Multineuron firing patterns are often observed, yet are predicted to be rare by models that assume independent firing. To explain these correlated network states, two groups recently applied a second-order maximum entropy model that used only observed firing rates and pairwise interactions as parameters (Schneidman et al., 2006; Shlens et al., 2006). Interestingly, with these minimal assumptions they predicted 90 -99% of network correlations. If generally applicable, this approach could vastly simplify analyses of complex networks. However, this initial work was done largely on retinal tissue, and its applicability to cortical circuits is mostly unknown. This work also did not address the temporal evolution of correlated states. To investigate these issues, we applied the model to multielectrode data containing spontaneous spikes or local field potentials from cortical slices and cultures. The model worked slightly less well in cortex than in retina, accounting for 88 Ϯ 7% (mean Ϯ SD) of network correlations. In addition, in 8 of 13 preparations, the observed sequences of correlated states were significantly longer than predicted by concatenating states from the model. This suggested that temporal dependencies are a common feature of cortical network activity, and should be considered in future models. We found a significant relationship between strong pairwise temporal correlations and observed sequence length, suggesting that pairwise temporal correlations may allow the model to be extended into the temporal domain. We conclude that although a second-order maximum entropy model successfully predicts correlated states in cortical networks, it should be extended to account for temporal correlations observed between states.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

D-Mannitol is the predominant carbon compound in conidiospores of the filamentous fungus Aspergillus niger and makes up 10 to 15% of the dry weight. A number of physiological functions have been ascribed to mannitol, including serving as a reserve carbon source, as an antioxidant, and to store reducing power. In this study, we cloned and characterized the A. niger mpdA gene, which encodes mannitol 1-phosphate dehydrogenase (MPD), the first enzyme in the mannitol biosynthesis pathway. The mpdA promoter contains putative binding sites for the development-specific transcription factors BRLA and ABAA. Furthermore, increased expression of mpdA in sporulating mycelium suggests that mannitol biosynthesis is, to a certain extent, developmentally regulated in A. niger. Inactivation of mpdA abolished mannitol biosynthesis in growing mycelium and reduced the mannitol level in conidiospores to 30% that in the wild type, indicating that MPD and mannitol 1-phosphate phosphatase form the major metabolic pathway for mannitol biosynthesis in A. niger. The viability of spores after prolonged storage and germination kinetics were normal in an mpdA null mutant, indicating that mannitol does not play an essential role as a reserve carbon source in A. niger conidia. However, conidiospores of a ⌬mpdA strain were extremely sensitive to a variety of stress conditions, including high temperature, oxidative stress and, to a lesser extent, freezing and lyophilization. Since mannitol supplied in the medium during sporulation repaired this deficiency, mannitol appears to be essential for the protection of A. niger spores against cell damage under these stress conditions. Polyols or polyhydroxyalcohols are present in all organisms, from bacteria to animals. In particular, plants and fungi are known to accumulate high levels of polyols intracellularly, up to several hundred millimoles per liter. The filamentous fungus Aspergillus niger produces a number of different polyols, including glycerol, erythritol, and D-mannitol (41). The intracellular concentrations of the individual polyols in A. niger depend on growth conditions and developmental stage, suggesting that polyols have important functions in fungal physiology.The hexitol D-mannitol is accumulated by vascular plants (37) and many fungal species (21). In A. niger conidiospores, D-mannitol is the predominant carbon-containing compound and makes up 10 to 15% of the dry weight (41). The metabolic pathway for mannitol biosynthesis in ascomycete and deuteromycete fungi most likely consists of two steps (19) (Fig. 1). Fructose 6-phosphate is reduced to mannitol 1-phosphate by NAD ϩ -dependent mannitol 1-phosphate dehydrogenase (MPD), followed by the dephosphorylation of mannitol 1-phosphate, which is catalyzed by mannitol 1-phosphate phosphatase (MPP), yielding mannitol. The presence of MPD and MPP in several fungi, such as A. niger (22), Aspergillus nidulans (34), and Alternaria alternata (18), has been demonstrated, but these enzymes are also responsible for mannitol biosynthesis in protozoa...

SUMMARYPurpose: To determine if the clinical characteristics of nonepileptic seizures (NES) are different in children younger than 13 years age as compared to adolescents. Methods: Retrospective review of medical records and video-EEGs (VEEG) of all patients with NES confirmed on VEEG monitoring was performed. Results: Sixty-eight (3.5%) of 1,967 patients monitored with VEEG had a clinical diagnosis of NES. Fifty-nine of 68 patients had their habitual event recorded. Mean age at the time of the VEEG diagnosis was 13 years 4 months. Twenty-two patients were less than 13 years (group A) and 37 were 13 years and older (group B). The male to female ratio was equal in group A, with female predominance seen in group B. NES commonly manifested as subtle motor activity in group A (p < 0.01) and prominent motor activity in group B (p < 0.001). Difficulties at school, family discord, and interpersonal conflicts, were frequent stressors in both groups. Sexual abuse was the least frequent. Depression was more common in group B; cognitive dysfunction (p < 0.001) and epilepsy (p < 0.01) were more common in group A. Conclusions: Differences in clinical semiology and predisposing factors may help identify young children and adolescents who might be at risk for the development of NES. KEY WORDS: Pseudoseizures-Epilepsy-Video-EEG.Nonepileptic seizures (NES) are nonepileptic events that may resemble epileptic seizures, but are not associated with abnormal cortical electrical discharges. They should be suspected whenever the attacks are frequent despite appropriate medical management, have atypical clinical features and are exacerbated by stress and when EEGs are repeatedly normal. A variety of terms have been used in the literature to describe these events, including pseudoseizures, psychogenic seizures, hysterical epilepsy, pseudoepileptic seizures, and nonphysiologic or functional seizures. We prefer to use the term NES, because it is more neutral. NES have been described extensively in the literature mainly in adults and less frequently in chil-

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Purpose Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. Methods Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. Results We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. Conclusions We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy.

FrpB is an outer membrane transporter from Neisseria meningitidis, the causative agent of meningococcal meningitis. It is a member of the TonB-dependent transporter (TBDT) family and is responsible for iron uptake into the periplasm. FrpB is subject to a high degree of antigenic variation, principally through a region of hypervariable sequence exposed at the cell surface. From the crystal structures of two FrpB antigenic variants, we identify a bound ferric ion within the structure which induces structural changes on binding which are consistent with it being the transported substrate. Binding experiments, followed by elemental analysis, verified that FrpB binds Fe3+ with high affinity. EPR spectra of the bound Fe3+ ion confirmed that its chemical environment was consistent with that observed in the crystal structure. Fe3+ binding was reduced or abolished on mutation of the Fe3+-chelating residues. FrpB orthologs were identified in other Gram-negative bacteria which showed absolute conservation of the coordinating residues, suggesting the existence of a specific TBDT sub-family dedicated to the transport of Fe3+. The region of antigenic hypervariability lies in a separate, external sub-domain, whose structure is conserved in both the F3-3 and F5-1 variants, despite their sequence divergence. We conclude that the antigenic sub-domain has arisen separately as a result of immune selection pressure to distract the immune response from the primary transport function. This would enable FrpB to function as a transporter independently of antibody binding, by using the antigenic sub-domain as a ‘molecular decoy’ to distract immune surveillance.

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