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Results for “protocol optimization”

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PubMed2005

Magnetic resonance imaging of the hip: detection of labral and chondral abnormalities using noncontrast imaging.

PURPOSE: Traditional imaging techniques have limited ability to detect subtle chondral and labral injuries of the hip. We performed a retrospective review of patients who underwent magnetic resonance imaging (MRI) of the hip and subsequent hip arthroscopy in order to evaluate the ability of optimized, noncontrast MRI to identify tears of the acetabular labrum and defects in articular cartilage. TYPE OF STUDY: Retrospective review of a consecutive sample. METHODS: Between January 1997 and July 2000, 92 patients had MRI of the hip, followed by arthroscopic surgery of that hip by 1 of 2 surgeons (R.B., D.E.P.). Two musculoskeletal MR radiologists blinded to the initial MRI and surgical findings, independently interpreted the studies, looking for the location and degree of articular cartilage and acetabular labral pathology. RESULTS: Of the 92 patients studied, each of 2 radiologists correctly identified 83 (94%) and 84 (95%) of the 88 labral tears present at surgery, respectively. There was 92% interobserver agreement on the MRI studies. For articular cartilage defects on the femoral head and acetabulum, there was good agreement (92% and 86% within 1 grade) between MRI and surgical grading and between the 2 MR readers (kappa of 0.8 for femoral head cartilage and 0.7 for acetabular cartilage). CONCLUSIONS: This study shows that noncontrast MRI of the hip, using an optimized protocol, can noninvasively identify labral and chondral pathology. Such information may facilitate deciding which patients warrant surgical intervention, thus preserving hip arthroscopy as a therapeutic tool. LEVEL OF EVIDENCE: Level II, Development of Diagnostic Criteria Study.

Acetabulum↗

PubMed2006

Efficient amplification and cloning of near full-length hepatitis C virus genome from clinical samples.

Long RT-PCR (LRP) amplification of RNA templates is sometimes difficult compared to long PCR of DNA templates. Among RNA templates, hepatitis C virus (HCV) represents an excellent example to challenge the potential of LRP technology due to its extensive secondary structures and its difficulty to be readily cultured in vitro. The only source for viral genome amplification is clinical samples in which HCV is usually present at low titers. We have created a comprehensive optimization protocol that allows robust amplification of a 9.1 kb fragment of HCV, followed by efficient cloning into a novel vector. Detailed analyses indicate the lack of potential LRP-mediated recombination and the preservation of viral diversity. Thus, our LRP protocol could be applied for the amplification of other difficult RNA templates and may facilitate RNA virus research such as linked viral mutations and reverse genetics.

Amino Acid Sequence↗

PubMed2006

B cell depletion therapy in rheumatic disease.

B cell depletion therapy was introduced for auto-antibody associated rheumatic disease in 1998. Encouraging pilot studies in rheumatoid arthritis were followed by randomised controlled trials confirming major benefit. Licensing for use in patients unable to benefit from tumour necrosis factor alpha (TNFalpha) neutralising agents is envisaged shortly. Open studies in other disorders, in particular systemic lupus erythematosus (SLE), have also suggested benefit and its use in life-threatening situations is becoming widespread. Toxicity appears to compare favourably with other agents, but respiratory problems may be more common. Repeated therapy is effective, but may lead to hypogammaglobulinemia. Rituximab is currently the main agent used but other agents are in development. Optimal protocols are not well characterised and will probably be different for different conditions.

Antibodies, Monoclonal↗

PubMed2005

Interfacial ternary complex DNA/Ca/lipids at anionic vesicle surfaces.

The electroporative transfer of gene DNA and other bioactive substances into tissue cells by electric pulses gains increasing importance in the new disciplines of electrochemotherapy and electrogenetherapy. The efficiency of the electrotransfer depends crucially on the adsorption of the gene DNA and oligonucleotides to the plasma cell membranes. Here it is shown that the adsorption of larger oligonucleotides such as fragments (ca. 300 bp) of sonicated calf-thymus DNA, to anionic lipids of unilamellar vesicles (diameter Phi=300+/-90 nm) is greatly enhanced by divalent cations such as Ca(2+)-ions. Applying centrifugation, bound and free DNA are monitored optically at the wavelength lambda=260 nm. Using arsenazo III as a Ca(2+)-indicator and atomic absorption spectroscopy (AAS), Ca(2+)-titrations of DNA and vesicles yield the individual equilibrium constants of Ca(2+)- and DNA-binding not only for the binary complexes: Ca/lipids, Ca/DNA and DNA/lipids, respectively, but also for the various processes to form the ternary complex DNA/Ca/lipids. The data provide the basis for goal-directed optimization protocols for the adsorption and thus efficient electrotransfer of oligonucleotides and polynucleotides into cells.

