Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease.

Germline mutations in CYBB, the human gene encoding the gp91(phox) subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This 'experiment of nature' indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria.

Dissociating electrophysiological correlates of contextual and perceptual learning in a visual search task.

Perceptual learning and contextual learning are two types of implicit visual learning that can co-occur in the same tasks. For example, to find an animal in the woods, you need to know where to look in the environment (contextual learning) and you must be able to discriminate its features (perceptual learning). However, contextual and perceptual learning are typically studied using distinct experimental paradigms, and little is known regarding their comparative neural mechanisms. In this study, we investigated contextual and perceptual learning in 12 healthy adult humans as they performed the same visual search task, and we examined psychophysical and electrophysiological (event-related potentials) measures of learning. Participants were trained to look for a visual stimulus, a small line with a specific orientation, presented among distractors. We found better performance for the trained target orientation as compared to an untrained control orientation, reflecting specificity of perceptual learning for the orientation of trained elements. This orientation specificity effect was associated with changes in the C1 component. We also found better performance for repeated spatial configurations as compared to novel ones, reflecting contextual learning. This context-specific effect was associated with the N2pc component. Taken together, these results suggest that contextual and perceptual learning are distinct visual learning phenomena that have different behavioral and electrophysiological characteristics.

Network dynamics of the brain and influence of the epileptic seizure onset zone.

The human brain is a dynamic networked system. Patients with partial epileptic seizures have focal regions that periodically diverge from normal brain network dynamics during seizures. We studied the evolution of brain connectivity before, during, and after seizures with graph-theoretic techniques on continuous electrocorticographic (ECoG) recordings (5.4 ± 1.7 d per patient, mean ± SD) from 12 patients with temporal, occipital, or frontal lobe partial onset seizures. Each electrode was considered a node in a graph, and edges between pairs of nodes were weighted by their coherence within a frequency band. The leading eigenvector of the connectivity matrix, which captures network structure, was tracked over time and clustered to uncover a finite set of brain network states. Across patients, we found that (i) the network connectivity is structured and defines a finite set of brain states, (ii) seizures are characterized by a consistent sequence of states, (iii) a subset of nodes is isolated from the network at seizure onset and becomes more connected with the network toward seizure termination, and (iv) the isolated nodes may identify the seizure onset zone with high specificity and sensitivity. To localize a seizure, clinicians visually inspect seizures recorded from multiple intracranial electrode contacts, a time-consuming process that may not always result in definitive localization. We show that network metrics computed from all ECoG channels capture the dynamics of the seizure onset zone as it diverges from normal overall network structure. This suggests that a state space model can be used to help localize the seizure onset zone in ECoG recordings.

Microbial eukaryote communities exhibit robust biogeographical patterns along a gradient of Patagonian and Antarctic lakes.

Microbial eukaryotes play important roles in aquatic ecosystem functioning. Unravelling their distribution patterns and biogeography provides important baseline information to infer the underlying mechanisms that regulate the biodiversity and complexity of ecosystems. We studied the distribution patterns and factors driving diversity gradients in microeukaryote communities (total, abundant, uncommon and rare community composition) along a latitudinal gradient of lakes distributed from Argentinean Patagonia to Maritime Antarctica using both denaturing gradient gel electrophoresis (DGGE) and high-throughput sequencing (Illumina HiSeq). DGGE and abundant Illumina operational taxonomic units (OTUs) showed both decreasing richness with latitude and significant differences between Patagonian and Antarctic lakes communities. In contrast, total richness did not change significantly across the latitudinal gradient, although evenness and diversity indices were significantly higher in Patagonian lakes. Beta-diversity was characterized by a high species turnover, influenced by both environmental and geographical descriptors, although this pattern faded in the rare community. Our results suggest the co-existence of a 'core biosphere' containing reduced number of abundant/dominant OTUs on which classical ecological rules apply, together with a much larger seedbank of rare OTUs driven by stochastic and reduced dispersal processes. These findings shed new light on the biogeographical patterns and forces structuring inland microeukaryote composition across broad spatial scales.

