SEARCH · PubMed Health

Results for “protocol optimization”

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

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

At least 271 records · Page 15Linked to original sources

PubMedDate not supplied

[Early detection and intervention of hearing impairment in Cuba: outcome after 20 years].

INTRODUCTION: Infant hearing loss is a highly prevalent disorder which untreated can severely disrupt normal brain development. As a result there is a significant delay in language acquisition as well as many cognitive and emotional problems in the child. Over the last decades important advances have occurred in the available technology for early detection and assessment of hearing impairment. Therefore many countries worldwide have become aware of the need for hearing screening programs. The optimal protocols depending on the local conditions of health care as well as the availability of technological and human resources. AIM: To summarize the results obtained over the last 20 years by an ongoing hearing screening protocol. DEVELOPMENT: Data on the coverage program, sensitivity and specificity, age of identification of hearing losses, diagnostic and intervention stages will be summarized and discussed. Also the long terms effects of early detection on the child cognitive and language development are analyzed. Finally, the possible role of a new technique based on the recording of multiple auditory steady state potentials with Cuban equipment (AUDIX system) was evaluated within this context. CONCLUSIONS: The Cuban targeting multiple high risk hearing screening program is a useful alternative to early detection of hearing losses. The average detection age of hearing loss was 10 months during the period of optimal functioning. Cognitive, emotional and linguistic development are improved by early detection and intervention. The multiple auditory steady state responses can provide valuable audiometric information within a screening context.

Child↗

PubMed1997

Optimization of transfection of human endothelial cells.

Recently developed transfection methods for mammalian cells provide a powerful means for the study of gene function. Unfortunately, human endothelial cells were relative refractory to the classic transfection techniques. In this study we compared the usability of calcium phosphate, DEAE-dextran transfection, transferrinfection, lipofection, and electroporation for the transfection of early passage HUVECs and for the human endothelial cell lines ECV 304 and EA.hy 926. The classic transfection methods resulted in no or only marginal expression of the reporter gene E. coli beta-galactosidase. For lipofection experiments we compared the commercially available liposome formulations DOTAP and Transfectam with liposomes prepared of dimethyldioctadecylammoniumbromide (DDAB) or 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE) as the cationic lipid compound and dioleylphosphatidylethanolamine (DOPE) or Azolectin (a crude fraction of soybean lipids, commercially available as phosphatidylcholine II) as neutral co-lipid. Because the protocol for the chemical synthesis of DMRIE has not been published yet, we developed a protocol for the chemical synthesis of this cationic lipid. With transfection protocols optimized for each cell line, we could achieve transfection efficiencies up to 2%. Compared to the other methods used, the lipofection proved to be a reliable technique for the efficient transfection of the human endothelial cell lines ECV 304 and EA.hy 926. Although we achieved a maximum transfection efficiency of 0.45% for the lipofection of HUVEC, the electroporation seemed to be the better choice for these cells.

Cell Line↗

PubMed2003

FDS: flexible ligand and receptor docking with a continuum solvent model and soft-core energy function.

