenotypic (e.g., microfluidic-based bacterial culture, and digital imaging of single cells) and molecular (e.g., multiplex PCR, hybridization probes, nanoparticles, synthetic biology tools, mass spectrometry, and sequencing technologies) methods. We also discuss representative point-of-care (POC) systems that integrate sample processing, fluid handling, and detection for rapid bacterial ID/AST. Finally, we highlight major remaining challenges and discuss potential future endeavors toward improving clinical outcomes with rapid bacterial ID/AST technologies. PMID:28850804
Rapid Immunochromatographic Detection of Serum Anti-α-Galactosidase A Antibodies in Fabry Patients after Enzyme Replacement Therapy

PubMed Central

Nakano, Sachie; Tsukimura, Takahiro; Togawa, Tadayasu; Ohashi, Toya; Kobayashi, Masahisa; Takayama, Katsuyoshi; Kobayashi, Yukuharu; Abiko, Hiroshi; Satou, Masatsugu; Nakahata, Tohru; Warnock, David G.; Sakuraba, Hitoshi; Shibasaki, Futoshi

2015-01-01

We developed an immunochromatography-based assay for detecting antibodies against recombinant α-galactosidase A proteins in serum. The evaluation of 29 serum samples from Fabry patients, who had received enzyme replacement therapy with agalsidase alpha and/or agalsidase beta, was performed by means of this assay method, and the results clearly revealed that the patients exhibited the same level of antibodies against both agalsidase alpha and agalsidase beta, regardless of the species of recombinant α-galactosidase A used for enzyme replacement therapy. A conventional enzyme-linked immunosorbent assay supported the results. Considering these, enzyme replacement therapy with agalsidase alpha or agalsidase beta would generate antibodies against the common epitopes in both agalsidase alpha and agalsidase beta. Most of the patients who showed immunopositive reaction exhibited classic Fabry phenotype and harbored gene mutations affecting biosynthesis of α-galactosidase A. As immunochromatography is a handy and simple assay system which can be available at bedside, this assay method would be extremely useful for quick evaluation or first screening of serum antibodies against agalsidase alpha or agalsidase beta in Fabry disease with enzyme replacement therapy. PMID:26083343
Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing.

PubMed

Sahoo, Malaya K; Holubar, Marisa; Huang, ChunHong; Mohamed-Hadley, Alisha; Liu, Yuanyuan; Waggoner, Jesse J; Troy, Stephanie B; Garcia-Garcia, Lourdes; Ferreyra-Reyes, Leticia; Maldonado, Yvonne; Pinsky, Benjamin A

2017-07-01

Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of <1%, depending on read depth. Sequencing of viral nucleic acids from the stool of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (<5%) distributed across the 5' UTR and P1 genomic region in all three Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication. Copyright © 2017 Sahoo et al.
Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing.

PubMed

Li, Yiyan; Yang, Xing; Zhao, Weian

2017-12-01

Rapid bacterial identification (ID) and antibiotic susceptibility testing (AST) are in great demand due to the rise of drug-resistant bacteria. Conventional culture-based AST methods suffer from a long turnaround time. By necessity, physicians often have to treat patients empirically with antibiotics, which has led to an inappropriate use of antibiotics, an elevated mortality rate and healthcare costs, and antibiotic resistance. Recent advances in miniaturization and automation provide promising solutions for rapid bacterial ID/AST profiling, which will potentially make a significant impact in the clinical management of infectious diseases and antibiotic stewardship in the coming years. In this review, we summarize and analyze representative emerging micro- and nanotechnologies, as well as automated systems for bacterial ID/AST, including both phenotypic (e.g., microfluidic-based bacterial culture, and digital imaging of single cells) and molecular (e.g., multiplex PCR, hybridization probes, nanoparticles, synthetic biology tools, mass spectrometry, and sequencing technologies) methods. We also discuss representative point-of-care (POC) systems that integrate sample processing, fluid handling, and detection for rapid bacterial ID/AST. Finally, we highlight major remaining challenges and discuss potential future endeavors toward improving clinical outcomes with rapid bacterial ID/AST technologies.
Genetic modification of stem cells for improved therapy of the infarcted myocardium.

PubMed

Haider, Husnain Kh; Mustafa, Anique; Feng, Yuliang; Ashraf, Muhammad

2011-10-03

The conventional treatment modalities for ischemic heart disease only provide symptomatic relief to the patient without repairing and regenerating the damaged myocardium. Stem cell transplantation has emerged as a promising alternative therapeutic approach for cardiovascular diseases. Stem cells possess the potential of differentiation to adopt morphofunctional cardiac and vasculogenic phenotypes to repopulate the scar tissue and restore regional blood flow in the ischemic myocardium. These beneficial therapeutic effects make stem cell transplantation the method of choice for the treatment of ischemic heart disease. The efficacy of stem cell transplantation may be augmented by genetic manipulation of the cells prior to transplantation. Not only will insertion of therapeutic transgene(s) into the stem cells support the survival and differentiation of cells in the unfavorable microenvironment of the ischemic myocardium, but also the genetically manipulated stem cells will serve as a source of the transgene expression product in the heart for therapeutic benefits. We provide an overview of the extensively studied stem cell types for cardiac regeneration, the various methods in which these cells have been genetically manipulated and rationale of genetic modification of stem cells for use in regenerative cardiovascular therapeutics.
Chiral Antioxidant-based Gold Nanoclusters Reprogram DNA Epigenetic Patterns

PubMed Central

Ma, Yue; Fu, Hualin; Zhang, Chunlei; Cheng, Shangli; Gao, Jie; Wang, Zhen; Jin, Weilin; Conde, João; Cui, Daxiang

2016-01-01

Epigenetic modifications sit ‘on top of’ the genome and influence DNA transcription, which can force a significant impact on cellular behavior and phenotype and, consequently human development and disease. Conventional methods for evaluating epigenetic modifications have inherent limitations and, hence, new methods based on nanoscale devices are needed. Here, we found that antioxidant (glutathione) chiral gold nanoclusters induce a decrease of 5-hydroxymethylcytosine (5hmC), which is an important epigenetic marker that associates with gene transcription regulation. This epigenetic change was triggered partially through ROS activation and oxidation generated by the treatment with glutathione chiral gold nanoclusters, which may inhibit the activity of TET proteins catalyzing the conversion of 5-methylcytosine (5mC) to 5hmC. In addition, these chiral gold nanoclusters can downregulate TET1 and TET2 mRNA expression. Alteration of TET-5hmC signaling will then affect several downstream targets and be involved in many aspects of cell behavior. We demonstrate for the first time that antioxidant-based chiral gold nanomaterials have a direct effect on epigenetic process of TET-5hmC pathways and reveal critical DNA demethylation patterns. PMID:27633378
Identification of salivary Lactobacillus rhamnosus species by DNA profiling and a specific probe.

PubMed

Richard, B; Groisillier, A; Badet, C; Dorignac, G; Lonvaud-Funel, A

2001-03-01

The Lactobacillus genus has been shown to be associated with the dental carious process, but little is known about the species related to the decay, although Lactobacillus rhamnosus is suspected to be the most implicated species. Conventional identification methods based on biochemical criteria lead to ambiguous results, since the Lactobacillus species found in saliva are phenotypically close. To clarify the role of this genus in the evolution of carious disease, this work aimed to find a rapid and reliable method for identifying the L. rhamnosus species. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some wild strains isolated from children's saliva. DNA profiling using semirandom polymorphic DNA amplification (semi-RAPD) generated specific patterns for L. rhamnosus ATCC 7469. The profiles of all L. rhamnosus strains tested were similar and could be grouped; these strains shared four common fragments. Wild strains first identified with classic methods shared common patterns with the L. rhamnosus species and could be reclassified. One fragment of the profile was purified, cloned, used as a probe and found to be specific to the L. rhamnosus species. These results may help to localize this species within its ecological niche and to elucidate the progression of the carious process.
Allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR), a powerful method for diagnosing loss of imprinting of the 11p15 region in Russell Silver and Beckwith Wiedemann syndromes.

PubMed

Azzi, Salah; Steunou, Virginie; Rousseau, Alexandra; Rossignol, Sylvie; Thibaud, Nathalie; Danton, Fabienne; Le Jule, Marilyne; Gicquel, Christine; Le Bouc, Yves; Netchine, Irène

2011-02-01

Many human syndromes involve a loss of imprinting (LOI) due to a loss (LOM) or a gain of DNA methylation (GOM). Most LOI occur as mosaics and can therefore be difficult to detect with conventional methods. The human imprinted 11p15 region is crucial for the control of fetal growth, and LOI at this locus is associated with two clinical disorders with opposite phenotypes: Beckwith-Wiedemann syndrome (BWS), characterized by fetal overgrowth and a high risk of tumors, and Russell-Silver syndrome (RSS), characterized by intrauterine and postnatal growth restriction. Until recently, we have been using Southern blotting for the diagnosis of RSS and BWS. We describe here a powerful quantitative technique, allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR), for the diagnosis of these two complex disorders. We first checked the specificity of the probes and primers used for ASMM RTQ-PCR. We then carried out statistical validation for this method, on both retrospective and prospective populations of patients. This analysis demonstrated that ASMM RTQ-PCR is more sensitive than Southern blotting for detecting low degree of LOI. Moreover, ASMM RTQ-PCR is a very rapid, reliable, simple, safe, and cost effective method. © 2011 Wiley-Liss, Inc.
High-resolution melting (HRM) re-analysis of a polyposis patients cohort reveals previously undetected heterozygous and mosaic APC gene mutations.

PubMed

Out, Astrid A; van Minderhout, Ivonne J H M; van der Stoep, Nienke; van Bommel, Lysette S R; Kluijt, Irma; Aalfs, Cora; Voorendt, Marsha; Vossen, Rolf H A M; Nielsen, Maartje; Vasen, Hans F A; Morreau, Hans; Devilee, Peter; Tops, Carli M J; Hes, Frederik J

2015-06-01

Familial adenomatous polyposis is most frequently caused by pathogenic variants in either the APC gene or the MUTYH gene. The detection rate of pathogenic variants depends on the severity of the phenotype and sensitivity of the screening method, including sensitivity for mosaic variants. For 171 patients with multiple colorectal polyps without previously detectable pathogenic variant, APC was reanalyzed in leukocyte DNA by one uniform technique: high-resolution melting (HRM) analysis. Serial dilution of heterozygous DNA resulted in a lowest detectable allelic fraction of 6% for the majority of variants. HRM analysis and subsequent sequencing detected pathogenic fully heterozygous APC variants in 10 (6%) of the patients and pathogenic mosaic variants in 2 (1%). All these variants were previously missed by various conventional scanning methods. In parallel, HRM APC scanning was applied to DNA isolated from polyp tissue of two additional patients with apparently sporadic polyposis and without detectable pathogenic APC variant in leukocyte DNA. In both patients a pathogenic mosaic APC variant was present in multiple polyps. The detection of pathogenic APC variants in 7% of the patients, including mosaics, illustrates the usefulness of a complete APC gene reanalysis of previously tested patients, by a supplementary scanning method. HRM is a sensitive and fast pre-screening method for reliable detection of heterozygous and mosaic variants, which can be applied to leukocyte and polyp derived DNA.
Prediction and Validation of Disease Genes Using HeteSim Scores.

PubMed

Zeng, Xiangxiang; Liao, Yuanlu; Liu, Yuansheng; Zou, Quan

2017-01-01

Deciphering the gene disease association is an important goal in biomedical research. In this paper, we use a novel relevance measure, called HeteSim, to prioritize candidate disease genes. Two methods based on heterogeneous networks constructed using protein-protein interaction, gene-phenotype associations, and phenotype-phenotype similarity, are presented. In HeteSim_MultiPath (HSMP), HeteSim scores of different paths are combined with a constant that dampens the contributions of longer paths. In HeteSim_SVM (HSSVM), HeteSim scores are combined with a machine learning method. The 3-fold experiments show that our non-machine learning method HSMP performs better than the existing non-machine learning methods, our machine learning method HSSVM obtains similar accuracy with the best existing machine learning method CATAPULT. From the analysis of the top 10 predicted genes for different diseases, we found that HSSVM avoid the disadvantage of the existing machine learning based methods, which always predict similar genes for different diseases. The data sets and Matlab code for the two methods are freely available for download at http://lab.malab.cn/data/HeteSim/index.jsp.
Study design and rationale for Optimal aNtiplatelet pharmacotherapy guided by bedSIDE genetic or functional TESTing in elective percutaneous coronary intervention patients (ONSIDE TEST): a prospective, open-label, randomised parallel-group multicentre trial (NCT01930773).

PubMed

Kołtowski, Łukasz; Aradi, Daniel; Huczek, Zenon; Tomaniak, Mariusz; Sibbing, Dirk; Filipiak, Krzysztof J; Kochman, Janusz; Balsam, Paweł; Opolski, Grzegorz

2016-01-01

High platelet reactivity (HPR) and presence of CYP2C19 loss-of-function alleles are associated with higher risk for periprocedural myocardial infarction in clopidogrel-treated patients undergoing percutaneous coronary intervention (PCI). It is unknown whether personalised treatment based on platelet function testing or genotyping can prevent such complications. The ONSIDE-TEST is a multicentre, prospective, open-label, randomised controlled clinical trial aiming to assess if optimisation of antiplatelet therapy based on either phenotyping or genotyping is superior to conventional care. Patients will be randomised into phenotyping, genotyping, or control arms. In the phenotyping group, patients will be tested with the VerifyNow P2Y12 assay before PCI, and patients with a platelet reactivity unit greater than 208 will be switched over to prasugrel, while others will continue on clopidogrel therapy. In the genotyping group, carriers of the *2 loss-of-function allele will receive prasugrel for PCI, while wild-type subjects will be treated with clopidogrel. Patients in the control arm will be treated with standard-dose clopidogrel. The primary endpoint of the study is the prevalence of periprocedural myocardial injury within 24 h after PCI in the controls as compared to the phenotyping and genotyping group. Secondary endpoints include cardiac death, myocardial infarction, definite or probable stent thrombosis, or urgent repeat revascularisation within 30 days of PCI. Primary safety outcome is Bleeding Academic Research Consortium (BARC) type 3 and 5 bleeding during 30 days of PCI. The ONSIDE TEST trial is expected to verify the clinical utility of an individualised antiplatelet strategy in preventing periprocedural myocardial injury by either phenotyping or genotyping. ClinicalTrials.gov: NCT01930773.
Adult siblings with homozygous G6PC3 mutations expand our understanding of the severe congenital neutropenia type 4 (SCN4) phenotype.

PubMed

Fernandez, Bridget A; Green, Jane S; Bursey, Ford; Barrett, Brendan; MacMillan, Andrée; McColl, Sarah; Fernandez, Sara; Rahman, Proton; Mahoney, Krista; Pereira, Sergio L; Scherer, Stephen W; Boycott, Kym M; Woods, Michael O

2012-11-21

Severe congenital neutropenia type 4 (SCN4) is an autosomal recessive disorder caused by mutations in the third subunit of the enzyme glucose-6-phosphatase (G6PC3). Its core features are congenital neutropenia and a prominent venous skin pattern, and affected individuals have variable birth defects. Oculocutaneous albinism type 4 (OCA4) is caused by autosomal recessive mutations in SLC45A2. We report a sister and brother from Newfoundland, Canada with complex phenotypes. The sister was previously reported by Cullinane et al., 2011. We performed homozygosity mapping, next generation sequencing and conventional Sanger sequencing to identify mutations that cause the phenotype in this family. We have also summarized clinical data from 49 previously reported SCN4 cases with overlapping phenotypes and interpret the medical histories of these siblings in the context of the literature. The siblings' phenotype is due in part to a homozygous mutation in G6PC3, [c.829C > T, p.Gln277X]. Their ages are 38 and 37 years respectively and they are the oldest SCN4 patients published to date. Both presented with congenital neutropenia and later developed Crohn disease. We suggest that the latter is a previously unrecognized SCN4 manifestation and that not all affected individuals have an intellectual disability. The sister also has a homozygous mutation in SLC45A2, which explains her severe oculocutaneous hypopigmentation. Her brother carried one SLC45A2 mutation and was diagnosed with "partial OCA" in childhood. This family highlights that apparently novel syndromes can in fact be caused by two known autosomal recessive disorders.
Genomic prediction in animals and plants: simulation of data, validation, reporting, and benchmarking.

PubMed

Daetwyler, Hans D; Calus, Mario P L; Pong-Wong, Ricardo; de Los Campos, Gustavo; Hickey, John M

2013-02-01

The genomic prediction of phenotypes and breeding values in animals and plants has developed rapidly into its own research field. Results of genomic prediction studies are often difficult to compare because data simulation varies, real or simulated data are not fully described, and not all relevant results are reported. In addition, some new methods have been compared only in limited genetic architectures, leading to potentially misleading conclusions. In this article we review simulation procedures, discuss validation and reporting of results, and apply benchmark procedures for a variety of genomic prediction methods in simulated and real example data. Plant and animal breeding programs are being transformed by the use of genomic data, which are becoming widely available and cost-effective to predict genetic merit. A large number of genomic prediction studies have been published using both simulated and real data. The relative novelty of this area of research has made the development of scientific conventions difficult with regard to description of the real data, simulation of genomes, validation and reporting of results, and forward in time methods. In this review article we discuss the generation of simulated genotype and phenotype data, using approaches such as the coalescent and forward in time simulation. We outline ways to validate simulated data and genomic prediction results, including cross-validation. The accuracy and bias of genomic prediction are highlighted as performance indicators that should be reported. We suggest that a measure of relatedness between the reference and validation individuals be reported, as its impact on the accuracy of genomic prediction is substantial. A large number of methods were compared in example simulated and real (pine and wheat) data sets, all of which are publicly available. In our limited simulations, most methods performed similarly in traits with a large number of quantitative trait loci (QTL), whereas in traits with fewer QTL variable selection did have some advantages. In the real data sets examined here all methods had very similar accuracies. We conclude that no single method can serve as a benchmark for genomic prediction. We recommend comparing accuracy and bias of new methods to results from genomic best linear prediction and a variable selection approach (e.g., BayesB), because, together, these methods are appropriate for a range of genetic architectures. An accompanying article in this issue provides a comprehensive review of genomic prediction methods and discusses a selection of topics related to application of genomic prediction in plants and animals.
Genomic Prediction in Animals and Plants: Simulation of Data, Validation, Reporting, and Benchmarking

PubMed Central

Daetwyler, Hans D.; Calus, Mario P. L.; Pong-Wong, Ricardo; de los Campos, Gustavo; Hickey, John M.

