Determining the mechanical properties of a radiochromic silicone-based 3D dosimeter.

New treatment modalities in radiotherapy (RT) enable delivery of highly conformal dose distributions in patients. This creates a need for precise dose verification in three dimensions (3D). A radiochromic silicone-based 3D dosimetry system has recently been developed. Such a dosimeter can be used for dose verification in deformed geometries, which requires knowledge of the dosimeter's mechanical properties. In this study we have characterized the dosimeter's elastic behaviour under tensile and compressive stress. In addition, the dose response under strain was determined. It was found that the dosimeter behaved as an incompressible hyperelastic material with a non-linear stress/strain curve and with no observable hysteresis or plastic deformation even at high strains. The volume was found to be constant within a 2% margin at deformations up to 60%. Furthermore, it was observed that the dosimeter returned to its original geometry within a 2% margin when irradiated under stress, and that the change in optical density per centimeter was constant regardless of the strain during irradiation. In conclusion, we have shown that this radiochromic silicone-based dosimeter's mechanical properties make it a viable candidate for dose verification in deformable 3D geometries.

On the presence and immunoregulatory functions of extracellular microRNAs in the trematode Fasciola hepatica.

Liver flukes represent a paraphyletic group of endoparasitic flatworms that significantly affect man either indirectly due to economic damage on livestock or directly as pathogens. A range of studies have focussed on how these macroscopic organisms can evade the immune system and live inside a hostile environment such as the mammalian liver and bile ducts. Recently, microRNAs, a class of short noncoding gene regulators, have been proposed as likely candidates to play roles in this scenario. MicroRNAs (miRNAs) are key players in development and pathogenicity and are highly conserved between metazoans: identical miRNAs can be found in flatworms and mammalians. Interestingly, miRNAs are enriched in extracellular vesicles (EVs) which are secreted by most cells. EVs constitute an important mode of parasite/host interaction, and recent data illustrate that miRNAs play a vital part. We have demonstrated the presence of miRNAs in the EVs of the trematode species Dicrocoelium dendriticum and Fasciola hepatica (Fhe) and identified potential immune-regulatory miRNAs with targets in the host. After our initial identification of miRNAs expressed by F. hepatica, an assembled genome and additional miRNA data became available. This has enabled us to update the known complement of miRNAs in EVs and speculate on potential immune-regulatory functions that we review here.

Eliminating the dose-rate effect in a radiochromic silicone-based 3D dosimeter.

Comprehensive dose verification, such as 3D dosimetry, may be required for safe introduction and use of advanced treatment modalities in radiotherapy. A radiochromic silicone-based 3D dosimetry system has recently been suggested, though its clinical use has so far been limited by a considerable dose-rate dependency of the dose response. In this study we have investigated the dose-rate dependency with respect to the chemical composition of the dosimeter. We found that this dependency was reduced with increasing dye concentration, and the dose response was observed to be identical for dosimeters irradiated with 2 and 6 Gy min(-1) at concentrations of 0.26% (w/w) dye and 1% (w/w) dye solvent. Furthermore, for the optimized dosimeter formulation, no dose-rate effect was observed due to the attenuation of the beam fluence with depth. However, the temporal stability of the dose response decreased with dye concentration; the response was reduced by (62 ± 1)% within approximately 20 h upon irradiation, at the optimal chemical composition and storage at room temperature. In conclusion, this study presents a chemical composition for a dose-rate independent silicone dosimeter which has considerably improved the clinical applicability of such dosimeters, but at the cost of a decreased stability.

Quaternary structures of a catalytic subunit-regulatory subunit dimeric complex and the holoenzyme of the cAMP-dependent protein kinase by neutron contrast variation.

Chimeric molecules of the cAMP-dependent protein kinase (PKA) holoenzyme (R2C2) and of a Delta1-91RC dimer were reconstituted using deuterated regulatory (R) and protiated catalytic (C) subunits. Small angle scattering with contrast variation has revealed the shapes and dispositions of R and C in the reconstituted complexes, leading to low resolution models for both forms. The crystal structures of C and a truncation mutant of R fit well within the molecular boundaries of the RC dimer model. The area of interaction between R and C is small, seemingly poised for dissociation upon a conformational transition within R induced by cAMP binding. Within the RC dimer, C has a "closed" conformation similar to that seen for C with a bound pseudosubstrate peptide. The model for the PKA holoenzyme has an extended dumbbell shape. The interconnecting bar is formed from the dimerization domains of the R subunits, arranged in an antiparallel configuration, while each lobe contains the cAMP-binding domains of one R interacting with one C. Our studies suggest that the PKA structure may be flexible via a hinge movement of each dumbbell lobe with respect to the dimerization domain. Sequence comparisons suggest that this hinge might be a property of the RII PKA isoforms.

