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PubMed2005

A lesion stabilization method for coronary angiography.

A method to make a coronary artery segment of interest appear stationary when viewing a sequence of angiographic images is proposed. The purpose of this method is to facilitate the assessment of lesions caused by coronary artery disease by improving detectability. A description of the stabilization algorithm based on template matching is given. Stabilization was performed on 41 clinical coronary angiograms exhibiting various stenoses and was successful in 39/41 cases. A quantitative analysis of stabilization errors was performed by introducing simulated moving vessels of decreasing contrast into sequences of clinical images.

Algorithms↗

PubMed2000

Comparing HPLC and UV spectrophotometric analysis methods for determining the stability of sorbic acid in nonionic creams containing lactic acid.

This paper describes a comparison between ultraviolet (UV) spectrophotometric and high-performance liquid chromatographic (HPLC) methods of analysis for the determination of sorbic acid in nonionic creams containing lactic acid. Sorbic acid is an antimycotic agent and is used as a preservative in pharmaceuticals, cosmetics, and food products. UV spectrophotometric analysis was done by calculating the concentration of remaining sorbic acid from the absorbance values and the molar extinction coefficient EM258 = 24,080. A decrease in absorbance at 258 nm was accompanied by a simultaneous increase in total carbonyls and monoaldehyde content and the appearance of a very weak absorption maximum between 215 and 225 nm. HPLC analysis was done with a Hypersil BDS C8 column with detection at 254 nm and employing a mobile phase consisting of a mixture of buffer and methanol (7:3 v/v) at a pH of 2.25. The buffer consisted of 0.85% H2SO4 in 17.5 mM KH2PO4. The validation results, together with statistical treatment of the data, demonstrated the reliability of both procedures. A drawback of the UV methods was, however, its lack of adequate measurement of sorbic acid stability at higher temperatures. For these assays, the HPLC method was found to be adequate, and it should therefore be used to obtain accurate stability data for sorbic acid in creams.

Chromatography, High Pressure Liquid↗

PubMed2004

Validating internal controls for quantitative plant gene expression studies.

BACKGROUND: Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. RESULTS: Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. CONCLUSION: Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.

Analysis of Variance↗

PubMed2004

Development and characterization of a scalable controlled precipitation process to enhance the dissolution of poorly water-soluble drugs.

PURPOSE: Poorly water-soluble compounds are being found with increasing frequency among pharmacologically active new chemical entities, which is a major concern to the pharmaceutical industry. Some particle engineering technologies have been shown to enhance the dissolution of many promising new compounds that perform poorly in formulation and clinical studies (Rogers et. al., Drug Dev Ind Pharm 27:1003-1015). One novel technology, controlled precipitation, shows significant potential for enhancing the dissolution of poorly soluble compounds. In this study, controlled precipitation is introduced; and process variables, such as mixing zone temperature, are investigated. Finally, scale-up of controlled precipitation from milligram or gram to kilogram quantities is demonstrated. METHODS: Dissolution enhancement capabilities were established using two poorly water-soluble model drugs, danazol and naproxen. Stabilized drug particles from controlled precipitation were compared to milled, physical blend, and bulk drug controls using particle size analysis (Coulter), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), dissolution testing (USP Apparatus 2), and residual solvent analysis. RESULTS: Stabilized nano- and microparticles were produced from controlled precipitation. XRD and SEM analyses confirmed that the drug particles were crystalline. Furthermore, the stabilized particles from controlled precipitation exhibited significantly enhanced dissolution properties. Residual solvent levels were below FDA limits. CONCLUSIONS: Controlled precipitation is a viable and scalable technology that can be used to enhance the dissolution of poorly water-soluble pharmaceutical compounds.

Chemistry, Pharmaceutical↗

PubMed2002

Efficient trans-cleavage by the Schistosoma mansoni SMalpha1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus.

The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMalpha DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme-product dissociation step. The optimal temperature for trans-cleavage was 70 degrees C. This result was confirmed when both the SMalpha1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMalpha1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMalpha1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo.

Animals↗

PubMedDate not supplied

Cost: benefit of point-of-care blood gas analysis vs. laboratory measurement during stabilization prior to transport.

INTRODUCTION: This study was conducted to determine whether point-of-care testing, using the iSTAT Portable Clinical Analyzer, would reduce time at the referring hospital required to stabilize ventilated pediatric patients prior to interfacility, air-medical transport. METHODS: The following data were collected prospectively: (1) When a blood gas analysis was ordered; (2) If it was necessary to call in a technician; (3) Waiting time for blood to be drawn; and (4) Waiting time for results. The cost-efficacy of point-of-care testing was calculated based on: (1) Three minutes for a transport team member to draw a sample and obtain a result using the iSTAT (unit cost 8,000 CDN dollars); (2) Lab technician call-back (minimum two hours at 90 dollars); (3) Paramedic overtime (by the minute at 49 dollars/hour); and (4) Cost of charter aircraft wait time (200 dollars per hour) for every hour beyond four hours. RESULTS: Data were collected on 46 ventilated patients over a three month period. A blood gas analysis was ordered on 35 patients. Laboratory technicians were called in for 17 (49%). For 12 (34%) patients, there was a wait for the sample to be drawn, and for 23 (66%), there was a wait for results to become available. Total time waiting to obtain laboratory gases was 526 minutes compared with a calculated 105 minutes using point-of-care testing. An iSTAT cartridge cost of 420 dollars would not have been different from laboratory costs. Cost-saving on technician callback (1,530 dollars), paramedic overtime (690 dollars) and aircraft time waiting charges (2,000 dollars) would have totaled (4,220 dollars). From this study, the cost of point-of-care equipment could be recouped in 101 patients if aircraft charges apply or 192 patients if no aircraft costs are involved. For 11 cases, ventilator adjustments were made subsequently during transport, and for six patients, point-of-care testing, if in place, would have been used to optimize transport care. CONCLUSION: The data from the present study indicate significant cost-efficacy from use of this technology to reduce stabilization times, and support the potential to improve quality of care during air medical interfacility transport.

Blood Gas Analysis↗

PubMed2000

[Tissue engineering of vascularized bone and soft tissue transplants].

The utilization of in vitro angiogenesis in tissue engineering might be useful in order to establish an artificial vascular network. However, it remains unclear how far the in vitro preformation of vascular structures may contribute to the perfusion of larger artificial tissue aggregates regarding the improvement of oxygenation and nutrition. In an in vitro study, we developed a model of a vascularized tissue. Stromal cells of a target tissue, e.g., adipose tissue or bone tissue, were expanded in vitro and seeded onto microcarriers or microparticles. Densely covered microcarriers were brought into a fibrin matrix together with endothelial cells. In order to demonstrate the formation and stabilization of capillary-like structures, UEA-I labeled specimens were evaluated using laser scanning microscopy and digital image analysis. The stabilization of capillary-like structures was better with stromal cells from bone marrow than from adipose tissue. In one of the culture aggregates, the total length of capillary-like structures increased after 6 weeks of cultivation to up to 140 mm/mm3. Additional tests were performed utilizing hyperbaric oxygenation. In the oxygenation group, a significant increase in the length of capillary-like structures was found. The method implies the option of coculturing different tissue elements and of an in vitro preformation of vascularized tissues.

Bone Transplantation↗

PubMed2006

Time course of ozone-induced changes in breathing pattern in healthy exercising humans.

We examined the time course of O3-induced changes in breathing pattern in 97 healthy human subjects (70 men and 27 women). One- to five-minute averages of breathing frequency (f(B)) and minute ventilation (Ve) were used to generate plots of cumulative breaths and cumulative exposure volume vs. time and cumulative exposure volume vs. cumulative breaths. Analysis revealed a three-phase response; delay, no response detected; onset, f(B) began to increase; response, f(B) stabilized. Regression analysis was used to identify four parameters: time to onset, number of breaths at onset, cumulative inhaled dose of ozone at onset of O3-induced tachypnea, and the percent change in f(B). The effect of altering O3 concentration, Ve, atropine treatment, and indomethacin treatment were examined. We found that the lower the O3 concentration, the greater the number of breaths at onset of tachypnea at a fixed ventilation, whereas number of breaths at onset of tachypnea remains unchanged when Ve is altered and O3 concentration is fixed. The cumulative inhaled dose of O3 at onset of tachypnea remained constant and showed no relationship with the magnitude of percent change in f(B). Atropine did not affect any of the derived parameters, whereas indomethacin did not affect time to onset, number of breaths at onset, or cumulative inhaled dose of O3 at onset of tachypnea but did attenuate percent change in f(B). The results are discussed in the context of dose response and intrinsic mechanisms of action.

Adolescent↗

PubMed1984

Carbon monoxide in blood: an improved microliter blood-sample collection system, with rapid analysis by gas chromatography.

We examined the sensitive assay for carboxyhemoglobin based on reaction with K3Fe(CN)6 and gas chromatography of the liberated CO. Our improvements included increased baseline stability, shorter analysis time, and simpler standardization. EDTA-containing Vacutainer Tubes (lavender-stoppered) increase the carboxyhemoglobin content of blood stored in them. The carboxyhemoglobin content of blood stored in capillary tubes containing solid heparin and saponin remained stable for two weeks. Using our improved procedures, we measured the carboxyhemoglobin content of blood from adults and neonates collected via venipuncture or heel or fingersticks. We observed no significant difference in carboxyhemoglobin content of blood obtained by venipuncture or heel stick for premature infants, 0.19 +/- 0.04 vs 0.18 +/- 0.03 mL of CO per 100 mL of blood, respectively (mean +/- SD). Nonsmoking adults (n = 19) had CO values (mean +/- SD) of 0.19 +/- 0.03 and 0.17 +/- 0.04 mL per 100 mL of blood, and smoking adults (n = 7) gave CO values of 0.96 +/- 0.49 and 0.91 +/- 0.49 mL/dL, for venipuncture and fingerstick, respectively.

Adult↗

PubMed1994

Complications after transpedicular stabilization of the spine. A survivorship analysis of 163 cases.

OBJECTIVES: The authors studied complications of transpedicular stabilization methods. SUMMARY OF BACKGROUND DATA: One hundred and sixty-three consecutive transpedicular stabilization procedures were performed between January 1987 and December 1991. The indications for stabilization were trauma (33 cases), metastatic spinal disorder (30 cases), spinal stenosis (33 cases), spondylolisthesis (27 cases), ankylosing spondylitis (6 cases), low back pain (22 cases), and miscellaneous (12 cases). METHODS: Patients records and the entire series of radiographs for each case were scrutinized by independent observers. All per- and postoperative complications, including implant loosening and fatigue, were recorded. Clinical and radiographic survivorship analyses of the implants were performed. RESULTS: Early complications were unusual and none were associated with permanent morbidity. The probability of not having the implant removed in the first postoperative year was 85%. There was a 40% risk of radiographic failure, defined as loosening or implant fatigue, at 6 months. The outcome was more favorable in cases in which anterior vertebral interbody fusion was also performed. CONCLUSIONS: Transpedicular fixation is a safe procedure with a low incidence of serious per- and early postoperative complications. The mechanical durability of transpedicular fixators used alone is a cause for concern.

Adolescent↗

PubMed2000

[Effects of standardized optokinetic stimuli on standing stability].

BACKGROUND: Human standing stability is ensured by means of a sensory-motor control system. Proprioceptivity is most important among sensory afferences. At this time, less is known about the effect of standardized visual stimuli on motor programs towards support of body stability. These investigations were performed with special consideration for different strains on bunions and heels of both feet. METHODS: 42 healthy individuals (averaged age 29.6 years) were investigated with relaxed standing on the static force platform of Portable Multiplate System P. M. S. with optokinetic stimuli in horizontal, vertical and torsional direction (speed 80 degrees/s). Conducted by computer-analysis, the stability index, varying foot-pressure on heels and bunions, Fourier spectral analysis, and weight distribution index were counted. The eye movements were controlled with help of the PENG device. RESULTS: The optokinetic stimulations led to partial statistically significant impairments of stability particularly with torsional stimuli, yet notably improved stability with left stripes pattern. The sensory conflict between visual and other sensory inputs led to a statistically significant shift of sway frequencies to the higher ranges. On stimulation, the distribution of pressure to the feet changed. The preponderance to rest on the left side was equalized. The heels were considerably strained more with visual stimuli in all directions. Individuals felt more agreeable, toward stripes vertically directed while torsionally directed stripes elicited particular strain. CONCLUSION: Sensory conflicts between visual and other sensory inputs for maintaining the upright body position were found to impair the stability. Nevertheless these conflicts were partially compensated by motor programs more straining to the heels.

Adolescent↗

PubMed2000

Photoencapsulation of chondrocytes in poly(ethylene oxide)-based semi-interpenetrating networks.

A photopolymerizing hydrogel system provides an efficient method to encapsulate cells. The present work describes the in vitro analysis of bovine and ovine chondrocytes encapsulated in a poly(ethylene oxide)-dimethacrylate and poly(ethylene glycol) semi-interpenetrating network using a photopolymerization process. One day after encapsulation, (3-[4,5-dimethylthiazol-2-y1]-2, 5-diphenyl-2H-tetrazolium bromide) (MTT) and light microscopy showed chondrocyte survival and a dispersed cell population composed of ovoid and elongated cells. Biochemical analysis demonstrated proteoglycan and collagen contents that increased over 2 weeks of static incubation. Cell content of the gels initially decreased and stabilized. Biomechanical analysis demonstrated the presence of a functional extracellular matrix with equilibrium moduli, dynamic stiffness, and streaming potentials that increased with time. These findings suggest the feasibility of photoencapsulation for tissue engineering and drug delivery purposes.

Animals↗

PubMed1997

Chemotherapeutic purine analogs alter the level of interferon-beta mRNA induced by poly I-poly C in cultured osteosarcoma cells.

The purine nucleoside analogs fludarabine, 2-chlorodeoxyadenosine, and 2'-deoxycoformycin exhibit impressive activity in lymphoproliferative malignancies of adults and children. Their mechanism of action is not clear. Studies have suggested that their use is associated with significant myelosuppression, immunosuppression, and in some circumstances, increased infection with viral and opportunistic pathogens. Because interferons (IFNs) are known to have immunomodulatory activity as well as potent antiproliferative and antiviral activity, we examined whether the chemotherapeutic purine nucleoside analogs alter interferon-beta (IFN-B) gene expression in MG63 in human osteosarcoma cells. Northern blot analysis showed a dose-dependent inhibition of IFN-B mRNA accumulation in response to a known inducer (Poly I-Poly C) all three purine analogs. Hybridization analysis also revealed that inhibition of IFN-beta mRNA accumulation by the purine analogs is not a result of decreased mRNA stability. Further analysis of gene expression by PCR differential display indicated that the effect of the purine analogs was restricted to only a limited number of inducible genes. The data suggest that these molecules alter the signaling process involved in regulating the expression of specific genes, including IFN-beta. These findings predict that the use of purine nucleoside analogs may reduce IFN production in vivo and thereby abrogate host defenses against infectious pathogens.

Antineoplastic Agents↗

PubMed1997

Immobilization of L-asparaginase into a biocompatible poly(ethylene glycol)-albumin hydrogel: evaluation of performance in vivo.

The L-asparaginase of Escherichia coli (ASNase) is currently used in combination with antineoplastic drugs to treat various lymphoblastic leukaemias. However, its use is limited by severe immunological reactions and the short serum half-life associated with the enzyme. Immobilization of ASNase into a biocompatible matrix can greatly decrease the immunogenicity of the enzyme, increase its half-life in vivo and its therapeutic index. Thus the E. coli ASNase was immobilized in a biocompatible hydrogel made of rat serum albumin and poly(ethylene glycol) (PEG; molecular mass 10 kDa). The effectiveness of this enzymic bioreactor to deplete serum L-asparagine was evaluated after its peritoneal implantation in rats. Seven units of immobilized ASNase/rat depleted serum asparagine to an undetectable level (< 1 microM) during 6 days, while 5 units of immobilized ASNase/rat decreased the level of serum asparagine by 85-90% during at least 2 days. Under both conditions asparagine levels returned to normal about 10 days after surgery, and hydrogels still retained 80% of their enzymic activity when assayed in vitro. After 10-14 days in vivo, hydrogels became opaque and surrounded by a fibrotic capsule with a few inflammatory sites. Nevertheless, the enzymic hydrogel showed great stability in vivo, and, after 4 months of implantation, 12% of the initial ASNase activity was still present. At 6 months, histological analysis showed stabilization of the fibrotic capsule thickness. Assays on the levels of ASNase and asparagine synthetase indicated an induction of the latter activity, mainly in the pancreas when compared with the level observed in spleen or liver. ELISA tests at 28 days and 120 days showed the presence of anti-ASNase (and, in lower amounts, anti-PEG) antibodies in sera of implanted rats. As observed with other enzyme-immobilization systems used in vivo, the formation of fibroblast-like cell layers around the implant, which block the translocation of the substrate into the enzymic matrix, is the major factor affecting the performance and longevity of the bioreactor.

