SEARCH · PubMed Health

Results for “Automated phenotyping”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 559 records · Page 31Linked to original sources

PubMed2001

Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy.

BACKGROUND: There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical scale method to prepare autologous DCs for cancer clinical trials. METHODS: PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 x 10(6) cells/mL for 5-7 days at 37 degrees C, with 5% CO(2), with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat in activated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus. RESULTS: DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0 +/- 3.7 x 10(8) fresh or cryopreserved autologous monocytes, DC yield was 2.1 +/- 1.0 x 10(8) cells, or 29% of the starting monocyte number. DISCUSSION: In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 x 10(6) cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.

Cell Culture Techniques↗

PubMed2006

Bio-Ontology and text: bridging the modeling gap.

MOTIVATION: Natural language processing (NLP) techniques are increasingly being used in biology to automate the capture of new biological discoveries in text, which are being reported at a rapid rate. Yet, information represented in NLP data structures is classically very different from information organized with ontologies as found in model organisms or genetic databases. To facilitate the computational reuse and integration of information buried in unstructured text with that of genetic databases, we propose and evaluate a translational schema that represents a comprehensive set of phenotypic and genetic entities, as well as their closely related biomedical entities and relations as expressed in natural language. In addition, the schema connects different scales of biological information, and provides mappings from the textual information to existing ontologies, which are essential in biology for integration, organization, dissemination and knowledge management of heterogeneous phenotypic information. A common comprehensive representation for otherwise heterogeneous phenotypic and genetic datasets, such as the one proposed, is critical for advancing systems biology because it enables acquisition and reuse of unprecedented volumes of diverse types of knowledge and information from text. RESULTS: A novel representational schema, PGschema, was developed that enables translation of phenotypic, genetic and their closely related information found in textual narratives to a well-defined data structure comprising phenotypic and genetic concepts from established ontologies along with modifiers and relationships. Evaluation for coverage of a selected set of entities showed that 90% of the information could be represented (95% confidence interval: 86-93%; n = 268). Moreover, PGschema can be expressed automatically in an XML format using natural language techniques to process the text. To our knowledge, we are providing the first evaluation of a translational schema for NLP that contains declarative knowledge about genes and their associated biomedical data (e.g. phenotypes). AVAILABILITY: http://zellig.cpmc.columbia.edu/PGschema

Abstracting and Indexing↗

PubMed2002

HGVbase: a human sequence variation database emphasizing data quality and a broad spectrum of data sources.

HGVbase (Human Genome Variation database; http://hgvbase.cgb.ki.se, formerly known as HGBASE) is an academic effort to provide a high quality and non-redundant database of available genomic variation data of all types, mostly comprising single nucleotide polymorphisms (SNPs). Records include neutral polymorphisms as well as disease-related mutations. Online search tools facilitate data interrogation by sequence similarity and keyword queries, and searching by genome coordinates is now being implemented. Downloads are freely available in XML, Fasta, SRS, SQL and tagged-text file formats. Each entry is presented in the context of its surrounding sequence and many records are related to neighboring human genes and affected features therein. Population allele frequencies are included wherever available. Thorough semi-automated data checking ensures internal consistency and addresses common errors in the source information. To keep pace with recent growth in the field, we have developed tools for fully automated annotation. All variants have been uniquely mapped to the draft genome sequence and are referenced to positions in EMBL/GenBank files. Data utility is enhanced by provision of genotyping assays and functional predictions. Recent data structure extensions allow the capture of haplotype and genotype information, and a new initiative (along with BiSC and HUGO-MDI) aims to create a central repository for the broad collection of clinical mutations and associated disease phenotypes of interest.

Base Sequence↗

PubMed2005

The ChemoFx assay: an ex vivo cell culture assay for predicting anticancer drug responses.

We provide a detailed description of the ChemoFx Assay, a phenotype-based cell culture assay for predicting anticancer drug responses in individual cancer patients. The ChemoFx Assay is based on the outgrowth and short-term primary culture of epithelial cells derived from pieces of solid tumor adenocarcinomas that are obtained at the time of tumor resection. Malignant epithelial cells are grown attached in wells of microtiter plates and treated with six escalating doses of chemotherapeutic drug. Using an operator-controlled automated image analysis system, cell kill is measured microscopically by counting the number of live cells remaining after dead cells have detached and are subsequently rinsed away. A dose-response graph is automatically generated by comparing the number of cells in drug-treated wells with those in control wells.