The docking of flexible small molecule ligands to large flexible protein targets is addressed in this article using a two-stage simulation-based method. The methodology presented is a hybrid approach where the first component is a dock of the ligand to the protein binding site, based on deriving sets of simultaneously satisfied intermolecular hydrogen bonds using graph theory and a recursive distance geometry algorithm. The output structures are reduced in number by cluster analysis based on distance similarities. These structures are submitted to a modified Monte Carlo algorithm using the AMBER-AA molecular mechanics force field with the Generalized Born/Surface Area (GB/SA) continuum model. This solvent model is not only less expensive than an explicit representation, but also yields increased sampling. Sampling is also increased using a rotamer library to direct some of the protein side-chain movements along with large dihedral moves. Finally, a softening function for the nonbonded force field terms is used, enabling the potential energy function to be slowly turned on throughout the course of the simulation. The docking procedure is optimized, and the results are presented for a single complex of the arabinose binding protein. It was found that for a rigid receptor model, the X-ray binding geometry was reproduced and uniquely identified based on the associated potential energy. However, when side-chain flexibility was included, although the X-ray structure was identified, it was one of three possible binding geometries that were energetically indistinguishable. These results suggest that on relaxing the constraint on receptor flexibility, the docking energy hypersurface changes from being funnel-like to rugged. A further 14 complexes were then examined using the optimized protocol. For each complex the docking methodology was tested for a fully flexible ligand, both with and without protein side-chain flexibility. For the rigid protein docking, 13 out of the 15 test cases were able to find the experimental binding mode; this number was reduced to 11 for the flexible protein docking. However, of these 11, in the majority of cases the experimental binding mode was not uniquely identified, but was present in a cluster of low energy structures that were energetically indistinguishable. These results not only support the presence of a rugged docking energy hypersurface, but also suggest that it may be necessary to consider the possibility of more than one binding conformation during ligand optimization.

Algorithms↗

PubMed2004

Laser microdissection of immunolabeled astrocytes allows quantification of astrocytic gene expression.

Astrocytes represent the major glial cell population within the central nervous system. In order to elucidate the function of astrocytes under physiological conditions and during the course of neurological disease, astrocytic gene expression profiling is necessary. However, since astrocytes form an intimately connected network with neurons and other cell types in the brain, gene expression analysis of astrocytes with a sufficient degree of cellular specificity is difficult. Here we are presenting a rapid and, thus, RNA preserving immunostaining protocol for the detection of astrocytes in rodent brain. This protocol can readily be combined with laser microdissection (Leica AS LMD platform) and quantitative RT-PCR (qPCR). Employing this method, we studied changes in glial fibrillary acidic protein (GFAP) expression in astrocytes of mouse entorhinal cortex following entorhinal cortex lesion. Using laser microdissection, astrocytes (n = 60) were collected in the tissue surrounding the lesion, the entorhinal cortex contralateral to the lesion, and in unlesioned control animals. Changes in GFAP mRNA were quantified using qPCR. GFAP mRNA levels were 82-fold higher in astrocytes of lesioned animals at the site of the lesion compared to GFAP mRNA levels in entorhinal cortex astrocytes of control mice. GFAP mRNA levels were only slightly elevated at the contralateral side (lesioned animals). This optimized protocol for immunolabeling and laser microdissection of astrocytes followed by qPCR allows quantification of astrocytic gene expression levels with a high degree of cellular specificity. It may similarly be employed in different settings where other cell types need to be identified and microdissected for gene expression profiling.

Animals↗

PubMed2000

The preparation of periapical lesions for flow cytometry.

AIM: To devise an optimal protocol and to analyse the leucocyte composition of periapical (PA) lesions by flow cytometry. METHODOLOGY: PA lesions were mechanically agitated, with and without proteolysis. This was with either 0.2% collagenase alone, or in combination with 0.02% DNA-ase in serial incubations until all tissue was digested. The efficacy of each method was assessed by counting total cell yield and cell viability. Phenotype stability was gauged by the percentage of peripheral blood leucocytes (PBL) which expressed CD45RB, CD3, CD20, CD4 and CD8 before and after mechanical and collagenase treatment. RESULTS: Disaggregation of PA lesions was superior if collagenase was present, but cell clumping was problematic unless the DNA-ase was also added, and serial digestion with this combination produced optimal cell yield and viability. Nevertheless, the total number of cells released rarely exceeded 105, though viability was in excess of 80%. Mechanical agitation and proteolysis adversely affected PBL phenotypes, but collagenase digestion limited to 10 min caused least damage. Flow cytometric analysis of disaggregated PA lesions failed to identify more than 7.9% (mean, range 6-10%) CD45RB + cells. CONCLUSIONS: Because of the necessity for single cell suspensions, flow cytometry is not easily applied to the analysis of leucocytes in PA lesions, and further refinements in tissue disaggregation and cell preparation are required.

