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Background : The current coronavirus disease 2019 (COVID-19) pandemic began in Ireland with the first confirmed positive case in March 2020. In the early stages of the pandemic clinicians and researchers in two affiliated Dublin hospitals identified the need for a COVID-19 biobanking initiative to support and enhance research into the disease. Through large scale analysis of clinical, regional, and genetic characteristics of COVID-19 patients, biobanks have helped identify, and so protect, at risk patient groups The STTAR Bioresource has been created to collect and store data and linked biological samples from patients with SARS-CoV-2 infection and healthy and disease controls. Aim : The primary objective of this study is to build a biobank, to understand the clinical characteristics and natural history of COVID-19 infection with the long-term goal of research into improved disease understanding, diagnostic tests and treatments. Methods : This is a prospective dual-site cohort study across two tertiary acute university teaching hospitals. Patients are recruited from inpatient wards or outpatient clinics. Patients with confirmed COVID-19 infection as well as healthy and specific disease control groups are recruited.  Biological samples are collected and a case report form detailing demographic and medical background is entered into the bespoke secure online Dendrite database. Impact : The results of this study will be used to inform national and international strategy on health service provision and disease management related to COVID-19. In common with other biobanks, study end points  evolve over time as new research questions emerge. They currently include patient survival, occurrence of severe complications of the disease or its therapy, occurrence of persistent symptoms following recovery from the acute illness and vaccine responses., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 O'Doherty L et al.)

Background: Simulation is reported as an appropriate replacement for a significant number of clinical hours in pregraduate programmes. To increase access for learners, educators have looked to understanding and improving learning in observer roles. Studies report equivalent learning outcomes and less stress in observer roles. However, reports on the prevalence, use and perceived value of observer roles from the educator's perspective are lacking., Methods: An exploratory survey for Australian and New Zealand (ANZ) simulation educators based on literature findings was developed and piloted with a small sample (n=10) of like subjects for language, clarity, skip logic and completion time. The final survey comprised 36 questions. Quantitative data were analysed using Pearson's chi-squared test, Welch's ANOVA and exploratory factor analysis. Select qualitative data were analysed using content analysis and summarised with frequency counts and categorisation., Results: Two hundred and sixty-seven surveys were completed, with 221 meeting criteria for analysis. The observer role is widely used in ANZ and most learners experience both hands-on and observer roles. The location of observers is dependent upon several factors including facility design, learner immersion, scenario design and observer involvement. Verbal briefings and/or other guides are provided to 89% of observers to direct their focus and 98% participate in the debrief. Educators value observer roles but tend to believe the best learning is hands-on., Conclusions: The learning in observer roles is less valued by educators than hands-on roles. Focused observation provides opportunities for noticing and attributing meaning, an essential skill for clinical practice. Learning spaces require consideration of scenario design and learning objectives. Scenario design should include objectives for observer roles and incorporate the observer into all phases of simulation. Attention to these areas will help promote the value of the different type of learning available in observer roles., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Effective communication between patients-clinicians, supervisors-learners and facilitators-participants within a simulation is a key priority in health profession education. There is a plethora of frameworks and recommendations to guide communication in each of these contexts, and they represent separate discourses with separate communities of practice and literature. Finding common ground within these frameworks has the potential to minimise cognitive load and maximise efficiency, which presents an opportunity to consolidate messages, strategies and skills throughout a communication curriculum and the possibility of expanding the research agenda regarding communication, feedback and debriefing in productive ways., Methods: A meta-synthesis of the feedback, debriefing and clinical communication literature was conducted to achieve these objectives., Results: Our analysis revealed that the concepts underlying the framework can be usefully categorised as stages, goals, strategies, micro-skills and meta-skills. Guidelines for conversations typically shared a common structure, and strategies aligned with a stage. Core transferrable communication skills (i.e., micro-skills) were identified across various types of conversation, and the major differences between frameworks were related to the way that power was distributed in the conversation and the evolution of conversations along the along the path of redistributing power. As part of the synthesis, an overarching framework "prepare-EMPOWER enact" was developed to capture these shared principles across discourses., Conclusions: Adopting frameworks for work-based communication that promote dialogue and empower individuals to contribute may represent an important step towards learner-centred education and person-centred care for patients.

