Management of intrahepatic and perihilar cholangiocarcinomas: Guidelines of the French Association for the Study of the Liver (AFEF).

Intrahepatic cholangiocarcinoma (iCCA) is the second most common malignant primary liver cancer. iCCA may develop on an underlying chronic liver disease and its incidence is growing in relation with the epidemics of obesity and metabolic diseases. In contrast, perihilar cholangiocarcinoma (pCCA) may follow a history of chronic inflammatory diseases of the biliary tract. The initial management of CCAs is often complex and requires multidisciplinary expertise. The French Association for the Study of the Liver wished to organize guidelines in order to summarize the best evidence available about several key points in iCCA and pCCA. These guidelines have been elaborated based on the level of evidence available in the literature and each recommendation has been analysed, discussed and voted by the panel of experts. They describe the epidemiology of CCA as well as how patients with iCCA or pCCA should be managed from diagnosis to treatment. The most recent developments of personalized medicine and use of targeted therapies are also highlighted.

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition.

BACKGROUND: Transposable elements are ubiquitous and play a fundamental role in shaping genomes during evolution. Since excessive transposition can be mutagenic, mechanisms exist in the cells to keep these mobile elements under control. Although many cellular factors regulating the mobility of the retrovirus-like transposon Ty1 in Saccharomyces cerevisiae have been identified in genetic screens, only very few of them interact physically with Ty1 integrase (IN). RESULTS: Here, we perform a proteomic screen to establish Ty1 IN interactome. Among the 265 potential interacting partners, we focus our study on the conserved CK2 kinase. We confirm the interaction between IN and CK2, demonstrate that IN is a substrate of CK2 in vitro and identify the modified residues. We find that Ty1 IN is phosphorylated in vivo and that these modifications are dependent in part on CK2. No significant change in Ty1 retromobility could be observed when we introduce phospho-ablative mutations that prevent IN phosphorylation by CK2 in vitro. However, the absence of CK2 holoenzyme results in a strong stimulation of Ty1 retrotransposition, characterized by an increase in Ty1 mRNA and protein levels and a high accumulation of cDNA. CONCLUSION: Our study shows that Ty1 IN is phosphorylated, as observed for retroviral INs and highlights an important role of CK2 in the regulation of Ty1 retrotransposition. In addition, the proteomic approach enabled the identification of many new Ty1 IN interacting partners, whose potential role in the control of Ty1 mobility will be interesting to study.

Escherichia coli RNase E can efficiently replace RNase Y in Bacillus subtilis.

RNase Y and RNase E are disparate endoribonucleases that govern global mRNA turnover/processing in the two evolutionary distant bacteria Bacillus subtilis and Escherichia coli, respectively. The two enzymes share a similar in vitro cleavage specificity and subcellular localization. To evaluate the potential equivalence in biological function between the two enzymes in vivo we analyzed whether and to what extent RNase E is able to replace RNase Y in B. subtilis. Full-length RNase E almost completely restores wild type growth of the rny mutant. This is matched by a surprising reversal of transcript profiles both of individual genes and on a genome-wide scale. The single most important parameter to efficient complementation is the requirement for RNase E to localize to the inner membrane while truncation of the C-terminal sequences corresponding to the degradosome scaffold has only a minor effect. We also compared the in vitro cleavage activity for the major decay initiating ribonucleases Y, E and J and show that no conclusions can be drawn with respect to their activity in vivo. Our data confirm the notion that RNase Y and RNase E have evolved through convergent evolution towards a low specificity endonuclease activity universally important in bacteria.

Impact of RNase E and RNase J on Global mRNA Metabolism in the Cyanobacterium Synechocystis PCC6803.

mRNA levels result from an equilibrium between transcription and degradation. Ribonucleases (RNases) facilitate the turnover of mRNA, which is an important way of controlling gene expression, allowing the cells to adjust transcript levels to a changing environment. In contrast to the heterotrophic model bacteria Escherichia coli and Bacillus subtilis, RNA decay has not been studied in detail in cyanobacteria. Synechocystis sp. PCC6803 encodes orthologs of both E. coli and B. subtilis RNases, including RNase E and RNase J, respectively. We show that in vitro Sy RNases E and J have an endonucleolytic cleavage specificity that is very similar between them and also compared to orthologous enzymes from E. coli, B. subtilis, and Chlamydomonas. Moreover, Sy RNase J displays a robust 5'-exoribonuclease activity similar to B. subtilis RNase J1, but unlike the evolutionarily related RNase J in chloroplasts. Both nucleases are essential and gene deletions could not be fully segregated in Synechocystis. We generated partially disrupted strains of Sy RNase E and J that were stable enough to allow for their growth and characterization. A transcriptome analysis of these strains partially depleted for RNases E and J, respectively, allowed to observe effects on specific transcripts. RNase E altered the expression of a larger number of chromosomal genes and antisense RNAs compared to RNase J, which rather affects endogenous plasmid encoded transcripts. Our results provide the first description of the main transcriptomic changes induced by the partial depletion of two essential ribonucleases in cyanobacteria.

Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.

Zif268/Egr1 gain of function facilitates hippocampal synaptic plasticity and long-term spatial recognition memory.

It is well established that Zif268/Egr1, a member of the Egr family of transcription factors, is critical for the consolidation of several forms of memory; however, it is as yet uncertain whether increasing expression of Zif268 in neurons can facilitate memory formation. Here, we used an inducible transgenic mouse model to specifically induce Zif268 overexpression in forebrain neurons and examined the effect on recognition memory and hippocampal synaptic transmission and plasticity. We found that Zif268 overexpression during the establishment of memory for objects did not change the ability to form a long-term memory of objects, but enhanced the capacity to form a long-term memory of the spatial location of objects. This enhancement was paralleled by increased long-term potentiation in the dentate gyrus of the hippocampus and by increased activity-dependent expression of Zif268 and selected Zif268 target genes. These results provide novel evidence that transcriptional mechanisms engaging Zif268 contribute to determining the strength of newly encoded memories.

Defective synaptic transmission and structure in the dentate gyrus and selective fear memory impairment in the Rsk2 mutant mouse model of Coffin-Lowry syndrome.

The Coffin-Lowry syndrome (CLS) is a syndromic form of intellectual disability caused by loss-of-function of the RSK2 serine/threonine kinase encoded by the rsk2 gene. Rsk2 knockout mice, a murine model of CLS, exhibit spatial learning and memory impairments, yet the underlying neural mechanisms are unknown. In the current study, we examined the performance of Rsk2 knockout mice in cued, trace and contextual fear memory paradigms and identified selective deficits in the consolidation and reconsolidation of hippocampal-dependent fear memories as task difficulty and hippocampal demand increase. Electrophysiological, biochemical and electron microscopy analyses were carried out in the dentate gyrus of the hippocampus to explore potential alterations in neuronal functions and structure. In vivo and in vitro electrophysiology revealed impaired synaptic transmission, decreased network excitability and reduced AMPA and NMDA conductance in Rsk2 knockout mice. In the absence of RSK2, standard measures of short-term and long-term potentiation (LTP) were normal, however LTP-induced CREB phosphorylation and expression of the transcription factors EGR1/ZIF268 were reduced and that of the scaffolding protein SHANK3 was blocked, indicating impaired activity-dependent gene regulation. At the structural level, the density of perforated and non-perforated synapses and of multiple spine boutons was not altered, however, a clear enlargement of spine neck width and post-synaptic densities indicates altered synapse ultrastructure. These findings show that RSK2 loss-of-function is associated in the dentate gyrus with multi-level alterations that encompass modifications of glutamate receptor channel properties, synaptic transmission, plasticity-associated gene expression and spine morphology, providing novel insights into the mechanisms contributing to cognitive impairments in CLS.

RecA-promoted, RecFOR-independent progressive disassembly of replisomes stalled by helicase inactivation.

In all organisms, replication impairment is a recognized source of genomic instability, raising an increasing interest in the fate of inactivated replication forks. We used Escherichia coli strains with a temperature-inactivated replicative helicase (DnaB) and in vivo single-molecule microscopy to quantify the detailed molecular processing of stalled replication forks. After helicase inactivation, RecA binds to blocked replication forks and is essential for the rapid release of hPol III. The entire holoenzyme is disrupted little by little, with some components lost in few minutes, while others are stable in 70% of cells for at least 1 hr. Although replisome dissociation is delayed in a recA mutant, it is not affected by RecF or RecO inactivation. RecFOR are required for full RecA filaments formation, and we propose that polymerase clearance can be catalyzed by short, RecFOR-independent RecA filaments. Our results identify a function for the universally conserved, central recombination protein RecA.

Upregulation of brain utrophin does not rescue behavioral alterations in dystrophin-deficient mice.

