A homozygous missense mutation in SCNN1A is responsible for a transient neonatal form of pseudohypoaldosteronism type 1.

Pseudohypoaldosteronism type 1 (PHA1) is a monogenic disorder of mineralocorticoid resistance characterized by salt wasting, hyperkalemia, high aldosterone levels, and failure to thrive. An autosomal recessive form (AR-PHA1) is caused by mutations in the epithelial sodium channel ENaC with usually severe and persisting multiorgan symptoms. The autosomal dominant form of PHA1 (AD-PHA1) is due to mutations in the mineralocorticoid receptor causing milder and transient symptoms restricted to the kidney. We identified a homozygous missense mutation in the SCNN1A gene (c.727T>C/p.Ser(243)Pro), encoding α-subunit of ENaC (α-ENaC) in a prematurely born boy with a severe salt-losing syndrome. The patient improved rapidly under treatment, and dietary salt supplementation could be stopped after 6 mo. Interestingly, the patient's sibling born at term and harboring the same homozygous Ser(243)Pro mutation showed no symptom of salt-losing nephropathy. In vitro expression of the αSer(243)Pro ENaC mutant revealed a slight but significant decrease in ENaC activity that is exacerbated in the presence of high Na(+) load. Our study provides the first evidence that ENaC activity is critical for the maintenance of salt balance in the immature kidney of preterm babies. Together with previous studies, it shows that, when the kidney is fully mature, the severity of the symptoms of AR-PHA1 is related to the degree of the ENaC loss of function. Finally, this study identifies a novel functional domain in the extracellular loop of ENaC.

Quantifying intracellular protein binding thermodynamics during mechanotransduction based on FRET spectroscopy.

Mechanical force modulates myriad cellular functions including migration, alignment, proliferation, and gene transcription. Mechanotransduction, the transmission of mechanical forces and its translation into biochemical signals, may be mediated by force induced protein conformation changes, subsequently modulating protein signaling. For the paxillin and focal adhesion kinase interaction, we demonstrate that force-induced changes in protein complex conformation, dissociation constant, and binding Gibbs free energy can be quantified by lifetime-resolved fluorescence energy transfer microscopy combined with intensity imaging calibrated by fluorescence correlation spectroscopy. Comparison with in vitro data shows that this interaction is allosteric in vivo. Further, spatially resolved imaging and inhibitor assays show that this protein interaction and its mechano-sensitivity are equal in the cytosol and in the focal adhesions complexes indicating that the mechano-sensitivity of this interaction must be mediated by soluble factors but not based on protein tyrosine phosphorylation.

Substance P and calbindin D-28k-immunoreactivity in primary sensory neurons of chick embryos: differential neuronal birthdates and transient co-localization.

During the ontogenesis of dorsal root ganglia (DRG), the immunoreactivity to substance P (SP) and calbindin D-28k (CaBP) appears in chickens at embryonic day 5 (E5) and E10 respectively. To establish the birthdates of primary sensory neurons expressing SP or CaBP, chick embryos were given repetitive intra-amniotic injections of [3H]-thymidine. The neuroblasts giving rise to SP-expressing neurons were labeled up to E6 while those generating CaBP-immunoreactive neurons stopped to incorporate [3H]-thymidine before E5.5. This finding indicates that neurons exhibiting distinct phenotypes may originate from neuroblasts which arrest to proliferate at close but distinct stages of development. To determine whether SP and CaBP are co-expressed or not in DRG neurons, chick embryos at E12, E18, and chickens two weeks after hatching were perfused and fixed to detect simultaneously SP- and CaBP-immunoreactivity in DRG sections. The results showed that SP and CaBP were transiently co-expressed by a subset of neurons at E12. Later, however, the SP-immunoreactivity was gradually lost by these ganglion cells, so that the SP- and CaBP-immunoreaction defined two distinct neuronal subpopulations after hatching. In conclusion, most CaBP-immunoreactive DRG cells derive from a subset of neurons in which SP and CaBP are transiently co-localized.

Current status of a model system: the gene Gp-9 and its association with social organization in fire ants.