2013-01-01

The genomic prediction of phenotypes and breeding values in animals and plants has developed rapidly into its own research field. Results of genomic prediction studies are often difficult to compare because data simulation varies, real or simulated data are not fully described, and not all relevant results are reported. In addition, some new methods have been compared only in limited genetic architectures, leading to potentially misleading conclusions. In this article we review simulation procedures, discuss validation and reporting of results, and apply benchmark procedures for a variety of genomic prediction methods in simulated and real example data. Plant and animal breeding programs are being transformed by the use of genomic data, which are becoming widely available and cost-effective to predict genetic merit. A large number of genomic prediction studies have been published using both simulated and real data. The relative novelty of this area of research has made the development of scientific conventions difficult with regard to description of the real data, simulation of genomes, validation and reporting of results, and forward in time methods. In this review article we discuss the generation of simulated genotype and phenotype data, using approaches such as the coalescent and forward in time simulation. We outline ways to validate simulated data and genomic prediction results, including cross-validation. The accuracy and bias of genomic prediction are highlighted as performance indicators that should be reported. We suggest that a measure of relatedness between the reference and validation individuals be reported, as its impact on the accuracy of genomic prediction is substantial. A large number of methods were compared in example simulated and real (pine and wheat) data sets, all of which are publicly available. In our limited simulations, most methods performed similarly in traits with a large number of quantitative trait loci (QTL), whereas in traits with fewer QTL variable selection did have some advantages. In the real data sets examined here all methods had very similar accuracies. We conclude that no single method can serve as a benchmark for genomic prediction. We recommend comparing accuracy and bias of new methods to results from genomic best linear prediction and a variable selection approach (e.g., BayesB), because, together, these methods are appropriate for a range of genetic architectures. An accompanying article in this issue provides a comprehensive review of genomic prediction methods and discusses a selection of topics related to application of genomic prediction in plants and animals. PMID:23222650
Structural and Functional Phenotyping of the Failing Heart: Is the Left Ventricular Ejection Fraction Obsolete?

PubMed

Bristow, Michael R; Kao, David P; Breathett, Khadijah K; Altman, Natasha L; Gorcsan, John; Gill, Edward A; Lowes, Brian D; Gilbert, Edward M; Quaife, Robert A; Mann, Douglas L

2017-11-01

Diagnosis, prognosis, treatment, and development of new therapies for diseases or syndromes depend on a reliable means of identifying phenotypes associated with distinct predictive probabilities for these various objectives. Left ventricular ejection fraction (LVEF) provides the current basis for combined functional and structural phenotyping in heart failure by classifying patients as those with heart failure with reduced ejection fraction (HFrEF) and those with heart failure with preserved ejection fraction (HFpEF). Recently the utility of LVEF as the major phenotypic determinant of heart failure has been challenged based on its load dependency and measurement variability. We review the history of the development and adoption of LVEF as a critical measurement of LV function and structure and demonstrate that, in chronic heart failure, load dependency is not an important practical issue, and we provide hemodynamic and molecular biomarker evidence that LVEF is superior or equal to more unwieldy methods of identifying phenotypes of ventricular remodeling. We conclude that, because it reliably measures both left ventricular function and structure, LVEF remains the best current method of assessing pathologic remodeling in heart failure in both individual clinical and multicenter group settings. Because of the present and future importance of left ventricular phenotyping in heart failure, LVEF should be measured by using the most accurate technology and methodologic refinements available, and improved characterization methods should continue to be sought. Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.
A European multi-centre External Quality Assessment (EQA) study on phenotypic and genotypic methods used for Herpes Simplex Virus (HSV) drug resistance testing.

PubMed

Afshar, Baharak; Bibby, David F; Piorkowska, Renata; Ohemeng-Kumi, Natasha; Snoeck, Robert; Andrei, Graciela; Gillemot, Sarah; Morfin, Florence; Frobert, Emilie; Burrel, Sonia; Boutolleau, David; Crowley, Brendan; Mbisa, Jean L

2017-11-01

Herpes Simplex Virus (HSV) drug resistance is a significant public health concern among immunocompromised individuals. Phenotypic assays are considered the gold standard method for detecting HSV drug resistance. However, plaque reduction assays (PRAs) are technically demanding, often with long turnaround times of up to four weeks. In contrast, genotypic tests can be performed within a few days. The development and coordination of the first European External Quality Assessment (EQA) study to evaluate phenotypic and genotypic methods used for HSV drug resistance testing in specialised reference laboratories. Four HSV-1 or HSV-2 strains with different antiviral susceptibility profiles were isolated from clinical samples. Isolates were quantified by qPCR, and aliquoted in culture medium. One isolate was distributed at two dilutions to help assess assay sensitivity. The panel was distributed to five European centres with a six-week deadline for the return of phenotypic and genotypic results, together with clinical reports. Four out of five participating labs returned results by the deadline. Limited results were later available from the fifth lab. Phenotypic and genotypic data were largely, but not completely, concordant. An unusual resistance profile shown by one of the samples was explained by the detection of a mixed virus population after extensive further investigation by one of the centres. Discordant clinical outputs reflecting the diversity of phenotypic methodologies demonstrated the utility of this exercise. With emerging genotypic technologies looking to supplant phenotyping, there is a need for curated public databases, accessible interpretation tools and standardised control materials for quality management. By establishing a network of testing laboratories, we hope that this EQA scheme will facilitate ongoing progress in this area. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.
Rhabdoid and Undifferentiated Phenotype in Renal Cell Carcinoma: Analysis of 32 Cases Indicating a Distinctive Common Pathway of Dedifferentiation Frequently Associated With SWI/SNF Complex Deficiency.

PubMed

Agaimy, Abbas; Cheng, Liang; Egevad, Lars; Feyerabend, Bernd; Hes, Ondřej; Keck, Bastian; Pizzolitto, Stefano; Sioletic, Stefano; Wullich, Bernd; Hartmann, Arndt

2017-02-01

Undifferentiated (anaplastic) and rhabdoid cell features are increasingly recognized as adverse prognostic findings in renal cell carcinoma (RCC), but their molecular pathogenesis has not been studied sufficiently. Recent studies identified alterations in the Switch Sucrose nonfermentable (SWI/SNF) chromatin remodeling complex as molecular mechanisms underlying dedifferentiation and rhabdoid features in carcinomas of different organs. We herein have analyzed 32 undifferentiated RCCs having in common an undifferentiated (anaplastic) phenotype, prominent rhabdoid features, or both, irrespective of the presence or absence of conventional RCC component. Cases were stained with 6 SWI/SNF pathway members (SMARCB1, SMARCA2, SMARCA4, ARID1A, SMARCC1, and SMARCC2) in addition to conventional RCC markers. Patients were 20 males and 12 females aged 32 to 85 years (mean, 59). A total of 22/27 patients with known stage presented with ≥pT3. A differentiated component varying from microscopic to major component was detected in 20/32 cases (16 clear cell and 2 cases each chromophobe and papillary RCC). The undifferentiated component varied from rhabdoid dyscohesive cells to large epithelioid to small monotonous anaplastic cells. Variable loss of at least 1 SWI/SNF complex subunit was noted in the undifferentiated/rhabdoid component of 21/32 cases (65%) compared with intact or reduced expression in the differentiated component. A total of 15/17 patients (88%) with follow-up died of metastatic disease (mostly within 1 y). Only 2 patients were disease free at last follow-up (1 and 6 y). No difference in survival, age distribution, or sex was observed between the SWI/SNF-deficient and the SWI/SNF-intact group. This is the first study exploring the role of SWI/SNF deficiency as a potential mechanism underlying undifferentiated and rhabdoid phenotype in RCC. Our results highlight the association between the aggressive rhabdoid phenotype and the SWI/SNF complex deficiency, consistent with studies on similar neoplasms in other organs. Thorough sampling of such tumors that are usually huge and locally advanced is necessary for recognizing the clone of origin and hence for proper subtyping and also for differentiating them from undifferentiated urothelial carcinoma.
HCS-Neurons: identifying phenotypic changes in multi-neuron images upon drug treatments of high-content screening.

PubMed

Charoenkwan, Phasit; Hwang, Eric; Cutler, Robert W; Lee, Hua-Chin; Ko, Li-Wei; Huang, Hui-Ling; Ho, Shinn-Ying

2013-01-01

High-content screening (HCS) has become a powerful tool for drug discovery. However, the discovery of drugs targeting neurons is still hampered by the inability to accurately identify and quantify the phenotypic changes of multiple neurons in a single image (named multi-neuron image) of a high-content screen. Therefore, it is desirable to develop an automated image analysis method for analyzing multi-neuron images. We propose an automated analysis method with novel descriptors of neuromorphology features for analyzing HCS-based multi-neuron images, called HCS-neurons. To observe multiple phenotypic changes of neurons, we propose two kinds of descriptors which are neuron feature descriptor (NFD) of 13 neuromorphology features, e.g., neurite length, and generic feature descriptors (GFDs), e.g., Haralick texture. HCS-neurons can 1) automatically extract all quantitative phenotype features in both NFD and GFDs, 2) identify statistically significant phenotypic changes upon drug treatments using ANOVA and regression analysis, and 3) generate an accurate classifier to group neurons treated by different drug concentrations using support vector machine and an intelligent feature selection method. To evaluate HCS-neurons, we treated P19 neurons with nocodazole (a microtubule depolymerizing drug which has been shown to impair neurite development) at six concentrations ranging from 0 to 1000 ng/mL. The experimental results show that all the 13 features of NFD have statistically significant difference with respect to changes in various levels of nocodazole drug concentrations (NDC) and the phenotypic changes of neurites were consistent to the known effect of nocodazole in promoting neurite retraction. Three identified features, total neurite length, average neurite length, and average neurite area were able to achieve an independent test accuracy of 90.28% for the six-dosage classification problem. This NFD module and neuron image datasets are provided as a freely downloadable MatLab project at http://iclab.life.nctu.edu.tw/HCS-Neurons. Few automatic methods focus on analyzing multi-neuron images collected from HCS used in drug discovery. We provided an automatic HCS-based method for generating accurate classifiers to classify neurons based on their phenotypic changes upon drug treatments. The proposed HCS-neurons method is helpful in identifying and classifying chemical or biological molecules that alter the morphology of a group of neurons in HCS.
Tumour-cytolytic human monocyte-derived macrophages: a simple and efficient method for the generation and long-term cultivation as non-adherent cells in a serum-free medium.

PubMed

Streck, R J; Hurley, E L; Epstein, D A; Pauly, J L

1992-01-01

We report a simple and efficient culture procedure for the generation of tumour-cytolytic human monocyte-derived macrophages (MAC). In this method, normal human peripheral blood mononuclear cells, isolated using a conventional Ficoll-Hypaque density gradient procedure, are cultured as a heterogenous leukocyte population in Teflon or other hydrophobic cultureware, in a commercially available serum-free culture medium (M-SFM) that has been formulated specifically for the cultivation and ex vivo stimulation of human monocytes and MAC, and in the absence of exogenous mitogens, antigens, cytokines or other stimulants. This procedure features a negative-selection technique that takes advantage of the differential survival of blood leukocytes. Using the prescribed in vitro conditions, lymphocytes survived relatively poorly, whereas monocytes differentiated in the absence of exogenous stimulants into mature tumour-cytolytic MAC. The MAC were present as non-adherent, single cells that expressed good viability (greater than 95%) for a prolonged period (greater than 60 days). When compared to conventional procedures for generating MAC, the prescribed technique is thought to offer several important advantages in that it: (a) eliminates the tedious and cumbersome monocyte isolation procedures, thus providing a significant savings not only in time and money but also in eliminating repetitive cell manipulations that have often been associated with damage to monocyte morphology and/or function; (b) reduces the loss of monocyte subsets that are not recovered during specific isolation procedures; (c) facilitates harvesting a single cell, non-adherent suspension of immunocompetent MAC suitable for various examinations including analyses defining MAC morphology, cytochemistry, phenotype and function; and (d) eliminates variability and artifacts associated with different sera that are utilised frequently as medium supplements. The utility of the prescribed method is illustrated by the results of ongoing studies in which scanning electron microscopy and confocal laser scanning microscopy are being used to define MAC function in different immunological reactions, and examples of these observations are presented herein.
Genomic landscapes of endogenous retroviruses unveil intricate genetics of conventional and genetically-engineered laboratory mouse strains.

PubMed

Lee, Kang-Hoon; Lim, Debora; Chiu, Sophia; Greenhalgh, David; Cho, Kiho

2016-04-01

Laboratory strains of mice, both conventional and genetically engineered, have been introduced as critical components of a broad range of studies investigating normal and disease biology. Currently, the genetic identity of laboratory mice is primarily confirmed by surveying polymorphisms in selected sets of "conventional" genes and/or microsatellites in the absence of a single completely sequenced mouse genome. First, we examined variations in the genomic landscapes of transposable repetitive elements, named the TREome, in conventional and genetically engineered mouse strains using murine leukemia virus-type endogenous retroviruses (MLV-ERVs) as a probe. A survey of the genomes from 56 conventional strains revealed strain-specific TREome landscapes, and certain families (e.g., C57BL) of strains were discernible with defined patterns. Interestingly, the TREome landscapes of C3H/HeJ (toll-like receptor-4 [TLR4] mutant) inbred mice were different from its control C3H/HeOuJ (TLR4 wild-type) strain. In addition, a CD14 knock-out strain had a distinct TREome landscape compared to its control/backcross C57BL/6J strain. Second, an examination of superantigen (SAg, a "TREome gene") coding sequences of mouse mammary tumor virus-type ERVs in the genomes of the 46 conventional strains revealed a high diversity, suggesting a potential role of SAgs in strain-specific immune phenotypes. The findings from this study indicate that unexplored and intricate genomic variations exist in laboratory mouse strains, both conventional and genetically engineered. The TREome-based high-resolution genetics surveillance system for laboratory mice would contribute to efficient study design with quality control and accurate data interpretation. This genetics system can be easily adapted to other species ranging from plants to humans. Copyright © 2016 Elsevier Inc. All rights reserved.

SU-F-R-52: A Comparison of the Performance of Radiomic Features From Free Breathing and 4DCT Scans in Predicting Disease Recurrence in Lung Cancer SBRT Patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huynh, E; Coroller, T; Narayan, V

Purpose: There is a clinical need to identify patients who are at highest risk of recurrence after being treated with stereotactic body radiation therapy (SBRT). Radiomics offers a non-invasive approach by extracting quantitative features from medical images based on tumor phenotype that is predictive of an outcome. Lung cancer patients treated with SBRT routinely undergo free breathing (FB image) and 4DCT (average intensity projection (AIP) image) scans for treatment planning to account for organ motion. The aim of the current study is to evaluate and compare the prognostic performance of radiomic features extracted from FB and AIP images in lungmore » cancer patients treated with SBRT to identify which image type would generate an optimal predictive model for recurrence. Methods: FB and AIP images of 113 Stage I-II NSCLC patients treated with SBRT were analysed. The prognostic performance of radiomic features for distant metastasis (DM) was evaluated by their concordance index (CI). Radiomic features were compared with conventional imaging metrics (e.g. diameter). All p-values were corrected for multiple testing using the false discovery rate. Results: All patients received SBRT and 20.4% of patients developed DM. From each image type (FB or AIP), nineteen radiomic features were selected based on stability and variance. Both image types had five common and fourteen different radiomic features. One FB (CI=0.70) and five AIP (CI range=0.65–0.68) radiomic features were significantly prognostic for DM (p<0.05). None of the conventional features derived from FB images (range CI=0.60–0.61) were significant but all AIP conventional features were (range CI=0.64–0.66). Conclusion: Features extracted from different types of CT scans have varying prognostic performances. AIP images contain more prognostic radiomic features for DM than FB images. These methods can provide personalized medicine approaches at low cost, as FB and AIP data are readily available within a large number of radiation oncology departments. R.M. had consulting interest with Amgen (ended in 2015).« less
Mapping Phenotypic Information in Heterogeneous Textual Sources to a Domain-Specific Terminological Resource

PubMed Central

Ananiadou, Sophia

2016-01-01

Biomedical literature articles and narrative content from Electronic Health Records (EHRs) both constitute rich sources of disease-phenotype information. Phenotype concepts may be mentioned in text in multiple ways, using phrases with a variety of structures. This variability stems partly from the different backgrounds of the authors, but also from the different writing styles typically used in each text type. Since EHR narrative reports and literature articles contain different but complementary types of valuable information, combining details from each text type can help to uncover new disease-phenotype associations. However, the alternative ways in which the same concept may be mentioned in each source constitutes a barrier to the automatic integration of information. Accordingly, identification of the unique concepts represented by phrases in text can help to bridge the gap between text types. We describe our development of a novel method, PhenoNorm, which integrates a number of different similarity measures to allow automatic linking of phenotype concept mentions to known concepts in the UMLS Metathesaurus, a biomedical terminological resource. PhenoNorm was developed using the PhenoCHF corpus—a collection of literature articles and narratives in EHRs, annotated for phenotypic information relating to congestive heart failure (CHF). We evaluate the performance of PhenoNorm in linking CHF-related phenotype mentions to Metathesaurus concepts, using a newly enriched version of PhenoCHF, in which each phenotype mention has an expert-verified link to a concept in the UMLS Metathesaurus. We show that PhenoNorm outperforms a number of alternative methods applied to the same task. Furthermore, we demonstrate PhenoNorm’s wider utility, by evaluating its ability to link mentions of various other types of medically-related information, occurring in texts covering wider subject areas, to concepts in different terminological resources. We show that PhenoNorm can maintain performance levels, and that its accuracy compares favourably to other methods applied to these tasks. PMID:27643689
Hierarchical compression of Caenorhabditis elegans locomotion reveals phenotypic differences in the organization of behaviour.

