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Results for “Automated phenotyping”

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At least 649 records · Page 36Linked to original sources

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↗

PubMed2002

Two novel variants in the thyroxine-binding globulin (TBG) gene behind the diagnosis of TBG deficiency.

OBJECTIVE: Search for germline mutations in the thyroxine-binding globulin (TBG) gene of two unrelated Portuguese females of Caucasian origin in whom the diagnosis of TBG deficiency was suspected because of suppressed TSH despite marginally low total thyroxine and tri-iodothyronine. DESIGN AND METHODS: Screening for germline mutations was conducted by non-radioactive PCR-SSCP analysis. The variants documented by this approach were characterized by sequencing. Moreover, in order to define whether they were mutations or polymorphisms we looked for the same variants analysing 100 alleles at random. To achieve this goal we used, alternatively, restriction analysis and the minisequencing method with an automated capillary electrophoresis system and fluorescent-labelled dideoxynucleotides. RESULTS AND CONCLUSIONS: Two novel variants, one in each patient, were identified. One, involved codon 23 (TCA-->TAA) and the other, codon 223 (CAA-->TAA). Analysis of 50 DNA samples, randomly chosen, revealed that all were homozygous for the wild variant at codon 23. One of them was heterozygous for the variant CAA-->TAA at codon 223. This sample was found to correspond to a Caucasian female in whom serum TBG proved to be not detected. Since both variants identified result in stop codons likely to induce truncated TBG proteins, they are probably responsible for the TBG phenotype observed in the individuals studied.

Base Sequence↗

PubMed2026

PAT: An Image Analysis Tool for Automated Scoring of Pollen in Alexander-Stained Anthers.

Quantitative pollen viability analysis is a critical but labor-intensive step in plant reproductive biology. Existing deep-learning Segment Anything Models (SAM) fail to reliably segment viable pollen in Alexander-stained anthers. To address this, we fine-tuned an existing Cellpose-SAM model for pollen segmentation. We integrated it into PAT (Pollen Analysis Tool), a cross-platform desktop application. PAT features instance segmentation with interactive quality control, an in-app model retraining module, and publication-ready statistical outputs. We deployed PAT in an EMS suppressor screen of semi-sterile Arabidopsis smg7-6 mutants, enabling efficient candidate prioritization for whole-genome sequencing and mapping of the candidate mutation. This screen led to the identification of a point mutation in CAP-D2 (capd2-2), a Condensin I subunit, that rescues the smg7-6 meiotic phenotype. Notably, mutation in a Condensin II subunits (CAP-D3 and CAP-H2) does not confer rescue. Further characterization suggests the capd2-2 allele is hypomorphic, showing no defects in vegetative growth, chromocenter compaction, or transposable element silencing. Collectively, we demonstrate that accessible AI tools have the potential to bridge gaps in plant phenotyping and accelerate the pace of biological discovery.

Alexander staining↗

PubMed1998

Rapid, efficient method for multiplex amplification from filter paper.

Guthrie cards derived from the New York State Newborn Screening Program were utilized to develop a rapid, economical method for amplifying multiple genes to detect mutations that impact public health. These specimens are untraceable to the donor because identifiers are removed and discarded; therefore, these pilot studies were carried out anonymously. The sample preparation requires minimal manipulation, is amenable to automation, and is useful in laboratories which routinely process large numbers of samples, such as those in typical newborn screening laboratories. Multiple gene fragments may be amplified from a 1 mm punch which contains less than 1 microl of whole blood. The blood spots used in these studies contain sufficient material for up to 25 amplification reactions which multiplex at least four different gene fragments each. Since sufficient material remains on the card after the routine testing is complete, this simple assay can greatly expand the efficacy of current newborn screening programs by permitting DNA diagnosis of some disorders when indicated, particularly those in which genotype-phenotype correlations are useful. In addition to newborn screening specimens, this method is also applicable to whole blood from adults after phlebotomy and from lymphoblastoid cell lines. Use of filter paper for DNA analysis is particularly useful for shipped specimens or for population studies whose subjects are refractory to phlebotomy.

Alleles↗

PubMed2001

Beryllium sensitivity is linked to HLA-DP genotype.

Chronic beryllium disease (CBD) appears to arise from a combination of both exposure and genetic risk factors. A distinguishing feature of CBD is beryllium hypersensitivity, which can be measured in vitro by a lymphocyte proliferation test. The objective of this study was to determine whether certain allelic variations of the HLA-DPB1 gene, which had been observed previously in CBD, could be found in a group of individuals having beryllium hypersensitivity, but no symptoms of CBD. A flow cytometry-based Lymphocyte Proliferation Test combined with immunophenotyping (Immuno-LPT) was used to detect CD4+ and CD8+ T cell proliferation in response to in vitro stimulation with beryllium. The HLA-DPB1 haplotypes of the same individuals were determined by automated DNA sequencing. Twenty-two out of 25 beryllium-sensitive, non-CBD individuals were found to be carriers of the HLA-DPB1 gene having a substitution of a glutamic acid at position 69 in Exon 2 (Glu69), and a significantly high percentage (24%) were Glu69 homozygotes. Most of the CD4+ responders on the Immuno-LPT (10/14) carried rare, non-*0201 Glu69 DPB1 alleles; while most of the non-CD4+ responders (9/11) were common Glu69 carriers (*0201 or *0202) or non-Glu69 individuals (non-Glu69/non-Glu69). This is the first direct evidence that HLA-DP genotype is linked to a phenotypic response that occurs in beryllium sensitization in the absence of clinical CBD.

Berylliosis↗

PubMed1990

An automated method to prepare cell suspensions from human biopsy samples for immunophenotyping by flow cytometry.

A comparison was made between a manual and automated method for preparing single cell suspensions from various types of human biopsy samples. The automated method uses the shearing action of fluid movement generated by a reciprocating paddle system. When compared on 11 samples, the automated instrument always isolated cells in less time, usually requiring only 15-30 seconds in contrast to 10-15 minutes for the manual method. The time comparisons involved "set up" time of the required minor equipment as well as the time to make a complete single cell suspension from the portion of the biopsy sample sent to the Flow Cytometry Lab so extra cells could be used for other purposes and cryogenically stored for future reference. Cells isolated by the automated method from various B-lymphocytic and T-lymphocytic malignancies were still viable and could be successfully immunophenotyped by flow cytometry. The immunophenotyping results were compared for both cell isolation methods on seven of these samples, and the results were comparable. The automated technique has now been used to satisfactorily immunophenotype more than 50 biopsy samples. The automated method should represent a significant aid for clinical flow cytometry laboratories performing immunophenotyping tests by efficiently preparing single cell suspensions in a few seconds instead of minutes. Furthermore, the automated method uses a closed sterile bag system that helps minimize the exposure of personnel to potential infectious material present in biopsy samples and prevents external contamination of cell suspensions. The automated technique proven successful for immunophenotyping may also be helpful for related procedures involving hematopoietic malignancies such as DNA content analysis and cell functional assays by flow cytometry, as well as other assays such as tissue typing, gene probe, and in vitro chemosensitivity assays.

Antigens, CD↗

PubMed1994

Detection of RET proto-oncogene point mutations in paraffin-embedded pheochromocytoma specimens by nonradioactive single-strand conformation polymorphism analysis and direct sequencing.

The suitability of formalin-fixed and paraffin-embedded tumor material was evaluated for molecular analysis of the RET proto-oncogene. We analyzed exons 10, 11, and 16 for point mutations in seven sporadic and six multiple endocrine neoplasia (MEN) 2A-associated pheochromocytomas by a nonradioactive single-strand conformation polymorphism assay followed by nonradioactive direct sequencing of PCR-amplified DNA using an automated DNA sequencer. All MEN 2A-associated pheochromocytomas contained a heterozygous missense germline mutation within cystine codons of the cysteine-rich extracellular domain encoded by exons 10 and 11. Mutations were located in codon 619 (TGC-->TCC; Cys-->Ser) in one, in codon 635 (TGC-->CGC; Cys--Arg) in three, and in codon 635 (TGC-->TAC; Cys-->Tyr) in two pheochromocytomas. No tumor-specific (somatic) mutations were detected in exons 10, 11, and 16 of the sporadic pheochromocytomas. These data support recent findings that germline point mutations that are clustered in distinct cysteine codons of the RET proto-oncogene are involved in the neoplastic phenotype of the MEN 2A syndrome. Our results demonstrate that both nonradioactive single-strand conformation polymorphism and direct sequencing are suitable methods to detect single base substitutions in DNA extracted from archival material.

Adolescent↗

PubMed1977

Nature and nurture in the expression of diabetes mellitus and its vascular manifestations.

The prevailing concept of etiologic heterogeneity for the diabetes mellitus syndrome is one of multiple genetic factors interacting with a variety of environmental influences. Variation in expression of the disorder, particularly the need for insulin, does not correlate with known etiologic distinctions. There is much evidence for genetic heterogeneity, as well as phenotypic variation when etiology can be presumed to be identical. The vascular manifestations of diabetes include microangiopathy unique to diabetes and larger vessel disease that differs from that of normal aging only by its prematurity. There is as much evidence for heterogeneity of the vascular expression as there is for glucose intolerance. Approximately 25% of persons with insulin-dependent diabetes may never develop the microvascular disease. The pathogenesis of vascular disease in diabetes may involve a number of abnormalities of plasma, circulating cells, and vascular tissue. Were absolute control of glycemia possible, some of the contributing factors involved in vasculopathy would possibly be alleviated. In the absence of automated physiologic insulin replacement the potential deleterious effect of our current methods of treatment might be reduced by specific inhibition of excess catecholamine, growth hormone and/or glucagon responses.

Aging↗

PubMed2003

From annotated genomes to metabolic flux models and kinetic parameter fitting.

Significant advances in system-level modeling of cellular behavior can be achieved based on constraints derived from genomic information and on optimality hypotheses. For steady-state models of metabolic networks, mass conservation and reaction stoichiometry impose linear constraints on metabolic fluxes. Different objectives, such as maximization of growth rate or minimization of flux distance from a reference state, can be tested in different organisms and conditions. In particular, we have suggested that the metabolic properties of mutant bacterial strains are best described by an algorithm that performs a minimization of metabolic adjustment (MOMA) upon gene deletion. The increasing availability of many annotated genomes paves the way for a systematic application of these flux balance methods to a large variety of organisms. However, such a high throughput goal crucially depends on our capacity to build metabolic flux models in a fully automated fashion. Here we describe a pipeline for generating models from annotated genomes and discuss the current obstacles to full automation. In addition, we propose a framework for the integration of flux modeling results and high throughput proteomic data, which can potentially help in the inference of whole-cell kinetic parameters.

Algorithms↗

PubMed2000

BRCA1 mutations and clinicopathological features in a sample of Italian women with early-onset breast cancer.

Breast cancer in young women is uncommon and often presents with unfavourable biopathological features. Although early age at onset could suggest a genetic susceptibility to cancer, the appropriateness of BRCA1 testing for women with early-onset breast cancer and modest family history (FH) is controversial. 40 Women diagnosed with breast cancer at the age of 35 years or less, unselected for FH, were screened for germ line BRCA1 mutations by automated sequencing of exons 2, 5, 6, 11, 13 and 20. Overall, deleterious mutations were evidenced in 6 (15%) patients. With regard to FH, mutations were detected in 14%, 11% and 29% of women with none, weak and strong FH, respectively. Large tumour size, grade 3, lack of oestrogen receptors and high proliferation rate were significantly more common in mutation carriers (MC). Our data support both the appropriateness of testing young breast cancer patients and the frequency of unfavourable features in BRCA1-related breast cancer. It is hypothesised that BRCA1 mutations partially justify the high rate of aggressive breast cancer in young patients and that combining age and breast cancer phenotype could help to identify probable MC.

Adult↗

PubMed2001

Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina.

Staphylococcus aureus is the most prevalent pathogen causing mastitis of dairy ruminants. This study was developed to ascertain the genotypes and genealogical relationship among strains isolated from milk of bovines with mastitis in Argentina. Molecular epidemiological analysis of S. aureus was performed on 112 isolates from 21 districts. Clonality was assessed by SmaI pulsed-field gel electrophoresis (PFGE) typing, automated EcoRI ribotyping and restriction enzyme analysis of plasmid (REAP) DNA profiles. A total of 22 band patterns distributed in four clusters were found by SmaI PFGE analysis. The similarity of clusters 2, 3 and 4 with cluster 1 was 0.73, 0.69 and 0.33, respectively, and 101 of 112 isolates belonged in cluster 1. PFGE band patterns from 42 isolates within cluster I were indistinguishable from each other (type A). The second largest group of isolates with indistinguishable PFGE band patterns was subtype A11, which was composed of 19 isolates. Automated ribotyping assigned the 112 isolates into 13 ribotypes. Among these, the most prevalent ribotypes I and VI were composed of 49 and 35 isolates respectively. Although there was certain correspondence between PFGE genotypes and ribotypes, further discrimination was achieved by combining both methods. REAP DNA profile analysis was useful to provide even further discrimination between isolates with identical PFGE genotype and ribotype. The most prevalent S. aureus strains A/I and A11/VI were widely distributed in the country and were not restricted to individual nearby locations. Prevalence of these two strains varied consecutively within a period of 8 years. Whether the shift in type prevalence was due to selection of a phenotypic trait remains undisclosed.

Animals↗

PubMed2002

Increased gyrification in Williams syndrome: evidence using 3D MRI methods.

Understanding patterns of gyrification in neurogenetic disorders helps to uncover the neurodevelopmental etiology underlying behavioral phenotypes. This is particularly true in Williams syndrome (WS), a condition caused by de novo deletion of approximately 1 to 2 Mb in the 7q11.23 region. Individuals with WS characteristically possess an unusual dissociation between deficits in visual-spatial ability and relative preservations in language, music, and social drive. A preliminary postmortem study reported anomalous gyri and sulci in individuals with WS. The present study examined gyrification patterns in 17 participants with WS (10 females, 7 males; mean age 28 years 11 months, SD 8 years 6 months) and 17 age- and sex-matched typically developing control participants (mean age 29 years 1 month, SD 8 years 1 month) using new automated techniques in MRI. Significantly increased cortical gyrification was found globally with abnormalities being more marked in the right parietal (p=0.0227), right occipital (p=0.0249), and left frontal (p=0.0086) regions. These results suggest that one or more genes in the 7q11.23 region are involved during the critical period when cortical folding occurs, and may be related to the hypothesized dorsal/ventral dissociation in this condition.

Adult↗

PubMed2006

Characterization of methicillin-resistant Staphylococcus aureus from Ulaanbaatar, Mongolia.

In order to expand current knowledge of the types of methicillin-resistant Staphylococcus aureus (MRSA) strains circulating in central Asia, six MRSA strains collected from hospitals in Ulaanbaatar, Mongolia during 2000-2002 were examined. Three strains possessed a staphylococcal cassette chromosome mec (SCCmec) element of type IV c, were sequence type (ST) 154 according to multilocus sequence typing (MLST), and contained lukS-lukF (Panton-Valentine leukocidin). Another three strains contained a SCCmec element of type III and were MLST type ST 239. Using automated ribotyping, the six MRSA strains were divided into four different EcoRI ribotypes, and two groups of isolates were distinguished by means of SmaI-macrorestriction patterns. In comparison to other countries, the incidence of MRSA in Mongolia is low.

Anti-Bacterial Agents↗

PubMedDate not supplied

Primary structure of a chloramphenicol acetyltransferase specified by R plasmids.

Naturally occurring isolates of chloramphenicol-resistant bacteria commonly synthesise chloramphenicol acetyltransferase (EC 2.3.28; CAT) in amounts which are sufficient to account for the resistance phenotype and often harbour plasmids which carry the structural gene for CAT. The findings of CAT in such diverse prokaryotes as Proteus mirabilis, Agrobacterium tumefaciens, Streptomyces sp., and a soil Flavobacterium has led to speculation concerning the origin and evolution of the more commonly observed CAT variants specified by plasmids in clinically important bacteria. To provide a more solid basis for studying the evolution and spread of CAT within prokaryotes we chose to determine the complete amino acid sequence of a type I variant of CAT, the variant known to be associated with most F-like plasmids conferring chloramphenicol resistance. The sequence has been determined by combining the results obtained from manual and automated sequential degradation with those obtained by mass spectrometry of peptides generated by enzymatic digestion. The directly determined primary structure is identical with that predicted by the DNA sequence analysis of the chloramphenicol resistance transponson Tn9 known to specify a type I variant of chloramphenicol acetyltransferase.

Acetyltransferases↗

PubMed1992

Application of PhastSystem to the resolution of bovine milk proteins on urea-polyacrylamide gel electrophoresis.

Optimal conditions were established for alkaline urea-PAGE using modified precast, ultrathin gradient gels on the automated PhastSystem. Profiles of milk proteins showed that the caseins and whey proteins resolved extremely well. Major bands were observed for alpha s1-casein and beta-casein, and alpha s2-casein appeared as a well-resolved doublet. In contrast, kappa-casein separated from other caseins as a faint doublet, and purified kappa-casein appeared as one major and one minor band. Whey proteins (serum albumin, alpha-lactalbumin, beta-lactoglobulin) separated into broad bands resolved from each other and from the caseins. Partially (40%) dephosphorylated whole casein showed multiple bands for alpha s1-casein and beta-casein at different levels of phosphorylation. Separation of genetic phenotypes was observed for beta-lactoglobulin A and B; alpha s1-casein A, B, and C; and beta-casein A, B, and C. Electrophoretic patterns of milk proteins extracted from cheese samples varied among the different types of cheeses. Our modified procedure provides researchers with a rapid technique to separate both caseins and whey proteins on the same urea gel according to their charge to mass ratios.

Animals↗

PubMed1999

Quality control in the endoplasmic reticulum: lessons from hereditary myeloperoxidase deficiency.

The optimal level of oxygen-dependent microbicidal activity in human neutrophils depends on the generation of highly toxic products, including hypochlorous acid, by hydrogen peroxide in the presence of chloride anion and the neutrophil granule protein myeloperoxidase (MPO). The biosynthesis of MPO is normally restricted to the promyelocytic stage of myeloid development and includes N-linked glycosylation, heme insertion, proteolytic processing, subunit dimerization, and eventual targeting to the azurophilic granule. In the endoplasmic reticulum, MPO precursors interact transiently with calreticulin and calnexin, presumably in their capacity as molecular chaperones. In light of the important role of the MPO-H2O2-chloride system in human host defense, the relatively high prevalence of inherited MPO deficiency was an unanticipated insight provided by the widespread use of automated flow cytometry for the enumeration of leukocytes in clinical specimens. In many cases of inherited MPO deficiency, affected neutrophils have immunochemical evidence of precursor protein but lack the subunits of mature MPO, peroxidase activity, or the ability to chlorinate target proteins. To date, four genotypes have been reported to cause inherited MPO deficiency, each of which results in missense mutations. In the genotype Y173C, the mutant precursor is retained in the endoplasmic reticulum by virtue of its prolonged interaction with calnexin, and it eventually undergoes degradation in the 20S proteasome. In this way, the quality control system operating in the endoplasmic reticulum retrieves malfolded MPO precursors from the biosynthetic pathway and creates the biochemical phenotype of MPO deficiency. Thus MPO deficiency caused by Y173C joins the ranks of cystic fibrosis, protein C deficiency, and other genetic disorders that reflect abnormalities in protein folding.

Amino Acid Sequence↗

PubMed2005

Biological ontologies in rice databases. An introduction to the activities in Gramene and Oryzabase.

An enormous amount of information and materials in the field of biology has been accumulating, such as nucleotide and amino acid sequences, gene and protein functions, mutants and their phenotypes, and literature references, produced by the rapid development in this field. Effective use of the information may strongly promote biological studies, and may lead to many important findings. It is, however, time-consuming and laborious for individual researchers to collect information from individual original sites and to rearrange it for their own purpose. A concept, ontology, has been introduced in biology to support and encourage researchers to share and reuse information among biological databases. Ontology has a glossary, named dynamic controlled vocabulary, in which relationships between terms are defined. Since each term is strictly defined and identified with an ID number, a set of data represented in biological ontology is easily accessible to automated information processing, even if the data sets are across several databases and/or different organisms. In this mini-review, we introduce activities in Gramene and Oryzabase, which provide biological ontologies for Oryza sativa (rice).

Databases, Genetic↗

PubMed2004

Red cell freezing and its impact on the supply chain.

Red blood cells (RBC) can be frozen in glycerol solutions and stored for many years. Thawed RBC must have the glycerol removed, but the recovered cells have normal survival in humans. Freezing has been used to store RBC of rare phenotypes for more than 40 years. In the 1960s and 1970s, when medical technology and blood use were expanding rapidly and liquid whole blood and RBC storage were limited to 3 weeks, many attempts were made to expand the use of frozen RBC for meeting the needs for a stable blood supply and to have RBC reserves for emergencies. These attempts have largely been abandoned because of the cost of freezing, storing and processing, better management of the larger and longer lived RBC inventory, concerns about the safety of stored RBC that have not received the most up-to-date testing and the losses associated with the short shelf life of thawed RBC. New automated frozen RBC processing systems will potentially allow extending the outdate of thawed RBC to 2 weeks, but will not materially effect the costs or losses associated with the use of frozen RBC. RBC freezing will have little effect on the logistics of blood supply.

Blood Banks↗