3-dimensional imaging modalities for phenotyping genetically engineered mice.

A variety of 3-dimensional (3D) digital imaging modalities are available for whole-body assessment of genetically engineered mice: magnetic resonance microscopy (MRM), X-ray microcomputed tomography (microCT), optical projection tomography (OPT), episcopic and cryoimaging, and ultrasound biomicroscopy (UBM). Embryo and adult mouse phenotyping can be accomplished at microscopy or near microscopy spatial resolutions using these modalities. MRM and microCT are particularly well-suited for evaluating structural information at the organ level, whereas episcopic and OPT imaging provide structural and functional information from molecular fluorescence imaging at the cellular level. UBM can be used to monitor embryonic development longitudinally in utero. Specimens are not significantly altered during preparation, and structures can be viewed in their native orientations. Technologies for rapid automated data acquisition and high-throughput phenotyping have been developed and continually improve as this exciting field evolves.

Seasonal and sex-specific mRNA levels of key endocrine genes in adult yellow perch (Perca flavescens) from Lake Erie.

To better understand the endocrine mechanisms that underlie sexually dimorphic growth (females grow faster) in yellow perch (Perca flavescens), real-time quantitative polymerase chain reaction (qPCR) was used to measure pituitary, liver, and ovary mRNA levels of genes related to growth and reproduction-sex in this species. Adult perch were collected from Lake Erie and body mass, age, gonadosomatic index (I (G)), hepatosomatic index (I (H)), and gene expression for growth hormone (GH), prolactin, somatolactin, insulin-like growth factor Ib (IGF-Ib), estrogen receptor alpha (esr1), estrogen receptor betaa (esr2a), and aromatase (cyp19a1a) were measured. Females had higher body mass, I (H), and liver esr1 mRNA level than males, while males had higher liver IGF-Ib, liver esr2a, and liver cyp19a1a mRNA levels. In both sexes, season had a significant effect on GH and liver IGF-Ib mRNAs with higher levels occurring in spring, which also corresponded with higher liver cyp19a1a mRNA levels. For females, I (G), liver esr1, and ovary cyp19a1a mRNA levels were higher in autumn than the spring, and ovary cyp19a1a mRNA levels showed a significant negative correlation with pituitary GH and liver IGF-Ib mRNA levels. The most significant (p

Embryo technologies and animal health - consequences for the animal following ovum pick-up, in vitro embryo production and somatic cell nuclear transfer.

Mammalian reproductive technologies that aim either to complement or to transcend conventional livestock breeding options have contributed to some of the most remarkable achievements in the field of reproductive biology in recent decades. In so doing they have extended our horizons in two distinct dimensions, the first concerning what it is technically possible to achieve and the second relating to the time-frame within which an individual's life-long developmental capability is initially established and ultimately realized or undermined. Our impressions of the benefits and values, or otherwise, of technologies such as in vitro embryo production and nuclear transfer are rightly influenced by the extent to which they impinge on the health of animals either subjected to or derived from them. Here, we consider some of the health implications of oocyte/embryo-centric technologies applied to farm livestock.

Fatty acid esterification in the yolk sac membrane of the avian embryo.

The transfer of lipid from the yolk to the avian embryo is mediated by the yolk sac membrane (YSM). Some, but not all, of the published morphological evidence supports the view that the lipid undergoes a cycle of hydrolysis and re-esterification during translocation across the YSM. The present study aims to test this view by investigating the capacity of the YSM to perform esterification of free fatty acids to form acyl-lipids. YSM pieces (area vasculosa), obtained from the chicken embryo at day 10 of development, were incubated in vitro in medium containing [14C]-palmitic acid. Radioactivity was rapidly incorporated into the tissue lipid indicating a high capacity for esterification. The incorporation was linear with time during the 1-h incubation. Approximately 84% of the incorporated label was recovered in triacylglycerol, 12% was incorporated into phospholipid and less than 1% was detected in cholesteryl ester. [14C]-palmitic acid was incorporated primarily at the sn-1/3 positions in the triacylglycerol molecule and at the sn-1 position of phospholipid. The incorporation of label into tissue pieces obtained from the non-vascularized peripheral region of the YSM (area vitellina) was much more limited than that observed for the area vasculosa. The results support the hypothesis that yolk lipid is hydrolyzed and re-esterified during transfer across the YSM.

