The role of phospholipase D in modulating the MTOR signaling pathway in polycystic kidney disease

The mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in polycystic kidney disease (PKD). Emerging evidence suggests that phospholipase D (PLD) and its product phosphatidic acid (PA) regulate mTOR activity. In this study, we assessed in vitro the regulatory function of PLD and PA on the mTOR signaling pathway in PKD. We found that the basal level of PLD activity was elevated in PKD cells. Targeting PLD by small molecule inhibitors reduced cell proliferation and blocked mTOR signaling, whereas exogenous PA stimulated mTOR signaling and abolished the inhibitory effect of PLD on PKD cell proliferation. We also show that blocking PLD activity enhanced the sensitivity of PKD cells to rapamycin and that combining PLD inhibitors and rapamycin synergistically inhibited PKD cell proliferation. Furthermore, we demonstrate that targeting mTOR did not induce autophagy, whereas targeting PLD induced autophagosome formation. Taken together, our findings suggest that deregulated mTOR pathway activation is mediated partly by increased PLD signaling in PKD cells. Targeting PLD isoforms with pharmacological inhibitors may represent a new therapeutic strategy in PKD.

Patient self-management of oral anticoagulation with vitamin k antagonists in everyday practice: efficacy and safety in a nationwide long-term prospective cohort study.

Patient self-management (PSM) of oral anticoagulation is under discussion, because evidence from real-life settings is missing. Using data from a nationwide, prospective cohort study in Switzerland, we assessed overall long-term efficacy and safety of PSM and examined subgroups. Data of 1140 patients (5818.9 patient-years) were analysed and no patient were lost to follow-up. Median follow-up was 4.3 years (range 0.2-12.8 years). Median age at the time of training was 54.2 years (range 18.2-85.2) and 34.6% were women. All-cause mortality was 1.4 per 100 patient-years (95% CI 1.1-1.7) with a higher rate in patients with atrial fibrillation (2.5; 1.6-3.7; p<0.001), patients>50 years of age (2.0; 1.6-2.6; p<0.001), and men (1.6; 1.2-2.1; p = 0.036). The rate of thromboembolic events was 0.4 (0.2-0.6) and independent from indications, sex and age. Major bleeding were observed in 1.1 (0.9-1.5) per 100 patient-years. Efficacy was comparable to standard care and new oral anticoagulants in a network meta-analysis. PSM of properly trained patients is effective and safe in a long-term real-life setting and robust across clinical subgroups. Adoption in various clinical settings, including those with limited access to medical care or rural areas is warranted.

Anatomic pathways of peripancreatic fluid draining to mediastinum in recurrent acute pancreatitis: visible human project and CT study

BACKGROUND In past reports, researchers have seldom attached importance to achievements in transforming digital anatomy to radiological diagnosis. However, investigators have been able to illustrate communication relationships in the retroperitoneal space by drawing potential routes in computerized tomography (CT) images or a virtual anatomical atlas. We established a new imaging anatomy research method for comparisons of the communication relationships of the retroperitoneal space in combination with the Visible Human Project and CT images. Specifically, the anatomic pathways of peripancreatic fluid extension to the mediastinum that may potentially transform into fistulas were studied. METHODS We explored potential pathways to the mediastinum based on American and Chinese Visible Human Project datasets. These drainage pathways to the mediastinum were confirmed or corrected in CT images of 51 patients with recurrent acute pancreatitis in 2011. We also investigated whether additional routes to the mediastinum were displayed in CT images that were not in Visible Human Project images. PRINCIPAL FINDINGS All hypothesized routes to the mediastinum displayed in Visible Human Project images, except for routes from the retromesenteric plane to the bilateral retrorenal plane across the bilateral fascial trifurcation and further to the retrocrural space via the aortic hiatus, were confirmed in CT images. In addition, route 13 via the narrow space between the left costal and crural diaphragm into the retrocrural space was demonstrated for the first time in CT images. CONCLUSION This type of exploration model related to imaging anatomy may be used to support research on the communication relationships of abdominal spaces, mediastinal spaces, cervical fascial spaces and other areas of the body.

DAT1 polymorphism determines L-DOPA effects on learning about others' prosociality.

