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From 2006 to 2011, Roslin Cells Ltd derived 17 human embryonic stem cells (hESC) while developing (RCM1, RC-2 to -8, -10) and implementing (RC-9, -11 to -17) quality assured standards of operation in a facility operating in compliance with European Union (EU) directives and United Kingdom (UK) licensure for procurement, processing and storage of human cells as source material for clinical application, and targeted to comply with an EU Good Manufacturing Practice specification. Here we describe the evolution and specification of the facility, its operation and outputs, complementing hESC resource details communicated in Stem Cell Research Lab Resources.
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Técnicas de Cultura de Células/normas , Células-Tronco Embrionárias Humanas/citologia , Diferenciação Celular , Células Cultivadas , Regulamentação Governamental , Humanos , Controle de QualidadeRESUMO
Preimplantation genetic diagnosis (PGD) aims to test the embryo for specific conditions before implantation in couples at risk of transmitting genetic abnormality to their offspring. The couple must undergo IVF procedures to generate embryos in vitro. The embryos can be biopsied at either the zygote, cleavage or blastocyst stage. Preimplantation genetic screening uses the same technology to screen for chromosome abnormalities in embryos from patients undergoing IVF procedures as a method of embryo selection. Fluorescence in-situ hybridization was originally used for chromosome analysis, but has now been replaced by array comparative genomic hybridization or next generation sequencing. For the diagnosis of single gene defects, polymerase chain reaction is used and has become highly developed; however, single nucleotide polymorphism arrays for karyomapping have recently been introduced. A partnership between IVF laboratories and diagnostic centres is required to carry out PGD and preimplantation genetic screening. Accreditation of PGD diagnostic laboratories is important. Accreditation gives IVF centres an assurance that the diagnostic tests conform to specified standards. ISO 15189 is an international laboratory standard specific for medical laboratories. A requirement for accreditation is to participate in external quality assessment schemes.
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Diagnóstico Pré-Implantação/normas , Controle de Qualidade , Acreditação , Auditoria Clínica , Erros de Diagnóstico , Fertilização in vitro , Humanos , Hibridização in Situ Fluorescente , Cariotipagem/métodos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo ÚnicoAssuntos
Transtornos Cromossômicos/diagnóstico , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase , Diagnóstico Pré-Natal/métodos , Aberrações Cromossômicas , Análise Custo-Benefício , Feminino , Humanos , Hibridização in Situ Fluorescente/economia , Cariotipagem , Reação em Cadeia da Polimerase/economia , Gravidez , Diagnóstico Pré-Natal/economia , Sensibilidade e EspecificidadeRESUMO
Rett syndrome (RTT) is an X-linked dominant neurological disorder, which appears to be the most common genetic cause of profound combined intellectual and physical disability in Caucasian females. This syndrome has been associated with mutations of the MECP2 gene, a transcriptional repressor of unknown target genes. We report a detailed mutational analysis of a large cohort of RTT patients from the UK and Italy. This study has permitted us to produce a hot spot map of the mutations identified. Bioinformatic analysis of the mutations, taking advantage of structural and evolutionary data, leads us to postulate the existence of a new functional domain in the MeCP2 protein, conserved among brain-specific regulatory factors.
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Proteínas Cromossômicas não Histona , Mapeamento Cromossômico , Biologia Computacional , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Mutação/genética , Proteínas Repressoras , Síndrome de Rett/genética , Adolescente , Adulto , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Criança , Pré-Escolar , Proteínas de Ligação a DNA/metabolismo , Feminino , Fatores de Transcrição Forkhead , Humanos , Lactente , Recém-Nascido , Itália , Proteína 2 de Ligação a Metil-CpG , Dados de Sequência Molecular , Proteínas Nucleares/genética , Estrutura Terciária de Proteína/genética , Fatores de Transcrição/genética , Reino UnidoRESUMO
Signal (dot) counting in fluorescence in-situ hybridization (FISH) images that relies on an automatic focusing method for obtaining clearly defined images is a time-consuming procedure prone to errors. Our recently developed system has dispensed with automatic focusing, and instead relies on a neural network classifying focused and unfocused signals into valid and artefact data, respectively, and thereby discriminating between in- and out-of-focus images. However, to train the classifier accurate labelling of the image signals is required. GELFISH is a Graphical Environment for Labelling FISH images that enables the rejection of unanalysable nuclei and labelling of FISH signals simply and rapidly. GELFISH is flexible and can be modified easily for additional FISH applications. Also, implemented using popular software, the environment can be employed on any computer by any user. Finally, GELFISH is proposed in controlling a classifier-based dot counting system.
