Pipeline for illumination correction of images for high-throughput microscopy.

The presence of systematic noise in images in high-throughput microscopy experiments can significantly impact the accuracy of downstream results. Among the most common sources of systematic noise is non-homogeneous illumination across the image field. This often adds an unacceptable level of noise, obscures true quantitative differences and precludes biological experiments that rely on accurate fluorescence intensity measurements. In this paper, we seek to quantify the improvement in the quality of high-content screen readouts due to software-based illumination correction. We present a straightforward illumination correction pipeline that has been used by our group across many experiments. We test the pipeline on real-world high-throughput image sets and evaluate the performance of the pipeline at two levels: (a) Z'-factor to evaluate the effect of the image correction on a univariate readout, representative of a typical high-content screen, and (b) classification accuracy on phenotypic signatures derived from the images, representative of an experiment involving more complex data mining. We find that applying the proposed post-hoc correction method improves performance in both experiments, even when illumination correction has already been applied using software associated with the instrument. To facilitate the ready application and future development of illumination correction methods, we have made our complete test data sets as well as open-source image analysis pipelines publicly available. This software-based solution has the potential to improve outcomes for a wide-variety of image-based HTS experiments.

Effects of dexpanthenol with or without Aloe vera extract on radiation-induced oral mucositis: preclinical studies.

PURPOSE: To define the effect of dexpanthenol with or without Aloe vera extract on radiation-induced oral mucositis. MATERIALS AND METHODS: Mouse tongue mucosal ulceration was analysed as the clinically relevant endpoint. Graded single or fractionated dose irradiation (10 x 3 Gy/2 weeks, graded test doses on day 14) were combined with topical administration of dexpanthenol or a base, with or without Aloe vera extract. The formulations were applied for 14 days (single dose) or 24 days after the first fraction. RESULTS: Single dose irradiation resulted in an ED50 (dose at which a positive mucosal response was expected in 50% of the animals irradiated) of 11.9+/-1.2 Gy. None of the formulations yielded a significant change in incidence or time course of ulceration. Test irradiation after 10 x 3 Gy gave an ED50 of 9.0+/-0.1 Gy. Base treatment increased the ED50-values to 10.5+/-0.8 Gy (p = 0.0095) and 9.9+/-0.7 Gy (p = 0.0445) without or with Aloe vera. Dexpanthenol resulted in ED50 values of 9.5+/-0.1 Gy without Aloe vera (p > 0.05), and of 10.9+/-0.9 Gy (p = 0.0035) with Aloe vera. The latent time to ulceration was prolonged, compared to the control (6.3 days) without Aloe vera (8.0-8.2 days, p < 0.001) and with dexpanthenol and Aloe vera (7.3 days, p = 0.0239). CONCLUSIONS: With single dose irradiation, neither dexpanthenol nor Aloe vera extract significantly changed the oral mucosal radiation response. With fractionated irradiation, drug administration significantly increased the isoeffective radiation doses, independent of dexpanthenol or Aloe vera content. Neither dexpanthenol nor Aloe vera display a prophylactic potential.

Experimental and theoretical analysis of phase singularity dynamics in cardiac tissue.

INTRODUCTION: Quantitative analysis of complex self-excitatory wave patterns, such as cardiac fibrillation and other high-order reentry, requires the development of new tools for identifying and tracking the most important features of the activation, such as phase singularities. METHODS AND RESULTS: Image processing operations can be used to detect the phase singularity at the tip of a spiral wave. The phase space behavior of a spatiotemporal sequence of data may be reconstructed using time-series analysis. The phase singularities then are localized efficiently by computing the topologic charge density as the curl of the spatial phase gradient. We analyzed the singularity interaction dynamics of both experimentally observed and numerically simulated instances of quatrefoil reentry and found that the singularity behavior in the experimental preparations can be classified into three categories on the basis of how their separation changes with time. CONCLUSION: Topologic charge densities can be calculated easily and efficiently to reveal phase singularity behavior. However, the differences between theoretical and experimental observations of singularity separation distances indicate the need for more sophisticated numerical models.