The neuromuscular junction (NMJ) is one of the best-studied cholinergic synapses. Inherited defects of peripheral neurotransmission result in congenital myasthenic syndromes (CMSs), a clinically and genetically heterogeneous group of rare diseases with fluctuating fatigable muscle weakness as the clinical hallmark. Whole-exome sequencing and Sanger sequencing in six unrelated families identified compound heterozygous and homozygous mutations in SLC5A7 encoding the presynaptic sodium-dependent high-affinity choline transporter 1 (CHT), which is known to be mutated in one dominant form of distal motor neuronopathy (DHMN7A). We identified 11 recessive mutations in SLC5A7 that were associated with a spectrum of severe muscle weakness ranging from a lethal antenatal form of arthrogryposis and severe hypotonia to a neonatal form of CMS with episodic apnea and a favorable prognosis when well managed at the clinical level. As expected given the critical role of CHT for multisystemic cholinergic neurotransmission, autonomic dysfunctions were reported in the antenatal form and cognitive impairment was noticed in half of the persons with the neonatal form. The missense mutations induced a near complete loss of function of CHT activity in cell models. At the human NMJ, a delay in synaptic maturation and an altered maintenance were observed in the antenatal and neonatal forms, respectively. Increased synaptic expression of butyrylcholinesterase was also observed, exposing the dysfunction of cholinergic metabolism when CHT is deficient in vivo. This work broadens the clinical spectrum of human diseases resulting from reduced CHT activity and highlights the complexity of cholinergic metabolism at the synapse., (Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: Simulation is widely used in health professional education. The convention that learners are actively involved may limit access to this educational method. The aim of this paper is to review the evidence for learning methods that employ directed observation as an alternative to hands-on participation in scenario-based simulation training. We sought studies that included either direct comparison of the learning outcomes of observers with those of active participants or identified factors important for the engagement of observers in simulation. We systematically searched health and education databases and reviewed journals and bibliographies for studies investigating or referring to observer roles in simulation using mannequins, simulated patients or role play simulations. A quality framework was used to rate the studies., Methods: We sought studies that included either direct comparison of the learning outcomes of observers with those of active participants or identified factors important for the engagement of observers in simulation. We systematically searched health and education databases and reviewed journals and bibliographies for studies investigating or referring to observer roles in simulation using mannequins, simulated patients or role play simulations. A quality framework was used to rate the studies., Results: Nine studies met the inclusion criteria. Five studies suggest learning outcomes in observer roles are as good or better than hands-on roles in simulation. Four studies document learner satisfaction in observer roles. Five studies used a tool to guide observers. Eight studies involved observers in the debrief. Learning and satisfaction in observer roles is closely associated with observer tools, learner engagement, role clarity and contribution to the debrief. Learners that valued observer roles described them as affording an overarching view, examination of details from a distance, and meaningful feedback during the debrief. Learners who did not value observer roles described them as passive, or boring when compared to hands-on engagement in the simulation encounter., Conclusions: Learning outcomes and role satisfaction for observers is improved through learner engagement and the use of observer tools. The value that students attach to observer roles appear contingent on role clarity, use of observer tools, and inclusion of observers' perspectives in the debrief.

Phloem, a plant tissue responsible for long-distance molecular transport, harbours specific junctions, sieve areas, between the conducting cells. To date, little is known about the molecular framework related to the biogenesis of these sieve areas. Here we identify mutations at the CHER1/AtCTL1 locus of Arabidopsis thaliana. The mutations cause several phenotypic abnormalities, including reduced pore density and altered pore structure in the sieve areas associated with impaired phloem function. CHER1 encodes a member of a poorly characterized choline transporter-like protein family in plants and animals. We show that CHER1 facilitates choline transport, localizes to the trans-Golgi network, and during cytokinesis is associated with the phragmoplast. Consistent with its function in the elaboration of the sieve areas, CHER1 has a sustained, polar localization in the forming sieve plates. Our results indicate that the regulation of choline levels is crucial for phloem development and conductivity in plants.

Purpose: The Central Retinal Enrichment Supplementation Trials (CREST) aim to investigate the potential impact of macular pigment (MP) enrichment, following supplementation with a formulation containing 10 mg lutein (L), 2 mg zeaxanthin (Z) and 10 mg meso-zeaxanthin (MZ), on visual function in normal subjects (Trial 1) and in subjects with early age-related macular degeneration (AMD; Trial 2)., Methods: CREST is a single center, double-blind, randomized clinical trial. Trial 1 (12-month follow-up) subjects are randomly assigned to a formulation containing 10 mg L, 10 mg MZ and 2 mg Z (n = 60) or placebo (n = 60). Trial 2 (24-month follow-up) subjects are randomly assigned to a formulation containing 10 mg L, 10 mg MZ, 2 mg Z plus 500 mg vitamin C, 400 IU vitamin E, 25 mg zinc and 2 mg copper (Intervention A; n = 75) or 10 mg L and 2 mg Z plus 500 mg vitamin C, 400 IU vitamin E, 25 mg zinc and 2 mg copper (Intervention B; n = 75). Contrast sensitivity (CS) at 6 cycles per degree represents the primary outcome measure in each trial. Secondary outcomes include: CS at other spatial frequencies, MP, best-corrected visual acuity, glare disability, photostress recovery, light scatter, cognitive function, foveal architecture, serum carotenoid concentrations, and subjective visual function. For Trial 2, AMD morphology, reading speed and reading acuity are also being recorded., Conclusions: CREST is the first study to investigate the impact of supplementation with all three macular carotenoids in the context of a large, double-blind, randomized clinical trial.