PubMed

Gomez-Marin, Alex; Stephens, Greg J; Brown, André E X

2016-08-01

Regularities in animal behaviour offer insights into the underlying organizational and functional principles of nervous systems and automated tracking provides the opportunity to extract features of behaviour directly from large-scale video data. Yet how to effectively analyse such behavioural data remains an open question. Here, we explore whether a minimum description length principle can be exploited to identify meaningful behaviours and phenotypes. We apply a dictionary compression algorithm to behavioural sequences from the nematode worm Caenorhabditis elegans freely crawling on an agar plate both with and without food and during chemotaxis. We find that the motifs identified by the compression algorithm are rare but relevant for comparisons between worms in different environments, suggesting that hierarchical compression can be a useful step in behaviour analysis. We also use compressibility as a new quantitative phenotype and find that the behaviour of wild-isolated strains of C. elegans is more compressible than that of the laboratory strain N2 as well as the majority of mutant strains examined. Importantly, in distinction to more conventional phenotypes such as overall motor activity or aggregation behaviour, the increased compressibility of wild isolates is not explained by the loss of function of the gene npr-1, which suggests that erratic locomotion is a laboratory-derived trait with a novel genetic basis. Because hierarchical compression can be applied to any sequence, we anticipate that compressibility can offer insights into the organization of behaviour in other animals including humans. © 2016 The Authors.
Microbiota-induced obesity requires farnesoid X receptor

PubMed Central

Parséus, Ava; Sommer, Nina; Sommer, Felix; Caesar, Robert; Molinaro, Antonio; Ståhlman, Marcus; Greiner, Thomas U; Perkins, Rosie; Bäckhed, Fredrik

2017-01-01

Objective The gut microbiota has been implicated as an environmental factor that modulates obesity, and recent evidence suggests that microbiota-mediated changes in bile acid profiles and signalling through the bile acid nuclear receptor farnesoid X receptor (FXR) contribute to impaired host metabolism. Here we investigated if the gut microbiota modulates obesity and associated phenotypes through FXR. Design We fed germ-free (GF) and conventionally raised (CONV-R) wild-type and Fxr−/− mice a high-fat diet (HFD) for 10 weeks. We monitored weight gain and glucose metabolism and analysed the gut microbiota and bile acid composition, beta-cell mass, accumulation of macrophages in adipose tissue, liver steatosis, and expression of target genes in adipose tissue and liver. We also transferred the microbiota of wild-type and Fxr-deficient mice to GF wild-type mice. Results The gut microbiota promoted weight gain and hepatic steatosis in an FXR-dependent manner, and the bile acid profiles and composition of faecal microbiota differed between Fxr−/− and wild-type mice. The obese phenotype in colonised wild-type mice was associated with increased beta-cell mass, increased adipose inflammation, increased steatosis and expression of genes involved in lipid uptake. By transferring the caecal microbiota from HFD-fed Fxr−/− and wild-type mice into GF mice, we showed that the obesity phenotype was transferable. Conclusions Our results indicate that the gut microbiota promotes diet-induced obesity and associated phenotypes through FXR, and that FXR may contribute to increased adiposity by altering the microbiota composition. PMID:26740296
Effect of chorioamnionitis on regulatory T cells in moderate/late preterm neonates.

PubMed

Rueda, Cesar M; Wells, Casey B; Gisslen, Tate; Jobe, Alan H; Kallapur, Suhas G; Chougnet, Claire A

2015-01-01

Regulatory T-cells (Treg) have a protective role for the control of immune activation and tissue damage. The effects of chorioamnionitis (chorio) on Treg in moderate/late preterm newborns are not known. We hypothesized that infants exposed to chorio would have decreased Treg frequency and/or function. We isolated mononuclear cells from adult peripheral blood and cord blood from term and moderate/late preterm infants who were classified for severity of chorio exposure. Mononuclear cells were analyzed by flow cytometry for Treg frequency and phenotype. Treg suppression of activation of conventional T-cells (Tcon) was also quantified. Treg frequencies were similar in all groups of neonates, but lower than that found in adults. Newborn Treg had a naïve phenotype, with decreased levels of CD45RO, HLA-DR, CD39 and TIGIT compared to adult Treg and chorio did not affect the phenotype. Treg from preterm newborns exposed to severe chorio had higher expression of Ki67 compared to the other groups. Treg from preterm newborns were less suppressive than Treg from adults or term, and the level of suppression was reduced with severe chorio. Relative to term, Treg frequency and phenotype were not affected by prematurity and chorio but their functionality was decreased. Lower Treg activity may contribute to inflammation in newborns that is often associated with chorioamnionitis. Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
Pathway-Based Factor Analysis of Gene Expression Data Produces Highly Heritable Phenotypes That Associate with Age

PubMed Central

Anand Brown, Andrew; Ding, Zhihao; Viñuela, Ana; Glass, Dan; Parts, Leopold; Spector, Tim; Winn, John; Durbin, Richard

2015-01-01

Statistical factor analysis methods have previously been used to remove noise components from high-dimensional data prior to genetic association mapping and, in a guided fashion, to summarize biologically relevant sources of variation. Here, we show how the derived factors summarizing pathway expression can be used to analyze the relationships between expression, heritability, and aging. We used skin gene expression data from 647 twins from the MuTHER Consortium and applied factor analysis to concisely summarize patterns of gene expression to remove broad confounding influences and to produce concise pathway-level phenotypes. We derived 930 “pathway phenotypes” that summarized patterns of variation across 186 KEGG pathways (five phenotypes per pathway). We identified 69 significant associations of age with phenotype from 57 distinct KEGG pathways at a stringent Bonferroni threshold (P<5.38×10−5). These phenotypes are more heritable (h2=0.32) than gene expression levels. On average, expression levels of 16% of genes within these pathways are associated with age. Several significant pathways relate to metabolizing sugars and fatty acids; others relate to insulin signaling. We have demonstrated that factor analysis methods combined with biological knowledge can produce more reliable phenotypes with less stochastic noise than the individual gene expression levels, which increases our power to discover biologically relevant associations. These phenotypes could also be applied to discover associations with other environmental factors. PMID:25758824
Analysis of mammalian gene function through broad based phenotypic screens across a consortium of mouse clinics

PubMed Central

Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl MJ; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie

2015-01-01

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems. PMID:26214591
Grape colour phenotyping: development of a method based on the reflectance spectrum.

PubMed

Rustioni, Laura; Basilico, Roberto; Fiori, Simone; Leoni, Alessandra; Maghradze, David; Failla, Osvaldo

2013-01-01

The colour of fruit is an important quality factor for cultivar classification and phenotyping techniques. Besides the subjective visual evaluation, new instruments and techniques can be used. This work aims at developping an objective, fast, easy and non-destructive method as a useful support for evaluating grapes' colour under different cultural and environmental conditions, as well as for breeding process and germplasm evaluation, supporting the plant characterization and the biodiversity preservation. Colours of 120 grape varieties were studied using reflectance spectra. The classification was realized using cluster and discriminant analysis. Reflectance of the whole berries surface was also compared with absorption properties of single skin extracts. A phenotyping method based on the reflectance spectra was developed, producing reliable colour classifications. A cultivar-independent index for pigment content evaluation has also been obtained. This work allowed the classification of the berry colour using an objective method. Copyright © 2013 John Wiley & Sons, Ltd.
A comparison of the clinical effectiveness of spinal orthoses manufactured using the conventional manual method and CAD/CAM method in the management of AIS.

PubMed

Wong, M S; Cheng, C Y; Ng, B K W; Lam, T P; Chiu, S W

2006-01-01

Spinal orthoses are commonly prescribed to patients with moderate AIS for prevention of further deterioration. In a conventional manufacturing method, plaster bandages are used to get patient's body contour and plaster cast is rectified manually. With the introduction of CAD/CAM system, a series of automated processes from body scanning to digital rectification and milling of positive model can be performed in a fast and accurate fashion. This project is to study the impact of CAD/CAM method as compared with the conventional method. In assessing the 147 recruited subjects fitted with spinal orthoses (43 subjects using conventional method and 104 subjects using CAD/CAM method), significant decreases (p<0.05) were found in the Cobb angles when comparing the pre-intervention data with that of the first year of intervention. Regarding the learning curve, Orthotists are getting more competent with the CAD/CAM technique in four years time. The mean productivity of the CAD/CAM method is 2.75 times higher than that of the conventional method. The CAD/CAM method could achieve similar clinical outcomes and with its high efficiency, could be considered as substitute for conventional methods in fabricating spinal orthoses for patients with AIS.
Tissue Doppler imaging for detection of radial and longitudinal myocardial dysfunction in a family of cats affected by dystrophin-deficient hypertrophic muscular dystrophy.

PubMed

Chetboul, Valérie; Blot, Stephane; Sampedrano, Carolina Carlos; Thibaud, Jean-Laurent; Granger, Nicolas; Tissier, Renaud; Bruneval, Patrick; Gaschen, Frederic; Gouni, Vassiliki; Nicolle, Audrey P; Pouchelon, Jean-Louis

2006-01-01

Diagnosis of feline hypertrophic cardiomyopathy currently is based on the presence of myocardial hypertrophy detected using conventional echocardiography. The accuracy of tissue Doppler imaging (TDI) for earlier detection of the disease has never been described. The objective of this sudy was to quantify left ventricular free wall (LVFW) velocities in cats with hypertrophic muscular dystrophy (HFMD) during preclinical cardiomyopathy using TDI. The study animals included 22 healthy controls and 7 cats belonging to a family of cats with HFMD (2 affected adult males, 2 heterozygous adult females, one 2.5-month-old affected male kitten, and 2 phenotypically normal female kittens from the same litter). All cats were examined via conventional echocardiography and 2-dimensional color TDI. No LVFW hypertrophy was detected in the 2 carriers or in the affected kitten when using conventional echocardiography and histologic examination, respectively. The LVFW also was normal for 1 affected male and at the upper limit of normal for the 2nd male. Conversely, LVFW dysfunction was detected in all affected and carrier cats with HFMD when using TDI. TDI consistently detects LVFW dysfunction in cats with HFMD despite the absence of myocardial hypertrophy. Therefore, TDI appears more sensitive than conventional echocardiography in detecting regional myocardial abnormalities.
Optimizing complex phenotypes through model-guided multiplex genome engineering

DOE PAGES

Kuznetsov, Gleb; Goodman, Daniel B.; Filsinger, Gabriel T.; ...

2017-05-25

Here, we present a method for identifying genomic modifications that optimize a complex phenotype through multiplex genome engineering and predictive modeling. We apply our method to identify six single nucleotide mutations that recover 59% of the fitness defect exhibited by the 63-codon E. coli strain C321.ΔA. By introducing targeted combinations of changes in multiplex we generate rich genotypic and phenotypic diversity and characterize clones using whole-genome sequencing and doubling time measurements. Regularized multivariate linear regression accurately quantifies individual allelic effects and overcomes bias from hitchhiking mutations and context-dependence of genome editing efficiency that would confound other strategies.
Optimizing complex phenotypes through model-guided multiplex genome engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuznetsov, Gleb; Goodman, Daniel B.; Filsinger, Gabriel T.

Here, we present a method for identifying genomic modifications that optimize a complex phenotype through multiplex genome engineering and predictive modeling. We apply our method to identify six single nucleotide mutations that recover 59% of the fitness defect exhibited by the 63-codon E. coli strain C321.ΔA. By introducing targeted combinations of changes in multiplex we generate rich genotypic and phenotypic diversity and characterize clones using whole-genome sequencing and doubling time measurements. Regularized multivariate linear regression accurately quantifies individual allelic effects and overcomes bias from hitchhiking mutations and context-dependence of genome editing efficiency that would confound other strategies.
FTIR spectroscopy in medical mycology: applications to the differentiation and typing of Candida.

PubMed

Toubas, Dominique; Essendoubi, Mohammed; Adt, Isabelle; Pinon, Jean-Michel; Manfait, Michel; Sockalingum, Ganesh D

2007-03-01

The incidence of fungal infections, in particular candidiasis and aspergillosis, has considerably increased during the last three decades. This is mainly due to advances in medical treatments and technologies. In high risk patients (e.g. in haematology or intensive care), the prognosis of invasive candidiasis is relatively poor. Therefore, a rapid and correct identification of the infectious agent is important for an efficient and prompt therapy. Most clinical laboratories rely on conventional identification methods that are based on morphological, physiological and nutritional characteristics. However, these have their limitations because they are time-consuming and not always very accurate. Moreover, molecular methods may be required to determine the genetic relationship between the infectious strains, for instance in Candida outbreaks. In addition, the latter methods require time, expensive consumables and highly trained staff to be performed adequately. In this study, we have applied the FTIR spectroscopic approach to different situations encountered in routine mycological diagnosis. We show the potentials of this phenotypic approach, used in parallel with routine identification methods, for the differentiation of 3 frequently encountered Candida species (C. albicans, C. glabrata and C. krusei) by using both suspensions and microcolonies. This approach, developed for an early discrimination, may help in the initial choice of antifungal treatment. Furthermore, we demonstrate the feasibility of the method for intraspecies comparison (typing) of 3 Candida species (C. albicans, C. glabrata and C. parapsilosis), particularly when an outbreak is suspected.
Comparison of methods for identifying causative bacterial microorganisms in presumed acute endophthalmitis: conventional culture, blood culture, and PCR.

PubMed

Pongsachareonnont, Pear; Honglertnapakul, Worawalun; Chatsuwan, Tanittha

2017-02-21

Identification of bacterial pathogens in endophthalmitis is important to inform antibiotic selection and treatment decisions. Hemoculture bottles and polymerase chain reaction (PCR) analysis have been proposed to offer good detection sensitivity. This study compared the sensitivity and accuracy of a blood culture system, a PCR approach, and conventional culture methods for identification of causative bacteria in cases of acute endophthalmitis. Twenty-nine patients with a diagnosis of presumed acute bacterial endophthalmitis who underwent vitreous specimen collection at King Chulalongkorn Memorial Hospital were enrolled in this study. Forty-one specimens were collected. Each specimen was divided into three parts, and each part was analyzed using one of three microbial identification techniques: conventional plate culture, blood culture, and polymerase chain reaction and sequencing. The results of the three methods were then compared. Bacteria were identified in 15 of the 41 specimens (36.5%). Five (12.2%) specimens were positive by conventional culture methods, 11 (26.8%) were positive by hemoculture, and 11 (26.8%) were positive by PCR. Cohen's kappa analysis revealed p-values for conventional methods vs. hemoculture, conventional methods vs. PCR, and hemoculture vs. PCR of 0.057, 0.33, and 0.009, respectively. Higher detection rates of Enterococcus faecalis were observed for hemoculture and PCR than for conventional methods. Blood culture bottles and PCR detection may facilitate bacterial identification in cases of presumed acute endophthalmitis. These techniques should be used in addition to conventional plate culture methods because they provide a greater degree of sensitivity than conventional plate culture alone for the detection of specific microorganisms such as E. faecalis. Thai Clinical Trial Register No. TCTR20110000024 .
A Comparison of Programed Instruction with Conventional Methods for Teaching Two Units of Eighth Grade Science.

ERIC Educational Resources Information Center

Eshleman, Winston Hull

Compared were programed materials and conventional methods for teaching two units of eighth grade science. Programed materials used were linear programed books requiring constructed responses. The conventional methods included textbook study, written exercises, lectures, discussions, demonstrations, experiments, chalkboard drawings, films,…
Comparative Evaluation of Multiplex PCR and Routine Laboratory Phenotypic Methods for Detection of Carbapenemases among Gram Negative Bacilli.

PubMed

Solanki, Rachana; Vanjari, Lavanya; Subramanian, Sreevidya; B, Aparna; E, Nagapriyanka; Lakshmi, Vemu

2014-12-01

Carbapenem resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of Multiplex PCR for rapid detection of carbapenemase genes among carbapenem resistant Gram negative bacteria in comparison with the existing phenotypic methods like modified Hodge test (MHT), combined disc test (CDT) and automated methods. A total of 100 Carbapenem resistant clinical isolates, [Escherichia coli (25), Klebsiella pneumoniae (35) P. aeruginosa (18) and Acinetobacter baumannii (22)] were screened for the presence of carbapenemases (bla NDM-1, bla VIM , blaIMP and blaKPC genes) by phenotype methods such as the modified Hodge test (MHT) and combined disc test (CDT) and the molecular methods such as Multiplex PCR. Seventy of the 100 isolates were MHT positive while, 65 isolates were positive by CDT. All the CDT positive isolates with EDTA and APB were Metallo betalactamase (MBL) and K. pneumoniae carbapenemase (KPC) producers respectively. bla NDM-1 was present as a lone gene in 44 isolates. In 14 isolates bla NDM-1 gene was present with blaKPC gene, and in one isolate bla NDM-1 gene was present with blaVIM , gene. Only one E. coli isolate had a lone blaKPC gene. We didn't find bla IMP gene in any of the isolates. Neither of the genes could be detected in 35 isolates. Accurate detection of the genes related with carbapenemase production by Molecular methods like Multiplex PCR overcome the limitations of the phenotypic methods and Automated systems.
Capitalizing on fine milk composition for breeding and management of dairy cows.

