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Results for “protocol optimization”

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PubMed1983

The control issue in biofeedback training.

Well-delineated models and procedural descriptions are noticeably missing from the clinical biofeedback literature dealing with the training of physiological self-control. As a consequence, optimal protocols for the assessment and training of self-control of physiological functions have not eventuated. The present article reviews evidence suggesting that researchers have (a) confused self-control training, assessment, and measurement of tonic response levels, (b) neglected to determine the degree of acquired physiological self-control, and (c) failed to report the relationship between level of acquired physiological control and clinical outcome. The article also proposes an assessment and training paradigm that may be useful in reducing these methodological problems in future biofeedback research.

Arousal↗

PubMed1992

Survival of human oocytes cryopreserved with or without the cumulus in 1,2-propanediol.

BACKGROUND: Although cryopreservation of human preembryos has been carried out with success, the cryostorage of oocytes, which pose fewer controversial moral, ethical, and legal problems has been much less successful. Various attempts to cryopreserve human oocytes have been mostly unsuccessful and the search for an optimal protocol for oocyte cryopreservation remains elusive. We therefore undertook this study to determine the effect of oocyte cryostorage in 1,2-propanediol. METHOD: Mature human oocytes with or without their cumuli were cryopreserved in precooled 1,2-propanediol, then thawed and inseminated with sperms for in vitro fertilization. The outcome of insemination and subsequent embryonic development were also recorded and compared. RESULTS: Postthaw cryosurvival rate was significantly better when cryostorage was carried out with the oocyte cumulus intact as compared to those oocytes denuded of their cumuli (54 versus 27%, respectively; P < 0.05). Eight (44%) of 18 surviving postthaw oocytes with intact cumuli were fertilized normally, with cleavage in six, as compared to two (25%) and one, respectively, of those denuded of their cumulus prior to cryostorage. Development to the blastocyst stage was achieved in three embryos derived from oocytes with an intact cumulus at cryostorage. CONCLUSION: We conclude that 1,2-propanediol can be used with success in oocyte cryopreservation, although the issue of parthenogenecity is still to be resolved. Oocyte's with intact cumulus survive cryostorage better than those without it.

Adult↗

PubMed1987

Establishment of primary cell cultures: experiences with 155 cell strains.

Cell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: Disintegration, culture media supplemented with basal additions, special supplements (growth factors, hormones), and attachment factors. The proliferation rates of the attained cell strains were evaluated by determination of cell doubling times. Procedures for how to obtain a relatively high plating efficiency (approx. 70% in our series of 219 attempts) of primary growth in vitro are described: (1) Mechanical disintegration is superior to enzymatic digestion. If mechanical treatment alone did not produce a sufficient number of viable cells, additional digestion with collagenase/dispase revealed a higher number of proliferating primary cultures than with trypsin. (2) Proliferation of cell cultures from normal and tumorous tissues of epithelial origin was superior in Leibovitz L 15 medium (58 of 87 (67%) cases). Cultures from mesenchymal tissues and tumors were found to have shortest cell doubling times in MEM and RPMI 1640 (16 of 23 (70%) cases). The media were supplemented with the basal additions indicated. (3) In approx. 30% of the cases special supplements like growth factors or hormones increased cell replication, although they were almost always not essential for cell growth. (4) Attachment factors only rarely contributed to the initiation of primary monolayer cultures. The application of various culture conditions does not lead to a protocol optimal for all tissues, for all probes of the same type of tumor, or for all tumor specimens of unique differentiation.

Cell Adhesion↗

PubMed2000

Muscle glycogen recovery after exercise measured by 13C-magnetic resonance spectroscopy in humans: effect of nutritional solutions.

The rate of glycogen resynthesis in human skeletal muscle after glycogen-depleting exercise is known to depend on carbohydrate intake and is reported to reach a plateau after an adequate amount of carbohydrate (CHO) consumption. Efforts to maximize the rate of glycogen storage by changing the type and form of CHO, as well as by adding proteins or lipids have yielded inconsistent results. The objective of this study was to assess whether isocaloric addition of proteins and arginine to a CHO diet in the first 4 h after an endurance exercise would increase the rate of glycogen synthesis. The CHO solution, given twice at a 2 h interval according to earlier optimized protocols, contained 1.7 g CHO/kg(body weieght) The effects of this solution were compared to those of an isocaloric solution containing 1.2 g CHO/kg(body weight) plus 0.5 g protein/kg(body weight) (including 5 g arginine). Glycogen was measured in quadriceps muscle in vivo with natural abundance 13C-magnetic resonance spectroscopy before exercise and twice after exercise, before and at the end of a 4-h period following the intake of one of the solutions. Eight subjects took part in a randomized cross-over trial separated by at least 1 week. Glycogen synthesis was found to be significantly increased with both regimes compared to a zero-caloric placebo diet, but no significant difference in glycogen resynthesis was found between the CHO-only diet and the one supplemented by proteins and arginine. It is estimated that significance would have been reached for an increase of 34%, while the effectively measured synthesis rates only differed by 5%.

