Fast Optimized Cluster Algorithm for Localizations (FOCAL): a spatial cluster analysis for super-resolved microscopy.

MOTIVATION: Single-molecule localization microscopy (SMLM) microscopy provides images of cellular structure at a resolution an order of magnitude below what can be achieved by conventional diffraction limited techniques. The concomitantly larger data sets generated by SMLM require increasingly efficient image analysis software. Density based clustering algorithms, with the most ubiquitous being DBSCAN, are commonly used to quantitatively assess sub-cellular assemblies. DBSCAN, however, is slow, scaling with the number of localizations like O(n log (n)) at best, and it's performance is highly dependent upon a subjectively selected choice of parameters. RESULTS: We have developed a grid-based clustering algorithm FOCAL, which explicitly accounts for several dominant artifacts arising in SMLM image reconstructions. FOCAL is fast and efficient, scaling like O(n), and only has one set parameter. We assess DBSCAN and FOCAL on experimental dSTORM data of clusters of eukaryotic RNAP II and PALM data of the bacterial protein H-NS, then provide a detailed comparison via simulation. FOCAL performs comparable and often superior to DBSCAN while yielding a significantly faster analysis. Additionally, FOCAL provides a novel method for filtering out of focus clusters from complex SMLM images. AVAILABILITY AND IMPLEMENTATION: The data and code are available at: http://www.utm.utoronto.ca/milsteinlab/resources/Software/FOCAL/ CONTACT: josh.milstein@utoronto.ca SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An exploration of connections between a mother and her unborn in preterm labor.

The purpose of this special feature is to elaborate on a unique physician-patient interaction between a neonatologist and a pediatrician established during the pediatrician's personal experience with the onset of labor between 22 and 23 weeks' gestation. As the two are both versed in conventional aspects of neonatal care including prenatal counseling, the neonatologist thought that he had more to offer the expectant mother holistically. Thus, he decided against approaching her as an information source and contribute to the customary data-dumping format that can be both frightening and dehumanizing. Instead, he wanted to focus on her individual experience of pregnancy and the healing aspects of care. The most important lesson received from the interaction is the value of being present with another. This sense of 'being with' applied to the neonatologist and the mother from their initial phone conversation to their in person times, and to the mother with her unborn permitting interaction in a meaningful way. Fostering each of those connections in alternative ways was valuable in this prenatal experience.

On the role of DNA biomechanics in the regulation of gene expression.

DNA is traditionally seen as a linear sequence of instructions for cellular functions that are expressed through biochemical processes. Cellular DNA, however, is also organized as a complex hierarchical structure with a mosaic of mechanical features, and a growing body of evidence is now emerging to imply that these mechanical features are connected to genetic function. Mechanical tension, for instance, which must be felt by DNA within the heavily constrained and continually fluctuating cellular environment, can affect a number of regulatory processes implicating a role for biomechanics in gene expression complementary to that of biochemical regulation. In this article, we review evidence for such mechanical pathways of genetic regulation.

Bead size effects on protein-mediated DNA looping in tethered-particle motion experiments.

Tethered particle motion (TPM) has become an important tool for single-molecule studies of biomolecules; however, concerns remain that the method may alter the dynamics of the biophysical process under study. We investigate the effect of the attached microsphere on an illustrative biological example: the formation and breakdown of protein-mediated DNA loops in the lac repressor system. By comparing data from a conventional TPM experiment with 800 nm polystyrene beads and dark-field TPM using 50 nm Au nanoparticles, we found that the lifetimes of the looped and unlooped states are only weakly modified, less than two-fold, by the presence of the large bead. This is consistent with our expectation of weak excluded-volume effects and hydrodynamic surface interactions from the cover glass and microsphere.

Protein-mediated DNA loop formation and breakdown in a fluctuating environment.

