Treatment with the Bacterial Toxin CNF1 Selectively Rescues Cognitive and Brain Mitochondrial Deficits in a Female Mouse Model of Rett Syndrome Carrying a MeCP2-Null Mutation

Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene and a major cause of intellectual disability in females. No cure exists for RTT. We previously reported that the behavioural phenotype and brain mitochondria dysfunction are widely rescued by a single intracerebroventricular injection of the bacterial toxin CNF1 in a RTT mouse model carrying a truncating mutation of the MeCP2 gene (MeCP2-308 mice). Given the heterogeneity of MECP2 mutations in RTT patients, we tested the CNF1 therapeutic efficacy in a mouse model carrying a null mutation (MeCP2-Bird mice). CNF1 selectively rescued cognitive defects, without improving other RTT-related behavioural alterations, and restored brain mitochondrial respiratory chain complex activity in MeCP2-Bird mice. To shed light on the molecular mechanisms underlying the differential CNF1 effects on the behavioural phenotype, we compared treatment effects on relevant signalling cascades in the brain of the two RTT models. CNF1 provided a significant boost of the mTOR activation in MeCP2-308 hippocampus, which was not observed in the MeCP2-Bird model, possibly explaining the differential effects of CNF1. These results demonstrate that CNF1 efficacy depends on the mutation beared by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across RTT models.     

Delayed Ventricular Repolarization and Sodium Channel Current Modification in a Mouse Model of Rett Syndrome

Rett syndrome (RTT) is a severe developmental disorder that is strongly linked to mutations in the MECP2 gene. RTT has been associated with sudden unexplained death and ECG QT interval prolongation. There are mixed reports regarding QT prolongation in mouse models of RTT, with some evidence that loss of Mecp2 function enhances cardiac late Na current, I_Na,Late. The present study was undertaken in order to investigate both ECG and ventricular AP characteristics in the Mecp2^Null/Y male murine RTT model and to interrogate both fast I_Na and I_Na,Late in myocytes from the model. ECG recordings from 8–10-week-old Mecp2^Null/Y male mice revealed prolongation of the QT and rate corrected QT (QTc) intervals and QRS widening compared to wild-type (WT) controls. Action potentials (APs) from Mecp2^Null/Y myocytes exhibited longer APD₇₅ and APD₉₀ values, increased triangulation and instability. I_Na,Late was also significantly larger in Mecp2^Null/Y than WT myocytes and was insensitive to the Nav1.8 inhibitor A-803467. Selective recordings of fast I_Na revealed a decrease in peak current amplitude without significant voltage shifts in activation or inactivation V_0.5. Fast I_Na ‘window current’ was reduced in RTT myocytes; small but significant alterations of inactivation and reactivation time-courses were detected. Effects of two I_Na,Late inhibitors, ranolazine and GS-6615 (eleclazine), were investigated. Treatment with 30 µM ranolazine produced similar levels of inhibition of I_Na,Late in WT and Mecp2^Null/Y myocytes, but produced ventricular AP prolongation not abbreviation. In contrast, 10 µM GS-6615 both inhibited I_Na,Late and shortened ventricular AP duration. The observed changes in I_Na and I_Na,Late can account for the corresponding ECG changes in this RTT model. GS-6615 merits further investigation as a potential treatment for QT prolongation in RTT.     

Camelina sativa Oil,Fatty Fish,and Lean Fish Do Not Markedly Affect Urinary Prostanoids in Subjects with Impaired Glucose Metabolism