Adult↗

PubMed1994

Polybrene/DMSO-assisted gene transfer. Generating stable transfectants with nanogram amounts of DNA.

Polybrene/DMSO-assisted gene transfer is a simple and versatile transfection strategy capable of producing high numbers of stable transfectants from adherent monolayer cultures with low (nanogram) quantities of exogenous DNA. The procedure involves two stages: adsorption and internalization. The former is mediated by polybrene (a polycation polymer) and favors the uniform coating of target cells with polybrene-DNA complexes. Following adsorption, the cells are permeabilized by a brief exposure to dimethyl sulfoxide (DMSO) to facilitate the uptake of DNA complexes. Diverse cell types can be exposed to a wide range of polybrene concentrations without adverse effects. By contrast, the key determinant of success is the DMSO permeabilization regime, which must be configured independently for each cell line. Protocols optimized for gene transfer in murine and human fibroblasts are presented along with a guide for the rapid optimization of the method. The advantages and limitations of the method are also discussed.

3T3 Cells↗

PubMedDate not supplied

Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.).

Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

Agrobacterium tumefaciens↗

PubMed1994

Genetic transformation of plants by protoplast electroporation.

This article describes an optimized protocol for the electroporation of tobacco mesophyll protoplasts together with notes and data on the effects of various parameters and suggestions for work with protoplasts of other species. In this protocol, electroporation is achieved by means of electrical pulses from a high-voltage, capacitive-discharge unit. Procedures are described for measurement of protoplast viability with Evan's blue, the detection of transient expression of CAT and GUS gene plasmid constructs, and for the recovery of stable transformants based on selection for kanamycin resistance.

Cell Survival↗

PubMed2002

Biochemical response after 3-d conformal radiotherapy of localized prostate cancer to a total dose of 66 gy 4-year results.

BACKGROUND: Since the introduction of 3-D conformal radiotherapy (CRT) doses of </= 70 Gy have been used in many European countries. In this analysis, the impact of a short-term neoadjuvant hormonal treatment in combination with CRT to a moderate dose level of 66 Gy was examined. PATIENTS AND METHODS: From January 1994 to February 1999 397 patients were treated for carcinoma of the prostate. In 279 patients a definitive curative treatment (T1 = 38, T2 = 165, T3 = 50, Tx = 11) with or without androgen deprivation was performed. 164 patients with radiotherapy of the prostate +/- seminal vesicles to a total dose of 66 Gy (n = 109) alone or in combination with a short-term hormonal treatment (n = 55) were included in this analysis. Biochemical relapse was defined as three rising PSA values or reintroduction of hormonal treatment. A low-risk subgroup was defined for patients with maximum serum PSA level </= 10 and cT</=2 and G</=2, all other patients were summarized as high-risk patients. RESULTS: The median follow-up of alive patients was 40 months (12-72 months). There was a total of 29/164 deaths, two were cause-specific and 27 were considered unrelated to prostate cancer. The 4-year rates of no biochemical evidence of disease for all patients was 58%. For the high-risk group the 4-year rates could be improved with borderline significance from 35% to 66% (p = 0.057) by additional neoadjuvant hormonal treatment. In contrast for the low-risk group no significant improvement was observed: 73% and 82%, respectively (p = 0.5). CONCLUSION: Especially in high-risk patients doses </= 70 Gy for radiotherapy alone seem not to be sufficient for curative treatment. Additional hormonal treatment and doses >/= 70 Gy should be considered. As a consequence of our earlier analysis a prospective multicenter treatment optimization protocol has been initiated in 1999. The protocol includes a risk-adapted dose increase from 70 Gy in low-risk patients to 74 Gy in high-risk patients including short-term androgen ablation.

Adenocarcinoma↗

PubMed1997

[MR angiography of thoracic blood vessels].

