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Results for “Field phenotyping”

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PubMed2006

Multicolor in vivo time-lapse imaging at cellular resolution by stereomicroscopy.

Intravital time-lapse imaging has altered significantly many long-standing rules of biological mechanisms, but being apparatus-intense and laborious, time-lapse imaging remained mostly restricted to specialized labs. We show that recently introduced, fully automated fluorescence stereomicroscopes represent cost-effective but powerful means of imaging dynamic events ranging from observing embryogenesis over several days to detailed tissue rearrangements and fast blood cell rolling in vivo. When combined with deconvolution approaches, even subcellular resolution in several colors can be achieved. Using three-dimensional image recording, we show the spatial reconstruction of expression patterns. Furthermore, by combining three-dimensional image recording over time with subsequent deconvolution analysis, we demonstrate that subcellular dynamics such as axonal pathfinding can be resolved. These findings promise that time-lapse imaging using a stereomicroscope will become a hands-on standard method for phenotype analysis in many fields of biology.

Animals↗
PubMed1978

HLA antigens-risk factors for primary open-angle glaucoma?

The comparison of the HLA A, B, and C phenotypes of 70 patients with primary open-angle glaucoma (POAG) with the phenotypes of 450 healthy control individuals and the rather discordant data published by other investigators show that there is no genetical influence of HLA A, HLA B, or HLA C genes on POAG and on the clinical complications associated with this disease.

Adult↗
PubMed1989

Rapid enzyme kinetic assays of individual Drosophila and comparisons of field-caught D. melanogaster and D. simulans.

Techniques for performing numerous enzyme kinetic assays with minimum time and effort would be valuable to studies of the evolutionary genetics of metabolic control and the quantitative genetics of determinants of kinetic parameters. Microtiter plate readers have been used for a variety of repetitious analytical techniques, and instruments are available that can take repetitive readings with sufficient speed to perform kinetic assays. The ability of these instruments to assay rapidly the kinetic properties of small samples makes them potentially useful for a number of problems in population genetics. While the ability to handle large numbers of samples is very attractive, the small sample volumes and optical imprecision of microtiter plates result in some sacrifice in accuracy. This paper presents methods for performing kinetic assays on individual field-caught Drosophila, quantifies the precision of these methods, and characterizes differences among Drosophila melanogaster and D. simulans from samples caught in California and Pennsylvania. Comparisons between field-caught and laboratory reared D. melanogaster show that most of the characters are very similar, with the exception of alpha GPDH, which has a threefold higher mean activity among field-caught flies. The phenotypic correlations are presented with a brief discussion of their relevance to assessing the evolution of metabolic control of these enzymes.

Animals↗
PubMed2006

Genotype-by-sex interaction in the aetiology of type 2 diabetes mellitus: support for sex-specific quantitative trait loci in Hypertension Genetic Epidemiology Network participants.

AIMS/HYPOTHESIS: While there are sex-related differences in both the prevalence of type 2 diabetes mellitus and disease risk factors, there is only limited research on sex-specific influences on type 2 diabetes aetiology within the same study population. Thus, we assessed genotype-by-sex interaction using a liability threshold model in an attempt to localise sex-specific type 2 diabetes quantitative trait loci (QTLs). SUBJECTS, MATERIALS AND METHODS: Hypertensive siblings and their offspring and/or parents in the Hypertension Genetic Epidemiology Network of the Family Blood Pressure Program were recruited from five field centres. The diabetic phenotype was adjusted for race, study centre, age and non-linear age effects. In total, 567 diabetic individuals were identified in 385 families. Variance component linkage analyses in the combined sample and stratified by sex and race were performed (SOLAR program) using race-specific marker allele frequencies derived from a random sample of participants at each centre. RESULTS: We observed a QTL-specific genotype-by-sex interaction (p=0.009) on chromosome 17 at 31 cM, with females displaying a robust adjusted logarithm of odds (LOD) of 3.0 compared with 0.2 in males and 1.3 in the combined sample. Three additional regions demonstrating suggestive evidence for linkage were detected: chromosomes 2 and 5 in the female sample and chromosome 22 (adjusted LOD=1.9) in the combined sample. CONCLUSIONS/INTERPRETATION: These findings suggest that multiple genes may regulate susceptibility to type 2 diabetes, demonstrating the importance of considering the interaction of genes and environment in the aetiology of common complex traits.

