Oscillatory synchronization in large-scale cortical networks predicts perception

Normal brain function requires the dynamic interaction of functionally specialized but widely distributed cortical regions. Long-range synchronization of oscillatory signals has been suggested to mediate these interactions within large-scale cortical networks, but direct evidence is sparse. Here we show that oscillatory synchronization is organized in such large-scale networks. We implemented an analysis approach that allows for imaging synchronized cortical networks and applied this technique to EEG recordings in humans. We identified two networks: beta-band synchronization (~20 Hz) in a fronto-parieto-occipital network and gamma-band synchronization (~80 Hz) in a centro-temporal network. Strong perceptual correlates support their functional relevance: the strength of synchronization within these networks predicted the subjects' perception of an ambiguous audiovisual stimulus as well as the integration of auditory and visual information. Our results provide evidence that oscillatory neuronal synchronization mediates neuronal communication within frequency-specific, large-scale cortical networks.     

A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella)

The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b₅ on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions.     

Specific elimination of effector memory CD4+ T cells due to enhanced Fas signaling complex formation and association with lipid raft microdomains

Elimination of autoreactive CD4⁺ T cells through the death receptor Fas/CD95 is an important mechanism of immunological self-tolerance. Fas deficiency results in systemic autoimmunity, yet does not affect the kinetics of T-cell responses to acute antigen exposure or infection. Here we show that Fas and TCR-induced apoptosis are largely restricted to CD4⁺ T cells with an effector memory phenotype (effector memory T cells (T_EM)), whereas central memory and activated naïve CD4⁺ T cells are relatively resistant to both. Sensitivity of T_EM to Fas-induced apoptosis depends on enrichment of Fas in lipid raft microdomains, and is linked to more efficient formation of the Fas death-inducing signaling complex. These results explain how Fas can cull T cells reactive against self-antigens without affecting acute immune responses. This work also identifies Fas-induced apoptosis as a possible immunotherapeutic strategy to eliminate T_EM linked to the pathogenesis of a number of autoimmune diseases.     

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

We previously reported that HSV-2 R1, the R1 subunit (ICP10; UL39) of herpes simplex virus type-2 ribonucleotide reductase, protects cells against apoptosis induced by the death receptor (DR) ligands tumor necrosis factor-alpha- (TNFα) and Fas ligand (FasL) by interrupting DR-mediated signaling at, or upstream of, caspase-8 activation. Further investigation of the molecular mechanism underlying HSV-2 R1 protection showed that extracellular-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K)/Akt, NF-κB and JNK survival pathways do not play a major role in this antiapoptotic function. Interaction studies revealed that HSV-2 R1 interacted constitutively with caspase-8. The HSV-2 R1 deletion mutant R1(1-834)-GFP and Epstein–Barr virus (EBV) R1, which did not protect against apoptosis induced by DR ligands, did not interact with caspase-8, indicating that interaction is required for protection. HSV-2 R1 impaired caspase-8 activation induced by caspase-8 over-expression, suggesting that interaction between the two proteins prevents caspase-8 dimerization/activation. HSV-2 R1 bound to caspase-8 directly through its prodomain but did not interact with either its caspase domain or Fas-associated death domain protein (FADD). Interaction between HSV-2 R1 and caspase-8 disrupted FADD-caspase-8 binding. We further demonstrated that individually expressed HSV-1 R1 (ICP6) shares, with HSV-2 R1, the ability to bind caspase-8 and to protect cells against DR-induced apoptosis. Finally, as the long-lived Fas protein remained stable during the early period of infection, experiments with the HSV-1 UL39 deletion mutant ICP6∆ showed that HSV-1 R1 could be essential for the protection of HSV-1-infected cells against FasL.     

The future of direct-to-consumer clinical genetic tests

In light of the meeting of the US Food and Drug Administration (FDA) in March 2011 to discuss the regulation of clinical direct-to-consumer (DTC) genetic tests, we have invited five experts to consider the best means of overseeing the ordering and interpretation of these tests. Should these tests be regulated? If so, who, if anyone, should communicate results to consumers?     