Discovery and structural diversity of the hepatitis C virus NS3/4A serine protease inhibitor series leading to clinical candidate IDX320.

Exploration of the P2 region by mimicking the proline motif found in BILN2061 resulted in the discovery of two series of potent HCV NS3/4A protease inhibitors. X-ray crystal structure of the ligand in contact with the NS3/4A protein and modulation of the quinoline heterocyclic region by structure based design and modeling allowed for the optimization of enzyme potency and cellular activity. This research led to the selection of clinical candidate IDX320 having good genotype coverage and pharmacokinetic properties in various species.

Alpha-band EEG activity in perceptual learning.

In studies of perceptual learning (PL), subjects are typically highly trained across many sessions to achieve perceptual benefits on the stimuli in those tasks. There is currently significant debate regarding what sources of brain plasticity underlie these PL-based learning improvements. Here we investigate the hypothesis that PL, among other mechanisms, leads to task automaticity, especially in the presence of the trained stimuli. To investigate this hypothesis, we trained participants for eight sessions to find an oriented target in a field of near-oriented distractors and examined alpha-band activity, which modulates with attention to visual stimuli, as a possible measure of automaticity. Alpha-band activity was acquired via electroencephalogram (EEG), before and after training, as participants performed the task with trained and untrained stimuli. Results show that participants underwent significant learning in this task (as assessed by threshold, accuracy, and reaction time improvements) and that alpha power increased during the pre-stimulus period and then underwent greater desynchronization at the time of stimulus presentation following training. However, these changes in alpha-band activity were not specific to the trained stimuli, with similar patterns of posttraining alpha power for trained and untrained stimuli. These data are consistent with the view that participants were more efficient at focusing resources at the time of stimulus presentation and are consistent with a greater automaticity of task performance. These findings have implications for PL, as transfer effects from trained to untrained stimuli may partially depend on differential effort of the individual at the time of stimulus processing.

Immunohistochemical application of a highly sensitive and specific murine monoclonal antibody recognising the extracellular domain of the human hepatocyte growth factor receptor (MET).

AIMS: Development of novel targeted therapies directed against hepatocyte growth factor (HGF) or its receptor (MET) necessitates the availability of quality diagnostics to facilitate their safe and effective use. Limitations of some commercially available anti-MET antibodies have prompted development of the highly sensitive and specific clone A2H2-3. Here we report its analytical properties when applied by an automated immunohistochemistry method. METHODS AND RESULTS: Excellent antibody specificity was demonstrated by immunoblot, ELISA, and IHC evaluation of characterised cell lines including NIH3T3 overexpressing the related kinase MST1R (RON). Sensitivity was confirmed by measurements of MET in cell lines or characterised tissues. IHC correlated well with FISH and quantitative RT-PCR assessments of MET (P < 0.001). Good total agreement (89%) was observed with the anti-MET antibody clone SP44 using whole-tissue sections, but poor positive agreement (21-47%) was seen in tissue microarray cores. Multiple lots displayed appropriate reproducibility (R(2)  > 0.9). Prevalence of MET positivity by IHC was higher in non-squamous cell NSCLC, MET or EGFR amplified cases, and in tumours harbouring abnormalities in EGFR exon 19 or 21. CONCLUSIONS: The anti-MET antibody clone A2H2-3 displays excellent specificity and sensitivity. These properties make it suitable for clinical trial investigations and development as a potential companion diagnostic.

A triple dissociation between learning of target, distractors, and spatial contexts.