Anions↗

PubMed2005

Preparation of inert magnetic nano-particles for the directed immobilization of antibodies.

Various activated supports (cyanogen bromide, glutaraldehyde, epoxy-chelates, primary amino) were evaluated for the immobilization of IgG anti-horseradish peroxidase. Cyanogen bromide and glutaraldehyde supports greatly reduced the recognition capacity of the antigen, probably due to the incorrect orientation of the antibody on the support. Hetero-functional epoxy-chelate and immobilization by the sugar chain on primary amino groups had little effect on high recognition of the antigen (near to the theoretically expected value). However, the immobilization by the sugar chain resulted in a higher adsorption rate of horseradish peroxidase, possibly due to a favourable orientation on a flexible spacer arm). Antibodies immobilized on aminated surfaces showed two major drawbacks. Firstly, the biological activity of the immobilized antibody sharply decreased over several days when stored at low ionic strength, although this effect could be partially reversed by incubation at high ionic strength. Secondly, a high level of non-specific proteins adsorption on the support surface was observed. Both problems could be successfully resolved by controlling the coating of the support with aldehyde-aspartic-dextran. We propose that the loss of biological activity was related to the ionic adsorption of the immobilized antibody on the support surface, leading to a blocking of the recognition areas. This optimized protocol was applied to the immobilization of IgG anti-horseradish peroxidase from rabbit on magnetic nano-particles. A 10 microg preparation of nano-particles was able to capture more than 75% of the 0.1 microgram of recombinant horseradish peroxidase present in 10 L of crude protein extract (1g/L) from Escherichia coli.

Antibodies↗

PubMed2004

Delineating bone's interstitial fluid pathway in vivo.

Although interstitial fluid flow has been suggested to play a role in bone adaptation and metabolism, the constituents and ultrastructure of this interstitial fluid pathway are not well understood. Bone's lacunar-canalicular porosity is generally believed to be a continuous interstitial fluid pathway through which osteocytes sense external mechanical loading as well as obtain nutrients and dispose of wastes. Recent electron microscopy studies have suggested that a fiber matrix surrounds the osteocytic cell processes and fills this pericellular fluid space. However, studies injecting tracer molecules into the bone vasculature have provided conflicting results about the pore size or the fiber spacing of the interstitial fluid pathway. In addition, whether the smaller collagen-apatite porosity in adult bone is also a continuous fluid pathway is still unclear. To delineate bone's interstitial fluid pathway, four tracers of various size were injected into rats: reactive red (approximately 1 nm), microperoxidase (MP, approximately 2 nm), horseradish peroxidase (HRP, approximately 6 nm), and ferritin (approximately 10 nm). Five minutes after injection, the tibiae were harvested and processed using histological protocols optimized to minimize processing time to reduce possible redistribution of tracer molecules. The number of blood vessels and osteocytic lacunae labeled with the tracers per unit bone area was then measured for mid-diaphysial cross-sections of the tibia. While none of the tracers was detected within the mineralized bone matrix (the collagen-apatite porosity) using light microscopy, all the tracers except ferritin were found to pass through the canaliculi and appear in the osteocytic lacunae. These results indicate that while small tracers (<6 nm) readily pass through the lacunar-canalicular porosity in the absence of mechanical loading, there appears to be an upper limit or cutoff size between 6 and 10 nm for molecular movement from bone capillaries to osteocytic lacunae in rat long bone. This range of pore size contains the most likely fiber spacing (approximately 7 nm) that has been proposed for the lacunar-canalicular annular space based on the presence of a proteoglycan fiber matrix surrounding the osteocyte.

Animals↗

PubMed2005

Identification and quantitative determination of diphenylarsenic compounds in abandoned toxic smoke canisters.