Artefacts arising from head movements have been a considerable obstacle in the deployment of automatic event detection systems in ambulatory EEG. Recently, gyroscopes have been identified as a useful modality for providing complementary information to the head movement artefact detection task. In this work, a comprehensive data fusion analysis is conducted to investigate how EEG and gyroscope signals can be most effectively combined to provide a more accurate detection of head-movement artefacts in the EEG. To this end, several methods of combining these physiological and physical signals at the feature, decision and score fusion levels are examined. Results show that combination at the feature, score and decision levels is successful in improving classifier performance when compared to individual EEG or gyroscope classifiers, thus confirming that EEG and gyroscope signals carry complementary information regarding the detection of head-movement artefacts in the EEG. Feature fusion and the score fusion using the sum-rule provided the greatest improvement in artefact detection. By extending multimodal head-movement artefact detection to the score and decision fusion domains, it is possible to implement multimodal artefact detection in environments where gyroscope signals are intermittently available., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Contamination of EEG signals by artefacts arising from head movements has been a serious obstacle in the deployment of automatic neurological event detection systems in ambulatory EEG. In this paper, we present work on categorizing these head-movement artefacts as one distinct class and on using support vector machines to automatically detect their presence. The use of additional physical signals in detecting head-movement artefacts is also investigated by means of support vector machines classifiers implemented with gyroscope waveforms. Finally, the combination of features extracted from EEG and gyroscope signals is explored in order to design an algorithm which incorporates both physical and physiological signals in accurately detecting artefacts arising from head-movements., (Copyright © 2012 IPEM. Published by Elsevier Ltd. All rights reserved.)

The Na(+)-independent, high affinity choline carrier system proposed to supply choline for the synthesis of cell membrane phospholipids was recently associated with SLC44 family members (SLC44A1-5) also called choline-like transporter family. SLC44A1 is widely expressed throughout the nervous system in both neurons and oligodendrocytes, while SLC44A2-4 are mainly detected in peripheral tissues. The subcellular localization of the proteins was mainly addressed for SLC44A1 through the development of specific antibodies. SLC44A1 is detected in both the plasma and mitochondrial membranes where the protein is able to transport choline at high affinity and in a Na(+)-independent manner. The physiological relevance of SLC44A1 as a choline carrier is indicated by its likely involvement in membrane synthesis for cell growth or repair, and also by its role in phospholipid production for the generation of lung surfactant. Moreover, an autoimmune disease has been related to the blockade of SLC44A2 function, which results in the alteration of hair cells in the inner ear and leads to autoimmune hearing loss. In the alloimmune syndrome called transfusion-related acute lung injury, antibodies to SLC44A2 cause a deleterious aggregation of granulocytes. Therefore transporters of the SLC44 family represent attractive and promising targets for therapeutic and diagnostic applications regarding both immune and degenerative diseases., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Cystinosin, the lysosomal cystine exporter defective in cystinosis, is the founding member of a family of heptahelical membrane proteins related to bacteriorhodopsin and characterized by a duplicated motif termed the PQ loop. PQ-loop proteins are more frequent in eukaryotes than in prokaryotes; except for cystinosin, their molecular function remains elusive. In this study, we report that three yeast PQ-loop proteins of unknown function, Ypq1, Ypq2, and Ypq3, localize to the vacuolar membrane and are involved in homeostasis of cationic amino acids (CAAs). We also show that PQLC2, a mammalian PQ-loop protein closely related to yeast Ypq proteins, localizes to lysosomes and catalyzes a robust, electrogenic transport that is selective for CAAs and strongly activated at low extracytosolic pH. Heterologous expression of PQLC2 at the yeast vacuole rescues the resistance phenotype of an ypq2 mutant to canavanine, a toxic analog of arginine efficiently transported by PQLC2. Finally, PQLC2 transports a lysine-like mixed disulfide that serves as a chemical intermediate in cysteamine therapy of cystinosis, and PQLC2 gene silencing trapped this intermediate in cystinotic cells. We conclude that PQLC2 and Ypq1-3 proteins are lysosomal/vacuolar exporters of CAAs and suggest that small-molecule transport is a conserved feature of the PQ-loop protein family, in agreement with its distant similarity to SWEET sugar transporters and to the mitochondrial pyruvate carrier. The elucidation of PQLC2 function may help improve cysteamine therapy. It may also clarify the origin of CAA abnormalities in Batten disease.

Choline is an essential nutrient necessary for synthesis of membrane phospholipids, cell signalling molecules and acetylcholine. The aim of this study was to detect and characterize the choline transporter-like 1 (CTL1/SLC44A1) protein in CNS tissues and the hybrid neuroblastoma x glioma cell line NG108-15, which synthesizes acetylcholine and has high affinity choline transport but does not express the cholinergic high affinity choline transporter 1. The presence of CTL1 protein in NG108-15 cells was confirmed using our antibody G103 which recognizes the C-terminal domain of human CTL1. Three different cognate small interfering RNAs were used to decrease CTL1 mRNA in NG108-15 cells, causing lowered CTL1 protein expression, choline uptake and cell growth. None of the small interfering RNAs influenced carnitine transport, demonstrating the absence of major non-specific effects. In parental C6 cells knockdown of CTL1 also reduced high affinity choline transport. Our results support the concept that CTL1 protein is necessary for the high affinity choline transport which supplies choline for cell growth. The presence of CTL1 protein in rat and human CNS regions, where it is found in neuronal, glial and endothelial cells, suggests that malfunction of this transporter could have important implications in nervous system development and repair following injury, and in neurodegenerative diseases.

Cell volume controls many functions and is itself regulated. To study cell volume regulations, the mean volume of C6-BU-1 rat glioma cells was electronically measured under isotonic and anisotonic conditions. Two isotonic solutions were used containing either normal (solution 1) or low (solution 2) NaCl. Anisotonicity was induced by changing NaCl or sucrose concentrations in solutions 1 and 2, respectively. The cells behaved like perfect osmometers when the tonicity was increased. In contrast, just after hypotonic challenges, the cell volume was smaller than that predicted by a perfect osmometer. This deviation reveals a new mechanism, which we call the volume increase limitation (VIL). When hypotonicity was induced by decreasing NaCl, a classical slow regulatory volume decrease (RVD) was also observed in addition to VIL. The cells expressed aquaporin-1 sensitive to HgCl(2) and decreased by siRNA, which both reduced fast volume changes. In this study, we show that: (1) RVD is proportional to the change in external Cl(-) concentration and is inhibited by Cl(-) channel and K(+)-Cl(-) cotransporter blockers; (2) cell swelling due to the influx of H(2)O through aquaporins shows rectification with decreasing osmolarity and is sensitive to the internal Na(+) concentration; (3) VIL is linearly related with hypotonicity and is abolished in solutions 1 and 2 by the Na(+) ionophore monensin and in solution 1 by the Na(+)-K(+) ATPase inhibitor ouabain. These results suggest that VIL is triggered by the decrease in internal Na(+) caused by hyponatrema and cell swelling. In addition to RVD, VIL should protect cells during hyposmotic stress.

The amount of carbon stored in savannas represents a significant uncertainty in global carbon budgets, primarily because fire causes actual biomass to differ from potential biomass. We analyzed the structural response of woody plants to long-term experimental burning in savannas. The experiment uses a randomized block design to examine fire exclusion and the season and frequency of burn in 192 7-ha experimental plots located in four different savanna ecosystems. Although previous studies would lead us to expect tree density to respond to the fire regime, our results, obtained from four different savanna ecosystems, suggest that the density of woody individuals was unresponsive to fire. The relative dominance of small trees was, however, highly responsive to fire regime. The observed shift in the structure of tree populations has potentially large impacts on the carbon balance. However, the response of tree biomass to fire of the different savannas studied were different, making it difficult to generalize about the extent to which fire can be used to manipulate carbon sequestration in savannas. This study provides evidence that savannas are demographically resilient to fire, but structurally responsive.

Study Objectives: To prospectively evaluate compliance with current interhospital trauma transfer guidelines in South West Sydney, before and after an implementation programme was instituted., Methods: A scoring system was developed to assess compliance with the 11 main components of the guideline. Baseline compliance was measured during an initial 3-month period (pre), followed by an implementation programme to alert staff at referring hospitals to the presence of the guidelines. Following this, compliance was again measured over 3 months (post)., Results: Twenty-two patients were transferred during the pre-implementation phase and 35 patients during the post-phase. Overall compliance with the guidelines increased from 62 to 67%. Mean pre-hospital compliance rose from 75 to 95%, and referring hospital compliance rose from 59 to 63%. While there was an improvement in compliance with the use of the dedicated trauma hotline (86-97%), the use of a transfer checklist (41-53%), and appropriateness of transfer (95-100%), none of these reached statistical significance., Conclusion: Practice guidelines have been developed to optimise the process of interhospital trauma transfers. An implementation programme met with limited success in improving compliance with the guidelines. Further work is needed to ensure awareness of these guidelines, with ongoing monitoring to ensure best practice and optimal patient outcome.

Immunophilins are ubiquitous enzymes responsible for proline isomerisation during protein synthesis and for the chaperoning of several membrane proteins. These activities can be blocked by the immunosuppressants cyclosporin A, FK506 and rapamycin. It has been shown that all three immunosuppressants have neurotrophic activity and can modulate neurotransmitter release, but the molecular basis of these effects is currently unknown. Here, we show that synapsin I, a synaptic vesicle-associated protein, can be purified from Torpedo cholinergic synaptosomes through its affinity to cyclophilin B, an immunophilin that is particularly abundant in brain. The interaction is direct and conserved in mammals, and shows a dissociation constant of about 0.5 microM in vitro. The binding between the two proteins can be disrupted by cyclosporin A and inhibited by physiological concentrations of ATP. Furthermore, cyclophilin B co-localizes with synapsin I in rat synaptic vesicle fractions and its levels in synaptic vesicle-containing fractions are decreased in synapsin knockout mice. These results suggest that immunophilins are involved in the complex protein networks operating at the presynaptic level and implicate the interaction between cyclophilin B and synapsins in presynaptic function.

For decades, the connection between cell size and division has been the subject of controversy. While in yeast, cell size checkpoints coordinate cellular growth with cell-cycle progression, it has been recently shown that large and small Schwann cells proliferate at the same rate (Conlon and Raff, 2003, J Biol 2:7). From this point of view, it is important to know whether normal and tumoral cells are similar. During continuous culture of NG108-15 neuroblastoma x glioma cells, the rate of proliferation, cell size, and external pH changed in parallel. At constant pH, the cell size-proliferation relationship followed a bell-shaped curve, so that proliferation was optimal within a cell volume window. In contrast, external acidification decreased proliferation independently of cell size. Using electrophysiological techniques, we showed that changes in cell size were dependent on both the uptake of nutrients and the passive influx of ions. Furthermore, an increase in cell size was associated with an increase in total proteins/cell. Another way to influence cell growth and proliferation is to alter the activity of the PI-3 kinase and target of rapamycin (TOR) signaling pathway. In NG108-15 cells, pharmacological inhibition of these proteins with LY 294002 and rapamycin respectively decreased proliferation but did not modify cell size. In contrast, aphidicolin treated cells did not proliferate, but they continued to increase in size. Altogether these results indicate that the proliferation of NG108-15 cells is controlled by both cell size-dependent and independent mechanisms that include extracellular pH and PI-3 kinase activity., (2004 Wiley-Liss, Inc.)

The presumptive choline transporter, CTL1, was initially identified through functional complementation of a triple yeast mutant (ctr ise URA3delta) with deficiencies in both choline transport and choline neosynthesis under selective conditions that cause perturbations in membrane synthesis and growth. After transformation of these yeasts with a heterologous yeast expression library made from Torpedo electric lobe cDNAs, several colonies showed increased growth but only one clone increased the accumulation of external choline. The corresponding full-length cDNA was isolated and encodes a protein with 10 transmembrane domains. Northern analysis of Torpedo mRNA indicates that CTL1 is expressed at high levels in the spinal cord and brain. In Xenopus oocytes, Torpedo CTL1 expression was associated with the appearance of sodium independent high-affinity choline uptake. We propose that CTL1 plays a role in providing choline for membrane synthesis in the nervous system.

We initially isolated CTL1 from the electric lobe of brain through functional complementation of a yeast mutant deficient in choline transport. Here, we present the first characterization of an antibody to the C-terminal of CTL1. When full length torpedo CTL1 was expressed in oocytes, a broad 60 kDa band appeared concomitant with the detection of immunoreactivity at the plasma membrane. In, the native protein was prominent throughout the CNS and along the electric nerves. CTL1 immunolabeling was particularly conspicuous in the myelin sheath surrounding the electric nerve and in central myelinated structures. The association of the presumptive choline transporter, CTL1, with myelin membranes suggests a role for this new protein in lipid production.

Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine. We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation. The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast. The gene was called CTL1 for its choline transporter-like properties. The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3. 5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon. In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain. High levels of rCTL1 were also identified in the mucosal cell layer of the colon. Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2-4). These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons.

A size-fractionated torpedo electric lobe cDNA library was screened for the neuronal choline transporter by functional expression in oocytes. A clone, TLC2B, was isolated that induced a component of choline uptake that was hemicholinium-3 sensitive and inhibited by the substitution of lithium for sodium at low choline concentrations. However, [3H]choline uptake by both injected and non-injected oocytes were characterized by high affinity constants, suggesting that TLC2B could be affecting a native choline transporter. Indeed, hemicholinium-3 sensitive choline uptake could also be induced by preincubation of non-injected oocytes with a protein kinase C inhibitor, H-7. By sequence analysis and immuno-precipitation, the peptide produced by injection of TLC2B cRNA was identified as a soluble 24 kDa C-terminal fragment of the neuronal protein, synaptotagmin. Full length synaptotagmin was, however, ineffective in the functional test. The peptide encoded by TLC2B corresponds to the second protein kinase C-homologous domain of torpedo synaptotagmin, and like other soluble C2 domain peptides, was capable of calcium-dependent translocation to membranes. Its action on choline uptake in oocytes was, however, abolished by the addition of calcium in the presence of a calcium ionophore. These results suggest that the interaction of certain C2 domains, such as the C-terminal domain of synaptotagmin, with more specific targets may be anulled in the presence of calcium due to its absorption to membrane phospholipids.