Adult↗
PubMed2000

Therapy in myotonic disorders and in muscle channelopathies.

Myotonia and muscle weakness are cardinal features of myotonic disorders including the myotonic dystrophies and the non-dystrophic myotonias. Despite the recent progress in molecular genetics of these myotonic disorders, the precise mechanisms responsible for myotonia and for permanent or episodic muscle weakness are still unclear. Treatment has been mostly symptomatic, independent of the disease process involved. Moreover, there have been few randomized controlled trials of treatment for myotonic disorders and consequently no standardized treatment regimens are available. We present a review of selected treatment trials in the myotonic disorders and in muscle channelopathies, and discuss, on the basis of our experience in the myotonic disorders, the limits and advantages of treatment trials in this field. Future genotype-phenotype correlations using the patch-clamp technique are also illustrated.

Calcium Channels↗
PubMed2006

A large-scale collection of phenotypic data describing an insertional mutant population to facilitate functional analysis of rice genes.

In order to facilitate the functional analysis of rice genes, we produced about 50,000 insertion lines with the endogenous retrotransposon Tos17. Phenotypes of these lines in the M2 generation were observed in the field and characterized based on 53 phenotype descriptors. Nearly half of the lines showed more than one mutant phenotype. The most frequently observed phenotype was low fertility, followed by dwarfism. Phenotype data with photographs of each line are stored in the Tos17 mutant panel web-based database with a dataset of sequences flanking Tos17 insertion points in the rice genome (http://tos.nias.affrc.go.jp/). This combination of phenotypic and flanking sequence data will stimulate the functional analysis of rice genes.

DNA Transposable Elements↗
PubMed1987

The segment polarity gene costal-2 in Drosophila. I. The organization of both primary and secondary embryonic fields may be affected.

A series of loss of function alleles at the costal-2 locus is described. Embryos mutant for lethal alleles that are derived from a mutant female germ line display polarity defects on the larval segments. A posterior part of the segmental denticle belt is missing and in its place is a mirror-image duplication of the anterior part including the segment boundary. Maternally rescued embryos are lethal but have normal morphology. Hypomorphic alleles escape to adults that display pattern duplications on the wings and halteres. Dominant gain of function alleles at the Costal-1 locus are also described and data are presented that argue that these are neomorphic and act in trans to impair functioning of costal-2. Some wild-type isoalleles of costal-2 are particularly sensitive to interference from Costal-1 mutations and different combinations of these alleles with Costal-1 can lead to embryos in which the primary embryonic field is disrupted (bicaudal phenotype) and adults with pattern duplications on the anterior compartment of most body segments.

Abdomen↗
PubMed1996

Cell membrane permeability and mitochondrial dysfunction-inducing activities in cell-free supernatants from Serpulina hyodysenteriae serotypes 1 and 2.

Membrane permeability (MP) and mitochondrial dysfunction-inducing (MDI) activities were detected in cell-free supernatants (CFS) of Serpulina hyodysenteriae, using either hemoglobin release from porcine red blood cells (RBC) or cytoplasmic lactate dehydrogenase release from porcine peripheral blood lymphocytes (PBL), and reduction of the 3-(4,5-dimethylthiazoyl-2-y1)-2,5-diphenyltetrazolium bromide dye by porcine PBL. The MP and MDI activities of CFS correlated with each other for serotype 1 and 2 isolates taken at different population densities; however, the kinetics of toxin production varied between each serotype. The loss of enteropathogenicity of two field isolates with nonpathogenic phenotypes and pathogenic isolates passaged up to 45 times in vitro was not attributable to a loss of either membrane permeability or mitochondrial dysfunction-inducing activity of cell-free supernatants. Results from this study suggested the potential for two separate toxins being involved in the pathogenesis of swine dysentery, with the MDI activity correlating with age susceptibility to clinical disease.

