Highly Improved Electrospray Ionization-Mass Spectrometry Detection of G-Quadruplex-Folded Oligonucleotides and Their Complexes with Small Molecules

G-quadruplexes are nucleic acids structures stabilized by physiological concentration of potassium ions. Because low stability G-quadruplexes are hardly detectable by mass spectrometry, we optimized solvent conditions: isopropanol in a triethylamine/hexafluoroisopropanol mixture highly increased G-quadruplex sensitivity with no modification of the physiological G-quadruplex conformation. G-quadruplexes/G-quadruplex-ligand complexes were also correctly detected at concentration as low as 40 nM. Detection of the physiological conformation of G4s and their complexes opens up the possibility to perform high-throughput screening of G-quadruplex ligands for the development of drug molecules effective against critical human diseases

Pathogenesis and Progression of Multiple Sclerosis: The Role of Arachidonic Acid–Mediated Neuroinflammation

Multiple sclerosis is characterized by inflammatory processes occurring within the central nervous system. In multiple sclerosis, inflammation could be either a physiological response secondary to the immune system activation or a phenomenon triggered by primary cytodegeneration of neurons and/ or oligodendrocytes without the involvement of immune cells. The arachidonic acid metabolism is activated via cyclooxygenases (COXs) and lipoxygenases (LOXs) in postmortem brain samples and in the cerebrospinal fluid of multiple sclerosis patients. It has been hypothesized that the arachidonic acid–mediated neuroinflammation could play a role in the pathogenic mechanisms triggering demyelination, oligodendrocyte loss, axonal pathology and, ultimately, motor dysfunctions, which are hallmarks of multiple sclerosis. COX-2 and 5-LOX selective inhibitors efficiently inhibit each of the hallmarks mentioned above in different animal models of multiple sclerosis. Thus, it is suggested that the arachidonic acid pathway represents a potential pharmacological target to ameliorate multiple sclerosis pathology and symptoms

The cellular protein nucleolin preferentially binds long-looped G-quadruplex nucleic acids

open5noBACKGROUND: G-quadruplexes (G4s) are four-stranded nucleic acid structures that form in G-rich sequences. Nucleolin (NCL) is a cellular protein reported for its functions upon G4 recognition, such as induction of neurodegenerative diseases, tumor and virus mechanisms activation. We here aimed at defining NCL/G4 binding determinants. METHODS: Electrophoresis mobility shift assay was used to detect NCL/G4 binding; circular dichroism to assess G4 folding, topology and stability; dimethylsulfate footprinting to detect G bases involved in G4 folding. RESULTS: The purified full-length human NCL was initially tested on telomeric G4 target sequences to allow for modulation of loop, conformation, length, G-tract number, stability. G4s in promoter regions with more complex sequences were next employed. We found that NCL binding to G4s heavily relies on G4 loop length, independently of the conformation and oligonucleotide/loop sequence. Low stability G4s are preferred. When alternative G4 conformations are possible, those with longer loops are preferred upon binding to NCL, even if G-tracts need to be spared from G4 folding. CONCLUSIONS: Our data provide insight into how G4s and the associated proteins may control the ON/OFF molecular switch to several pathological processes, including neurodegeneration, tumor and virus activation. Understanding these regulatory determinants is the first step towards the development of targeted therapies. GENERAL SIGNIFICANCE: The indication that NCL binding preferentially stimulates and induces folding of G4s containing long loops suggests NCL ability to modify the overall structure and steric hindrance of the involved nucleic acid regions. This protein-induced modification of the G4 structure may represent a cellular mechanosensor mechanism to molecular signaling and disease pathogenesis.openLago, Sara; Tosoni, Elena; Nadai, Matteo; Palumbo, Manlio; Richter, Sara NLago, Sara; Tosoni, Elena; Nadai, Matteo; Palumbo, Manlio; Richter, Sar

Experimental in vivo models of multiple sclerosis: state of the art

Multiple sclerosis is a multifactorial and heterogeneous neurological disease; hence, several experimental animal models had to be developed to mimic the different features of human pathology. Three main classes of animal models have been developed:experimental autoimmune encephalomyelitis (EAE), cupri- zone intoxication, and Theiler’s murine encephalomyelitis virus (TMEV) infection. The EAE model is the most versatile as it allows the reproduction of different patterns of multiple sclerosis; it is mostly relevant for relapsing-remitting multiple sclerosis and has allowed the development of several first-line, disease-modifying drugs for the treatment of multiple sclerosis. The other two models are less flexi- ble than the EAE model and, to date, have not led to the discovery of any clinically relevant therapies. The cuprizone model mostly mimics the acute and chronic courses of multiple sclerosis, and it may represent a useful tool to develop novel therapies to protect oligodendrocytes and stimulate remyelination. Finally, the TMEV infection is the reference model to specifically study viral-mediated mecha- nisms of acute and primary progressive multiple sclerosis

Alterations of brain eicosanoid synthetic pathway in multiple sclerosis and in animal models of demyelination: Role of cyclooxygenase-2

Inflammation is a physiological response to exogenous and endogenous stimuli and, together with demyelination and immune system activation, is one of the key features of multiple sclerosis (MS). Arachidonic acid (AA) metabolism by cyclooxygenase (COX) and lipoxygenase (LO) enzymes leads to the production of proinflammatory eicosanoids, and stimulates cytokine production and activation of microglia and astrocytes, thereby contributing to MS pathology. Current therapies target the immune system but do not specifically target AA-related inflammatory pathway. Corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs) are frequently associated with immunomodulatory therapies to treat flu-like adverse effects. Few clinical and mounting preclinical data in MS show that AA metabolism contributes to immune system activation, demyelination and motor disabilities, and administration of NSAIDs reduces these symptoms. The beneficial effect of NSAIDs seems to be a prerogative of COX-2 selective inhibitors and suggests that NSAIDs selective for COX-2 may be more effective than mixed COX-1/2 inhibitors