Dystrophin, the protein responsible for X-linked Duchenne muscular dystrophy (DMD), is normally expressed in both muscle and brain, which explains that its loss also leads to cognitive deficits. The utrophin protein, an autosomal homolog, is a natural candidate for dystrophin replacement in patients. Pharmacological upregulation of endogenous utrophin improves muscle physiology in dystrophin-deficient mdx mice, and represents a potential therapeutic tool that has the advantage of allowing delivery to various organs following peripheral injections. Whether this could alleviate cognitive deficits, however, has not been explored. Here, we first investigated basal expression of all utrophins and dystrophins in the brain of mdx mice and found no evidence for spontaneous compensation by utrophins. Then, we show that systemic chronic, spaced injections of arginine butyrate (AB) alleviate muscle alterations and upregulate utrophin expression in the adult brain of mdx mice. AB selectively upregulated brain utrophin Up395, while reducing expression of Up113 and Up71. This, however, was not associated with a significant improvement of behavioral functions typically affected in mdx mice, which include exploration, emotional reactivity, spatial and fear memories. We suggest that AB did not overcome behavioral and cognitive dysfunctions because the regional and cellular expression of utrophins did not coincide with dystrophin expression in untreated mice, nor did it in AB-treated mice. While treatments based on the modulation of utrophin may alleviate DMD phenotypes in certain organs and tissues that coexpress dystrophins and utrophins in the same cells, improvement of cognitive functions would likely require acting on specific dystrophin-dependent mechanisms.

Distinctive features of Egr transcription factor regulation and DNA binding activity in CA1 of the hippocampus in synaptic plasticity and consolidation and reconsolidation of fear memory.

Activity-dependent regulation of Egr1/Zif268, a transcription factor (TF) of the Egr family, is essential for stabilization of dentate gyrus synaptic plasticity and consolidation and reconsolidation of several forms of memory. The gene can be rapidly induced in selective brain circuits after certain types of learning or after recall. Here, we focused on area CA1 and examined regulation of Egr1, Egr2, and Egr3 mRNA and protein, and their DNA binding activity to the Egr response element (ERE) at different times after LTP in vivo and after learning and recall of a fear memory. We found LTP in CA1 leads to rapid induction of the three Egrs, however only Egr1 protein was overexpressed without a co-ordinated change in binding activity, indicating a fundamental difference between CA1 and dentate gyrus LTP. Our investigations in fear memory reveal that both learning and retrieval lead to an increase in binding of constitutively expressed Egr1 and Egr3 to the ERE, but not Egr2. Memory recall was also associated with increased Egr1 protein translation. The nature and temporal dynamics of these changes and tests for interactions between TFs suggest that in addition to ERE-mediated transcription, Egr1 in CA1 may interact with the TF c-Fos to regulate genes via other DNA response elements.

Contribution of Egr1/zif268 to Activity-Dependent Arc/Arg3.1 Transcription in the Dentate Gyrus and Area CA1 of the Hippocampus.

Egr1, a member of the Egr family of transcription factors, and Arc are immediate early genes known to play major roles in synaptic plasticity and memory. Despite evidence that Egr family members can control Arc transcriptional regulation, demonstration of a selective role of Egr1 alone is lacking. We investigated the extent to which activity-dependent Arc expression is dependent on Egr1 by analyzing Arc mRNA expression using fluorescence insitu hybridization in the dorsal dentate gyrus and CA1 of wild-type (WT) and Egr1 knockout mice. Following electroconvulsive shock, we found biphasic expression of Arc in area CA1 in mice, consisting in a rapid (30 min) and transient wave followed by a second late-phase of expression (8 h), and a single but prolonged wave of expression in the dentate gyrus. Egr1 deficiency abolished the latest, but not the early wave of Arc expression in CA1, and curtailed that of the dentate gyrus. Since the early wave of Arc expression was not affected in Egr1 mutant mice, we next analyzed behaviorally induced Arc expression patterns as an index of neural ensemble activation in the dentate gyrus and area CA1 of WT and Egr1 mutant mice. Spatial exploration of novel or familiar environments induced in mice a single early and transient wave of Arc expression in the dentate gyrus and area CA1, which were not affected in Egr1 mutant mice. Analyses of Arc-expressing cells revealed that exploration recruits similar size dentate gyrus and CA1 neural ensembles in WT and Egr1 knockout mice. These findings suggest that hippocampal neural ensembles are normally activated immediately following spatial exploration in Egr1 knockout mice, indicating normal hippocampal encoding of information. They also provide evidence that in condition of strong activation Egr1 alone can control late-phases of activity-dependent Arc transcription in the dentate gyrus and area CA1 of the hippocampus.

Rescue of a dystrophin-like protein by exon skipping normalizes synaptic plasticity in the hippocampus of the mdx mouse.

Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin, a protein that fulfills important functions in both muscle and brain. The mdx mouse model of DMD, which also lacks dystrophin, shows a marked reduction in γ-aminobutyric acid type A (GABA(A))-receptor clustering in central inhibitory synapses and enhanced long-term potentiation (LTP) at CA3-CA1 synapses of the hippocampus. We have recently shown that U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors are able to induce skipping of the mutated exon 23 and to rescue expression of a functional dystrophin-like product both in the muscle and nervous tissue in vivo. In the brain, this rescue was accompanied by restoration of both the size and number of hippocampal GABA(A)-receptor clustering. Here, we report that 25.2±8% of re-expression two months after intrahippocampal injection of rAAV reverses the abnormally enhanced LTP phenotype at CA3-CA1 synapses of mdx mice. These results suggests that dystrophin expression indirectly influences synaptic plasticity through modulation of GABA(A)-receptor clustering and that re-expression of the otherwise deficient protein in the adult can significantly alleviate alteration of neural functions in DMD.

Paroxysmal nocturnal hemoglobinuria in Budd-Chiari syndrome: findings from a cohort study.

BACKGROUND/AIMS: A well recognized cause of Budd-Chiari syndrome (BCS) is paroxysmal nocturnal hemoglobinuria (PNH). PNH is an acquired disorder of hematopoietic stem cells, characterized by intravascular hemolysis and venous thrombosis. Testing for this hematological disorder should be considered in all BCS patients. METHODS: Using data from the EN-Vie study, a multi-center study of 163 patients with BCS, we investigated the relationship between BCS and PNH in 15 patients with combined disease and compared the results to 62 BCS patients in whom PNH was excluded. RESULTS: Median follow-up for the study group (n=77) was 20 months (range 0-44 months). BCS patients with PNH presented with a significantly higher percentage of additional splanchnic vein thrombosis (SVT) as compared to BCS patients without PNH (47% vs. 10%, p=0.002). During follow-up, type and frequency of interventions for BCS was similar between both groups. Six patients with BCS and PNH were successfully treated with a transjugular intrahepatic portosystemic shunt (TIPS). Of 15 patients with PNH, six underwent allogenic stem cell transplantation after diagnosis of BCS. PNH was successfully cured in five cases. There was no significant difference in survival between BCS patients with and without PNH. CONCLUSIONS: This study shows that despite a higher frequency of additional SVT, short-term prognosis of BCS patients with PNH does not differ from BCS patients without PNH. Treatment with TIPS can be safely performed in patients with PNH. Stem cell transplantation appears to be a feasible treatment option for PNH in BCS patients.

In vivo bioluminescence imaging of Ca signalling in the brain of Drosophila.

Many different cells' signalling pathways are universally regulated by Ca(2+) concentration [Ca(2+)] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+) signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+) signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+) imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+) reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+)] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+) response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+)] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca(2+)] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+) stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+) signalling pathways and for functional mapping of neurophysiological processes in the fly brain.

Aiming at minimal invasiveness as a therapeutic strategy for Budd-Chiari syndrome.

The 1-year spontaneous mortality rate in patients with Budd-Chiari syndrome (BCS) approaches 70%. No prospective assessment of indications and impact on survival of current therapeutic procedures has been performed. We evaluated a therapeutic strategy uniformly applied during the last 8 years in a single referral center. Fifty-one consecutive patients first received anticoagulation and were treated for associated diseases. Symptomatic patients were considered for hepatic vein recanalization; then for transjugular intrahepatic portosystemic shunt (TIPS), and finally for liver transplantation. The absence of a complete response led to the next procedure. Assessment was according to the strategy, whether procedures were technically applicable and successful. At entry, median (range) Child-Pugh score and Clichy prognostic index were 8 (5-12), and 5.4 (3.1-7.7), respectively. A complete response was achieved on medical therapy alone in 9 patients; after recanalization in 6, TIPS in 20, liver transplantation in 9, and retransplantation in 1. Of the 41 patients considered for recanalization, the procedure was not feasible in 27 and technically unsuccessful in 3. Of the 34 patients considered for TIPS, the procedure was considered not feasible in 9 and technically unsuccessful in 4. At 1 year of follow-up, a complete response to TIPS was achieved in 84%. One- and 5-year survival from starting anticoagulation were 96% (95% CI, 90-100) and 89% (95% CI, 79-100), respectively. In conclusion, excellent survival can be achieved in BCS patients when therapeutic procedures are introduced by order of increasing invasiveness, based on the response to previous therapy rather than on the severity of the patient's condition.