Clinical validation of an automated boundary tracking algorithm on cardiac MR images.

The goal of this research was to develop an automated algorithm for tracking the borders of the left ventricle (LV) in a cine-MRI gradient-echo temporal data set. The algorithm was validated on four patient populations: healthy volunteers and patients with dilated cardiomyopathy (DCM), left ventricular hypertrophy (LVH), or left ventricular aneurysm (LVA). A full tomographic set (approximately 11 slices/case) of short-axis images through systole was obtained for each patient. Initial endocardial and epicardial contours for the end-diastolic (ED) and end-systolic (ES) frames were manually traced on the computer by an experienced radiologist. The ED tracings were used as the starting point for the algorithm. The borders were tracked through each phase of the temporal data set, until the ES frame was reached (approximately 7 phases/slice). Peak gradients along equally spaced chords calculated perpendicular to a centerline determined midway between the endocardial and epicardial borders were used for border detection. This approach was tested by comparing the LV epicardial and endocardial volumes calculated at ES to those based on the manual tracings. The results of the algorithm compared favorably with both the endocardial (r2 = 0.72 - 0.98) and epicardial (r2 = 0.96 - 0.99) volumes of the tracer.

Dynamin inhibits phosphatidylinositol 3-kinase in hematopoietic cells.

Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in late stages of endocytosis as well as in cellular proliferation and transformation. The SH3 domain of its regulatory p85 subunit stimulates the GTPase activity of dynamin in vitro. Dynamin is a GTPase enzyme required for endocytosis of activated growth factor receptors. An interaction between these proteins has not been demonstrated in vivo. Here, we report that dynamin associates with PI 3-kinase in hematopoietic cells. We detected both p85 and PI 3-kinase activity in dynamin immune complexes from IL-3-dependent BaF3 cells. However, this association was significantly reduced in BaF3 cells transformed with the BCR/abl oncogene. After transformation only a 4-fold increase in PI 3-kinase activity was detected in dynamin immune complexes, whereas grb2 associated activity was elevated 20-fold. Furthermore, dynamin inhibited the activity of both purified recombinant and immunoprecipitated PI 3-kinase. In BaF3 cells expressing a temperature-sensitive mutant of BCR/abl, a significant decrease in p85 and dynamin association was observed 4 h after the induction of BCR/abl activity. In contrast, in IL-3-stimulated parental BaF3 cells, this association was increased. Our results demonstrate an in vivo association of PI 3-kinase with dynamin and this interaction regulates the activity of PI 3-kinase.

Automatic segmentation of mammographic density.

RATIONALE AND OBJECTIVES: The purpose of this study was to evaluate a completely automatic method, based on Kittler's optimal threshold, to estimate breast density by using the mammographers' definition. MATERIALS AND METHODS: Thirty-two normal, right-craniocaudal-view mammograms of women aged 37-86 years were digitized. The whole breast area was segmented by using Kittler's optimal threshold procedure, and the dense portions were then segmented by using a modified version of Kittler's method. Segmentation results were validated by three independent mammographers who provided a signed percentage (in steps of 5%) to indicate the difference between their own visual estimation of the dense portions and the results obtained with the algorithm. The difference between the algorithm measurements and the mammographers' measurements was compared to the interobserver differences. RESULTS: A high correlation was found between the algorithm measured density and the mammographers' measurements. Spearman correlations ranged from 0.92 to 0.95 (P < .001). Algorithm-measured density differed from the mammographers' measurements by an average of 6.9% (ie, average of the absolute differences). In contrast, mammographers' measurements differed between themselves by an average of 5.4%. CONCLUSION: The difference between density as measured with the algorithm and as measured by the mammographers is similar to the differences observed between mammographers. This algorithm could be useful in providing clinically accurate estimates of breast density.

Sodium iodide symporter (NIS) gene expression in human placenta.