Systematic study of sequence motifs for RNA trans splicing in Trypanosoma brucei

mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5' splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts], 3' splice sites (3'SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3'SS, we constructed a luciferase-beta-galactosidase double-reporter system. By testing approximately 90 sequences, we demonstrated that the optimum poly(Y) tract length is approximately 25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3'SS is important, and an AC dinucleotide at positions -3 and -4 can lead to a 20-fold decrease in trans splicing. However, efficient trans splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3'SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome.     

SNPs in the neural cell adhesion molecule 1 gene (NCAM1) may be associated with human neural tube defects

Neural tube defects (NTDs) are common birth defects, occurring in approximately 1/1,000 births; both genetic and environmental factors are implicated. To date, no major genetic risk factors have been identified. Throughout development, cell adhesion molecules are strongly implicated in cell–cell interactions, and may play a role in the formation and closure of the neural tube. To evaluate the role of neural cell adhesion molecule 1 (NCAM1) in risk of human NTDs, we screened for novel single-nucleotide polymorphisms (SNPs) within the gene. Eleven SNPs across NCAM1 were genotyped using TaqMan. We utilized a family-based approach to evaluate evidence for association and/or linkage disequilibrium. We evaluated American Caucasian simplex lumbosacral myelomeningocele families (n=132 families) using the family based association test (FBAT) and the pedigree disequilibrium test (PDT). Association analysis revealed a significant association between risk for NTDs and intronic SNP rs2298526 using both the FBAT test (P=0.0018) and the PDT (P=0.0025). Using the HBAT version of the FBAT to look for haplotype association, all pairwise comparisons with SNP rs2298526 were also significant. A replication study set, consisting of 72 additional families showed no significant association; however, the overall trend for overtransmission of the less common allele of SNP rs2298526 remained significant in the combined sample set. In addition, we analyzed the expression pattern of the NCAM1 protein in human embryos, and while NCAM1 is not expressed within the neural tube at the time of closure, it is expressed in the surrounding and later in differentiated neurons of the CNS. These results suggest variations in NCAM1 may influence risk for human NTDs.Other members of NTD Collaborative Group involved in this study are listed in the appendix     

SAR and biological evaluation of novel trans-3,4-dimethyl-4-arylpiperidine derivatives as opioid antagonists

The phenolic hydroxy group of opiate-derived ligands is of known importance for biological activity. We have developed a SAR study around LY255582 by comparing the effect of the hydroxy group in the 2- and 4-position of the phenyl ring. Also, we have proved that the 3-position of the phenyl ring is optimal for opioid activity. Furthermore, we have successfully replaced the hydroxy group in LY255582 by carbamate and carboxamide groups. The new analogs have high affinity for the opioid receptors comparable to the corresponding phenol. Carboxamide analog 12 has an improved metabolism profile and proved to be efficacious in in vivo studies.     

Synthesis and characterization of mPEG-PLA prodrug micelles

Polymeric prodrugs of mPEG-PLA-haloperidol (methoxypoly(ethylene glycol)-b-poly(lactic acid)) can self-assemble into nanoscale micelle-like structures in aqueous solutions. mPEG-PLA-haloperidol was prepared and characterized using 1H and 13C NMR. The conjugation efficiency was found to be 64.8 +/- 21%. Micelles that form spontaneously upon solubilization of the mPEG-PLA and the polymeric prodrugs in water were characterized using a variety of techniques. The mPEG-PLA and prodrug micelles were found to have diameters of 28.73 +/- 1.45 and 49.67 +/- 4.29 nm, respectively, using dynamic light scattering (DLS). The micelle size and polydispersity were also evaluated with cryogenic transmission electron microscopy (cryo-TEM) and were consistent with the DLS results. Cryo-TEM and proton NMR confirmed that the micelles were spherical in shape. DLS was also used to determine the aggregation numbers of the micelles. The aggregation numbers ranged from 351 to 603. The change in aggregation number was dependent on the total drug incorporation into the micelle core. Critical micelle concentrations were determined for the various micelle/drug formulations and found to range from 3 to 14 microg/mL. Finally, drug was incorporated into the micelle core using the conjugate, free drug with a saturated aqueous phase during production, or a combination of both techniques. Drug incorporation could be increased from 3% to 20% (w/w) using the different formulations.