Antigens, CD↗

PubMed2002

Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls.

BACKGROUND: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas the flow-FISH technique requires relatively few cells (10(5)) and can be completed in a single day. A further advantage of the flow-FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to obtain accurate and reproducible results using the flow-FISH technique, we systematically explored the influence of various steps in the protocol on telomere length values and established an acceptable range for the most critical parameters. METHODS: Isolated leukocytes from whole blood are denatured by heat and 70%/75% formamide, then hybridized with or without a telomere-specific fluorescein isothiocyante (FITC)-conjugated peptide nucleic acid probe (PNA). Unbound telomere PNA is washed away, the DNA is counterstained, and telomere fluorescence is measured on a flow cytometer using an argon ion laser (488 nm) to excite FITC. For each sample, duplicates of telomere PNA-stained and unstained tubes are analyzed. RESULTS: Cell counts and flow-FISH telomere length measurements were performed on leukocytes and thymocytes of humans and other species. Leukocyte suspensions were prepared by two red blood cell lysis steps with ammonium chloride. Optimal denaturation of DNA was achieved by heating at 85-87 degrees C for 15 min in a solution containing 70%/75% formamide. Hybridization was performed at room temperature with a 0.3 microg/ml telomere-PNA probe for at least 60-90 min. Unbound telomere-PNA probe was diluted at least 4,000-40,000 times with wash steps containing 70%/75% formamide at room temperature. LDS 751 and DAPI were suitable as DNA counterstains as they did not show significant interference with telomere length measurement. CONCLUSIONS: The use of flow-FISH for telomere length measurements in nucleated blood cells requires tight adherence to an optimized protocol. The method described here can be used to determine rapidly the telomere length in subsets of nucleated blood cells.

Animals↗

PubMed1995

Factors affecting the electroporation of Bacillus subtilis.

Bacillus subtilis 168 trp- was found to be transformable with the tetracycline resistance plasmid pAB124 by electroporation of whole cells, inconsistently and at very low frequencies. Supplementation of the growth medium with glycine, or particularly DL-threonine, produced cells that could be electrotransformed much more efficiently at frequencies up to 2.5 x 10(3) transformants per microgram plasmid DNA. Transformation was optimal with cells grown in medium containing a racemic mixture of the D- and L-isomers of threonine, and no transformants were obtained when pure forms of the D- and L-threonine isomers were used. The cell walls of B. subtilis grown in the presence or absence of D-, L- and DL-threonine had a similar amino acid composition which did not include threonine. A more complex biochemical explanation of the enhancement of electroporation by growth in DL-threonine is likely, and this is discussed. Lysozyme treatments to weaken the cell wall and possibly mimic the effect of DL-threonine did not yield any transformants. The effects of buffer composition and culture incubation time were also determined and the electroporation protocol optimized accordingly. The response of a range of other B. subtilis strains to electroporation by the method produced was found to be variable. In all cases, transformation was verified by recovery of the plasmid DNA from putative transformants.

Amino Acids↗

PubMed2006

Systematic QM/MM investigation of factors that affect the cytochrome P450-catalyzed hydrogen abstraction of camphor.

The hydrogen abstraction reaction of camphor in cytochrome P450(cam) has been investigated in the native enzyme environment by combined quantum mechanical/molecular mechanical (QM/MM) calculations and in the gas phase by density functional calculations. This work has been motivated by contradictory published QM/MM results. In an attempt to pinpoint the origin of these discrepancies, we have systematically studied the factors that may affect the computed barriers, including the QM/MM setup, the optimization procedures, and the choice of QM region, basis set, and protonation states. It is found that the ChemShell and QSite programs used in the published QM/MM calculations yield similar results at given geometries, and that the discrepancies mainly arise from two technical issues (optimization protocols and initial system preparation) that need to be well controlled in QM/MM work. In the course of these systematic investigations, new mechanistic insights have been gained. The crystallographic water 903 placed near the oxo atom of Compound I lowers the hydrogen abstraction barrier by ca. 4 kcal/mol, and thus acts as a catalyst for this reaction. Spin density may appear at the A-propionate side chain of the heme if the carboxylate group is not properly screened, which might be expected to happen during protein dynamics, but not in static equilibrium situations. There is no clear correlation between the computed A-propionate spin density and the hydrogen abstraction barrier, and hence, no support for a previously proposed side-chain mediated transition state stabilization mechanism. Standard QM/MM optimizations yield an A-propionate environment close to the X-ray structure only for protonated Asp297, and not for deprotonated Asp297, but the computed barriers are similar in both cases. An X-ray like A-propionate environment can also be obtained when deprotonated Asp297 is included in the QM region and His355 is singly protonated, but this Compound II-type species with a closed-shell porphyrin ring has a higher hydrogen abstraction barrier and should thus not be mechanistically relevant.

Binding Sites↗

PubMed2002

Development and validation of a method for using breast core needle biopsies for gene expression microarray analyses.

PURPOSE: Gene expression microarray technologies have the potential to define molecular profiles that may identify specific phenotypes(diagnosis), establish a patient's expected clinical outcome (prognosis), and indicate the likelihood of a beneficial effect of a specific therapy (prediction). We wished to develop optimal tissue acquisition, processing, and analysis procedures for exploring the gene expression profiles of breast core needle biopsies representing cancer and noncancer tissues. EXPERIMENTAL DESIGN: Human breast cancer xenografts were used to evaluate several processing methods for prospectively collecting adequate amounts of high-quality RNA for gene expression microarray studies. Samples were assessed for the preservation of tissue architecture and the quality and quantity of RNA recovered. An optimized protocol was applied to a small study of core needle breast biopsies from patients, in which we compared the molecular profiles from cancer with those from noncancer biopsies. Gene expression data were obtained using Research Genetics, Inc. Named Genes cDNA microarrays. Data were visualized using simple hierarchical clustering and a novel principal component analysis-based multidimensional scaling. Data dimensionality was reduced by simple statistical approaches. Predictive neural networks were built using a multilayer perceptron and evaluated in an independent data set from snap-frozen mastectomy specimens. RESULTS: Processing tissue through RNALater preserves tissue architecture when biopsies are washed for 5 min on ice with ice-cold PBS before histopathological analysis. Cell margins are clear, tissue folding and fragmentation are not observed, and integrity of the cores is maintained, allowing optimal pathological interpretation and preservation of important diagnostic information. Adequate concentrations of high-quality RNA are recovered; 51 of 55 biopsies produced a median of 1.34 microg of total RNA (range, 100 ng to 12.60 microg). Snap-freezing or the use of RNALater does not affect RNA recovery or the molecular profiles obtained from biopsies. The neural network predictors accurately discriminate between predominantly cancer and noncancer breast biopsies. CONCLUSIONS: The approaches generated in these studies provide a simple, safe, and effective method for prospectively acquiring and processing breast core needle biopsies for gene expression studies. Gene expression data from these studies can be used to build accurate predictive models that separate different molecular profiles. The data establish the use and effectiveness of these approaches for future prospective studies.

Animals↗

PubMed2004

Evaluation of plasmid DNA removal from lentiviral vectors by benzonase treatment.

To improve the purity of lentiviral vector supernatants for clinical studies we have evaluated plasmid DNA removal from lentiviral vectors and also the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol. Optimal conditions of plasmid DNA removal by benzonase treatment were established by varying the temperature, time, and benzonase concentrations in the reaction mix and were determined to be 50 units of benzonase per milliliter of vector supernatant at 37 degrees C, for 15 min. No plasmid DNA was detected, suggesting efficient plasmid degradation was achieved under these experimental conditions. The infectious titer of benzonase-treated lentiviral vector (RRL-CMV-GFP) was nearly identical to the titer of untreated vector (2.3 +/- 0.3 x 10(6) transduction units per milliliter (TU/ml) and 2.7 +/- 0.3 x 10(6) TU/ml, respectively). Analysis of plasmid DNA in concentrated lentiviral vectors shows that concentration substantially decreases the amount of DNA per TU. Analysis of the extent of plasmid DNA associated with transduced CD34 cells in an ex vivo transduction protocol suggests that a minimal amount of plasmid is transferred to transduced cells if the vector supernatant was not previously treated with benzonase. In conclusion, benzonase treatment is effective in eliminating plasmid DNA from vector supernatants and treatment does not affect infectious titers. However, because there is minimal transfer of plasmid DNA to transduced cells under ex vivo transduction conditions, DNA removal from lentiviral vectors may not be essential for all ex vivo clinical applications.