Dietary fatty acids are suggested to affect oxidative stress; however, results from interventions have been inconclusive. The aim was to examine if fatty fish, lean fish, and Camelina sativa oil (CSO) affect the urinary prostanoid levels in subjects with impaired glucose metabolism. Altogether 79 participants aged 43–72 years completed a randomized controlled study lasting 12 weeks. There were four parallel groups, fatty fish, lean fish (four fish meals/week in both), CSO providing 10 g/day alpha-linolenic acid (ALA), and control diet with limited fish and ALA containing oil consumption. Urinary prostanoids (prostaglandin F_2α, 5-F_2t-isoprostanes and 15-F_2t-isoprostane metabolites, isofuran, 8-F_3t-isoprostanes, and 4 - (RS)-4-F_4t-neuroprostane) of 72 participants (age: mean (±SD) 58.9 ± 6.5 years; body mass index: 29.3 ± 2.5 kg/m²) collected over 12-h were measured using liquid chromatography tandem-mass spectrometry. Plasma phospholipid fatty acids were determined using gas chromatography. Our study showed that the proportion of ALA in plasma phospholipids increased in the CSO group (overall difference among the groups p-value <0.001). In the fatty fish group, proportions of eicosapentaenoic and docosahexaenoic acids increased (overall p-value <0.001 for both). Prostaglandin F_2α was higher in the CSO group than in the control group (p < 0.05), however, there were no other significant changes in urinary excretion of other prostanoids among the study groups. At baseline, arachidonic acid in plasma phospholipids was positively (r = 0.247, p < 0.05) and ALA negatively (r = −0.326, p < 0.05) associated with urinary total isoprostanes. In conclusion, CSO, fatty fish, and lean fish consumption do not cause major changes in oxidative stress markers in subjects with impaired glucose tolerance.     

Measurement of Enzymatic and Nonenzymatic Polyunsaturated Fatty Acid Oxidation Products in Plasma and Urine of Macular Degeneration Using LC-QTOF-MS/MS

Oxidized polyunsaturated fatty acids (PUFA) are associated to pathogenesis of diseases including cardiovascular and neurodegeneration. The novel products are not only biomarkers but also lipid mediators in gene regulation and signaling pathways. Herein, simultaneous quantitation of 28 products derived from nonenzymatic and enzymatic oxidation of PUFA i.e. 5-, 15-F_2t-isoprostanes, 7-, 17-F_2t-dihomo-isoprostanes, 7-, 17-F_2t-dihomo-isofurans, 5-, 8-, 18-F_3t-isoprostanes, 4-, 10-, 13-, 14-, 20-F_4t-neuroprostanes, 5-, 8-, 9-, 11-,12-, 15-, 20-HETE, 4-, 7-, 11-, 14-, 17-HDHA, RvE1, and NPD1 using LC-(ESI)-QTOF-MS/MS was developed. These products were measurable in a single sample and the analytical time was relative short (~15 min). Furthermore, we showed that the use of internal standards is a requisite to normalize matrix effects and preparation loss for the quantitation. Validation assays indicated the method to be robust for plasma and mid-stream urine sample analysis in particular from those of age-related macular degeneration subjects, where the accuracy of quantitation displayed good repeatability.     

The P2X7R-NLRP3 and AIM2 Inflammasome Platforms Mark the Complexity/Severity of Viral or Metabolic Liver Damage

P2X7R-NLRP3 and AIM2 inflammasomes activate caspase-1 and the release of cytokines involved in viral-related liver disease. Little is known about their role in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steato-hepatitis (NASH). We characterized the role of inflammasomes in NAFLD, NASH, and HCV. Gene expression and subcellular localization of P2X7R/P2X4R-NLRP3 and AIM2 inflammasome components were examined in histopathological preparations of 46 patients with biopsy-proven viral and metabolic liver disease using real-time PCR and immunofluorescence. P2X7R, P2X4R, and Caspase-1 are two- to five-fold more expressed in patients with NAFLD/NASH associated with chronic HCV infection than those with metabolic damage only (p ≤ 0.01 for all comparisons). The AIM2 inflammasome is 4.4 times more expressed in patients with chronic HCV infection, regardless of coexistent metabolic abnormalities (p = 0.0006). IL-2, a cytokine playing a pivotal role during chronic HCV infection, showed a similar expression in HCV and NASH patients (p = 0.77) but was virtually absent in NAFLD. The P2X7R-NLRP3 complex prevailed in infiltrating macrophages, while AIM2 was localized in Kupffer cells. Caspase-1 expression correlated with elastography-based liver fibrosis (r = 0.35, p = 0.02), whereas P2X7R, P2X4R, NRLP3, Caspase-1, and IL-2 expression correlated with circulating markers of disease severity. P2X7R and P2X4R play a major role in liver inflammation accompanying chronic HCV infection, especially when combined with metabolic damage, while AIM2 is specifically expressed in chronic viral hepatitis. We describe for the first time the hepatic expression of IL-2 in NASH, so far considered a peculiarity of HCV-related liver damage.     

