A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis upon metabolic demands. Detection of these contact sites at the nanometer scale over time in living cells is challenging. We developed a tool kit for detecting contact sites based on fluorogen-activated bimolecular complementation at CONtact sites, FABCON, using a reversible, low-affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic regulation.

A fluorogenic complementation tool kit for interrogating lipid droplet-organelle interaction.

Contact sites between lipid droplets and other organelles are essential for cellular lipid and energy homeostasis. Detection of these contact sites at nanometer scale over time in living cells is challenging. Here, we developed a tool kit for detecting contact sites based on Fluorogen-Activated Bimolecular complementation at CONtact sites, FABCON, using a reversible, low affinity split fluorescent protein, splitFAST. FABCON labels contact sites with minimal perturbation to organelle interaction. Via FABCON, we quantitatively demonstrated that endoplasmic reticulum (ER)- and mitochondria (mito)-lipid droplet contact sites are dynamic foci in distinct metabolic conditions, such as during lipid droplet biogenesis and consumption. An automated analysis pipeline further classified individual contact sites into distinct subgroups based on size, likely reflecting differential regulation and function. Moreover, FABCON is generalizable to visualize a repertoire of organelle contact sites including ER-mito. Altogether, FABCON reveals insights into the dynamic regulation of lipid droplet-organelle contact sites and generates new hypotheses for further mechanistical interrogation during metabolic switch.

Differences in the effects of school meals on children's cognitive performance according to gender, household education and baseline reading skills.

BACKGROUND/OBJECTIVES: We previously found that the OPUS School Meal Study improved reading and increased errors related to inattention and impulsivity. This study explored whether the cognitive effects differed according to gender, household education and reading proficiency at baseline. SUBJECTS/METHODS: This is a cluster-randomised cross-over trial comparing Nordic school meals with packed lunch from home (control) for 3 months each among 834 children aged 8 to 11 years. At baseline and at the end of each dietary period, we assessed children's performance in reading, mathematics and the d2-test of attention. Interactions were evaluated using mixed models. Analyses included 739 children. RESULTS: At baseline, boys and children from households without academic education were poorer readers and had a higher d2-error%. Effects on dietary intake were similar in subgroups. However, the effect of the intervention on test outcomes was stronger in boys, in children from households with academic education and in children with normal/good baseline reading proficiency. Overall, this resulted in increased socioeconomic inequality in reading performance and reduced inequality in impulsivity. Contrary to this, the gender difference decreased in reading and increased in impulsivity. Finally, the gap between poor and normal/good readers was increased in reading and decreased for d2-error%. CONCLUSIONS: The effects of healthy school meals on reading, impulsivity and inattention were modified by gender, household education and baseline reading proficiency. The differential effects might be related to environmental aspects of the intervention and deserves to be investigated further in future school meal trials.

Associations between adiposity, hormones, and gains in height, whole-body height-adjusted bone size, and size-adjusted bone mineral content in 8- to 11-year-old children.