Living cells provide a fluctuating, out-of-equilibrium environment in which genes must coordinate cellular function. DNA looping, which is a common means of regulating transcription, is very much a stochastic process; the loops arise from the thermal motion of the DNA and other fluctuations of the cellular environment. We present single-molecule measurements of DNA loop formation and breakdown when an artificial fluctuating force, applied to mimic a fluctuating cellular environment, is imposed on the DNA. We show that loop formation is greatly enhanced in the presence of noise of only a fraction of k_{B}T, yet find that hypothetical regulatory schemes that employ mechanical tension in the DNA-as a sensitive switch to control transcription-can be surprisingly robust due to a fortuitous cancellation of noise effects.

Femtonewton entropic forces can control the formation of protein-mediated DNA loops.

We show that minuscule entropic forces, on the order of 100 fN, can prevent the formation of DNA loops-a ubiquitous means of regulating the expression of genes. We observe a tenfold decrease in the rate of LacI-mediated DNA loop formation when a tension of 200 fN is applied to the substrate DNA, biasing the thermal fluctuations that drive loop formation and breakdown events. Conversely, once looped, the DNA-protein complex is insensitive to applied force. Our measurements are in excellent agreement with a simple polymer model of loop formation in DNA, and show that an antiparallel topology is the preferred LacI-DNA loop conformation for a generic loop-forming construct.

Methylphenidate-induced impulsivity: pharmacological antagonism by beta-adrenoreceptor blockade.

Noradrenaline-dopamine interactions mediate increases in locomotor activity, development of sensitisation and subjective effects of psychostimulant drugs. However, the modulatory effects of noradrenaline on psychostimulant-induced impulsivity are less clear. This article examined the relative roles of noradrenaline and dopamine in the modulation of methylphenidate-induced impulsive responding in rats performing the 5-choice serial reaction time task. Experiment 1 examined the systemic antagonism of methylphenidate-induced impulsivity with either propranolol, a beta-adrenoreceptor blocker, or prazosin, an alpha1-adrenoreceptor antagonist, which antagonises the locomotor activating effects of amphetamine. Propranolol completely abolished methylphenidate-induced impulsivity. This effect was centrally rather than peripherally mediated, as nadolol, a peripheral beta-blocker failed to affect methylphenidate-induced premature responding. Prazosin partially attenuated the methylphenidate-mediated increase in premature responding. A second experiment examined the effects of selective anti-D beta H saporin-induced cortical noradrenaline depletion on methylphenidate-induced impulsivity. Contrary to the effects of beta-adrenoreceptor blockade, cortical noradrenergic depletion did not alter methylphenidate-induced impulsivity. Other experiments examined the comparative effects of selective dopamine and serotonin receptor blockade. D4 dopamine receptor blockade with systemically administered L-745,870 also attenuated methylphenidate-induced impulsivity. The other antagonists had no effect on methylphenidate-induced impulsivity. Taken together, these studies provide evidence for a modulatory role of beta-adrenoreceptors on methylphenidate-induced impulsive responding.

A statewide quality improvement collaborative to reduce neonatal central line-associated blood stream infections.

OBJECTIVE: The objective of this study was to reduce central line-associated blood stream infections (CLABSIs) among 13 collaborating regional neonatal intensive care units by 25%. We tested the hypothesis that change could be attributed to the quality improvement collaborative by testing for 'special cause' variation. STUDY DESIGN: Our prevention project included five features: (1) leadership commitment, (2) potentially best practices, (3) collaborative processes, (4) audit and feedback tools and (5) quality improvement techniques. Baseline (1 January 2006 to 30 August 2006) data were compared with the intervention (1 September 2006 to 30 June 2007) and post-intervention (1 July 2007 to 30 December 2007) periods and analyzed using statistical process control (SPC) methods. RESULT: We detected special cause variation, suggesting that the collaborative was associated with reduced infection rates, from 4.32 to 3.22 per 1000 line days (a 25% decrease) when comparing the baseline with the follow-up period. CONCLUSION: The collaborative's process was associated with fewer infections. SPC suggested that systematic changes occurred. The remaining challenges include sustaining or even further reducing the infection rate.