Knowledge of the exact nature of the constituents of abandoned chemical weapons (ACW) is a prerequisite for their orderly destruction. Here we report the development of analytical procedures to identify diphenylchloroarsine (DA/Clark I), diphenylcyanoarsine (DC/Clark II) and related substances employed in one of the munitions known as "Red canister". Both DA and DC are relatively unstable under conventional analytical procedures without thiol derivatization. Unfortunately however, thiol drivatization affords the same volatile organo-arsenic derivative from several different diphenylarsenic compounds, making it impossible to identify and quantify the original compounds. Further, diminishing the analytical interference caused by the celluloid powder used as a stacking material in the weapons, is also essential for accurate analysis. In this study, extraction and instrumental conditions have been evaluated and an optimal protocol was determined. The analysis of Red canister samples following this protocol showed that most of the DA and DC associated with pumice had degraded to bis(diphenylarsine)oxide (BDPAO), while those associated with celluloid were dominantly degraded to diphenylarsinic acid (DPAA).

Arsenicals↗

PubMed2006

Gas chromatographic determination of amino acids via one-step phase-transfer catalytic pentafluorobenzylation-preconcentration.

The gas chromatographic determination of amino acids via their simultaneous extraction, preconcentration and pentafluorobenzylation is reported. Using phase-transfer catalysis (PTC), the amino acids under study were transformed to their pentafluorobenzyl adducts. The method was tested for different catalysts and tetrabutylammonium bromide provided favorable features in comparison to the other PTCs. The derivatization procedure was optimized and the best reaction conditions are given. With the exception of arginine, 19 amino acids were converted to volatile derivatives and analyzed with GC/MS and GC/FID at low concentration levels with acceptable sensitivity and good reproducibility. The LODs were found to range from 0.7 to 2.3microM for the GC/MS analyses and from 1.7 to 6.9microM for GC/FID analyses. The method practicability and applicability were confirmed by the analysis of urine, fruit juice and wheat flour for the determination of the amino acids under study. Protein-bound amino acids were analyzed after an alkaline hydrolysis step with 5M NaOH applying this method to wheat flour with an overall procedure duration less than 12h. The optimized protocol was applied to these samples without any pretreatment and their amino acid concentrations were calculated from the appropriate calibration plots.

Amino Acids↗

PubMed2004

Enzyme-linked immunosorbent assay of total inhibin: direct determination based on inhibin alpha subunit-specific monoclonal antibodies.

OBJECTIVE: Inhibin circulates in various molecular weight forms. Alpha (alpha)-subunit-directed total inhibin immunoassays, which detect all forms of alpha subunits plus the alpha/beta inhibin dimers, have been found valuable in the diagnosis and monitoring of ovarian cancer. Because of the dependency of the published methods on boiling sample pre-treatment with SDS and unavailability of a commercial assay, we developed an enzyme-linked immunosorbent assay (ELISA) for direct determination of total inhibin. DESIGN AND METHODS: Method development involved a pair of well-characterized inhibin alpha subunit-directed antibodies and determination of the effects of various assay parameters. Selection of the optimized protocol was guided by the outcome of comparative sample analysis using previously reported boiling sample pre-treatment reagents and protocols. RESULTS: We report development of a simplified ELISA for total inhibin. Method evaluation data demonstrated acceptable analytical performance characteristics with detection limit of 2 ng/l (recombinant inhibin-A), dynamic range of 12.5-500 ng/l, and intra- and inter-assay imprecision of 2.3-4.6% and 3.3-5.1% at total inhibin concentrations of approximately 60-400 ng/l, respectively. The mean (+/-SD) recovery from spiked serum samples averaged 109 +/- 14% and recovery in response to serial sample dilution was 99 +/- 10%. Serum values by the direct method (n = 40) correlated strongly with those obtained after sample pre-treatment by boiling with SDS (r = 0.97). As expected, the total inhibin immunoreactivity in human follicular fluid fractionated by HPLC gel filtration in multiple immunoreactive peaks (8-250 kDa). In serum samples from postmenopausal women with ovarian cancer, the assay detected significantly higher total inhibin levels than in samples from normal postmenopausal controls. CONCLUSION: The development of a fast and simplified ELISA should facilitate wider investigations of pathophysiology and diagnostic potential of total inhibin measurement.

Adolescent↗

PubMed2006

Photocoupling of fibronectin to titanium surfaces influences keratinocyte adhesion, pellicle formation and thrombogenicity.

OBJECTIVES: Coating of implant surfaces with biomolecules can influence basic host responses and enhance subsequent tissue integration. The biological factors have to be immobilized on the implant material. Human fibronectin (Fn) was used as a model protein and covalently coupled to titanium (Ti) surfaces via silanization and an anthraquinone linker. The impact on several aspects of initial host/biomaterial interactions (keratinocyte adhesion, platelet interactions and pellicle formation) was studied. METHODS: Coupling efficiency was characterized by immunological techniques. The effects of coupled Fn on initial host/biomaterial interactions were assessed. Cell adhesion and spreading were investigated by fluorescent staining, pellicle formation by an acoustic sensor system (quartz crystal microbalance with dissipation, QCM-D), and platelet adhesion as one parameter mediating the inflammatory response by scanning electron microscopy (SEM) and immunological assays. RESULTS: Coupling efficiency was related to irradiation time used for photochemical coupling of the UV-activated anthraquinone to the silanized Ti surface. With an optimized protocol, the amount of Fn coupled to the surface could be almost doubled compared to standard dip-coating methods. On the anthraquinone-coupled Fn coatings, cell adhesion and spreading of human keratinocytes was significantly enhanced. Online detection of pellicle formation revealed strong reversibility of saliva protein adhesion on Fn coated surfaces compared to the pure Ti surface. Furthermore, the Fn coated Ti showed a low thrombogenicity. SIGNIFICANCE: This study suggests that anthraquinone-coupled biological coatings may be useful for biofunctionalization of Ti dental implants by enhancement of soft tissue re-integration (restoration of the epithelial seal) combined with diminished pellicle formation.

Anthraquinones↗

PubMed2026

Non-tobacco nicotine dependence and postoperative complications after total ankle arthroplasty: A propensity-matched cohort study.

BACKGROUND: The clinical impact of non-tobacco nicotine dependence (NTND) is poorly defined. This study evaluated the association between NTND and complications following total ankle arthroplasty (TAA). METHODS: A retrospective cohort study using the TriNetX Research Network was performed. Adults undergoing primary TAA (Current Procedural Terminology [CPT] 27702) between 2010 and 2025 were included. Patients were categorized into NTND (International Classification of Diseases, Tenth Revision [ICD-10]: F17, excluding tobacco-specific codes) and nonsmoker cohorts. Propensity score matching (1:1) yielded 939 NTND patients and 939 controls. Ninety-day medical and wound complications and ≥ 2-year mechanical outcomes were assessed. RESULTS: NTND patients had higher rates of 90-day readmission (11.7% vs 6.5%; OR 1.9), wound disruption (4.3% vs 2.6%; OR 1.7), surgical site infection (3.1% vs 1.6%; OR 2.0), and sepsis (2.4% vs 1.1%; OR 2.3). At a minimum 2-year follow-up, NTND was not associated with increased risk of mechanical complications. CONCLUSION: These findings challenge the assumption that smokeless nicotine products are benign in the perioperative setting and support incorporating NTND screening and cessation counseling into preoperative optimization protocols. Future prospective studies are warranted to further characterize the dose-dependent effects of non-tobacco nicotine exposure and to evaluate the impact of perioperative cessation strategies on outcomes following TAA. LEVEL OF EVIDENCE: IV.

Humans↗

PubMed2006

Challenging protein purification from anammox bacteria.

The anaerobic ammonium oxidation (anammox) is a fascinating microbial pathway contributing to the global biogeochemical nitrogen cycle. The anammox pathway of nitrogen conversion can only be elucidated after the responsible proteins have been purified and characterised. The anammox bacteria have a complex cell envelope consisting of protein and lipopolysaccharide and they grow in dense cell aggregates. Preparing cell extract and purifying proteins from the cell aggregates is hampered by the extracellular polymeric material and by gel formation. It was demonstrated that protein-protein (i.e. disulfide formation) as well as protein-polysaccharide interaction caused this gel formation in extracts. Cell extract gelled upon freezing/thawing and boiling. Additionally, proteins aggregated on various chromatography media upon concentration and during desalting. The polysaccharides clogged the matrix of chromatographic materials and the pores of ultrafiltration membranes. The precipitation of proteins and polysaccharides caused very low resolution and streaking on SDS- and two-dimensional polyacrylamide gels. The present work describes the potential causes for gel formation in anammox cell extracts. Optimized protocols for sample preparation for polyacrylamide gel electrophoresis and ion exchange chromatography are presented. High-resolution gel electrophoresis of the cell extract was achieved after clarification from polymeric substances with denaturating phenol extraction and the purification of a 10 kDa cytochrome c is presented as an example.