The Ewing's sarcoma cell line ICB 112 was examined in detail for a cholinergic phenotype. Choline acetyltransferase activity (12.3 +/- 2.9 nmol/h/mg of protein) was associated with the presence of multiple mRNA species labeled with a human choline acetyltransferase riboprobe. Choline was taken up by the cells by a high-affinity, hemicholinium-3-sensitive transporter that was partially inhibited when lithium replaced sodium in the incubation medium; the choline taken up was quickly incorporated into both acetylcholine and phosphorylcholine. High-affinity binding sites for vesamicol, an inhibitor of vesicular acetylcholine transport, were also present. The mRNAs for synaptotagmin (p65) and the 15-kDa proteolipid were readily detected and were identical in size to those observed in cholinergic regions of the human brain. Cumulative acetylcholine efflux was increased by raising the extracellular potassium level or the addition of a calcium ionophore, but the time course of stimulated efflux was slow and persistent. These results show that this morphologically undifferentiated cell line is capable of acetylcholine synthesis and expresses markers for synaptic vesicles as well as proteins implicated in calcium-dependent release but lacks an organized release mechanism.

A large spectrum of ocular pathologic features have been described in infantile cystinosis. Reported symptoms may require corneal transplantation and therapeutic efforts to solubilize cystine crystals with hourly administration of cysteamine (mercaptamine) drops. French Canada has the highest incidence of cystinosis in the world. We studied 18 patients with cystinosis who appeared to have much milder ocular involvement than that in other reported series. No operative interventions were required. Relatively mild photophobia was the most common ocular manifestation of cystinosis.

Eleven pediatric patients with nephrosis and focal segmental glomerulosclerosis were treated with long-term (8-38 months) ciclosporin A in combination with steroids. All had abnormal height-velocity curves and multiple hospitalizations for complications of nephrosis. Eight patients attained remission with a dramatic improvement in growth and decrease in necessity for hospitalization for therapy of nephrosis complications, while maintaining adequate renal function. Three nonresponders developed end-stage renal disease. Long-term ciclosporin A therapy may be of benefit in steroid-resistant nephrosis in childhood.

Formation of posterior subcapsular cataracts is a known complication of systemic corticosteroid therapy. However, the relation between steroid dosage, cumulative dose and type of steroid on one hand and the subsequent formation of cataract on the other is unclear. We carried out a study to determine the incidence of posterior subcapsular cataracts in 64 children who had undergone renal transplantation and to attempt to determine what factors were associated with cataract formation. Cataracts were detected in 17 (26%) of the patients. The steroid dosage, cumulative dose and duration of therapy were not associated with cataract formation. There was a significant difference in the distribution of HLA-CW3 antigen between the patients with cataracts and those without cataracts. The reason for the link between corticosteroid therapy and formation of posterior subcapsular cataracts remains unclear.

Hemolytic-uremic syndrome (HUS) of childhood is a triad of acute hemolytic anemia, thrombocytopenia, and acute renal failure associated with a gastrointestinal prodrome. From 1977 to 1988, 134 patients with HUS were admitted to this institution. All patients presented with abdominal pain and diarrhea, which was virtually always bloody. Seventy-eight patients (60%) required dialysis. Five patients died (4%). One patient died as a result of colon perforation, the other four patients died of other nonsurgical complications of HUS. Three patients underwent exploratory laparotomy. One patient had a hemoperitoneum from mesenteric and transmural bleeding of the entire intraabdominal colon. Another patient had undergone surgery elsewhere for presumed intussusception with pancolitis found at exploration. Fourteen days postoperatively, he had a spontaneous perforation of the transverse colon. The third patient presented with pancolitis and perforation of the transverse colon. Despite surgical intervention he died on the sixth postoperative day. One other patient was treated conservatively for pancreatitis, which developed 3 weeks after her presentation with HUS. Complications requiring surgical intervention in HUS are rare, potentially lethal, and usually involve the colon.

The major pathological findings in 23 patients with hereditary tyrosinemia type I seen at the Hôpital Sainte-Justine over a 23-year period are reviewed in combination with findings in the literature. Hepatic and renal alterations are given special emphasis. Hepatic changes differ in the acute and chronic forms of the disease. The former is characterized by alterations shared by several hepatopathies of infancy, whereas the latter is characterized by established cirrhosis, frequently of a mixed macro- and micronodular type, with a frightening propensity for the development of hepatocellular carcinoma. Renal changes reflect tubular injury, resulting in Fanconi syndrome, with tubular dilatation, nephrocalcinosis, and involution of epithelial cells. A significant proportion of patients also reveal some degree of glomerulosclerosis and interstitial fibrosis, indicating at least the need for careful assessment and follow-up of renal function, particularly in light of the adverse renal effects of immunosuppressive regimens used in liver transplantation.

High dose loop diuretic therapy was administered at time of admission to hospital or at time of diagnosis to 54 patients with childhood hemolytic uremic syndrome. Forty-one patients maintained a diuresis sufficient to avoid the necessity for dialysis. When compared to an earlier time period the dialysis rate fell from 82% to 24% with furosemide therapy. Though a change in disease severity may explain the decrease in necessity for dialysis, a salutary effect of furosemide therapy may also be responsible.