Adenocarcinoma↗

PubMed1995

Molecular diagnosis of mucopolysaccharidosis type II (Hunter syndrome) by automated sequencing and computer-assisted interpretation: toward mutation mapping of the iduronate-2-sulfatase gene.

Virtually all mutations causing Hunter syndrome (mucopolysaccharidosis type II) are expected to be new mutations. Therefore, as a means of molecular diagnosis, we developed a rapid method to sequence the entire iduronate-2-sulfatase (IDS) coding region. PCR amplicons representing the IDS cDNA were sequenced with an automatic instrument, and output was analyzed by computer-assisted interpretation of tracings, using Staden programs on a Sun computer. Mutations were found in 10 of 11 patients studied. Unique missense mutations were identified in five patients: H229Y (685C-->T, severe phenotype); P358R (1073C-->G, severe); R468W (1402C-->T, mild); P469H (1406C-->A, mild); and Y523C (1568A-->G, mild). Non-sense mutations were identified in two patients: R172X (514C-->T, severe) and Q389X (1165C-->T, severe). Two other patients with severe disease had insertions of 1 and 14 bp, in exons 3 and 6, respectively. In another patient with severe disease, the predominant (> 95%) IDS message resulted from aberrant splicing, which skipped exon 3. In this last case, consensus sequences for splice sites in exon 3 were intact, but a 395 C-->G mutation was identified 24 bp upstream from the 3' splice site of exon 3. This mutation created a cryptic 5' splice site with a better consensus sequence for 5' splice sites than the natural 5' splice site of intron 3. A minor population of the IDS message was processed by using this cryptic splice site; however, no correctly spliced message was detected in leukocytes from this patient. The mutational topology of the IDS gene is presented.

Amino Acid Sequence↗

PubMed2006

Small molecule screening by imaging.

Useful small molecule tools can be discovered in imaging screens that measure phenotype in single cells or small organisms. Recent examples include identification of small molecule inhibitors of processes such as cell migration, cytokinesis, mitotic spindle length determination, melanogenesis, aggresome formation, membrane transport and nuclear export. Imaging screens are currently limited by challenges in the areas of image analysis and target identification. We discuss the use of model organisms such as zebrafish in screens and review different methods of target identification. The emerging field of automated image analysis is also introduced.

Animals↗

PubMed2001

Electronic detection of single-base mismatches in DNA with ferrocene-modified probes.

Genotyping and gene-expression monitoring is critical to the study of the association between genetics and drug response (pharmacogenomics) and the association of sequence variation with heritable phenotypes. Recently, we developed an entirely electronic method for the detection of DNA hybridization events by the site-specific incorporation of ferrocenyl derivatives into DNA oligonucleotides. To perform rapid and accurate point mutation detection employing this methodology, two types of metal-containing signaling probes with varying redox potentials are required. In this report we describe a new ferrocene-containing phosphoramidite 9 that provides a range of detectable redox potentials. Using automated DNA/RNA synthesis techniques the two ferrocenyl complexes were inserted at various positions along oligonucleotide probes. Thermal stability analysis of these metal-containing DNA oligonucleotides indicates that incorporation of 9 resulted in no destabilization of the duplex. A mixture of oligonucleotides containing compounds 9 and I was analyzed by alternating current voltammetry (ACV) monitored at the 1st harmonic. The data demonstrate that the two ferrocenyl oligonucleotide derivatives can be distinguished electrochemically. A CMS-DNA array was prepared on an array of gold electrodes on a printed circuit board substrate with a self-assembled mixed monolayer, coupled to an electronic detection system. Experiments for the detection of a single-base match utilizing two signaling probes were carried out. The results demonstrate that rapid and accurate detection of a single-base mismatch can be achieved by using these dual-signaling probes on CMS-DNA chips.

Base Pair Mismatch↗

PubMedDate not supplied

Expansion of activated lymphocytes obtained from renal cell carcinoma in an automated hollow fiber bioreactor.

Immunotherapy using IL-2 alone or combined with activated lymphocytes has been promising for metastatic renal cell carcinoma. Cytotoxic lymphocytes can be isolated from tumors, expanded in vitro with IL-2, and adoptively transferred back into the tumor-bearing host. These cells can also be transduced with the genes coding for cytokines for local delivery to tumor sites. A major drawback in adoptive immunotherapy is the cumbersome and expensive culture technology associated with the growth of large numbers of cells required for their therapeutic effect. To reduce the cost, resources, and manpower, we have developed the methodology for lymphocyte activation and expansion in the automated hollow fiber bioreactor IMMUNO*STAR Cell Expander (ACT BIOMEDICAL, INC). Tumor Infiltrating Lymphocytes (TIL) isolated from human renal cell carcinoma tumor specimens were inoculated at a number of 10(8) cells in a small bioreactor of 30 ml extracapillary space volume. We have determined the medium flow rates and culture conditions to obtain a significant and repeated expansion of TIL at weekly intervals. The lymphocytes cultured in the bioreactor demonstrated the same phenotype and cytotoxic activity as those expanded in parallel in tissue culture plates. Lymphocyte expansion in the hollow fiber bioreactor required lower volumes of medium, human serum, IL-2 and minimal labor. This technology may facilitate the use of adoptive immunotherapy for the treatment of refractory malignancies.

Biotechnology↗

PubMed2001

Issues in the epidemiology of melanoma.

Cutaneous melanoma is a significant health problem throughout the developing world. Primary and secondary prevention are discussed. The wavelengths of the ultraviolet radiation spectrum and their association with melanoma are discussed. Although excessive sun exposure during childhood is a critical risk factor, excessive sun exposure during adult years is also important. The major risk factors for melanoma--numerous or atypical moles and a sun-sensitive phenotype--are genetic. Their interaction with sun exposure is currently being examined, as well as the interaction of other genetic factors, such as alterations in the melanocortin receptor and the familial melanoma gene, INK4A. Secondary prevention strategies include self-examination and physician examination. New technologies are being developed to supplement visual examination of suspected lesions. These technologies are discussed in detail and include digital photography, digital dermoscopy, confocal scanning laser microscopy and automated diagnosis systems.

Humans↗

PubMed2003

Identification of Phoenix dactylifera L. varieties based on amplified fragment length polymorphism (AFLP) markers.

The amplified fragment length polymorphism (AFLP) technique was applied to identify palm varieties. Fluorescence labelled primers were used in selective amplifications and the amplified fragments were detected on capillary gel electrophoresis using an automated DNA sequencer with the analysis fragment option. This is a rapid and efficient technique for detecting a large number of DNA markers on the date palm. Phoenix dactylifera L. varieties Bou-Fegous, Medjool, and E-528 from Estación Phoenix (Elche), Spain, were analysed, yielding a total of 310 AFLP fragments derived from five primer combinations. The process for regenerating the date palm cultivars from in vitro tissue culture should yield individuals phenotypically and genetically identical to the explant they are derived from. The AFLP markers obtained were successfully used for comparing and identifying vitroplants of palm.

Arecaceae↗

PubMed1993

Structure-function relationship studies in human cholinesterases reveal genomic origins for individual variations in cholinergic drug responses.

1. Due to their involvement in the termination of neurotransmission at cholinergic synapses and neuromuscular junctions, cholinesterases are the target proteins for numerous drugs of neuro-psychopharmacology importance. 2. In order to perform structure-function relationship studies on human cholinesterases with respect to such drugs, a set of expression vectors was engineered, all of which include cloned cDNA inserts encoding various forms of human acetyl- and butyrylcholinesterase. These vectors were designed to be transcribed in vitro into their corresponding mRNA products which, when microinjected into Xenopus oocytes, are efficiently translated to yield their catalytically active enzymes, each with its distinct substrate specificity and sensitivity to selective inhibitors. 3. A fully automated microtiter plate assay for evaluating the inhibition of said enzymes by tested cholinergic drugs and/or poisons has been developed, in conjunction with computerized data analysis, which offers prediction of such inhibition data on the authentic human enzymes and their natural or mutagenized variants. 4. Thus, it was found that asp70-->gly substitution renders butyrylcholinesterase succinylcholine insensitive and resistant to oxime reactivation while ser 425-->Pro with gly70 gives rise to the "atypical" butyrylcholinesterase phenotype, abolishing dibucaine binding. 5. Furthermore, differences in cholinesterase affinities to physostigmine, ecothiophate and bambuterol were shown in these natural variants. 6. Definition of key residues important for drug interactions may initiate rational design of more specific cholinesterase inhibitors, with fewer side effects. This, in turn, offers therapeutic potential in the treatment of clinical syndromes such as Alzheimer's and Parkinson's disease, glaucoma and myasthenia gravis.