Through the introduction of newly invented high-performance gradient systems to MRI, which enable for echoplanar imaging (EPI), also magnetic resonance angiography (MRA) has gained an entirely new field of applications and techniques. Ultrafast imaging techniques in MRA allow the investigation of larger vascular areas within a single breath-hold-period. Artifacts like motion induced signal misregistrations, dephasing or saturation of the vascular signal are minimized by extremely short echo times. The technique thus requires the intravenous application of a contrast media bolus, usually a gadolinium compound, which is in standard clinical use. Coordination of the bolus injection and the timing of the data acquisition is crucial for optimal results. The first pass evaluation of the contrast media resembles CTA to a certain extend. Due to the fast measurement and the high contrast in contrast-enhanced MRA (CE-MRA) new applications and indications are developed like MRA of the pulmonary vessels. The paper offers considerations and trials for optimization of thoracical CE-MRA. Besides parameter constellation also bolus-optimization is described with respect to the dedicated anatomical premises. Investigations on volunteers and on patients build a basis for suggestions of optimized CE-MRA procedures. To date, a final estimation of the clinical value of the new technique cannot be given since ongoing improvements change the optimal protocol frequently and the potential of further developments is high.

Adolescent↗

PubMed1999

Dopamine neurons in a simple GDNF-treated meso-striatal organotypic co-culture model.

Neurodegeneration of dopamine neurons in the ventral mesencephalon projecting to the dorsal striatum (meso-striatal system) plays a major role in Parkinson's disease. The aim of this study was to establish a simple organotypic, in vitro co-culture model for investigating the survival of dopamine neurons stimulated by the novel growth factor, glial-cell-line-derived neurotrophic factor. This model should allow investigation of the effects of the dopaminergic neurotoxin, 6-hydroxydopamine, on the expression of the transcription factor c-fos and on TUNEL staining in vitro. The dopaminotrophic factor, glial-cell-line-derived neurotrophic factor, markedly enhanced dopamine tissue levels and dopamine neuron number. Nerve-fiber ingrowth of dopamine neurons into its striatal target was found to be enhanced with glial-cell-line-derived neurotrophic factor. Using an optimized protocol, it was shown that the neurotoxin 6-hydroxydopamine selectively destructed dopamine neurons. C-fos-like immunoreactivity was enhanced in the mesencephalic part of the co-slices 3 h after application of the neurotoxin. The TUNEL staining occurred 2-5 days after the application of the neurotoxin, but did not seem to be related to dopamine neurons. In conclusion, the organotypic co-culture model provides a simple model for studying survival of dopamine neurons and for observing expression of genes and proteins that could be related to Parkinson's disease. This simple model is useful for screening novel drugs and growth factors and may markedly reduce severe animal experiments.

Animals↗

PubMed2003

Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount.

We describe an optimized protocol for the transient transformation of tobacco protoplasts mediated by polyethylene-glycol (PEG). As expected, the quantitative beta-glucuronidase (Gus) activity driven by pCaMVGus was dependent on the amount of plasmid used. Nevertheless, we demonstrate by an immunodetection method that transformation efficiency did not depend on the amount of plasmid used but on the limitation imposed by cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1-10 microg per test). Finally, we show that, when we used two plasmid types in a mixture at a concentration ranging from 0.1 to 10 microg for each, all transformed cells expressed proteins encoded by both plasmids. Transient expression and co-transformation experiments are routinely used methods and, probably, the major results from this work were assumed by many researchers in this field, but our data experimentally support this assumption.

Plasmids↗

PubMed2003

Use of high-concentration contrast media in multiple-detector-row CT: principles and rationale.

Contrast-medium-enhanced multiple-detector-row CT (MDCT) is a powerful technique for vascular and hepatic imaging. With increasingly faster acquisition speeds, which have become possible with latest 8- and 16-channel scanner systems, contrast medium delivery is becoming increasingly difficult. This article reviews the pharmacokinetic and physiologic principles of vascular and hepatic enhancement following the intravenous injection of iodinated contrast medium. The effects of user-selectable injection parameters, such as the injection rate, the injection duration, and the contrast medium concentration on arterial and parenchymal enhancement are elucidated. Equipped with this knowledge, rational injection strategies for CT angiographic protocols for scanners with different acquisition speeds are derived. Furthermore, injection and timing protocols, optimized for hepatic MDCT during the early arterial, late arterial, and parenchymal phases, are developed.

Contrast Media↗

PubMed2003

Multidetector-row CT: cardiosurgery indications.

This article critically evaluates the role of multidetector-row CT before and after cardiosurgical procedures. In addition, technical aspects, such as scan protocols, optimal image reconstruction intervals, image reformation techniques, and data evaluation, are presented and briefly discussed.

Aortic Valve↗

PubMed2002

An improved method for whole-mount in situ hybridization of Heterodera schachtii juveniles.