Effect of oncotic pressure on apolipoprotein A-I metabolism in the rat.

The nephrotic syndrome is characterized by reduced plasma albumin and colloid osmotic pressure (pi), urinary protein loss and hyperlipidemia. High-density lipoprotein (HDL) and the level of apo A-I, the principal apolipoprotein in HDL, is increased in nephrotic rats and rats with hereditary analbuminemia (NAR)--animals with virtually no albumin in plasma and reduced plasma pi, but without proteinuria, suggesting that urinary protein loss is not responsible for increased plasma apo A-I levels. We conducted these studies to determine the mechanism responsible for increased plasma apo A-I levels in the nephrotic syndrome and NAR and to determine whether reduced plasma pi or albumin was responsible for increased apo A-I. We first measured the clearance of 125I apo A-I HDL in NAR and rats with passive Heymann nephritis (HN) compared with normal Sprague Dawley (SD) control. Both the clearance of apo A-I and fractional apo A-I turnover rate (FTR) were significantly reduced both in HN (7.40 +/- 2.18% plasma pool/hr) and NAR (5.63 +/- 1.12) compared with SD (9.87 +/- 0.75). Total apo A-I turnover rate, which in steady state equals apo A-I synthesis rate, was also significantly increased in both HN (487 +/- 127 micrograms/100 g body weight/hr) and NAR (253 +/- 16), compared with SD (216 +/- 19). Thus decreased apo A-I catabolism and increased synthesis both contributed to increased apo A-I levels in HN and NAR. We then infused either f3p4roncotic human albumin or ficoll into two additional groups of HN for days in quantities sufficient to maintain plasma pi within the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein AI levels are increased in part as a consequence of reduced catabolism in nephrotic rats.

Apolipoprotein AI (apo AI) synthesis, measured as the turnover of 125I-labeled apo AI-labeled high-density lipoprotein (HDL), was increased significantly in rats with Heymann nephritis (HN) vs. control Sprague-Dawley (SD) rats. However, fractional apo AI catabolic rate was also significantly less in HN vs. SD. We used 125I-apo AI tyramine cellobiose HDL, a marker retained at the catabolic site, to establish where apo AI catabolism decreased in six HN rats, seven rats with adriamycin (Adria)-induced nephrosis, and six control SD. Total renal apo AI catabolism, plus urinary losses, were the same in all three groups, despite significant urinary apo AI in HN and Adria rats. Apo AI catabolism was reduced in skin in both nephrotic groups, accounting for approximately 44% of reduced in apo AI catabolism. Thus a significant fraction of apo AI is catabolized in skin of normal male rats. Reduced apo AI catabolism in skin contributes to increased plasma levels in nephrotic rats.

Financing of independent and assisted-living retirement communities by nonprofits.

The article provides reasons for decreased activity in the development of market rate senior housing and briefly describes the criteria lenders use to assess risk. It summarizes the various financing options available for nonprofits to finance senior housing projects. Options discussed include tax-exempt bond financing: different supportive senior housing options administered by the Department of Housing and Urban Development, such as Sections 202, 232, and 221 (d)(3) and (4); the HOME Investment Partnership Program (HOME Program); and low-income housing tax credits.

Inhibin activity in the mare and stallion.