Animals↗

PubMed2003

Interpretation systems for genotypic drug resistance of HIV-1.

Genotypic assays are widely used tools for determining human immunodeficiency virus type 1 (HIV-1) drug resistance and for guiding treatment changes in patients failing antiretroviral therapy. Several systems have been developed to interpret the complex influence of key amino acid substitutions of the enzymes targeted by therapy on the phenotypic susceptibility or clinical response to available antiretroviral agents. This overview identifies 21 systems giving an interpretation on how amino acid substitutions affect phenotypic drug susceptibility or clinical activity of anti-HIV-1 agents. There was substantial variability in the mechanisms underlying the interpretations, the nature of the systems, their intended use, the source type of their knowledge base, and their update and output. Most of the systems could be accessed for free on the internet, functioned as rule-based algorithms updated by experts and at least partially based on literature evidence, and offered an automated report through a software. Nevertheless, the rule base was not always clarified. An update of the rules and the clinical validation of the systems are presented to help in the critical evaluation of their possible use. Importantly, only 8 systems were intended for clinical use and 5 of these had at least partially undergone clinical validation.

Anti-HIV Agents↗

PubMed2005

Use of time-saving flow cytometry for rapid determination of resistance of human cytomegalovirus to ganciclovir.

There are two ways to assess the susceptibility of human cytomegalovirus (HCMV) to ganciclovir (GCV): one is a genotypic test that detects resistance-related mutations and the other is a phenotypic test that actually assesses susceptibility. The advantages of genotyping the UL 97 gene are its rapidity and accuracy. However, to detect novel mutations or mutations affecting the UL 54 DNA polymerase, a phenotypic test such as the plaque reduction assay (PRA) is also required. To avoid the shortcomings of PRA such as its time-consuming nature and labor-intensiveness, we developed a time-saving fluorescence-activated cell sorting (TS-FACS) technique. We obtained a GCV 50% inhibitory concentration (IC(50)) from five clinical isolates and an HCMV laboratory strain (AD169) and compared the results with those from the PRA. The laboratory strain and three clinical isolates were sensitive to GCV. Although there was a minor discrepancy in the case of one of the three isolates, the GCV IC(50) values obtained by TS-FACS analysis correlated well with the results of the PRA. The remaining two isolates were resistant to GCV; one was GCV resistant due to the mutation M 460 V, and the GCV IC(50) results obtained by TS-FACS analysis and by PRA were also comparable. The advantages of TS-FACS analysis are the shorter time required, the possibility of automation, and its comparability to PRA, considered the gold standard. Thus, TS-FACS analysis may be useful as an alternative to PRA in the clinic.

Amino Acid Sequence↗

PubMed2002

High-throughput behavioral screening method for detecting auditory response defects in zebrafish.

We have developed an automated, high-throughput behavioral screening method for detecting hearing defects in zebrafish. Our assay monitors a rapid escape reflex in response to a loud sound. With this approach, 36 adult zebrafish, restrained in visually isolated compartments, can be simultaneously assessed for responsiveness to near-field 400 Hz sinusoidal tone bursts. Automated, objective determinations of responses are achieved with a computer program that obtains images at precise times relative to the acoustic stimulus. Images taken with a CCD video camera before and after stimulus presentation are subtracted to reveal a response to the sound. Up to 108 fish can be screened per hour. Over 6500 fish were tested to validate the reliability of the assay. We found that 1% of these animals displayed hearing deficits. The phenotypes of non-responders were further assessed with radiological analysis for defects in the gross morphology of the auditory system. Nearly all of those showed abnormalities in conductive elements of the auditory system: the swim bladder or Weberian ossicles.

Acoustic Stimulation↗

PubMedDate not supplied

[Generalized epilepsy with febrile seizures plus: clinical and genetic analysis of three Serbian families].