The placenta must allow the passage of iodide from the maternal to the fetal circulation for synthesis of thyroxine by the fetal thyroid. The thyroid sodium iodide symporter (NIS) was cloned in 1996 and, although widely distributed among epithelial tissues, early studies failed to detect it in placenta. We demonstrated NIS mRNA in human placenta and in the human choriocarcinoma cell line, JAr. NIS protein was localized to trophoblasts, with a tendency to apical distribution, in sections of human placenta immunostained with a monoclonal antibody against hNIS. We conclude that NIS is expressed in placenta and may mediate placental iodide transport.

Regional heterogeneity of function in nonischemic dilated cardiomyopathy.

OBJECTIVE: To quantify regional three-dimensional (3D) motion and myocardial strain using magnetic resonance (MR) tissue tagging in patients with non-ischemic dilated cardiomyopathy (DCM). METHODS: MR grid tagged images were obtained in multiple short- and long-axis planes in thirteen DCM patients. Regional 3D displacements and strains were calculated with the aid of a finite element model. Five of the patients were also imaged after LV volume reduction by partial left ventriculectomy (PLV), combined with mitral and tricuspid valve repair. RESULTS: DCM patients showed consistent, marked regional heterogeneity. Systolic lengthening occurred in the septum in both circumferential (%S(C) -5+/-7%) and longitudinal (%S(L) -2+/-5%) shortening components (negative values indicating lengthening). In contrast, the lateral wall showed relatively normal systolic shortening (%S(C) 12+/-6% and %S(L) 6+/-5%, P<0.001 lateral vs. septal walls). A geometric estimate of regional stress was correlated with shortening on a regional basis, but could not account for the differences in shortening between regions. In the five patients imaged post-PLV, septal function recovered (%S(C) 9+/-5%,%S(L) 6+/-5%, P<0.02 pre vs. post) with normalization of wall stress, whereas lateral wall shortening was reduced (%S(C) 7+/-6%,%S(L) 3+/-3%, P<0.02 pre vs. post) around the site of surgical resection. CONCLUSIONS: A consistent pattern of regional heterogeneity of myocardial strain was seen in all patients. Reduced function may be related to increased wall stress, since recovery of septal function is possible after PLV. However, simple geometric stress determinants are not sufficient to explain the functional heterogeneity observed.

Different transporters for tri-iodothyronine (T(3)) and thyroxine (T(4)) in the human choriocarcinoma cell line, JAR.

We investigated transport systems for tri-iodothyronine (T(3)) and thyroxine (T(4)) in the human choriocarcinoma cell line, JAR, using a range of structurally similar compounds to determine whether these thyroid hormones are transported by common or different mechanisms. Saturable T(3) but not saturable T(4) uptake was inhibited by a wide range of aromatic compounds (nitrendipine, nifedipine, verapamil, meclofenamic acid, mefenamic acid, diazepam, phenytoin). Nitrendipine and diazepam were the most effective inhibitors of saturable thyroid hormone uptake. Nitrendipine decreased the K(m) for T(4) uptake from a control value of around 500 nM to around 300 nM (n=6). In contrast, the K(m) for T(3) uptake was increased from a control value of around 300 nM to around 750 nM (n=4). Diazepam had similar effects. This divergent shift in affinity for the uptake of T(3) and T(4) suggested that separate uptake systems exist for these two thyroid hormones. This provides evidence for at least two transporters mediating uptake of T(3) and T(4) in JAR cells: a specific T(4) transporter that does not interact with T(3) or structurally similar compounds; and a shared iodothyronine transporter that interacts with T(3), T(4), nitrendipine and diazepam.

Can critically timed sleep deprivation be useful in pregnancy and postpartum depressions?

BACKGROUND: The aim of this study was to test the efficacy of critically timed sleep deprivation in major mood disorders (MMD) occurring during pregnancy and postpartum. METHODS: Nine women who met DSM-IV criteria for a MMD with onset during pregnancy or within 1 year postpartum underwent a trial of either early-night sleep deprivation (ESD), in which they were sleep deprived in the early part of one night and slept from 03:00-07:00 h, or late-night sleep deprivation (LSD), in which they were deprived of sleep in the latter part of one night and slept from 21:00-01:00 h. Mood was assessed before the night of sleep deprivation, after the night of sleep deprivation, and after a night of recovery sleep (sleep 22:30-06:30 h) by trained clinicians, blind to treatment condition, using standardized scales. RESULTS: More patients responded to LSD (nine of 11 trials: 82%) compared with ESD (two of six trials: 33%) and they responded more after a night of recovery sleep (nine of 11 nights: 82%) than after a night of sleep deprivation (six of 11 nights: 55%). Pregnant women were the only responders to ESD and the only nonresponders to LSD. LIMITATIONS: The small and heterogeneous sample size prevents us from making more definitive conclusions based on statistical analyses. CONCLUSIONS: Although the findings are preliminary, the results suggest that with further study, critically timed sleep deprivation interventions may benefit women with pregnancy or postpartum major mood disorders and potentially provide a viable alternative treatment modality for those women who are not candidates for pharmacologic or psychotherapeutic interventions. Such interventions are needed to help prevent the devastating effects of depression during pregnancy and the postpartum period on the mother, infant, her family and society.