Age Factors↗
PubMed2005

Unusual implication of biopsy forceps in outbreaks of Pseudomonas aeruginosa infections and pseudo-infections related to bronchoscopy.

Between January and April 2003, a sudden increase in positive respiratory tract specimens for Pseudomonas aeruginosa was observed in an intensive care unit of the University Teaching Hospital of Montpellier, France. Most of the strains were cultured from bronchoalveolar lavage fluid samples, suggesting that bronchoscopic procedures could be implicated. The relationships between isolates were investigated by antibiotyping and pulsed-field gel electrophoresis. Both phenotypic and molecular markers allowed identification of two consecutive nosocomial outbreaks of respiratory infections related to two different bronchoscopes. These two outbreaks implicated nine and seven patients, respectively. Four of these 16 patients had true infections and recovered with antibiotic therapy. Inspection of both bronchoscopes revealed a damaged internal channel caused by defective biopsy forceps. These defects led to improper cleaning and disinfection of the bronchoscopes despite adherence to all current reprocessing procedures. The two outbreaks were controlled after replacing the inner channels of the bronchoscopes and switching from use of re-usable to disposable biopsy forceps. These outbreaks emphasize the need to establish surveillance procedures for detecting contamination of bronchoscopes, and the importance of recording each endoscopic procedure to facilitate further investigations if needed.

Bronchoalveolar Lavage Fluid↗
PubMed2004

Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea.

Reliable methods for disease severity assessment are of crucial importance in the study of plant pathogen interactions, either for disease diagnostic on the field or to assess phenotypical differences in plants or pathogen strains. Currently, most of the assays used in fungal disease diagnostic rely on visual assessment of the symptoms, lesion diameter measurement or spore counting. However, these tests are tedious and often cannot discriminate between slightly different levels of resistance. Besides, they are not well suited to assess fungal development in the early phases of the infection, before macroscopical symptoms are visible or before sporulation. We describe here a pathogenicity assay based on the relative quantification of fungal and plant DNA in infected Arabidopsis thaliana leaves by means of real-time quantitative PCR. We show that it allows to monitor quantitatively the growth of the fungi Alternaria brassicicola and Botrytis cinerea in a sensitive and reliable way. Although highly sensitive, this test also exhibits a high robustness, which is crucial to significantly discriminate between lines displaying slightly different levels of resistance. Therefore, it allows to assess fungal development from the very first stages of infection and provides a fast and very practical alternative to currently described assays for phenotyping either plant mutant lines or fungal strains.

Alternaria↗
PubMed2001

Changes in properties of adenosine transporters in Trypanosoma evansi and modes of selection of resistance to the melaminophenyl arsenical drug, Mel Cy.

Resistance to arsenical drugs in trypanosomes has been linked to changes in adenosine uptake. The transport of melaminophenyl arsenicals into Trypanosoma brucei was shown to be mediated by an unusual adenosine nucleoside transporter, P2 (Carter and Fairlamb, 1993), and the loss of this transporter is associated with resistance to melaminophenyl arsenicals in these parasites. To further understand the mechanisms of arsenical resistance, we generated several lines of Mel Cy-resistant T. evansi from a drug-sensitive isolate using both in vivo and in vitro selection methods. Uptake of the melaminophenyl arsenical, Mel Cy on the P2 transporter was studied in the drug-sensitive as well as Mel Cy-resistant parasites, by means of inhibition of Mel Cy-induced lysis of trypanosomes, in an in vitro lysis assay. Adenosine uptake was also investigated using competition inhibition assays. Our study shows that T. evansi, TREU 1840, possesses the P1/P2 adenosine transport system as reported in T. brucei and T. equiperdum. However, in T. evansi, the P2 transporter is the larger transport process instead of the P1. The P2 transporter in T. evansi mediated the uptake of Mel Cy in the drug-sensitive parasites. The P2 was retained in all the arsenical-resistant T. evansi lines studied. However, the activity of the transporter was reduced to different extents in the different-resistant lines. The residual P2 activity related well to the levels of drug resistance in each line, suggesting that P2 activity could be an important marker for arsenical resistance. Furthermore, important differences were observed between the in vivo- and the in vitro-selected arsenical-resistant parasites suggesting that there may be differences in resistance phenotypes selected on the field.