PubMed

Gengler, N; Soyeurt, H; Dehareng, F; Bastin, C; Colinet, F; Hammami, H; Vanrobays, M-L; Lainé, A; Vanderick, S; Grelet, C; Vanlierde, A; Froidmont, E; Dardenne, P

2016-05-01

The challenge of managing and breeding dairy cows is permanently adapting to changing production circumstances under socio-economic constraints. If managing and breeding address different timeframes of action, both need relevant phenotypes that allow for precise monitoring of the status of the cows, and their health, behavior, and well-being as well as their environmental impact and the quality of their products (i.e., milk and subsequently dairy products). Milk composition has been identified as an important source of information because it could reflect, at least partially, all these elements. Major conventional milk components such as fat, protein, urea, and lactose contents are routinely predicted by mid-infrared (MIR) spectrometry and have been widely used for these purposes. But, milk composition is much more complex and other nonconventional milk components, potentially predicted by MIR, might be informative. Such new milk-based phenotypes should be considered given that they are cheap, rapidly obtained, usable on a large scale, robust, and reliable. In a first approach, new phenotypes can be predicted from MIR spectra using techniques based on classical prediction equations. This method was used successfully for many novel traits (e.g., fatty acids, lactoferrin, minerals, milk technological properties, citrate) that can be then useful for management and breeding purposes. An innovation was to consider the longitudinal nature of the relationship between the trait of interest and the MIR spectra (e.g., to predict methane from MIR). By avoiding intermediate steps, prediction errors can be minimized when traits of interest (e.g., methane, energy balance, ketosis) are predicted directly from MIR spectra. In a second approach, research is ongoing to detect and exploit patterns in an innovative manner, by comparing observed with expected MIR spectra directly (e.g., pregnancy). All of these traits can then be used to define best practices, adjust feeding and health management, improve animal welfare, improve milk quality, and mitigate environmental impact. Under the condition that MIR data are available on a large scale, phenotypes for these traits will allow genetic and genomic evaluations. Introduction of novel traits into the breeding objectives will need additional research to clarify socio-economic weights and genetic correlations with other traits of interest. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
Phenotypes in phylogeography: Species’ traits, environmental variation, and vertebrate diversification

PubMed Central

Bell, Rayna C.; Mason, Nicholas A.

2016-01-01

Almost 30 y ago, the field of intraspecific phylogeography laid the foundation for spatially explicit and genealogically informed studies of population divergence. With new methods and markers, the focus in phylogeography shifted to previously unrecognized geographic genetic variation, thus reducing the attention paid to phenotypic variation in those same diverging lineages. Although phenotypic differences among lineages once provided the main data for studies of evolutionary change, the mechanisms shaping phenotypic differentiation and their integration with intraspecific genetic structure have been underexplored in phylogeographic studies. However, phenotypes are targets of selection and play important roles in species performance, recognition, and diversification. Here, we focus on three questions. First, how can phenotypes elucidate mechanisms underlying concordant or idiosyncratic responses of vertebrate species evolving in shared landscapes? Second, what mechanisms underlie the concordance or discordance of phenotypic and phylogeographic differentiation? Third, how can phylogeography contribute to our understanding of functional phenotypic evolution? We demonstrate that the integration of phenotypic data extends the reach of phylogeography to explain the origin and maintenance of biodiversity. Finally, we stress the importance of natural history collections as sources of high-quality phenotypic data that span temporal and spatial axes. PMID:27432983
Robust inference in summary data Mendelian randomization via the zero modal pleiotropy assumption.

PubMed

Hartwig, Fernando Pires; Davey Smith, George; Bowden, Jack

2017-12-01

Mendelian randomization (MR) is being increasingly used to strengthen causal inference in observational studies. Availability of summary data of genetic associations for a variety of phenotypes from large genome-wide association studies (GWAS) allows straightforward application of MR using summary data methods, typically in a two-sample design. In addition to the conventional inverse variance weighting (IVW) method, recently developed summary data MR methods, such as the MR-Egger and weighted median approaches, allow a relaxation of the instrumental variable assumptions. Here, a new method - the mode-based estimate (MBE) - is proposed to obtain a single causal effect estimate from multiple genetic instruments. The MBE is consistent when the largest number of similar (identical in infinite samples) individual-instrument causal effect estimates comes from valid instruments, even if the majority of instruments are invalid. We evaluate the performance of the method in simulations designed to mimic the two-sample summary data setting, and demonstrate its use by investigating the causal effect of plasma lipid fractions and urate levels on coronary heart disease risk. The MBE presented less bias and lower type-I error rates than other methods under the null in many situations. Its power to detect a causal effect was smaller compared with the IVW and weighted median methods, but was larger than that of MR-Egger regression, with sample size requirements typically smaller than those available from GWAS consortia. The MBE relaxes the instrumental variable assumptions, and should be used in combination with other approaches in sensitivity analyses. © The Author 2017. Published by Oxford University Press on behalf of the International Epidemiological Association
Φ-score: A cell-to-cell phenotypic scoring method for sensitive and selective hit discovery in cell-based assays.

PubMed

Guyon, Laurent; Lajaunie, Christian; Fer, Frédéric; Bhajun, Ricky; Sulpice, Eric; Pinna, Guillaume; Campalans, Anna; Radicella, J Pablo; Rouillier, Philippe; Mary, Mélissa; Combe, Stéphanie; Obeid, Patricia; Vert, Jean-Philippe; Gidrol, Xavier

2015-09-18

Phenotypic screening monitors phenotypic changes induced by perturbations, including those generated by drugs or RNA interference. Currently-used methods for scoring screen hits have proven to be problematic, particularly when applied to physiologically relevant conditions such as low cell numbers or inefficient transfection. Here, we describe the Φ-score, which is a novel scoring method for the identification of phenotypic modifiers or hits in cell-based screens. Φ-score performance was assessed with simulations, a validation experiment and its application to gene identification in a large-scale RNAi screen. Using robust statistics and a variance model, we demonstrated that the Φ-score showed better sensitivity, selectivity and reproducibility compared to classical approaches. The improved performance of the Φ-score paves the way for cell-based screening of primary cells, which are often difficult to obtain from patients in sufficient numbers. We also describe a dedicated merging procedure to pool scores from small interfering RNAs targeting the same gene so as to provide improved visualization and hit selection.

Φ-score: A cell-to-cell phenotypic scoring method for sensitive and selective hit discovery in cell-based assays

PubMed Central

Guyon, Laurent; Lajaunie, Christian; fer, Frédéric; bhajun, Ricky; sulpice, Eric; pinna, Guillaume; campalans, Anna; radicella, J. Pablo; rouillier, Philippe; mary, Mélissa; combe, Stéphanie; obeid, Patricia; vert, Jean-Philippe; gidrol, Xavier

2015-01-01

Phenotypic screening monitors phenotypic changes induced by perturbations, including those generated by drugs or RNA interference. Currently-used methods for scoring screen hits have proven to be problematic, particularly when applied to physiologically relevant conditions such as low cell numbers or inefficient transfection. Here, we describe the Φ-score, which is a novel scoring method for the identification of phenotypic modifiers or hits in cell-based screens. Φ-score performance was assessed with simulations, a validation experiment and its application to gene identification in a large-scale RNAi screen. Using robust statistics and a variance model, we demonstrated that the Φ-score showed better sensitivity, selectivity and reproducibility compared to classical approaches. The improved performance of the Φ-score paves the way for cell-based screening of primary cells, which are often difficult to obtain from patients in sufficient numbers. We also describe a dedicated merging procedure to pool scores from small interfering RNAs targeting the same gene so as to provide improved visualization and hit selection. PMID:26382112
Laplacian normalization and random walk on heterogeneous networks for disease-gene prioritization.

PubMed

Zhao, Zhi-Qin; Han, Guo-Sheng; Yu, Zu-Guo; Li, Jinyan

2015-08-01

Random walk on heterogeneous networks is a recently emerging approach to effective disease gene prioritization. Laplacian normalization is a technique capable of normalizing the weight of edges in a network. We use this technique to normalize the gene matrix and the phenotype matrix before the construction of the heterogeneous network, and also use this idea to define the transition matrices of the heterogeneous network. Our method has remarkably better performance than the existing methods for recovering known gene-phenotype relationships. The Shannon information entropy of the distribution of the transition probabilities in our networks is found to be smaller than the networks constructed by the existing methods, implying that a higher number of top-ranked genes can be verified as disease genes. In fact, the most probable gene-phenotype relationships ranked within top 3 or top 5 in our gene lists can be confirmed by the OMIM database for many cases. Our algorithms have shown remarkably superior performance over the state-of-the-art algorithms for recovering gene-phenotype relationships. All Matlab codes can be available upon email request. Copyright © 2015 Elsevier Ltd. All rights reserved.
Family-Based Rare Variant Association Analysis: A Fast and Efficient Method of Multivariate Phenotype Association Analysis.

PubMed

Wang, Longfei; Lee, Sungyoung; Gim, Jungsoo; Qiao, Dandi; Cho, Michael; Elston, Robert C; Silverman, Edwin K; Won, Sungho

2016-09-01

Family-based designs have been repeatedly shown to be powerful in detecting the significant rare variants associated with human diseases. Furthermore, human diseases are often defined by the outcomes of multiple phenotypes, and thus we expect multivariate family-based analyses may be very efficient in detecting associations with rare variants. However, few statistical methods implementing this strategy have been developed for family-based designs. In this report, we describe one such implementation: the multivariate family-based rare variant association tool (mFARVAT). mFARVAT is a quasi-likelihood-based score test for rare variant association analysis with multiple phenotypes, and tests both homogeneous and heterogeneous effects of each variant on multiple phenotypes. Simulation results show that the proposed method is generally robust and efficient for various disease models, and we identify some promising candidate genes associated with chronic obstructive pulmonary disease. The software of mFARVAT is freely available at http://healthstat.snu.ac.kr/software/mfarvat/, implemented in C++ and supported on Linux and MS Windows. © 2016 WILEY PERIODICALS, INC.
An Adaptive Association Test for Multiple Phenotypes with GWAS Summary Statistics.

PubMed

Kim, Junghi; Bai, Yun; Pan, Wei

2015-12-01

We study the problem of testing for single marker-multiple phenotype associations based on genome-wide association study (GWAS) summary statistics without access to individual-level genotype and phenotype data. For most published GWASs, because obtaining summary data is substantially easier than accessing individual-level phenotype and genotype data, while often multiple correlated traits have been collected, the problem studied here has become increasingly important. We propose a powerful adaptive test and compare its performance with some existing tests. We illustrate its applications to analyses of a meta-analyzed GWAS dataset with three blood lipid traits and another with sex-stratified anthropometric traits, and further demonstrate its potential power gain over some existing methods through realistic simulation studies. We start from the situation with only one set of (possibly meta-analyzed) genome-wide summary statistics, then extend the method to meta-analysis of multiple sets of genome-wide summary statistics, each from one GWAS. We expect the proposed test to be useful in practice as more powerful than or complementary to existing methods. © 2015 WILEY PERIODICALS, INC.
From the patient to the clinical mycology laboratory: how can we optimise microscopy and culture methods for mould identification?

PubMed

Vyzantiadis, Timoleon-Achilleas A; Johnson, Elizabeth M; Kibbler, Christopher C

2012-06-01

The identification of fungi relies mainly on morphological criteria. However, there is a need for robust and definitive phenotypic identification procedures in order to evaluate continuously evolving molecular methods. For the future, there is an emerging consensus that a combined (phenotypic and molecular) approach is more powerful for fungal identification, especially for moulds. Most of the procedures used for phenotypic identification are based on experience rather than comparative studies of effectiveness or performance and there is a need for standardisation among mycology laboratories. This review summarises and evaluates the evidence for the major existing phenotypic identification procedures for the predominant causes of opportunistic mould infection. We have concentrated mainly on Aspergillus, Fusarium and mucoraceous mould species, as these are the most important clinically and the ones for which there are the most molecular taxonomic data.
Identification of functional modules that correlate with phenotypic difference: the influence of network topology

PubMed Central

2010-01-01

One of the important challenges to post-genomic biology is relating observed phenotypic alterations to the underlying collective alterations in genes. Current inferential methods, however, invariably omit large bodies of information on the relationships between genes. We present a method that takes account of such information - expressed in terms of the topology of a correlation network - and we apply the method in the context of current procedures for gene set enrichment analysis. PMID:20187943
Rare Variant Association Test with Multiple Phenotypes

PubMed Central

Lee, Selyeong; Won, Sungho; Kim, Young Jin; Kim, Yongkang; Kim, Bong-Jo; Park, Taesung

2016-01-01

Although genome-wide association studies (GWAS) have now discovered thousands of genetic variants associated with common traits, such variants cannot explain the large degree of “missing heritability,” likely due to rare variants. The advent of next generation sequencing technology has allowed rare variant detection and association with common traits, often by investigating specific genomic regions for rare variant effects on a trait. Although multiply correlated phenotypes are often concurrently observed in GWAS, most studies analyze only single phenotypes, which may lessen statistical power. To increase power, multivariate analyses, which consider correlations between multiple phenotypes, can be used. However, few existing multi-variant analyses can identify rare variants for assessing multiple phenotypes. Here, we propose Multivariate Association Analysis using Score Statistics (MAAUSS), to identify rare variants associated with multiple phenotypes, based on the widely used Sequence Kernel Association Test (SKAT) for a single phenotype. We applied MAAUSS to Whole Exome Sequencing (WES) data from a Korean population of 1,058 subjects, to discover genes associated with multiple traits of liver function. We then assessed validation of those genes by a replication study, using an independent dataset of 3,445 individuals. Notably, we detected the gene ZNF620 among five significant genes. We then performed a simulation study to compare MAAUSS's performance with existing methods. Overall, MAAUSS successfully conserved type 1 error rates and in many cases, had a higher power than the existing methods. This study illustrates a feasible and straightforward approach for identifying rare variants correlated with multiple phenotypes, with likely relevance to missing heritability. PMID:28039885
Comparison of performance due to guided hyperlearning, unguided hyperlearning, and conventional learning in mathematics: an empirical study

NASA Astrophysics Data System (ADS)

Fathurrohman, Maman; Porter, Anne; Worthy, Annette L.

2014-07-01

In this paper, the use of guided hyperlearning, unguided hyperlearning, and conventional learning methods in mathematics are compared. The design of the research involved a quasi-experiment with a modified single-factor multiple treatment design comparing the three learning methods, guided hyperlearning, unguided hyperlearning, and conventional learning. The participants were from three first-year university classes, numbering 115 students in total. Each group received guided, unguided, or conventional learning methods in one of the three different topics, namely number systems, functions, and graphing. The students' academic performance differed according to the type of learning. Evaluation of the three methods revealed that only guided hyperlearning and conventional learning were appropriate methods for the psychomotor aspects of drawing in the graphing topic. There was no significant difference between the methods when learning the cognitive aspects involved in the number systems topic and the functions topic.
Ictal SPECT using an attachable automated injector: clinical usefulness in the prediction of ictal onset zone.

PubMed

Lee, Jung-Ju; Lee, Sang Kun; Choi, Jang Wuk; Kim, Dong-Wook; Park, Kyung Il; Kim, Bom Sahn; Kang, Hyejin; Lee, Dong Soo; Lee, Seo-Young; Kim, Sung Hun; Chung, Chun Kee; Nam, Hyeon Woo; Kim, Kwang Ki

2009-12-01

Ictal single-photon emission computed tomography (SPECT) is a valuable method for localizing the ictal onset zone in the presurgical evaluation of patients with intractable epilepsy. Conventional methods used to localize the ictal onset zone have problems with time lag from seizure onset to injection. To evaluate the clinical usefulness of a method that we developed, which involves an attachable automated injector (AAI), in reducing time lag and improving the ability to localize the zone of seizure onset. Patients admitted to the epilepsy monitoring unit (EMU) between January 1, 2003, and June 30, 2008, were included. The definition of ictal onset zone was made by comprehensive review of medical records, magnetic resonance imaging (MRI), data from video electroencephalography (EEG) monitoring, and invasive EEG monitoring if available. We comprehensively evaluated the time lag to injection and the image patterns of ictal SPECT using traditional visual analysis, statistical parametric mapping-assisted, and subtraction ictal SPECT coregistered to an MRI-assisted means of analysis. Image patterns were classified as localizing, lateralizing, and nonlateralizing. The whole number of patients was 99: 48 in the conventional group and 51 in the AAI group. The mean (SD) delay time to injection from seizure onset was 12.4+/-12.0 s in the group injected by our AAI method and 40.4+/-26.3 s in the group injected by the conventional method (P=0.000). The mean delay time to injection from seizure detection was 3.2+/-2.5 s in the group injected by the AAI method and 21.4+/-9.7 s in the group injected by the conventional method (P=0.000). The AAI method was superior to the conventional method in localizing the area of seizure onset (36 out of 51 with AAI method vs. 21 out of 48 with conventional method, P=0.009), especially in non-temporal lobe epilepsy (non-TLE) patients (17 out of 27 with AAI method vs. 3 out of 13 with conventional method, P=0.041), and in lateralizing the seizure onset hemisphere (47 out of 51 with AAI method vs. 33 out of 48 with conventional method, P=0.004). The AAI method was superior to the conventional method in reducing the time lag of tracer injection and in localizing and lateralizing the ictal onset zone, especially in patients with non-TLE.
Scalable parallel elastic-plastic finite element analysis using a quasi-Newton method with a balancing domain decomposition preconditioner

NASA Astrophysics Data System (ADS)

Yusa, Yasunori; Okada, Hiroshi; Yamada, Tomonori; Yoshimura, Shinobu

2018-04-01

A domain decomposition method for large-scale elastic-plastic problems is proposed. The proposed method is based on a quasi-Newton method in conjunction with a balancing domain decomposition preconditioner. The use of a quasi-Newton method overcomes two problems associated with the conventional domain decomposition method based on the Newton-Raphson method: (1) avoidance of a double-loop iteration algorithm, which generally has large computational complexity, and (2) consideration of the local concentration of nonlinear deformation, which is observed in elastic-plastic problems with stress concentration. Moreover, the application of a balancing domain decomposition preconditioner ensures scalability. Using the conventional and proposed domain decomposition methods, several numerical tests, including weak scaling tests, were performed. The convergence performance of the proposed method is comparable to that of the conventional method. In particular, in elastic-plastic analysis, the proposed method exhibits better convergence performance than the conventional method.
Advanced Echocardiography in Adult Zebrafish Reveals Delayed Recovery of Heart Function after Myocardial Cryoinjury

PubMed Central

Kossack, Mandy; Juergensen, Lonny; Fuchs, Dieter; Katus, Hugo A.; Hassel, David

2015-01-01

Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage. PMID:25853735
Capturing Human Naïve Pluripotency in the Embryo and in the Dish.