Indomethacin-associated neutropenia with subsequent Gram-negative sepsis in a preterm infant. Cause or coincidence?

A preterm male infant with a patent ductus arteriosus developed neutropenia during treatment with indomethacin. Afterward, the mother described her own history of indomethacin-associated neutropenia. During the recovery from the neutropenia, the infant became septic with bacteremia caused by Enterobacter cloacae. Although indomethacin-related neutropenia has been described in adults, no case in a neonate has been reported. If neutropenia occurs after indomethacin therapy in a neonate, a familial history of indomethacin-associated neutropenia should be sought and the increased risk of infection should be considered.

Signatures of resonance superfluidity in a quantum Fermi gas.

We predict a direct and observable signature of the superfluid phase in a quantum Fermi gas, in a temperature regime already accessible in current experiments. We apply the theory of resonance superfluidity to a gas confined in a harmonic potential and demonstrate that a significant increase in density will be observed in the vicinity of the trap center.

Hemodynamic effects of positive end-expiratory pressure during partial liquid ventilation in newborn lambs.

BACKGROUND/PURPOSE: The aim of this study was to compare the effect of positive end-expiratory pressure (PEEP) application on hemodynamics, lung mechanics, and oxygenation in the intact newborn lung during conventional ventilation (CV) and partial liquid ventilation (PLV) at functional residual capacity (FRC). CV or PLV modes of ventilation do not affect hemodynamics nor the optimum PEEP for oxygenation. METHODS: Seven newborn lambs (1 to 3 days old) were instrumented to measure pulmonary hemodynamics and airway mechanics. Each lamb was used as their own control to compare different modes of ventilation (CV followed by PLV) under graded variations of PEEP (4, 8, 12, and 16 cm H(2)O) on the influence on pulmonary blood flow and pulmonary vascular resistance. RESULTS: There was a significant drop in pulmonary blood flow (PBF) from baseline (PEEP of 4 cm H(2)O on CV, 1,229 +/- 377 mL/min) in both modes of ventilation on a PEEP of 16 cm H(2)O (CV, 750 +/- 318 mL/min v PLV, 926 +/- 396 mL/min, respectively; P <.05). Peak inspiratory pressure (PIP) was higher on PLV at PEEP states of 4 cm H(2)O (16.5 +/- 1.3 cm H(2)O to 10.6 +/- 2.1 cm H(2)O; P <.05) and 8 cm H(2)O (18.8 +/- 2.2 cm H(2)O to 15.1 +/- 2.6 cm H(2)O; P <.05) when compared with CV. Conversely, PIP required to maintain the pCO(2) was lower on PLV at PEEP states of 12 (22.5 +/- 3.6 cm H(2)O to 24.2 +/- 3.8 cm H(2)O; P <.05) and 16 cm H(2)O (27.0 +/- 1.6 cm H(2)O to 34.0 +/- 5.9 cm H(2)O; P <.05). CONCLUSIONS: Hemodynamically, CO is impaired at a PEEP above 12 cm H(2)O in intact lungs. PFC at FRC does provide an advantage in lung mechanics more than 10 to 12 cm H(2)O of PEEP by decreasing the amount PIP needed to achieve the similar levels of gas exchange and minute ventilation, implying a reduced risk for barotrauma with chronic ventilation. Thus, selection of the appropriate level of PEEP appears to be important if PLV is to be utilized at FRC. The best strategy for PLV, including the selection of PEEP, remains to be determined.

Gastroschisis and Hirschsprung's disease: a rare combination.

The authors report on a neonate with gastroschisis repaired at birth who later had abdominal distension, emesis, feeding intolerance, and an abnormal stooling pattern. Total colon and partial small bowel aganglionosis (TCAS), or Hirschsprung's disease, was diagnosed subsequently. This is the first report of this combination of gastrointestinal anomalies. J Pediatr Surg 36:638-640.