New Labdane-Type Diterpenoids and Anti-Inflammatory Constituents from Hedychium coronarium

Four new labdane-type diterpenoids: hedychicoronarin (1), peroxycoronarin D (2), 7β-hydroxycalcaratarin A (3), and (E)-7β-hydroxy-6-oxo-labda-8(17),12-diene-15,16-dial (4), have been isolated from the rhizomes of Hedychium coronarium, together with 13 known compounds (5–17). The structures of these new compounds were determined through spectroscopic and MS analyses. Compounds 3, 5, 6, and 10 exhibited inhibition (IC₅₀ values ≤4.52 μg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 3–6, 10, and 11 inhibited fMLP/CB-induced elastase release with IC₅₀ values ≤6.17 μg/mL.     

Complex formation between S−6 and organic polysulphides in dimethylacetamide

In a dipolar aprotic medium (dimethylacetamide), organic sulphides or polysulphides RS_xR (R = alkyl or phenyl group, x = 1, 2, 3) are not reduced into thiolate ions by S₃⁻ or S²⁻₈, electrogenerated from sulphur, but give complexes RS_xR, S²⁻₆ by shifting the equilibrium between S₃⁻ and S²⁻₆ ions. Complexes stability constants, visible spectra and rates of formation are given. A mechanism for the complex formation is proposed form the kinetic studies.     

Music-Based Intervention Ameliorates Mecp2-Loss-Mediated Sociability Repression in Mice through the Prefrontal Cortex FNDC5/BDNF Pathway

Patients with Rett syndrome (RTT) show severe difficulties with communication, social withdrawl, and learning. Music-based interventions improve social interaction, communication skills, eye contact, and physical skills and reduce seizure frequency in patients with RTT. This study aimed to investigate the mechanism by which music-based interventions compromise sociability impairments in mecp2 ^null/y mice as an experimental RTT model. Male mecp2 ^null/y mice and wild-type mice (24 days old) were randomly divided into control, noise, and music-based intervention groups. Mice were exposed to music or noise for 6 h/day for 3 consecutive weeks. Behavioral patterns, including anxiety, spontaneous exploration, and sociability, were characterized using open-field and three-chamber tests. BDNF, TrkB receptor motif, and FNDC5 expression in the prefrontal cortex (PFC), hippocampus, basal ganglia, and amygdala were probed using RT-PCR or immunoblotting. mecp2 ^null/y mice showed less locomotion in an open field than wild-type mice. The social novelty rather than the sociability of these animals increased following a music-based intervention, suggesting that music influenced the mecp2-deletion-induced social interaction repression rather than motor deficit. Mechanically, the loss of BDNF signaling in the prefrontal cortex and hippocampal regions, but not in the basal ganglia and amygdala, was compromised following the music-based intervention in mecp2 ^null/y mice, whereas TrkB signaling was not significantly changed in either region. FNDC5 expression in the prefrontal cortex region in mecp2 ^null/y mice also increased following the music-based intervention. Collective evidence reveals that music-based interventions improve mecp2-loss-induced social dysfunction. BDNF and FNDC5 signaling in the prefrontal cortex region mediates the music-based-intervention promotion of social interactions. This study gives new insight into the mechanisms underlying the improvement of social behaviors in mice suffering from experimental Rett syndrome following a music-based intervention.     