Ammonia↗

PubMed2004

Measurement of eotaxin (CCL11) in induced sputum supernatants: validation and detection in asthma.

BACKGROUND: Induced sputum is widely used in asthma research; however, for many mediators, the detection methods have not been validated. OBJECTIVE: We sought to optimize the method of detection of eotaxin, an important chemokine acting through the CCR3 receptor on eosinophils, basophils, and T(H)2 cells. METHODS: Induced sputum from normal and asthmatic subjects was processed with dithioerythritol (DTE) or PBS; recovery of eotaxin was assessed by means of ELISA before and after spiking with recombinant eotaxin. Furthermore, the effects of removing DTE by means of ultrafiltration or the addition of protease inhibitors and high-speed centrifugation on endogenous levels and spiking recovery of eotaxin were assessed. RESULTS: Endogenous eotaxin was undetectable in DTE-processed samples, with a mean of only 30% (SD, 13%) spike recovery. DTE had no effect on the immunoassay capture antibody but dramatically reduced the detection of recombinant eotaxin. Removal of DTE from sputum before immunoassay did not improve detection, although it restored the recovery of a subsequent eotaxin spike. In contrast, PBS-processed sputum resulted in an eotaxin spike recovery of 101% (SD, 20%). Addition of protease inhibitors or high-speed centrifugation had no effect on eotaxin detection. By using this optimized protocol, eotaxin levels in PBS-processed sputum samples were found to be significantly increased in asthmatic sputum (P<.05). CONCLUSION: Measurement of eotaxin by means of immunoassay is adversely affected by DTE, possibly through irreversible denaturation of epitopes, which makes eotaxin undetectable by using the immunoassay antibody. Sputum samples should be processed into PBS for assessment of eotaxin, which is present at increased levels in asthmatic sputum.

Adult↗

PubMed2006

Purification and characterization of cassiicolin, the toxin produced by Corynespora cassiicola, causal agent of the leaf fall disease of rubber tree.

Cassiicolin, a phytotoxin produced by the necrotrophic fungus Corynespora cassiicola, was purified to homogeneity from a rubber tree isolate. The optimized protocol involves reverse phase chromatography followed by size exclusion chromatography, with monitoring of the toxicity on detached rubber tree leaves. Cassiicolin appeared to be a peptide composed of 27 amino acids, glycosylated on the second residue, with a N-terminal pyroglutamic acid and 6 cysteines involved in disulfide bonds. Its molecular mass was estimated to be 2885 Da. No significant sequence homology with other proteins could be found. The availability of pure toxin in sufficient amount is a prerequisite for its structure determination, which is a key step in the understanding of the aggression mechanism.

Amino Acid Sequence↗

PubMed2004

WGA-grafted PLGA-nanospheres: preparation and association with Caco-2 single cells.

Biotech drugs are poorly absorbed, highly susceptible to loss of activity, and require formulations with protective effects. Their incorporation into nanospheres is a promising approach since colloidal formulations were shown to be absorbed. Due to rather low absorption rates the surface engineering with absorption enhancing agents is a current challenge. Thus, an optimized protocol relying on carbodiimide-mediated covalent binding of biorecognitive ligands to the surface of biodegradable and biocompatible poly(D,L-lactic-co-glycolic)acid (PLGA) nanospheres was established. Important parameters such as retainment of particle size distribution and biorecognitive characteristics of the ligand as well as storage stability were considered. As exemplified by nanoparticles grafted with wheat germ agglutinin (WGA) as a carbohydrate binding ligand and Caco-2 single cells, flow cytometry as well as confocal laser scanning microscopy revealed improved cytoassociation of the nanoscaled carriers as compared to the unmodified ones. As detected by flow cytometry, the binding of WGA-decorated nanospheres considerably increases the roughness of the cell surface with increasing ligand density. Additionally, the biorecognitive nanoparticles were not toxic in the Caco-2 model. The protocol presented for surface modification of nanospheres promises high versatility of application in search for biorecognitive ligands enhancing the cytoadhesion, cytoinvasion as well as transcellular transport of colloidal carriers.

