Intranasal fentanyl and inhaled nitrous oxide for fracture reduction: The FAN observational study.

INTRODUCTION: Procedural sedation and analgesia (PSA) are frequently used for fracture reduction in pediatric emergency departments (ED). Combining intranasal (IN) fentanyl with inhalation of nitrous oxide (N2O) allow for short recovery time and obviates painful and time-consuming IV access insertions. METHODS: We performed a bicentric, prospective, observational cohort study. Patients aged 4-18years were included if they received combined PSA with IN fentanyl and N2O for the reduction of mildly/moderately displaced fracture or of dislocation. Facial Pain Scale Revised (FPS-R) and Face, Leg, Activity, Cry, Consolability (FLACC) scores were used to evaluate pain and anxiety before, during and after procedure. University of Michigan Sedation Score (UMSS), adverse events, detailed side effects and satisfaction of patients, parents and medical staff were recorded at discharge. A follow up telephone call was made after 24-72h. RESULTS: 90 patients were included. There was no difference in FPS-R during the procedure (median score 2 versus 2), but the FLACC score was significantly higher as compared to before (median score 4 versus 0, Δ 2, 95% CI 0, 2). Median UMSS was 1 (95% CI 1, 2). We recorded no serious adverse events. Rate of vomiting was 12% (11/84). Satisfaction was high among participants responding to this question 85/88 (97%) of parents, 74/83 (89%) of patients and 82/85 (96%) of physicians would want the same sedation again. CONCLUSION: PSA with IN fentanyl and N2O is effective and safe for the reduction of mildly/moderately displaced fracture or dislocation, and has a high satisfaction rate.

Cystamine/cysteamine rescues the dopaminergic system and shows neurorestorative properties in an animal model of Parkinson's disease.

The neuroprotective properties of cystamine identified in pre-clinical studies have fast-tracked this compound to clinical trials in Huntington's disease, showing tolerability and benefits on motor symptoms. We tested whether cystamine could have such properties in a Parkinson's disease murine model and now provide evidence that it can not only prevent the neurodegenerative process but also can reverse motor impairments created by a 6-hydroxydopamine lesion 3 weeks post-surgery. Importantly, we report that cystamine has neurorestorative properties 5 weeks post-lesion as seen on the number of nigral dopaminergic neurons which is comparable with treatments of cysteamine, the reduced form of cystamine used in the clinic, as well as rasagiline, increasingly prescribed in early parkinsonism. All three compounds induced neurite arborization of the remaining dopaminergic cells which was further confirmed in ex vivo dopaminergic explants derived from Pitx3-GFP mice. The disease-modifying effects displayed by cystamine/cysteamine would encourage clinical testing.

New lithium metal polymer solid state battery for an ultrahigh energy: nano C-LiFePO4 versus nano Li1.2V3O8.

Novel lithium metal polymer solid state batteries with nano C-LiFePO4 and nano Li1.2V3O8 counter-electrodes (average particle size 200 nm) were studied for the first time by in situ SEM and impedance during cycling. The kinetics of Li-motion during cycling is analyzed self-consistently together with the electrochemical properties. We show that the cycling life of the nano Li1.2V3O8 is limited by the dissolution of the vanadium in the electrolyte, which explains the choice of nano C-LiFePO4 (1300 cycles at 100% DOD): with this olivine, no dissolution is observed. In combination with lithium metal, at high loading and with a stable SEI an ultrahigh energy density battery was thus newly developed in our laboratory.

Effect of treatment for paediatric cancers on balance: what do we know? A review of the evidence.

This review aims to explore the literature investigating balance outcomes in survivors of childhood cancer. A structured search of five databases resulted in 16 articles included in this review. Nearly all were classified as Level 4 evidence using the updated Oxford Centre for Evidence-Based Medicine Levels of Evidence. Balance abilities have been investigated solely in survivors of acute lymphoblastic leukaemia or central nervous system tumours. The literature tends to support the idea that survivors present with balance difficulties but the results need to be closely scrutinised. Several studies report results using the same experimental group, while other studies use balance outcome measures that have not had their psychometric properties assessed with this population. There are also few studies that evaluate dynamic balance abilities in survivors of paediatric cancers, which may be more influential on functional tasks. Furthermore, very few of the included studies investigate how the found balance deficits affect this population's daily lives, which would be necessary in order to determine if intervention should be geared towards this area. Directions for future research should also include multi-centred, clinically oriented trials to evaluate balance abilities in survivors of childhood cancers compared with healthy control subjects in order to strengthen the literature.

FOXO1 and c-jun transcription factors mRNA are modulated in endometriosis.

Endometriosis is a polygenic gynaecological condition affecting 5-15% of women of childbearing age. Major symptoms of the disease are pelvic pain and infertility. No clear link has been established between symptoms and the stage of the disease. Although some aspects have begun to be clarified, clinical understanding of endometriosis remains partial at the molecular level. In this perspective, we targeted isolation of differentially expressed genes in the eutopic endometrial tissue. Our assumption was that the endometrial cells of patients presented an unusual gene expression profile, allowing their implantation and survival in an ectopic site, leading to endometriotic lesions. Here, we report that mRNA steady-state levels of two key transcription factors are modulated in endometriosis. FOXO1 (also known as FKHR) levels were 1.6-fold lower in endometriosis compared to the control group at the onset of the secretory phase (day 15-21), while c-jun mRNA was present at higher amounts in endometriosis (1.5-fold) at the proliferative phase of the menstrual cycle. These results were derived from a large sample composed of 157 control subjects and 209 patients with endometriosis. Gene profiling was conducted by real-time quantitative PCR, and data were quality controlled before statistical analysis. Whether protein levels are affected as well remains to be investigated.

Large scale validation of human N-myc downstream-regulated gene (NDRG)-1 expression in endometrium during the menstrual cycle.

A major challenge in the comprehension of the endometrial transformations leading to the completion of each menstrual cycle in humans is in the identification of specific molecular pathways underlying these monthly turnovers. Towards this goal we compared, by the differential display technique, the relative expression of mRNA in endometrial biopsies harvested in individuals (n = 48) either at the proliferative or the secretory phase of the menstrual cycle. We isolated a cDNA fragment homologous to NDRG1 (N-myc Downstream-Regulated Gene-1) that is present in markedly higher amounts in the secretory phase. Northern blot analysis and quantitative real time PCR experiments confirmed this result in distinct cohorts of individuals (44 and 560 respectively). A closer examination of data showed that the highest mRNA levels were found during the range of 25-28 days of the uterine cycle. Consistent with the mRNA data, the temporal profile of the NDRG1 protein showed a 15-fold increase during the secretory phase, as demonstrated by using semi-quantitative dot blot analyses (n = 92). Immunohistochemical localization revealed that NDRG1 was expressed both in epithelial and stromal cells. This large scale validation of the NDRG1 mRNA and protein increase in endometrium during the secretory phase is consistent with its differentiation-related function described in other tissues and its potential involvement in the window of implantation of the human endometrium, as suggested by previous chip-based evidence.

Respiratory day hospital: a novel approach to acute respiratory care.

In 1996 we established a day hospital dedicated to acute respiratory care, as an alternative to emergency department and inpatient treatment. The unit is staffed by respirologists, family physicians and specialized nurses; patients have access to all standard inpatient treatments and services. Between 1996/97 and 1998/99 the annual number of admissions to the day hospital increased from 658 to 922. By 1998/99 more than 75% of patients were referred for acute treatment, with a mean stay of 2.3 days. The most common diagnoses were asthma and chronic obstructive pulmonary disease, which accounted for 58% and 32% respectively of treatment-related admissions. Treatment most often involved intravenous corticosteroid therapy and inhaled bronchodilator therapy. Between 1996/97 and 1998/9 the proportion of patients requiring transfer to overnight care decreased from 22% to 14%; complications and unscheduled return visits were rare. We believe that a respiratory day hospital provides a useful alternative to emergency department and inpatient care.

Genomic organization of the X-linked inhibitor of apoptosis and identification of a novel testis-specific transcript.

Here we report the genomic organization and mapping of the X-linked inhibitor of apoptosis gene (BIRC4, also known as XIAP and hILP) and the identification of a closely related transcript. BIRC4 is located on Xq25 and is composed of seven exons. The intron/exon structure is highly conserved between the mouse homologue and its human counterpart. Four bands cross-react with a BIRC4 coding region probe on a genomic Southern blot. One of these cross-reactive bands encodes an intronless gene that expresses a 2.2-kb transcript solely in the testis. This testis-specific transcript contains a putative open reading frame (ORF) that is homologous to the carboxy-terminal end of BIRC4; overexpression of this ORF shows protective effects against BAX-induced apoptosis.

The palmitoylation state of the beta(2)-adrenergic receptor regulates the synergistic action of cyclic AMP-dependent protein kinase and beta-adrenergic receptor kinase involved in its phosphorylation and desensitization.