When we perform any task, we engage a diverse set of processes. These processes can be optimized with learning. While there exists substantial research that probes specific aspects of learning, there is a scarcity of research regarding interactions between different types of learning. Here, we investigate possible interactions between Perceptual Learning (PL) and Contextual Learning (CL), two types of implicit learning that have garnered much attention in the psychological sciences and that often co-occur in natural settings. PL increases sensitivity to features of task targets and distractors and is thought to involve improvements in low-level perceptual processing. CL regards learning of regularities in the environment (such as spatial relations between objects) and is consistent with improvements in higher level perceptual processes. Surprisingly, we found CL, PL for target features, and PL for distractor features to be independent. This triple dissociation demonstrates how different learning processes may operate in parallel as tasks are mastered.

Dynamic training of a novelty classifier algorithm for real-time detection of early seizure onset.

OBJECTIVE: To develop an adaptive framework for seizure detection in real-time that is practical to use in the Epilepsy Monitoring Unit (EMU) as a warning signal, and whose output helps characterize epileptiform activity. METHODS: Our algorithm was tested on intracranial EEG from epilepsy patients admitted to the EMU for presurgical evaluation. Our framework uses a one-class Support Vector Machine (SVM) that is being trained dynamically according to past activity in all available channels to classify the novelty of the current activity. In this study we compared multiple configurations using a one-class SVM to assess if there is significance over specific neural features or electrode locations. RESULTS: Our results show that the algorithm reaches a sensitivity of 87% for early-onset seizure detection and of 97.7% as a generic seizure detection. CONCLUSIONS: Our algorithm is capable of running in real-time and achieving a high performance for early seizure-onset detection with a low false positive rate and robustness in detection of different type of seizure-onset patterns. SIGNIFICANCE: This algorithm offers a solution to warning systems in the EMU as well as a tool for seizure characterization during post-hoc analysis of intracranial EEG data for surgical resection of the epileptogenic network.

p47phox Phox homology domain regulates plasma membrane but not phagosome neutrophil NADPH oxidase activation.

The assembly of cytosolic subunits p47(phox), p67(phox), and p40(phox) with flavocytochrome b(558) at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47(phox) subunit plays a critical role in oxidase assembly. Recent studies showed that the p47(phox) Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47(phox) PX domain in neutrophils is unclear, we investigated its role using p47(phox) knock-out (KO) mouse neutrophils to express human p47(phox) and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47(phox) proteins were expressed at levels similar to endogenous murine p47(phox), with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47(phox) rescued p47(phox) KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47(phox) with Arg(90) substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47(phox) Arg(90) mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47(phox) PX domain for neutrophil NADPH oxidase activation.

A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity.

Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91(phox) and p22(phox), which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47(phox) and p67(phox). A fifth subunit, p40(phox), plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40(phox), in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40(phox)R105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40(phox)-deficient granulocytes, with premature loss of p40(phox)R105Q from phagosomes. Thus, p40(phox) binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.

Improving early seizure detection.

Over the last decade, the search for a method able to reliably predict seizures hours in advance has been largely replaced by the more realistic goal of very early detection of seizure onset, which would allow therapeutic or warning devices to be triggered prior to the onset of disabling clinical symptoms. We explore in this article the steps along the pathway from data acquisition to closed-loop applications that can and should be considered to design the most efficient early seizure detection. Microelectrodes, high-frequency oscillations, high sampling rate, high-density arrays, and modern analysis techniques are all elements of the recording and detection process that in combination with modeling studies can provide new insights into the dynamics of seizure onsets. Each of these steps needs to be considered if detection devices that will favorably impact the quality of life of patients are to be implemented. This article is part of a Supplemental Special Issue entitled The Future of Automated Seizure Detection and Prediction.

Aberrant regulation of hematopoiesis by T cells in BAZF-deficient mice.