UNLABELLED: We examined fat-independent associations of hormones with height and whole-body bone size and mineral content in 633 school children. IGF-1 and osteocalcin predict growth in height, while fat, osteocalcin, and in girls also, IGF-1 predict growth in bone size. Leptin and ghrelin are inversely associated with bone size in girls. INTRODUCTION: Obesity causes larger bone size and bone mass, but the role of hormones in this up-regulation of bone in obesity is not well elucidated. We examined longitudinal associations between baseline body fat mass (FM), and fat-independent fasting levels of ghrelin, adiponectin, leptin, insulin, insulin-like growth factor-I (IGF-1), osteocalcin, and intact parathyroid hormone, and subsequent changes in height and in whole-body height-adjusted bone area "BAheight" and size-adjusted bone mineral content "BMCsize" in 8- to 11-year-olds. METHODS: Analyses were carried out separately for boys (n = 325) and girls (n = 308) including data from baseline, 3 and 6 months from OPUS School Meal Study. RESULTS: In both sexes: gain in BAheight was positively associated with baseline FM (≥2.05 cm(2)/kg, both p ≤ 0.003). Furthermore, gain in height was positively associated with baseline IGF-1 (≥0.02 cm/ng/ml, p = 0.001) and osteocalcin (≥0.13 cm/ng/ml, p ≤ 0.009); and gain in BAheight was positively associated with baseline osteocalcin (≥0.35 cm(2)/ng/ml, p ≤ 0.019). In girls only, gain in BAheight was also positively associated with baseline IGF-1 (0.06 cm(2)/ng/ml, p = 0.017) and inversely associated with both baseline ghrelin (-0.01 cm(2)/pg/ml, p = 0.001) and leptin (-1.21 cm(2)/µg/ml, p = 0.005). In boys, gain in BMCsize was positively associated with osteocalcin (0.18 g/ng/ml, p = 0.030). CONCLUSIONS: This large longitudinal study suggests that in 8- to 11-year-old children, IGF-1 and osteocalcin predict growth in height, while FM, osteocalcin, and in girls also, IGF-1 predict growth in BAheight. Fat-independent inverse associations of leptin and ghrelin with BAheight in girls' are contrary to proposed growth-stimulating effects of leptin. Osteocalcin in boys predicts gain in BMCsize.

CYP6B cytochrome p450 monooxygenases from Papilio canadensis and Papilio glaucus: potential contributions of sequence divergence to host plant associations.

Two groups of furanocoumarin-inducible cytochrome p450 genes, the CYP6B4 group and the CYP6B17 group, characterized in two closely related tiger swallowtails, Papilio glaucus and Papilio canadensis, are induced to different extents, with generally higher levels of CYP6B transcripts in P. glaucus. To investigate the evolutionary history of these CYP6B genes in the context of their association with furanocoumarin detoxification, we isolated thirteen CYP6B genes from these species. Each of these genes contains an intron at a conserved position (1334 nucleotides from the translation start site), which varies in length due to three insertion/deletions. The proximal 5' end flanking sequence from the transcription initiation site is highly conserved (91-98% nt identity). The sequence 5' to -640 is significantly variable due largely to the presence of three insertion/deletions. The sequence at the 3' end of this region contains a putative xenobiotic response element to xanthotoxin (XRE-xan), important for basal and xanthotoxin-inducible transcription of the P. polyxenes CYP6B1v3 gene, and multiple elements known to regulate vertebrate phase I and II promoters, including an XRE-AhR (Xenobiotic Response Element to Aryl hydrocarbon Receptor), an OCT-1 element (octamer protein binding site), an ARE (Antioxidant Response Element), an EcRE (Ecdysone Response Element), and an imperfect PXR (Pregnane X Receptor) responsive element (PRE). Our analyses of CYP6B genes in these two species indicate that these genes are in an early stage of divergence and that differential exposure of these two species to chemically distinct host plants resulting from geographical isolation has had functional impacts not only on the coding regions of these genes but also on their promoter regions. Thus, changes in p450 regulation as well as catalytic activity may play a role in the evolution of host plant associations in herbivorous insects.

Endoscopic dacryocystorhinostomy in children.

BACKGROUND: Persistent nasolacrimal duct obstruction (NLDO) often requires treatment by probing, intubation, or balloon dacryoplasty. Refractory cases have been managed by external dacryocystorhinostomy (DCR), which leaves a scar; however, this procedure is generally avoided in young children. Endoscopic DCR has been successfully performed in adults and described in children. We report the success of this procedure in a series of pediatric patients. METHODS: A retrospective review of all endoscopic lacrimal procedures performed in a 3-year period was undertaken. Seventeen children (22 ducts) with persistent NLDO after at least one failed probing, with or without silicone tube placement, underwent endoscopic DCR. Follow-up ranged from 6 to 36 months, and success was defined as resolution of tearing and discharge by follow-up clinical evaluation and by parental history. RESULTS: All but 2 patients (88%) with NLDO showed complete resolution of tearing and discharge. These 2 patients had recurrent symptoms after the Crawford tubes were removed and required revision endoscopic DCR. No complications from this procedure were noted. CONCLUSIONS: Endoscopic DCR is a safe and effective means of treating persistent NLDO in infants and young children when simple probing, intubation, or balloon procedures have failed. The team ophthalmology-otolaryngology endoscopic approach provides a highly successful alternative for patients with a persistent distal obstruction that might otherwise require an external procedure.