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Citometria por Imagem/métodos , Aumento da Imagem/métodos , Hibridização in Situ Fluorescente/métodos , Software , Artefatos , Núcleo Celular , Corantes Fluorescentes , Citometria por Imagem/instrumentação , Aumento da Imagem/instrumentação , Hibridização in Situ Fluorescente/instrumentação , Redes Neurais de ComputaçãoRESUMO
Education programs have been developed to promote adherence to recommended breast cancer screening guidelines. Few studies have assessed the degree to which ethnic subgroups are perceiving and acting on the proffered information. Such assessment is vital to the creation of efficient public health interventions. This paper describes the reported breast cancer knowledge, attitudes, and screening behaviors of 194 American Asian Indian women. While monthly breast self exam adherence was low, only 40.7%, 61.3% of women 40 and older, and 70% of women 50 and older, reported having had a mammogram within the past 12 months. These rates for annual mammography screening are high relative to many other ethnic groups. While the results are encouraging, the respondents may not be representative of all Asian Indian women. The majority of these women reported that their breast cancer knowledge is inadequate. They were willing to be called upon to share with others any knowledge they gained. There is a clear opportunity for public health nurses to provide Asian Indian women with a more comprehensive understanding of breast health and disease. Those women can then share their health knowledge with other women within their ethnic group.
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Asiático/psicologia , Neoplasias da Mama/prevenção & controle , Conhecimentos, Atitudes e Prática em Saúde , Cooperação do Paciente/etnologia , Adulto , Idoso , Neoplasias da Mama/etnologia , Neoplasias da Mama/psicologia , Autoexame de Mama/estatística & dados numéricos , California , Feminino , Educação em Saúde , Promoção da Saúde , Humanos , Índia/etnologia , Mamografia/estatística & dados numéricos , Pessoa de Meia-Idade , Inquéritos e QuestionáriosRESUMO
Rett syndrome is an X-linked dominant neurological disorder, which appears to be the commonest genetic cause of profound combined intellectual and physical disability in Caucasian females. Recently, this syndrome has been associated with mutations of the MECP2 gene, a transcriptional repressor of still unknown target genes. Here we report a detailed mutational analysis of 62 patients from UK and Italian archives, representing the first comparative study among different populations and one of the largest number of cases so far analyzed. We review the literature on MECP2 mutations in Rett syndrome. This analysis has permitted us to produce a map of the recurrent mutations identified in this and previous studies. Bioinformatic analysis of the mutations, taking advantage of structural and evolutionary data, leads us to postulate the existence of a new functional domain in the MeCP2 protein, which is conserved among brain-specific regulatory factors.
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Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/genética , Proteínas Repressoras , Síndrome de Rett/etnologia , Síndrome de Rett/genética , Adolescente , Adulto , Sequência de Aminoácidos , Sequência de Bases , Pré-Escolar , Sequência Conservada , Análise Mutacional de DNA , Proteínas de Ligação a DNA/química , Evolução Molecular , Éxons , Feminino , Mutação da Fase de Leitura , Heterozigoto , Humanos , Lactente , Íntrons , Itália , Proteína 2 de Ligação a Metil-CpG , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Mutação de Sentido Incorreto , Linhagem , Polimorfismo Conformacional de Fita Simples , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Reino UnidoRESUMO
BACKGROUND: Previous systems for dot (signal) counting in fluorescence in situ hybridization (FISH) images have relied on an auto-focusing method for obtaining a clearly defined image. Because signals are distributed in three dimensions within the nucleus and artifacts such as debris and background fluorescence can attract the focusing method, valid signals can be left unfocused or unseen. This leads to dot counting errors, which increase with the number of probes. METHODS: The approach described here dispenses with auto-focusing, and instead relies on a neural network (NN) classifier that discriminates between in and out-of-focus images taken at different focal planes of the same field of view. Discrimination is performed by the NN, which classifies signals of each image as valid data or artifacts (due to out of focusing). The image that contains no artifacts is the in-focus image selected for dot count proportion estimation. RESULTS: Using an NN classifier and a set of features to represent signals improves upon previous discrimination schemes that are based on nonadaptable decision boundaries and single-feature signal representation. Moreover, the classifier is not limited by the number of probes. Three classification strategies, two of them hierarchical, have been examined and found to achieve each between 83% and 87% accuracy on unseen data. Screening, while performing dot counting, of in and out-of-focus images based on signal classification suggests an accurate and efficient alternative to that obtained using an auto-focusing mechanism.