The BAZF (BCL-6b) protein is highly similar to the BCL-6 transcriptional repressor. While BCL-6 has been characterized extensively, relatively little is known about the normal function of BAZF. In order to understand the physiological role of BAZF, we created BAZF-deficient mice. Unlike BCL-6-deficient mice, BAZF-deficient mice are healthy and normal in size. However, BAZF-deficient mice have a hematopoietic progenitor phenotype that is almost identical to that of BCL-6-deficient mice. Compared to wild-type mice, both BAZF-deficient and BCL-6-deficient mice have greatly reduced numbers of cycling hematopoietic progenitor cells (HPC) in the BM and greatly increased numbers of cycling HPC in the spleen. In contrast to HPC from wild-type mice, HPC from BAZF-deficient and BCL-6-deficient mice are resistant to chemokine-induced myelosuppression and do not show a synergistic growth response to granulocyte-macrophage colony-stimulating factor plus stem cell factor. Depletion of CD8 T cells in BAZF-deficient mice reverses several of the hematopoietic defects in these mice. Since both BAZF- and BCL-6-deficient mice have defects in CD8 T-cell differentiation, we hypothesize that both BCL-6 and BAZF regulate HPC homeostasis by an indirect pathway involving CD8 T cells.

Controversies in epilepsy: debates held during the Fourth International Workshop on Seizure Prediction.

Debates on six controversial topics were held during the Fourth International Workshop on Seizure Prediction (IWSP4) convened in Kansas City, KS, USA, July 4-7, 2009. The topics were (1) Ictogenesis: Focus versus Network? (2) Spikes and Seizures: Step-relatives or Siblings? (3) Ictogenesis: A Result of Hyposynchrony? (4) Can Focal Seizures Be Caused by Excessive Inhibition? (5) Do High-Frequency Oscillations Provide Relevant Independent Information? (6) Phase Synchronization: Is It Worthwhile as Measured? This article, written by the IWSP4 organizing committee and the debaters, summarizes the arguments presented during the debates.

Can a cure be achieved with taxane-based chemotherapy plus surgery in patients with primary mediastinal non-seminomatous germ cell tumors and progression or relapse despite first-line chemotherapy?

Primary mediastinal non-seminomatous germ cell tumors (NSCGTs) have a poor prognosis in the International Germ Cell Cancer Collaborative Group (IGCCCG) classification. There is no clear standard of treatment at relapse. Between 1995 and 2005, 13 patients experienced progression or relapse, and 1 patient was cured with a taxane-based chemotherapy plus surgical resection at our institution.

Analysis of dynamics and propagation of parietal cingulate seizures with secondary mesial temporal involvement.

Cingulate-onset seizures, particularly those originating from parietal cingulate regions, are inadequately described and confounded by patterns of propagation. We analyzed scalp and depth electrode recordings in a patient whose seizures originated from a lesion in the right posterior cingulate region and produced secondary seizure activity in ipsilateral mesial temporal structures. Analyses included the matching pursuit (MP) method of time-frequency decomposition and the Gabor atom density (GAD) measure of signal complexity. Although scalp recordings suggested a right temporal onset, seizures recorded with depth electrodes clearly began in the parietal cingulate region before producing a secondary discharge in ipsilateral mesial structures. GAD revealed a significant increase in complexity during ictal cingulate activity and a consistent pattern of subsequent complexity changes in the hippocampus 30 seconds later. MP and GAD measures were valuable supplements to confirm the stereotyped pattern of both time-frequency changes and complexity. This provides additional evidence for pathways between the parietal cingulate region and mesial temporal structures and raises questions as to whether parietal cingulate seizures can produce clinical symptoms independent of regional or remote propagation.

Differences in the temporal processing between identification and categorization of durations: A behavioral and ERP study.

This study examined how different forms of decision-making modulate time perception. Participants performed temporal bisection and generalization tasks, requiring them to either categorize a stimulus duration as more similar to short or long standards (bisection), or identify whether or not a duration was the same as a previously-presented standard (generalization). They responded faster in the bisection task than in the generalization one for long durations. This behavioral effect was accompanied by modulation of event-related potentials (ERPs). More specifically, between 500 ms and 600 ms after stimulus offset, a late positive component (LPC), appearing in the centro-parietal region, showed lower amplitude in the bisection task than in the generalization one, for long durations, mirroring the behavioral result. Before (200-500 ms) and after (600-800 ms) this window, the amplitude of the LPC was globally larger in the generalization paradigm, independently of the presented duration. Finally, the LPC amplitude was higher for long durations than for shorter ones at the beginning of the component (between 200 and 300 ms after stimulus extinction) and was then higher for short durations than for longer ones (between 300 and 600 ms after offset), indicating that the decision about the former stimuli was made earlier than for the latter ones. Taken together, these results indicate that the categorization of durations engages fewer cognitive resources than their identification.