A wide spectrum of renal diseases ranging from glomerulopathic lesions to tumors may be associated with HIV infection. This infection may also complicate the course of dialysis and renal transplantation. We review the renal complications of HIV infection.

Experience with percutaneous gastrojejunostomy is described in children on chronic cycler peritoneal dialysis. We recommend this mode of enteral alimentation for children requiring caloric supplementation.

Peritoneal dialysis catheters are at risk for exit-site contamination when swimming or bathing. Also in pre-toilet trained children fecal and urine contamination can occur. The use of Tegaderm dressings prevents contamination and thus minimizes restriction of activity in children.

Between 1971 and 1980, 55 children with urolithiasis were assessed and treated. Pediatric urolithiasis accounted for 1:4090 hospital admissions. Forty percent of patients had no identifiable predisposition to urolithiasis. Eight patients presented with hematuria in the absence of renal colic suggesting that pediatric urolithiasis, although uncommon, is an important cause of painless hematuria.

Constipation was confirmed by history, rectal examination, and rectal manometric studies in 22 of 25 children with enuresis. Treatment of constipation resulted in resolution of enuresis. Uninhibited bladder contractions, observed in enuretic constipated children, were also noted in children with constipation alone, suggesting that constipation is a commonly unrecognized etiologic factor in enuresis.

Echocardiographic assessment of cardiac function was made on 24 children with chronic renal failure of varying etiology and severity. In 20 patients without evidence of cardiac failure, parameters of left ventricular performance as represented by PEP/LVET and mean velocity of circumferential fiber shortening were within normal limits in the majority of patients. In addition, ejection fraction and shortening fraction were, in most children, within the 95% confidence limits for their age. In 4 patients who presented with congestive heart failure, marked left ventricular dilatation was noted in association with decreased shortening and ejection fractions and depressed mean velocity of circumferential fiber shortening. Also the PEP/LVET in these patients suggested the presence of a uremic cardiomyopathic condition. These studies, in addition to our own studies on children who have undergone fistula construction, hemodialysis, and transplantation, suggest that cardiac performance, in the majority of pediatric patients with end-stage renal disease, is well maintained and that the major factor involved in reducing exercise tolerance is the presence of uremic anemia. Only a minority of patients may develop severe uremic heart disease.

The oligo-anuria of the hemolytic uremic syndrome is attributed to the presence of a renal lesion which is predominantly glomerulopathic but which may have a vasculopathic component of varying severity. Fourteen children, four of whom had anuric, four oliguric and six non oliguric acute renal failure were treated with intravenous fluids and high dose intravenous furosemide therapy. Polyuria was induced in all, obviating the need for dialysis. We hypothesize that oligo-anuria in this syndrome may be due to the previously recognized hyperuricemia causing a urate nephropathy superimposed on the glomerulopathy thus explaining its possible amenability to fluid and diuretic therapy.

Four patients with total aganglionosis of the intestine had renal abnormalities. Two patients had cortical subcapsular cysts, one had unilateral renal agenesis and one other had unilateral renal dysplasia. The consistency of renal anomalies with total aganglionosis suggests the presence of a common genetic origin.

In our study, 32 nephrotic children with focal glomerular sclerosis were observed for an average period of 8 years (ranging 1-19 years of age). Of the 32, 25 children showed histological lesions of focal segmental sclerosis (FSG) and 7 of focal global glomerulosclerosis (FGG). All patients were reevaluated in 1979, creating the most recent status as follows: for children with FSG, 6 (24%) are in remission, 10 (40%) have a relatively normal renal function but exhibit either a persisting proteinuria (PP) or a recurrent nephrotic syndrome (NS), 1 (4%) is in chronic renal failure, 5 (20%) required dialysis and transplantation, and 3 (12%) died from non-renal causes. For children with FGG, 4 (57%) are in remission, 2 (29%) have a good renal function but display either PP or NS, and 1 patient (14%) is in chronic renal failure. The long-term observation of our study shows a more favorable prognosis than the one reported in researched literature. We believe that such results reflect a difference in the type of population encountered in our institution. Our population of patients represents less a highly referred population than the one of the centers who reported similar long-term studies, and we believe therefore that our study may represent a wider spectrum of the natural history of the disease. Moreover, the group of patients with PP or NS does not show a progressive decrease of glomerular filtration rate with time, which suggests that the disease may progress in a stepladder fashion.

Six patients who had neonatal renal vein thrombosis underwent re-evaluation of renal function 21 months to 12 years after initial diagnosis. Plasma creatinine clearances were normal in all patients as was urinary concentrating ability and urinalysis. Five patients had elevated systolic and diastolic blood pressures. However plasma renin activities were normal. Intravenous pyelography and renal scintigraphy revealed morphologic abnormalities in four patients. This study emphasizes that after apparent full recovery from neonatal renal vein thrombosis, arterial hypertension may persist and warrants careful long-term follow-up.