Despite that a wealth of evidence links striatal dopamine to individualś reward learning performance in non-social environments, the neurochemical underpinnings of such learning during social interaction are unknown. Here, we show that the administration of 300 mg of the dopamine precursor L-DOPA to 200 healthy male subjects influences learning about a partners' prosocial preferences in a novel social interaction task, which is akin to a repeated trust game. We found learning to be modulated by a well-established genetic marker of striatal dopamine levels, the 40-bp variable number tandem repeats polymorphism of the dopamine transporter (DAT1 polymorphism). In particular, we found that L-DOPA improves learning in 10/10R genoype subjects, who are assumed to have lower endogenous striatal dopamine levels and impairs learning in 9/10R genotype subjects, who are assumed to have higher endogenous dopamine levels. These findings provide first evidence for a critical role of dopamine in learning whether an interaction partner has a prosocial or a selfish personality. The applied pharmacogenetic approach may open doors to new ways of studying psychiatric disorders such as psychosis, which is characterized by distorted perceptions of others' prosocial attitudes.

Validation of network communicability metrics for the analysis of brain structural networks.

Computational network analysis provides new methods to analyze the brain's structural organization based on diffusion imaging tractography data. Networks are characterized by global and local metrics that have recently given promising insights into diagnosis and the further understanding of psychiatric and neurologic disorders. Most of these metrics are based on the idea that information in a network flows along the shortest paths. In contrast to this notion, communicability is a broader measure of connectivity which assumes that information could flow along all possible paths between two nodes. In our work, the features of network metrics related to communicability were explored for the first time in the healthy structural brain network. In addition, the sensitivity of such metrics was analysed using simulated lesions to specific nodes and network connections. Results showed advantages of communicability over conventional metrics in detecting densely connected nodes as well as subsets of nodes vulnerable to lesions. In addition, communicability centrality was shown to be widely affected by the lesions and the changes were negatively correlated with the distance from lesion site. In summary, our analysis suggests that communicability metrics that may provide an insight into the integrative properties of the structural brain network and that these metrics may be useful for the analysis of brain networks in the presence of lesions. Nevertheless, the interpretation of communicability is not straightforward; hence these metrics should be used as a supplement to the more standard connectivity network metrics.

Selection signatures in worldwide sheep populations.

The diversity of populations in domestic species offers great opportunities to study genome response to selection. The recently published Sheep HapMap dataset is a great example of characterization of the world wide genetic diversity in sheep. In this study, we re-analyzed the Sheep HapMap dataset to identify selection signatures in worldwide sheep populations. Compared to previous analyses, we made use of statistical methods that (i) take account of the hierarchical structure of sheep populations, (ii) make use of linkage disequilibrium information and (iii) focus specifically on either recent or older selection signatures. We show that this allows pinpointing several new selection signatures in the sheep genome and distinguishing those related to modern breeding objectives and to earlier post-domestication constraints. The newly identified regions, together with the ones previously identified, reveal the extensive genome response to selection on morphology, color and adaptation to new environments.

Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia.

INTRODUCTION Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses. RESULTS For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patient's bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n=88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n=91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n=91), which reflect the total neoplastic burden, revealed four patient groups with different survival. CONCLUSION AND PERSPECTIVE Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.

Transcriptional profiles of cytokine/chemokine factors of immune cell-homing to the parasitic lesions: a comprehensive one-year course study in the liver of E. multilocularis-infected mice.

Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-β. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.

Potassium and the excitability properties of normal human motor axons in vivo.

Hyperkalemia is an important cause of membrane depolarization in renal failure. A recent theoretical model of axonal excitability explains the effects of potassium on threshold electrotonus, but predicts changes in superexcitability in the opposite direction to those observed. To resolve this contradiction we assessed the relationship between serum potassium and motor axon excitability properties in 38 volunteers with normal potassium levels. Most threshold electrotonus measures were strongly correlated with potassium, and superexcitability decreased at higher potassium levels (P = 0.016), contrary to the existing model. Improved modelling of potassium effects was achieved by making the potassium currents obey the constant-field theory, and by making the potassium permeabilities proportional to external potassium, as has been observed in vitro. This new model also accounted well for the changes in superexcitability and other excitability measures previously reported in renal failure. These results demonstrate the importance of taking potassium levels into account when assessing axonal membrane dysfunction by excitability testing, and provide evidence that potassium currents are activated by external potassium in vivo.