Although palmitoylation of the beta(2)-adrenergic receptor (beta(2)AR), as well as its phosphorylation by the cyclic AMP-dependant protein kinase (PKA) and the beta-adrenergic receptor kinase (beta ARK), are known to play important roles in agonist-promoted desensitization, their relative contribution and mutual regulatory influences are still poorly understood. In this study, we investigated the role that the carboxyl tail PKA site (Ser(345,346)) of the beta(2)AR plays in its rapid agonist-promoted phosphorylation and desensitization. Mutation of this site (Ala(345,346)beta(2)AR) significantly reduced the rate and extent of the rapid desensitization promoted by sustained treatment with the agonist isoproterenol. The direct contribution of Ser(345,346) in desensitization was then studied by mutating all other putative PKA and beta ARK phosphorylation sites (Ala(261,262)beta ARK(-)beta(2)AR). We found this mutant receptor to be phosphorylated upon receptor activation but not following direct activation of PKA, suggesting a role in receptor-specific (homologous) but not heterologous phosphorylation. However, despite its phosphorylated state, Ala(261,262)beta ARK(-)beta(2)AR did not undergo rapid desensitization upon agonist treatment, indicating that phosphorylation of Ser(345,346) alone is not sufficient to promote desensitization. Taken with the observation that mutation of either Ser(345,346) or of the beta ARK phosphorylation sites prevented both the hyper-phosphorylation and constitutive desensitization of a palmitoylation-less mutant (Gly(341)beta(2)AR), our data suggest a concerted/synergistic action of the two kinases that depends on the palmitoylation state of the receptor. Consistent with this notion, in vitro phosphorylation of Gly(341)beta(2)AR by the catalytic subunit of PKA facilitated further phosphorylation of the receptor by purified beta ARK. Our study therefore allows us to propose a coordinated mechanism by which sequential depalmitoylation, and phosphorylation by PKA and beta ARK lead to the functional uncoupling and desensitization of the ss(2)AR.

Activation of the beta(2)-adrenergic receptor-Galpha(s) complex leads to rapid depalmitoylation and inhibition of repalmitoylation of both the receptor and Galpha(s).

Palmitoylation is unique among lipid modifications in that it is reversible. In recent years, dynamic palmitoylation of G protein alpha subunits and of their cognate receptors has attracted considerable attention. However, very little is known concerning the acylation/deacylation cycle of the proteins in relation to their activity status. In particular, the relative contribution of the activation and desensitization of the signaling unit to the regulation of the receptors and G proteins palmitoylation state is unknown. To address this issue, we took advantage of the fact that a fusion protein composed of the stimulatory alpha subunit of trimeric G protein (Galpha(s)) covalently attached to the beta(2)-adrenergic receptor (beta(2)AR) as a carboxyl-terminal extension (beta(2)AR-Galpha(s)) can be stimulated by agonists but does not undergo rapid inactivation, desensitization, or internalization. When expressed in Sf9 cells, both the receptor and the Galpha(s) moieties of the fusion protein were found to be palmitoylated via thioester linkage. Stimulation with the beta-adrenergic agonist isoproterenol led to a rapid depalmitoylation of both the beta(2)AR and Galpha(s) and inhibited repalmitoylation. The extent of depalmitoylation induced by a series of agonists was correlated (0.99) with their intrinsic efficacy to stimulate the adenylyl cyclase activity. However, forskolin-stimulated cAMP production did not affect the palmitoylation state of beta(2)AR-Galpha(s), indicating that the agonist-promoted depalmitoylation is linked to conformational changes and not to second messenger generation. Given that, upon activation, the fusion protein mimics the activated receptor-G protein complex but cannot undergo desensitization, the data demonstrate that early steps in the activation process lead to the depalmitoylation of both receptor and G protein and that repalmitoylation requires later events that cannot be accommodated by the activated fusion protein.

The late expression factors 8 and 9 and possibly the phosphoprotein p78/83 of Autographa californica multicapsid nucleopolyhedrovirus are components of the virus-induced RNA polymerase.