Following an episode of rheumatic carditis, severe mitral incompetence developed in a 9-year-old girl. A mitral annuloplasty succeeded for a short time in ameliorating her symptoms of cardiac failure. However, mitral incompetence recurred and was accompanied by severe anemia and hemosiderinuria. Distortion of erythrocytes was evident on a peripheral blood smear. A second mitral annuloplasty resulted in resolution of the hemolytic anemia.

The uptake of acetate and its incorporation into acetylcholine were measured under various conditions in nerve terminals isolated from the electric organ in order to characterize acetate uptake and to study the relationship between acetate uptake and acetylcholine synthesis in a pure cholinergic preparation. It was found that increasing extracellular choline up to 10(?4) M had no effect on either acetate uptake or the conversion of acetate to ACh, while the addition of hemicholinium-3 to the incubation medium led to decreases in both parameters. Hence, it appears that endogenous levels of choline are sufficient to support ongoing acetylcholine synthesis in this preparation and that this synthesis depends to some extent on the uptake of extracellular choline. Nonetheless, in the absence of choline uptake, both the uptake of acetate and the conversion of acetate to acetylcholine remained substantial, indicating that internal sources of choline as well can be used for acetylcholine synthesis. Acetate uptake displayed a marked requirement for external Na(+) and was decreased following depolarization of the synaptosomes by an elevated K(+) concentration. The conversion of acetate to acetylcholine followed a similar pattern, except that a small reduction in acetylcholine synthesis was observed in the absence of external Ca(2+), while acetate uptake was unaffected. The addition of ATP, AMP-PNP or phosphate to the incubation medium caused an increase in both the uptake and incorporation of acetate, but adenosine had no effect on either of these functions. Choline uptake, meanwhile, was unchanged in the presence of ATP, phosphate or adenosine. Acetate uptake appears to be more closely linked to its intracellular metabolism than to the transmembrane movement of choline itself. The mechanism by which acetate crosses the nerve terminal membrane has not been established, but the possibility that acetate is a substrate for a monocarboxylate transport system such as has been described in other systems can be ruled out as inhibitors of anion permeability do not block acetate uptake in this preparation.

The observation that constipation alone may induce uninhibited bladder contractions in children and is associated with recurrent urinary tract infection in childhood suggests an etiologic association. Rectal distension due to faecal retention in chronic functional constipation causes bladder distortion and may cause stimulation of detrusor stretch receptors resulting in detrusor peroneal dyssynergism. Distortion of the trigonal area may result in failure of ureteral valve competence and allow for vesico-ureteric reflux.

We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high-affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high-affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug-treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil-treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.

Eight children with acute leukemia in relapse were hypokalemic during their hospital course. All had accompanying hypophosphatemia, and three had mild metabolic alkalosis. Potassium chloride supplementation in each case resulted in resolution of the electrolyte and acid-base disturbances. These findings were not present in patients with conditions newly diagnosed or those in remission. The pathogenesis of the electrolyte and acidbase disturbances was not evident and was not related to antibiotic or cytotoxic drug therapy, but may have been related to the patients' poor nutritional status. Seven of the eight patients died within six months of the hypokalemic episode. Hypokalemia may be a common accompaniment of terminal leukemia.

Many previous series reporting the construction of Brescia-Cimino fistulae in children describe a high rate of immediate and late patency failure. Using microsurgery, we have constructed 23 Brescia-Cimino fistulae in 23 children, 10 weighing less than 20 kg and 13 weighing more than 20 kg. An immediate patency rate of 100% was achieved in both weight groups with a late patency rate of 90%-100%, in those under and over 20 kg, respectively. We recommend using microsurgery for all such angioaccess procedures.

Advantage was taken of a preparation of proteoliposomes made using Torpedo presynaptic membranes in which both the internal and external media can be controlled to investigate the effects of membrane potential and the Na+ gradient on choline transport activity. Under control conditions, Na+ outside and K+ inside, choline was concentrated by proteoliposomes and this phenomenon was sensitive to hemicholinium-3 and high levels of external choline. While proteoliposomes showed no permeability towards K+ spontaneously, in the presence of valinomycin a transmembrane potential was developed. The rate of transport was higher, the greater the inside negative potential. Both the affinity and the maximal velocity of high affinity transport rose in the presence of a potential. Likewise, the affinity and velocity of this transporter increased with increasing external Na+. Increasing internal Na+, on the other hand, caused a decrease in affinity and had little effect on the maximal velocity. The low affinity component was much less, if at all, affected by these changes. These results are consistent with a model of high affinity choline transport in which Na+ binds before choline and the carrier-Na+-choline complex is positively charged. However, these results do not provide a direct explanation for choline transport activation by nerve activity, underlining the need to study the effects of parameters other than membrane potential and the Na+ gradient on choline transport activity.