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PubMed2027

Protocol for Detecting and Sequencing Chikungunya Virus from Field-Collected Mosquitoes.

Arboviral diseases represent a major public health challenge, especially in tropical regions where environmental conditions may favor the proliferation and spread of mosquito vectors. Thus, early and accurate detection of chikungunya virus (CHIKV) in mosquito populations can be a valuable tool for effective surveillance of circulating variants and for identifying new viral introductions. Given the challenges of detecting arboviruses in field-captured mosquitoes, we describe an integrated workflow for CHIKV molecular detection and whole-genome sequencing. This protocol includes mosquito homogenization using a bead-based mechanical disruptor, RNA extraction using TRIzol reagent with minor modifications, molecular screening using CHIKV-specific RT-qPCR, and whole-genome amplification followed by sequencing on Illumina platforms. Despite the protocol being optimized for individual mosquitoes, it results in high-quality RNA suitable for both entomological surveillance and genomic analysis. As this protocol allows recovery of complete CHIKV genomes from mosquito specimens, it can serve as a basis for genomic epidemiology studies, enabling monitoring of viral diversity and lineage dynamics, and facilitating early detection of emerging variants to support timely and targeted public health interventions in endemic and at-risk regions.

Animals↗
PubMed1999

Identification and optimization of regeneration conditions for affinity-based biosensor assays. A multivariate cocktail approach.

A general regeneration, identification, and optimization (RO) protocol for Biacore systems was developed. The RO protocol uses six multi-ingredient stock solutions that represent the six most common chemical properties employed as regeneration agents. The regeneration effect of different regeneration cocktails of these six stock solutions were tested iteratively until a satisfactory result was obtained. The RO protocol was designed with an ease-of-use and multivariate approach. The RO protocol was tested on 13 different antibody-antigen systems. For 10 of these, only the first screening session was tested. For 9 of the 13 systems, the RO-protocol screening session identified cocktails that removed more than 90% of the bound analyte in a 30 s pulse. For 5 systems, the RO protocol identified cocktails that regenerated the surface completely and that were more gentle than previously used regeneration conditions. Furthermore, the regeneration optimization results can be interpreted as a characterization of the interacting molecules. The relevance of testing cocktails was justified by the fact that at least one cocktail was significantly better than all diluted stock solutions for all tested model systems. By using the multivariate approach, the risk of missing relevant combinations of stock solutions was minimized. This resulted in an unexpected discovery of excellent properties of EDTA as an additive in regeneration cocktails containing chaotropic agents and ions in high concentration.

Biosensing Techniques↗
PubMed1991

A qualitative approach to cell growth modeling and simulation for cancer chemotherapy.

A qualitative model of cell growth, based on qualitative process theory, is presented. The model can be used to analyze the effects of the interaction of antiproliferative drugs on cells when the effects of each specific drug are known, mainly when designing multi-drug protocols for optimal cancer treatment.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2026

Establishment of an efficient Agrobacterium-mediated genetic transformation protocol for Saccharum officinarum using Black Cheribon as a model genotype.

Efficient Agrobacterium-mediated transformation (AMT) is vital for the biotechnological improvement of sugarcane (Saccharum spp.). Saccharum officinarum is the main ancestor of all modern cultivars, yet little research has been conducted on its AMT system. In this work, an efficient AMT protocol for S. officinarum was developed, with Black Cheribon as the model genotype owing to its superior tissue culture performance and regeneration capacity. The optimized agro-infection protocol comprised the following main parameters: concentration of acetosyringone (AS) in Agrobacterium culture, concentration of AS for infection, Agrobacterium concentration at OD600 = 0.4, infection time of 30 minutes, vacuum infiltration time of 10 minutes and co-cultivation time of 3 days. To further improve transformation efficiency, 0.5 mg/L thidiazuron and 200 mg/L citric acid were added to the regeneration medium, which enhanced the regeneration of shoots. A modified stage-dependent selection strategy (FlexII) was established by using glufosinate-ammonium at concentrations of 2.0, 1.0, and 0.75 mg/L in the callus proliferation, shoot regeneration, and rooting stages, respectively. This strategy was more successful than the minimum inhibitory concentration-based strategy in S. officinarum transformation. The optimized protocol further boosted the transformation efficiency of Black Cheribon from 1.12% to 7.17%. The resulting transgenic lines were confirmed by PCR amplification of T-DNA regions and immunochromatographic detection of Bar protein expression in primary transformants, respectively. These results provide a sound technical foundation for the functional genomics and biotechnological optimization of S. officinarum germplasm, and may serve as a reference for future transformation studies in other sugarcane germplasm.