Albumins↗

PubMed1987

The chicken receptor for endocytosis of glycoproteins contains a cluster of N-acetylglucosamine-binding sites.

The oligomeric state of the chicken hepatic receptor for N-acetylglucosamine-terminated glycoproteins (the chicken hepatic lectin) has been examined in detergent solution, in various membrane preparations, and in hepatocytes. In detergent solution, the cross-linking reagent, 1,5-difluoro-2,4-dinitrobenzene produces covalent complexes containing up to six receptor polypeptides. This result, along with hydrodynamic studies of the receptor-detergent complex, indicates that the purified receptor is a hexamer. Analysis of large proteolytic fragments of the receptor reveals that portions of the receptor polypeptide near the membrane anchor are essential for hexamer stability. This analysis also demonstrates that each receptor polypeptide has an N-acetylglucosamine-binding site, indicating that the native hexameric receptor contains a cluster of six such sites. Immunoblot analysis of membrane fractions and cells cross-linked with 1,5-difluoro-2,4-dinitrobenzene or dimethyl adipimidate reveals that the receptor is also oligomeric in intact cells and in subcellular fractions representing cell surface and internalized receptor. Although the pattern of cross-linking observed in membranes differs from that observed with purified receptor, experiments indicate that the differences may be explained by the presence of membrane components which compete with receptor for reaction with cross-linking reagent. The presence of a cluster of carbohydrate-binding sites in the hepatocyte membrane can account for the preferential endocytosis of multivalent glycoprotein ligands by hepatocytes.

Acetylglucosamine↗

PubMed2005

Failure of the PAXgene Blood RNA System to maintain mRNA stability in whole blood.

In multicentre studies of malignant and inflammatory diseases, whole blood, cell or tissue samples are often collected for analyses of gene expression to predict or monitor treatment effects. For correct analysis, sample stability during handling and transport is crucial. In developing the logistics for multicentre studies in malignant melanoma and inflammatory bowel disease, we found poor stability of a number of transcripts using the PAXgene Blood RNA System, which was advertised to maintain RNA stability for several days at room temperature. The results indicate that general statements on sample stability are not reliable and have to be verified for the specific transcripts of interest.

Artifacts↗

PubMed2002

Analysis of the factors that stabilize a designed two-stranded antiparallel beta-sheet.

Autonomously folding beta-hairpins (two-strand antiparallel beta-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a beta-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in beta-hairpin stability by comparing 1 with (D)P--> N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the (D)PG segment decreases the entropic cost of beta-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of (D)PG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed beta-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.

Amino Acid Sequence↗

PubMed2004

Fluorescence and CD spectroscopic analysis of the alpha-chymotrypsin stabilization by the ionic liquid, 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide.

The stability of alpha-chymotrypsin in the ionic liquid, 1-ethyl-3-methyl-imidizolium bis[(trifluoromethyl)sulfonyl]amide ([emim][NTf2]), was studied at 30 and 50 degrees C and compared with the stability in other liquid media, such as water, 3 M sorbitol, and 1-propanol. The kinetic analysis of the enzyme stability pointed to the clear denaturative effect of 1-propanol, while both 3M sorbitol and [emim][NTf2] displayed a strong stabilizing power. For the first time, it is shown that enzyme stabilization by ionic liquids seems to be related to the associated structural changes of the protein that can be observed by differential scanning calorimetry (DSC) and fluorescence and circular dichroism (CD). The [emim][NTf2] enhanced both the melting temperature and heat capacity of the enzyme compared to the other media assayed. The fluorescence spectra clearly showed the ability of [emim][NTf2] to compact the native structural conformation of alpha-chymotrypsin, preventing the usual thermal unfolding which occurs in other media. Changes in the secondary structure of this beta/beta protein, as quantified by the CD spectra, pointed to the great enhancement (up 40% with respect to that in water) of beta-strands in the presence of the ionic liquid, which reflects its stabilization power.

Chymotrypsin↗