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Citometria por Imagem/métodos , Aumento da Imagem/métodos , Hibridização in Situ Fluorescente/métodos , Líquido Amniótico/química , Líquido Amniótico/citologia , Artefatos , Núcleo Celular/química , Corantes Fluorescentes/química , Humanos , Citometria por Imagem/instrumentação , Aumento da Imagem/instrumentação , Hibridização in Situ Fluorescente/classificação , Hibridização in Situ Fluorescente/instrumentação , Redes Neurais de ComputaçãoRESUMO
Culturally and linguistically compatible university students were trained as community health educators to provide breast cancer education and screening information to shoppers at Asian grocery stores. Information about early detection of breast cancer was shared with 8,877 women, who reported speaking 40 different languages. Baseline surveys were completed by 1,202 women; 779 took part in the follow-up survey. The survey questions assessed baseline knowledge, attitudes, and screening behaviors regarding breast cancer, tested the efficacy of the intervention, and sought barriers to accessing screening services. Screening adherence at baseline was low, but reported screening compliance had increased by follow-up. This study confirms the cost-effectiveness of student health educators and Asian grocery store sites as venues to reach the diverse age, ethnic, and socioeconomic segments of the Asian community, while demonstrating the community's receptiveness to the dissemination of health information and introducing bilingual students to health education and research careers.
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Asiático , Neoplasias da Mama/prevenção & controle , Serviços de Saúde Comunitária , Educação em Saúde , Comércio , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Estados UnidosRESUMO
BACKGROUND: African Americans suffer a disproportionate burden of illness and premature mortality. METHODS: A health education program delivered via cosmetologists was pilot tested as a supplement to other programs seeking to reach this community with information designed to remedy this inequality. Eight cosmetologists were randomized to either an active or a passive educational intervention arm, with the active arm (experimental arm) focused on breast cancer early detection. RESULTS: Both cosmetologists and clients found this an acceptable intervention. Nearly all women in the study demonstrated that they had heard the mainstream messages about the value of breast cancer early detection, but a considerable proportion appeared not to realize breast cancer's high level of morbidity and mortality within their own community. CONCLUSION: The results suggest this approach is worthy of further evaluation.
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Atitude Frente a Saúde/etnologia , Indústria da Beleza , Negro ou Afro-Americano/estatística & dados numéricos , Neoplasias da Mama/prevenção & controle , Promoção da Saúde/organização & administração , Adulto , Idoso , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Colorado , Feminino , Inquéritos Epidemiológicos , Humanos , Pessoa de Meia-Idade , Projetos Piloto , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Valores de Referência , Estatísticas não ParamétricasRESUMO
The reflectivity of a gallium/silica interface formed on an optical flat or at the tip of a cleaved optical fiber can be reduced in a reversible fashion when the interface is excited by a few milliwatts of laser power. This phenomenon occurs at temperatures just below gallium's melting point. We believe that the effect can be attributed to light-induced structuring at the interface.
