Biocomplexity and Fractality in the Search of Biomarkers of Aging and Pathology: Focus on Mitochondrial DNA and Alzheimer's Disease

Alzheimer's disease (AD) represents one major health concern for our growing elderly population. It accounts for increasing impairment of cognitive capacity followed by loss of executive function in late stage. AD pathogenesis is multifaceted and difficult to pinpoint, and understanding AD etiology will be critical to effectively diagnose and treat the disease. An interesting hypothesis concerning AD development postulates a cause-effect relationship between accumulation of mitochondrial DNA (mtDNA) mutations and neurodegenerative changes associated with this pathology. Here we propose a computerized method for an easy and fast mtDNA mutations-based characterization of AD. The method has been built taking into account the complexity of living being and fractal properties of many anatomic and physiologic structures, including mtDNA. Dealing with mtDNA mutations as gaps in the nucleotide sequence, fractal lacunarity appears a suitable tool to differentiate between aging and AD. Therefore, Chaos Game Representation method has been used to display DNA fractal properties after adapting the algorithm to visualize also heteroplasmic mutations. Parameter β from our fractal lacunarity method, based on hyperbola model function, has been measured to quantitatively characterize AD on the basis of mtDNA mutations. Results from this pilot study to develop the method show that fractal lacunarity parameter β of mtDNA is statistically different in AD patients when compared to age-matched controls. Fractal lacunarity analysis represents a useful tool to analyze mtDNA mutations. Lacunarity parameter β is able to characterize individual mutation profile of mitochondrial genome and appears a promising index to discriminate between AD and aging

Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma ‘anandamide amidohydrolase’

AbstractThe endogenous cannabimimetic substance, anandamide (N-arachidonoyl-ethanolamine) and the recently isolated sleep-inducing factor, oleoyl-amide (cis-9,10-octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of ‘anandamide amidohydrolase’ from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [14C]anandamide was found in both microsomal and 10,000 × g pellet fractions. The latter fractions, which displayed the highest Vmax for anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl-amide, with an apparent Km of 9.0 ± 2.2 μM. [14C]anandamide- and [14C]oleoyl-amide-hydrolysing activities: (i) exhibited identical pH- and temperature-dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A2 inhibitor arachidonyl-trifluoromethyl-ketone with the same IC50 (3 μM); (iv) were competitively inhibited by both anandamide (or other polyunsaturated fatty acid-ethanolamides) and oleoyl-amide. Proteins solubilised from 10,000 × g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]anandamide and [14C]oleoyl-amide. These data suggest that ‘anandamide amidohydrolase’ enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep-inducing factor oleoyl-amide

Targeting mGlu5 metabotropic glutamate receptors in the treatment of cognitive dysfunction in a mouse model of phenylketonuria

We studied group-I metabotropic glutamate (mGlu) receptors in Pah(enu2) (ENU2) mice, which mimic the genetics and neurobiology of human phenylketonuria (PKU), a metabolic disorder characterized, if untreated, by autism, and intellectual disability (ID). Male ENU2 mice showed increased mGlu5 receptor protein levels in the hippocampus and corpus striatum (but not in the prefrontal cortex) whereas the transcript of the mGlu5 receptor was unchanged. No changes in mGlu1 receptor mRNA and protein levels were found in any of the three brain regions of ENU2 mice. We extended the analysis to Homer proteins, which act as scaffolds by linking mGlu1 and mGlu5 receptors to effector proteins. Expression of the long isoforms of Homer was significantly reduced in the hippocampus of ENU2 mice, whereas levels of the short Homer isoform (Homer 1a) were unchanged. mGlu5 receptors were less associated to immunoprecipitated Homer in the hippocampus of ENU2 mice. The lack of mGlu5 receptor-mediated long-term depression (LTD) in wild-type mice (of BTBR strain) precluded the analysis of hippocampal synaptic plasticity in ENU2 mice. We therefore performed a behavioral analysis to examine whether pharmacological blockade of mGlu5 receptors could correct behavioral abnormalities in ENU2 mice. Using the same apparatus we sequentially assessed locomotor activity, object exploration, and spatial object recognition (spatial novelty test) after displacing some of the objects from their original position in the arena. Systemic treatment with the mGlu5 receptor antagonist, MPEP (20 mg/kg, i.p.), had a striking effect in the spatial novelty test by substantially increasing the time spent in exploring the displaced objects in ENU2 mice (but not in wild-type mice). These suggest a role for mGlu5 receptors in the pathophysiology of ID in PKU and suggest that, also in adult untreated animals, cognitive dysfunction may be improved by targeting these receptors with an appropriate therapy

Derivation of canine hepatocyte in vitro models to study Branched-Chain Amino Acid effects on liver functions.

Branched chain amino acids (BCAA), have been shown to affect human gene expression, proteinmetabolism, apoptosis, and regeneration of hepatocytes. Furthermore, they have been demonstratedto inhibit proliferation of liver cancer cells in vitro, and to be essential for lymphocyte proliferation.In veterinary medicine, the use of BCAAs as integration of a normal dietary plan, is likely to be a validchoice for the same benefit found in human clinical nutrition, although this aspect is still debated.Indeed, long-term oral supplementation with BCAAs in the prevention of liver fibrosis and injury in thedog's liver is still unclear. Aim of the present study will be to determine how BCAAs preserve liverfunctions in vitro. To this purpose we have selected and set up three different in vitro models: hepaticdog cells and canine hepatocellular carcinoma cells plated in 2D monolayer and hepatic dog cellscultured onto 3D scaffolds, obtained from decellularized rabbit liver. All cells adhered and proliferatedonce plated. Cells grown in monolayer quickly entered G0 end arrested growth, ELISA test confirmedtheir ability to produce albumin. Cells grown on scaffold vigorously replicated and showed theircapability to recellularize ECM rabbit liver. These results, although preliminary, demonstrate that theculture conditions used well preserved the original phenotype and function and further support thepossibility to use in vitro models to successfully study BCAA efficacy in dog