Data analysis for detection and localization of multiple abnormalities with application to mammography.

RATIONALE AND OBJECTIVES: In assessing diagnostic accuracy it is often essential to determine the reader's ability both to detect and to correctly locate multiple abnormalities per patient. The authors developed a new approach for the detection and localization of multiple abnormalities and compared it with other approaches. MATERIALS AND METHODS: The new approach involves partitioning the image into multiple regions of interest (ROIs). The reader assigns a confidence score to each ROI. Statistical methods for clustered data are used to assess and compare reader accuracy. The authors applied this new method to a reader-performance study of conventional film images and digitized images used to detect and locate malignant breast cancer lesions. RESULTS: The ROI-based approach, the free-response receiver operating characteristic (FROC) curve, and the patient-based approach handle the estimation of the false-positive rate (FPR) quite differently. These differences affect the measures of the respective areas under the curves. In the ROI-based approach the denominator is the number of ROIs without a malignant lesion. In the FROC approach the average number of false-positive findings per patient is plotted on the x axis of the curve. In contrast, the patient-based approach mishandles the FPR by ignoring multiple detection and/or localization errors in the same patient. The FROC approach does not lend itself easily to statistical evaluations. CONCLUSION: The ROI-based approach appropriately captures both the detection and localization tasks. The interpretation of the ROI-based accuracy measures is simple and clinically relevant. There are statistical methods for estimating and comparing ROI-based estimates of accuracy.

Comparing the performance of mammographic enhancement algorithms: a preference study.

OBJECTIVE: The objective of this study was to compare the performance of four image enhancement algorithms on secondarily digitized (i.e., digitized from film) mammograms containing masses and microcalcifications of known pathology in a clinical soft-copy display setting. MATERIALS AND METHODS: Four different image processing algorithms (adaptive unsharp masking, contrast-limited adaptive histogram equalization, adaptive neighborhood contrast enhancement, and wavelet-based enhancement) were applied to one image of secondarily digitized mammograms of forty cases (10 each of benign and malignant masses and 10 each of benign and malignant microcalcifications). The four enhanced images and the one unenhanced image were displayed randomly across three high-resolution monitors. Four expert mammographers ranked the unenhanced and the four enhanced images from 1 (best) to 5 (worst). RESULTS: For microcalcifications, the adaptive neighborhood contrast enhancement algorithm was the most preferred in 49% of the interpretations, the wavelet-based enhancement in 28%, and the unenhanced image in 13%. For masses, the unenhanced image was the most preferred in 58% of cases, followed by the unsharp masking algorithm (28%). CONCLUSION: Appropriate image enhancement improves the visibility of microcalcifications. Among the different algorithms, the adaptive neighborhood contrast enhancement algorithm was preferred most often. For masses, no significant improvement was observed with any of these image processing approaches compared with the unenhanced image. Different image processing approaches may need to be used, depending on the type of lesion. This study has implications for the practice of digital mammography.

Clinical evaluation of wavelet-compressed digitized screen-film mammography.

RATIONALE AND OBJECTIVES: The authors compared diagnostic accuracy and callback rates with conventional screen-film mammograms and wavelet-compressed digitized images. MATERIALS AND METHODS: Sixty sets of mammograms (four views per case) were digitized at a spatial resolution of 100 microm. The images were wavelet compressed to a mean compression ratio of 8:1 and reviewed by three mammographers. Five regions were evaluated in each breast. Suspicion of malignancy was graded on a scale of 0% to 100%, and receiver operating characteristic (ROC) analysis was performed. Callback rates were calculated by using the American College of Radiology's Breast Imaging Reporting and Data System lexicon scale. RESULTS: The mean diagnostic accuracy with compressed and conventional images was 0.832 and 0.860, respectively. The upper 95% confidence bound for the difference in ROC areas was 0.061. The mean false-positive rate at a fixed sensitivity of 0.90 was 0.041 for compressed images and 0.059 for conventional images. The mean callback rates for normal, benign, and malignant regions were 0.023, 0.305, and 0.677, respectively, for compressed images and 0.036, 0.447, and 0.750, respectively, for conventional images. The upper 95% confidence bound for the (absolute) differences in callback rates was 0.012 for normal regions, 0.163 for benign regions, and 0.138 for malignant regions. CONCLUSION: Diagnostic accuracies were equivalent for both compressed and conventional images. The mean false-positive rate at fixed sensitivity was much better with the compressed images. However, the callback rates for malignant lesions were lower when the compressed images were used.

Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids.

Dynamin I is phosphorylated in nerve terminals exclusively in the cytosolic compartment and in vitro by protein kinase C (PKC). Dephosphorylation is required for synaptic vesicle retrieval, suggesting that its phosphorylation affects its subcellular localization. An in vitro phospholipid binding assay was established that prevents lipid vesiculation and dynamin lipid insertion into the lipid. Dynamin I bound the phospholipid in a concentration-dependent and saturable manner, with an apparent affinity of 230 +/- 51 nM. Optimal binding occurred with mixtures of phosphatidylserine and phosphatidylcholine of 1:3 with little binding to phosphatidylcholine or phosphatidylserine alone. Phospholipid binding was abolished after dynamin I phosphorylation by PKC and was restored after dephosphorylation by calcineurin. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry revealed the phosphorylation site in PKCalpha-phosphorylated dynamin I as a single site at Ser-795, located near a binding site for the SH3 domain of p85, the regulatory subunit of phosphatidylinositol 3-kinase. However, phosphorylation had no effect on dynamin binding to a bacterially expressed p85-SH3 domain. Thus, phosphorylation of dynamin I on Ser-795 prevents its association with phospholipid, providing a basis for the cytosolic localization of the minor pool of phospho-dynamin I that mediates synaptic vesicle retrieval in nerve terminals.

Trafficking of Glut4-green fluorescent protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface glut4 levels.

Insulin stimulates glucose transport in adipose and muscle tissue by stimulating the movement ('translocation') of an intracellular pool of glucose transporters (the Glut4 isoform) to the plasma membrane. We have engineered a series of chimaeras between Glut4 and green fluorescent protein (GFP) from Aequoria victoria and expressed these proteins in 3T3-L1 adipocytes by microinjection of plasmid cDNA. In the absence of insulin, GFP-Glut4 is localized intracellularly within a perinuclear compartment and multiple intracellular punctate structures. In response to insulin, chimaeric GFP-Glut4 species exhibit a profound redistribution to the cell surface with kinetics comparable with the endogenous protein. The intracellular localization of GFP-Glut4 overlaps partially with compartments labelled with Texas Red transferrin, but is largely distinct from intracellular structures identified using Lysotracker-Red(R). K(+)-depletion resulted in the accumulation of GFP-Glut4 at the cell surface, but to an lesser extent than that observed in response to insulin. In contrast with native Glut4, removal of the insulin stimulus or treatment of insulin-stimulated cells with phosphatidylinositol 3'-kinase inhibitors did not result in re-internalization of the chimaeric GFP-Glut4 from the plasma membrane, suggesting that the recycling properties of this species differ from the native Glut4 molecule. We suggest that the recycling pathway utilized by GFP-Glut4 in the absence of insulin is distinct from that used to internalize GFP-Glut4 from the plasma membrane after withdrawal of the insulin stimulus, which may reflect distinct pathways for internalization of endogenous Glut4 in the presence or absence of insulin.

Film-screen versus digitized mammography: assessment of clinical equivalence.

OBJECTIVE: The purpose of this study was to determine whether diagnostic accuracy and callback rates using digitized film images are equivalent to those using film-screen mammograms. MATERIALS AND METHODS: Sixty sets of mammograms (four views per case) were digitized at a spatial resolution of 100 microm. The images were reviewed by seven mammographers. Five regions were evaluated in each breast. Each region was scored on a scale of 0 100% for suspicion of malignancy, and a receiver operating characteristic analysis was performed. Callback rates were calculated using a published lexicon scale. RESULTS: The observers' mean diagnostic accuracies using films and digitized images were 0.872 and 0.848, respectively. The upper 95% confidence boundary on the difference in accuracy was 0.066. The mean callback rate for normal, benign, and malignant areas using films versus digitized images was 0.048 versus 0.055, 0.498 versus 0.441, and 0.786 versus 0.737, respectively. The upper 95% confidence boundary for the absolute difference in callback rates was 0.037, 0.026, and 0.130 for normal, benign, and malignant areas, respectively. CONCLUSION: The diagnostic accuracies of the digitized images and films were similar; however, an increase in callback rates of 0.037 (i.e., upper 95% confidence boundary) for normal results and a reduction in the callback rates of 0.130 for malignant lesions is important. The use of digitized film images, at a spatial resolution of 100 microm, may compromise patient treatment in clinical practice.

Correlation of valve closing to visualization of single-leg separation in radiographic images of Björk-Shiley convexo-concave heart valves.

BACKGROUND AND AIM OF THE STUDY: The study goal was to determine whether the visualization of single-leg separation (SLS) in cineangiographic sequences of Björk-Shiley convexo-concave heart valves could be correlated to the position of the occluder disk within the cardiac cycle. METHODS: Images from ten patient cases with SLS valves were reviewed by three experts, who identified the image frames within a cine sequence that appeared suspicious for SLS. The position of the occluder disk, the frame rate, and the length of the cardiac cycle were noted relative to these image frames. RESULTS: The probability of detecting a SLS was not significantly correlated to any of these factors. CONCLUSIONS: Visualization of SLS in cineangiographic images is limited to a few frames within an imaging sequence. It appears that other features within the image play a larger role in a clinician's ability to detect a fracture than do the cardiac dynamics of the system.

The surface coat of infective larvae of Trichinella spiralis.

The surface coat of the infective larvae of the parasitic nematode Trichinella spiralis was characterized with respect to its biophysical properties, morphology and composition. Labelling of larvae with the fluorescent surface probe PKH26 was lost after activation (by incubation in mammalian medium containing trypsin and bile), or following pronase treatment. Electron microscopical examination revealed that pronase treatment resulted in the loss of an amorphous surface layer only, further demonstrating the specificity of PKH26 for the larval surface coat. Surface coat shedding was inhibited by sodium azide and carbonyl cyanide, or by incubation of larvae at 4 degrees C, suggesting the shedding process required metabolic energy. Pre-labelled, unactivated larvae demonstrated continuous slow surface coat shedding and could be re-labelled with PKH26, indicating that the shed coat is replaced in these parasites. However, pre-labelled larvae which were activated failed to re-label with the probe, suggesting that activation provides an irreversible trigger for surface changes. PKH26, therefore, is a useful marker for larval activation. Examination of the shed coat material by scanning electron microscopy revealed 2 types of morphologies; one comprising thin multilaminate sheets and the other of amorphous material with ridges producing a fingerprint-like motif. Western- and lectin-blotting of the shed coat material demonstrated 2 prominent entities; a 90 kDa glycoprotein, which bound Datura stramonium agglutinin and was resistant to N- and O-glycanase treatment and a 47-60 kDa set of protein(s). Analysis of the surface lipids by electrospray mass spectometry revealed the presence of lysophosphatidic acid (lysoPA, C14:2) and an unidentifiable component of 339.4 Da. These two lipids constituted 36.9% and 36% by mass of surface coat lipids respectively. The presence of lysoPA was confirmed by thin layer chromatography, which also detected phosphatidic acid (PA). The polar lipids detected in solvent rinses of intact parasites by electrospray mass spectrometry were PI (C48:4), PE (C40:4 and C38:4), PS (C40:4), lysoPC (C20:2 and C18:2) and lysoPA (C14:2). These observations are discussed with respect to the role of the surface coat and its shedding in the T. spiralis host-parasite relationship.