From P-type to N-type: Peripheral fluorination of axially substituted silicon phthalocyanines enables fine tuning of charge transport

Silicon phthalocyanines (R₂-SiPcs) are a family of promising tunable materials for organic electronic applications. We report the chemistry of the synthesis of axially substituted fluorinated SiPcs (tb-Ph)₂-F_xSiPc (where X = 0, 4, 8, or 16) and explore how the degree of fluorination effects optical and electronic properties. A new treatment with boron trichloride was included to obtain Cl₂-F_XSiPcs from F₂-F_XSiPcs, activating the axial position for further functionalization. We observed that as the degree of fluorination increased, so did the electron affinity of the compounds, leading to a drop in frontier orbital levels, as measured by electrochemistry and ultraviolet photoelectron spectroscopy (UPS). The deeper energy levels enabled successful (tb-Ph)₂-F₄SiPc and poly [[6,7-difluoro[(2-hexyldecyl)oxy]-[5,8-quinoxalinediyl]-2,5-thiophenediyl]] (PTQ10) blends for organic photovoltaics and photodetectors. All four compounds were incorporated in organic thin-film transistors (OTFTs), where the degree of fluorination influenced device operation, changing it from p-type conduction for (tb-Ph)₂-F₀SiPc, to ambipolar for (tb-Ph)₂-F₄SiPc, and n-type for (tb-Ph)₂-F₈SiPc and (tb-Ph)₂-F₁₆SiPc. The OTFT devices made with (tb-Ph)₂-F₁₆SiPc achieved a low average threshold voltage of 7.0 V in N₂ and retained its n-type mobility when exposed to air.     

K2Sr4Nb10O30-based dielectric ceramics having the tetragonal tungsten bronze structure and temperature-stable high permittivity

This study concerns K₂Sr₄Nb₁₀O₃₀-based (KSN) dielectric ceramics, having the tetragonal tungsten bronze structure (TTB), which display a high permittivity and flat ε′_r=f(T) curves meeting the X7R specification. The series K₂Sr₄(Mg_xNb_10–x)O_30-(3/2)x exhibit these properties, as well as K₂Sr₄Nb_9·78O_29·45 and K₂(Sr_3·25Pb_0·75)Nb_9·78O_29·45 when sintered with MgO or ZrO₂. Both X-ray and electron diffraction reveal the former composition to be a single phase material with the TTB structure. In this material, the presence of disorder, resulting from a large variation in the local K/Sr ratio and the presence of Zr on the octahedral niobium sites, is primarily responsible for the X7R ε′_r=f(T) curve with ε′_r,25°C=5000.     

A study of curative interactions in cis-1,4-polyisoprene. X. The cis-1,4-polyisoprene/sulfur/zinc dimethyldithiocarbamate and cis-1,4-polyisoprene/sulfur/zinc dimethyldithiocarbamate/ZnO systems

Aspects of the mechanism of zinc dimethyldithiocarbamate (ZDMC)-accelerated sulfur vulcanization were discussed. The trends in the efficiency parameter E, confirmed that crosslinking is preceded by the formation of pendent groups RS_xSX [R = polyisoprenyl, X = Me₂NC(S)] in ZDMC-based systems. The index x in RS_xX was calculated as 5.82 in the cis-1,4-polyisoprene (IR)/sulfur/ZDMC/ZnO compound at the initial stages of curing, compared to 3.23 in the absence of ZnO. The high value of x supports the postulation that elemental sulfur and ZDMC react at the early stages of vulcanization, to form the active sulphurating agent XS_xSZnSSX. Crosslinks form by either a disproportionation reaction between two α-methylic or α-methylenic pendent groups RS_xX, or a reaction between a pendent group RS_xX and the rubber chain—these routes are the same as that suggested for the IR/tetramethylthiuram disulfide (TMTD)/ZnO compound. The beneficial role of ZnO and zinc stearate is shown, as in the case of ZnO in the IR/TMTD/ZnO system, to be related to their ability to trap dimethyldithiocarbamic acid, which formed in the generation of pendent groups and crosslinks. ZnS is inactive in this regard. The formation of ZnS is characteristic of natural rubber/sulfur/ZDMC/ZnO systems, as opposed to IR/TMTD/ZnO mixtures where little ZnS forms.     