Attempts were made to identify some of the subunits of the baculovirus-induced RNA polymerase following purification of its enzymatic activity by conventional chromatography. Polymerase activity was extracted from lysates of insect cells infected with Autographa californica multicapsid nucleopolyhedrovirus by polyethylenimine precipitation and subsequently purified by phosphocellulose, anion exchange, poly(A) Sepharose affinity, and gel filtration chromatography. The presence of the polymerase was monitored by its alpha-amanitin-resistant activity in in vitro transcription assays. A number of polypeptides associated with the enzymatic activity were identified. Peptide-specific antibodies were generated against a variety of late-expression factors (LEFs) and these antibodies, along with antisera directed against several other baculovirus proteins, were used in an immunoblot analysis of the purified polymerase. The results revealed that both the viral helicase (p143) and the virogenic stroma protein, pp31, copurify with the baculovirus-induced RNA polymerase activity through several chromatographic steps and may be loosely associated with the RNA polymerase. LEF8, LEF9 and p78/83, a nucleocapsid-associated phosphoprotein, were found to associate with the viral-induced polymerase activity. LEF8 and LEF9 contain regions of sequence homology with components of other DNA-directed RNA polymerases, while a portion of p78/83 exhibits some homology to the sigma factor of bacterial RNA polymerase, the RAP30 protein found in the mammalian transcription complex TFIIF, and the RAP94 polypeptide associated with vaccinia virus RNA polymerase. The p78/83 protein has previously been shown by our laboratory to be a capsid protein, but it may also play some role with the RNA polymerase. These results represent a first attempt to identify specific components of the RNA polymerase associated with infections of insect cells by A. californica nucleopolyhedrovirus.

Biliopancreatic Diversion with a New Type of Gastrectomy: Some Previous Conclusions Revisited.

BACKGROUND: In 1990, we modified Scopinaro's billopancreatic diversion (BPD); instead of a distal gastrectomy and gastroileal anastomosis, a parietal gastrectomy was performed with nutrients diverted through a duodenal switch. Also, the length of the common channel (50 cm) was doubled to 100 cm, while the nutrient limb remained 250 cm. In 1991, we reported initial results after 16 months, weight loss was as expected following BPD, but patients reported fewer side-effects and the prevalence of excessive malabsorption was less. This cohort of patients had their duodenum stapled shut to construct the duodenal switch. This staple-line failed insidiously in some patients, allowing the duodenum to recanalize partially or completely. This resulted in an incomplete BPD. METHODS: Since 1992, the duodenal switch has been constructed with a complete transaction of the duodenum to prevent recanalization. We report here on the first 61 patients who underwent this definitive procedure. RESULTS: At 16 months, we observed a mean weight loss of 84% of initial excess weight, the number of daily stools at 2.9 +/- 1.6 and the prevalence of diarrhea at 10%. Twenty per cent of patients experienced mild anemia, hypocalcemia, or hypoalbuminemia, which required added supplements. CONCLUSIONS: BPD with parietal gastrectomy, duodenal switch and longer common channel improved weight loss and decreased gastrointestinal side-effects without an increased prevalence of excessive malabsorption. The parietal gastrectomy may contribute to weight loss by increasing satiety, and decreasing side-effects by regulating gastric emptying.

Biliopancreatic Diversion, with Distal Gastrectomy, 250 cm and 50 cm Limbs: Long-term Results.

BACKGROUND: Since 1984, biliopancreatic diversion (BPD) has been our procedure of choice in the treatment of morbid obesity. Better understanding of long-term outcome following BPD is needed. METHODS: We report the results of our first consecutive 92 patients who underwent BPD more than 5 years ago. Of these 92, only 82 were available for a recent formal evaluation after a mean of 79 months. RESULTS: Weight loss, was maintained over the years at 62% of initial excess weight; the success rate for losing more than 50% of initial excess weight was 72%. The gastrointestinal side-effects decreased with time, but diarrhea was still present in 13%. The average number of daily stools was 3 +/- 1.0. Of the patients, 76% were free from any gastrointestinal side-effects, taking normal diet and having normal stools. Malabsorption, however, was still present. A third of patients had laboratory values slightly below normal levels for hemoglobin, albumin and calcium. These values were mostly without clinical manifestation and were well tolerated by the patients. Regarding associated diseases, 75% were cured or improved following BPD. In 14 patients, reoperation was required to improve diarrhea or serum albumin. In these patients, the common channel was lengthened from 50 to 100 cm. The revision was successful in 11 and did not cause significant weight gain. CONCLUSIONS: BPD, as proposed by Scopinaro, was an efficient surgical treatment of morbid obesity that allowed normal eating habits and despite malabsorption was well tolerated by the great majority of patients.

The carbamyl phosphate synthetase promoter contains multiple binding sites for C/EBP-related proteins.