PubMed

Zimmerlin, Ludovic; Park, Tea Soon; Zambidis, Elias T

2017-08-15

Although human embryonic stem cells (hESCs) were first derived almost 20 years ago, it was only recently acknowledged that they share closer molecular and functional identity to postimplantation lineage-primed murine epiblast stem cells than to naïve preimplantation inner cell mass-derived mouse ESCs (mESCs). A myriad of transcriptional, epigenetic, biochemical, and metabolic attributes have now been described that distinguish naïve and primed pluripotent states in both rodents and humans. Conventional hESCs and human induced pluripotent stem cells (hiPSCs) appear to lack many of the defining hallmarks of naïve mESCs. These include important features of the naïve ground state murine epiblast, such as an open epigenetic architecture, reduced lineage-primed gene expression, and chimera and germline competence following injection into a recipient blastocyst-stage embryo. Several transgenic and chemical methods were recently reported that appear to revert conventional human PSCs to mESC-like ground states. However, it remains unclear if subtle deviations in global transcription, cell signaling dependencies, and extent of epigenetic/metabolic shifts in these various human naïve-reverted pluripotent states represent true functional differences or alternatively the existence of distinct human pluripotent states along a spectrum. In this study, we review the current understanding and developmental features of various human pluripotency-associated phenotypes and discuss potential biological mechanisms that may support stable maintenance of an authentic epiblast-like ground state of human pluripotency.
Dominant Epistasis Between Two Quantitative Trait Loci Governing Sporulation Efficiency in Yeast Saccharomyces cerevisiae

PubMed Central

Bergman, Juraj; Mitrikeski, Petar T.

2015-01-01

Summary Sporulation efficiency in the yeast Saccharomyces cerevisiae is a well-established model for studying quantitative traits. A variety of genes and nucleotides causing different sporulation efficiencies in laboratory, as well as in wild strains, has already been extensively characterised (mainly by reciprocal hemizygosity analysis and nucleotide exchange methods). We applied a different strategy in order to analyze the variation in sporulation efficiency of laboratory yeast strains. Coupling classical quantitative genetic analysis with simulations of phenotypic distributions (a method we call phenotype modelling) enabled us to obtain a detailed picture of the quantitative trait loci (QTLs) relationships underlying the phenotypic variation of this trait. Using this approach, we were able to uncover a dominant epistatic inheritance of loci governing the phenotype. Moreover, a molecular analysis of known causative quantitative trait genes and nucleotides allowed for the detection of novel alleles, potentially responsible for the observed phenotypic variation. Based on the molecular data, we hypothesise that the observed dominant epistatic relationship could be caused by the interaction of multiple quantitative trait nucleotides distributed across a 60--kb QTL region located on chromosome XIV and the RME1 locus on chromosome VII. Furthermore, we propose a model of molecular pathways which possibly underlie the phenotypic variation of this trait. PMID:27904371
Emergent surgical airway: comparison of the three-step method and conventional cricothyroidotomy utilizing high-fidelity simulation.

PubMed

Quick, Jacob A; MacIntyre, Allan D; Barnes, Stephen L

2014-02-01

Surgical airway creation has a high potential for disaster. Conventional methods can be cumbersome and require special instruments. A simple method utilizing three steps and readily available equipment exists, but has yet to be adequately tested. Our objective was to compare conventional cricothyroidotomy with the three-step method utilizing high-fidelity simulation. Utilizing a high-fidelity simulator, 12 experienced flight nurses and paramedics performed both methods after a didactic lecture, simulator briefing, and demonstration of each technique. Six participants performed the three-step method first, and the remaining 6 performed the conventional method first. Each participant was filmed and timed. We analyzed videos with respect to the number of hand repositions, number of airway instrumentations, and technical complications. Times to successful completion were measured from incision to balloon inflation. The three-step method was completed faster (52.1 s vs. 87.3 s; p = 0.007) as compared with conventional surgical cricothyroidotomy. The two methods did not differ statistically regarding number of hand movements (3.75 vs. 5.25; p = 0.12) or instrumentations of the airway (1.08 vs. 1.33; p = 0.07). The three-step method resulted in 100% successful airway placement on the first attempt, compared with 75% of the conventional method (p = 0.11). Technical complications occurred more with the conventional method (33% vs. 0%; p = 0.05). The three-step method, using an elastic bougie with an endotracheal tube, was shown to require fewer total hand movements, took less time to complete, resulted in more successful airway placement, and had fewer complications compared with traditional cricothyroidotomy. Published by Elsevier Inc.
Inferring causal relationships between phenotypes using summary statistics from genome-wide association studies.

PubMed

Meng, Xiang-He; Shen, Hui; Chen, Xiang-Ding; Xiao, Hong-Mei; Deng, Hong-Wen

2018-03-01

Genome-wide association studies (GWAS) have successfully identified numerous genetic variants associated with diverse complex phenotypes and diseases, and provided tremendous opportunities for further analyses using summary association statistics. Recently, Pickrell et al. developed a robust method for causal inference using independent putative causal SNPs. However, this method may fail to infer the causal relationship between two phenotypes when only a limited number of independent putative causal SNPs identified. Here, we extended Pickrell's method to make it more applicable for the general situations. We extended the causal inference method by replacing the putative causal SNPs with the lead SNPs (the set of the most significant SNPs in each independent locus) and tested the performance of our extended method using both simulation and empirical data. Simulations suggested that when the same number of genetic variants is used, our extended method had similar distribution of test statistic under the null model as well as comparable power under the causal model compared with the original method by Pickrell et al. But in practice, our extended method would generally be more powerful because the number of independent lead SNPs was often larger than the number of independent putative causal SNPs. And including more SNPs, on the other hand, would not cause more false positives. By applying our extended method to summary statistics from GWAS for blood metabolites and femoral neck bone mineral density (FN-BMD), we successfully identified ten blood metabolites that may causally influence FN-BMD. We extended a causal inference method for inferring putative causal relationship between two phenotypes using summary statistics from GWAS, and identified a number of potential causal metabolites for FN-BMD, which may provide novel insights into the pathophysiological mechanisms underlying osteoporosis.
Mimvec: a deep learning approach for analyzing the human phenome.

PubMed

Gan, Mingxin; Li, Wenran; Zeng, Wanwen; Wang, Xiaojian; Jiang, Rui

2017-09-21

The human phenome has been widely used with a variety of genomic data sources in the inference of disease genes. However, most existing methods thus far derive phenotype similarity based on the analysis of biomedical databases by using the traditional term frequency-inverse document frequency (TF-IDF) formulation. This framework, though intuitive, not only ignores semantic relationships between words but also tends to produce high-dimensional vectors, and hence lacks the ability to precisely capture intrinsic semantic characteristics of biomedical documents. To overcome these limitations, we propose a framework called mimvec to analyze the human phenome by making use of the state-of-the-art deep learning technique in natural language processing. We converted 24,061 records in the Online Mendelian Inheritance in Man (OMIM) database to low-dimensional vectors using our method. We demonstrated that the vector presentation not only effectively enabled classification of phenotype records against gene ones, but also succeeded in discriminating diseases of different inheritance styles and different mechanisms. We further derived pairwise phenotype similarities between 7988 human inherited diseases using their vector presentations. With a joint analysis of this phenome with multiple genomic data, we showed that phenotype overlap indeed implied genotype overlap. We finally used the derived phenotype similarities with genomic data to prioritize candidate genes and demonstrated advantages of this method over existing ones. Our method is capable of not only capturing semantic relationships between words in biomedical records but also alleviating the dimensional disaster accompanying the traditional TF-IDF framework. With the approaching of precision medicine, there will be abundant electronic records of medicine and health awaiting for deep analysis, and we expect to see a wide spectrum of applications borrowing the idea of our method in the near future.
Quality Control Test for Sequence-Phenotype Assignments

PubMed Central

Ortiz, Maria Teresa Lara; Rosario, Pablo Benjamín Leon; Luna-Nevarez, Pablo; Gamez, Alba Savin; Martínez-del Campo, Ana; Del Rio, Gabriel

2015-01-01

Relating a gene mutation to a phenotype is a common task in different disciplines such as protein biochemistry. In this endeavour, it is common to find false relationships arising from mutations introduced by cells that may be depurated using a phenotypic assay; yet, such phenotypic assays may introduce additional false relationships arising from experimental errors. Here we introduce the use of high-throughput DNA sequencers and statistical analysis aimed to identify incorrect DNA sequence-phenotype assignments and observed that 10–20% of these false assignments are expected in large screenings aimed to identify critical residues for protein function. We further show that this level of incorrect DNA sequence-phenotype assignments may significantly alter our understanding about the structure-function relationship of proteins. We have made available an implementation of our method at http://bis.ifc.unam.mx/en/software/chispas. PMID:25700273
Progeny Clustering: A Method to Identify Biological Phenotypes

PubMed Central

Hu, Chenyue W.; Kornblau, Steven M.; Slater, John H.; Qutub, Amina A.

2015-01-01

Estimating the optimal number of clusters is a major challenge in applying cluster analysis to any type of dataset, especially to biomedical datasets, which are high-dimensional and complex. Here, we introduce an improved method, Progeny Clustering, which is stability-based and exceptionally efficient in computing, to find the ideal number of clusters. The algorithm employs a novel Progeny Sampling method to reconstruct cluster identity, a co-occurrence probability matrix to assess the clustering stability, and a set of reference datasets to overcome inherent biases in the algorithm and data space. Our method was shown successful and robust when applied to two synthetic datasets (datasets of two-dimensions and ten-dimensions containing eight dimensions of pure noise), two standard biological datasets (the Iris dataset and Rat CNS dataset) and two biological datasets (a cell phenotype dataset and an acute myeloid leukemia (AML) reverse phase protein array (RPPA) dataset). Progeny Clustering outperformed some popular clustering evaluation methods in the ten-dimensional synthetic dataset as well as in the cell phenotype dataset, and it was the only method that successfully discovered clinically meaningful patient groupings in the AML RPPA dataset. PMID:26267476
Usefulness of the automatic quantitative estimation tool for cerebral blood flow: clinical assessment of the application software tool AQCEL.

PubMed

Momose, Mitsuhiro; Takaki, Akihiro; Matsushita, Tsuyoshi; Yanagisawa, Shin; Yano, Kesato; Miyasaka, Tadashi; Ogura, Yuka; Kadoya, Masumi

2011-01-01

AQCEL enables automatic reconstruction of single-photon emission computed tomogram (SPECT) without image degradation and quantitative analysis of cerebral blood flow (CBF) after the input of simple parameters. We ascertained the usefulness and quality of images obtained by the application software AQCEL in clinical practice. Twelve patients underwent brain perfusion SPECT using technetium-99m ethyl cysteinate dimer at rest and after acetazolamide (ACZ) loading. Images reconstructed using AQCEL were compared with those reconstructed using conventional filtered back projection (FBP) method for qualitative estimation. Two experienced nuclear medicine physicians interpreted the image quality using the following visual scores: 0, same; 1, slightly superior; 2, superior. For quantitative estimation, the mean CBF values of the normal hemisphere of the 12 patients using ACZ calculated by the AQCEL method were compared with those calculated by the conventional method. The CBF values of the 24 regions of the 3-dimensional stereotaxic region of interest template (3DSRT) calculated by the AQCEL method at rest and after ACZ loading were compared to those calculated by the conventional method. No significant qualitative difference was observed between the AQCEL and conventional FBP methods in the rest study. The average score by the AQCEL method was 0.25 ± 0.45 and that by the conventional method was 0.17 ± 0.39 (P = 0.34). There was a significant qualitative difference between the AQCEL and conventional methods in the ACZ loading study. The average score for AQCEL was 0.83 ± 0.58 and that for the conventional method was 0.08 ± 0.29 (P = 0.003). During quantitative estimation using ACZ, the mean CBF values of 12 patients calculated by the AQCEL method were 3-8% higher than those calculated by the conventional method. The square of the correlation coefficient between these methods was 0.995. While comparing the 24 3DSRT regions of 12 patients, the squares of the correlation coefficient between AQCEL and conventional methods were 0.973 and 0.986 for the normal and affected sides at rest, respectively, and 0.977 and 0.984 for the normal and affected sides after ACZ loading, respectively. The quality of images reconstructed using the application software AQCEL were superior to that obtained using conventional method after ACZ loading, and high correlations were shown in quantity at rest and after ACZ loading. This software can be applied to clinical practice and is a useful tool for improvement of reproducibility and throughput.
“Forward Genetics” as a Method to Maximize Power and Cost-Efficiency in Studies of Human Complex Traits

PubMed Central

Derks, E. M.; Dolan, C. V.; Kahn, R. S.; Ophoff, R. A.

2010-01-01

There is increasing interest in methods to disentangle the relationship between genotype and (endo)phenotypes in human complex traits. We present a population-based method of increasing the power and cost-efficiency of studies by selecting random individuals with a particular genotype and then assessing the accompanying quantitative phenotypes. Using statistical derivations, power- and cost graphs we show that such a “forward genetics” approach can lead to a marked reduction in sample size and costs. This approach is particularly apt for implementing in epidemiological studies for which DNA is already available but the phenotyping costs are high. Electronic supplementary material The online version of this article (doi:10.1007/s10519-010-9348-y) contains supplementary material, which is available to authorized users. PMID:20232132

Machining and characterization of self-reinforced polymers

NASA Astrophysics Data System (ADS)

Deepa, A.; Padmanabhan, K.; Kuppan, P.

2017-11-01

This Paper focuses on obtaining the mechanical properties and the effect of the different machining techniques on self-reinforced composites sample and to derive the best machining method with remarkable properties. Each sample was tested by the Tensile and Flexural tests, fabricated using hot compaction test and those loads were calculated. These composites are machined using conventional methods because of lack of advanced machinery in most of the industries. The advanced non-conventional methods like Abrasive water jet machining were used. These machining techniques are used to get the better output for the composite materials with good mechanical properties compared to conventional methods. But the use of non-conventional methods causes the changes in the work piece, tool properties and more economical compared to the conventional methods. Finding out the best method ideal for the designing of these Self Reinforced Composites with and without defects and the use of Scanning Electron Microscope (SEM) analysis for the comparing the microstructure of the PP and PE samples concludes our process.
Phenotype Instance Verification and Evaluation Tool (PIVET): A Scaled Phenotype Evidence Generation Framework Using Web-Based Medical Literature.

PubMed

Henderson, Jette; Ke, Junyuan; Ho, Joyce C; Ghosh, Joydeep; Wallace, Byron C

2018-05-04

Researchers are developing methods to automatically extract clinically relevant and useful patient characteristics from raw healthcare datasets. These characteristics, often capturing essential properties of patients with common medical conditions, are called computational phenotypes. Being generated by automated or semiautomated, data-driven methods, such potential phenotypes need to be validated as clinically meaningful (or not) before they are acceptable for use in decision making. The objective of this study was to present Phenotype Instance Verification and Evaluation Tool (PIVET), a framework that uses co-occurrence analysis on an online corpus of publically available medical journal articles to build clinical relevance evidence sets for user-supplied phenotypes. PIVET adopts a conceptual framework similar to the pioneering prototype tool PheKnow-Cloud that was developed for the phenotype validation task. PIVET completely refactors each part of the PheKnow-Cloud pipeline to deliver vast improvements in speed without sacrificing the quality of the insights PheKnow-Cloud achieved. PIVET leverages indexing in NoSQL databases to efficiently generate evidence sets. Specifically, PIVET uses a succinct representation of the phenotypes that corresponds to the index on the corpus database and an optimized co-occurrence algorithm inspired by the Aho-Corasick algorithm. We compare PIVET's phenotype representation with PheKnow-Cloud's by using PheKnow-Cloud's experimental setup. In PIVET's framework, we also introduce a statistical model trained on domain expert-verified phenotypes to automatically classify phenotypes as clinically relevant or not. Additionally, we show how the classification model can be used to examine user-supplied phenotypes in an online, rather than batch, manner. PIVET maintains the discriminative power of PheKnow-Cloud in terms of identifying clinically relevant phenotypes for the same corpus with which PheKnow-Cloud was originally developed, but PIVET's analysis is an order of magnitude faster than that of PheKnow-Cloud. Not only is PIVET much faster, it can be scaled to a larger corpus and still retain speed. We evaluated multiple classification models on top of the PIVET framework and found ridge regression to perform best, realizing an average F1 score of 0.91 when predicting clinically relevant phenotypes. Our study shows that PIVET improves on the most notable existing computational tool for phenotype validation in terms of speed and automation and is comparable in terms of accuracy. ©Jette Henderson, Junyuan Ke, Joyce C Ho, Joydeep Ghosh, Byron C Wallace. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 04.05.2018.
Incomplete Timothy syndrome secondary to a mosaic mutation of the CACNA1C gene diagnosed using next-generation sequencing.

PubMed

Baurand, Amandine; Falcon-Eicher, Sylvie; Laurent, Gabriel; Villain, Elisabeth; Bonnet, Caroline; Thauvin-Robinet, Christel; Jacquot, Caroline; Eicher, Jean-Christophe; Gourraud, Jean-Baptiste; Schmitt, Sébastien; Bézieau, Stéphane; Giraud, Mathilde; Dumont, Solenne; Kuentz, Paul; Probst, Vincent; Burguet, Antoine; Kyndt, Florence; Faivre, Laurence

2017-02-01

Autosomal dominant genetic diseases can occur de novo and in the form of somatic mosaicism, which can give rise to a less severe phenotype, and make diagnosis more difficult given the sensitivity limits of the methods used. We report the case of female child with a history of surgery for syndactyly of the hands and feet, who was admitted at 6 years of age to a pediatric intensive care unit following cardiac arrest. The electrocardiogram (ECG) showed a long QT interval that on occasions reached 500 ms. Despite the absence of facial dysmorphism and the presence of normal psychomotor development, a diagnosis of Timothy syndrome was made given the association of syndactyly and the ECG features. Sanger sequencing of the CACNA1C gene, followed by sequencing of the genes KCNQ1, KCNH2, KCNE1, KCNE2, were negative. The subsequent analysis of a panel of genes responsible for hereditary cardiac rhythm disorders using Haloplex technology revealed a recurrent mosaic p.Gly406Arg missense mutation of the CACNA1C gene in 18% of the cells. This mosaicism can explain the negative Sanger analysis and the less complete phenotype in this patient. Given the other cases in the literature, mosaic mutations in Timothy syndrome appear more common than previously thought. This case demonstrates the importance of using next-generation sequencing to identify mosaic mutations when the clinical picture supports a specific mutation that is not identified using conventional testing. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Readmission prediction via deep contextual embedding of clinical concepts.