Adenovirus-mediated CTLA4Ig or CD40Ig gene transfer delays pancreatic islet rejection in a rat-to-mouse xenotransplantation model after systemic but not local expression.

Transient costimulation signal blockade of either CD28/CD80-86 interactions and/or CD40/CD154 interactions can prevent islet rejection in some models of both allo- and xenotransplantation. We have used adenoviruses coding for CTLA4Ig or CD40Ig and compared the efficacy of genetic modification of islets to systemic production through either intramuscular (i.m.) or intravenous (i.v.) injection of these vectors in a rat-to-mouse islet transplantation model. When gene transfer was performed into islets, a high level of primary nonfunction was induced. Furthermore, transduced functional grafts were rejected with the same kinetics as nontransduced islets. In contrast, i.m. AdCTLA4Ig and i.v. AdCD40Ig significantly delayed rejection (mean survival time of 54 +/- 26.9 and 67.6 +/- 44.9 days, respectively, vs. 24.3 +/- 9.7 days for unmodified islets, p < 0.05). Combination of ex vivo AdCTLA4Ig islet transduction and i.v. AdCD40Ig did not improve graft survival further. In conclusion, islet graft transduction with adenoviruses coding for costimulation inhibitors resulted in local expression with low serum concentrations of CTLA4Ig or CD40Ig and was unable to protect islet xenografts from rejection. In contrast, i.m. or i.v. gene transfer resulted in high serum concentrations of these molecules and was highly efficient in prolonging xenograft survival. These results contrast with the efficacy of AdCTLA4Ig we observed in a rat islet allotransplantation model and suggest that islet xenograft rejection might be more difficult to control.

Drosophila CAKI/CMG protein, a homolog of human CASK, is essential for regulation of neurotransmitter vesicle release.

Vertebrate CASK is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins. CASK is present in the nervous system where it binds to neurexin, a transmembrane protein localized in the presynaptic membrane. The Drosophila homologue of CASK is CAKI or CAMGUK. CAKI is expressed in the nervous system of larvae and adult flies. In adult flies, the expression of caki is particularly evident in the visual brain regions. To elucidate the functional role of CASK, we employed a caki null mutant in the model organism Drosophila melanogaster. By means of electrophysiological methods, we analyzed, in adult flies, the spontaneous and evoked neurotransmitter release at the neuromuscular junction (NMJ) as well as the functional status of the giant fiber pathway and of the visual system. We found that in caki mutants, when synaptic activity is modified, the spontaneous neurotransmitter release of the indirect flight muscle NMJ was increased, the response of the giant fiber pathway to continuous stimulation was impaired, and electroretinographic responses to single and continuous repetitive stimuli were altered and optomotor behavior was abnormal. These results support the involvement of CAKI in neurotransmitter release and nervous system function.

A tamponade leading to death after radiofrequency ablation of hepatocellular carcinoma.

A case of hemorrhagic cardiac tamponade after radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC) leading to death is presented. The complication occurred during a procedure performed under general anesthesia with an expandable needle system for a 2-cm HCC sited in the second segment of the liver close to the diaphragm. Thermal damage to the organs surrounding the liver are major complications of liver tumor RFA. For lesions that are adjacent to the cardiac cavities, a discussion of better therapeutic options remains necessary and has to take into account the effectiveness and complication rate of each technique.

Fluorescent activated cell sorting (FACS): a rapid and reliable method to estimate the number of neurons in a mixed population.

Cells derived from the central nervous system (CNS) are usually characterised by manual counting on slides after specific immunolabelling. In this study, we investigated the possibility of using flow cytometry to determine the proportion of neurons, astrocytes or microglial cells in primary cultures. We show that parameters other than physical features are necessary to discriminate between these different cell types because of some overlap in their size and granulosity. We then used specific antibodies against intracellular markers such as Tuj-1 or GFAP to discriminate neurons from astrocytes by flow cytometry. The labelling was specific and reliable, allowing quantitative studies. Indeed, we did not find any significant difference in the number of Tuj-1 and GFAP-positive cells in primary cultures of neuronal and glial cells as determined by manual counting on slides or flow cytometry. More importantly, similar data were obtained in mixed populations, indicating that flow cytometry can be used for quantitative studies of heterogeneous cultures. The flow cytometry therefore appears to be a reliable method for the phenotypic characterisation of CNS-derived cells. This technique which enables a rapid analysis of numerous samples, might be particularly interesting for the study of neural stem cell differentiation.