Experimental study of photon-counting CT neural network material decomposition under conditions of pulse pileup.

Purpose: We investigated the performance of a neural network (NN) material decomposition method under varying pileup conditions. Approach: Experiments were performed at tube current settings that provided count rates incident on the detector through air equal to 9%, 14%, 27%, 40%, and 54% of the maximum detector count rate. An NN was trained for each count-rate level using transmission measurements through known thicknesses of basis materials (PMMA and aluminum). The NN trained for each count-rate level was applied to x-ray transmission measurements through test materials and to CT data of a rod phantom. Material decomposition error was evaluated as the distance in basis material space between the estimated thicknesses and ground truth. Results: There was no clear trend between count-rate level and material decomposition error for all test materials except neoprene. As an example result, Teflon error was 0.33 cm at the 9% count-rate level and 0.12 cm at the 54% count-rate level for the x-ray transmission experiments. Decomposition error increased with count-rate level for the neoprene test case, with 0.65-cm error at 9% count-rate level and 1.14-cm error at the 54% count-rate level. In the CT study, material decomposition error decreased with increasing incident count rate. For example, the material decomposition error for Teflon was 0.089, 0.066, 0.054 at count-rate levels of 14%, 27%, and 40%, respectively. Conclusions: Results demonstrate over a range of incident count-rate levels that an NN trained at a specific count-rate level can learn the relationship between photon-counting spectral measurements and basis material thicknesses.

Bone Disease in the Common Marmoset: Radiographic and Histological Findings.

The common marmoset (Callithrix jacchus) is a New World primate that is used in biomedical research due to its small size and relative ease of handling compared with larger primates. Although bone disease in common marmosets is well recognized, there are very few detailed descriptions in the literature that cover the range of lesions seen in these animals. For all animals used to model human disease, it is important to be aware of background lesions that may affect the interpretation of study findings. This retrospective study details bone diseases encountered in marmoset breeding colonies at 2 different institutions. Affected marmosets at Johns Hopkins University had lesions compatible with diagnoses of rickets, fibrous osteodystrophy and osteopenia. Affected marmosets at the Wisconsin National Primate Research Center exhibited severe lesions of osteoclastic bone resorption and remodeling that had an unusual distribution and were not easily categorized into a known disease entity. The purpose of this report is to document these naturally occurring skeletal lesions of common marmosets and suggest an approach to evaluating skeletal disease in prospective studies of these animals that will allow the most accurate diagnoses.

Estrogen-dependent sushi domain containing 3 regulates cytoskeleton organization and migration in breast cancer cells.

Aromatase inhibitors (AIs) are the standard endocrine therapy for postmenopausal breast cancer; however, currently used biomarkers, such as, estrogen receptor-alpha/progesterone receptor (ERα/PR), predict only slightly more than half of the potential responders to AI treatment. To identify novel markers of AI responsiveness, a genome-wide microarray analysis was performed using primary breast tumor samples from 50 postmenopausal women who later developed metastatic breast cancer. Sushi domain containing 3 (SUSD3) is a significantly differentially expressed gene, with 3.38-fold higher mRNA levels in AI-responsive breast tumors vs non-responders (P<0.001). SUSD3 was highly expressed in ERα-positive breast tumors and treatment with estradiol increased SUSD3 expression in ERα-positive breast cancer cells. Treatment with an antiestrogen or ERα knockdown abolished basal and estradiol-dependent SUSD3 expression. Recruitment of ERα upstream of the transcription start site of SUSD3 was demonstrated by chromatin immunoprecipitation-PCR. Flow cytometric analysis of SUSD3-knockdown cells revealed blunted estradiol effects on progression into S and M phases. SUSD3 was localized to the plasma membrane of breast cancer cells. SUSD3 knockdown decreased the appearance of actin-rich protrusions, stress fibers and large basal focal adhesions, while increasing the presence of cortical actin concomitant with a decrease in Rho and focal adhesion kinase activity. SUSD3-deficient cells demonstrated diminished cell spreading, cell-cell adhesion and motility. In conclusion, SUSD3 is a novel promoter of estrogen-dependent cell proliferation and regulator of cell-cell and cell-substrate interactions and migration in breast cancer. It may serve as a novel predictor of response to endocrine therapy and potential therapeutic target.