The Gp-9 gene in fire ants represents an important model system for studying the evolution of social organization in insects as well as a rich source of information relevant to other major evolutionary topics. An important feature of this system is that polymorphism in social organization is completely associated with allelic variation at Gp-9, such that single-queen colonies (monogyne form) include only inhabitants bearing B-like alleles while multiple-queen colonies (polygyne form) additionally include inhabitants bearing b-like alleles. A recent study of this system by Leal and Ishida (2008) made two major claims, the validity and significance of which we examine here. After reviewing existing literature, analyzing the methods and results of Leal and Ishida (2008), and generating new data from one of their study sites, we conclude that their claim that polygyny can occur in Solenopsis invicta in the U.S.A. in the absence of expression of the b-like allele Gp-9(b) is unfounded. Moreover, we argue that available information on insect OBPs (the family of proteins to which GP-9 belongs), on the evolutionary/population genetics of Gp-9, and on pheromonal/behavioral control of fire ant colony queen number fails to support their view that GP-9 plays no role in the chemosensory-mediated communication that underpins regulation of social organization. Our analyses lead us to conclude that there are no new reasons to question the existing consensus view of the Gp-9 system outlined in Gotzek and Ross (2007).

Prognostic in Vivo Biomarkers for Lesion Development after Cerebral Ischemia

A better prediction of the outcome after ischemia and estimation of onset time at early time points would greatly facilitate clinical decisions. Therefore, the aim of the present study was to use magnetic resonance spectroscopy to identify neurochemical markers for outcome prediction at early time points after ischemia.ICR-CD1 mice were subjected to 10-minute, 30-minute or permanent middle cerebral artery occlusion (MCAO). The regional cerebral blood flow (CBF) was monitored in all animals by laser-Doppler flowmetry. All MR studies were carried out in a horizontal 14.1T magnet. Fast spin echo images with T2-weighted parameters were Bacquired to localize the volume of interest and evaluate the lesion size. Immediately after adjustment of field inhomogeneities, localized 1H MRS was applied to obtain the neurochemical profile from the striatum (6-8 μl) or the cortex (2.2-2.5 μl). Six animals (sham group) underwent nearly identical procedures without MCAO.By comparing the evolution of several metabolites in ischemia of varying severity, we observed that glutamine increases early after transient ischemia independently of severity, but decreases in permanent ischemia. On the opposite, GABA increased in permanent ischemia and decreased in transient. We also observed a decrease in the sum of N-acetyl aspartate + glutamate + taurine in all irreversibly damaged tissues, independently of reperfusion and severity. Finally, we have observed that some metabolites decrease exponentially after ischemia. This exponential decrease could be used to determine the time of ischemia onset in permanent ischemia.In Conclusion, magnetic resonance spectroscopy can be used as a prognostic and diagnostic tool to monitor reperfusion, identify reversibly and irreversibly damaged tissue and evaluate the time of ischemia onset. If these Results can be translated to stroke patients, this technique would greatly improve the diagnosis and help with clinical decisions.

Congenital diaphragmatic hernia: current status and review of the literature.

Treatment of congenital diaphragmatic hernia (CDH) challenges obstetricians, pediatric surgeons, and neonatologists. Persistent pulmonary hypertension (PPHT) associated with lung hypoplasia in CDH leads to a high mortality rate at birth. PPHT is principally due to an increased muscularization of the arterioles. Management of CDH has been greatly improved by the introduction of prenatal surgical intervention with tracheal obstruction (TO) and by more appropriate postnatal care. TO appears to accelerate fetal lung growth and to increase the number of capillary vessels and alveoli. Improvement of postnatal care over the last years is mainly due to the avoidance of lung injury by applying low peak inflation pressure during ventilation. The benefits of other drugs or technical improvements such as the use of inhaled nitric oxide or extracorporeal membrane oxygenation (ECMO) are still being debated and no single strategy is accepted worldwide. Despite intensive clinical and experimental research, the treatment of newborn with CDH remains difficult.

Sleep function: current questions and new approaches.