Adult↗

PubMed2004

Interleukin-2 receptor antagonists and aggressive steroid minimization strategies for kidney transplant patients.

Steroid withdrawal during the first week after transplantation surgery, or complete avoidance of steroids, offers potential benefits. The interleukin-2 (IL-2) receptor antibodies, basiliximab and daclizumab, can enable aggressive steroid minimization protocols that are efficacious while reducing toxicity. A multicenter, randomized trial of kidney transplant recipients has shown the incidence of biopsy-proven acute rejection with basiliximab, cyclosporine and mycophenolate mofetil with steroids withdrawn at day 5 to be similar to a conventional triple-therapy regimen. A single perioperative dose of corticosteroids with an IL-2 receptor antagonist also seems as efficacious as standard steroid therapy. Corticosteroid-minimization with IL-2 receptor antagonists has also been investigated with sirolimus-containing regimens and has shown excellent outcomes. Experience with complete steroid avoidance, using an IL-2 receptor antagonist, is limited, but initial results are promising, particularly in pediatric patients. Administration of an IL-2 receptor antagonist with aggressive steroid minimization in selected, well-monitored patients seems reasonable, but further trials are required to define optimal protocols.

Adrenal Cortex Hormones↗

PubMed2002

PCR detection of Tetramicra brevifilum (Microspora) infection in turbot (Scophthalmus maximus L.) musculature.

This study investigated the spatial distribution of Tetramicra brevifilum spores in the musculature of infected turbot Scophthalmus maximus, with the aim of identifying the most appropriate body locations for diagnostic assays. A PCR protocol optimized for the detection of T. brevifilum spores in turbot muscle is also described. In fish showing low- and moderate-intensity infection, the spatial distribution of spores was best fitted by a negative binomial distribution, indicating a clumped spatial pattern; the negative binomial coefficient k was lower for fish with low-intensity infection, indicating a more markedly clumped pattern in these fish. In fish with high-intensity infection, the spatial distribution of spores was best fitted by the Poisson distribution, indicating a random pattern. In both low- and moderate-intensity infection, spores were present at highest density in the musculature adjoining the dorsal fins. Samples for PCR were therefore obtained from this location. PCR amplification was of the small subunit ribosomal DNA (SSUrDNA), using a pair of species-specific primers that amplify the 1250 bp product. The PCR protocol developed showed better sensitivity than microscopical techniques (detection rate by microscopy 25%, versus 42% by PCR), suggesting that it may be useful for routine screening for Tetramicra brevifilum infection in cultured turbot.

Animals↗

PubMed1990

Refractory and relapsing Hodgkin's disease: role of high-dose chemotherapy with bone marrow transplantation.