Tissue mineral concentrations and osteochondrosis lesions in prolific sows across parities 0 through 7.

Mortality in swine herds is often associated with lameness, and trace minerals are implicated in maintaining integrity of skeletal tissues. The objectives of this study were to determine if prolific sows displayed evidence of trace mineral depletion with age and to determine the prevalence of osteochondrosis (OC) lesions. Reduced mineral concentrations with age would support recommendations for an increase in the amount of dietary minerals. Tissue samples were collected from 66 sows selected to represent a cross-sectional profile of a prolific herd fed diets with inorganic sources of trace minerals fortified at concentrations typically found in commercial diets. Females ranged from nulliparous (parity 0) to parity 7 with a lifetime average of 12.9 ± 0.5 pigs born alive per litter. Minerals were assessed in humerus, scapula, ovary, liver, and muscle (psoas major) tissues. Percent bone ash increased (P < 0.05) with parity from 64 to 66% but differed among bone sections. The Ca (39.0%) and P (18.9%) concentrations in bone ash were essentially constant in all sections and parities. Bone Cu, Fe, Mn, and Zn concentrations varied among sections, but differences due to parity (P < 0.05) were only detected in Fe. Bone Fe decreased from approximately 49 µg/g ash in parity 0 and 1 sows to approximately 29 µg/g ash in parity 7, likely reflecting loss of hemopoietic tissue with age. No evidence was detected in liver for depletion of trace minerals across parity; however, liver Cu and Zn concentrations tended to increase with age. Liver Mn concentrations varied with parity, but no consistent trend with parity was evident. Ovary Cu and Mn concentrations varied dramatically as a function of the reproductive status, but no evidence was detected for depletion with parity. Articular surfaces of the distal scapula and proximal and distal humerus were evaluated grossly for prevalence of OC; bones were then sectioned to evaluate lesions in subchondral bone and physis. Incidence of OC lesions on the articular-epiphyseal cartilage complex varied among bone sites, but differences across parities were not detected. In a subset of sows with subchondral bone lesions, the lesions appeared severe enough to contribute to clinical lameness, particularly in the distal humerus site. However, none of the sows exhibited lameness at slaughter. As no reductions in mineral concentrations with age were detected, recommendations to increase dietary mineral supplementation with age were not supported.

Effects of immunization against alpha-inhibin using two adjuvants on daily sperm production and hormone concentrations in ram lambs.

Twenty-five ram lambs were immunized against alpha-inhibin peptide emulsified in Freund's adjuvant (FRA), Emulsigen (EML) containing an oligodeoxynucleotide as an immunostimulant, or adjuvant without alpha-inhibin antigen (control). Four immunizations were administered during an 85-d period, after which testes were obtained for determination of daily sperm production (DSP) and histological evaluation. alpha-Inhibin antibody (Ab) titers were 70-fold greater in lambs treated with FRA than in EML-treated ram lambs. alpha-Inhibin immunization had no effect on testes weight or on plasma concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Mean DSP/g tended (P=0.1) to be greater in alpha-inhibin-immunized (EML=17.6x10(6); FRA=15.8x10(6)) ram lambs than in control animals (14.4x10(6)). One of the 8 control ram lambs had an elevated DSP/g, which was a statistical outlier. Without data from this lamb, DSP/g was increased (P<0.01) in alpha-inhibin-immunized ram lambs by 28% over controls. No association was found between the titer of alpha-inhibin Ab developed and DSP/g. Histologically, the percentage of testicular area occupied by seminiferous tubules differed (P=0.01) by treatment and was greatest (82%) in EML-treated ram alpha-inhibin-immunized lambs and lowest (74%) in control animals. Percentage tubular area and DSP/g were correlated (r=0.57, P=0.003). Findings show that (1) the extent of the increase in DSP/g is not dependent on the titer of alpha-inhibin Ab; (2) the increase in DSP/g is achieved through an increase in the mass of seminiferous tubules; and (3) FRA elicits a greater alpha-inhibin Ab titer than EML containing an oligodeoxynucleotide.

