960 resultados para CULTURE METHOD
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As opposed to culture on standard tissue-treated plastic, cell culture on three-dimensional scaffolds impedes additional challenges with respect to substrate preparation, cell seeding, culture maintenance, and analysis. We herewith present a general route for the culture of primary cells, differentiated cells, or stem cells on plasma-coated, electrospun scaffolds. We describe a method to prepare and fix the scaffolds in culture wells and discuss a convenient method for cell seeding and subsequent analysis by scanning electron microscopy or immunohistology.
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This project was an experiment in widening the traditional borders of study in the field and looking at the phenomenon of Gothic taste in many genres and kinds of art. The Gothic taste was a major element in the cultural image of the Enlightenment both in western Europe and in Russia. It was an essential component in the world outlook of an educated person and without studying this phenomenon it is impossible to fully understand the thinking of artistic professionals, amateurs and users in Russian society in the 18th century. Mr. Khatchatourov first analysed the reasons for the importance of Gothic taste in the culture of the European Enlightenment and then studied its linguistic and lexicographic evolution in 18th century Russian culture. He sought to determine the semantic context which actively formed the human mind set in the Enlightenment, including potential users and producers of articles in the Gothic taste. He then looked at the process of absorption of this concept by those forms of art which express it most strongly, in particular architecture and the theatre. His study was based on a comprehensive historical and culturological study using a wide range of sources, a formal stylistic method approach considering the interaction of non-classical styles of the Enlightenment with the dominant classicism, and an iconographic approach which revealed the essential aspects in a new image synthesis of the culture of the Enlightenment.
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This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and/or demonstrated positive anti-VHSV-antibody titres (83 sites, 121 positive blood samples) in a serum plaque neutralization test (SPNT). The RT-PCR technique confirmed the four VHSV-positive tissue sample pools detected by virus isolation and additionally identified one VHSV-positive sample that showed positive anti-VHSV-AB titres, but was negative in virus isolation. With IHNV, RT-PCR detected two positive samples not identified by virus isolation while in these fish the SPNT result had been questionable. One of the IHNV-positive samples represents the first detection of IHNV-RNA in wild brown trout in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus isolation. However, in a titration experiment under laboratory conditions, the sensitivity of RT-PCR was not significantly higher when compared with virus isolation.
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Many mechanisms involved in the pathogenesis of chronic enteropathies or host-pathogen interactions in canine intestine have not been elucidated so far. Next to the clinical and in vivo research tools, an in vitro model of canine intestinal cell culture would be very helpful for studies at the cellular level. Therefore, the purpose of this study was to establish and characterize a primary canine duodenal epithelial cell culture. Neonatal duodenum was disrupted with trypsin-ethylenediaminetetraacetic acid (EDTA) and the mucosa scraped off and digested with collagenase and dispase. After centrifugation on a 2% sorbitol gradient, the cells were incubated at 37 degrees C in OptiMEM supplemented with Primocin, epidermal growth factor, insulin, hydrocortisone, and 10% fetal calf serum (FCS). After 24 h, the FCS concentration was reduced to 2.5%, and the temperature decreased to 33 degrees C. With this method, the cultures were growing to confluent monolayers within 5-6 d and remained viable for an average of 2 wk. Their epithelial nature was confirmed by electron microscopy and immunofluorescence staining using antibodies directed against specific cytokeratins, desmosomes, and tight junctions. The intestinal cells proliferated, as evidenced by immunolabeling with a Ki-67 antibody, and cryptal cell subpopulations could be identified. Furthermore, alkaline phosphatase and sucrase activity were detected.
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INTRODUCTION: The incidence of bloodstream infection (BSI) in extracorporeal life support (ECLS) is reported between 0.9 and 19.5%. In January 2006, the Extracorporeal Life Support Organization (ELSO) reported an overall incidence of 8.78% distributed as follows: respiratory: 6.5% (neonatal), 20.8% (pediatric); cardiac: 8.2% (neonatal) and 12.6% (pediatric). METHOD: At BC Children's Hospital (BCCH) daily surveillance blood cultures (BC) are performed and antibiotic prophylaxis is not routinely recommended. Positive BC (BC+) were reviewed, including resistance profiles, collection time of BC+, time to positivity and mortality. White blood cell count, absolute neutrophile count, immature/total ratio, platelet count, fibrinogen and lactate were analyzed 48, 24 and 0 h prior to BSI. A univariate linear regression analysis was performed. RESULTS: From 1999 to 2005, 89 patients underwent ECLS. After exclusion, 84 patients were reviewed. The attack rate was 22.6% (19 BSI) and 13.1% after exclusion of coagulase-negative staphylococci (n = 8). BSI patients were significantly longer on ECLS (157 h) compared to the no-BSI group (127 h, 95% CI: 106-148). Six BSI patients died on ECLS (35%; 4 congenital diaphragmatic hernias, 1 hypoplastic left heart syndrome and 1 after a tetralogy repair). BCCH survival on ECLS was 71 and 58% at discharge, which is comparable to previous reports. No patient died primarily because of BSI. No BSI predictor was identified, although lactate may show a decreasing trend before BSI (P = 0.102). CONCLUSION: Compared with ELSO, the studied BSI incidence was higher with a comparable mortality. We speculate that our BSI rate is explained by underreporting of "contaminants" in the literature, the use of broad-spectrum antibiotic prophylaxis and a higher yield with daily monitoring BC. We support daily surveillance blood cultures as an alternative to antibiotic prophylaxis in the management of patients on ECLS.