Adenine↗
PubMed2002

An evaluation of the role of LIG4 in genomic instability and adaptive mutagenesis in Candida albicans.

The non-homologous end-joining (NHEJ) pathway of DNA recombination is important for genomic stability in animal cells, since the absence of Ku70, Ku80, Lig4 or Xrcc4 results in non-reciprocal translocation and chromosome fragmentation. The role of LIG4 in the genomic instability of Candida albicans has been analyzed. We have found that both cell transformation and 5'-fluoroorotic acid selection steps used to obtain several lig4 mutants (LIG4/lig4 Ura(+); LIG4/lig4 Ura(-); lig4/lig4 Ura(+); lig4/lig4 Ura(-); and revertant lig4/LIG4 Ura(+)) resulted in significant alterations in chromosome R (ChrR). However, this effect is not specific for LIG4, since disruption of SHE9, a gene unrelated to recombination, also caused alterations in the mobility of ChrR. On the other hand, we could not detect reciprocal or non-reciprocal translocations between non-homologous chromosomes in several lig4 mutants. Furthermore, propagation of these mutants in rich medium did not cause other alterations in the mobility of ChrR. Adaptive mutagenesis of C. albicans, determined by the appearance of L-sorbose-utilizing mutants on L-sorbose plates, was also independent of the presence of Lig4 and occurred by monosomy of Chr5. Accordingly, the NHEJ pathway does not appear to be involved in the adaptive mutagenesis mediated by alterations in chromosome copy number.

Candida albicans↗
PubMed1996

Comparison of three molecular methods for typing and subtyping pathogenic Yersinia enterocolitica strains.

The efficiency of pulsed-field gel electrophoresis (PFGE), ribotyping and restriction enzyme analysis of the virulence plasmid (REAP) for typing and subtyping strains of Yersinia enterocolitica was compared. All three techniques gave concordant results, and the strains studied could be separated into three distinct clusters: (1) heterogeneous strains of biotype 1A and serotype O5 (1A/O5); (2) one 3/O3 strain and all 2/O9 strains; and (3) all 4/O3, 2/O5 and two 3/O3 strains. Within cluster 3, the 2/O5 and 3/O3 strains were related more closely to each other than to the 4/O3 isolates. With ribotyping, PFGE and phage-typing, the 4/O3 isolates were subdivided into two homogeneous groups, corresponding to strains of phage type IXb and strains of other phage types, respectively. Similarly, ribotyping and PFGE subdivided the 2/O9 strains into two conserved groups (I and II), but REAP gave a different subdivision and identified a new REAP pattern (P3). The three techniques confirmed the clear distinction between the heterogeneous group of non-pathogenic 1A/O5 strains and the well conserved group of pathogenic 2/O5 strains. Additional plasmids were identified in some 3/O3 strains. Combined, the results indicated that REAP (with EcoRI) and ribotyping (with EcoRV) are valuable alternatives to bioserotype determination, and PFGE is the most suitable technique for epidemiological tracing.

Animals↗
PubMed2024

Causal Mediation Analysis for Integrating Exposure, Genomic, and Phenotype Data.

Causal mediation analysis provides an attractive framework for integrating diverse types of exposure, genomic, and phenotype data. Recently, this field has seen a surge of interest, largely driven by the increasing need for causal mediation analyses in health and social sciences. This article aims to provide a review of recent developments in mediation analysis, encompassing mediation analysis of a single mediator and a large number of mediators, as well as mediation analysis with multiple exposures and mediators. Our review focuses on the recent advancements in statistical inference for causal mediation analysis, especially in the context of high-dimensional mediation analysis. We delve into the complexities of testing mediation effects, especially addressing the challenge of testing a large number of composite null hypotheses. Through extensive simulation studies, we compare the existing methods across a range of scenarios. We also include an analysis of data from the Normative Aging Study, which examines DNA methylation CpG sites as potential mediators of the effect of smoking status on lung function. We discuss the pros and cons of these methods and future research directions.

causal inference↗
PubMed2006

Cell and animal models of mtDNA biology: progress and prospects.

The past two decades have witnessed an evolving understanding of the mitochondrial genome's (mtDNA) role in basic biology and disease. From the recognition that mutations in mtDNA can be responsible for human disease to recent efforts showing that mtDNA mutations accumulate over time and may be responsible for some phenotypes of aging, the field of mitochondrial genetics has greatly benefited from the creation of cell and animal models of mtDNA mutation. In this review, we critically discuss the past two decades of efforts and insights gained from cell and animal models of mtDNA mutation. We attempt to reconcile the varied and at times contradictory findings by highlighting the various methodologies employed and using human mtDNA disease as a guide to better understanding of cell and animal mtDNA models. We end with a discussion of scientific and therapeutic challenges and prospects for the future of mtDNA transfection and gene therapy.

Animals↗
PubMed2002

Distinctive neurophysiological properties of embryonic trigeminal and geniculate neurons in culture.

Neurons in trigeminal and geniculate ganglia extend neurites that share contiguous target tissue fields in the fungiform papillae and taste buds of the mammalian tongue and thereby have principal roles in lingual somatosensation and gustation. Although functional differentiation of these neurons is central to formation of lingual sensory circuits, there is little known about electrophysiological properties of developing trigeminal and geniculate ganglia or the extrinsic factors that might regulate neural development. We used whole cell recordings from embryonic day 16 rat ganglia, maintained in culture as explants for 3-10 days with neurotrophin support to characterize basic properties of trigeminal and geniculate neurons over time in vitro and in comparison to each other. Each ganglion was cultured with the neurotrophin that supports maximal neuron survival and that would be encountered by growing neurites at highest concentration in target fields. Resting membrane potential and time constant did not alter over days in culture, whereas membrane resistance decreased and capacitance increased in association with small increases in trigeminal and geniculate soma size. Small gradual differences in action potential properties were observed for both ganglion types, including an increase in threshold current to elicit an action potential and a decrease in duration and increase in rise and fall slopes so that action potentials became shorter and sharper with time in culture. Using a period of 5-8 days in culture when neural properties are generally stable, we compared trigeminal and geniculate ganglia and revealed major differences between these embryonic ganglia in passive membrane and action potential characteristics. Geniculate neurons had lower resting membrane potential and higher input resistance and smaller, shorter, and sharper action potentials with lower thresholds than trigeminal neurons. Whereas all trigeminal neurons produced a single action potential at threshold depolarization, 35% of geniculate neurons fired repetitively. Furthermore, all trigeminal neurons produced TTX-resistant action potentials, but geniculate action potentials were abolished in the presence of low concentrations of TTX. Both trigeminal and geniculate neurons had inflections on the falling phase of the action potential that were reduced in the presence of various pharmacological blockers of calcium channel activation. Use of nifedipine, omega-conotoxin-MVIIA and GVIA, and omega-agatoxin-TK indicated that currents through L-, N-, and P/Q- type calcium channels participate in the action potential inflection in embryonic trigeminal and geniculate neurons. The data on passive membrane, action potential, and ion channel characteristics demonstrate clear differences between trigeminal and geniculate ganglion neurons at an embryonic stage when target tissues are innervated but receptor organs have not developed or are still immature. Therefore these electrophysiological distinctions between embryonic ganglia are present before neural activity from differentiated receptive fields can influence functional phenotype.

Action Potentials↗