The rod photoreceptors in retinopathy of prematurity: an electroretinographic study.

OBJECTIVE: To test the hypothesis that the more severe the acute phase retinopathy of prematurity (ROP) was in the preterm weeks, the more severely compromised is rod photoreceptor function after the ROP has resolved. METHODS: Electroretinographic (ERG) responses were recorded from 25 dark-adapted children (ages 2.5 months' postterm to 14 years) categorized by maximum, acute phase ROP (None to Very Severe). From the ERG a-wave "S," a sensitivity parameter for the rod photoreceptor response, and R(mp3), the saturated amplitude of the rod photoreceptor response were calculated using a model of the activation of rod phototransduction. The patients' results were compared with those of healthy controls (n = 71). RESULTS: Among those in the None, Mild, Moderate, and Severe categories, both S and R(mp3) varied significantly with severity of acute phase ROP. In the Very Severe category, ERG responses were too attenuated to calculate S and R(mp3). CONCLUSIONS: The rod photoreceptors must be involved in ROP. The more severe the acute phase ROP, the more severe is the compromise of the processes involved in the activation of phototransduction in the rods.

Management of orbital subperiosteal abscess in children.

OBJECTIVES: To present guidelines for the management of an orbital subperiosteal abscess (SPA) in children and to assess the efficacy and safety of transnasal endoscopic drainage of an orbital SPA. SETTING: Tertiary care children's hospital. PATIENTS: Nineteen patients treated for an SPA between July 1997 and December 1999. The age of the patients ranged from 17 months to 14 years (mean, 6 years). The male-female ratio was 10:9. Treatment modalities included transnasal endoscopic drainage (n = 11), external drainage (n = 3), and intravenous antibiotics alone (n = 5). RESULTS: Bilateral pansinusitis was the most common cause. All patients received an initial trial of intravenous antibiotics. Based on the Fisher exact test, no statistically significant differences were detected for age, sex, presence of gaze restriction, and radiographic findings. Based on multiple logistic regression, degree of proptosis was the only significant multivariate predictor of surgery (P =.003). The estimated probability of surgery was 6% when there was no proptosis, and 92% for 2 mm of proptosis. The location of the SPA determined the route of surgical drainage. Eleven patients with a medially based SPA underwent drainage via the transnasal endoscopic approach, and 3 with a superior SPA underwent drainage externally. The external approach was associated with a longer hospital stay (median, 7 days) than either the endoscopic or the intravenous antibiotic approach (median, 5 days).

Expression of CYP6B1 and CYP6B3 cytochrome P450 monooxygenases and furanocoumarin metabolism in different tissues of Papilio polyxenes (Lepidoptera: Papilionidae).

The CYP6B1 and CYP6B3 cytochrome P450 monooxygenases in the midgut of the black swallowtail participate in the metabolism of toxic furanocoumarins present in its host plants. In this study, biochemical analyses indicate that the fat body metabolizes significant amounts of the linear furanocoumarins bergapten and xanthotoxin after larvae feed on xanthotoxin. Northern analyses of the combined CYP6B1/3 transcript expression patterns indicate that transcripts in this P450 subfamily are induced in the midgut and fat body by xanthotoxin. Semi-quantitative RT-PCR analyses of individual CYP6B1/CYP6B3 mRNAs indicate that CYP6B1 transcripts are induced by xanthotoxin in all tissues examined and that CYP6B3 transcripts are induced in the fat body only. These results indicate that the fat body participates in the P450-mediated metabolism of excess furanocoumarins unmetabolized by the midgut. Although transcripts of both genes were detected and CYP6B1 transcripts were induced by xanthotoxin in the integument, furanocoumarin metabolism was not detected. Comparison of these P450 promoters with the promoters of alcohol dehydrogenase genes expressed in the fat bodies of several Drosophila species suggest that the xanthotoxin inducibilities of these P450 genes in fat bodies are regulated by elements other than those modulating expression of Adh genes.