Trans-Cinnamic Acid Increases Adiponectin and the Phosphorylation of AMP-Activated Protein Kinase through G-Protein-Coupled Receptor Signaling in 3T3-L1 Adipocytes

Adiponectin and intracellular 5′adenosine monophosphate-activated protein kinase (AMPK) are important modulators of glucose and fat metabolism. Cinnamon exerts beneficial effects by improving insulin sensitivity and blood lipids, e.g., through increasing adiponectin concentrations and AMPK activation. The underlying mechanism is unknown. The G_i/G_o-protein-coupled receptor (GPR) 109A stimulates adiponectin secretion after binding its ligand niacin. Trans-cinnamic acid (tCA), a compound of cinnamon is another ligand. We hypothesize whether AMPK activation and adiponectin secretion by tCA is transmitted by GPR signaling. Differentiated 3T3-L1 cells were incubated with pertussis toxin (PTX), an inhibitor of G_i/G_o-protein-coupling, and treated with different tCA concentrations. Treatment with tCA increased adiponectin and the pAMPK/AMPK ratio (p ≤ 0.001). PTX incubation abolished the increased pAMPK/AMPK ratio and adiponectin secretion. The latter remained increased compared to controls (p ≤ 0.002). tCA treatment stimulated adiponectin secretion and AMPK activation; the inhibitory effect of PTX suggests GPR is involved in tCA stimulated signaling.     

New Coumarin Derivatives and Other Constituents from the Stem Bark of Zanthoxylum avicennae: Effects on Neutrophil Pro-Inflammatory Responses

Three new coumarin derivatives, 8-formylalloxanthoxyletin (1), avicennone (2), and (Z)-avicennone (3), have been isolated from the stem bark of Zanthoxylum avicennae (Z. avicennae), together with 15 known compounds (4–18). The structures of these new compounds were determined through spectroscopic and MS analyses. Compounds 1, 4, 9, 12, and 15 exhibited inhibition (half maximal inhibitory concentration (IC₅₀) values ≤7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 2, 4, 8 and 9 inhibited fMLP/CB-induced elastase release with IC₅₀ values ≤8.17 µg/mL. This investigation reveals bioactive isolates (especially 1, 2, 4, 8, 9, 12 and 15) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.     

A novel facile synthesis and characterization of molybdenum nanowires

We describe a straightforward technique to synthesize pure Mo nanowires (NWs) from Mo₆S_yI_z (8,2 <y + z ≤ 10) NWs as precursor templates. The structural transformations occur when Mo₆S_yI_z NWs are annealed in Ar/H₂ mixture leading to the formation of pure Mo NWs with similar structures as initial morphologies. Detailed microscopic characterizations show that large diameters (>15 nm) Mo NWs are highly porous, while small diameters (<7 nm) are made of solid nanocrystalline grains. We find NW of diameter 4 nm can carry up to 30 μA current without suffering structural degradation. Moreover, NWs can be elastically deformed over several cycles without signs of plastic deformation.     