Three-dimensional surface reconstruction and fluorescent visualization of cardiac activation.

Optical imaging of transmembrane potentials in cardiac tissue is a rapidly growing technique in cardiac electrophysiology. Traditional studies typically use a monocular imaging setup, thus limiting investigation to a restricted region of tissue. However, studies of large-scale wavefront dynamics, especially those during fibrillation and defibrillation, would benefit from visualization of the entire epicardial surface. To solve this problem, a panoramic cardiac visualization algorithm was developed which performs the two tasks of reconstruction of the surface geometry of the heart, and representation of the panoramic fluorescence information as a texture mapping onto the geometry that was previously created. This system permits measurement of epicardial electrodynamics over a geometrically realistic representation of the actual heart being studied. To verify the accuracy of the algorithm, the procedure was applied to synthetic images of a patterned ball; further verification was provided by application of the algorithm to a model heart placed in the experimental setup. Both sets of images produced mean registration image errors on the order of 2 pixels, corresponding to roughly 3 mm on the geometry. We demonstrate the algorithm by visualizing epicardial wavefronts on an isolated, perfused rabbit heart.

Efficacy of postoperative follow-up telephone calls for patients who underwent adenotonsillectomy.

OBJECTIVE: To evaluate the efficacy and cost-effectiveness of postoperative follow-up telephone calls among pediatric patients who underwent adenotonsillectomy. DESIGN: Prospective study with a follow-up questionnaire administered by telephone. SETTING: Tertiary-care children's hospital. PATIENTS: One hundred thirty-four children between the ages of 4 and 18 years who underwent adenotonsillectomy between December 1997 and June 1998 and did not have associated cardiac, pulmonary, bleeding, or syndromic disorders were included in this pilot study. INTERVENTION: Parents of these patients were given the opportunity to participate in our study, and it was emphasized that, at any time during the child's care, if the parent desired a follow-up visit or if the child experienced any symptoms that caused concern, the parent should contact the clinic for a follow-up appointment. A telephone call was placed 3 to 4 weeks postoperatively by an otolaryngology nurse, and a questionnaire was filled out using the parents' responses. MAIN OUTCOME MEASURES: The incidence rates of voice change, velopharyngeal insufficiency, bleeding, constipation, dehydration, and pain were measured. Parent satisfaction, patient safety, and cost-benefit were also evaluated. RESULTS: Less than 5% of patients reported temporary velopharyngeal insufficiency, while 2% of patients required operative intervention for bleeding episodes and 1% required hospitalization. Voice change, reported by approximately 70% of all patients, was the most common complaint, but it resolved in all instances. Pain was reported to be most severe on postoperative day 1. Ninety-six percent of parents requested no further follow-up visit. CONCLUSIONS: Our pilot study revealed that a follow-up telephone call is a safe and cost-effective method of postoperative management for pediatric patients who have undergone adenotonsillectomy and that this method of follow-up is also desirable to parents.

Synthesis and SAR of a novel, potent and structurally simple LTD4 antagonist of the quinoline class.

The two geminal ethyl groups in the succinic acid moiety of CGP57698 (4-[3-(7-fluoro-2-quinolinyl-methoxy)phenyl-amino]-2,2-diethyl-4-oxo- butanoic acid) are responsible for the high in vitro and in vivo potency of this peptidoleukotriene antagonist of the quinoline type. The synthesis and structure activity relationships of CGP57698 and its analogs are described.

Membrane refractoriness and excitation induced in cardiac fibers by monophasic and biphasic shocks.

INTRODUCTION: This modeling study examines the effect of low-intensity monophasic and biphasic waveforms on the response of a refractory cardiac fiber to the defibrillation shock. METHODS AND RESULTS: Two cardiac fiber representations are considered in this study: a continuous fiber and a discrete fiber that incorporates gap junctions. Each fiber is undergoing a propagating action potential. Shocks of various strengths and coupling intervals are delivered extracellularly at fiber ends during the relative refractory period. In a continuous fiber, monophasic shock strengths of three times the diastolic threshold either elicit no response or, for coupling intervals above 380 msec, reinitiate propagation. In contrast, biphasic shocks of same strength are capable of terminating the existing wavefronts by either invoking a nonpropagating response (coupling intervals 370 to 382 msec) that prolongs the refractory period or inducing wavefront collision (coupling intervals above 400 msec). The fiber response is similar for other shock strengths and when cellular discontinuity is accounted for. Thus, for a refractory fiber, biphasic shocks have only a small "vulnerable" window of coupling intervals over which propagation is reinitiated. CONCLUSION: At short coupling intervals, a significant extension of refractoriness is generated at regions where the biphasic shock induced hyperpolarization followed by depolarization. At large coupling intervals, the enhanced efficacy of biphasic shocks is associated with their ability to induce wavefront collision, thus decreasing the probability of reinitiating fibrillation. Overall, the defibrillation shock affects the tissue through the induced large-scale hyperpolarization and depolarization, and not through the small-scale transmembrane potential oscillations at cell ends.

Cochlear implantation in children.

Cochlear implantation in the pediatric population is no longer considered experimental practice since the Food and Drug Administration (FDA) approved the Nucleus 22 Channel cochlear implant in 1990. Today, cochlear implantation is a viable option for selected children with profound hearing loss to achieve potential language development. Not every child is a candidate, however, nor can implantation rectify the underlying cause of deafness or restore normal hearing function. For successful outcomes in proper candidates, rigorous pre-surgical evaluation and screening followed by long-term rehabilitation and education are necessary for both child and family. The close collaboration of an interdisciplinary team is essential throughout the process.

Effect of leukotriene D4 on ciliary activity in human, guinea-pig and rat respiratory mucosa.

Leukotriene D4 (LTD4) is a potent bronchoconstrictor and inflammatory mediator in asthma. Data concerning the effects of LTD4 on ciliary function in the respiratory tract are sparse and contradictory. The purpose of the present study was to clarify the effects of LTD4 on mucociliary activity using the tracheal mucosa of two laboratory animal species, guinea-pig and rat, as well as human nasal mucosa. The ciliary beat frequency (CBF) was measured photoelectrically and determined by Fast Fourier Transform computer analysis. Additionally the structure of ciliated epithelia of guinea-pig trachea after LTD4-immersion was investigated using a scanning electron microscope (SEM). In all tissues, LTD4 increased CBF showing a bell-shaped dose-response curve. The maximum effect was 75 +/- 30% in guinea-pig at 10(-9) mol/l, 119 +/- 49% in rat at 10(-7) mol/l, and 86 +/- 28% at 10(-6) mol/l in human tissue. In guinea-pig tracheal mucosa, there was an indication of an increase in the amount of mucus and disorientation of cilia were seen by SEM after immersion in LTD4. These findings suggest that LTD4 stimulates ciliary activity, but impairs the orientation of cilia.

Characterization of 24-h cortisol release in obese and non-obese hyperandrogenic women.

Excessive androgen output is a well-recognized feature of adrenocortical oversecretion in women with ovarian hyperandrogenism, or polycystic ovary disease (PCOD). However, evidence of a concomitant alteration of cortisol secretion is lacking even though obesity per se, a common clinical feature of PCOD, has been shown to be associated with cortisol oversecretion. To clarify whether a subtle alteration in cortisol secretion exists, a study of 24-h episodic cortisol release and post-prandial cortisol responses was undertaken in eight women with PCOD and eight normal women comprising equal numbers of obese and non-obese subjects. All four groups showed normal biphasic 24-h cortisol secretion profiles but cortisol pulse frequency was increased in the PCOD groups. Independently, both hyperandrogenism and obesity were associated with an accelerated cortisol clearance rate. These changes, together with normal or only slightly elevated 24-h cortisol integrated area under the curve, suggest an increased compensatory cortisol production in women with PCOD. Furthermore, subjects with PCOD and subjects with obesity showed different post-prandial cortisol responses to normal non-obese women. In conclusion, these subtle cortisol abnormalities may be a manifestation of altered central regulation of the hypothalamic-pituitary-adrenal axis and peripheral metabolic abnormalities, and may be linked to the pathophysiology of PCOD.

Strategies in the design of peptidoleukotriene antagonists.

The research of the last two decades in the field of SRS-A and peptidoleukotriene (pLT) antagonists has provided information for the design of potent pLT antagonists, which share some or all of the following structural elements: (1) a lipophilic anchor, which fits into the lipophilic pocket of the LTD4 receptor; (2) a central lipophilic unit mimicking the tetraene system of LTD4; (3) one or two acidic groups, as mimics of the cysteinyl-glycine unit and/or the carboxylic group in the eicosanoid backbone of LTD4; (4) spacers connecting these elements. Potent pLT antagonists lacking a second polar binding group compensate by stronger interaction in other regions of the receptor. Identification of pLT antagonists is based on lead optimisation, preparation of pLT analogs and on the knowledge of the pLT receptor.

Human immunodeficiency virus-1 infection in an infertile population.

OBJECTIVE: The aim of our study was to determine the prevalence of human immunodeficiency virus-1 (HIV-1) in an infertile population. DESIGN: The study design included a retrospective anonymous survey of clinical data and screening for HIV-1 antibody by enzyme-linked immunoabsorbent assay (ELISA) in stored sera. Samples that were ELISA-positive were further tested by Western blot assay. Frequency distributions were analyzed by Fisher's exact test. SETTING: University tertiary care center. PARTICIPANTS: Based on availability of stored frozen sera, the total study population included 182 of 304 consecutively registered infertile couples. RESULTS: Seventy-five percent of the study population were found to have one or more risk factors for HIV infection. Of the 252 sera tested, 10 were repeatedly reactive by ELISA, and Western blot testing confirmed HIV-1 infection in one woman and two men. CONCLUSIONS: This relatively high HIV-1 seroprevalence (male: 2.6%; female: 0.6%) in a low-middle class infertile population emphasizes the urgent need to implement on-site HIV-infection counseling aimed at preventing the spread of disease to the healthy partner and fetus and to discuss therapeutic and reproductive options.

Circadian, ultradian, and episodic gonadotropin and prolactin secretion in human pseudocyesis.

Six women with pseudocyesis were studied by 15-min blood sampling for 12 to 24 h to determine their gonadotropin and PRL secretory profiles aiming to clarify the endocrine alterations in this form of hypothalamic amenorrhea. Clinical and biochemical evidence of hyperandrogenism was found in 4 patients. Persistent hyperprolactinemia was present only in one patient. Significant circadian and ultradian periodicities were identified by time series analysis in the 12-24 h profiles of FSH, LH and PRL secretion. Pulse analysis by the Van Cauter (UL-TRA.JN) method revealed a 24-h mean LH interpulse interval of 91 +/- 21 min with a mean LH amplitude of 5.4 +/- 0.8 IU/l. There was a significantly lower pulse frequency at night than during the daytime. The mean 24-h PRL interpulse interval and pulse amplitude were 134 +/- 22 min and 9.2 +/- 1.8 IU/l, respectively. Both FSH and LH mean levels were higher during the daytime than at night, while the reverse was true for PRL values. Decreased LH pulse frequency and amplitude emerged as the most distinctive findings. Antecedent hypothalamic-pituitary aberrations due to other endocrinopathies and the timing of the hormonal assessment (e.g. recovery phase) may explain, at least in part, the reported heterogeneity of neuroendocrinologic findings in pseudocyesis.

Inhibition of complement-factor-5a-induced inflammatory reactions by prostaglandin E2 in experimental meningitis.