The mammalian brain oscillates through three distinct global activity states: wakefulness, non-rapid eye movement (NREM) sleep and REM sleep. The regulation and function of these 'vigilance' or 'behavioural' states can be investigated over a broad range of temporal and spatial scales and at different levels of functional organization, i.e. from gene expression to memory, in single neurons, cortical columns or the whole brain and organism. We summarize some basic questions that have arisen from recent approaches in the quest for the functions of sleep. Whereas traditionally sleep was viewed to be regulated through top-down control mechanisms, recent approaches have emphasized that sleep is emerging locally and regulated in a use-dependent (homeostatic) manner. Traditional markers of sleep homeostasis, such as the electroencephalogram slow-wave activity, have been linked to changes in connectivity and plasticity in local neuronal networks. Thus waking experience-induced local network changes may be sensed by the sleep homeostatic process and used to mediate sleep-dependent events, benefiting network stabilization and memory consolidation. Although many questions remain unanswered, the available data suggest that sleep function will best be understood by an analysis which integrates sleep's many functional levels with its local homeostatic regulation.

Stress ulcer prophylaxis in non-critically ill patients: a prospective evaluation of current practice in a general surgery department

Objective: There is little evidence regarding the benefit of stress ulcer prophylaxis (SUP) outside critical care setting. Over-prescription of SUP is not devoid of risks. This prospective study aimed to evaluate the use of proton pump inhibitors (PPIs) for SUP in a general surgery department.Methods: Data collection was performed prospectively during an 8-week period on patients hospitalized in a general surgery department (58 beds) by pharmacists. Patients with a PPI prescription for the treatment of ulcers, gastro-oesophageal reflux disease, oesophagitis or epigastric pain were excluded. Patients admitted twice during the study period were not re-included. The American Society of Health-System Pharmacists guidelines on SUP were used to assess the appropriateness of de novo PPI prescriptions.Results: Among 255 consecutive patients in the study, 138 (54%) received a prophylaxis with PPI, of which 86 (62%) were de novo PPI prescriptions. One-hundred twenty-nine patients (94%) received esomeprazole (according to the hospital drug policy). The most frequent dosage was 40 mg/day. Use of PPI for SUP was evaluated in 67 patients. Fifty-three patients (79%) had no risk factors for SUP. Twelve and 2 patients had one or two risk factors, respectively. At discharge, PPI prophylaxis was continued in 34% of patients with a de novo PPI prescription.Conclusion: This study highlights the overuse of PPIs in non-ICU patients and the inappropriate continuation of PPI prescriptions at discharge.Treatment

Pre-hospital tracheal intubation in patients with traumatic brain injury: systematic review of current evidence.

BACKGROUND: We reviewed the current evidence on the benefit and harm of pre-hospital tracheal intubation and mechanical ventilation after traumatic brain injury (TBI). METHODS: We conducted a systematic literature search up to December 2007 without language restriction to identify interventional and observational studies comparing pre-hospital intubation with other airway management (e.g. bag-valve-mask or oxygen administration) in patients with TBI. Information on study design, population, interventions, and outcomes was abstracted by two investigators and cross-checked by two others. Seventeen studies were included with data for 15,335 patients collected from 1985 to 2004. There were 12 retrospective analyses of trauma registries or hospital databases, three cohort studies, one case-control study, and one controlled trial. Using Brain Trauma Foundation classification of evidence, there were 14 class 3 studies, three class 2 studies, and no class 1 study. Six studies were of adults, five of children, and three of both; age groups were unclear in three studies. Maximum follow-up was up to 6 months or hospital discharge. RESULTS: In 13 studies, the unadjusted odds ratios (ORs) for an effect of pre-hospital intubation on in-hospital mortality ranged from 0.17 (favouring control interventions) to 2.43 (favouring pre-hospital intubation); adjusted ORs ranged from 0.24 to 1.42. Estimates for functional outcomes after TBI were equivocal. Three studies indicated higher risk of pneumonia associated with pre-hospital (when compared with in-hospital) intubation. CONCLUSIONS: Overall, the available evidence did not support any benefit from pre-hospital intubation and mechanical ventilation after TBI. Additional arguments need to be taken into account, including medical and procedural aspects.

