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Sacred conceptions: clinical theodicies, uncertain science, and technologies of procreation in India.

This article argues that the rapid transfer of assisted conception technologies, such as in vitro fertilization, to India is not restricted merely to the modalities of offering potential biomedical resolution of infertility but includes, more crucially, how clinicians and infertile consumers assimilate the "Western technoscience" of conception. The article draws on a larger multisite ethnographic study of infertility and assisted conception in India's five major cities and is principally based on narratives of clinicians and infertile couples and on clinic-based ethnographic observations. In this article I contend that the success or failure of assisted conception, when situated in the universe of Hindu faith, becomes a powerful critique of the "incompleteness" of the "Western" science of conception. Situating this contention in the broader context of a clinician's faith, I assert that assisted conception--by conjoining seemingly disparate domains of the traditional and the modern, the sacred and the profane, the human and the superhuman, science and religion--produces clinical theodicies that help explain and contain the tentativeness permeating the conception technologies. The article concludes by arguing that this enchanted version of a thoroughly disenchanted worldview of biomedicine is part of a larger cultural process of indigenization of biomedicine in India.

Attitude to Health↗

Transfer of DNA tests from research to routine laboratories: some lessons from the French experience in the case of Duchenne muscular dystrophy.

In the last few years, the activity of research laboratories has led to the emergence of new DNA diagnostic tests in France. They permit the origin of genetic diseases to be identified and provide an answer concerning the detection of carriers and prevention. Nevertheless, given this, new actors have emerged on the health care scene: the research workers who developed the tests and who work in public research laboratories. The economic question of the transfer of the test practice from research to hospital laboratories is the main topic of this paper, taking Duchenne Muscular Dystrophy (DMD) DNA diagnostic tests as the example. After a presentation of the complexity of DNA tests for DMD, the fact that financial and human constraints do not allow the actors to continue to produce the DNA tests is discussed. The financial role of the non-profit-making associations is then explained and leads to the conclusion that a more global policy on DNA tests, such as carried out in the UK and the Netherlands, should be adopted in France by the Social Security if it wants DNA testing activity to be pursued.

Clinical Laboratory Techniques↗

Polymorphisms in the promoter regions of FAS and FASL genes as candidate genetic factors conferring susceptibility to endometriosis.

Although the pathophysiological mechanisms leading to endometriosis remain unknown, several hypothesis have been proposed, including a dysregulation of the normal apoptotic process which takes place in the endometrium. One of the apoptotic pathways playing a crucial role in the programmed cell death within the endometrium is the Fas-FasL system. In this study we have performed a case-control analysis in order to evaluate three polymorphisms located within FAS (-1377G>A and -670A>G) and FASL (-843C>T) genes, as susceptibility factors for endometriosis. We have analysed a series of women with endometriosis compared respectively to a group of women without symptoms of the disease, and to a group of confirmed unaffected women. The genotyping of the three variants was carried out by Fluorescence Resonance Energy Transfer (FRET) technology, and statistical analysis was performed using chi2 test with Yates correction. Our results show that the differences in the distribution of the polymorphic variants were not statistically significant when the group of patients was compared to the other groups. Thus, it seems to indicate that the variants here analysed are not involved in the pathogenesis of the disease in our population. However this does not let us to completely exclude such genes as potential candidates for the disease. A complete genetic analysis of the genes involved in the intricate regulatory system of the apoptosis may lead to the identification of susceptibility factors for the disease and a better understanding of its etiology.

Adult↗

Nuclear transfer in cats and its application.

Nuclear transfer (NT) technology is typically used for generating identical individuals, but it is also a powerful resource for understanding the cellular and molecular aspects of nuclear reprogramming. Most recently, the procedure has been used in humans for producing patient-specific embryonic stem cells. The successful application of NT in cats was demonstrated by the birth of domestic and non-domestic cloned kittens at a similar level of efficiency to that reported for other mammalian species. In cats, it has been demonstrated that either in vivo or in vitro matured oocytes can be used as donor cytoplasts. The length of in vitro oocyte maturation affects in vitro development of reconstructed embryos, and oocytes matured in vitro for shorter periods of time are the preferred source of donor cytoplasts. For NT, cat somatic cells can be synchronized into the G0/G1 phase of the cell cycle by using different methods of cell synchronization without affecting the frequency of in vitro development of cloned embryos. Also, embryo development to the blastocyst stage in vitro is not influenced by cell type, but the effect of cell type on the percentage of normal offspring produced requires evaluation. Inter-species NT has potential application for preserving endangered felids, as live offspring of male and female African wildcats (AWC, Felis silvestris lybica) have been born and pregnancies have been produced after transferring black-footed cat (Felis nigripes) cloned embryos into domestic cat (Felis silvestris catus) recipients. Also, successful in vitro embryo development to the blastocyst stage has been achieved after inter-generic NT of somatic cells of non-domestic felids into domestic cat oocytes, but no viable progeny have been obtained. Thus, while cat cytoplasm induces early nuclear remodeling of cell nuclei from a different genus, the high incidence of early embryo developmental arrest may be caused by abnormal nuclear reprogramming. Fetal resorption and abortions were frequently observed at various stages of pregnancy after transfer of AWC cloned embryos into domestic cat recipients. Abnormalities, such as abdominal organ exteriorization and respiratory failure and septicemia were the main causes of death in neonatal cloned kittens. Nonetheless, several live domestic and AWC cloned kittens have been born that are seemingly normal and healthy. It is important to continue evaluating these animals throughout their lives and to examine their capability for natural reproduction.

Animals↗

[Social capital, from sociology to epidemiology: critical analysis of a transfer across disciplines].

In recent years, social capital has emerged in epidemiological studies as a new concept, improving our understanding of the relationships between social inequalities and health inequalities. This concept, borrowed from social sciences, has three distinct sociological sources. However, only the most recent theory, which emphasizes the role of civic trust and is useful for analysis at community level, has been used in epidemiological studies. Social capital poses three kinds of problem: i) theoretical problems, because it is defined by its effects rather than by its causes, and because it is presumed that these effects are positive, although they can in fact be negative; ii) methodological problems, because of the heterogeneity of empirical scales, from micro to macro, and because of the diversity of its semantic content, including contradictions; iii) political problems, because of the emphasis placed on individual responsibility and due to the imposition of a model of civic virtue, to the detriment of structural analysis.

Causality↗

Serological status and abortion rate in progeny obtained by natural service or embryo transfer from Neospora caninum-seropositive cows.

Bovine neosporosis is an important disease in dairy herds in Argentina. Embryo transfer (ET) technology offers the opportunity to minimize vertical transmission of neosporosis. The objective of this work was to compare the serological status and the incidence of abortion between heifers obtained via natural service and heifers obtained by ET from Neospora-infected cows. Progeny from eight Neospora-infected cows belonging to a commercial dairy farm where ET was routinely performed were studied. Between 1996 and 2001, 16 and 15 heifers were obtained by natural service and ET, respectively. Serostatus and reproductive performance were recorded in both progeny. Serostatus was determined by indirect fluorescent antibody test (IFAT). All heifers obtained by natural service were seropositive and six of 16 pregnancies ended in abortion. All heifers obtained by ET were seronegative and only two abortions of 15 pregnancies were recorded.

Abortion, Veterinary↗

Isolation of transfected fibroblast clones for use in nuclear transfer and transgene detection in cattle embryos.

Transgenesis in cattle has provided numerous opportunities for livestock production. The development of nuclear transfer (NT) technology has improved the production of transgenic livestock. However, the isolation of pure colonies from a single transfection event remains laborious and can be a constraint in the production of transgenic livestock. We used 96-well cell culture plates to isolate cell lineages obtained from a single fibroblast transfected with the pCi-Neo plasmid. Since single mammalian cells do not grow well in fresh medium, we evaluated the use of conditioned medium. The neomycin phosphotransferase gene was detected in isolated colonies and NT embryos were produced from these cells. Multiplex-PCR assays were performed to detect the transfected fragment as well as autosomal satellite DNA in single NT embryos. This approach provided a reliable method for isolating transfected mammalian cells and for diagnosing the incorporation of desirable vectors in NT embryos. This method can reduce the time and cost of transgenic livestock production.

Animals↗

Improving the fit between research and practice in health promotion: overcoming structural barriers.

This paper is based on the proposition that transfer of knowledge between researchers and practitioners concerning effective health promotion interventions in less than optimal. It considers how evidence concerning effectiveness in health promotion is established through a sequence of research studies, and how such evidence is applied by health promotion practitioners and policy markers in deciding what to do and what to found when addressing public health problems. There are too few rewards for researchers to pursue research with potential for widespread application, and to systematically develop promising interventions to a stage of field dissemination. Practitioners often find themselves in the position of tackling a public health problem where evidence of efficacy is either lacking, or has to be considered alongside a desire to respond to expressed community needs, or the need to respond to political imperative. Some of the major structural barriers which prevent the flow of research into practice are considered, and different approaches to improving the fit between research and practice are proposed.

Health Promotion↗

Getting new evidence into medicine.

The rate of transfer of the knowledge gained from health and medical research into evidence-based practice is determined by many factors. Preconditions for the uptake of new evidence are the availability of good evidence, ready access to the evidence, a supportive organisational environment, and effective mechanisms for promoting knowledge uptake. Evidence-based medicine is being promoted in Australia by a body of enthusiastic clinicians, public health practitioners and consumer advocates, supported by initiatives from national, State and local healthcare services and professional bodies. The short to medium term future of evidence-based medicine in Australia is likely to be shaped by three major factors: a reduction in cost and technical barriers which limit access to computerised databases; a trend towards shared decision-making between clinicians and patients; and increased demand for information to fill the gaps in research-based evidence on specific problems.

Australia↗

Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.

Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.

Animals↗

Research translation: a new frontier for clinical laboratories.

Translational research and translational medicine (referred to hereafter as translational research) are interchangeable terms that underline the pressing need to translate into practical benefits for those affected by disease the extensive investments divested by the private and public sectors in biomedical research. For people more directly involved in clinical practice (physicians, clinical laboratory professionals and patients), translational research responds to the need to accelerate the capture of benefits of research, closing the gap between what we know and what we practice. This basically means the transfer of diagnostic and therapeutic advances proven effective in large, well-conducted trials (and, therefore, evidence-based) to daily medical practice. Translational research should be regarded as a two-way road: bench to bedside, and bedside to bench. In particular, to make possible a more effective translation process, a new road map should be implemented through interaction and cooperation between basic researchers, clinicians, laboratory professionals and manufacturers. Some examples of recent developments in clinical laboratory testing, including markers of cardiovascular diseases, clinical proteomics and recombinant allergens, may explain the importance of careful evaluation of all variables that allow the introduction of such new insights into clinical practice to assure better clinical outcomes. The vital role of laboratory medicine in the delivery of safer and more effective healthcare requires more careful evaluation not only of the analytical characteristics, but also of any other variable that may affect the clinical usefulness and diagnostic performances of laboratory tests, thus allowing more accurate interpretation and utilization of laboratory information.

Albuminuria↗

New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells.

Until recently, precise modification of the animal genome by gene targeting was restricted to the mouse because germline competent embryonic stem cells are not available in any other mammalian species. Nuclear transfer (NT) technology now provides an alternative route for cell-based transgenesis in domestic species, offering new opportunities in genetic modification. Livestock that produce human therapeutic proteins in their milk, have organs suitable for xenotransplantation, or that could provide resistance to diseases such as spongiform encephalopathies have been produced by NT from engineered, cultured somatic cells. However, improvements in the efficiency of somatic cell gene targeting and a greater understanding of the reprogramming events that occur during NT are required for the routine application of what is currently an inefficient process. The ability to reprogramme and genetically manipulate cells will also be crucial for full exploitation of human embryonic stem (hES) cells, which offer unparalleled opportunities in human health and biotechnology. Particularly pertinent are directed differentiation of hES lines to specific cell lineages, production of cells that evade the patient's immune system and ensuring the safety of ensuing transplants. This review will discuss some of the successes, applications and challenges facing gene targeting in livestock and hES cells.

Animals↗

Interisland and interarchipelago transfer of stone tools in prehistoric Polynesia.

Tracing interisland and interarchipelago movements of people and artifacts in prehistoric Polynesia has posed a challenge to archaeologists due to the lack of pottery and obsidian, two materials most readily used in studies of prehistoric trade or exchange. Here we report the application of nondestructive energy-dispersive x-ray fluorescence (EDXRF) analysis to the sourcing of Polynesian artifacts made from basalt, one of the most ubiquitous materials in Polynesian archaeological sites. We have compared excavated and surface-collected basalt adzes and adze flakes from two sites in Samoa (site AS-13-1) and the Cook Islands (site MAN-44), with source basalts from known prehistoric quarries in these archipelagoes. In both cases, we are able to demonstrate the importing of basalt adzes from Tutuila Island, a distance of 100 km to Ofu Island, and of 1600 km to Mangaia Island. These findings are of considerable significance for Polynesian prehistory, as they demonstrate the movement of objects not only between islands in the same group (where communities were culturally and linguistically related) but also between distant island groups. Further applications of EDXRF analysis should greatly aid archaeologists in their efforts to reconstruct ancient trade and exchange networks, not only in Polynesia but also in other regions where basalt was a major material for artifact production.

Animals↗

Exonuclease III protection assay with FRET probe for detecting DNA-binding proteins.

We describe a new method for the assay of sequence-specific DNA-binding proteins in this paper. In this method, the sensitive fluorescence resonance energy transfer (FRET) technology is combined with the common DNA footprinting assay in order to develop a simple, rapid and high-throughput approach for quantitatively detecting the sequence-specific DNA-binding proteins. We named this method as exonuclease III (ExoIII) protection assay with FRET probe. The FRET probe used in this assay was a duplex DNA which was designed to contain one FRET pair in the center and two flanking protein-binding sites. During protein detection, if a target protein exists, it will bind to the two protein-binding sites of the FRET probe and thus protect the FRET pair from ExoIII digestion, resulting in high FRET. However, if the target protein does not exist, the FRET pair on the naked FRET probe will be degraded by ExoIII, resulting in low FRET. Three kinds of recombinant transcription factors including NF-kappaB, SP1 and p50, and the target protein of NF-kappaB in HeLa cell nuclear extracts, were successfully detected by the assay. This assay can be extensively used in biomedical research targeted at DNA-binding proteins.

Cell Nucleus↗

RETRACTED: Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst.

Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.

Animals↗

Functional assay of voltage-gated sodium channels using membrane potential-sensitive dyes.

The discovery of novel therapeutic agents that act on voltage-gated sodium channels requires the establishment of high-capacity screening assays that can reliably measure the activity of these proteins. Fluorescence resonance energy transfer (FRET) technology using membrane potential-sensitive dyes has been shown to provide a readout of voltage-gated sodium channel activity in stably transfected cell lines. Due to the inherent rapid inactivation of sodium channels, these assays require the presence of a channel activator to prolong channel opening. Because sodium channel activators and test compounds may share related binding sites on the protein, the assay protocol is critical for the proper identification of channel inhibitors. In this study, high throughput, functional assays for the voltage-gated sodium channels, hNa(V)1.5 and hNa(V)1.7, are described. In these assays, channels stably expressed in HEK cells are preincubated with test compound in physiological medium and then exposed to a sodium channel activator that slows channel inactivation. Sodium ion movement through open channels causes membrane depolarization that can be measured with a FRET dye membrane potential-sensing system, providing a large and reproducible signal. Unlike previous assays, the signal obtained in the agonist initiation assay is sensitive to all sodium channel modulators that were tested and can be used in high throughput mode, as well as in support of Medicinal Chemistry efforts for lead optimization.

Cell Line↗

[Analysis of Toll-like receptor 4 and myeloid differentiation protein-2 interaction with fluorescence resonance energy transfer].

OBJECTIVE: To study the interaction between Toll-like receptor (TLR) 4 and myeloid differentiation protein-2 (MD-2) in living cells using fluorescence resonance energy transfer (FRET) technology. METHODS: The coding sequences of TLR4 and MD-2 (without the signal peptide sequence) were amplified by PCR and cloned into enhanced cyan fluorescence protein (CFP) and enhanced yellow fluorescence protein (YFP) expression vectors carrying TLR4 signal peptides (pECFP-C1-SP and pEYFP-C1-SP). HEK293 cells were transfected respectively or together with the reconstructed plasmids verified by enzyme digestion and sequence analysis, and the expression and sublocalization of these fluorescence proteins in the cells were observed using fluorescence microscope. FRET in the cells coexpressing CFP-TLR4 and YFP-MD-2 was detected using routine and acceptor photobleaching method. RESULTS: The reconstructed plasmids were expressed in HEK293 cells. The cyan or yellow fluorescence was located in the cytoplasm, mainly around the nucleus in the cells transfected with pECFP/TLR4 or pEYFP/MD-2, and both the cyan and yellow fluorescence located mainly in the membrane and occasional in the cytoplasm of cells cotransfected with pECFP/TLR4 and pEYFP/MD-2. Routine or acceptor photobleaching detected FRET phenomena in cells coexpressing CFP-TLR4 and YFP-MD-2, suggesting direct interaction between TLR4 and MD-2. CONCLUSION: This study provides direct evidence of the interaction between TLR4 and MD-2 in living cells.

Cell Line↗

Pharmacology and signaling properties of epidermal growth factor receptor isoforms studied by bioluminescence resonance energy transfer.

We have developed a new assay for measuring epidermal growth factor receptor (EGFR) activation using the bioluminescence resonance energy transfer (BRET) technology, which directly measures the recruitment of signaling proteins to activated EGFR. Our results demonstrate that EGFR BRET assays precisely measure the pharmacology and signaling properties of EGFR expressed in human embryonic kidney 293T cells. EGFR BRET assays are highly sensitive to known EGFR ligands [pEC50 of epidermal growth factor (EGF)=10.1+/-0.09], consistent with previous pharmacological methods for measuring EGFR activation. We applied EGFR BRET assays to study the characteristics of somatic EGFR mutations that were recently identified in lung cancer. In agreement with recent reports, we detected constitutively active mutant EGFR isoforms, which predominantly signal through the phosphatidylinositol-3-kinase/Akt pathway. The EGFR inhibitors Iressa or Tarceva are severalfold more potent in inhibiting constitutive activity of mutant EGFR isoforms compared with wild-type EGFR. Notable, our results reveal that most of the mutant EGFR isoforms tested were significantly impaired in their response to EGF. The highest level of constitutive activity and nearly complete loss of epidermal growth factor responsiveness was detected in isoforms that carry the activating mutation L858R and the secondary resistance mutation T790M. In summary, our study reveals that somatic mutations in EGFR quantitatively differ in pharmacology and signaling properties, which suggest the possibility of differential clinical responsiveness to treatment with EGFR inhibitors. Furthermore, we demonstrate that the EGFR BRET assays are a useful tool to study the pharmacology of ligand-induced interaction between EGFR and signaling pathway-specifying adapter proteins.

Cell Line↗