Agrobacterium↗
PubMedDate not supplied

Factors influencing vascular and hepatic enhancement at CT: experimental study on injection protocol using a canine model.

PURPOSE: The purpose of this work was to evaluate the effects of contrast medium injection parameters on aortic, portal vein, and hepatic enhancement at spiral CT and to assess optimal injection protocol for hepatic CT. METHOD: Ten 15 kg dogs underwent single level dynamic CT through the hepatic hilum at 5 s intervals just after the injection of contrast medium for 3 min. With use of different volumes (1, 2, and 3 ml/kg), injection rates (0.5, 1, and 2 ml/s), and concentrations (150, 200, and 300 mg/ml), a total of 270 spiral CT scans were performed. In each scan, time-attenuation curves of aorta, portal vein, and liver were obtained. The degree of maximum contrast enhancement (Imax), time to maximum enhancement (Tmax), and time to equilibrium phase (Teq) for to each injection protocol were analyzed. RESULTS: Alterations in contrast material volume, injection rate, and concentration had significant impact on contrast enhancement of the liver. With increasing volume of contrast medium, Imax, Tmax, and Teq of aorta, portal vein, and liver increased (p < 0.005). With increasing rate of injection, on the other hand, Imax of aorta and liver increased (p < 0.05), but Tmax and Teq decreased (p < 0.005). Change of concentration of contrast medium had a significant effect on Imax of vessels (p < 0.05). CONCLUSION: Maximum contrast enhancement of liver and vessels was influenced mainly by injection volume of contrast medium and the time to peak enhancement by injection rate of contrast medium. Under given amounts of contrast medium, therefore, the strategy of increasing volume by dilution and faster injection might give better Imax values without penalty for the duration of an optimal temporal window (Tmax and Teq).

Animals↗
PubMed2000

Adult rat and human bone marrow stromal cells differentiate into neurons.

Bone marrow stromal cells exhibit multiple traits of a stem cell population. They can be greatly expanded in vitro and induced to differentiate into multiple mesenchymal cell types. However, differentiation to non-mesenchymal fates has not been demonstrated. Here, adult rat stromal cells were expanded as undifferentiated cells in culture for more than 20 passages, indicating their proliferative capacity. A simple treatment protocol induced the stromal cells to exhibit a neuronal phenotype, expressing neuron-specific enolase, NeuN, neurofilament-M, and tau. With an optimal differentiation protocol, almost 80% of the cells expressed NSE and NF-M. The refractile cell bodies extended long processes terminating in typical growth cones and filopodia. The differentiating cells expressed nestin, characteristic of neuronal precursor stem cells, at 5 hr, but the trait was undetectable at 6 days. In contrast, expression of trkA, the nerve growth factor receptor, persisted from 5 hr through 6 days. Clonal cell lines, established from single cells, proliferated, yielding both undifferentiated and neuronal cells. Human marrow stromal cells subjected to this protocol also differentiated into neurons. Consequently, adult marrow stromal cells can be induced to overcome their mesenchymal commitment and may constitute an abundant and accessible cellular reservoir for the treatment of a variety of neurologic diseases.

Animals↗
PubMed1991

An improved method of loading pH-sensitive liposomes with soluble proteins for class I restricted antigen presentation.

We have recently shown that ovalbumin (OVA) entrapped in pH-sensitive liposomes could sensitize mouse thymoma cells for lysis by MHC class I-restricted cytotoxic T lymphocytes (CTL) (Reddy et al. (1991) J. Immunol. Methods, 141, 157-163). The present studies were designed to optimize the antigen delivery system. A simple freeze-thaw method was developed to load OVA into pH-sensitive liposomes, and the protocol was optimized in terms of the choice of buffer, pH and ionic strength of the medium, lipid composition, lipid and OVA concentrations and the number of freeze-thaw cycles. Under optimized conditions, approximately 25% of OVA could be entrapped in pH-sensitive liposomes at 172 micrograms protein/mg lipid. This compares to only about 5% entrapment (70 micrograms protein/mg lipid) using the previous method. OVA loaded to pH-sensitive liposomes using the improved method led to a sensitive measure of CTL activity. The approach promises to be suitable to measure CTL against less available soluble antigens such as viral proteins.