A Computational and Experimental Study of the Conformers of Pyrrolidinium Ionic Liquid Cations Containing an Ethoxy Group in the Alkyl Side Chain

We investigate the conformers of the N-methoxyethyl-N-methylpyrrolidinium (PYR1(2O1)) and N-ethoxyethyl-Nmethylpyrrolidinium (PYR1(2O2)) ionic liquid cations by means of DFT calculations at the B3LYP/6-31G∗∗ level and we calculate their infrared vibration frequencies. The comparison with the absorbance spectra of two ionic liquids containing these ions indicates good performance of such a combination of theory and basis set.The lowest energy conformer of each pyrrolidinium cation displays equatorial-envelope geometry; however, in contrast with the prototypical PYR14, the main alkyl side chain is not in an all-trans configuration, but it tends to be bent. Moreover, calculations indicate that the LUMO orbital extends more along the alkyl side chain in PYR1(2O1) and PYR1(2O2) than in the parent ion 1-butyl-1-methylpyrrolidinium (PYR14)

Cuprizone induced-demyelination in mice alters brain expression of genes involved in arachidonic acid metabolism .

Chronic feeding with the copper chelator cuprizone in mice causes oligodendrocyte death and subsequent reversible demyelination. Although the mechanism of demyelination is unknown, activation of glia is integral to the process. Since metabolism of arachidonic acid (AA) is involved in glial activation, we hypothesized that cuprizone exposure would alter expression of AA cascade genes. Mice were fed 0.2 % cuprizone in the diet for 6 weeks and then returned to a normal diet. Histochemistry with the myelin stains Black Gold and Fluoromyelin demonstrated that frank demyelination and influx of glial cells into the corpus collosum begins at week 3 and peaks at week 5. A decrease in myelin and oligodendrocyte markers, accompanied by increased expression of markers of microglia (CD11b) and astrocytes (glial acidic fibrillary protein), was evident at week one. Gene expression of cyclooxygenase-2 and 15-lipoxygenase (LOX) was also changed at week one, suggesting that these genes are either involved in or respond to early demyelination. Expression of 5-LOX was not changed during early demyelination but it peaked during week 5, when glial markers and frank demyelination also reached their peak, suggesting that 5-LOX expression is a consequence of the massive influx of inflammatory cells into the area of demyelination. Our study is the first to demonstrate that multiple enzymes involved in arachidonic acid metabolism are altered in the cuprizone model of demyelination and remyelination. These data may help to develop new therapeutic targets to treat human demyelinating diseases, such as multiple sclerosis. Supported by the Intramural Research Program of the NIH, NIA

Genes and Aggressive Behavior: Epigenetic Mechanisms Underlying Individual Susceptibility to Aversive Environments

Over the last two decades, the study of the relationship between nature and nurture in shaping human behavior has encountered a renewed interest. Behavioral genetics showed that distinct polymorphisms of genes that code for proteins that control neurotransmitter metabolic and synaptic function are associated with individual vulnerability to aversive experiences, such as stressful and traumatic life events, and may result in an increased risk of developing psychopathologies associated with violence. On the other hand, recent studies indicate that experiencing aversive events modulates gene expression by introducing stable changes to DNA without modifying its sequence, a mechanism known as “epigenetics”. For example, experiencing adversities during periods of maximal sensitivity to the environment, such as prenatal life, infancy and early adolescence, may introduce lasting epigenetic marks in genes that affect maturational processes in brain, thus favoring the emergence of dysfunctional behaviors, including exaggerate aggression in adulthood. The present review discusses data from recent research, both in humans and animals, concerning the epigenetic regulation of four genes belonging to the neuroendocrine, serotonergic and oxytocinergic pathways—Nuclear receptor subfamily 3-group C-member 1 (NR3C1), oxytocin receptor (OXTR), solute carrier-family 6 member 4 (SLC6A4) and monoamine oxidase A (MAOA)—and their role in modulating vulnerability to proactive and reactive aggressive behavior. Behavioral genetics and epigenetics are shedding a new light on the fine interaction between genes and environment, by providing a novel tool to understand the molecular events that underlie aggression. Overall, the findings from these studies carry important implications not only for neuroscience, but also for social sciences, including ethics, philosophy and law

Osteocytes Specific GSK3 Inhibition Affects In Vitro Osteogenic Differentiation.

Osteocytes, the most important regulators of bone processes, are producers of molecules (usually proteins) that act as signals in order to communicate with nearby cells. These factors control cell division (proliferation), differentiation, and survival. Substantial evidence showed different signaling pathways activated by osteocytes and involved in osteoblast differentiation, in particular in the last decade, when the Wingless-related integration site (WNT) pathway assumed a critical large importance. WNT activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism, making GSK3 a potential therapeutic target for bone diseases. In our study, we hypothesized an important role of the osteocyte MLO-Y4 conditioned medium in controlling the differentiation process of osteoblast cell line 2T3. We found an effect of diminished differentiation capability of 2T3 upon conditioning with medium from murine long bone osteocyte-Y4 cells (MLO-Y4) pre-treated with GSK3 inhibitor CHIR2201. The novel observations of this study provide knowledge about the inhibition of GSK3 in MLO-Y4 cells. This strategy could be used as a plausible target in osteocytes in order to regulate bone resorption mediated by a loss of osteoblasts activity through a paracrine loop