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A probe was generated from the YAC clone 831B9 that was suitable for the prenatal detection of trisomy 21 using fluorescence in situ hybridization (FISH). This probe was initially tested on a series of 650 unselected amniotic fluid samples prior to the karyotype being available. 630 were correctly identified as having two copies and 13 samples were correctly scored as having three copies of chromosome 21. Seven samples failed to produce a result. A trial was then initiated, reporting to clinicians the interphase FISH results before cytogenetic analysis had been performed. During the first 18 months of this trial 1504 samples were tested: 1467 were correctly identified as disomic and 35 samples were correctly scored as trisomic for chromosome 21. Two samples failed to produce a result. A chromosome 18 specific probe (LI.84) was employed where there was a relevant clinical indication (181 samples) and 10 samples were correctly scored as having three copies of chromosome 18. Thus, this approach appears to be reliable and is popular with both clinicians and patients due to the speed of the result. However, it does not replace chromosomal analysis on cultured cells, which detected a range of abnormalities besides the trisomies and triploidies detected by FISH.
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Cromossomos Humanos Par 18 , Síndrome de Down/diagnóstico , Hibridização in Situ Fluorescente/métodos , Trissomia/diagnóstico , Líquido Amniótico/citologia , Humanos , Microscopia de Fluorescência/métodos , Diagnóstico Pré-Natal , Estudos ProspectivosRESUMO
The gallium/silica interface optical nonlinearity associated with a light-induced structural phase transition from a-gallium to a more reflective, more metallic phase shows an exceptionally broadband spectral response. It allows 40% deep nanosecond/microsecond cross-wavelength intensity modulation between signals at 1.3 and 1.55aem.
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BACKGROUND: Prenatal diagnosis of chromosomal abnormality requires cytogenetic analysis of amniotic fetal cells. The necessary culture time delays diagnosis, is expensive, and requires substantial scientific expertise. In a masked prospective study, we investigated the feasibility of PCR amplification of chromosome 21 markers for the prenatal diagnosis of Down's syndrome. METHODS: The study population consisted of 2167 pregnant women, undergoing amniocentesis for prenatal diagnosis. In this cohort at least 1.5 mL amniotic fluid was available surplus to the requirements for traditional diagnostic methods. DNA was extracted from the surplus amniotic fluid and amplified in fluorescence-based PCR reactions, with three small-tandem-repeat markers located on chromosome 21. The products of the reactions were analysed on a DNA sequencer to identify the presence of two or three copies of chromosome 21. FINDINGS: In 2083 (97.4%) of 2139 samples of amniotic fluid that were not macroscopically blood-stained, two DNA markers gave an informative and correct result, identifying 2053 fetuses as normal and 30 as having trisomy 21 Down's syndrome (as confirmed by cytogenetic analysis). An extra marker was informative in 32 of 41 other clear samples. Thus a total of 99.6% informative results was achieved with these three markers. Macroscopically blood-stained samples (28 [1.3%]) were unsuitable for DNA testing. They gave a typical but non-informative result. There were no false-positive or false-negative results. INTERPRETATION: The PCR-based DNA diagnostic test has great potential for improved prenatal diagnosis of Down's syndrome, with the advantage that results may be available within a day.
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Amniocentese , DNA/análise , Síndrome de Down/diagnóstico , Reação em Cadeia da Polimerase , Amniocentese/métodos , Feminino , Humanos , Gravidez , Estudos ProspectivosRESUMO
A very strong (~10(-8) esu) femtosecond cubic nonlinearity in metallic indium that increased sharply in magnitude as the temperature approached the melting point was observed.
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We have observed that a light beam that passed through an optically active crystal of Bi(12)SiO(20) and that was then ref lected exactly back through it did not recover its initial polarization orientation. The nonreciprocal component of the rotation was of the order of 2 x 10(-3) of the reciprocal, single-pass rotation. This nonreciprocity is unambiguous evidence of broken reversality of the light-matter interaction process.
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The effects of zinc on neuronal excitability in rodent hippocampal slices were examined. In a paired-pulse protocol, the second population spike increased appreciably in the presence of zinc, whereas the first spike and the size of both population excitatory post-synaptic potentials remained unaffected. Changes in the second population spike produced by zinc were most pronounced when the afferents were stimulated with paired-pulses separated by 8-40 ms. The magnitude of altered excitability increased with the concentration of zinc in the perfusate. A long exposure to zinc in physiological concentration caused an epileptiform discharge followed by a period of depression. The effects of zinc could be mimicked with 1-3 microM bicuculline. We conclude that the integrity of the hippocampal inhibitory system is particularly vulnerable to zinc.