Bacterial diskospondylitis associated with posterior paresis/paralysis in North American farmed mink (Mustela vison).

Posterior paresis/paralysis in farmed mink is responsible for significant morbidity and mortality, with individual farms reporting the loss of as many as 700 animals each year. Although this disease has been recognized by North American mink farmers for approximately 40 years, there are few published reports focusing on this entity. The objective of this study was to investigate the etiology and pathogenesis of the disease. Complete necropsy examinations were done on 40 clinically affected mink, ranging from 7 to 10 weeks of age, and on three normal animals in the same age range from two mink farms. Thirty-two of the 40 clinically affected animals had an isolated vertebral lesion characterized by bone lysis and proliferation that usually was centered on an intervertebral disk space in the midthoracic area. An inflammatory reaction, composed primarily of neutrophils, was present within the vertebral sections in 25 of the 40 affected animals (62.5%), and the presence of gram-positive cocci was confirmed in 8 of 10 animals (80%) in which bacterial organisms were observed histologically. Bacterial cultures from 15 affected animals yielded Streptococcus sp. from the intervertebral disk space in 13 of 15 (86.7%) animals and from heart blood in 6 of 8 (75%). A farm visit revealed no history or evidence of traumatic wounds as a source of infection in these animals, and the diet appeared to be adequate for skeletal development. We conclude that posterior paresis/paralysis in farmed mink is associated with bacterial diskospondylitis, likely occurring secondary to bacteremia/septicemia.

An improved resazurin-based cytotoxicity assay for hepatic cells.

A simple resazurin-based cytotoxicity assay is presented for screening of cytotoxicity in hepatocytes and liver cell lines. Human hepatoma (HepG2) cells in 96-well culture plates were exposed to known toxic (cisplatin, 5-fluorouracil, ethionine, flufenamic acid, and diflunisal) and control (transplatin, 5-chlorouracil, methionine, and acetylsalicylic acid) compounds for 1-3 days, and resazurin (5 micromol/L) was added. A conventional short-term (1 h) assay was first performed, where cytotoxicity is indicated by decreased reduction of resazurin to its fluorescent product resorufin. Our improved assay consists of additionally measuring fluorescence 2-4 days later, when cytotoxicity is indicated by a striking increase in the concentration of resorufin, resulting from two distinct processes. First, viable liver-derived cells slowly convert resorufin to nonfluorescent metabolites. Fluorescence of control cell wells decreased to background during a 2- to 4-day exposure to resazurin. This metabolism of resorufin was largely blocked by dicumarol and to lesser extents by disulfiram and SKF525a. Second, dead or dying cells slowly convert resazurin to resorufin but do not further metabolize resorufin; thus this fluorescent metabolite accumulates to high levels in wells with dead cells by 2 to 4 days. A similar increase in fluorescence associated with cytotoxicity was observed in primary cultures of rat hepatocytes using the long-term resazurin-based assay. In addition to an improved signal relative to the short-term assay, the inversion of the fluorescent signal from high = alive short-term to high = dead long-term allows determination of two independent cytotoxicity endpoints after addition of one innocuous vital dye.

Cytochrome P450 induction in rat hepatocytes assessed by quantitative real-time reverse-transcription polymerase chain reaction and the RNA invasive cleavage assay.