An optimized protocol is presented to visualize gene expression in the sedentary beet cyst nematode, Heterodera schachtii, by whole-mount in situ hybridization. Two different probes were used for genes with known expression pattern in other nematodes. Vacuum infiltration of the fixative significantly increased its efficiency and resulted in a nicely preserved morphology. Additional modifications were introduced to simplify and standardize the process.

Animals↗

PubMed1999

Novel therapeutic strategies in scleroderma.

Optimal management for scleroderma (systemic sclerosis) is likely to require treatment of the underlying disease process, which remains incompletely understood, and also of the organ-based complications of this heterogeneous condition. Clinical trials evaluating several potential agents have been completed recently, including D-penicillamine and interferon alpha. Unfortunately none of these studies has suggested significant efficacy. This article focuses on new treatment approaches using existing therapeutic agents, such as prostacyclin, and considers the potential usefulness of new agents (eg, relaxin, halofuginone) or strategies such as intensive immunosuppression with peripheral stem cell rescue. Ultimately, a better understanding of disease pathogenesis may facilitate the development of targeted therapy against key events or mediators, but for the present better evaluation of existing agents and a focus on optimizing protocols for organ-based complications, such as pulmonary vascular disease or hypertensive renal crisis, are important goals.

Clinical Trials as Topic↗

PubMed2026

Liquid fermentation and an adapted cetyltrimethylammonium bromide (CTAB) method enable sequencing-grade DNA extraction from Phanerodontia australis.

High-quality genomic DNA extraction from basidiomycete fungi remains challenging due to polysaccharide co-purification and nuclease-mediated DNA degradation. We systematically compared 22 experimental conditions combining five DNA extraction methods (one Dellaporta-based method, three CTAB-based variants, and one commercial kit) with five mycelium preparation techniques (scalpel scraping, fungal suspension, silica bead homogenization, liquid fermentation, and liquid nitrogen treatment) for Phanerodontia australis (BRM62389). DNA quality was assessed by 1% agarose gel electrophoresis, NanoDrop spectrophotometry, and Qubit fluorimetry. The optimal protocol combined liquid-fermentation mycelium with an adapted CTAB method, yielding Qubit-quantified concentrations of 34.2 and 62.6 ng/µL (samples S21 and S22), 260/280 ratios of 2.06 and 2.05, 260/230 ratios of 1.88 and 1.85, and no detectable DNA degradation by agarose gel electrophoresis. This protocol enabled whole-genome sequencing, yielding a 37.62 Mb assembly with 98.5% completeness as assessed by BUSCO v5 (basidiomycota_odb10 lineage dataset, n = 1,764). Liquid fermentation is associated with the production of younger, actively growing hyphae with reduced cell wall thickness, and the adapted CTAB method effectively removed residual polysaccharides. This protocol provides a reliable, cost-effective approach for obtaining sequencing-grade DNA from P. australis.

Basidiomycota↗

PubMed1993

Immortalization of antigen selected B cells.

This paper reports the generation of monoclonal antibody producing hybridomas from a small number of antigen-specific B cells selected by panning on antigen-coated dishes and rosetting with antigen-coupled paramagnetic beads. Anti-HIV positive B cells from spleen could be recovered by panning with an efficiency of 5% and a purity of 24%. Immunobead selection of anti-HIV positive B cells from the same mice yielded a recovery of 17% and a purity of 7%. Various experimental conditions with respect to the selection of specific B cells were investigated, leading to an optimized protocol for the isolation of a limited subset of B cells. The selected cells retained their property to produce immunoglobulins and could be clonally expanded in the presence of human T cell supernatant and irradiated murine thymoma helper cells to generate sufficient cells for a mini-electrofusion with NS-1 myeloma cells. Up to 78 specific hybridomas could be generated from one anti-HIV positive B cell. An overall efficiency of specific B cell immortalization of up to 10% was obtained.

Animals↗

PubMed1994

Efficient transfer of antibodies into mammalian cells by electroporation.

An optimized protocol for electroporation is described which is suitable for introducing antibodies into mammalian cells. The method results in the uptake of detectable amounts of antibodies in 80% of the cells and in 40% large amounts are introduced. As an example, cell cycle activity (transition from the G1 to S phase) was inhibited by the introduction of monoclonal antibodies against G1-specific cyclin D1 into CV-1 and MCF7 cells. This specific antibody mediated inhibition of cellular function did not affect the viability of the cells since they recovered from the inhibition after some time. While approaching the efficiency of microinjection, the new protocol for electroporation of antibodies additionally permits treatment of the larger number of cells which are required for biochemical analyses.

Animals↗