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Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Zinco/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Estimulação Elétrica/métodos , Eletrofisiologia , Epilepsia/induzido quimicamente , Epilepsia/fisiopatologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos CBA , Ratos , Ratos WistarRESUMO
Guinea pigs, reared from birth in an environment of omnidirectional white noise, fail to develop a map of auditory space in the deeper layers of the superior colliculus. Collicular responses from such noise-reared animals reveal large auditory spatial receptive fields. The representation of auditory space in the colliculus shows no topographic order. Exposing developing animals to the noise environment only for restricted time periods showed that animals reared normally up to 26 days after birth (DAB) and then placed in the noise chamber could not construct spatial maps, whereas animals reared normally to 30 DAB and then placed in the noise chamber until the terminal mapping experiment could construct topographically organized spatial maps with local receptive fields. Limiting the noise exposure to the period between 26 and 30 DAB was sufficient to prevent spatial map formation. The failure to form a map of auditory space did not reflect environmental damage to the cochlea or the functional organization of the primary auditory pathway. The response thresholds of cochlear microphonics and of auditory responses in both the inferior and superior colliculus were normal in noise-reared animals. Similarly normal were the tonotopic organization and frequency tuning characteristics of inferior collicular neurons. The rearing environment thus appears to exert a selective effect upon the maturation of the superior collicular map of auditory space. We attribute this effect to the masking, by the omnidirectional broad-band noise, of discrete localized auditory stimuli. Cues deriving from these latter stimuli would appear to be necessary for the elaboration of the map of auditory space. This auditory experience operates during a 4 day crucial developmental period from 26 to 30 DAB. This is the same developmental time window as that during which visual experience is required for the construction of the map.
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1. The responses of Purkinje cells to short duration (pulse) ionophoretic applications of L-aspartate (L-asp), L-glutamate (L-glu), N-methyl DL-aspartate (NMDLA) and quisqualic acid in their dendritic fields were studied in vitro on sagittal slices of lobules IX and X of the adult rat cerebellum.2. Pulse application of L-asp or L-glu evoked transient and dose-dependent increases in the firing rate of the simple spikes recorded extracellularly as single units. When the ionophoretic electrode was positioned in the dendritic field of the Purkinje cells, the lowest thresholds for L-glu and L-asp mediated excitations of the cells were as low as 25 and 35 pC respectively, with a latency for maximal responses as brief as 7 ms.3. In intracellular recordings these excitatory responses consisted of depolarizations of up to 18 mV in amplitude and with depolarizing slopes up to 0.52 mV/ms. They were generally unaccompanied by changes in cell input resistance in contrast to the marked decrease which occurred in response to steady applications of large doses of L-asp and L-glu.4. The spatial distribution of the excitatory sites confirmed that the dendritic sensitivity to L-glu was greater than that of the soma and showed that the same was true for L-asp. In 34% of cells the sensitivity for L-asp declined markedly in the upper region of the molecular layer, whereas it remained high for L-glu; no such differential sensitivity was detected in the remaining 66% of cells.5. Inhibitory responses, antagonized by 10(-5) M-bicuculline in the bath, were also induced in Purkinje cells by L-glu and L-asp when the ionophoretic electrode was withdrawn from the excitatory sites by as little as 8 mum and up to 40 mum upward or downward along the track of parallel fibres or positioned as far as 250 mum laterally.6. Whenever it was applied in the molecular layer, the pulse application of NMDLA elicited no excitatory response in Purkinje cells recorded extra or intracellularly. However, slow depolarizations accompanied by a slight increase in cell input resistance were obtained with steady applications of 20-50 nA of the drug for 20-30 s.7. In contrast, pulse application of quisqualic acid appeared to have the same type of fast excitatory effect on Purkinje cells as L-asp and L-glu, but its potency was greater and its action more prolonged. Furthermore, its steady application led to an abrupt and marked decrease in cell membrane resistance.8. The excitatory effects of L-asp, L-glu and quisqualic acid were antagonized by L-glutamic acid diethyl ester more consistently than by D-alpha-aminoadipate, suggesting together with previous observations that L-asp and L-glu act on Purkinje cells via quisqualic acid rather than via NMDLA receptors.