PubMed

Xiao, Cao; Ma, Tengfei; Dieng, Adji B; Blei, David M; Wang, Fei

2018-01-01

Hospital readmission costs a lot of money every year. Many hospital readmissions are avoidable, and excessive hospital readmissions could also be harmful to the patients. Accurate prediction of hospital readmission can effectively help reduce the readmission risk. However, the complex relationship between readmission and potential risk factors makes readmission prediction a difficult task. The main goal of this paper is to explore deep learning models to distill such complex relationships and make accurate predictions. We propose CONTENT, a deep model that predicts hospital readmissions via learning interpretable patient representations by capturing both local and global contexts from patient Electronic Health Records (EHR) through a hybrid Topic Recurrent Neural Network (TopicRNN) model. The experiment was conducted using the EHR of a real world Congestive Heart Failure (CHF) cohort of 5,393 patients. The proposed model outperforms state-of-the-art methods in readmission prediction (e.g. 0.6103 ± 0.0130 vs. second best 0.5998 ± 0.0124 in terms of ROC-AUC). The derived patient representations were further utilized for patient phenotyping. The learned phenotypes provide more precise understanding of readmission risks. Embedding both local and global context in patient representation not only improves prediction performance, but also brings interpretable insights of understanding readmission risks for heterogeneous chronic clinical conditions. This is the first of its kind model that integrates the power of both conventional deep neural network and the probabilistic generative models for highly interpretable deep patient representation learning. Experimental results and case studies demonstrate the improved performance and interpretability of the model.
Biochemical markers and protein pattern analysis for canine coagulase-positive staphylococci and their distribution on dog skin.

PubMed

Chanchaithong, Pattrarat; Prapasarakul, Nuvee

2011-08-01

Coagulase-positive staphylococci (CoPS) including S. pseudintermedius, S. schleiferi subsp. coagulans and S. aureus are etiological agents of dermatitis in companion animals and can be zoonotic pathogens. To date no consensual biochemical marker for routine microbiological identification of these species has been identified. The aim of this study was to evaluate biochemical markers and compare the results with the approved molecular method, multiplex-PCR (M-PCR), and confirm their species-specific phenotypic characteristic by using SDS-PAGE. The distribution and frequency of CoPS species were also determined. Three hundred and thirty-seven canine CoPS isolates were obtained from the nasal mucosa, perineum and groins of 66 healthy dogs and were identified by the M-PCR as S. aureus (n=5), S. pseudintermedius (n=263) and S. schleiferi subsp. coagulans (n=69). Selected biochemical tests including the Voges-Proskauer test, mannitol broth fermentation, the assimilation of maltose, galactose, trahalose and lactose using broth medium, were successfully used to distinguish the three species of canine CoPS from other CoPS species. Additionally, species-specific protein patterns were also found to be useful for phenotypic differentiation, with good agreement with the results of M-PCR and the use of biochemical markers. S. aureus occured infrequently on dog skin while co-colonization with S. pseudintermedius and S. schleiferi subsp. coagulans was observed. We propose the use of consensual biochemical markers of canine CoPS with the presence of the unique protein patterns as an alternative tool for conventional laboratory use. Copyright © 2011 Elsevier B.V. All rights reserved.
MALDI-TOF mass spectrometry proteomic phenotyping of clinically relevant fungi.

PubMed

Putignani, Lorenza; Del Chierico, Federica; Onori, Manuela; Mancinelli, Livia; Argentieri, Marta; Bernaschi, Paola; Coltella, Luana; Lucignano, Barbara; Pansani, Laura; Ranno, Stefania; Russo, Cristina; Urbani, Andrea; Federici, Giorgio; Menichella, Donato

2011-03-01

Proteomics is particularly suitable for characterising human pathogens with high life cycle complexity, such as fungi. Protein content and expression levels may be affected by growth states and life cycle morphs and correlate to species and strain variation. Identification and typing of fungi by conventional methods are often difficult, time-consuming and frequently, for unusual species, inconclusive. Proteomic phenotypes from MALDI-TOF MS were employed as analytical and typing expression profiling of yeast, yeast-like species and strain variants in order to achieve a microbial proteomics population study. Spectra from 303 clinical isolates were generated and processed by standard pattern matching with a MALDI-TOF Biotyper (MT). Identifications (IDs) were compared to a reference biochemical-based system (Vitek-2) and, when discordant, MT IDs were verified with genotyping IDs, obtained by sequencing the 25-28S rRNA hypervariable D2 region. Spectra were converted into virtual gel-like formats, and hierarchical clustering analysis was performed for 274 Candida profiles to investigate species and strain typing correlation. MT provided 257/303 IDs consistent with Vitek-2 ones. However, amongst 26/303 discordant MT IDs, only 5 appeared "true". No MT identification was achieved for 20/303 isolates for incompleteness of database species variants. Candida spectra clustering agreed with identified species and topology of Candida albicans and Candida parapsilosis specific dendrograms. MT IDs show a high analytical performance and profiling heterogeneity which seems to complement or even outclass existing typing tools. This variability reflects the high biological complexity of yeasts and may be properly exploited to provide epidemiological tracing and infection dispersion patterns.
Biochemical fingerprinting of Vibrio parahaemolyticus by the PhenePlate system: comparison between pandemic and non-pandemic serotypes.

PubMed

Rahman, Mokhlasur; Bhuiyan, N A; Kuhn, I; Ramamurthy, T; Rahman, M; Mollby, R; Nair, G Balakrish

2006-10-01

During recent years a pandemic clone of Vibrio parahaemolyticus has emerged. Isolates of this clone are distributed among several serotypes, but are genotypically related. In the present study, a phenotyping method (biochemical fingerprinting) was used to characterize pandemic and non-pandemic isolates belonging to V. parahaemolyticus. It was found that the pandemic isolates showed a high level of phenotypic homogeneity and a majority of the pandemic isolates belonged to the same biochemical phenotype, whereas non-pandemic V. parahemolyticus isolates were more heterogeneous. In conclusion, biochemical fingerprinting of V. parahaemolyticus can be used as a first screening method to differentiate between pandemic and non-pandemic isolates of V. parahaemolyticus.
A case of hairy cell leukemia variant.

PubMed

Găman, Amelia Maria; Dobrea, Camelia Marioara; Găman, Mihnea Alexandru

2015-01-01

Hairy cell leukemia variant (HCLv) is a rare B-cell chronic lymphoproliferative disorder with features of the classic HCL but presenting some particularities, a poor response to conventional therapy of classic HCL and a more aggressive course of disease with shorter survival than classic HCL. We present a case of a 52-year-old man hospitalized in July 2012 in the Clinic of Hematology of Craiova, Romania, having splenomegaly, leukocytosis with lymphocytosis, anemia and thrombocytopenia, without monocytopenia, which exposed, in the peripheral blood and bone marrow cells, intermediate morphology between hairy cells and prolymphocytes and immunophenotype of mature B-cell phenotype CD19, CD20, CD22, CD11c, CD103, low positive for CD25 and negative for CD3, diagnosed with HCL variant, with no response to conventional chemotherapy and interferon-alpha, an aggressive course of disease and a survival of less than a year from diagnosis.
Lessons from a Phenotyping Center Revealed by the Genome-Guided Mapping of Powdery Mildew Resistance Loci.

PubMed

Cadle-Davidson, Lance; Gadoury, David; Fresnedo-Ramírez, Jonathan; Yang, Shanshan; Barba, Paola; Sun, Qi; Demmings, Elizabeth M; Seem, Robert; Schaub, Michelle; Nowogrodzki, Anna; Kasinathan, Hema; Ledbetter, Craig; Reisch, Bruce I

2016-10-01

The genomics era brought unprecedented opportunities for genetic analysis of host resistance, but it came with the challenge that accurate and reproducible phenotypes are needed so that genomic results appropriately reflect biology. Phenotyping host resistance by natural infection in the field can produce variable results due to the uncontrolled environment, uneven distribution and genetics of the pathogen, and developmentally regulated resistance among other factors. To address these challenges, we developed highly controlled, standardized methodologies for phenotyping powdery mildew resistance in the context of a phenotyping center, receiving samples of up to 140 grapevine progeny per F 1 family. We applied these methodologies to F 1 families segregating for REN1- or REN2-mediated resistance and validated that some but not all bioassays identified the REN1 or REN2 locus. A point-intercept method (hyphal transects) to quantify colony density objectively at 8 or 9 days postinoculation proved to be the phenotypic response most reproducibly predicted by these resistance loci. Quantitative trait locus (QTL) mapping with genotyping-by-sequencing maps defined the REN1 and REN2 loci at relatively high resolution. In the reference PN40024 genome under each QTL, nucleotide-binding site-leucine-rich repeat candidate resistance genes were identified-one gene for REN1 and two genes for REN2. The methods described here for centralized resistance phenotyping and high-resolution genetic mapping can inform strategies for breeding resistance to powdery mildews and other pathogens on diverse, highly heterozygous hosts.
Prioritization of candidate disease genes by topological similarity between disease and protein diffusion profiles.

PubMed

Zhu, Jie; Qin, Yufang; Liu, Taigang; Wang, Jun; Zheng, Xiaoqi

2013-01-01

Identification of gene-phenotype relationships is a fundamental challenge in human health clinic. Based on the observation that genes causing the same or similar phenotypes tend to correlate with each other in the protein-protein interaction network, a lot of network-based approaches were proposed based on different underlying models. A recent comparative study showed that diffusion-based methods achieve the state-of-the-art predictive performance. In this paper, a new diffusion-based method was proposed to prioritize candidate disease genes. Diffusion profile of a disease was defined as the stationary distribution of candidate genes given a random walk with restart where similarities between phenotypes are incorporated. Then, candidate disease genes are prioritized by comparing their diffusion profiles with that of the disease. Finally, the effectiveness of our method was demonstrated through the leave-one-out cross-validation against control genes from artificial linkage intervals and randomly chosen genes. Comparative study showed that our method achieves improved performance compared to some classical diffusion-based methods. To further illustrate our method, we used our algorithm to predict new causing genes of 16 multifactorial diseases including Prostate cancer and Alzheimer's disease, and the top predictions were in good consistent with literature reports. Our study indicates that integration of multiple information sources, especially the phenotype similarity profile data, and introduction of global similarity measure between disease and gene diffusion profiles are helpful for prioritizing candidate disease genes. Programs and data are available upon request.
Corruption of the intra-gene DNA methylation architecture is a hallmark of cancer.

PubMed

Bartlett, Thomas E; Zaikin, Alexey; Olhede, Sofia C; West, James; Teschendorff, Andrew E; Widschwendter, Martin

2013-01-01

Epigenetic processes--including DNA methylation--are increasingly seen as having a fundamental role in chronic diseases like cancer. It is well known that methylation levels at particular genes or loci differ between normal and diseased tissue. Here we investigate whether the intra-gene methylation architecture is corrupted in cancer and whether the variability of levels of methylation of individual CpGs within a defined gene is able to discriminate cancerous from normal tissue, and is associated with heterogeneous tumour phenotype, as defined by gene expression. We analysed 270985 CpGs annotated to 18272 genes, in 3284 cancerous and 681 normal samples, corresponding to 14 different cancer types. In doing so, we found novel differences in intra-gene methylation pattern across phenotypes, particularly in those genes which are crucial for stem cell biology; our measures of intra-gene methylation architecture are a better determinant of phenotype than measures based on mean methylation level alone (K-S test [Formula: see text] in all 14 diseases tested). These per-gene methylation measures also represent a considerable reduction in complexity, compared to conventional per-CpG beta-values. Our findings strongly support the view that intra-gene methylation architecture has great clinical potential for the development of DNA-based cancer biomarkers.
Peripheral blood antigen presenting cell responses in otitis-prone and non-otitis-prone infants.

PubMed

Surendran, Naveen; Nicolosi, Ted; Kaur, Ravinder; Pichichero, Michael E

2016-01-01

Stringently defined otitis-prone (sOP) children represent a new classification of the otitis-prone condition. Previous studies showed dysfunction in Ab, B-cell memory and T-cell memory responses. We sought to determine whether there are defects in numbers, phenotype and/or function of professional APC in the peripheral blood of sOP infants. APC phenotypic counts, MHC II expression and intracellular cytokine levels were determined in response to TLR7/8 (R848) stimulation by flow cytometry. Innate immune mRNA expression was measured using RT-PCR and cytokines were measured using Luminex technology. Significant (P < 0.05) increases in the phenotypic counts of monocytes and conventional dendritic cells but not plasmacytoid DCs were observed in sOP compared with non-otitis-prone (NOP) age-matched infants. No significant differences in APC activation or function were observed. Expression of various TLRs, intracellular signaling molecules and downstream cytokines was also not found to be significantly different between sOP and NOP infants. Higher numbers of APCs in sOP infants suggest the possibility of a persistent mucosal inflammatory status. Transcriptional and cytokine profiles of PBMCs among sOP infants suggest their systemic innate responses are not different compared to NOP infants. © The Author(s) 2015.
Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

PubMed

Kameishi, Sumako; Umemoto, Terumasa; Matsuzaki, Yu; Fujita, Masako; Okano, Teruo; Kato, Takashi; Yamato, Masayuki

2016-05-06

Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells. Copyright © 2015 Elsevier Inc. All rights reserved.
Cancer Stem Cells: Cellular Plasticity, Niche, and its Clinical Relevance.

PubMed

Lee, Gina; Hall, Robert R; Ahmed, Atique U

2016-10-01

Cancer handles an estimated 7.6 million deaths worldwide per annum. A recent theory focuses on the role Cancer Stem Cells (CSCs) in driving tumorigenesis and disease progression. This theory hypothesizes that a population of the tumor cell with similar functional and phenotypic characteristics as normal tissue stem cells are responsible for formation and advancement of many human cancers. The CSCs subpopulation can differentiate into non-CSC tumor cells and promote phenotypic and functional heterogeneity within the tumor. The presence of CSCs has been reported in a number of human cancers including blood, breast, brain, colon, lung, pancreas prostate and liver. Although the origin of CSCs remains a mystery, recent reports suggest that the phenotypic characteristics of CSCs may be plastic and are influenced by the microenvironment specific for the individual tumor. Such factors unique to each tumor preserve the dynamic balance between CSCs to non-CSCs cell fate, as well as maintain the proper equilibrium. Alternating such equilibrium via dedifferentiation can result in aggressiveness, as CSCs are considered to be more resistant to the conventional cancer treatments of chemotherapy and radiation. Understanding how the tumoral microenvironment affects the plasticity driven CSC niche will be critical for developing a more effective treatment for cancer by eliminating its aggressive and recurring nature that now is believed to be perpetuated by CSCs.
Modulation of Macrophage Phenotype by Biodegradable Polyurethane Nanoparticles: Possible Relation between Macrophage Polarization and Immune Response of Nanoparticles.

PubMed

Huang, Yen-Jang; Hung, Kun-Che; Hung, Huey-Shan; Hsu, Shan-Hui

2018-06-13

Nanomaterials with surface functionalized by different chemical groups can either provoke or attenuate the immune responses of the nanomaterials, which is critical to their biomedical efficacies. In this study, we demonstrate that synthetic waterborne polyurethane nanoparticles (PU NPs) can inhibit the macrophage polarization toward the M1 phenotype but not M2 phenotype. The surface-functionalized PU NPs decrease the secretion levels of proinflammatory cytokines (TNF-α and IL-1β) for M1 macrophages. Specifically, PU NPs with carboxyl groups on the surface exhibit a greater extent of inhibition on M1 polarization than those with amine groups. These water-suspended PU NPs reduce the nuclear factor-κB (NF-κB) activation and suppress the subsequent NLR family pyrin domain containing 3 (NLRP3) inflammasome signals. Furthermore, the dried PU films assembled from PU NPs have a similar effect on macrophage polarization and present a smaller shifting foreign body reaction (FBR) in vivo than the conventional poly(l-lactic acid). Taken together, the biodegradable waterborne PU NPs demonstrate surface-dependent immunosuppressive properties and macrophage polarization effects. The findings suggest potential therapeutic applications of PU NPs in anti-inflammation and macrophage-related disorders and propose a mechanism for the low FBR observed for biodegradable PU materials.
Disease Modeling via Large-Scale Network Analysis

DTIC Science & Technology

2015-05-20

SECURITY CLASSIFICATION OF: A central goal of genetics is to learn how the genotype of an organism determines its phenotype. We address the implicit...guarantees for the methods. In the past, we have developed predictive methods general enough to apply to potentially any genetic trait, varying from... genetics is to learn how the genotype of an organism determines its phenotype. We address the implicit problem of predicting the association of genes with
Effect of the callipyge phenotype and cooking method on tenderness of several major lamb muscles.