The results of clinical and genetic analysis of three Serbian families (pedigrees) with autosomal dominant inheritance, incomplete penetrance and phenotypic features of GEFS+ are presented in this study. Mutation analysis of the SCN1A, SCN1B and GABRG2 genes was performed in all affected and some unaffected members of these three families. Twenty-six exons of SCN1A, five exons of SCN1B and nine exons of GABRG2 were individually amplified using primers based on intronic sequence. PCR products were sequenced in both forward and reverse directions. Subsequently, the samples were run and analyzed using 377 DNA automated sequencer. No consanguinity was noticed. The MM and OM family members live in Republic of Srpska while KS family originates from the central Serbia. No mutations of the exons of SCN1A, SCN1B and GABRG2 genes were found in tested subjects. Obligate carriers in MM family (III-1, III-2, and III-4) exhibit variable expressivity or incomplete penetrance rather than proof of polygenetic inheritance. OM pedigree follows autosomal dominant pattern despite reduced penetrance. Bilinear transmission may assume the possibility of multigenetic mode of inheritance in KS family. The fact that all affected members in three Serbian families were negative for mutations in SCN1A, SCN1B and GABRG2 genes strongly supports the hypothesis of significant genetic heterogeneity of GEFS+. Recognizing GEFS+ on clinical grounds contributes to more precise integration of this syndrome into already existing classification of epileptic syndromes.

Adolescent↗

PubMed2005

Fluorescent multiplex PCR--fast method for autosomal dominant spinocerebellar ataxias screening.

Expansion of CAG trinucleotide repeats has been shown to cause a number of autosomal dominant spinocerebellar ataxias such as SCA1, SCA2, SCA3/MJD, SCA6 and SCA7. These disorders are characterized by a wide inter- and intrafamiliar variation in clinical features. The same mutation can result in different phenotypes and the very similar phenotypes can be caused by different mutations. Therefore it is necessary to investigate more SCA genes (according to prevalence) to identify the causal elongation. We developed a fast and efficient screening method based on touchdown multiplex PCR with fluorescent labelled primers for the most common types of SCAs (SCA 1, 2, 3 and 7). It has been reliable in 113 probands tested. Fragment analysis was performed by using 6% denaturing polyacrylamide gel and employing the automated DNA sequencer. This method considerably shortens the process of molecular genetic screening of SCAs and might be used as a tip for designing other SCA screening sets.

Genetic Testing↗

PubMed1989

The use of DNA amplification for genetic counselling related diagnosis in haemophilia B.

A single base pair variation in the coding sequence of coagulation factor IX produces a protein polymorphism detectable with monoclonal antibodies and a restriction fragment length polymorphism (RFLP). This allows carrier and prenatal diagnoses in 48% of Caucasian families segregating for haemophilia B. However, this RFLP cannot be detected by standard Southern blotting, while the antibody assay may give equivocal results in some females and can only allow prenatal diagnoses on second trimester fetal blood samples. We show that, using the polymerase chain reaction, the polymorphic DNA segment can be amplified and directly tested for the presence of the alternative sequences by a non-radioactive procedure that has the advantage of speed (1-2 days), partial automation and applicability to first trimester diagnoses. We also show that the method gives results on a single drop of dried blood.

Base Sequence↗

PubMed1978

[Groupamatic 360 C1 and automated blood donor processing in a transfusion center].

Automation of donor management flow path is controlled by: --a 3 slip "port a punch" card, --the groupamatic unit with a result sorted out on punch paper tape, --the management computer off line connected to groupamatic. Data tracking at blood collection time is made by punching a card with the donor card used as a master card. Groupamatic performs: --a standard blood grouping with one run for registered donors and two runs for new donors, --a phenotyping with two runs, --a screening of irregular antibodies. Themanagement computer checks the correlation between the data of the two runs or the data of a single run and that of previous file. It updates the data resident in the central file and prints out: --the controls of the different blood group for the red cell panel, --The listing of error messages, --The listing of emergency call up, --The listing of collected blood units when arrived at the blood center, with quantitative and qualitative information such as: number of blood, units collected, donor addresses, etc., --Statistics, --Donor cards, --Diplomas.

Autoanalysis↗