Methylomic analysis of monozygotic twins discordant for childhood psychotic symptoms

Childhood psychotic symptoms are associated with increased rates of schizophrenia, other psychiatric disorders, and suicide attempts in adulthood; thus, elucidating early risk indicators is crucial to target prevention efforts. There is considerable discordance for psychotic symptoms between monozygotic twins, indicating that child-specific non-genetic factors must be involved. Epigenetic processes may constitute one of these factors and have not yet been investigated in relation to childhood psychotic symptoms. Therefore, this study explored whether differences in DNA methylation at age 10 were associated with monozygotic twin discordance for psychotic symptoms at age 12. The Environmental Risk (E-Risk) Longitudinal Twin Study cohort of 2,232 children (1,116 twin pairs) was assessed for age-12 psychotic symptoms and 24 monozygotic twin pairs discordant for symptoms were identified for methylomic comparison. Children provided buccal samples at ages 5 and 10. DNA was bisulfite modified and DNA methylation was quantified using the Infinium HumanMethylation450 array. Differentially methylated positions (DMPs) associated with psychotic symptoms were subsequently tested in post-mortem prefrontal cortex tissue from adult schizophrenia patients and age-matched controls. Site-specific DNA methylation differences were observed at age 10 between monozygotic twins discordant for age-12 psychotic symptoms. Similar DMPs were not found at age 5. The top-ranked psychosis-associated DMP (cg23933044), located in the promoter of the C5ORF42 gene, was also hypomethylated in post-mortem prefrontal cortex brain tissue from schizophrenia patients compared to unaffected controls. These data tentatively suggest that epigenetic variation in peripheral tissue is associated with childhood psychotic symptoms and may indicate susceptibility to schizophrenia and other mental health problems.     

A GpC-Rich Oligonucleotide Acts on Plasmacytoid Dendritic Cells To Promote Immune Suppression

Short synthetic oligodeoxynucleotides (ODNs) rich in CpG or GpG motifs have been considered as potential modulators of immunity in clinical settings. In this study, we show that a synthetic GpC-ODN conferred highly suppressive activity on mouse splenic plasmacytoid dendritic cells, demonstrable in vivo in a skin test assay. The underlying mechanism involved signaling by noncanonical NF-κB family members and TGF-β-dependent expression of the immunoregulatory enzyme IDO. Unlike CpG-ODNs, the effects of GpC-ODN required TLR7/TRIF-mediated but not TLR9/MyD88-mediated events, as do sensing of viral ssRNA and the drug imiquimod. Induction of IDO by a GpC-containing ODN could also be demonstrated in human dendritic cells, allowing those cells to assist FOXP3(+) T cell generation in vitro. Among potentially therapeutic ODNs, this study identifies GpC-rich sequences as novel activators of TLR7-mediated, IDO-dependent regulatory responses.     

Synthesis of new 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted and antiplatelet/antiphlogistic activities evaluation

In pursuing our research on some 2,4-diamino-benzopyranopyrimidines and 2-amino-5,6-dihydrobenzo[h]quinazolines, previously reported as antiplatelet and analgesic/anti-inflammatory agents respectively, we designed and synthesized a new series of 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted. The insertion of amino substituents at positions 2 and 4 of the benzoquinazoline scaffold resulted in compounds endowed with a potent ASA-like antiplatelet activity, combined with an anti-inflammatory activity comparable, in some cases, to that of indomethacin, used as a reference drug.     

Sulfatases are determinants of alveolar formation

Alveolar formation or alveolarization is orchestrated by a finely regulated and complex interaction between growth factors and extracellular matrix proteins. The lung parenchyma contains various extracellular matrix proteins including proteoglycans, which are composed of glycosaminoglycans (GAGs) linked to a protein core. Although GAGs are known to regulate growth factor distribution and activity according to their degree of sulfation the role of sulfated GAG in the respiratory system is not well understood. The degree of sulfation of GAGs is regulated in part, by sulfatases that remove sulfate groups. In vertebrates, the enzyme Sulfatase-Modifying Factor 1 (Sumf1) activates all sulfatases. Here we utilized mice lacking Sumf1(-/-) to study the importance of proteoglycan desulfation in lung development. The Sumf1(-/-) mice have normal lungs up until the onset of alveolarization at post-natal day 5 (P5). We detected increased deposition of sulfated GAG throughout the lung parenchyma and a decrease in alveolar septa formation. Moreover, stereological analysis showed that the alveolar volume is 20% larger in Sumf1(-/-) as compared to wild type (WT) mice at P10 and P30. Additionally, pulmonary function test was consistent with increased alveolar volume. Genetic experiments demonstrate that in Sumf1(-/-) mice arrest of alveolarization is independent of fibroblast growth factor signaling. In turn, the Sumf1(-/-) mice have increased transforming growth factor β (TGFβ) signaling and in vivo injection of TGFβ neutralizing antibody leads to normalization of alveolarization. Thus, absence of sulfatase activity increases sulfated GAG deposition in the lungs causing deregulation of TGFβ signaling and arrest of alveolarization.     

Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy

Glucocorticoid-induced tumor necrosis factor receptor (GITR) on T cells and its natural ligand, GITRL, on accessory cells contribute to the control of immune homeostasis. Here we show that reverse signaling through GITRL after engagement by soluble GITR initiates the immunoregulatory pathway of tryptophan catabolism in mouse plasmacytoid dendritic cells, by means of noncanonical NF-kappaB-dependent induction of indoleamine 2,3-dioxygenase (IDO). The synthetic glucocorticoid dexamethasone administered in vivo activated IDO through the symmetric induction of GITR in CD4(+) T cells and GITRL in plasmacytoid dendritic cells. The drug exerted IDO-dependent protection in a model of allergic airway inflammation. Modulation of tryptophan catabolism via the GITR-GITRL coreceptor system might represent an effective therapeutic target in immune regulation. Induction of IDO could be an important mechanism underlying the anti-inflammatory action of corticosteroids.     

A CaMKII/calcineurin switch controls the direction of Ca(2+)-dependent growth cone guidance

Axon pathfinding depends on attractive and repulsive turning of growth cones to extracellular cues. Localized cytosolic Ca2+ signals are known to mediate the bidirectional responses, but downstream mechanisms remain elusive. Here, we report that calcium-calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) phosphatase provide a switch-like mechanism to control the direction of Ca(2+)-dependent growth cone turning. A relatively large local Ca2+ elevation preferentially activates CaMKII to induce attraction, while a modest local Ca2+ signal predominantly acts through CaN and phosphatase-1 (PP1) to produce repulsion. The resting level of intracellular Ca2+ concentrations also affects CaMKII/CaN operation: a normal baseline allows distinct turning responses to different local Ca2+ signals, while a low baseline favors CaN-PP1 activation for repulsion. Moreover, the cAMP pathway negatively regulates CaN-PP1 signaling to inhibit repulsion. Finally, CaMKII/CaN-PP1 also mediates netrin-1 guidance. Together, these findings establish a complex Ca2+ mechanism that targets the balance of CaMKII/CaN-PP1 activation to control distinct growth cone responses.     

Role of the hinge peptide and the intersubunit interface in the swapping of N-termini in dimeric bovine seminal RNase.

Bovine seminal ribonuclease (BS-RNase) is the only known dimeric enzyme characterized by an equilibrium between two different 3D structures: MxM, with exchange (or swapping) of the N-terminal 1-20 residues, and M=M, without exchange. As a consequence, the hinge region 16-22 has a different tertiary structure in the two forms. In the native protein, the equilibrium ratio between MxM and M=M is about 7 : 3. Kinetic analysis of the swapping process for a recombinant sample shows that it folds mainly in the M=M form, then undergoes interconversion into the MxM form, reaching the same 7 : 3 equilibrium ratio. To investigate the role of the regions that are most affected structurally by the swapping, we expressed variant proteins by replacing two crucial residues with the corresponding ones from RNase A: Pro19, within the hinge peptide, and Leu28, located at the interface between subunits. We compared the structural properties of the monomeric forms of P19A-BS-RNase, L28Q-BS-RNase and P19A/L28Q-BS-RNase variants with those of the parent protein, and investigated the exchange kinetics of the corresponding dimers. The P19A mutation slightly increases the thermal stability of the monomer, but it does not alter the swapping tendency of the dimer. In contrast, the L28Q mutation significantly affects both the dimerization and swapping processes but not the thermal stability of the monomer. Overall, these results suggest that the structural determinants that control the exchange of N-terminal arms in BS-RNase may not be located within the hinge peptide, and point to a crucial role of the interface residues.     

Measurement of endogenous and TNFα-mediated H2O2 production in supernatants of SK-N-SH neuroblastoma cells with an enhanced chemiluminescence assay

A sensitive peroxidase-dependent luminol-enhanced chemiluminescence (ECL) assay for determination of hydrogen peroxide (H₂O₂) generation by tumour cells was established. This test system allows determination of H₂O₂ in concentrations as low as 25 pmol (50nmol/L) and yields results which are comparable to those obtained using a less sensitive photometric method and a previously described scopoletin flurorescence assay. After 3h incubation time 10⁴SK-N-SH neuroblastoma cells released 60° 5 pmol H₂O₂ in the supernatant and this level was significantly (p<0.025) increased by about 70% in the presence of 5pmol (100 ng/mL) recombinant tumour necrosis factor α (TNFα). In contrast, H₂O₂ production was slightly reduced by TNFα at a very low concentration of 0.5 fmol (0.01 ng/mL).