Animals↗
PubMed1996

Simultaneous enumeration of chromosomes 13, 18, 21, X, and Y in interphase cells for preimplantation genetic diagnosis of aneuploidy.

A fluorescence in situ hybridization (FISH) protocol to simultaneously enumerate chromosomes 13, 18, 21, X, and Y in interphase cell nuclei for application in preimplantation genetic diagnosis (PGD) of aneuploidy was tested. Strict scoring criteria were developed to minimize recording errors. The protocol used optimized probes for chromosome-specific DNA repeat and single-copy loci and showed a significantly higher efficiency (95%) than previously published protocols. The other purpose of this study was to differentiate between two signals originating from a single split target or from two targets in close proximity. These criteria were based on the FISH results obtained from the analysis of all or most of the cells from 50 chromosomally normal or mosaic human embryos, 20 aneuploid embryos, and five polyploid embryos donated for research. Subsequently, 183 human embryos underwent PGD of aneuploidy in one of their cells. In 64 embryos that were not transferred back to the uterus, the rest of cells were also analyzed, and the previous results were confirmed in 91% of these embryos, with 0% (0/21) of the embryos classified as normal embryos being abnormal and 14% (6/43) of the embryos classified as abnormal being normal. Compared to previous protocols, these criteria minimize the risk of transferring abnormal embryos after PGD analysis.

Aneuploidy↗
PubMed1995

High-throughput method for determination of apolipoprotein E genotypes with use of restriction digestion analysis by microplate array diagonal gel electrophoresis.

Molecular epidemiological research has identified the association of a common apolipoprotein E (apo E) isoform (E4 as opposed to E3), with risk both of coronary artery disease and of Alzheimer dementia. In addition, the role of apo E genotype (usually E2/E2) in Type III hyperlipidemia is well known. However, both for diagnostic and research purposes, apo E genotyping is cumbersome. The preferred approach is electrophoretic sizing of restriction digestion fragments, enabling simultaneous analysis of the two codons (112 and 158) that represent the six common genotypes (E2/E2; E2/E3; E2/E4; E3/E3; E3/E4; E4/E4). However, the consequent demands of high-yield PCR, high-resolution, high-throughput electrophoresis, and sufficient detection sensitivity have left shortfalls in published protocols. In conjunction with a high-throughput electrophoresis system we described recently, microplate array diagonal gel electrophoresis (MADGE), we have constructed extensively optimized, simplified protocols for DNA isolation from mouthwash samples for PCR setup and high-yield PCR, for restriction digestion, and for subsequent MADGE gel image analysis. The integral system enables one worker to readily undertake apo E genotyping of as many as hundreds of DNA samples per day, without special equipment.

Apolipoproteins E↗
PubMed1997

13C-urea breath test in Helicobacter pylori diagnosis and eradication. Correlation to histology, origin of 'false' results, and influence of food intake.

BACKGROUND: Which protocol is optimal for the 13C-urea breath test (UBT) for Helicobacter pylori detection is controversial. This study aimed to characterize a very simple UBT protocol for the clinical routine (two-point-analysis performed with 75 mg 13C-urea and citric acid) with special consideration of 'false' UBT results. RESULTS; UBT was evaluated in reference to histology (Warthin-Starry). In mismatching results re-gastroscopy was performed. By UBT, 74 of 77 patients with H. pylori-positive histology were detected (sensitivity, 96%). The false-negative UBTs were due to low colonization densities during spontaneous H. pylori elimination or pyloric obstruction. Seven of 49 patients with negative histology had a positive UBT, but re-gastroscopy showed that all of them had a positive histology when multiple antral biopsy specimens were taken (UBT specificity, 100%). UBT correlated only weakly with H. pylori colonization density. No correlation was found between UBT and gastric neutrophil and lymphocyte infiltration. UBT reproducibility was excellent (93 of 94 in a 6-month period). Non-fasting conditions induced a shift to lower UBT results in H. pylori-positive and to higher UBT results in negative patients, resulting in 2 of 10 false-positive and 1 of 10 false-negative UBTs. CONCLUSION: This simple version of the urea breath test combines the highest sensitivity with excellent reproducibility. It is superior to histologic detection of H. pylori in the clinical routine and an optimal tool for monitoring H. pylori eradication. Fasting conditions are required for the test.