Form and function of fetal and neonatal pulmonary arterial bifurcations.

Bifurcation is a basic form of vascular connection. It is composed of a parent vessel of diameter d(0), and two daughter vessels, d(1) and d(2), where d(0) > d(1) >/= d(2). Optimal values for the bifurcation area ratio, beta = (d(1)(2) + d(2)(2))/d(0)(2), and the junction exponent, x, in d(0)(x) = d(1)(x) + d(2)(x), are postulated to be universal in nature. However, we have hypothesized that the perinatal pulmonary arterial circulation is an exception. Arterial diameters were measured in pulmonary vascular casts of a fetal lamb (140 days gestation/145 days term) and a neonatal lamb (1 day old). The values for beta and x were evaluated in 10,970 fetal and 846 neonatal bifurcations sampled from the proximal and intermediate arterial regions. Mean values and confidence intervals (CI) for the fetus were beta = 0.890 (0.886-0.895 CI) and x = 1.75 (1.74-1.76 CI); and for the newborn were beta = 0.913 (0.90-0.93 CI) and x = 1. 79 (1.75-1.82 CI). These values are significantly different from Murray's law (beta > 1, x = 3) or the West-Brown-Enquist law (beta = 1, x = 2). Therefore, perinatal pulmonary bifurcation design appears to be distinctive and exceptional. The decreasing cross-sectional area with branching leads to the hemodynamic consequence of shear stress amplification. This structural organization may be important for facilitating vascular development at low flow rates; however, it may be the origin of unstable reactivity if elevated blood flow and pressure occurs.

Effects of correction of hypocalcemia on cardiac function in the newborn lamb.

The effect of correction of hypocalcemia on cardiac function in the neonate is unclear. Therefore, we evaluated changes in hemodynamic variables and cardiac contractility in a hypocalcemic neonatal lamb model. Baseline serum ionized hypocalcemia was induced via chelation with intravenous EGTA. Cardiac contractility, measured as end-systolic elastance, did not change with correction of hypocalcemia in this model. In contrast, the variable dP/dt(max), a load-dependent index of contractility, showed an increase with calcium administration while the end-diastolic volume decreased, suggesting that diastolic function or early systolic function had changed. Systemic vascular resistance, cardiac output, and heart rate did not change significantly with correction of hypocalcemia. The results of this study suggest that in our newborn lamb model, cardiac contractility is insensitive to ionized calcium and may reflect cardiac immaturity, but that there may be an effect of calcium administration on diastolic or early systolic function.

A nicotine conjugate vaccine reduces nicotine distribution to brain and attenuates its behavioral and cardiovascular effects in rats.

Vaccination of animals to elicit drug-specific antibodies, or the passive transfer of such antibodies from other animals, can reduce the behavioral effects of drugs such as cocaine and heroin. To study the potential application of this approach to treating nicotine dependence, IgG was isolated from rabbits immunized with a nicotine-protein conjugate vaccine. Anesthetized rats received immune IgG containing nicotine-specific antibodies (Nic-IgG) or control-IgG i.v.. Thirty minutes later, rats received nicotine at 0.03 mg/kg i.v., equivalent on an mg/kg basis to the nicotine intake from two cigarettes by a smoker. Compared to control-IgG, Nic-IgG reduced the brain nicotine concentration in a dose-related manner (65% reduction at the highest IgG dose). Pretreatment with Nic-IgG also reduced the distribution to brain of five repeated doses of nicotine (equivalent to the nicotine intake from 10 cigarettes) administered over 80 min. To study blood pressure effects, rats received control-IgG or Nic-IgG 1 day prior to administering nicotine. Nicotine-induced systolic blood pressure increases were attenuated by Nic-IgG in a dose-related manner, and were almost completely blocked by the highest Nic-IgG dose. Pretreatment with Nic-IgG also completely prevented the nicotine-induced stimulation of locomotor activity observed in rats receiving control-IgG. Nic-IgG did not prevent locomotor activation from cocaine, demonstrating its specificity for nicotine. These data demonstrate that the administration of nicotine-specific antibodies can reduce or prevent some of the pharmacokinetic, cardiovascular, and behavioral consequences of nicotine in rats. Effects were observed at nicotine doses and nicotine serum concentrations equal to or exceeding those typically associated with nicotine exposure in cigarette smokers. A potential role for immunization in the treatment of nicotine dependence is suggested.

Metastasis stage, adjuvant treatment, and residual tumor are prognostic factors for medulloblastoma in children: conclusions from the Children's Cancer Group 921 randomized phase III study.

PURPOSE: From 1986 to 1992, "eight-drugs-in-one-day" (8-in-1) chemotherapy both before and after radiation therapy (XRT) (54 Gy tumor/36 Gy neuraxis) was compared with vincristine, lomustine (CCNU), and prednisone (VCP) after XRT in children with untreated, high-stage medulloblastoma (MB). PATIENTS AND METHODS: Two hundred three eligible patients with an institutional diagnosis of MB were stratified by local invasion and metastatic stage (Chang T/M) and randomized to therapy. Median time at risk from study entry was 7.0 years. RESULTS: Survival and progression-free survival (PFS) +/- SE at 7 years were 55%+/-5% and 54%+/-5%, respectively. VCP was superior to 8-in-1 chemotherapy, with 5-year PFS rates of 63%+/-5% versus 45%+/-5%, respectively (P = .006). Upon central neuropathology review, 188 patients were confirmed as having MB and were the subjects for analyses of prognostic factors. Children aged 1.5 to younger than 3 years had inferior 5-year estimates of PFS, compared with children 3 years old or older (P = .0014; 32%+/-10% v 58%+/-4%, respectively). For MB patients 3 years of age or older, the prognostic effect of tumor spread (MO v M1 v M2+) on PFS was powerful (P = .0006); 5-year PFS rates were 70%+/-5%, 57%+/-10%, and 40%+/-8%, respectively. PFS distributions at 5 years for patients with M0 tumors with less than 1.5 cm2 of residual tumor, versus > or = 1.5 cm2 of residual tumor by scan, were significantly different (P = .023; 78%+/-6% v 54%+/-11%, respectively). CONCLUSION: VCP plus XRT is a superior adjuvant combination compared with 8-in-1 chemotherapy plus XRT. For patients with M0 tumors, residual tumor bulk (not extent of resection) is a predictor for PFS. Patients with M0 tumors, > or = 3 years with < or = 1.5 cm2 residual tumor, had a 78%+/-6% 5-year PFS rate. Children younger than 3 years old who received a reduced XRT dosage had the lowest survival rate.

Role of nitric oxide in cardiovascular responses to endotoxemia in neonatal lambs.

To determine whether systemic cardiovascular responses to gram-negative endotoxemia are mediated by nitric oxide, we evaluated time-dependent changes in contractility and hemodynamics in a neonatal sheep model subjected to nitric oxide synthesis inhibition with L-Name (Nomega-nitro-L-arginine methyl ester). Four groups were studied: control (C), endotoxin (E), endotoxin L-Name where the nitric oxide synthase inhibitor was given prior to endotoxin (ELN), and a control L-Name group pretreated with L-Name (CLN). The contractility, measured as end-systolic elastance (Ees), increased transiently in the E group and then returned to baseline. In contrast, Ees declined over time in the ELN group. In terms of peripheral hemodynamics, both the E and ELN groups demonstrated significant progressive decreases in blood pressure and vascular resistance. The results of this study suggest that nitric oxide contributes to the newborn contractile response of the heart to endotoxin, but does not appear to mediate the systemic vascular relaxation response.

Device evaluation and coverage policy in workers' compensation: examples from Washington State.