PubMed

Shackelford, S D; Wheeler, T L; Koohmaraie, M

1997-08-01

We conducted three experiments to determine the effects of the callipyge phenotype on the tenderness of several major lamb muscles and to determine the effect of method of cookery on the tenderness of callipyge lamb at 7 d postmortem. In Exp. 1, chops from normal (n = 23) and callipyge (n = 16) carcasses were open-hearth-broiled. Warner-Bratzler shear force values of longissimus, gluteus medius, semimembranosus, biceps femoris, semitendinosus, adductor, and quadriceps femoris were 123, 44, 28, 26, 19, 16, and 13% greater, respectively, for callipyge (P < .05). In Exp. 2, muscles from normal (n = 18) and callipyge (n = 18) carcasses were oven-roasted. Shear force of triceps brachii was 11% greater for callipyge (P < .001); however, phenotype did not affect shear force of supraspinatus (P = .87) or psoas major (P = .64). In Exp. 3, a trained sensory panel evaluated leg roasts and open-hearth-broiled leg chops from normal (n = 60) and callipyge lamb carcasses (n = 60). Callipyge chops were less tender than normal chops (P < .05). Regardless of callipyge phenotype, muscles were more (P < .05) tender when roasted; however, the effect of method of cookery on tenderness scores was greater for callipyge muscles than for normal muscles. Callipyge roasts and normal roasts had similar tenderness (P = .58), and callipyge roasts were more tender than normal chops (P < .05). Regardless of cooking method, callipyge samples were less juicy than normal samples (P < .05). These data demonstrate that the callipyge phenotype will likely reduce consumer satisfaction due to reduced tenderness and juiciness; however, reduced tenderness in callipyge leg muscles could be prevented by ovenroasting.
Analysis of the human diseasome using phenotype similarity between common, genetic, and infectious diseases

NASA Astrophysics Data System (ADS)

Hoehndorf, Robert; Schofield, Paul N.; Gkoutos, Georgios V.

2015-06-01

Phenotypes are the observable characteristics of an organism arising from its response to the environment. Phenotypes associated with engineered and natural genetic variation are widely recorded using phenotype ontologies in model organisms, as are signs and symptoms of human Mendelian diseases in databases such as OMIM and Orphanet. Exploiting these resources, several computational methods have been developed for integration and analysis of phenotype data to identify the genetic etiology of diseases or suggest plausible interventions. A similar resource would be highly useful not only for rare and Mendelian diseases, but also for common, complex and infectious diseases. We apply a semantic text-mining approach to identify the phenotypes (signs and symptoms) associated with over 6,000 diseases. We evaluate our text-mined phenotypes by demonstrating that they can correctly identify known disease-associated genes in mice and humans with high accuracy. Using a phenotypic similarity measure, we generate a human disease network in which diseases that have similar signs and symptoms cluster together, and we use this network to identify closely related diseases based on common etiological, anatomical as well as physiological underpinnings.
An omnibus test for family-based association studies with multiple SNPs and multiple phenotypes.

PubMed

Lasky-Su, Jessica; Murphy, Amy; McQueen, Matthew B; Weiss, Scott; Lange, Christoph

2010-06-01

We propose an omnibus family-based association test (MFBAT) that can be applied to multiple markers and multiple phenotypes and that has only one degree of freedom. The proposed test statistic extends current FBAT methodology to incorporate multiple markers as well as multiple phenotypes. Using simulation studies, power estimates for the proposed methodology are compared with the standard methodologies. On the basis of these simulations, we find that MFBAT substantially outperforms other methods, including haplotypic approaches and doing multiple tests with single single-nucleotide polymorphisms (SNPs) and single phenotypes. The practical relevance of the approach is illustrated by an application to asthma in which SNP/phenotype combinations are identified and reach overall significance that would not have been identified using other approaches. This methodology is directly applicable to cases in which there are multiple SNPs, such as candidate gene studies, cases in which there are multiple phenotypes, such as expression data, and cases in which there are multiple phenotypes and genotypes, such as genome-wide association studies that incorporate expression profiles as phenotypes. This program is available in the PBAT analysis package.
Detecting novel genes with sparse arrays

PubMed Central

Haiminen, Niina; Smit, Bart; Rautio, Jari; Vitikainen, Marika; Wiebe, Marilyn; Martinez, Diego; Chee, Christine; Kunkel, Joe; Sanchez, Charles; Nelson, Mary Anne; Pakula, Tiina; Saloheimo, Markku; Penttilä, Merja; Kivioja, Teemu

2014-01-01

Species-specific genes play an important role in defining the phenotype of an organism. However, current gene prediction methods can only efficiently find genes that share features such as sequence similarity or general sequence characteristics with previously known genes. Novel sequencing methods and tiling arrays can be used to find genes without prior information and they have demonstrated that novel genes can still be found from extensively studied model organisms. Unfortunately, these methods are expensive and thus are not easily applicable, e.g., to finding genes that are expressed only in very specific conditions. We demonstrate a method for finding novel genes with sparse arrays, applying it on the 33.9 Mb genome of the filamentous fungus Trichoderma reesei. Our computational method does not require normalisations between arrays and it takes into account the multiple-testing problem typical for analysis of microarray data. In contrast to tiling arrays, that use overlapping probes, only one 25mer microarray oligonucleotide probe was used for every 100 b. Thus, only relatively little space on a microarray slide was required to cover the intergenic regions of a genome. The analysis was done as a by-product of a conventional microarray experiment with no additional costs. We found at least 23 good candidates for novel transcripts that could code for proteins and all of which were expressed at high levels. Candidate genes were found to neighbour ire1 and cre1 and many other regulatory genes. Our simple, low-cost method can easily be applied to finding novel species-specific genes without prior knowledge of their sequence properties. PMID:20691772

MicroCT-based phenomics in the zebrafish skeleton reveals virtues of deep phenotyping in a distributed organ system.

PubMed

Hur, Matthew; Gistelinck, Charlotte A; Huber, Philippe; Lee, Jane; Thompson, Marjorie H; Monstad-Rios, Adrian T; Watson, Claire J; McMenamin, Sarah K; Willaert, Andy; Parichy, David M; Coucke, Paul; Kwon, Ronald Y

2017-09-08

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of phenotypic measures comprised of morphological and densitometric traits at a large number of sites within the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.
Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

PubMed

de Angelis, Martin Hrabě; Nicholson, George; Selloum, Mohammed; White, Jacqui; Morgan, Hugh; Ramirez-Solis, Ramiro; Sorg, Tania; Wells, Sara; Fuchs, Helmut; Fray, Martin; Adams, David J; Adams, Niels C; Adler, Thure; Aguilar-Pimentel, Antonio; Ali-Hadji, Dalila; Amann, Gregory; André, Philippe; Atkins, Sarah; Auburtin, Aurelie; Ayadi, Abdel; Becker, Julien; Becker, Lore; Bedu, Elodie; Bekeredjian, Raffi; Birling, Marie-Christine; Blake, Andrew; Bottomley, Joanna; Bowl, Mike; Brault, Véronique; Busch, Dirk H; Bussell, James N; Calzada-Wack, Julia; Cater, Heather; Champy, Marie-France; Charles, Philippe; Chevalier, Claire; Chiani, Francesco; Codner, Gemma F; Combe, Roy; Cox, Roger; Dalloneau, Emilie; Dierich, André; Di Fenza, Armida; Doe, Brendan; Duchon, Arnaud; Eickelberg, Oliver; Esapa, Chris T; El Fertak, Lahcen; Feigel, Tanja; Emelyanova, Irina; Estabel, Jeanne; Favor, Jack; Flenniken, Ann; Gambadoro, Alessia; Garrett, Lilian; Gates, Hilary; Gerdin, Anna-Karin; Gkoutos, George; Greenaway, Simon; Glasl, Lisa; Goetz, Patrice; Da Cruz, Isabelle Goncalves; Götz, Alexander; Graw, Jochen; Guimond, Alain; Hans, Wolfgang; Hicks, Geoff; Hölter, Sabine M; Höfler, Heinz; Hancock, John M; Hoehndorf, Robert; Hough, Tertius; Houghton, Richard; Hurt, Anja; Ivandic, Boris; Jacobs, Hughes; Jacquot, Sylvie; Jones, Nora; Karp, Natasha A; Katus, Hugo A; Kitchen, Sharon; Klein-Rodewald, Tanja; Klingenspor, Martin; Klopstock, Thomas; Lalanne, Valerie; Leblanc, Sophie; Lengger, Christoph; le Marchand, Elise; Ludwig, Tonia; Lux, Aline; McKerlie, Colin; Maier, Holger; Mandel, Jean-Louis; Marschall, Susan; Mark, Manuel; Melvin, David G; Meziane, Hamid; Micklich, Kateryna; Mittelhauser, Christophe; Monassier, Laurent; Moulaert, David; Muller, Stéphanie; Naton, Beatrix; Neff, Frauke; Nolan, Patrick M; Nutter, Lauryl Mj; Ollert, Markus; Pavlovic, Guillaume; Pellegata, Natalia S; Peter, Emilie; Petit-Demoulière, Benoit; Pickard, Amanda; Podrini, Christine; Potter, Paul; Pouilly, Laurent; Puk, Oliver; Richardson, David; Rousseau, Stephane; Quintanilla-Fend, Leticia; Quwailid, Mohamed M; Racz, Ildiko; Rathkolb, Birgit; Riet, Fabrice; Rossant, Janet; Roux, Michel; Rozman, Jan; Ryder, Ed; Salisbury, Jennifer; Santos, Luis; Schäble, Karl-Heinz; Schiller, Evelyn; Schrewe, Anja; Schulz, Holger; Steinkamp, Ralf; Simon, Michelle; Stewart, Michelle; Stöger, Claudia; Stöger, Tobias; Sun, Minxuan; Sunter, David; Teboul, Lydia; Tilly, Isabelle; Tocchini-Valentini, Glauco P; Tost, Monica; Treise, Irina; Vasseur, Laurent; Velot, Emilie; Vogt-Weisenhorn, Daniela; Wagner, Christelle; Walling, Alison; Weber, Bruno; Wendling, Olivia; Westerberg, Henrik; Willershäuser, Monja; Wolf, Eckhard; Wolter, Anne; Wood, Joe; Wurst, Wolfgang; Yildirim, Ali Önder; Zeh, Ramona; Zimmer, Andreas; Zimprich, Annemarie; Holmes, Chris; Steel, Karen P; Herault, Yann; Gailus-Durner, Valérie; Mallon, Ann-Marie; Brown, Steve Dm

2015-09-01

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.
Quantitative in vivo cell-surface receptor imaging in oncology: kinetic modeling & paired-agent principles from nuclear medicine and optical imaging

PubMed Central

Tichauer, Kenneth M.; Wang, Yu; Pogue, Brian W.; Liu, Jonathan T. C.

2015-01-01

The development of methods to accurately quantify cell-surface receptors in living tissues would have a seminal impact in oncology. For example, accurate measures of receptor density in vivo could enhance early detection or surgical resection of tumors via protein-based contrast, allowing removal of cancer with high phenotype specificity. Alternatively, accurate receptor expression estimation could be used as a biomarker to guide patient-specific clinical oncology targeting of the same molecular pathway. Unfortunately, conventional molecular contrast-based imaging approaches are not well adapted to accurately estimating the nanomolar-level cell-surface receptor concentrations in tumors, as most images are dominated by nonspecific sources of contrast such as high vascular permeability and lymphatic inhibition. This article reviews approaches for overcoming these limitations based upon tracer kinetic modeling and the use of emerging protocols to estimate binding potential and the related receptor concentration. Methods such as using single time point imaging or a reference-tissue approach tend to have low accuracy in tumors, whereas paired-agent methods or advanced kinetic analyses are more promising to eliminate the dominance of interstitial space in the signals. Nuclear medicine and optical molecular imaging are the primary modalities used, as they have the nanomolar level sensitivity needed to quantify cell-surface receptor concentrations present in tissue, although each likely has a different clinical niche. PMID:26134619
Identification of Balanced Chromosomal Rearrangements Previously Unknown Among Participants in the 1000 Genomes Project: Implications for Interpretation of Structural Variation in Genomes and the Future of Clinical Cytogenetics

PubMed Central

Dong, Zirui; Wang, Huilin; Chen, Haixiao; Jiang, Hui; Yuan, Jianying; Yang, Zhenjun; Wang, Wen-Jing; Xu, Fengping; Guo, Xiaosen; Cao, Ye; Zhu, Zhenzhen; Geng, Chunyu; Cheung, Wan Chee; Kwok, Yvonne K; Yang, Huangming; Leung, Tak Yeung; Morton, Cynthia C.; Cheung, Sau Wai; Choy, Kwong Wai

2017-01-01

Purpose Recent studies demonstrate that whole-genome sequencing (WGS) enables detection of cryptic rearrangements in apparently balanced chromosomal rearrangements (also known as balanced chromosomal abnormalities, BCAs) previously identified by conventional cytogenetic methods. We aimed to assess our analytical tool for detecting BCAs in The 1000 Genomes Project without knowing affected bands. Methods The 1000 Genomes Project provides an unprecedented integrated map of structural variants in phenotypically normal subjects, but there is no information on potential inclusion of subjects with apparently BCAs akin to those traditionally detected in diagnostic cytogenetics laboratories. We applied our analytical tool to 1,166 genomes from the 1000 Genomes Project with sufficient physical coverage (8.25-fold). Results Our approach detected four reciprocal balanced translocations and four inversions ranging in size from 57.9 kb to 13.3 Mb, all of which were confirmed by cytogenetic methods and PCR studies. One of DNAs has a subtle translocation that is not readily identified by chromosome analysis due to similar banding patterns and size of exchanged segments, and another results in disruption of all transcripts of an OMIM gene. Conclusions Our study demonstrates the extension of utilizing low-coverage WGS for unbiased detection of BCAs including translocations and inversions previously unknown in the 1000 Genomes Project. PMID:29095815
Detection of Candida species in pregnant Chinese women with a molecular beacon method.

PubMed

Zhai, Yanhong; Liu, Jing; Zhou, Li; Ji, Tongzhen; Meng, Lingxin; Gao, Yang; Liu, Ran; Wang, Xiao; Li, Lin; Lu, Binghuai; Cao, Zheng

2018-04-20

Candida pathogens are commonly found in women and can cause vulvovaginal candidiasis (VVC), whose infection rate is further increased during pregnancy. We aimed to study the Candida prevalence and strain distribution in pregnant Chinese women with a molecular beacon assay. From March 2016 to February 2017, a total of 993 pregnant women attending routine antenatal visits at the Beijing Obstetrics and Gynecology Hospital were enrolled. For Candida detection and identification, a unique molecular beacon assay was presented and compared with a traditional phenotypic method. Antifungal susceptibility was tested with the following agents: 5-flucytosine, amphotericin B, fluconazole, itraconazole and voriconazole. The prevalence of Candida was found to be 21.8 % when using the molecular method and 15.0 % when using the phenotypic method. The distribution of the Candida spp. was listed in order of decreasing prevalence: Candida albicans (79.8 %), Candida glabrata (13.5 %), Candida parapsilosis (3.7 %), Candida krusei (2.2 %) and Candida tropicalis (1.1 %). We found that 90.7 % of the Candida detection results were consistent between the molecular and the phenotypic methods. In the cases where the sequencing analyses for the Candida isolates resulted in inconsistent identification, the molecular method showed higher sensitivity than the phenotypic method (96.0 vs 64.6 %). C. albicans, C. glabrata and C. parapsilosis were essentially susceptible to all five antifungal agents tested, whereas C. tropicalis and C. krusei were susceptible to voriconazole and amphotericin B. By exhibiting good sensitivity and specificity, the molecular assay may offer a fast and accurate Candida screening platform for pregnant women.
Comprehensive detection of genes causing a phenotype using phenotype sequencing and pathway analysis.

PubMed

Harper, Marc; Gronenberg, Luisa; Liao, James; Lee, Christopher

2014-01-01

Discovering all the genetic causes of a phenotype is an important goal in functional genomics. We combine an experimental design for detecting independent genetic causes of a phenotype with a high-throughput sequencing analysis that maximizes sensitivity for comprehensively identifying them. Testing this approach on a set of 24 mutant strains generated for a metabolic phenotype with many known genetic causes, we show that this pathway-based phenotype sequencing analysis greatly improves sensitivity of detection compared with previous methods, and reveals a wide range of pathways that can cause this phenotype. We demonstrate our approach on a metabolic re-engineering phenotype, the PEP/OAA metabolic node in E. coli, which is crucial to a substantial number of metabolic pathways and under renewed interest for biofuel research. Out of 2157 mutations in these strains, pathway-phenoseq discriminated just five gene groups (12 genes) as statistically significant causes of the phenotype. Experimentally, these five gene groups, and the next two high-scoring pathway-phenoseq groups, either have a clear connection to the PEP metabolite level or offer an alternative path of producing oxaloacetate (OAA), and thus clearly explain the phenotype. These high-scoring gene groups also show strong evidence of positive selection pressure, compared with strictly neutral selection in the rest of the genome.
Robust and Sensitive Analysis of Mouse Knockout Phenotypes

PubMed Central

Karp, Natasha A.; Melvin, David; Mott, Richard F.

2012-01-01

A significant challenge of in-vivo studies is the identification of phenotypes with a method that is robust and reliable. The challenge arises from practical issues that lead to experimental designs which are not ideal. Breeding issues, particularly in the presence of fertility or fecundity problems, frequently lead to data being collected in multiple batches. This problem is acute in high throughput phenotyping programs. In addition, in a high throughput environment operational issues lead to controls not being measured on the same day as knockouts. We highlight how application of traditional methods, such as a Student’s t-Test or a 2-way ANOVA, in these situations give flawed results and should not be used. We explore the use of mixed models using worked examples from Sanger Mouse Genome Project focusing on Dual-Energy X-Ray Absorptiometry data for the analysis of mouse knockout data and compare to a reference range approach. We show that mixed model analysis is more sensitive and less prone to artefacts allowing the discovery of subtle quantitative phenotypes essential for correlating a gene’s function to human disease. We demonstrate how a mixed model approach has the additional advantage of being able to include covariates, such as body weight, to separate effect of genotype from these covariates. This is a particular issue in knockout studies, where body weight is a common phenotype and will enhance the precision of assigning phenotypes and the subsequent selection of lines for secondary phenotyping. The use of mixed models with in-vivo studies has value not only in improving the quality and sensitivity of the data analysis but also ethically as a method suitable for small batches which reduces the breeding burden of a colony. This will reduce the use of animals, increase throughput, and decrease cost whilst improving the quality and depth of knowledge gained. PMID:23300663
Phenotypic and genotypic study of biofilm formation in Enterococci isolated from urinary tract infections.