Preparation and Antibacterial Activity of Nano Copper Oxide- Loaded Zeolite 10X

Copper oxide nanosheet-loaded zeolite 10X nanocomposites (CuO-zeolite NCs) were successfully prepared by modifying zeolite 10X with CuSO₄ aqueous solution. The formation of copper oxide nanosheets on the surface of zeolite 10X was observed by SEM. The thickness of CuO nanosheets was about 30–40 nm, and the width ranged from 200 nm to 300 nm. The XRD patterns showed that the new diffraction peaks of copper oxide appeared at 35.6° and 38.8°. According to the XPS results, the Cu 2p_3/2 and Cu 2p_1/2 peaks in CuO-zeolite NC were centered at 934.1 eV and 953.8 eV, which could be attributed to Cu(II). The EDS analysis revealed that the energy spectra of calcium gradually decreased as the copper ion concentration increased during the preparation of CuO-zeolite NCs. Meanwhile, the energy spectra of copper increased gradually, and the highest content of copper in CuO-zeolite NCs reached 22.35 wt.%. The BET surface areas of zeolite 10X and CuO-zeolite NCs were 587 and 363 m²/g, respectively, based on the N₂ adsorption–desorption experiment. The antibacterial activities of CuO-zeolite NC were evaluated using Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The antibacterial activities were related to both copper ion content in CuO-zeolite NCs and the particle size of copper oxide. The results showed that nano CuO-loaded zeolite 10X inhibited the activity of E. coli and S. aureus. CuO-zeolite NCs are expected to be further used in antifouling coating.     

Modeling Rett Syndrome with Human Pluripotent Stem Cells: Mechanistic Outcomes and Future Clinical Perspectives

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Among many different roles, MeCP2 has a high phenotypic impact during the different stages of brain development. Thus, it is essential to intensively investigate the function of MeCP2, and its regulated targets, to better understand the mechanisms of the disease and inspire the development of possible therapeutic strategies. Several animal models have greatly contributed to these studies, but more recently human pluripotent stem cells (hPSCs) have been providing a promising alternative for the study of RTT. The rapid evolution in the field of hPSC culture allowed first the development of 2D-based neuronal differentiation protocols, and more recently the generation of 3D human brain organoid models, a more complex approach that better recapitulates human neurodevelopment in vitro. Modeling RTT using these culture platforms, either with patient-specific human induced pluripotent stem cells (hiPSCs) or genetically-modified hPSCs, has certainly contributed to a better understanding of the onset of RTT and the disease phenotype, ultimately allowing the development of high throughput drugs screening tests for potential clinical translation. In this review, we first provide a brief summary of the main neurological features of RTT and the impact of MeCP2 mutations in the neuropathophysiology of this disease. Then, we provide a thorough revision of the more recent advances and future prospects of RTT modeling with human neural cells derived from hPSCs, obtained using both 2D and organoids culture systems, and its contribution for the current and future clinical trials for RTT.     

A Next-Generation Sequencing Approach to Identify Gene Mutations in Early- and Late-Onset Hypertrophic Cardiomyopathy Patients of an Italian Cohort

Sequencing of sarcomere protein genes in patients fulfilling the clinical diagnostic criteria for hypertrophic cardiomyopathy (HCM) identifies a disease-causing mutation in 35% to 60% of cases. Age at diagnosis and family history may increase the yield of mutations screening. In order to assess whether Next-Generation Sequencing (NGS) may fulfil the molecular diagnostic needs in HCM, we included 17 HCM-related genes in a sequencing panel run on PGM IonTorrent. We selected 70 HCM patients, 35 with early (≤25 years) and 35 with late (≥65 years) diagnosis of disease onset. All samples had a 98.6% average of target regions, with coverage higher than 20× (mean coverage 620×). We identified 41 different mutations (seven of them novel) in nine genes: MYBPC3 (17/41 = 41%); MYH7 (10/41 = 24%); TNNT2, CAV3 and MYH6 (3/41 = 7.5% each); TNNI3 (2/41 = 5%); GLA, MYL2, and MYL3 (1/41=2.5% each). Mutation detection rate was 30/35 (85.7%) in early-onset and 8/35 (22.9%) in late-onset HCM patients, respectively (p < 0.0001). The overall detection rate for patients with positive family history was 84%, and 90.5% in patients with early disease onset. In our study NGS revealed higher mutations yield in patients with early onset and with a family history of HCM. Appropriate patient selection can increase the yield of genetic testing and make diagnostic testing cost-effective.