A stereotactic radiation therapy device for retinoblastoma using a noncircular collimator and intensity filter.

The proximity of the lens to the retina makes the treatment of retinoblastoma a challenge for external beam radiation therapy. The approximately 1 mm separation between the posterior edge of the lens and the anterior region of the retina causes a trade-off between coverage of the entire retina and excessive dose to the lens. A stereotactic, LINAC based, lens sparing technique for treating retinoblastoma is presented. The technique uses noncoplanar arcs with the lens at isocenter. A special noncircular collimator blocks the lens but it also causes the dose distribution to vary across the retina. A fluence modulation filter is used to reduce the dose inhomogeneity across the target. The resulting dose distribution is roughly hemispheric, providing both anterior coverage of the retina and lens blocking unlike conventional techniques. The method used to develop the collimator and filter assembly is presented. Dosimetry of the assembly was carried out using radiochromic film, and the results were entered in a treatment planning system. The dose distribution as measured in a phantom is provided and compared to calculations.

Dark-adapted thresholds in children with histories of mild retinopathy of prematurity.

PURPOSE: To test the hypothesis that rod-mediated visual thresholds at 10 degrees eccentricity are elevated in children with histories of mild retinopathy of prematurity (ROP). METHODS: Dark-adapted thresholds for detection of 50 msec, 2 degrees diameter spots at a 10 degrees eccentric site, and at a peripheral reference site, 30 degrees eccentric, were measured in 20 children with a history of mild ROP and known courses of refractive development. Ten myopic control subjects also were tested. The thresholds of the ROP and control subjects were compared. RESULTS: Six of the subjects with ROP had elevated thresholds at the 10 degrees site. High myopia had been present since age 18 months or younger in each of the six. The thresholds of all other subjects with ROP, whose courses of refractive development had been toward emmetropia, and the control subjects with myopia were normal. In subjects with ROP, the association of early, persistent high myopia and an elevated threshold at 10 degrees was significant (chi 2 = 20; P < 0.01). Among the subjects with ROP, refractive error and axial length were correlated. CONCLUSIONS: ROP or factors causing ROP appear to alter rod-mediated retinal function. The association of abnormal rod-mediated sensitivity and refractive development suggests that rod-mediated retinal function is involved in the regulation of eye growth in children with a history of mild ROP.

Extracorporeal membrane oxygenation causing asymmetric vasculopathy in neonatal infants.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is a form of cardiopulmonary bypass therapy used in term or near-term infants with severe cardiorespiratory disorders not responsive to conventional intensive care interventions. An ECMO-associated retinal vasculopathy has been described with little reference to the specific condition of the patient. We examined the eyes of 91 infants who underwent ECMO treatment. An assessment was made of the following: (1) when retinal changes occurred, (2) whether there was a particular systemic disorder or ECMO approach associated with these retinal findings, and (3) whether there may be ocular sequelae from this development. METHODS: Ninety-one neonates were treated with ECMO for meconium aspiration syndrome (MAS), primary persistent pulmonary hypertension of the newborn, sepsis, congenital diaphragmatic hernia (CDH), respiratory distress syndrome (RDS), and blood aspiration. Venoarterial bypass was performed in 73 patients. The remaining 18 patients underwent venovenous bypass. Ophthalmologic examinations were performed during bypass in 6 infants and within 3 weeks of ECMO in the remainder. RESULTS: Asymmetric retinopathy (left eye > right eye) was discovered in six infants with CDH and in one infant with RDS within a 2-week period after bypass, demonstrating venous tortuosity with or without intraretinal hemorrhages. One infant treated for MAS had a left eye intraretinal hemorrhage only. All patients with the noted retinal changes underwent venoarterial cannulation. Six of 9 patients with CDH had retinal findings noted compared with 1 of 10 patients with RDS and 1 of 35 patients with MAS. CONCLUSION: Because we were able to examine infants while they were receiving ECMO or shortly after termination of bypass, asymmetric vasculopathy was found in a greater percentage of our patients compared with a similar large case series. ECMO-associated retinal vasculopathy appeared to disproportionately occur in those patients with CDH who underwent venoarterial bypass. Further study of retinal vascular changes in patients with CDH should be performed to assess long-term effects.