Breath Tests↗
PubMed1995

Radiation doses to paediatric patients undergoing micturating cystourethrography examinations and potential reduction by radiation protection optimization.

Dose-area product values were measured in paediatric micturating cystourethrography examinations in two dedicated facilities. The results are compared, taking into account features of the X-ray rooms and differences in examination protocol. Several optimization procedures were simulated or implemented, in order to estimate the dose savings which could be attained. Potential savings identified in the dose-area values reach about 85% for both centres.

Adolescent↗
PubMed1998

[Multimodal therapy of rectal carcinoma].

Multimodal therapy for rectal carcinoma is--in contrast to colon cancer--largely based on adjuvant or neoadjuvant radiotherapy. Although radiotherapy reduces local recurrence rates, overall survival is hardly improved, while on the other hand specific complications of radiotherapy are recognized. Extended neoadjuvant treatment concepts including chemotherapy may improve long-term outcome, but to date this has not been demonstrated conclusively by randomized trials. New developments include intraoperative radiotherapy and the combination of hyperthermia with radiochemotherapy. Initial results from randomized studies are promising, but require confirmation by larger trials. At the same time the importance of optimal surgical treatment has been recognized as a prerequisite for favourable long-term results. Since this aspect has not received due attention in past protocols the optimal combination of surgery with other modalities and their time sequence cannot be determined presently.

Chemotherapy, Adjuvant↗
PubMed1990

Selective termination: clinical experience and residual risks.

Assisted reproductive technologies have aided thousands of couples, but complications have resulted in multifetal pregnancies creating a bitter irony for infertility patients. In an effort to increase the rate of intact survival, we have successfully performed transabdominal first-trimester selective termination procedures on 22 pregnancies including one octuplet, five quintuplet, twelve quadruplet, and four triplet gestations. There have been eight sets of twins, and two singletons delivered, seven twin pregnancies are ongoing, and one early and four late losses of pregnancies. With experience we now counsel as to a high likelihood of a technically successful procedure, but we still have concerns for late losses. We have tried to balance the arguments about the direct harms of performing selective termination and the obstetric risks of not performing selective termination. We believe that selective termination should not be considered a "social" procedure. Our data do not yet make clear whether one, two, or three is the optimal number of embryos to leave. Therefore, on the basis of both current obstetric risk factors and ethical reasoning we will continue to support our protocol of optimally leaving twins.

Abortion, Induced↗
PubMed2000

Use of flow cytometry to rapidly optimize the transfection of animal cells.

Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.

Animals↗
PubMed1999

Scan optimization of gadolinium contrast-enhanced three-dimensional MRA of peripheral arteries with multiple bolus injections and in vitro validation of stenosis quantification.

In this study, a T1-weighted three-dimensional (3D) spoiled gradient-echo scanning protocol was developed to image the complete arterial system of the pelvis and both legs along their entire length in patients with peripheral arterial disease. Three adjacent stations were to be acquired consecutively, with some overlap, to image the entire area of interest; per station one gadolinium (Gd) contrast bolus would be administered. In an in vitro phantom study, the scanning protocol was optimized. The optimal flip angle was found to be 50 degrees. Also, the optimal scan delay was chosen to be equal to the arrival time of the contrast bolus, thereby minimizing artifacts. Three contrast bolus injections showed sufficient enhancement of the vessels after image subtraction. Finally, stenosis quantification by manual caliper was performed by five observers in the magnetic resonance angiography (MRA) images and correlated with the percent diameter reduction determined by quantitative angiography from corresponding X-ray images. The MRA measurements were reproducible, and intra- and interobserver variabilities were statistically non-significant (p=0.54 and p=0.12, respectively). Stenosis quantification performed by four observers showed a good correlation with the X-ray-derived values (rp > 0.90, p < 0.02); the results from one observer were not significantly correlated. Five patients with proven peripheral disease were investigated with this new MRA scanning protocol, using standard hardware and software. The images were of good quality, which allowed adequate clinical evaluation; the original diagnoses obtained from X-ray examinations, were confirmed with MRA. In conclusion, peripheral arterial disease can be evaluated adequately with this magnetic resonance scanning protocol.