Thirty percent of adult patients with Hodgkin's disease fail primary treatment or relapse after treatment. Whereas overall mortality for Hodgkin's disease is about 20%, half the patients who relapse will die. Among patients with refractory or relapsing disease, about a third can be rescued by conventional salvage treatment. Unfortunately, except for patients with late relapse, remission after conventional salvage treatment is generally not of long duration. However, durable complete remissions can now be achieved in nearly a third of patients with refractory or relapsing disease by means of very aggressive (myeloablative) chemotherapy with consecutive autologous bone marrow transplantation (aBMT). The rate of durable complete remissions seems to be even higher if previous exposure to chemotherapeutic agents is not in excess of two different treatment protocols (optimal timing of aBMT) and if responsiveness to cytotoxic drugs is preserved (low degree of drug resistance). Bone marrow transplantation should be restricted to patients whose resulting long-term prognosis justifies such radical treatment. Reflecting ongoing clinical therapy-studies, in particular in Germany, the role of bone marrow transplantation in a general concept of salvage treatment should be pointed out. Patients should be considered candidates if they fail alternating primary chemotherapy or develop an early relapse after this treatment, but still show responsiveness to chemotherapeutic agents.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells defined by multilineage potential, ease to gene modification, and immunosuppressive ability, thus holding promise for tissue engineering, gene therapy, and immunotherapy. They exhibit a unique in vitro expansion capacity, which, however, does not compensate for the very low percentage in their niches given the vast numbers of cells required for the relative studies. Taking into consideration the lack of a uniform approach for MSC isolation and expansion, we attempted in this study, by comparing various culture conditions, to identify the optimal protocol for the large-scale production of MSCs while maintaining their multilineage and immunosuppressive capacities. Our data indicate that, apart from the quality of fetal calf serum, other culture parameters, including basal medium, glucose concentration, stable glutamine, bone marrow mononuclear cell plating density, MSC passaging density, and plastic surface quality, affect the final outcome. Furthermore, the use of basic fibroblast growth factor (bFGF), the most common growth supplement in MSC culture media, greatly increases the proliferation rate but also upregulates HLA-class I and induces low HLA-DR expression. However, not only does this upregulation not elicit significant in vitro allogeneic T cell responses, but also bFGF-cultured MSCs exhibit enhanced in vivo immunosuppressive potential. Besides, addition of bFGF affects MSC multilineage differentiation capacity, favoring differentiation toward the osteogenic lineage and limiting neurogenic potential. In conclusion, in this report we define the optimal culture conditions for the successful isolation and expansion of human MSCs in high numbers for subsequent cellular therapeutic approaches.

Cell Culture Techniques↗

PubMed1998

High-level gene transfer to cord blood progenitors using gibbon ape leukemia virus pseudotype retroviral vectors and an improved clinically applicable protocol.

The best methods for transducing hematopoietic progenitor cells usually involve either direct co-cultivation with virus-producing cells or human stromal supportive cells. However, these methods cannot be safely or easily applied to clinical use. Therefore, we aimed at improving retrovirus-mediated gene transfer into hematopoietic progenitors derived from cord blood CD34+ cells using viral supernatant to levels achieved at least with direct co-cultivation and under conditions that are suitable for clinical applications. In a first set of experiments, CD34+ cells were infected with supernatant containing amphotropic retroviral particles carrying the nls-lacZ reporter gene and the effects of centrifugation, cell adhesion to fibronectin, and Polybrene on the transduction of both clonogenic progenitors (CFC) and long-term culture initiating cells (LTC-IC) were studied. Transduction efficiency was evaluated on the percentage and total number of progenitors expressing the beta-galactosidase activity. Results show that a 48-hr infection of CD34+ cells with viral supernatant combining centrifugation at 1000 x g for 3 hr followed by adhesion to fibronectin allows transduction levels for both CFC and LTC-IC to be reached that are as good as using direct co-cultivation. In a second set of experiments, CD34+ cells were infected using this optimized protocol with pseudotyped retroviral particles carrying the gibbon ape leukemia virus (GALV) envelope protein. Under these conditions, between 50 and 100% of CFC and LTC-IC were transduced. Thus, we have developed a protocol capable of highly transducing cord blood progenitors under conditions suitable for a therapeutical use.

Antigens, CD34↗

PubMed1999

Different phases of renal enhancement: role in detecting and characterizing renal masses during helical CT.