The acceleration of drug discovery due to combinatorial chemistry and high-throughput screening methods has increased the numbers of candidate pharmaceuticals entering the drug development phase, and the capability to accurately predict whether drug candidates will induce various members of the drug-metabolizing cytochrome P450 (CYP) enzyme superfamily is currently of great interest to the pharmaceutical industry. In the present study, we describe the rapid and reliable analysis of CYP induction in a readily obtained model system (cultured rat hepatocytes) using both real-time quantitative reverse transcription-polymerase chain reaction (real-time RT-PCR) and the RNA invasive cleavage assay. The levels of members in the three primary inducible rat CYP subfamilies (CYP1A1, CYP2B1/2, and CYP3A1) were analyzed in untreated and induced (beta-naphthoflavone, phenobarbital, and hydrocortisone) hepatocyte cultures under various media conditions to screen for optimal CYP induction profiles. The fold inductions measured by real-time RT-PCR and the RNA invasive cleavage assay were also compared with enzyme activity measurements in parallel cultures using liquid chromatography/double mass spectrometry-based assays, and the sensitivity and the specificity of the two RNA analysis methods were compared. Using these techniques, various culture conditions were examined for optimizing induction of the three CYP subfamily members. Both real-time RT-PCR and the RNA invasive cleavage assay prove to be effective methods for determining the effects of drugs on specific CYPs in primary rat hepatocytes.

Nile Red binding to HepG2 cells: an improved assay for in vitro studies of hepatosteatosis.

Nile Red is a fluorescent dye used extensively to study fat accumulation in many types of cells; unfortunately protocols that work well for most cells are not effective for studying drug-induced lipid accumulation in cultured liver cells and hepatocyte-derived cell lines. Using human hepatoma (HepG2) cells, we have developed a simple Nile Red binding assay as a screen for steatosis-inducing compounds. Increases in Nile Red binding in response to known hepatotoxic compounds were observed after incubating treated cells with 1 microM Nile Red for several hours, washing away free Nile Red, and then allowing redistribution, and/or clearance of the lipid-indicator dye. Several compounds known to cause hepatic fat accumulation in vivo were examined and most robustly increased Nile Red binding in HepG2 cells. These include estrogen and other steroids, ethionine, cyclosporin A, and valproic acid. Required concentrations for increased Nile Red binding were generally three-fold or more lower than the cytotoxic concentration determined by a resazurin reduction assay in the same cells. Qualitatively similar Nile Red binding results were obtained when primary canine or rat hepatocytes were used. Morphological differences in Nile Red staining were observed by confocal fluorescence microscopy in HepG2 cells after treatment with different compounds and likely reflect distinct toxicological mechanisms.

Toxicogenomics-based discrimination of toxic mechanism in HepG2 human hepatoma cells.

The rapid discovery of sequence information from the Human Genome Project has exponentially increased the amount of data that can be retrieved from biomedical experiments. Gene expression profiling, through the use of microarray technology, is rapidly contributing to an improved understanding of global, coordinated cellular events in a variety of paradigms. In the field of toxicology, the potential application of toxicogenomics to indicate the toxicity of unknown compounds has been suggested but remains largely unsubstantiated to date. A major supposition of toxicogenomics is that global changes in the expression of individual mRNAs (i.e., the transcriptional responses of cells to toxicants) will be sufficiently distinct, robust, and reproducible to allow discrimination of toxicants from different classes. Definitive demonstration is still lacking for such specific "genetic fingerprints," as opposed to nonspecific general stress responses that may be indistinguishable between compounds and therefore not suitable as probes of toxic mechanisms. The present studies demonstrate a general application of toxicogenomics that distinguishes two mechanistically unrelated classes of toxicants (cytotoxic anti-inflammatory drugs and DNA-damaging agents) based solely upon a cluster-type analysis of genes differentially induced or repressed in cultured cells during exposure to these compounds. Initial comparisons of the expression patterns for 100 toxic compounds, using all approximately 250 genes on a DNA microarray ( approximately 2.5 million data points), failed to discriminate between toxicant classes. A major obstacle encountered in these studies was the lack of reproducible gene responses, presumably due to biological variability and technological limitations. Thus multiple replicate observations for the prototypical DNA damaging agent, cisplatin, and the non-steroidal anti-inflammatory drugs (NSAIDs) diflunisal and flufenamic acid were made, and a subset of genes yielding reproducible inductions/repressions was selected for comparison. Many of the "fingerprint genes" identified in these studies were consistent with previous observations reported in the literature (e. g., the well-characterized induction by cisplatin of p53-regulated transcripts such as p21(waf1/cip1) and PCNA [proliferating cell nuclear antigen]). These gene subsets not only discriminated among the three compounds in the learning set but also showed predictive value for the rest of the database ( approximately 100 compounds of various toxic mechanisms). Further refinement of the clustering strategy, using a computer-based optimization algorithm, yielded even better results and demonstrated that genes that ultimately best discriminated between DNA damage and NSAIDs were involved in such diverse processes as DNA repair, xenobiotic metabolism, transcriptional activation, structural maintenance, cell cycle control, signal transduction, and apoptosis. The determination of genes whose responses appropriately group and dissociate anti-inflammatory versus DNA-damaging agents provides an initial paradigm upon which to build for future, higher throughput-based identification of toxic compounds using gene expression patterns alone.