Binding Sites↗

PubMed2006

Evaluation of high-risk human papillomavirus types PCR detection in paired urine and cervical samples of women with abnormal cytology.

BACKGROUND: During the last decade, increasing efforts have focused on HPV detection in self-obtained samples, to increase the overall proportion of patients participating in cervical cancer screening procedures. OBJECTIVES: A clinical evaluation study of an optimized protocol for PCR detection of high-risk human papillomavirus (HPV) types in urine compared with cervical samples in consecutive women referred to the colposcopy clinic with abnormal cervical cytology. STUDY DESIGN: Paired urine and cervical specimens were collected from 100 consecutive women referred to the colposcopy clinic with abnormal cervical cytology and normal urine parameters. In-house and a commercial PCR method for the detection of HPV types 16 and 18, and a commercial multiplex PCR for HPV types 6, 11, 16, 18, and 33 were performed. All HPV cervix-positive/urine-negative paired urine samples were spiked with serial dilutions of cell lines infected with HPV 16 or 18 to test the sensitivity of HPV detection in these urine samples. RESULTS: In all but two cases HPV type 16 was detected. In cancer cases, the urine/cervix HPV detection sensitivity was 88.8%; in cases with high-grade lesions it was 76.5%; and in cases with low-grade lesions it was 45.5%. In all concordant cases the same HPV type was detected in both samples. The urine/cervix HPV detection sensitivity was higher when urine samples contained two or more epithelial cells per field in urine microscopy. HPV detection in 9 cervix-positive but urine-negative urine samples spiked with serial dilutions of HPV-positive cell lines showed that in these cases urine PCR inhibitors did not affect PCR amplification. CONCLUSIONS: A higher urine/cervix HPV detection sensitivity in cancer and high-grade lesions suggests that urine testing could be used to detect HPV mainly when these lesions are present.

Cell Line↗

PubMed2006

Analyzing the performance of conformational search programs on compound databases.

We have studied the sampling performance of conformational search programs using geometric and energetic criteria. Ideally, a conformational search algorithm should identify the largest possible number of low-energy structures (energy criterion) covering the widest possible range of molecular shapes (geometric criterion). Geometric analysis consisted in comparing the distribution of conformations within the generated ensembles by multidimensional scaling and by analysing the eigenvalue structure of the pairwise coordinate covariance matrices. The energetic comparison was carried out by assessing the energy distribution of conformers after minimizing them all using the same semi-empirical quantum mechanics optimization protocol. The present investigation focused on five conformational search programs: DGEOM, QXP, ROTATE, LMOD and OMEGA. We have applied these methodologies to a maximally diverse 604-compound subset of the LeadQuest library. The program LMOD performs best according to the energetic criterion, whereas a wider range of geometrically diverse conformations is sampled by the other programs, at the cost of higher median conformer energies. In terms of speed, OMEGA is fastest. We recommend the use of LMOD or OMEGA for high-quality conformational search applications.

Computer Simulation↗

PubMed2004

Use of bipolar parallel electrodes for well-controlled microstimulation in a mouse hippocampal brain slice.

In a hippocampal brain slice two types of stimulating electrodes [single (SE) or monopolar and parallel bipolar (PE)] were used to determine the optimal protocol for single pulse microstimulation. We show that even for a constant-current power source the amplitude of stimulating current (SC) is not constant, especially for short pulse widths (PW) (<200 micros). Recording the stimulating current and computing the amount of electric charge that is passed through the microelectrode gives the best estimate of the strength of electrical stimulation. For SE the evoked response is obstructed for a time interval larger than three times the PW. The stimulus artifact (SA) substantially decreases when a PE is used. The orientation of the stimulating current relative to the position of the targeted fibers (Schaffer collaterals) was controlled when using a PE. The use of PEs allowed the accurate recording of the physiological response that contains three clearly defined peaks. Stimulation can be elicited at PW as short as 30 micros when the main current is capacitive. The charge needed to elicit physiological responses was in the range of 1-40 nC (the lower values for the PE) suggesting that use of PEs is most advantageous for well-controlled microstimulation studies in brain slices.

Action Potentials↗