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The single-celled protozoan Trypanosoma brucei spp. is the causative agent of human African trypanosomiasis and nagana in cattle. Quantitative proteomics for the first time allowed for the characterization of the proteome from several different life stages of the parasite (1-3). To achieve this, stable isotope labeling by amino acids in cell culture (SILAC; (4)) was adapted to T. brucei spp. cultures. T. brucei cells grown in standard media with dialyzed fetal calf serum containing heavy isotope-labeled amino acids (arginine and lysine) show efficient incorporation of the labeled amino acids into the whole cell proteome (8-12 divisions) and no detectable amino acid conversions. The method can be applied to both of the major life stages of the parasite and in combination with RNAi or gene knock-out approaches.
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In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for induction with rifampicin were created. Williams' medium E showed the best results on morphology and viability of the hepatocytes and was therefore used for the following induction experiments. Cells cultured in Dulbecco's Modified Eagle Medium were not inducible. Incubation with rifampicin increased the CYP activity in two different hepatocyte preparations in a dose dependent manner (EC50=1.20 μM and 6.06 μM; Emax=4.1- and 3.4-fold induction). No increase in CYP activity was detected after incubation with dexamethasone or phenobarbital. The hepatocyte culture conditions established in this study proved to be valuable for investigation of the induction of equine CYPs. In further studies, other equine drugs can be evaluated for CYP induction with this in vitro system.
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Abstract OBJECTIVE: Signaling molecules derived from osteocytes have been proposed as a mechanism by which autografts contribute to bone regeneration. However, there have been no studies that determined the role of osteocytes in bone grafts. MATERIAL AND METHOD: Herein, it was examined whether bone chips and demineralized bone matrix release sclerostin and FGF-23, both of which are highly expressed by osteocytes. RESULTS: Bone grafts from seven donors were placed in culture medium. Immunoassay showed that bone chips released sclerostin (median 1.0 ng/ml) and FGF-23 (median 9.8 relative units/ml) within the first day, with declining levels overtime. Demineralized bone matrix also released detectable amounts of sclerostin into culture medium, while FGF-23 remained close to the detection limit. In vitro expanded isolated bone cells failed to release detectable amounts of sclerostin and FGF-23. CONCLUSION: These results suggest that autografts but also demineralized bone matrix can release signaling molecules that are characteristically produced by osteocytes. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. KEYWORDS: FGF-23; autologous bone; bone grafts; demineralized bone matrix; osteocytes; sclerostin
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A new method is presented that increases the sensitivity of ultrasound-based techniques for detection of bacteria. The technique was developed for the detection of catalase-positive microorganisms. It uses a bubble trapping medium containing hydrogen peroxide that is mixed with the sample for microbiological evaluation. The enzyme catalase is present in catalase-positive bacteria, which induces a rapid hydrolysis of hydrogen peroxide, forming bubbles which remain in the medium. This reaction results in the amplification of the mechanical changes that the microorganisms produce in the medium. The effect can be detected by means of ultrasonic wave amplitude continuous measurement since the bubbles increase the ultrasonic attenuation significantly. It is shown that microorganism concentrations of the order of 105 cells ml−1 can be detected using this method. This allows an improvement of three orders of magnitude in the ultrasonic detection threshold of microorganisms in conventional culture media, and is competitive with modern rapid microbiological methods. It can also be used for the characterization of the enzymatic activity.
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We describe a method to design dominant-negative proteins (D-N) to the basic helix–loop–helix–leucine zipper (B-HLHZip) family of sequence-specific DNA binding transcription factors. The D-Ns specifically heterodimerize with the B-HLHZip dimerization domain of the transcription factors and abolish DNA binding in an equimolar competition. Thermal denaturation studies indicate that a heterodimer between a Myc B-HLHZip domain and a D-N consisting of a 12-amino acid sequence appended onto the Max dimerization domain (A-Max) is −6.3 kcal⋅mol−1 more stable than the Myc:Max heterodimer. One molar equivalent of A-Max can totally abolish the DNA binding activity of a Myc:Max heterodimer. This acidic extension also has been appended onto the dimerization domain of the B-HLHZip protein Mitf, a member of the transcription factor enhancer binding subfamily, to produce A-Mitf. The heterodimer between A-Mitf and the B-HLHZip domain of Mitf is −3.7 kcal⋅mol−1 more stable than the Mitf homodimer. Cell culture studies show that A-Mitf can inhibit Mitf-dependent transactivation both in acidic extension and in a dimerization-dependent manner. A-Max can inhibit Myc-dependent foci formation twice as well as the Max dimerization domain (HLHZip). This strategy of producing D-Ns may be applicable to other B-HLHZip or B-HLH proteins because it provides a method to inhibit the DNA binding of these transcription factors in a dimerization-specific manner.