An overnight double antibody RIA, employing a rabbit antiserum raised to bovine 31 kDa inhibin (rAs-#1989, NICHD) and purified bovine 31 kDa inhibin (bINH-I-90/1, NICHD) as trace and standard, was validated to measure immunoreactive inhibin (iINH) concentrations in equine peripheral plasma, follicular fluid (FF), ovarian vein (OV) plasma, testicular tissue extracts (TTE) and testicular vein (TV) plasma. The dynamic relationship of iINH and follicle stimulating hormone (FSH) was investigated during the estrous cycle of the mare and the annual reproductive cycle of the stallion. In the RIA, parallel dose-response curves were observed between the bovine inhibin standard and serial dilutions of equine FF, OV, TTE, TV and plasma. The average recovery of a known amount of purified bovine inhibin added to gelding plasma was approximately 100%. In the inhibin bioassay, serial dilution of equine FF and TTE were observed to be parallel to the bovine inhibin standard. A five-fold difference (p < 0.05) between jugular and gonadal vein plasma iINH concentrations was observed in the mare and an eight-fold difference (p < 0.05) was observed in the stallion. Plasma levels of iINH in ovariectomized mares or geldings were undetectable in the RIA. Concentrations of FSH, estradiol and iINH changed significantly in the mare during the estrous cycle (p < 0.05). Immunoreactive inhibin levels were highest (0.54 +/- 0.06 ng/ml) on the day of ovulation, declined rapidly following ovulation and reached a nadir (0.21 +/- 0.03 ng/ml) on day 7 post-ovulation. Plasma iINH and estradiol concentrations followed a similar profile and were found to be positively correlated (r = 0.7064; p < 0.01), whereas iINH and FSH levels demonstrated an inverse relationship (r = -0.7359, p < 0.01) throughout the estrous cycle. Concentrations of FSH were also inversely related (-0.8498, p < 0.01) with estradiol during the cycle. In the stallion, plasma iINH and FSH levels changed significantly during the year (p < 0.05). The iINH profile reflected seasonal changes in testicular activity, with highest concentrations in late spring (3.37 +/- 0.44 ng/ml) and lowest concentrations in the fall (2.21 +/- 0.33 ng/ml). Plasma concentrations of iINH were positively correlated (r = 0.7691, p < 0.01) with FSH concentrations throughout the year. In conclusion, a specific and sensitive RIA for iINH has been validated for plasma and biological fluids in the horse. Furthermore, the gonads appear to be the source of bioactive and immunoreactive inhibin as observed in other species.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a domain that mediates vesicle aggregation reveals functional diversity of annexin repeats.

Annexins are structurally related proteins that bind phospholipids in a Ca2(+)-dependent manner and possess at least four conserved 70-amino acid repeat domains. The ability of certain annexins to promote contact between vesicle membranes in vitro has prompted the suggestion that these proteins regulate membrane traffic in exocytosis. We have previously found that annexins I and II promote contact between vesicles whereas annexin V does not. In order to understand the mechanism of annexin I-mediated vesicle-vesicle contact, we prepared a monoclonal antibody that specifically inhibits annexin I-mediated vesicle aggregation. We identified the domain of annexin I recognized by this monoclonal antibody by using it to screen an expression library containing random fragments of annexin I cDNA. The antibody identified a fragment encoding amino acids 41-118 (the first repeat plus 8 residues of the amino-terminal tail). We constructed a chimeric protein containing these amino acids of annexin I fused to the second, third, and fourth repeats of annexin V. Transfer of this domain conferred the ability to promote vesicle aggregation, confirming that this domain participates directly in mediating contact between vesicle membranes.

Purification and characterization of an abundant cytosolic protein from human neutrophils that promotes Ca2(+)-dependent aggregation of isolated specific granules.

Intracellular ionized calcium has been strongly implicated in mediating several responses of human neutrophils to stimulation. However, proteins that serve as effectors of these responses have not been well characterized. To identify proteins that might serve as mediators of the effects of Ca2+ in human neutrophils, we isolated proteins that bind to membrane phospholipids in a Ca2(+)-dependent manner. The most abundant of these, a protein of 33 kD, was readily purified to homogeneity, and was found to bind to phosphatidylserine vesicles in the presence of 2 microM ionized Ca2+. In addition, this purified protein promoted Ca2(+)-dependent aggregation of isolated specific granules from human neutrophils, indicating that it might mediate membrane-membrane contact during processes such as phagosome-lysosome fusion or degranulation. This protein was localized to the cytoplasm of unstimulated neutrophils and found to account for approximately 1% of the cytosol protein. Amino acid sequence of several peptides derived from the purified protein revealed that it is identical to lipocortin III, a recently described member of the annexin family that is scarce in other cells and tissues. The abundance of this protein, together with its Ca2(+)-dependent membrane effects, suggest that it mediates membrane-localized events in stimulated neutrophils, such as phagosome-lysosome fusion or degranulation.

Use of a novel strategy for the preparation and characterization of an antipeptide antibody capable of recognizing members of the annexin family.

Annexins are structurally-related proteins which bind phospholipids in a Ca2+-dependent manner. We have used a novel coupling strategy to prepare an antiserum directed against a 17-amino acid synthetic peptide that resembles the sequence of a highly-conserved portion of these proteins. This antipeptide serum specifically recognizes 5 of 6 human annexins on Western blots, despite differences between the protein and peptide sequences of 3 or 4 amino acids. The antiserum does not recognize endonexin II, whose sequence differs from that of the peptide by 6 amino acids. The availability of multiple proteins with known amino acid sequence has allowed analysis of structural requirements for recognition by this antibody. In some situations, use of such an antibody may allow the identification of a protein as a member of a family.

Lp(a) phenotyping by immunoblotting with polyclonal and monoclonal antibodies.

A new method that allows rapid phenotyping of genetic Lp(a) glycoprotein types in large numbers of samples is described. The method is based on sodium dodecyl sulfate gel electrophoresis of reduced serum or plasma in horizontal slab gels followed by immunoblotting with polyclonal anti-Lp(a) lipoprotein or monoclonal anti-Lp(a) glycoprotein antibodies. Phenotyping of 194 unrelated, healthy subjects resulted in Lp(a) allele frequencies of Lp(a)B = 0.013, Lp(a)S1 = 0.032, Lp(a)S2 = 0.106, Lp(a)S3 = 0.096, Lp(a)S4 = 0.156, and Lp(a)O = 0.600, and confirmed the recently recognized association of Lp(a) glycoprotein phenotype with Lp(a) lipoprotein concentration. The new procedure is suitable for large-scale population, genetic, and epidemiologic studies and may be important for atherosclerotic risk assessment.

Comparison of experimental and calculated relative sensitivity coefficients for spark-source mass-spectrometry of metals.

Sensitivity calibration has been performed for the spark-source mass-spectrometric analysis of iron, copper and aluminium matrices, with standard reference materials. The experimental relative sensitivity coefficients, corrected for discrimination effects in the mass spectrometer, are compared with values obtained with various empirical approaches to calculate relative sensitivity coefficients for an r.f. spark. The best correlation found is only of the order of 50%.

Sensitivity calibration for the spark-source mass-spectrometric analysis of aluminium and its alloys.

The analysis of aluminium and its alloys has been studied with use of five standards from Johnson Matthey and ten aluminium alloys from Aluminium Pechiney. The relative sensitivity coefficients for Mg, Al, Si, Ti, Cr, Mn, Ni, Cu, Zn, Sn and Pb were determined vs. iron as an internal standard. They were obtained, by using electrical detection, with a mean precision of 10% relative standard deviation. The sensitivity coefficients measured appear to be independent of the elemental concentration, but for some elements, especially the more volatile ones, remarkable changes were noticed when sparking conditions were altered and when the electrode temperature was changed by cooling with liquid nitrogen.

Mass-spectrometric multielement analysis of copper and copper-base alloys, with electrical detection.

Relative sensitivity coefficients have been determined for 21 elements in copper and its alloys, with iron as an internal standard, by spark-source mass-spectrometry with electrical detection and magnetic peak-switching. Twenty calibration standards ranging from pure copper to 60%-copper alloys were used. The sensitivity coefficients measured appear to be independent of the elemental concentration and are obtained with a mean precision of 15%.

Accuracy of analysis of steels by use of a spark-source mass spectrometer with electrical detection.

The precision and accuracy of spark-source mass spectrometry with electrical detection has been studied, with five steel standard reference materials (NBS-SRM 661-665). Two different modes of analysis have been evaluated, magnetic scanning with electrical detection of the individual ions in sequence, using the total ion-current as reference, and magnetic switching between masses, with current integration. Measurements of isotope abundances have been used to evaluate the precision. The relative sensitivity coefficients of Ti, V, Cr, Mn, Co, Ni, Cu, As, Zr, Nb, Mo, Sb, La, Ta and W have been determined vs. iron as an internal standard. The accuracy of analyses based on these experimentally measured relative sensitivity coefficients was confirmed by comparing the results for a pure iron sample with those obtained by neutron-activation analysis.

Relative sensitivity coefficients for the analysis of steel by spark-source mass-spectrometry.

Quantitative analysis by spark-source mass-spectrometry requires the knowledge of socalled sensitivity coefficients for the elements being determined. Five series of analyses have been carried out on five different steel standard reference materials (NBS-SRM 661-665), using photoplate detection. The relative sensitivity coefficients (S(R)) of Ti, V, Cr, Mn, Co, Ni, Cu, As, Zr, Nb, Mo, Sn, Sb, La, Ta and W were determined vs. iron as an internal standard. The S(R) values were independent of the elemental concentration. A relative standard deviation of about 15% was obtained. The accuracy as confirmed by comparing the results for a pure iron sample with those obtained by neutron-activation analysis was within the same limits.