Expression, purification, and structural insights for the human uric acid transporter, GLUT9, using the Xenopus laevis oocytes system.

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.

The Self-Expanding Symetis Acurate Does Not Increase Cerebral Microembolic Load When Compared to the Balloon-Expandable Edwards Sapien Prosthesis: A Transcranial Doppler Study in Patients Undergoing Transapical Aortic Valve Implantation

Objectives The aim of this study was to quantify potential differences in count, frequency and pattern of high-intensity transient signals (HITS) during transapical transcatheter aortic valve implantation (TA-TAVI), by comparing the Symetis Acurate TA (SA) with the balloon-expandable Edwards Sapien XT (ES) system. Background Recently, the Symetis Acurate TA revalving system has been introduced for TA-TAVI. The Symetis Acurate TA aortic bioprosthesis is self-expanding and is deployed by a specific two-step implantation technique. Whether this novel method increases the load of intraprocedural emboli, detected by transcranial Doppler ultrasound (TCD) as HITS, or not is not clear. Methods Twenty-two patients (n = 11 in each study arm, median logistic EuroScore 20%, median STS score 7%) displayed continuous TCD signals of good quality throughout the entire TA-TAVI procedure and were included in the final analysis. Data are presented as median with interquartile ranges. Results No significant differences were detected in total procedural or interval-related HITS load (SA: 303 [200; 594], ES: 499 [285; 941]; p = 0.16). With both devices, HITS peaked during prosthesis deployment (PD), whereas significantly fewer HITS occurred during instrumentation (SA: p = 0.002; ES: <0.001) or post-implantation PI (SA: p = 0.007; ES: <0.001). PD-associated HITS amounted to almost half of the total HITS load. One patient suffered new disabling stroke at 30 days. Thirty-day mortality amounted to 13.6% (3 of 22 patients). Conclusions Simplified transapical delivery using the self-expanding SA device does not increase HITS, despite of a two-step deployment technique with more interactions with the native aortic valve, when compared to the balloon-expandable ES valve. The similarity in HITS count, frequency and pattern with the two systems suggests a common mechanism for the release of cerebral microemboli.

Retrocrural space involvement on computed tomography as a predictor of mortality and disease severity in acute pancreatitis

BACKGROUND Because computed tomography (CT) has advantages for visualizing the manifestation of necrosis and local complications, a series of scoring systems based on CT manifestations have been developed for assessing the clinical outcomes of acute pancreatitis (AP), including the CT severity index (CTSI), modified CTSI, etc. Despite the internationally accepted CTSI having been successfully used to predict the overall mortality and disease severity of AP, recent literature has revealed the limitations of the CTSI. Using the Delphi method, we establish a new scoring system based on retrocrural space involvement (RCSI), and compared its effectiveness at evaluating the mortality and severity of AP with that of the CTSI. METHODS We reviewed CT images of 257 patients with AP taken within 3-5 days of admission in 2012. The RCSI scoring system, which includes assessment of infectious conditions involving the retrocrural space and the adjacent pleural cavity, was established using the Delphi method. Two radiologists independently assessed the RCSI and CTSI scores. The predictive points of the RCSI and CTSI scoring systems in evaluating the mortality and severity of AP were estimated using receiver operating characteristic (ROC) curves. PRINCIPAL FINDINGS The RCSI score can accurately predict the mortality and disease severity. The area under the ROC curve for the RCSI versus CTSI score was 0.962±0.011 versus 0.900±0.021 for predicting the mortality, and 0.888±0.025 versus 0.904±0.020 for predicting the severity of AP. Applying ROC analysis to our data showed that a RCSI score of 4 was the best cutoff value, above which mortality could be identified. CONCLUSION The Delphi method was innovatively adopted to establish a scoring system to predict the clinical outcome of AP. The RCSI scoring system can predict the mortality of AP better than the CTSI system, and the severity of AP equally as well.

Experimental infection of the pig with Mycobacterium ulcerans: a novel model for studying the pathogenesis of Buruli ulcer disease.