Effect of a fact sheet reminder on performance of the periodic health examination: a randomized controlled trial.

Physicians' performance of the periodic health examination is often incomplete. Performance rates may be low because physicians forget recommendations for specific periodic health examination components at the time of the patient encounter. We studied the effect of providing information about seven periodic health examination actions (cervical Pap smear, mammography, fecal occult blood testing, serum cholesterol level, and influenza, pneumococcal, and diphtheria-tetanus immunizations) on first-year medical residents' performance of these actions over a three-month period. We randomly selected 16 residents to receive periodic health examination recommendations, plus data supporting each recommendation, on their outpatient charts at the patient encounter. Thirteen residents who did not receive this information served as controls. Experimental and control groups achieved similar knowledge scores (0.53 versus 0.47, P = .48) and attitude scores (0.73 versus .078, P = .19) for preventive care measures on prestudy testing. The experimental group performed 10.5% of indicated periodic health examination actions, whereas the control group performed 5.8% of indicated actions (P = NS). These results suggest no clinically meaningful improvement in performance of periodic health examination actions, even when periodic health examination guidelines were available at the time of the physician-patient encounter.

Coronary sinus pressure has a direct effect on gradient for coronary perfusion.

In previous work from this laboratory we demonstrated that the coronary pressure-flow relationship exhibits a zero pressure intercept in the absence of the influence of the collateral circulation. In the present study we determined the effect of varying coronary sinus pressures on coronary perfusion. Specifically, we investigated whether coronary flow would cease when the coronary inflow pressure equaled the coronary sinus pressure. The study was performed while inflow perfusion pressure to all coronary vessels was changed simultaneously in order to reduce the influence of the collateral circulation while coronary sinus outflow was measured. Coronary pressure-flow relationships were obtained for coronary sinus pressures of 0, 10, and 20 mmHg. The results demonstrate a strong correlation between perfusion pressure and coronary sinus pressure (r2 = 0.994 +/- 0.001), which passed through the origin. We conclude that coronary sinus pressures between 0 and 20 mmHg have a direct influence on coronary perfusion.

Double blind comparison of alfentanil N2O and fentanyl N2O for outpatient surgical procedures.

Alfentanil - nitrous oxide and fentanyl - nitrous oxide techniques were compared in outpatients undergoing therapeutic abortion or dilatation and curettage. Thirty patients were studied in each group. Time to awakening was similar in both groups but patients who received alfentanil responded to verbal commands one minute sooner and were alert 1.5 minutes before those who received fentanyl. At ten minutes post anaesthesia the recovery scores were the same for both groups. Patients who received alfentanil were not street-worthy earlier than those who received fentanyl. During the procedure approximately two thirds of the patients moved. This movement was vigorous in 23 per cent of the patients who received alfentanil and in 30 per cent of those given fentanyl. We conclude that: (1) a more flexible dosage schedule is required in order to prevent disturbing movement of the patient during the procedure and (2) patients who received alfentanil were not street-worthy earlier than those who were given fentanyl.