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We developed a real-time detection (RTD) polymerase chain reaction (PCR) with rapid thermal cycling to detect and quantify Pseudomonas aeruginosa in wound biopsy samples. This method produced a linear quantitative detection range of 7 logs, with a lower detection limit of 103 colony-forming units (CFU)/g tissue or a few copies per reaction. The time from sample collection to result was less than 1h. RTD-PCR has potential for rapid quantitative detection of pathogens in critical care patients, enabling early and individualized treatment.
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The pathogenic protozoan parasite Entamoeba histolytica, the cause of amebic dysentery and amebic liver abscess, is an obligate anaerobe, and derives energy from the fermentation of glucose to ethanol with pyruvate and acetyl coenzyme A as intermediates. We have isolated EhADH2, a key enzyme in this pathway, that is a NAD+- and Fe2+-dependent bifunctional enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activities. EhADH2 is the only known eukaryotic member of a newly defined family of prokaryotic multifunctional enzymes, which includes the Escherichia coli AdhE enzyme, an enzyme required for anaerobic growth of E. coli. Because of the critical role of EhADH2 in the amebic fermentation pathway and the lack of known eukaryotic homologues of the EhADH2 enzyme, EhADH2 represents a potential target for antiamebic chemotherapy. However, screening of compounds for antiamebic activity is hampered by the cost of large scale growth of Ent. histolytica, and difficulties in quantitating drug efficacy in vitro. To approach this problem, we expressed the EhADH2 gene in a mutant strain of E. coli carrying a deletion of the adhE gene. Expression of EhADH2 restored the ability of the mutant E. coli strain to grow under anaerobic conditions. By screening compounds for the ability to inhibit the anaerobic growth of the E. coli/EhADH2 strain, we have developed a rapid assay for identifying compounds with anti-EhADH2 activity. Using bacteria to bypass the need for parasite culture in the initial screening process for anti-parasitic agents could greatly simplify and reduce the cost of identifying new therapeutic agents effective against parasitic diseases.
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This mixed method study aimed to redress the gap in the literature on academic service-learning partnerships, especially in Eastern settings. It utilized Enos and Morton's (2003) theoretical framework to explore these partnerships at the American University in Cairo (AUC). Seventy-nine community partners, administrators, faculty members, and students from a diverse range of age, citizenship, racial, educational, and professional backgrounds participated in the study. Qualitative interviews were conducted with members of these four groups, and a survey with both close-ended and open-ended questions administered to students yielded 61 responses. Qualitative analyses revealed that the primary motivators for partners' engagement in service-learning partnerships included contributing to the community, enhancing students' learning and growth, and achieving the civic mission of the University. These partnerships were characterized by short-term relationships with partners' aspiring to progress toward long-term commitments. The challenges to these partnerships included issues pertaining to the institution, partnering organizations, culture, politics, pedagogy, students, and faculty members. Key strategies for improving these partnerships included institutionalizing service-learning in the University and cultivating an institutional culture supportive of community engagement. Quantitative analyses showed statistically significant relationships between students' scores on the Community Awareness and Interpersonal Effectiveness scales and their overall participation in community service activities inside and outside the classroom, as well as a statistically significant difference between their scores on the Community Awareness scale and department offering service-learning courses. The study's outcomes underscore the role of the local culture in shaping service-learning partnerships, as well as the role of both curricular and extracurricular activities in boosting students' awareness of their community and interpersonal effectiveness. Cultivating a culture of community engagement and building support mechanisms for engaged scholarship are among the critical steps required by public policy-makers in Egypt to promote service-learning in Egyptian higher education. Institutionalizing service-learning partnerships at AUC and enhancing the visibility of these partnerships on campus and in the community are essential to the future growth of these collaborations. Future studies should explore factors affecting community partners' satisfaction with these partnerships, top-down and bottom-up support to service-learning, the value of reflection to faculty members, and the influence of students' economic backgrounds on their involvement in service-learning partnerships.
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Mode of access: Internet.
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Microfilm. Ann Arbor, Mich., Xerox University Microfilms, 1974. 1 reel. 35 mm. (American Culture Series, reel 617.3)