The promoter of the gene (CPS) encoding rat carbamyl phosphate synthetase I has been mapped 5' to a segment of about 525 nucleotides upstream from the transcription start point and, when analyzed in liver nuclear extracts, contained six well-defined protein-recognition elements, designated CPS sites I-VI. All six elements were recognized, with varying affinities, by CAAT and enhancer-binding protein (C/EBP alpha) produced in bacteria. Oligodeoxyribonucleotides corresponding to CPS site II or to the C/EBP alpha-recognition element of the ALB promoter, site D, competed with the six CPS-promoter elements in footprinting assays. However, mutagenesis of the C/EBP alpha-recognition element, 5'-GTTGCAAC, at the core of site II was sufficient to abolish transactivation of the CPS promoter by C/EBP alpha in co-transfected HepG2 cells. These findings indicate that the CPS promoter contains multiple recognition elements for factors with DNA-binding specificities similar to C/EBP proteins. Activation by C/EBP alpha, however, requires promoter site II.

Factors interacting with the rat carbamyl phosphate synthetase promoter in expressing and nonexpressing tissues.

The proximal promoter of the rat carbamyl phosphate synthetase-encoding gene (CPS) contains at least six potential cis-acting regulatory elements (sites I-VI), as judged by DNase I footprint analysis using rat liver nuclear extracts; all six regions bind proteins with DNA recognition properties similar to those of CAAT and enhancer-binding protein alpha (C/EBP alpha) [Lagacé et al., Gene 118 (1992) 231-238]. In contrast, nuclear extracts from kidney, brain and spleen contain proteins that recognize CPS promoter sites II, V and VI, but not sites I, III and IV. Mutation of the octameric sequence (5'-GTTGCAAC) within site II, which is a recognition element for C/EBP alpha, abolished binding of nuclear proteins to site II oligodeoxyribonucleotides (oligos) in all tissues. As well, the site II mutation reduced the level of in vitro transcription from the CPS promoter by about 50% in liver and spleen nuclear extracts, but had a negligible effect in brain and kidney extracts. The fact that promoter activity was observed in extracts of tissues that do not express the endogenous CPS gene (i.e., brain, kidney and spleen) indicates that these tissues, nevertheless, contain factors with the potential to activate transcription through a limited number of CPS promoter elements. Tissue-specific regulation, therefore, must involve steps to prevent these factors from acting on the endogenous CPS promoter in situ.

Activity of the carbamyl phosphate synthetase I promoter in liver nuclear extracts is dependent on a cis-acting C/EBP recognition element.

We have identified an essential cis element in the proximal promoter region of the rat carbamyl phosphate synthetase I (CPSI) gene that is requisite for promoter activity in liver nuclear extracts. Excess synthetic oligonucleotides specifying this region abolished promoter-dependent in vitro transcription. We show that C/EBP, a nuclear factor enriched in liver but found as well in other tissues, such as gut, fat, and lung, interacts with an inverted repeat, GTTGCAAC, at the core of the essential cis element. In brain, a tissue that did not express CPSI or contain significant levels of C/EBP, a different factor was capable of binding at or near the C/EBP recognition element. Activity of the CPSI promoter in liver nuclear extracts was also dependent on sequences 5' to the C/EBP motif; presumably, factors binding to elements within this upstream region are instrumental in restricting CPSI gene expression to liver and intestinal mucosa.

Rat carbamyl-phosphate synthetase I gene. Promoter sequence and tissue-specific transcriptional regulation in vitro.

The region flanking the 5'-end of the rat gene encoding the cytoplasmic precursor of carbamyl-phosphate synthetase I, a mitochondrial matrix enzyme, has been cloned and partially characterized. S1 nuclease and primer extension analyses position the starts of transcription 138-140 nucleotides upstream of the translation initiation codon. Exon 1 contains this untranslated sequence and extends downstream to include the coding region for the pre-enzyme signal peptide (38 amino acids) plus 4 amino acids from the amino terminus of the mature protein. The 5'-flanking sequence contains typical promoter elements, including putative TATA and CAAT motifs at -21 and -82 nucleotides, respectively. In addition, several copies of consensus sequences corresponding to the H4TF-1 recognition element, GATTTC, together with the enhancer-like octamer, ATTTGCAT, are also present. Carbamyl-phosphate synthetase I is a cell-type specific enzyme, being expressed only in hepatocytes and epithelial cells of the intestinal mucosa. It is also synthesized at relatively high levels in the hepatoma cell line, Hep G2. Employing pCPS2.1, a minigene containing the promoter and part of exon 1, we show that nuclear extracts from Hep G2 support accurate carbamyl-phosphate synthetase I gene transcription in vitro. No such activity was observed, however, in extracts from HeLa, a cell line which does not express carbamyl-phosphate synthetase I.