Early disruption of parvalbumin expression and perineuronal nets in the hippocampus of the Tg2576 mouse model of Alzheimer's disease can be rescued by enriched environment.

Recent findings show that parvalbumin (PV) interneuron function is impaired in Alzheimer's disease (AD), and that this impairment in PV function can be linked to network dysfunction and memory deficits. PV cells are often associated with a specific extracellular matrix, the perineuronal net (PNN). PNNs are believed to protect PV cell integrity, and whether the amyloidopathy affects PNNs remains unclear. Here, we evaluated the number of PV cells with and without PNNs in the hippocampus of the Tg2576 mouse model of AD at different stages of the disease. We show a deficit of PV+ and/or PV+/PNN+ cells in the areas CA1, CA2, and CA3 in Tg2576 as young as 3 months of age. Importantly, transient exposure to an enriched environment, which has proven long-lasting beneficial effects on memory in AD subjects, rescues the PV/PNN cell number deficits. We conclude that cognitive improvements induced by enriched environment in AD mouse models could be supported by a remodeling of hippocampal PV cell network and their PNNs.

[An unusual case of duplication of the spinal canal]. / Un cas inhabituel de duplication du canal rachidien.

We describe here a very rare congenital malformation, in which the vertebra Th 9 to 11 were divided into independent bodies and neural arches. The vertebral canal contained the spinal cord, whereas the space between bodies and arches was filled by nerve roots and a central remnant cord, not clearly connected to the main spinal cord. This malformation was considered as related to a fetal alcohol syndrome, with craniofacial and genitourinary abnormalities. The authors analyze, moreover the possible role of abnormal Sonic Hedgehog gene patterning in the pathogeny of this complex malformative sequence.

Cytotoxicity of seven naturally occurring phenolic compounds towards multi-factorial drug-resistant cancer cells.

INTRODUCTION: In medical oncology, multi-drug resistance (MDR) of cancer cells continues to be a major impediment. We are in quest of novel anti-proliferative agents to overcome drug-resistant tumor cells. METHODS: In the present study, we investigated the cytotoxicity of 7 naturally occurring phenolic compounds including two isoflavonoids alpinumisoflavone (1) and laburnetin (2), one biflavonoid amentoflavone (3), three lignans pycnanthulignene A (4), pycnanthulignene B (5), and syringaresinol (7) and one xanthone, euxanthone (6) against 9 drug-sensitive and MDR cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry. RESULTS: The IC50 values for the investigational phenolics ranged from 5.91 µM (towards leukemia CEM/ADR5000 cells) to 65.65 µM (towards drug-resistant breast adenocarcinoma MDA-MB-231-BCRP cells) for 1, 27.63 µM (towards leukemia CCRF-CEM cells) to 107.57 µM (towards MDA-MB-231-pcDNA cells) for 2, from 5.84 µM (towards CEM/ADR5000 cells) to 65.32 µM (towards colon carcinoma HCT116 (p53(-/-)) cells) for 4 and 0.20 µM (towards CCRF-CEM cells) to 195.12 µM (towards leukemia CEM/ADR5000) for doxorubicin. Phenolics 3, 5, 6 and 7 displayed selectivity cytotoxic effects on cancer cells lines. Compounds 1 and 4 induced apoptosis in CCRF-CEM cells, mediated by loss of MMP and increase ROS production. CONCLUSIONS: The studied phenolics and mostly isoflavonoid 1 and lignan 4 are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers.