Instrument for determining the complex shear modulus of soft-tissue-like materials from 10 to 300 Hz.

Accurate determination of the complex shear modulus of soft tissues and soft-tissue-like materials in the 10-300 Hz frequency range is very important to researchers in MR elastography and acoustic radiation force impulse (ARFI) imaging. A variety of instruments for making such measurements has been reported, but none of them is easily reproduced, and none have been tested to conform to causality via the Kramers-Kronig (K-K) relations. A promising linear oscillation instrument described in a previous brief report operates between 20 and 160 Hz, but results were not tested for conformity to the K-K relations. We have produced a similar instrument with our own version of the electronic components and have also accounted for instrumental effects on the data reduction, which is not addressed in the previous report. The improved instrument has been shown to conform to an accurate approximation of the K-K relations over the 10-300 Hz range. The K-K approximation is based on the Weichert mechanical circuit model. We also found that the sample thickness must be small enough to obtain agreement with a calibrated commercial rheometer. A complete description of the improved instrument is given, facilitating replication in other labs.

Hematocrit change in tropical scuba divers.

OBJECTIVE: Direct evidence that dehydration results from scuba diving is scanty. Increased hematocrit (Ht) is a commonly used proxy measure for dehydration. This study sought evidence that an increase in Ht occurs over the course of a scuba dive in tropical conditions. As a secondary outcome, evidence was sought that the degree of Ht increase is correlated to pressure exposure. METHODS: Twenty male and 21 female scuba divers were recruited at a remote tropical dive site. Water temperature was 30 degrees C (+/- 1 degrees C). Each diver gave venous blood relating to 1 dive only. Mean maximum dive depth was 13.6 m (+/- 3.7 m [SD]) and mean duration 39.5 minutes (+/- 4.5 minutes [SD]) using air as the breathing gas. Blood was taken at a mean of 12.4 minutes (+/- 3.5 minutes [SD]) before diving and a mean of 16.2 minutes (+/- 3.7 minutes [SD]) after diving. After centrifugation of microcapillaries, Ht was estimated on a visual plate reader. RESULTS: A paired Wilcoxon test showed evidence (P < .001) for a change in Ht. The mean difference between predive and postdive measurements was 0.0073 (95% confidence interval: 0.0104-0.0042), equating to a mean relative Ht increase of 1.78%. Similar results were found for the sexes individually. A correlation between maximum depth of dive and Ht increase was statistically significant, although the correlation itself was weak (P = .049, Spearman's r = .326). CONCLUSIONS: There is evidence of a statistically significant increase in Ht over the course of a single warm-water scuba dive. This increase is small and is within the range of error associated with the techniques of Ht estimation employed in this study. Depth exposure was found to correlate with Ht increase. In view of the small magnitude of change in the Ht, there is no reason to amend protocols for fluid resuscitation of recreational scuba divers suspected to have experienced decompression injury in tropical locations.

Temperature dependence of ultrasonic propagation speed and attenuation in excised canine liver tissue measured using transmitted and reflected pulses.

Previous reported data from our laboratory demonstrated the temperature dependence of propagation speed and attenuation of canine tissue in vitro at discrete temperatures ranging from 25 to 95 degrees C. However, concerns were raised regarding heating the same tissue specimen over the entire temperature range, a process that may introduce irreversible and, presumably, cumulative tissue degradation. In this paper propagation speed and attenuation vs temperature are measured using multiple groups of samples, each group heated to a different temperature. Sample thicknesses are measured directly using a technique that uses both transmitted and reflected ultrasound pulses. Results obtained using 3 and 5 MHz center frequencies demonstrate a propagation speed elevation of around 20 m/s in the 22-60 degrees C range, and a decrease of 15 m/s in the 60-90 degrees C range, in agreement with previous results where the same specimens were subjected to the entire temperature range. However, sound speed results reported here are slightly higher than those reported previously, probably due to more accurate measurements of sample thickness in the present experiments. Results also demonstrate that while the propagation speed varies with temperature, it is not a function of tissue coagulation. In contrast, the attenuation coefficient depends on both tissue coagulation effects and temperature elevation.

Tissue-mimicking oil-in-gelatin dispersions for use in heterogeneous elastography phantoms.

A ten-month study is presented of materials for use in heterogeneous elastography phantoms. The materials consist of gelatin with or without a suspension of microscopic safflower oil droplets. The highest volume percent of oil in the materials is 50%. Thimerosal acts as a preservative. The greater the safflower oil concentration, the lower the Young's modulus. Elastographic data for heterogeneous phantoms, in which the only variable is safflower oil concentration, demonstrate stability of inclusion geometry and elastic strain contrast. Young's modulus ratios (elastic contrasts) producible in a heterogeneous phantom are as high as 2.7. The phantoms are particularly useful for ultrasound elastography. They can also be employed in MR elastography, although the highest achievable ratio of longitudinal to transverse relaxation times is considerably less than is the case for soft tissues.

Nutritionally induced adipose hypertrophy in young pigs is transient and independent of changes in the expression of the obese and peroxisome proliferator activated receptor genes.

Previous studies have shown that piglets weaned to a liquid milk replacer (MR), rather than a typical dry diet (DD) regimen, have improved growth rates and deposit more energy as body fat. In the present study, we used this model to determine whether changes in the expression of genes linked to the regulation of adiposity were related to the accelerated fat accretion. We also determined whether the increase in body fat was sustained throughout a substantial proportion of the growth curve. At weaning (19 plus minus 2 days of age), 96 piglets were placed in 12 replicate pens per diet (4 pigs per pen, 2 barrows and 2 gilts), and fed a liquid MR or conventional DD regimen for 5 weeks. Thereafter, 6 barrows and 6 gilts pigs from each diet were killed for determination of whole body chemical composition (less gastrointestinal contents). The remaining pigs were assigned randomly to weight target groups (60, 85, and 110 kg), placed in individual pens, and fed a conventional dietary regimen until killed at their respective weight targets for tissue sampling and determination of whole body chemical composition. Over the 5-week period in which the MR was fed, the growth rate of the pigs consuming the MR exceeded that of the pigs fed the DD by 36% (P <.05). Fat gain in these pigs was increased to 1.8 times that of the pigs fed the DD, and percentage body fat was 45% greater (P <.05). Acetyl Co-A carboxylase (ACC) activity (per mg of adipose extract protein) was not different between the two diet groups at the conclusion of the 5-week period, or at 110 kg body weight. During the MR period, actual protein gain was increased (P <.05) 22% in the pigs fed the MR as well. By 110 kg of body weight, body fat was reduced (P <.05) by 7.7% (total fat mass) and 8.3% (percentage of body weight basis) in the pigs fed MR vs. the DD group. The expression of the peroxisome proliferator activated receptors (PPAR) alpha and gamma was not influenced by diet or by body weight. Expression of the obese gene was independent of diet, but was greater (P <.09) in pigs at 110 kg body weight than at 60 kg. These data provide additional evidence that piglets weaned to liquid diets have greater rates of growth and deposit more body fat, but that this difference subsides quickly when a typical dry dietary regimen is imposed. Furthermore, the biochemical changes responsible for the increased adiposity are independent of changes in the expression of the obese or PPAR genes, at least at the mRNA level.

Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay.

Canine infections with Leishmania infantum are important as a cause of serious disease in the dog and as a reservoir for human visceral leishmaniasis (VL). Accurate diagnosis of canine infections is essential to the veterinary community and for VL surveillance programs. A standardized ELISA using a purified recombinant antigen (rK39) specific to VL was compared to the immunofluorescent antibody test (IFAT) as the standard. The ELISA was developed, optimized and evaluated using sera from 6368 dogs. The standardized ELISA and IFAT results were highly concordant. The timing and pattern of ELISA and IFAT seroconversion in dogs followed prospectively after natural infections were very similar. Antibodies reacting with rK39 were more common in asymptomatic canine infections than reported for subclinical human VL. The rK39 ELISA is a relatively simple and rapid assay for assessing the infection status of dogs, and is an alternative to IFAT, especially when screening large numbers of samples.

Low-reflection-coefficient liquid interfaces for system characterization.

The use of liquid brominated hydrocarbons to form a planar reflecting interface with water is described. Gravity-based planar reflecting surfaces with known reflection coefficients can be used in system characterization for quantitative ultrasonics, and a set of surfaces with a range of reflection coefficients allows calibration of the output power and receiver gain of ultrasonic imaging systems. The substances reported here are immiscible in water and form interfaces with water, resulting in a broad range of acoustic reflection coefficients. Reflection coefficients were measured at temperatures from 18-24 degrees C for "pure" substances and for mixtures of two brominated hydrocarbons. Results show that reflection coefficients are weakly dependent on temperature and that, at a specific temperature, a significant range of arbitrarily small reflection coefficients is available, in the case of the mixtures, by the appropriate choice of weight-percents of the two brominated hydrocarbons.

Tissue mimicking materials for a multi-imaging modality prostate phantom.

Materials that simultaneously mimic soft tissue in vivo for magnetic resonance imaging (MRI), ultrasound (US), and computed tomography (CT) for use in a prostate phantom have been developed. Prostate and muscle mimicking materials contain water, agarose, lipid particles, protein, Cu++, EDTA, glass beads, and thimerosal (preservative). Fat was mimicked with safflower oil suffusing a random mesh (network) of polyurethane. Phantom material properties were measured at 22 degrees C. (22 degrees C is a typical room temperature at which phantoms are used.) The values of material properties should match, as well as possible, the values for tissues at body temperature, 37 degrees C. For MRI, the primary properties of interest are T1 and T2 relaxations times, for US they are the attenuation coefficient, propagation speed, and backscatter, and for CT, the x-ray attenuation. Considering the large number of parameters to be mimicked, rather good agreement was found with actual tissue values obtained from the literature. Using published values for prostate parenchyma, T1 and T2 at 37 degrees C and 40 MHz are estimated to be about 1,100 and 98 ms, respectively. The CT number for in vivo prostate is estimated to be 45 HU (Hounsfield units). The prostate mimicking material has a T1 of 937 ms and a T2 of 88 ms at 22 degrees C and 40 MHz; the propagation speed and attenuation coefficient slope are 1,540 m/s and 0.36 dB/cm/MHz, respectively, and the CT number of tissue mimicking prostate is 43 HU. Tissue mimicking (TM) muscle differs from TM prostate in the amount of dry weight agarose, Cu++, EDTA, and the quality and quantity of glass beads. The 18 microm glass beads used in TM muscle increase US backscatter and US attenuation; the presence of the beads also has some effect on T1 but no effect on T2. The composition of tissue-mimicking materials developed is such that different versions can be placed in direct contact with one another in a phantom with no long term change in US, MRI, or CT properties. Thus, anthropomorphic phantoms can be constructed.

Effective growth hormone therapy in a growth hormone deficient patient with Duchenne muscular dystropy without evidence of acceleration of the dystrophic process.

We describe a patient with Duchenne muscular dystropy and growth hormone deficiency in whom treatment with human growth hormone for 2 years resulted in improved growth velocity without any detrimental effect on muscle strength.

Changes in the expression of uncoupling proteins and lipases in porcine adipose tissue and skeletal muscle during feed deprivation*(1).

The hormone-sensitive and lipoprotein lipases are critical determinants of the metabolic adaptation to starvation. Additionally, the uncoupling proteins have emerged with potential roles in the metabolic adaptations required by energy deficiency. The objective of this study was to evaluate the expression (mRNA abundance) of uncoupling proteins 2 and 3 and that of hormone-sensitive and lipoprotein lipase in the adipose tissue and skeletal muscle of the pig in relationship to feed deprivation. Thirty-two male castrates (87 kg +/- 5%) were assigned at random to fed and feed-deprived treatment groups. After 96 hr, the pigs were euthanized and adipose and skeletal muscle tissue obtained for total RNA extraction and nuclease protection assays. Feed deprivation increased uncoupling protein 3 mRNA abundance 103-237% (P < 0.01) in longissimus and red and white semitendinosus muscle. In contrast, the increase in uncoupling protein 3 mRNA in adipose tissue was only 23% (P < 0.06), and adipose uncoupling protein 2 mRNA was not influenced (P > 0.66) by feed deprivation. The increased abundance of uncoupling protein 2 mRNA in the longissimus muscle of feed-deprived pigs was small (22%), but significant (P < 0.04). The expression of hormone-sensitive lipase was increased 46% and 64% (P < 0.04) in adipose tissue and longissimus muscle, respectively, by feed deprivation, whereas adipose lipoprotein lipase expression was reduced (P < 0.01) to 20% of that of the fed group. Longissimus lipoprotein lipase expression in the feed-deprived group was 37% of that of the fed group (P < 0.01), and similar reductions were detected in red and white semitendinosus muscle. Overall, these findings indicate that uncoupling protein 3 expression in skeletal muscle is quite sensitive to starvation in the pig, whereas uncoupling protein 2 changes are minimal. Furthermore, we conclude that hormone-sensitive lipase is upregulated at the mRNA level with prolonged feed deprivation, whereas lipoprotein lipase is downregulated.

Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1).

An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.

Interlaboratory comparison of ultrasonic backscatter, attenuation, and speed measurements.

In a study involving 10 different sites, independent results of measurements of ultrasonic properties on equivalent tissue-mimicking samples are reported and compared. The properties measured were propagation speed, attenuation coefficients, and backscatter coefficients. Reasonably good agreement exists for attenuation coefficients, but less satisfactory results were found for propagation speeds. As anticipated, agreement was not impressive in the case of backscatter coefficients. Results for four sites agreed rather well in both absolute values and frequency dependence, and results from other sites were lower by as much as an order of magnitude. The study is valuable for laboratories doing quantitative studies.

Soybean isoflavones, genistein and genistin, inhibit rat myoblast proliferation, fusion and myotube protein synthesis.

The isoflavones, genistein and genistin, are cytotoxic in vitro (e.g. , inhibition of cell proliferation), due in part to inhibition of protein tyrosine kinase and DNA topoisomerase activities. Normal cell functions associated with these enzymatic activities could potentially be impaired in animals through ingestion of soybean products. In this study, cultured rat myogenic cells (L8) were used to determine whether genistein or genistin influences myoblast proliferation and fusion, and myotube protein synthesis and degradation. Genistein or genistin was dissolved in dimethylsulfoxide and included in the culture medium at 0, 1, 10 or 100 micromol/L. Myoblast proliferation was measured by methyl-3H-thymidine incorporation over 48 h. Myoblast differentiation was evaluated by the number of nuclei in multinucleated myotubes. Myotube protein synthesis was measured by 2-h 3H-amino acid incorporation into the myosin and total protein pools after acute (2 h) or chronic (24 h) exposure to similar treatments; protein degradation was measured by measuring radioactivity in protein pools following a time course of protein breakdown after myotube proteins were prelabeled with 3H-amino acids. Genistein or genistin strongly inhibited in vitro myoblast proliferation (P < 0.001) and fusion (P < 0.001) in a dose-dependent manner with effective genistein concentration as low as 1 micromol/L. Genistein or genistin inhibited protein accretion in myotubes (P < 0.001). Decreased protein accretion is largely a result of inhibition on cellular (myofibrillar) protein synthesis rate. No adverse effect on protein degradation was observed. Results suggest that if sufficient circulating concentrations are reached in tissues of animals consuming soy products, genistein/genistin can potentially affect normal muscle growth and development.

Myostatin expression in porcine tissues: tissue specificity and developmental and postnatal regulation.

The objective of this study was to establish the developmental pattern and tissue specificity of porcine myostatin expression and to evaluate expression in skeletal muscle during circumstances in which muscle growth was altered. Northern blot analysis revealed two transcripts (1.5 and 0.8 kb). Myostatin mRNA was detected in whole fetuses at 21 and 35 days and was markedly increased (P < 0.05) by 49 days. At birth, mRNA abundance in longissimus muscle had declined significantly (P < 0.05) from that at day 105 of gestation and continued to decrease (P < 0.05) to its lowest level 2 wk postnatally (4 kg body wt). Myostatin expression was higher (P < 0. 05) at 55, 107, and 162 kg body wt than at 4 kg body wt. Postnatally, myostatin mRNA was detected in skeletal muscle and mammary gland. Expression at birth was 65% higher (P < 0.04) in longissimus muscle of low-birth-weight piglets (0.57 +/- 0.052 kg body wt) vs. normal (1.37 +/- 0.077 kg body wt) littermates, irrespective of gender. However, suppression of longissimus muscle growth by food deprivation (3 days) did not alter (P > 0.15) myostatin expression in either 4- or 7-wk-old piglets. Additionally, myostatin mRNA abundance was not changed by porcine growth hormone administration in growing animals. These data indicate that myostatin expression in skeletal muscle peaks prenatally and that greater expression is associated with low birth weight. Expression in mammary gland indicates a possible role for myostatin in mammary gland development and/or lactation.

Anthropomorphic 1H MRS head phantom.

An anthropomorphic 1H MRS head phantom has been developed which mimics the in vivo structure, metabolite concentrations, and relaxation times (for both water and metabolites) of human brain tissue. Different brain regions and two tumor types, fluid-containing ventricles, and air-filled sinus, and subcutaneous fat are all simulated. The main tissue-mimicking materials are gelatin/agar mixtures with metabolites and several other ingredients added. Their composition and method of production are thoroughly described. T1's and T2's of water in the phantom are very close to in vivo values, and metabolite T1's and T2's are considerably more realistic than those in aqueous solutions. Spectra and relaxation times for the pig brain were also acquired and compare well with those of the phantom. The realistic properties of this phantom should be useful for testing spectral quantitation and localization.

Liquid or solid ultrasonically tissue-mimicking materials with very low scatter.

A new tissue-mimicking material for ultrasound, using evaporated milk as the primary absorption component, is described. It has very low backscatter but still exhibits the 1540 m s-1 propagation speed and proportionality of attenuation coefficient and frequency over the diagnostic frequency range. The material can be produced in solid or liquid form with attenuation coefficient slopes spanning the range 0.1-0.7 dB cm-1 MHz-1. The liquid form is useful in phantoms where detailed beam patterns are to be determined, either involving translation of measurement devices in the liquid or phantoms with fibers present for causing the only detectable echoes. In the latter case, the liquid quality allows removal of liquid with one attenuation coefficient slope and replacement with another. The solid form may be more useful than the liquid for two reasons. First, many simulated lesions (including ones that produce essentially no internal echoes) can lie in the scan slice with positions extending over the entire image area without enhancement or shadowing effects being of concern. Second, the lack of significant backscatter from the material in the absence of added scatterers allows the backscatter coefficient to be varied over a considerable range. A critical result is that intrinsic material contrast between targets and surroundings can be accurately predicted in terms of the concentrations of added scatterers and, assuming all scatterers are of the same type, the contrast will be completely independent of frequency. Use of the fungicide thimerosal eliminates deterioration, and ultrasonic properties have been shown to be stable over 2.5 years.

Porcine somatotropin improves growth in finishing pigs without altering calpain 3 (p94) or alpha-actin mRNA abundance and has a differential effect on calpastatin transcription products.

The objective of this study was to determine whether the improvements in growth and efficiency of gain achieved by recombinant porcine somatotropin (pST) are associated with altered expression of the p94, calpastatin, or alpha-actin genes in porcine longissimus (LD) muscle. Forty-eight barrows (initial 64.2 to 67.4 kg BW) were assigned to four treatments (n = 12) arranged as a 2 x 2 factorial in a randomized complete block design. Factors were duration of treatment (3 or 6 wk) and pST administration (0 or 3 mg x pig(-1) x d(-1)). Plasma samples were obtained 24 h after the first pST injection and at the end of the each treatment period for assays of selected variables. The LD samples were obtained at 3 and 6 wk of pST treatment. Northern blot analysis of calpastatin expression in LD muscle revealed three distinct transcription products of approximately 8.5 (CPST I), 5.5 (CPST II), and 2.5 (CPST III) kb; CPST II was reduced (P < .02) 33 and 61% by pST at 3 and 6 wk, respectively, whereas CPST I and III were not influenced (P > .12). Neither alpha-actin nor p94 was responsive to pST injection. As expected, pST resulted in higher (50%, P < .02) plasma insulin within 24 h and one- and twofold higher (P < .01) concentrations at 3 and 6 wk, respectively. Glucose was increased (P < .01) at 3 (15%) and 6 (10%) wk, whereas urea nitrogen was reduced (32 to 36%, P < .01). The efficacy of pST was evident in that ADG was improved (P < .01) 11 to 13% independent of time. Likewise, feed intake was reduced (P < .01) 10 to 11% and gain: feed improved (P < .01) approximately 26% for pigs receiving pST independent of time. These data indicate that the enhanced muscle growth achieved by pST is not associated with altered expression of p94 or alpha-actin, or an increase in the abundance of any calpastatin transcription product.