The possible role of complement factor 5a (C5a) and prostaglandin E2 (PGE2) in cerebrospinal fluid (CSF) pleocytosis and protein accumulation was assessed in a rabbit model of meningitis. Intracisternally administered C5a caused a rapid, early influx of leukocytes into CSF that peaked at 1 h after injection; by 6 h, cell counts were slightly higher than those in controls. Administration of PGE2 or saline did not induce detectable CSF leukocytosis. Coadministration of PGE2 with C5a decreased CSF leukocytosis in a dose-related fashion. Protein concentration increased 30 min after administration of C5a, peaked after 1 h, and remained elevated for 6 h. PGE2 caused a dose-related increase in protein content after 2 h, whereas coadministration caused an inversely dose-related inhibition of the C5a-induced protein influx into CSF. These data suggest that PGE2 in the subarachnoid space exerts an inhibitory action on the C5a-mediated response that is probably not related to its direct effects on protein extravasation.

Platelet activating factor (PAF) is a potent stimulator of porcine tracheal fluid secretion in vitro.

Mucus hypersecretion is a major clinical feature of chronic obstructive lung diseases such as asthma. The possible role of the inflammatory and bronchoconstrictor ether lipid PAF (platelet activating factor) has been studied in isolated porcine trachea with the tantalum 'hillock' technique used to visualize fluid production from tracheal submucosal glands. PAF caused a rapid, dose-dependent (0.001-1 nM) stimulation of fluid secretion which could be detected after 5 min and which increased with time up to at least 15 min. The PAF-induced fluid secretion was unaffected by both antagonists of histamine, acetylcholine and leukotriene D4 and inhibitors of prostaglandin and leukotrienes synthesis. A purported PAF receptor antagonist (CV 3988) inhibited the PAF responses in a dose-dependent manner implying a receptor-mediated event. These results may be of relevance to the mucus hypersecretion seen in chronic airway diseases.

Nonsteroidal anti-inflammatory agents in the therapy for experimental pneumococcal meningitis.

An increased inflammatory mass in the subarachnoid space during bacterial meningitis may correlate with a poor outcome of disease. Using a rabbit model of pneumococcal meningitis, we sought to reduce this inflammatory process. The ability of the pneumococcal cell wall to cause death and to generate leukocytosis and abnormal chemistry in cerebrospinal fluid was prevented when animals were treated with inhibitors of cyclooxygenase pathway of arachidonate metabolism. Bacterial lysis by ampicillin led to release of cell wall that caused a significant, transient increase in meningeal inflammation. This inflammatory burst was also prevented by administering cyclooxygenase inhibitors concurrently with the antibiotic.

Stimulated release of neutral proteinases elastase and cathepsin G from inflammatory rat polymorphonuclear leukocytes.

Rat leukocytes from inflammatory peritoneal exudates respond in vitro to a variety of chemotactic and phagocytic stimuli by releasing both elastase and cathepsin G neutral proteinase enzyme activities. PAF, FMLP, and PMA stimulated a rapid, cytochalasin B-dependent, dose-related release of both enzymes; however, leukotriene B4 was inactive. It was not possible to measure the activity of zymosan-activated serum on these cells as rat serum contains high levels of proteinase inhibitors. The calcium ionophore A23187 stimulated a dose-related, time-dependent, cytochalasin B-independent enzyme release. Concanavalin A stimulated a weak, nondose-related release of enzyme activity. Zymosan and serum-coated zymosan stimulated enzyme secretion which was markedly inhibited by the presence of cytochalasin B. These data indicate that release of azurophillic granule neutral proteinases from rat inflammatory leukocytes can be detected and quantitated in vitro. This model could provide a test system for monitoring the pattern and specificity of enzyme release from azurophil granules. The ability of a variety of stimuli to induce proteolytic enzyme release from inflammatory neutrophils may be of considerable relevance to chronic inflammatory diseases.