Adult↗
PubMed1999

Half-side comparison of erythemogenic versus suberythemogenic UVA doses in oral photochemotherapy of psoriasis.

BACKGROUND: Early American and European multicenter trials on the efficacy of photochemotherapy (PUVA) for psoriasis have clearly shown that the treatment protocol has a crucial impact on the cumulative UVA dose required for clearing patients. Most, if not all, treatment protocols rely on the PUVA-induced erythema as a guideline for UVA dosimetry. OBJECTIVE: Our aim was to investigate whether phototoxic erythema is integral to an optimized PUVA protocol or reflects an unnecessary overexposure of patients. METHODS: A standard high-dose UVA regimen using minimal phototoxic doses (MPD) was compared against two different low-dose regimens. To this purpose a bilateral comparison study was performed on 31 patients and divided in two parts. In the first trial on 14 patients, half of each patient's body was irradiated at each visit with 1 MPD, whereas the other half received only two thirds of the MPD. In the second trial on 17 patients treatment with 1 MPD was compared against treatment with one half of the MPD. RESULTS: A total of 27 patients (12 patients in the first trial, 15 patients in the second trial) completed the study. In both trials the suberythemogenic doses were therapeutically as effective as the minimal phototoxic doses. CONCLUSION: We conclude that PUVA-induced erythema is not a prerequisite for effective psoriasis treatment and that a low-dose UVA regimen is a promising approach to increase the short- and long-term safety of photochemotherapy.

Administration, Oral↗
PubMed1996

Cryopreservation of an attenuated vaccine strain of the protozoan parasite Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite that infects birds and mammals, including humans. T. gondii T-263 is an attenuated mutant strain that is being developed as a live vaccine to protect cats from shedding oocysts. A cryopreservation procedure for T. gondii T-263 bradyzoites has been developed to meet the requirement for product stability. A Me2SO-based procedure for the cryopreservation of tachyzoites was used as a basis for process optimization. A modified cell culture plaque assay was used to determine the effects of selected cryobiological parameters on bradyzoite viability. The major parameters evaluated were: (i) cooling rates; (ii) intermediate plunge temperature; and (iii) thawing and dilution rates and temperatures. The optimized cryopreservation protocol comprised incubation in 12.5% Me2SO and 4% BSA for 30 min at room temperature, cooling at 1 degree C min-1 to -40 degrees C, followed by direct transfer into liquid nitrogen. Rapid thawing (approximately 120 degrees C min-1) followed by slow dilution of cryoprotectant over 15 min resulted in the highest survival. The optimized procedure increased survival 10,000-fold over that obtained using an established tachyzoite protocol. This procedure is to be adapted for the large-scale cryopreservation of T. gondii T-263 bradyzoites in individual vaccine doses.

Animals↗
PubMed1987

Cancer therapy by biological response modifiers.

Biological response modifiers (BRMs) are agents or approaches which can modify the biological response of the host to tumors and thereby are expected to augment the resistance to development or progression of cancer. Recent advances in the technology of genetic engineering and monoclonal antibodies have led to rapid progress in this field. There is an increasing number of genetically engineered cytokines, which appear promising for cancer treatment and are becoming available for clinical trials. These include the interferons, leukin-2, tumor necrosis factor, and colony-stimulating factors. For development of optimal therapeutic protocols with these and a variety of immunomodulatory agents, it appears necessary to develop a detailed understanding of the possible mechanisms of their antitumor effects, and to determine the optimal dose and schedule for altering the antitumor effector mechanisms. BRMs would also be expected to be quite useful in the prevention of cancer. Indeed, in a variety of animal tumor model systems, potent protective effects can be demonstrated. However, the strategies for clinical application of such information have yet to be adequately worked out. One more immediate application of this approach is for the prevention of metastatic spread of tumor cells. BRMs, which stimulate natural killer cell activity, have been shown to strongly protect against dissemination of tumors, and clinical strategies for this important aspect of cancer treatment are being developed.

Humans↗