ROLES OF ACID-SENSING ION CHANNELS (ASICS) IN PERIPHERAL NEUROPEPTIDE SECRETION AND PAIN

Acid-sensing ion channels (ASICs) are non-voltage-gated sodium channels activated by an extracellular acidification. They are widely expressed in neurons of the central and peripheral nervous system. ASICs have a role in learning, the expression of fear, in neuronal death after cerebral ischemia, and in pain sensation. Tissue damage leads to the release of inflammatory mediators. There is a subpopulation of sensory neurons which are able to release the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). Neurogenic inflammation refers to the process whereby peripheral release of the neuropeptides CGRP and SP induces vasodilation and extravasation of plasma proteins, respectively. Our laboratory has previously shown that calcium-permeable homomeric ASIC1a channels are present in a majority of CGRP- or SP-expressing small diameter sensory neurons. In the first part of my thesis, we tested the hypothesis that a local acidification can produce an ASIC-mediated calcium-dependant neuropeptide secretion. We have first verified the co-expression of ASICs and CGRP/SP using immunochemistry and in-situ hybridization on dissociated rat dorsal root ganglion (DRG) neurons. We found that most CGRP/SP-positive neurons also expressed ASIC1a and ASIC3 subunits. Calcium imaging experiments with Fura-2 dye showed that an extracellular acidification can induce an increase of intracellular Ca2+ concentration, which is essential for secretion. This increase of intracellular Ca2+ concentration is, at least in some cells, ASIC-dependent, as it can be prevented by amiloride, an ASIC antagonist, and by Psalmotoxin (PcTx1), a specific ASIC1a antagonist. We identified a sub-population of neurons whose acid-induced Ca2+ entry was completely abolished by amiloride, an amiloride-resistant population which does not express ASICs, but rather another acid-sensing channel, possibly transient receptor potential vanilloïde 1 (TRPV1), and a population expressing both H+-gated channel types. Voltage-gated calcium channels (Cavs) may also mediate Ca2+ entry. Co-application of the Cavs inhibitors (ω-conotoxin MVIIC, Mibefradil and Nifedipine) reduced the Ca2+ increase in neurons expressing ASICs during an acidification to pH 6. This indicates that ASICs can depolarise the neuron and activate Cavs. Homomeric ASIC1a are Ca2+-permeable and allow a direct entry of Ca2+ into the cell; other ASICs mediate an indirect entry of Ca2+ by inducing a membrane depolarisation that activates Cavs. We showed with a secretion assay that CGRP secretion can be induced by extracellular acidification in cultured rat DRG neurons. Amiloride and PcTx1 were not able to inhibit the secretion at acidic pH, but BCTC, a TRPV1 inhibitor was able to decrease the secretion induced by an extracellular acidification in our in vitro secretion assay. In conclusion, these results show that in DRG neurons a mild extracellular acidification can induce a calcium-dependent neuropeptide secretion. Even if our data show that ASICs can mediate an increase of intracellular Ca2+ concentration, this appears not to be sufficient to trigger neuropeptide secretion. TRPV1, a calcium channel whose activation induces a sustained current - in contrary of ASICs - played in our experimental conditions a predominant role in neurosecretion. In the second part of my thesis, we focused on the role of ASICs in neuropathic pain. We used the spared nerve injury (SNI) model which consists in a nerve injury that induces symptoms of neuropathic pain such as mechanical allodynia. We have previously shown that the SNI model modifies ASIC currents in dissociated rat DRG neurons. We hypothesized that ASICs could play a role in the development of mechanical allodynia. The SNI model was performed on ASIC1a, -2, and -3 knock-out mice and wild type littermates. We measured mechanical allodynia on these mice with calibrated von Frey filaments. There were no differences between the wild-type and the ASIC1, or ASIC2 knockout mice. ASIC3 null mice were less sensitive than wild type mice at 21 day after SNI, indicating a role for ASIC3. Finally, to investigate other possible roles of ASICs in the perception of the environment, we measured the baseline heat responses. We used two different models; the tail flick model and the hot plate model. ASIC1a null mice showed increased thermal allodynia behaviour in the hot plate test at three different temperatures (49, 52, 55°C) compared to their wild type littermates. On the contrary, ASIC2 null mice showed reduced thermal allodynia behaviour in the hot plate test compared to their wild type littermates at the three same temperatures. We conclude that ASIC1a and ASIC2 in mice can play a role in temperature sensing. It is currently not understood how ASICs are involved in temperature sensing and what the reason for the opposed effects in the two knockout models is.

Les méthodes d'études de cas en psychothérapie: perspectives actuelles [Methods of psychotherapy case studies: current perspectives].