The effects of fatigue on physician performance--an underestimated cause of physician impairment and increased patient risk.

Evidence is reviewed demonstrating the high level of drug and alcohol abuse and marital disharmony among physicians and the particularly high rate among anaesthetists. The relationship between these factors and the effects of fatigue is explored. The current evidence for reduction in physician performance and vigilance resulting from fatigue and sleep loss is reviewed. Supplementary indirect evidence is surveyed which suggests that increased experience may not compensate adequately for this reduced performance. Since hours of work can be controlled, it is essential that anaesthetists, their professional organizations and regulatory agencies ensure that pressure for efficiency does not result in fatigue and the consequent compromise of both patient and physician health and safety.

Verbal impairment in alcoholics.

Research utilizing psychometric and neuropsychological measures has shown that alcoholics are impaired on visual-spatial tasks. Recent studies have also shown alcoholics are impaired on novel verbal tasks as well. The present study was undertaken to determine the degree of impairment among alcoholics in visual-spatial versus verbal tasks. Limited support for the hypothesis that alcoholics are impaired in verbal abilities was obtained in that the WAIS Vocabulary subtest discriminated between alcoholics and nonalcoholics. These findings are discussed in light of both the tendency to interpret neuropsychological tests in a rather simple fashion and the need for further research into the verbal functioning of alcoholics.

Spinal melanotic clear-cell sarcoma: a light and electron microscopic study.

We report a melanotic spindle-cell tumor that arose from a thoracic spinal nerve root and metastasized to both lungs. The bulk of the tumor consisted of glycogen-rich clear cells and bore a striking resemblance on light and electron microscopy to at least one variant of the clear-cell sarcoma of tendons and aponeuroses. The presence of schwannoma-like areas noted in the primary tumor on light microscopy and the formation of a highly developed basal lamina noted on ultrastructural examination suggest that the tumor may be a partially dedifferentiated malignant melanotic schwannoma. This tumor is discussed in the context of a simple histogenetic classification of melanotic tumors.

Dynamic changes in circulating 1,25-dihydroxyvitamin D during reproduction in rats.

The concentrations of 1,25-dihydroxyvitamin D [1,25-(OH)2D], calcium, and phosphorus were measured in the serum of rats during pregnancy and at various stages of lactation. The concentration of 1,25-(OH)2D hormone increased almost two-fold during pregnancy and the latter part of lactation, but decreased to control levels or very low values immediately after birth and weaning, respectively. Furthermore, the concentration of 1,25-(OH)2D was inversely correlated with the concentration of calcium, suggesting that circulating 1,25-(OH)2D fluctuates in concert with calcium demands during the reproductive cycle. Parathyroidectomy in lactating rats caused a 70 percent inhibition of the normally observed 1,25-(OH)2D increase, indicating that parathyroid hormone, in response to changes in serum calcium, is a primary modulator of 1,25-(OH)2D during lactation.

Eye hue and the oculocardiac reflex.

During operations for squint nodal rhythm and other more serious arrhythmias such as heart block and multifocal ventricular extrasystoles, as well as the oculocardiac reflex, are shown to occur more frequently in patients with brown or hazel eyes than in patients with blue or grey eyes.

Anorexia nervosa: a combined therapeutic approach.

Ten female patients with a diagnosis of anorexia nervosa were treated with a combination of behavior modification and psychotherapy and, when appropriate, psychotropic medication. All patients had favorable weight gain and improvement in adjustment during hospitalization. Later crises for each patient thus far have not significantly affected their weight. Three case histories illustrate the method used. The interrelationship between weight gain and the process in psychotherapy is illustrated and the psychopathologic features typical of this patient population are discussed. We conclude that the combined therapeutic method described is an effective and appropriate approach to the treatment of patients with anorexia nervosa.