PubMed

Fallah, F; Yousefi, M; Pourmand, M R; Hashemi, A; Nazari Alam, A; Afshar, D

2017-07-01

Urinary tract infection (UTI) is one of the most frequent types of nosocomial and community acquired infections in humans. Management of multidrug-resistant Enterococci UTI due to the limited therapeutic options is a great challenge for physicians and clinical microbiologists. The role of bacterial biofilms in recurrent urinary tract infections and antimicrobial resistance has great importance for public health. The aim of this study was to investigate the antibiotic susceptibility pattern as well as the phenotypic and genotypic biofilm formation ability of Enterococci isolates from patients with UTI. A total of 57 isolates of Enterococci were collected from patients with UTI. Enterococcus species were identified using conventional microbiological methods. The antibiotic susceptibility patterns of the isolates were determined by the Kirby-Bauer disk-diffusion. The Modified Congo red agar (MCRA) and Microtiter plate methods used to assess the ability of biofilm formation. All enterococcal isolates were examined for determination of biofilm-related genes, esp, asa1 and ebpR using PCR method. Of 57 enterococcal isolates, 85.9% were recognized as E. faecalis and 14.1% of them were E. faecium. According to our results, linezolid, chloramphenicol and nitrofurantoin were the most effective agents against Enterococcus species. Overall, 26.5% of E. faecalis and 75% of E. faecium isolates were biofilm producers, respectively. Resistance to some antibiotics including penicillin G, ampicillin, vancomycin, nitrofurantoin and chloramphenicol, and ciprofloxacin was significantly higher among biofilm producers than non-biofilm producers Enterococci. The esp, asa1 and ebpR genes were present in 84.2%, 91.2% and 100% isolates. In this study, there was not a significant relationship between presence of these genes and biofilm formation. Our findings reinforce the role of biofilm formation in resistance to antimicrobial agents. Quinupristin/dalfopristin, tetracycline and rifampin may be used as an effective treatment for UTI caused by biofilm producers Enterococci. Our results suggest that biofilm formation is complex and depends on various factors but not just esp, asa1 and ebpR genes in Enterococcus strains. Copyright © 2017 Elsevier Ltd. All rights reserved.
A Fast, Reliable, and Sensitive Method for Detection and Quantification of Listeria monocytogenes and Escherichia coli O157:H7 in Ready-to-Eat Fresh-Cut Products by MPN-qPCR

PubMed Central

Russo, Pasquale; Botticella, Giuseppe; Capozzi, Vittorio; Massa, Salvatore; Spano, Giuseppe; Beneduce, Luciano

2014-01-01

In the present work we developed a MPN quantitative real-time PCR (MPN-qPCR) method for a fast and reliable detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed vegetables. In order to validate the proposed technique, the results were compared with conventional MPN followed by phenotypic and biochemical assays methods. When L. monocytogenes and E. coli O157:H7 were artificially inoculated in fresh-cut vegetables, a concentration as low as 1 CFU g−1 could be detected in 48 hours for both pathogens. qPCR alone allowed a limit of detection of 101 CFU g−1 after 2 hours of enrichment for L. monocytogenes and E. coli O157:H7. Since minimally processed ready-to-eat vegetables are characterized by very short shelf life, our method can potentially address the consistent reduction of time for microbial analysis, allowing a better management of quality control. Moreover, the occurrences of both pathogenic bacteria in mixed salad samples and fresh-cut melons were monitored in two production plants from the receipt of the raw materials to the early stages of shelf life. No sample was found to be contaminated by L. monocytogenes. One sample of raw mixed salad was found positive to an H7 enterohemorrhagic serotype. PMID:24949460
Renal F4/80+CD11c+ Mononuclear Phagocytes Display Phenotypic and Functional Characteristics of Macrophages in Health and in Adriamycin Nephropathy

PubMed Central

Wang, Yiping; Wang, Xin Maggie; Lu, Junyu; Lee, Vincent W.S.; Ye, Qianling; Nguyen, Hanh; Zheng, Guoping; Zhao, Ye; Alexander, Stephen I.; Harris, David C.H.

2015-01-01

Conventional markers of macrophages (Mфs) and dendritic cells (DCs) lack specificity and often overlap, leading to confusion and controversy regarding the precise function of these cells in kidney and other diseases. This study aimed to identify the phenotype and function of renal mononuclear phagocytes (rMPs) expressing key markers of both Mфs and DCs. F4/80+CD11c+ cells accounted for 45% of total rMPs in normal kidneys and in those from mice with Adriamycin nephropathy (AN). Despite expression of the DC marker CD11c, these double-positive rMPs displayed the features of Mфs, including Mф-like morphology, high expression of CD68, CD204, and CD206, and high phagocytic ability but low antigen-presenting ability. F4/80+CD11c+ cells were found in the cortex but not in the medulla of the kidney. In AN, F4/80+CD11c+ cells displayed an M1 Mф phenotype with high expression of inflammatory mediators and costimulatory factors. Adoptive transfer of F4/80+CD11c+ cells separated from diseased kidney aggravated renal injury in AN mice. Furthermore, adoptive transfer of common progenitors revealed that kidney F4/80+CD11c+ cells were derived predominantly from monocytes, but not from pre-DCs. In conclusion, renal F4/80+CD11c+ cells are a major subset of rMPs and display Mф-like phenotypic and functional characteristics in health and in AN. PMID:25012165
Immunogenic apoptosis in human acute myeloid leukemia (AML): primary human AML cells expose calreticulin and release heat shock protein (HSP) 70 and HSP90 during apoptosis.

PubMed

Fredly, Hanne; Ersvær, Elisabeth; Gjertsen, Bjørn-Tore; Bruserud, Oystein

2011-06-01

Several previous studies have demonstrated that both conventional cytotoxic drugs as well as targeted therapeutics can induce apoptosis in primary human acute myelogenous leukemia (AML) cells. However, the apoptotic phenotype of dying AML cells has been less extensively characterized. Even though specific antileukemic immune reactivity is important in AML, especially for allotransplanted patients, it has not been investigated whether dying primary human AML cells show phenotypic characteristics consistent with immunogenic apoptosis [calreticulin exposure, heat shock protein (HSP) release]. We therefore investigated whether in vitro cultured primary human acute myeloid leukemia (AML) cells show calreticulin exposure and HSP70/HSP90 release during spontaneous (stress-induced) apoptosis when cultured in medium alone and when cultured in the presence of antileukemic drugs. Both surface exposure of calreticulin and release of HSP70 and HSP90 was detected but showed a wide variation between patients. This variation was also maintained when the AML cells were cultured in the presence of cytotoxic drugs (cytarabine, daunorubicin, mitomycin), all-trans retinoic acid (ATRA) and valproic acid. Finally, AML cells collected during in vivo ATRA therapy showed increased calreticulin exposure during spontaneous in vitro apoptosis, suggesting that in vivo pharmacotherapy can modulate the apoptotic phenotype. To conclude, apoptotic AML cells can show phenotypic characteristics consistent with immunogenic apoptosis, but there is a wide variation between patients and the level of calreticulin exposure/HSP release seems to depend on individual patient characteristics rather than the apoptosis-inducing agent.
Renal F4/80+ CD11c+ mononuclear phagocytes display phenotypic and functional characteristics of macrophages in health and in adriamycin nephropathy.

PubMed

Cao, Qi; Wang, Yiping; Wang, Xin Maggie; Lu, Junyu; Lee, Vincent W S; Ye, Qianling; Nguyen, Hanh; Zheng, Guoping; Zhao, Ye; Alexander, Stephen I; Harris, David C H

2015-02-01

Conventional markers of macrophages (Mфs) and dendritic cells (DCs) lack specificity and often overlap, leading to confusion and controversy regarding the precise function of these cells in kidney and other diseases. This study aimed to identify the phenotype and function of renal mononuclear phagocytes (rMPs) expressing key markers of both Mфs and DCs. F4/80(+)CD11c(+) cells accounted for 45% of total rMPs in normal kidneys and in those from mice with Adriamycin nephropathy (AN). Despite expression of the DC marker CD11c, these double-positive rMPs displayed the features of Mфs, including Mф-like morphology, high expression of CD68, CD204, and CD206, and high phagocytic ability but low antigen-presenting ability. F4/80(+)CD11c(+) cells were found in the cortex but not in the medulla of the kidney. In AN, F4/80(+)CD11c(+) cells displayed an M1 Mф phenotype with high expression of inflammatory mediators and costimulatory factors. Adoptive transfer of F4/80(+)CD11c(+) cells separated from diseased kidney aggravated renal injury in AN mice. Furthermore, adoptive transfer of common progenitors revealed that kidney F4/80(+)CD11c(+) cells were derived predominantly from monocytes, but not from pre-DCs. In conclusion, renal F4/80(+)CD11c(+) cells are a major subset of rMPs and display Mф-like phenotypic and functional characteristics in health and in AN. Copyright © 2015 by the American Society of Nephrology.
Alternative methods for CYP2D6 phenotyping: comparison of dextromethorphan metabolic ratios from AUC, single point plasma, and urine.

PubMed

Chen, Rui; Wang, Haotian; Shi, Jun; Hu, Pei

2016-05-01

CYP2D6 is a high polymorphic enzyme. Determining its phenotype before CYP2D6 substrate treatment can avoid dose-dependent adverse events or therapeutic failures. Alternative phenotyping methods of CYP2D6 were compared to aluate the appropriate and precise time points for phenotyping after single-dose and ultiple-dose of 30-mg controlled-release (CR) dextromethorphan (DM) and to explore the antimodes for potential sampling methods. This was an open-label, single and multiple-dose study. 21 subjects were assigned to receive a single dose of CR DM 30 mg orally, followed by a 3-day washout period prior to oral administration of CR DM 30 mg every 12 hours for 6 days. Metabolic ratios (MRs) from AUC∞ after single dosing and from AUC0-12h at steady state were taken as the gold standard. The correlations of metabolic ratios of DM to dextrorphan (MRDM/DX) values based on different phenotyping methods were assessed. Linear regression formulas were derived to calculate the antimodes for potential sample methods. In the single-dose part of the study statistically significant correlations were found between MRDM/DX from AUC∞ and from serial plasma points from 1 to 30 hours or from urine (all p-values < 0.001). In the multiple-dose part, statistically significant correlations were found between MRDM/DX from AUC0-12h on day 6 and MRDM/DX from serial plasma points from 0 to 36 hours after the last dosing (all p-values < 0.001). Based on reported urinary antimode and linear regression analysis, the antimodes of AUC and plasma points were derived to profile the trend of antimodes as the drug concentrations changed. MRDM/DX from plasma points had good correlations with MRDM/DX from AUC. Plasma points from 1 to 30 hours after single dose of 30-mg CR DM and any plasma point at steady state after multiple doses of CR DM could potentially be used for phenotyping of CYP2D6.
Plant characterization of genetically modified maize hybrids MON-89Ø34-3 × MON-88Ø17-3, MON-89Ø34-3 × MON-ØØ6Ø3-6, and MON-ØØ6Ø3-6: alternatives for maize production in Mexico.

PubMed

Heredia Díaz, Oscar; Aldaba Meza, José Luis; Baltazar, Baltazar M; Bojórquez Bojórquez, Germán; Castro Espinoza, Luciano; Corrales Madrid, José Luis; de la Fuente Martínez, Juan Manuel; Durán Pompa, Héctor Abel; Alonso Escobedo, José; Espinoza Banda, Armando; Garzón Tiznado, José Antonio; González García, Juvencio; Guzmán Rodríguez, José Luis; Madueño Martínez, Jesús Ignacio; Martínez Carrillo, José Luis; Meng, Chen; Quiñones Pando, Francisco Javier; Rosales Robles, Enrique; Ruiz Hernández, Ignacio; Treviño Ramírez, José Elías; Uribe Montes, Hugo Raúl; Zavala García, Francisco

2017-02-01

Environmental risk assessment (ERA) of genetically modified (GM) crops is a process to evaluate whether the biotechnology trait(s) in a GM crop may result in increased pest potential or harm to the environment. In this analysis, two GM insect-resistant (IR) herbicide-tolerant maize hybrids (MON-89Ø34-3 × MON-88Ø17-3 and MON-89Ø34-3 × MON-ØØ6Ø3-6) and one herbicide-tolerant GM hybrid (MON-ØØ6Ø3-6) were compared with conventional maize hybrids of similar genetic backgrounds. Two sets of studies, Experimental Phase and Pilot Phase, were conducted across five ecological regions (ecoregions) in Mexico during 2009-2013, and data were subject to meta-analysis. Results from the Experimental Phase studies, which were used for ERA, indicated that the three GM hybrids were not different from conventional maize for early stand count, days-to-silking, days-to-anthesis, root lodging, stalk lodging, or final stand count. Statistically significant differences were observed for seedling vigor, ear height, plant height, grain moisture, and grain yield, particularly in the IR hybrids; however, none of these phenotypic differences are expected to contribute to a biological or ecological change that would result in an increased pest potential or ecological risk when cultivating these GM hybrids. Overall, results from the Experimental Phase studies are consistent with those from other world regions, confirming that there are no additional risks compared to conventional maize. Results from Pilot Phase studies indicated that, compared to conventional maize hybrids, no differences were detected for the agronomic and phenotypic characteristics measured on the three GM maize hybrids, with the exception of grain moisture and grain yield in the IR hybrids. Since MON-89Ø34-3 × MON-88Ø17-3 and MON-89Ø34-3 × MON-ØØ6Ø3-6 confer resistance to target insect pests, they are an alternative for farmers in Mexico to protect the crop from insect damage. Additionally, the herbicide tolerance conferred by all three GM hybrids enables more cost-effective weed management.
Comparison of Phenotypic and Genotypic Methods for Detection of Diphtheria Toxin among Isolates of Pathogenic Corynebacteria

PubMed Central

Efstratiou, Androulla; Engler, Kathryn H.; Dawes, Charlotte S.; Sesardic, Dorothea

1998-01-01

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the “gold standard” in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of C. diphtheriae possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended. PMID:9774560
Comparison of phenotypic and genotypic methods for detection of diphtheria toxin among isolates of pathogenic corynebacteria.

PubMed

Efstratiou, A; Engler, K H; Dawes, C S; Sesardic, D

1998-11-01

We have compared molecular, immunochemical, and cytotoxic assays for the detection of diphtheria toxin from 55 isolates of Corynebacterium diphtheriae and Corynebacterium ulcerans originally isolated in five different countries. The suitabilities and accuracies of these assays for the laboratory diagnosis of diphtheria were compared and evaluated against the "gold standard" in vivo methods. The in vivo and Vero cell cytotoxicity assays were accurate in their abilities to detect diphtheria toxin but were time-consuming; however, the cytotoxicity assay is a suitable in vitro alternative to the in vivo virulence test. There was complete concordance between all the phenotypic methods. Genotypic tests based upon PCR were rapid; however, PCR must be used with caution because some isolates of C. diphtheriae possessed toxin genes but failed to express a biologically active toxin. Therefore, phenotypic confirmation of toxigenicity for the microbiological diagnosis of diphtheria is recommended.
Using in Vitro Evolution and Whole Genome Analysis To Discover Next Generation Targets for Antimalarial Drug Discovery

PubMed Central

2018-01-01

Although many new anti-infectives have been discovered and developed solely using phenotypic cellular screening and assay optimization, most researchers recognize that structure-guided drug design is more practical and less costly. In addition, a greater chemical space can be interrogated with structure-guided drug design. The practicality of structure-guided drug design has launched a search for the targets of compounds discovered in phenotypic screens. One method that has been used extensively in malaria parasites for target discovery and chemical validation is in vitro evolution and whole genome analysis (IVIEWGA). Here, small molecules from phenotypic screens with demonstrated antiparasitic activity are used in genome-based target discovery methods. In this Review, we discuss the newest, most promising druggable targets discovered or further validated by evolution-based methods, as well as some exceptions. PMID:29451780
shinyGISPA: A web application for characterizing phenotype by gene sets using multiple omics data combinations.

PubMed

Dwivedi, Bhakti; Kowalski, Jeanne

2018-01-01

While many methods exist for integrating multi-omics data or defining gene sets, there is no one single tool that defines gene sets based on merging of multiple omics data sets. We present shinyGISPA, an open-source application with a user-friendly web-based interface to define genes according to their similarity in several molecular changes that are driving a disease phenotype. This tool was developed to help facilitate the usability of a previously published method, Gene Integrated Set Profile Analysis (GISPA), among researchers with limited computer-programming skills. The GISPA method allows the identification of multiple gene sets that may play a role in the characterization, clinical application, or functional relevance of a disease phenotype. The tool provides an automated workflow that is highly scalable and adaptable to applications that go beyond genomic data merging analysis. It is available at http://shinygispa.winship.emory.edu/shinyGISPA/.
shinyGISPA: A web application for characterizing phenotype by gene sets using multiple omics data combinations

PubMed Central

Dwivedi, Bhakti

2018-01-01

While many methods exist for integrating multi-omics data or defining gene sets, there is no one single tool that defines gene sets based on merging of multiple omics data sets. We present shinyGISPA, an open-source application with a user-friendly web-based interface to define genes according to their similarity in several molecular changes that are driving a disease phenotype. This tool was developed to help facilitate the usability of a previously published method, Gene Integrated Set Profile Analysis (GISPA), among researchers with limited computer-programming skills. The GISPA method allows the identification of multiple gene sets that may play a role in the characterization, clinical application, or functional relevance of a disease phenotype. The tool provides an automated workflow that is highly scalable and adaptable to applications that go beyond genomic data merging analysis. It is available at http://shinygispa.winship.emory.edu/shinyGISPA/. PMID:29415010
Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping

PubMed Central

Augustsson, Per; Karlsen, Jonas T.; Su, Hao-Wei; Bruus, Henrik; Voldman, Joel

2016-01-01

Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic focusing of cell lines and leukocytes, showing that acoustic properties provide phenotypic information independent of size. PMID:27180912

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