Workers' compensation health benefits are broader than general health benefits and include payment for medical and rehabilitation costs, associated indemnity (lost time) costs, and vocational rehabilitation (return-to-work) costs. In addition, cost liability is for the life of the claim (injury), rather than for each plan year. We examined device evaluation and coverage policy in workers' compensation over a 10-year period in Washington State. Most requests for device coverage in workers' compensation relate to the diagnosis, prognosis, or treatment of chronic musculoskeletal conditions. A number of specific problems have been recognized in making device coverage decisions within workers' compensation: (1) invasive devices with a high adverse event profile and history of poor outcomes could significantly increase both indemnity and medical costs; (2) many noninvasive devices, while having a low adverse event profile, have not proved effective for managing chronic musculoskeletal conditions relevant to injured workers; (3) some devices are marketed and billed as surrogate diagnostic tests for generally accepted, and more clearly proven, standard tests; (4) quality oversight of technology use among physicians may be inadequate; and (5) insurers' access to efficacy data adequate to make timely and appropriate coverage decisions in workers' compensation is often lacking. Emerging technology may substantially increase the costs of workers' compensation without significant evidence of health benefit for injured workers. To prevent ever-rising costs, we need to increase provider education and patient education and consent, involve the state medical society in coverage policy, and collect relevant outcomes data from healthcare providers.

Survival and prognostic factors following radiation therapy and chemotherapy for ependymomas in children: a report of the Children's Cancer Group.

OBJECT: Ependymomas in children continue to generate controversy regarding their histological diagnosis and grading. optimal management, and possible prognostic factors. To increase our knowledge of these tumors the authors addressed these issues in a cohort of children with prospectively staged ependymomas treated with radiotherapy and chemotherapy. METHODS: Children between the ages of 2 and 17.3 years harboring an intracranial ependymoma confirmed by a central review of the tumor's pathological characteristics were treated according to Children's Cancer Group Protocol 921 from 1986 to 1992. Treatment following surgery and postoperative tumor staging (including brain computerized tomography or magnetic resonance [MR] imaging, spinal MR imaging or myelography, and cerebrospinal fluid cytological investigation) included craniospinal irradiation with a local boost to the primary tumor and patient randomization to receive adjuvant chemotherapy with either 1) CCNU, vincristine, and prednisone, or 2) the eight-drugs-in-1-day regimen. Centralized review of the tumor pathological characteristics revealed 20 ependymomas and 12 anaplastic ependymomas in the 32 children included in the study. Diagnoses made at the individual institutions included anaplastic (malignant) ependymoma (15 patients), ependymoma (four patients), ependymoblastoma (nine patients), ependymoastrocytoma (one patient), and primitive neuroectodermal tumor (three patients), which were discordant with the centralized review diagnosis in 22 of 32 cases. Only three of the 32 patients had metastatic disease (two with M and one with M3 stages). At surgery, 47% of tumors were estimated to be totally resected. Among the 14 of 17 patients who suffered a relapse and were evaluated for site of relapse, 10 (71%) had an isolated local relapse, three (21%) had concurrent local and metastatic relapse, and only one (7%) had an isolated metastatic relapse. Kaplan-Meier estimates of 5-year progression-free survival (PFS) and overall survival rates were 50 +/- 10% and 64 +/- 9%, respectively. CONCLUSIONS: Predictors of PFS duration included an estimate of the extent of resection made at surgery (total compared with less than total, p = 0.0001) and the amount of residual tumor on postoperative imaging as verified by centralized radiological review (< or = 1.5 cm2 compared with > 1.5 cm2, p < 0.0001). No other factors, including centrally reviewed tumor histopathological type, location, metastasis and tumor (M and T) stages, patient age, race, gender, or chemotherapy treatment regimen significantly correlated with PFS duration. The pattern of predominantly local relapse and the important influence of residual tumor or the extent of resection on PFS duration confirms a prevailing impression that local disease control is the major factor in the prediction of outcome of ependymoma. Survival rates were comparable with those reported by other investigators who have treated patients with similar doses of radiation and no chemotherapy.