BACKGROUND Buruli ulcer (BU) is a slowly progressing, necrotising disease of the skin caused by infection with Mycobacterium ulcerans. Non-ulcerative manifestations are nodules, plaques and oedema, which may progress to ulceration of large parts of the skin. Histopathologically, BU is characterized by coagulative necrosis, fat cell ghosts, epidermal hyperplasia, clusters of extracellular acid fast bacilli (AFB) in the subcutaneous tissue and lack of major inflammatory infiltration. The mode of transmission of BU is not clear and there is only limited information on the early pathogenesis of the disease available. METHODOLOGY/PRINCIPAL FINDINGS For evaluating the potential of the pig as experimental infection model for BU, we infected pigs subcutaneously with different doses of M. ulcerans. The infected skin sites were excised 2.5 or 6.5 weeks after infection and processed for histopathological analysis. With doses of 2 × 10(7) and 2 × 10(6) colony forming units (CFU) we observed the development of nodular lesions that subsequently progressed to ulcerative or plaque-like lesions. At lower inoculation doses signs of infection found after 2.5 weeks had spontaneously resolved at 6.5 weeks. The observed macroscopic and histopathological changes closely resembled those found in M. ulcerans disease in humans. CONCLUSION/SIGNIFICANCE Our results demonstrate that the pig can be infected with M. ulcerans. Productive infection leads to the development of lesions that closely resemble human BU lesions. The pig infection model therefore has great potential for studying the early pathogenesis of BU and for the development of new therapeutic and prophylactic interventions.

Hippocampus Is Place of Interaction between Unconscious and Conscious Memories.

Recent evidence suggests that humans can form and later retrieve new semantic relations unconsciously by way of hippocampus-the key structure also recruited for conscious relational (episodic) memory. If the hippocampus subserves both conscious and unconscious relational encoding/retrieval, one would expect the hippocampus to be place of unconscious-conscious interactions during memory retrieval. We tested this hypothesis in an fMRI experiment probing the interaction between the unconscious and conscious retrieval of face-associated information. For the establishment of unconscious relational memories, we presented subliminal (masked) combinations of unfamiliar faces and written occupations ("actor" or "politician"). At test, we presented the former subliminal faces, but now supraliminally, as cues for the reactivation of the unconsciously associated occupations. We hypothesized that unconscious reactivation of the associated occupation-actor or politician-would facilitate or inhibit the subsequent conscious retrieval of a celebrity's occupation, which was also actor or politician. Depending on whether the reactivated unconscious occupation was congruent or incongruent to the celebrity's occupation, we expected either quicker or delayed conscious retrieval process. Conscious retrieval was quicker in the congruent relative to a neutral baseline condition but not delayed in the incongruent condition. fMRI data collected during subliminal face-occupation encoding confirmed previous evidence that the hippocampus was interacting with neocortical storage sites of semantic knowledge to support relational encoding. fMRI data collected at test revealed that the facilitated conscious retrieval was paralleled by deactivations in the hippocampus and neocortical storage sites of semantic knowledge. We assume that the unconscious reactivation has pre-activated overlapping relational representations in the hippocampus reducing the neural effort for conscious retrieval. This finding supports the notion of synergistic interactions between conscious and unconscious relational memories in a common, cohesive hippocampal-neocortical memory space.

The transcriptome of equine peripheral blood mononuclear cells.

Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To improve the horse genome annotation we performed RNA-seq on 561 samples of peripheral blood mononuclear cells (PBMCs) derived from 85 Warmblood horses. The mapped sequencing reads were used to build a new transcriptome assembly. The new assembly revealed many alternative isoforms associated to known genes or to those predicted by the Ensembl and/or Gnomon pipelines. We also identified 7,531 transcripts not associated with any horse gene annotated in public databases. Of these, 3,280 transcripts did not have a homologous match to any sequence deposited in the NCBI EST database suggesting horse specificity. The unknown transcripts were categorized as coding and noncoding based on predicted coding potential scores. Among them 230 transcripts had high coding potential score, at least 2 exons, and an open reading frame of at least 300 nt. We experimentally validated 9 new equine coding transcripts using RT-PCR and Sanger sequencing. Our results provide valuable detailed information on many transcripts yet to be annotated in the horse genome.