Bilateral infantile cataractogenesis in a patient with deficiency of complex I, a mitochondrial electron transport chain enzyme.

Progressive bilateral cataracts developed in infancy in a 5-month-old girl with deficiency of complex I, a mitochondrial electron transport chain enzyme. In the newborn period, she had severe lactic acidosis and the diagnosis of complex I deficiency was confirmed by mitochondrial respiratory chain assay on muscle biopsy. By 5 months, she had completely opaque nuclear sclerotic cataracts, with loss of fixation and the red reflex. She underwent bilateral, sequential cataract extraction. The lens aspirate was submitted for cytologic analysis and electron microscopy, which revealed increased intracellular glycogen and swollen mitochondria. To our knowledge the association of complex I deficiency with cataracts in infancy has not been reported previously. The diagnosis of a respiratory chain enzyme defect in infancy is an indication for early ophthalmic evaluation to identify cataracts that may result in visual loss. Conversely, the recognition of cataracts in infants with unexplained neurologic disease or metabolic acidosis may necessitate further evaluation for metabolic etiologies, including mitochondrial disorders.

The course of myopia in children with mild retinopathy of prematurity.

The courses of spherical equivalent in patients (n = 62) who had mild, non-cicatricial retinopathy of prematurity (ROP), and in those without a history of ROP (n = 25) were modeled as a linear function of age; an exponential model was also considered. A few (n = 5) without ROP have abnormal courses characterized by hyperopia in early infancy; none have poor acuity. Although the majority of patients with ROP have courses indistinguishable from those of term born controls, 27 (43.5%) have abnormal courses, most of which are toward myopia. Optotype acuities were significantly poorer among the ROP patients with abnormal than normal refractive courses. Thus abnormal refractive development and acuity deficits are associated in eyes that have had mild ROP.

Outcome of treatment for bilateral congenital cataracts.

The outcome of treatment for bilateral congenital cataracts was studied retrospectively in a group of 51 patients. Two major categories of lens opacities were identified. In the first category, the opacities were extensive and visual impairment was evident early in the first year. These cataracts often occurred in eyes with small corneal diameters and poorly dilating pupils. Postoperative strabismus was nearly universal; nystagmus developed in over 50%; and late onset open-angle glaucoma developed in 8 of the 29 patients studied. Early surgery did not seem to abort the development of nystagmus in this group of patients. In the second category, the lens opacities were partial, often lamellar in configuration, and visual impairment was less severe. Surgery was usually performed after 3 years of age, with good visual results if the opacities were symmetrical and there was no nystagmus. No deprivation amblyopia developed in this group, even when surgery was delayed into the second decade. Strabismus developed postoperatively in about a third, but so far, no delayed open-angle glaucoma has been identified.

Optic disc drusen and primary megalencephaly in children.

Three patients with primary megalencephaly were identified among 40 children seen in our clinic with pseudopapilledema due to optic disc drusen. These patients were genetically unrelated and did not have cutaneous hemangiomas, distinguishing their condition from the Riley-Smith syndrome. Although the nature of the relationship between optic disc drusen and primary megalencephaly is not clear, we feel that the two conditions have more than a chance association. A clinically important consideration is that the concomitant occurrence of pseudopapilledema due to optic disc drusen and a large head due to primary megalencephaly may lead to an erroneous diagnosis of increased intracranial pressure in children.