Recent results and perspectives on cosmology and fundamental physics from microwave surveys

Recent cosmic microwave background data in temperature and polarization have reached high precision in estimating all the parameters that describe the current so-called standard cosmological model. Recent results about the integrated Sachs-Wolfe effect from cosmic microwave background anisotropies, galaxy surveys, and their cross-correlations are presented. Looking at fine signatures in the cosmic microwave background, such as the lack of power at low multipoles, the primordial power spectrum and the bounds on non-Gaussianities, complemented by galaxy surveys, we discuss inflationary physics and the generation of primordial perturbations in the early Universe. Three important topics in particle physics, the bounds on neutrinos masses and parameters, on thermal axion mass and on the neutron lifetime derived from cosmological data are reviewed, with attention to the comparison with laboratory experiment results. Recent results from cosmic polarization rotation analyses aimed at testing the Einstein equivalence principle are presented. Finally, we discuss the perspectives of next radio facilities for the improvement of the analysis of future cosmic microwave background spectral distortion experiments.Comment: 27 pages, 9 figures. Review Article. International Journal of Modern Physics D, in press. [Will appear also on the proceedings of the Fourteenth Marcel Grossmann Meeting University of Rome "La Sapienza" - Rome, July 12-18, 2015 (http://www.icra.it/mg/mg14/), eds. Robert T. Jantzen, Kjell Rosquist, Remo Ruffini. World Scientific, Singapore

GILZ promotes production of peripherally induced Treg cells and mediates the crosstalk between glucocorticoids and TGF-β signaling

Summary: Regulatory T (Treg) cells expressing the transcription factor forkhead box P3 (FoxP3) control immune responses and prevent autoimmunity. Treatment with glucocorticoids (GCs) has been shown to increase Treg cell frequency, but the mechanisms of their action on Treg cell induction are largely unknown. Here, we report that glucocorticoid-induced leucine zipper (GILZ), a protein induced by GCs, promotes Treg cell production. In mice, GILZ overexpression causes an increase in Treg cell number, whereas GILZ deficiency results in impaired generation of peripheral Treg cells (pTreg), associated with increased spontaneous and experimental intestinal inflammation. Mechanistically, we found that GILZ is required for GCs to cooperate with TGF-β in FoxP3 induction, while it enhances TGF-β signaling by binding to and promoting Smad2 phosphorylation and activation of FoxP3 expression. Thus, our results establish an essential GILZ-mediated link between the anti-inflammatory action of GCs and the regulation of TGF-β-dependent pTreg production. : Peripherally induced Treg cells (pTreg) are generated outside of the thymus and regulate responses to foreign antigens. In this manuscript, Riccardi and colleagues demonstrate that glucocorticoid-induced protein GILZ controls generation of pTreg cells and colon homeostasis. GILZ promotes TGF-β-induced phosphorylation of Smad2 and the expression of FoxP3. Thus, GILZ mediates a synergy between glucocorticoids and TGF-β in pTreg cell induction. GILZ is essential for Treg induction by glucocorticoids and their anti-inflammatory activity

Homocysteine levels and C677T polymorphism of methylenetetrahydrofolate reductase in women with polycystic ovary syndrome

The aim of this study was to investigate the homocysteine (Hcy) levels and the C677T polymorphism of 5,10-methyl-enetetrahydrofolate reductase (MTHFR), a crucial factor of the Hey metabolism in young women with polycystic ovary syndrome (PCOS).Seventy young women with PCOS and another 70 healthy women with low folate intake were enrolled. Cases and controls were matched for age, body mass index, and allele frequency. Hey, vitamin B(12), and folate levels were measured, and a genetic analysis of 5,10-MTHFR at nucleotide 677 was performed in all subjects.No difference in mean Hcy levels was observed between PCOS women in comparison to the control group. Considering the different MTHFR polymorphism, no significant difference was found in serum Hey levels between subjects with PCOS and controls showing CC (10.4 +/- 3.1 us. 9.7 +/- 2.9 pmol/liter +/- SD) and CT genotypes (10.9 +/- 3.8 us. 11.0 +/- 3.2 pmol/liter SD). In subjects with a TT homozygous state, a significant (P < 0.05) difference was observed between PCOS and control women (11.5 +/- 3.9 as. 22.0 +/- 7.8 pmol/liter SD).In conclusion, our data show that in PCOS women, the serum Hey levels are normal, and the C677T polymorphism of MTHFR does not influence the Hey levels like in controls

Secondary Structures of MERS-CoV, SARS-CoV, and SARS-CoV-2 Spike Proteins Revealed by Infrared Vibrational Spectroscopy

All coronaviruses are characterized by spike glycoproteins whose S1 subunits contain the receptor binding domain (RBD). The RBD anchors the virus to the host cellular membrane to regulate the virus transmissibility and infectious process. Although the protein/receptor interaction mainly depends on the spike’s conformation, particularly on its S1 unit, their secondary structures are poorly known. In this paper, the S1 conformation was investigated for MERS-CoV, SARS-CoV, and SARS-CoV-2 at serological pH by measuring their Amide I infrared absorption bands. The SARS-CoV-2 S1 secondary structure revealed a strong difference compared to those of MERS-CoV and SARS-CoV, with a significant presence of extended β-sheets. Furthermore, the conformation of the SARS-CoV-2 S1 showed a significant change by moving from serological pH to mild acidic and alkaline pH conditions. Both results suggest the capability of infrared spectroscopy to follow the secondary structure adaptation of the SARS-CoV-2 S1 to different environments