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Results for “protocol optimization”

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PubMed2007

Mitochondrial DNA amplification success rate as a function of hair morphology.

This study examines the amplification success rate of mitochondrial DNA from human head hair with respect to their potential for forensic application. Mitochondrial DNA was isolated using a Chelex-based extraction method and amplified using the LINEAR ARRAY duplex PCR system. The particular focus of this study was to characterize the morphological features of human head hair in order to further the understanding of the factors that influence amplification success rate in hair tissue using the LINEAR ARRAY duplex PCR system. 2554 head hairs from 132 individuals representing four population groups were amplified. The hair samples were characterized as follows: 1251 were identified microscopically as telogen hairs and 1303 were classified as hairs without roots (removed before extraction). Amplification success was assessed as a function of several independent variables: morphological characteristics; telogen root versus no root; donor age; scalp origin; use of cosmetic hair treatments; and race of the donor. The results show that a positive correlation exists between amplification success and the presence of a telogen root. Combining the amplification success with either the original or optimized protocol, telogen hairs result in an overall success rate of 77.5% compared with 65% for hairs with no roots. Controlling for telogen hairs, the findings indicate that the overall success rate is independent of cosmetic hair treatments; medulla structure; shaft length, diameter, and volume; and scalp origin. Conversely, the age of the donor, the race of the donor, and hair pigmentation all contribute to a variation in amplification success rate.

Adolescent↗

PubMed1999

Transformation of a type 4 encapsulated strain of Streptococcus pneumoniae.

Streptococcus pneumoniae strain JNR.7/87 is a highly virulent, type 4 encapsulated Gram-positive bacterium whose transformability has not been tested previously, and whose genome is currently being sequenced. The strain was transformed at very low efficiency by addition of exogenous competence-stimulating peptide: However, the efficiency was too low and irreproducible to be useful in many genetic studies. Therefore, the effects on transformation efficiency of changing different components of competence-stimulating peptide-induced transformation have been examined. Screening of growth media was followed by optimization of pre-induction culture acidification, glycine concentration, and induction time. An optimized protocol was developed whereby S. pneumoniae strain JNR.7/87 was transformed reproducibly with a streptomycin resistance (SmR) marker at an efficiency of approximately 10(5) colony forming units per 10(8) cells.

Bacterial Proteins↗

PubMedDate not supplied

Prospects for exosomes in immunotherapy of cancer.

Exosomes are nanometer sized membrane vesicles invaginating from multivesicular bodies and secreted from epithelial and hematopoietic cells. They were first described "in vitro" but vesicles with the hallmarks of exosomes are present in vivo in germinal centers and biological fluids. Their protein and lipid composition are unique and could account for their expanding functions such as eradication of obsolete proteins, antigen presentation or "Trojan horses" for viruses or prions. Exosome secretion could be a regulated process participating in the transfer of molecules inbetween immune cells. Despite numerous questions pertaining to their biological relevance, the potential of dendritic cell derived-exosomes as cell-free cancer vaccines is currently being assessed. This review will summarize the composition and formation of exosomes, preclinical data, Phase I trials and optimization protocols for improving their immunogenicity in tumor bearing patients.

Animals↗

PubMed2005

Preclinical ex vivo expansion of G-CSF-mobilized peripheral blood stem cells: effects of serum-free media, cytokine combinations and chemotherapy.

OBJECTIVES: Ex vivo expansion of granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) is a promising approach for overcoming the developmental delay of bone marrow (BM) reconstitution after transplantation. This project investigated the effects of culture duration, serum-free media, cytokine combinations, and chemotherapy on the outcomes of expansion. METHODS: Enriched CD34+ cells were cultured for 8 or 10 d in serum-free media (QBSF-60 or X-Vivo 10) and four combinations of cytokines consisting of recombinant human pegylated-megakaryocyte growth and development factor, stem cell factor, flt-3 ligand, G-CSF, interleukin (IL)-6, platelet-derived growth factor (PDGF), and IL-1beta. RESULTS: Eight days of culture in QBSF-60 significantly supported efficient expansions of CD34+ cells, CD34+ CD38- cells, colony-forming units (CFU) of myeloid, erythroid, megakaryocytic, and mixed lineages to 3.76-, 14.4-, 28.3-, 24.0-, 38.1-, and 15.7-fold, respectively. Whilst PDGF or IL-6 enhanced the expansion of early, myeloid, and erythroid progenitors, IL-1beta specifically promoted the megakaryocytic lineage. Engraftment of human CD45+ cells were detectable in all non-obese diabetic/severe-combined immunodeficient mice transplanted with expanded PBSC from donor samples, being 5.80 +/- 3.34% of mouse BM cells. The expansion and engraftment capacity of CD34+ cells from subjects postchemotherapy were significantly compromised across the panel of progenitor cells. CONCLUSION: Our results provided an optimized protocol for PBSC expansion, applicable to ameliorating neutropenia and thrombocytopenia in post-BM transplant patients by the prompt provision of progenitor cells. For postchemotherapy patients, expansion products might provide committed progenitors for improving short-term engraftment, but not self-renewable stem cells.

ADP-ribosyl Cyclase↗

PubMedDate not supplied

Magnetic resonance arthrography of the canine stifle joint: technique and applications in eleven military dogs.

The stifle joints of eleven military working dogs were evaluated using conventional magnetic resonance (MR) imaging and MR arthrography. A protocol optimizing MR imaging of the canine stifle joint is discussed, as well as potential uses for administration of intra-articular gadolinium. The technique for performing MR arthrography is described, and post-contrast image findings are reviewed. MR arthrography was performed by using an intra-articular injection of diluted gadolinium. Consistently good quality images were obtained, and no complications were clinically detected following MR arthrography. Cranial cruciate ligament abnormalities were seen in six dogs, meniscal abnormalities were visualized in nine menisci, and synovitis and medial ligament strain were seen in eight dogs. Surgical and post-mortem confirmation of these findings is discussed in seven dogs. Although MR arthrography adds an invasive procedure to conventional MR imaging, it can provide useful information on pathologic changes in the canine stifle joint.

Animals↗

PubMedDate not supplied

Post insertion catheter care in peritoneal dialysis (PD) centres across Europe--Part 2: complication rates and individual patient outcomes.

The first part of this report, which looked at centre policy, showed that there was no consensus on the best way to manage a patient in the rest period between PD catheter insertion and the first use of the catheter for dialysis. This paper intends to investigate if the differences in policy had any effect on complication rate and individual patient outcomes. Data were included from 298 patients of 49 participating centres. The results revealed a high rate of catheter related complications, with half of the patients having been treated for complications including leakage (29%), malfunction (23%) or infection (10%), and a quarter of patients having been hospitalised for catheter problems. Leakage was more frequently observed in lean and obese patients and if the catheter was only immobilized for a short time period. Diabetes, having constipation at first use and having rested for less than 6 hours after catheter insertion were significant risk factors for malfunction. Infection seemed to be related to the type of catheter used and hygienic precautions (not significant) and showed a significant relationship with the frequency of dressing changes. There is still an important lack of evidence on which to develop an optimal protocol for PD catheter insertion and care before first use.

Adolescent↗

PubMed2000

A localization algorithm and error analysis for stereo x-ray image guidance.

Stereo x-ray radiography attracts increasing attention in major clinical applications. The purpose of this paper is to analyze the 3D localization error for breast biopsy procedures and provide guidelines for improving its accuracy. Our prototype is a CCD based digital stereo x-ray imaging system. The mathematical model consists of two x-ray sources and one stationary detector plane. A closed form least-squares solution is derived for 3D localization of feature points, particularly a biopsy needle tip, from a pair of 2D digital radiographs. Based on the least-squares formula and its first order approximation, the 3D localization error is analyzed in terms of object location, measurement error, separation between the two x-ray sources, and distance from the source to the detector. The stereo imaging and error estimation formulas are numerically simulated and experimentally validated. The data are in agreement with theoretical prediction. These results can be used for the purpose of system design and protocol optimization.

Algorithms↗

PubMed2006

A DNA microarray platform based on direct detection of rRNA for characterization of freshwater sediment-related prokaryotic communities.

A DNA microarray platform for the characterization of bacterial communities in freshwater sediments based on a heterogeneous set of 70 16S rRNA-targeted oligonucleotide probes and directly labeled environmental RNA was developed and evaluated. Application of a simple protocol for the efficient background blocking of aminosilane-coated slides resulted in an improved signal-to-noise ratio and a detection limit of 10 ng for particular 16S rRNA targets. An initial specificity test of the system using RNA from pure cultures of different phylogenetic lineages showed a fraction of false-positive signals of approximately 5% after protocol optimization and a marginal loss of correct positive signals. Subsequent microarray analysis of sediment-related community RNA from four different German river sites suggested low diversity for the groups targeted but indicated distinct differences in community composition. The results were supported by parallel fluorescence in situ hybridization in combination with sensitive catalyzed reporter deposition (CARD-FISH). In comparisons of the data of different sampling sites, specific detection of populations with relative cellular abundances down to 2% as well as a correlation of microarray signal intensities and population size is suggested. Our results demonstrate that DNA microarray technology allows for the fast and efficient precharacterization of complex bacterial communities by the use of standard single-cell hybridization probes and the direct detection of environmental rRNA, also in methodological challenging habitats such as heterogeneous lotic freshwater sediments.

Bacteria↗

PubMed2001

Simultaneous recovery of RNA and DNA from soils and sediments.

Recovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316-322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0 degrees C to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 microg g of soil(-1) dry weight), whereas the DNA yield ranged from 23 to 435 microg g(-1). In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification.

DNA, Bacterial↗

PubMed2001

Development and evaluation of functional gene arrays for detection of selected genes in the environment.

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65 degrees C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r(2) = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.

Bacteria↗

PubMed2002

Enrichment double-antibody sandwich indirect enzyme-linked immunosorbent assay that uses a specific monoclonal antibody for sensitive detection of Ralstonia solanacearum in asymptomatic potato tubers.

Sensitive and specific routine detection of Ralstonia solanacearum in symptomless potato tubers was achieved by efficient enrichment followed by a reliable double-antibody sandwich indirect enzyme-linked immunosorbent assay based on the specific monoclonal antibody 8B-IVIA. This monoclonal antibody reacted with 168 typical R. solanacearum strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato. The optimized protocol included an initial enrichment step consisting of shaking the samples in modified Wilbrink broth for 72 h at 29 degrees C. This step enabled specific detection by the enzyme-linked immunosorbent assay of 1 to 10 CFU of R. solanacearum per ml of initial potato extract. Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods.

Antibodies, Monoclonal↗

PubMed2004

Comparison of enzyme-linked immunosorbent assay, Western blotting, microagglutination, indirect immunofluorescence assay, and flow cytometry for serological diagnosis of tularemia.

The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.

Adolescent↗

PubMed2004

Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-PCR with an armored RNA internal control.

The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine alpha-casein per microl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine alpha-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens.

Enterovirus↗

PubMed1997

Genomic diversity and evolution of papillomaviruses in rhesus monkeys.

We are studying the diversity of and relationships among papillomaviruses (PVs) to understand the modes and timescales of PV evolution and in the hope of finding animal PVs that may serve as model systems for disease caused by human PVs (HPVs). Toward this goal, we have examined 326 genital samples from rhesus monkeys and long-tailed macaques with a PCR protocol optimized for detecting genital HPV types. In 28 of the rhesus monkey samples, we found amplicons derived from 12 different and novel PV genomes, RhPV-a to RhPV-m, with the likely taxonomic status of "type." The frequency with which novel RhPVs were detected suggests that rhesus monkeys may play host to PVs with a diversity similar to that of humans. In phylogenetic trees, all 12 of the different RhPVs and the previously described type RhPV-1 were members of the genital HPV supergroup and formed three minor branches distinct from the 11 branches formed by genital HPVs. We also identified a novel PV amplicon, MfPV-a, from a long-tailed macaque, a species belonging to the same genus as rhesus monkeys. MfPV-a turned out to be a close relative of five RhPVs. It appears that the evolution of primate lineages leading to the genus Macaca and to humans created transmission barriers for PVs, resulting in viral evolution closely linked to the host. Additional support for the linked-evolution hypothesis comes from considering the phylogenetic association of two other ape and monkey PVs with the genital HPVs, the supergroup formed by at least seven ungulate PVs, and the isolated phylogenetic position of the only known bird PV.

Amino Acid Sequence↗

PubMed1992

AAPM tutorial. CT image detail and noise.

Two important characteristics of the computed tomographic (CT) image that affect the ability to visualize anatomic structures and pathologic features are blur and noise. Increased blurring reduces the visibility of small objects (image detail); increased visual noise reduces the visibility of low-contrast objects. Sources of blurring in CT include the size of the sampling aperture (which can be regulated by the focal spot size and the detector size), the size of the voxels, and the reconstruction filter selected. Noise is caused by the variation in attenuation coefficients between voxels. Use of small voxels and edge-enhancing filters helps reduce blurring and improve visibility of fine details. However, small voxels absorb fewer photons and therefore result in increased noise. Noise can be reduced by using large voxels, increasing radiation dose, or using a smoothing filter, but this filter increases blurring. An optimized protocol for a specific clinical study must take these physical principles into account and be adjusted to give proper balance among detail, low noise, and patient exposure.

Radiographic Image Enhancement↗

PubMed1994

A C-fiber reflex modulated by heterotopic noxious somatic stimuli in the rat.

1. Electromyographic recordings were made from the biceps femoris muscle through a pair of noninsulated platinum/iridium needle electrodes in male Sprague-Dawley rats artificially ventilated and anesthetized with 0.8% halothane in a N2O-O2 mixture (2/3:1/3). The animals' ventilation, heart rates, and body temperatures were continuously monitored. Electrical stimuli (2-ms duration, 0.16 Hz) were delivered to the sural nerve territory through a pair of noninsulated platinum/iridium needle electrode inserted subcutaneously in the medial aspect of the 4th and the lateral part of the 5th toe. Such stimulation elicited a two-component reflex response in the ipsilateral biceps femoris muscle: The first had a short latency (17.5 +/- 2.3 ms), short duration (20.7 +/- 2.6 ms), and low threshold (1.5 +/- 0.6 mA), whereas the second had a longer latency (162.4 +/- 5.1 ms), longer duration (202.3 +/- 6.2 ms), and higher threshold (5.7 +/- 0.5 mA). 2. Lidocaine (0.02-0.1%; 0.1 ml), but not saline, injected subcutaneously over the proximal part of the sural nerve, produced a selective depression of the late component of the reflex response, whereas the first component remained unchanged. The conduction velocity of the afferent fibers was estimated from the stimulation needles in the sural nerve territory to the nerve's projection in the lumbar spinal cord: it was concluded that the second, late component of the reflex response was due to afferent signals transmitted via unmyelinated C-fibers, whereas the first component was related to activation of fine myelinated fibers (A delta group). 3. Electrical stimulation of the sural nerve was still able to elicit the two-component reflex responses in the ipsilateral biceps femoris muscle of chronic spinal rats, indicating that these responses were genuine reflex responses, transmitted completely through a spinal circuit. 4. The C-fiber reflex was recorded when the duration and frequency of the stimuli applied to the sural nerve varied within the 0.5- to 4-ms and 0.02- to 1-Hz ranges, respectively. It was concluded that a single 2-ms duration shock at an intensity of 1.2 times the C-fiber reflex threshold, delivered every 6 s (0.16 Hz), constituted an acceptable and optimal protocol for experiments in which the C-fiber reflex was studied as a function of time. These parameters were used throughout the subsequent experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferent Pathways↗

PubMed1998

Fluorescent in situ hybridization detection of HER-2/neu gene amplification in rhabdomyosarcoma.

Embryonal rhabdomyosarcoma is the most common malignant soft-tissue tumor in childhood, comprising 45-50% of childhood sarcomas. Cytogenetic studies of this tumor are rare. In view of the paucity of cytogenetic data on this cancer and based on the finding of HER-2/neu gene amplification in a number of cancers that was detected mostly using the traditional technique of immunohistochemistry, we decided to conduct a pilot study to investigate whether HER-2/neu gene amplification in this tumor can be detected using the newer technique of fluorescent in situ hybridization (FISH). Archival tissues of rhabdomyosarcoma were retrieved and FISH using an HER-2/neu probe was undertaken on formalin-fixed paraffin-embedded tissue sections using a protocol optimized for our laboratory at Rhode Island Hospital. Out of 9 cases of rhabdomyosarcoma studied to date, 1 case clearly showed HER-2/neu gene amplification. Thus, FISH is a sensitive technique suitable for the detection of oncogene amplification and the delineation of tumor heterogeneity in this tumor. Future experiments utilizing additional specimens from our centers as well as from other laboratories will be needed to extend the finding in the present pilot study.

Child↗

PubMed1997

Stage I and stage II infiltrating ductal carcinoma of the breast analyzed for chromosome 8 copy number using fluorescent in situ hybridization.

We previously reported the results of 30 informative samples (from a total of 34 specimens gathered) of archival breast cancer tissue, including infiltrating ductal carcinoma (NOS), ductal carcinoma in situ, lobular carcinoma, papillary carcinoma and benign lesions of the breast. The study was conducted using fluorescent in situ hybridization (FISH) and a chromosome 8 alpha-satellite probe. Subsequently, a total of 34 cases of infiltrating ductal carcinoma of the breast (NOS, 17 cases stage I and 17 cases stage II) were studied, again using interphase cytogenetics. The aim of the present study is to confirm and extend the results of our initial study of stage I and stage II disease. Towards this end, 36 additional specimens of formalin-fixed paraffin-embedded breast cancer tissue have been analyzed cytogenetically under blinded conditions for the frequency of abnormal chromosome 8 copy numbers using FISH and the previously described protocol optimized for our laboratory. Of these, 18 were stage I and 18 were stage II. The frequency of trisomy 8 among stage I tumors was found to be 28% (5 out of 18). The frequency of trisomy 8 among stage II tumors was found to be 61% (11 out of 18). These results, while less striking, are consistent with those reported in our initial study of stage I and stage II disease, where the frequencies of trisomy 8 among stage I and stage II tumors were 24% (4 out of 17) and 82% (14 out of 17). These results not only establish that chromosome 8 trisomy is a recurrent finding in breast cancer, but also confirm that a higher frequency of trisomy 8 was observed with a higher clinical stage (stage II) than with a lower stage (stage I). It will be of interest to extend the findings in stage I and stage II breast cancer to other stages as well.

Adult↗