There is a renewal of interest among psychotherapy researchers and psychotherapists towards psychotherapy case studies. This article presents two paradigms that have greatly influenced this increasing interest in psychotherapy case studies : the pragmatic case study and the theory-building case study paradigm. The origins, developments and key-concepts of both paradigms are presented, as well as their methodological and ethical specificities. Examples of case studies, along with models developed, are cited. The differential influence of the post-modern schools on both paradigms are presented, as well as their contribution to the field of methods of psychotherapy case studies discussed and assessed in terms of relevance for the researcher and the psychotherapist.

Current state of knowledge on neuroendocrine small bowel tumours: non-systematic review of the literature based on one case.

More than 60% of neuroendocrine tumours, also called carcinoids, are localised within the gastrointestinal tract. Small bowel neuroendocrine tumours have been diagnosed with increasing frequency over the past 35 years, being the second most frequent tumours of the small intestine. Ileal neuroendocrine tumours diagnosis is late because patients have non-specific symptoms. We have proposed to illustrate as an example the case of a patient, and on its basis, to make a brief review of the literature on small bowel neuroendocrine tumours, resuming several recent changes in the field, concerning classification criteria of these tumours and new recommendations and current advances in diagnosis and treatment. This patient came to our emergency department with a complete bowel obstruction, along with a 2-year history of peristaltic abdominal pain, vomits and diarrhoea episodes. During emergency laparotomy, an ileal stricture was observed, that showed to be a neuroendocrine tumour of the small bowel.

Identification of a potent MAR element from the mouse genome and assessment of its activity in stable and transient transfections.

Matrix attachment regions are DNA sequences found throughout eukaryotic genomes that are believed to define boundaries interfacing heterochromatin and euchromatin domains, thereby acting as epigenetic regulators. When included in expression vectors, MARs can improve and sustain transgene expression, and a search for more potent novel elements is therefore actively pursued to further improve recombinant protein production. Here we describe the isolation of new MARs from the mouse genome using a modified in silico analysis. One of these MARs was found to be a powerful activator of transgene expression in stable transfections. Interestingly, this MAR also increased GFP and/or immunoglobulin expression from some but not all expression vectors in transient transfections. This effect was attributed to the presence or absence of elements on the vector backbone, providing an explanation for earlier discrepancies as to the ability of this class of elements to affect transgene expression under such conditions.

Glutamatergic and GABAergic energy metabolism measured in the rat brain by (13) C NMR spectroscopy at 14.1 T.

Energy metabolism supports both inhibitory and excitatory neurotransmission processes. This study investigated the specific contribution of astrocytic metabolism to γ-aminobutyric acid (GABA) synthesis and inhibitory GABAergic neurotransmission that remained to be ilucidated in vivo. Therefore, we measured (13) C incorporation into brain metabolites by dynamic (13) C nuclear magnetic resonance spectroscopy at 14.1 T in rats under α-chloralose anaesthesia during infusion of [1,6-(13) C]glucose. The enhanced sensitivity at 14.1 T allowed to quantify incorporation of (13) C into the three aliphatic carbons of GABA non-invasively. Metabolic fluxes were determined with a mathematical model of brain metabolism comprising glial, glutamatergic and GABAergic compartments. GABA synthesis rate was 0.11 ± 0.01 μmol/g/min. GABA-glutamine cycle was 0.053 ± 0.003 μmol/g/min and accounted for 22 ± 1% of total neurotransmitter cycling between neurons and glia. Cerebral glucose oxidation was 0.47 ± 0.02 μmol/g/min, of which 35 ± 1% and 7 ± 1% was diverted to the glutamatergic and GABAergic tricarboxylic acid cycles, respectively. The remaining fraction of glucose oxidation was in glia, where 12 ± 1% of the TCA cycle flux was dedicated to oxidation of GABA. 16 ± 2% of glutamine synthesis was provided to GABAergic neurons. We conclude that substantial metabolic activity occurs in GABAergic neurons and that glial metabolism supports both glutamatergic and GABAergic neurons in the living rat brain. We performed (13) C NMR spectroscopy in vivo at high magnetic field (14.1 T) upon administration of [1,6-(13) C]glucose. This allowed to measure (13) C incorporation into the three aliphatic carbons of GABA in the rat brain, in addition to those of glutamate, glutamine and aspartate. These data were then modelled to determine fluxes of energy metabolism in GABAergic and glutamatergic neurons and glial cells.