Although helical CT offers greater flexibility in data acquisition and reconstruction than does conventional axial CT, new opportunities for error must be understood so that optimal protocols are used that minimize the likelihood of misdiagnosis. Most nonrenal abdominal helical CT scans are obtained late in the corticomedullary phase of renal enhancement because of the preferable enhancement of other parenchymal organs. CT evaluation of the kidneys during the corticomedullary phase or at an intermediate phase between the corticomedullary and nephrographic phases has significant limitations. Therefore, dedicated renal CT performed for the detection of suspected renal masses or for the characterization of known renal masses must include images obtained during later phases of enhancement (i.e., nephrographic or excretory phase). Nephrographic or excretory phase images appear to be similar to one another but superior to corticomedullary phase images in the ability to both detect and characterize renal masses. Corticomedullary phase images should always be obtained when information about the renal vasculature is desired or when there is a possibility that a detected renal mass may represent an aneurysm or an arteriovenous malformation or fistula. Optimal technique of helical CT for staging renal cancers may require use of both corticomedullary and nephrographic or excretory phase images, although work in this area is still preliminary.

Artifacts↗

PubMed1999

Amplification of MelanA messenger RNA in addition to tyrosinase increases sensitivity of melanoma cell detection in peripheral blood and is associated with the clinical stage and prognosis of malignant melanoma.

An improved protocol for reverse transcription-polymerase chain reaction (RT-PCR), amplifying tyrosinase and MelanA/MART-1 mRNA from peripheral blood, was used to test 340 blood samples from 225 patients with malignant melanoma for the presence of circulating tumour cells. Positive results for tyrosinase or MelanA were obtained in 19% of patients in stage I (n = 74), 31% in stage II (n = 45), 29% in stage III (n = 48) and 52% in stage IV (n = 58). Amplification of MelanA in addition to tyrosinase resulted in a 30% enhanced sensitivity of melanoma cell detection compared with amplification of tyrosinase alone. The sensitivity was further enhanced by analysis of at least two blood samples per patient and performing at least two PCR analyses per sample. During a median follow-up of 4 months, patients with a positive PCR showed a 2. 4-fold increased risk for relapse compared with PCR-negative patients. These data indicate that the detection of circulating melanoma cells in peripheral blood using our optimized protocol for RT-PCR correlated with the clinical stage of disease and is therefore likely to be a prognostic marker for recurrence. MelanA is a sensitive additional marker to tyrosinase in detecting micrometastases using RT-PCR.

Adolescent↗

PubMed2005

Testosterone secretion and pharmacological spermatozoal recovery in the cane toad (Bufo marinus).

The cane toad (Bufo marinus) was used as a model to study male anuran reproductive endocrinology and to develop a protocol for non-invasive sperm recovery. Circulating testosterone concentrations in 6-hourly samples did not vary significantly (P < 0.05) over a 24 h period although there was a tendency (P = 0.06) for testosterone to be elevated at 19:00 h relative to other times of the day, which may be related to the nocturnal activity pattern of this species. Testosterone secretion after intraperitoneal (IP) injection of either a GnRH agonist (5 microg IP) or hCG (1000 IU) was also examined. While the GnRH agonist did not produce a significant increase above basal plasma testosterone (0.29, 95% C.I. of 0.05-1.10 ng/ml), injection of hCG resulted in an increase (P < 0.01) of plasma testosterone with peak concentrations at approximately 120 min (4.17, 95% C.I. of 2.69-7.44 ng/ml) after injection. Non-invasive pharmaceutical sperm recovery was attempted following IP injection of graded doses of GnRH agonist, hCG or FSH. Urine was collected at 3, 6 and 12 h after treatment to assess sperm quality and quantity. The optimal protocol for sperm recovery in cane toads was injection of either 1000 or 2000 IU hCG; there was no significant difference in the quality of the spermic urine samples obtained using either dose of hCG or with respect to collection time. The findings indicated that hCG can be used to assess testicular steroidogenic status and also to induce sperm recovery in the cane toad. The hCG protocols developed in this study will have application in studies on the reproductive biology of rare and endangered male anurans.

Animals↗