Endogenous Opioids and Their Role in Stem Cell Biology and Tissue Rescue.

Opioids are considered the oldest drugs known by humans and have been used for sedation and pain relief for several centuries. Nowadays, endogenous opioid peptides are divided into four families: enkephalins, dynorphins, endorphins, and nociceptin/orphanin FQ. They exert their action through the opioid receptors (ORs), transmembrane proteins belonging to the super-family of G-protein-coupled receptors, and are expressed throughout the body; the receptors are the δ opioid receptor (DOR), µ opioid receptor (MOR), κ opioid receptor (KOR), and nociceptin/orphanin FQ receptor (NOP). Endogenous opioids are mainly studied in the central nervous system (CNS), but their role has been investigated in other organs, both in physiological and in pathological conditions. Here, we revise their role in stem cell (SC) biology, since these cells are a subject of great scientific interest due to their peculiar features and their involvement in cell-based therapies in regenerative medicine. In particular, we focus on endogenous opioids' ability to modulate SC proliferation, stress response (to oxidative stress, starvation, or damage following ischemia-reperfusion), and differentiation towards different lineages, such as neurogenesis, vasculogenesis, and cardiogenesis.

Aerobic training affects fatty acid composition of erythrocyte membranes.

The effect of exercise training on the fatty acid composition of erythrocyte membranes was evaluated in an experimental animal model where rats were subjected to a ten-wk aerobic training. Five groups of rats were compared: sedentary rats at 19 or 23 wks of age, rats trained at moderate or high intensity sacrificed at 19 wks of age, and rats trained at high intensity, and sacrificed following 4 weeks of sedentary life. We had already demonstrated that cardioprotection correlates with training intensity and partially persists in detrained rats. Main findings are that rats trained at higher intensity display consistent signs of lipid peroxidation but a lower ω6/ω3 ratio and a lower content of trans fatty acids when compared to rats trained at lower intensity and to older sedentary rats. Trans fatty acids negatively affect cell membrane fluidity and permeability. Detrained rats showed intermediate values. Gene expression evaluation of selected enzymes involved in lipid biosynthesis revealed some of the adaptive mechanisms leading to the maintenance of membrane fatty acid homeostasis following exercise. The decrease in the amount of trans fatty and in the inflammatory pathways (i.e. ω6/ω3 ratio) in high-intensity trained rats underscores the protective effect of high intensity aerobic training.

The Alteration of Chloride Homeostasis/GABAergic Signaling in Brain Disorders: Could Oxidative Stress Play a Role?

In neuronal precursors and immature neurons, the depolarizing (excitatory) effect of γ-Aminobutyric acid (GABA) signaling is associated with elevated [Cl-]i; as brain cells mature, a developmental switch occurs, leading to the decrease of [Cl-]i and to the hyperpolarizing (inhibitory) effect of GABAergic signaling. [Cl-]i is controlled by two chloride co-transporters: NKCC1, which causes Cl- to accumulate into the cells, and KCC2, which extrudes it. The ontogenetic upregulation of the latter determines the above-outlined switch; however, many other factors contribute to the correct [Cl-]i in mature neurons. The dysregulation of chloride homeostasis is involved in seizure generation and has been associated with schizophrenia, Down's Syndrome, Autism Spectrum Disorder, and other neurodevelopmental disorders. Recently, much effort has been put into developing new drugs intended to inhibit NKCC1 activity, while no attention has been paid to the origin of [Cl-]i dysregulation. Our study examines the pathophysiology of Cl- homeostasis and focuses on the impact of oxidative stress (OS) and inflammation on the activity of Cl- co-transporters, highlighting the relevance of OS in numerous brain abnormalities and diseases. This hypothesis supports the importance of primary prevention during pregnancy. It also integrates the therapeutic framework addressed to restore normal GABAergic signaling by counteracting the alteration in chloride homeostasis in central nervous system (CNS) cells, aiming at limiting the use of drugs that potentially pose a health risk.

Herb-Derived Products: Natural Tools to Delay and Counteract Stem Cell Senescence.

Cellular senescence plays a very important role in organismal aging increasing with age and in age-related diseases (ARDs). This process involves physiological, structural, biochemical, and molecular changes of cells, leading to a characteristic trait referred to "senescence-associated secretory phenotype (SASP)." In particular, with aging, stem cells (SCs) in situ exhibit a diminished capacity of self-renewal and show a decline in their functionality. The identification of interventions able to prevent the accumulation of senescent SCs in the organism or to pretreat cultured multipotent mesenchymal stromal cells (MSCs) prior to employing them for cell therapy is a main purpose of medical research. Many approaches have been investigated and resulted effective to prevent or counteract SC senescence in humans, as well as other animal models. In this work, we have reviewed the chance of using a number of herb-derived products as novel tools in the treatment of cell senescence, highlighting the efficacy of these agents, often still far from being clearly understood.

High predictive values of RBC membrane-based diagnostics by biophotonics in an integrated approach for Autism Spectrum Disorders.

Membranes attract attention in medicine, concerning lipidome composition and fatty acid correlation with neurological diseases. Hyperspectral dark field microscopy (HDFM), a biophotonic imaging using reflectance spectra, provides accurate characterization of healthy adult RBC identifying a library of 8 spectral end-members. Here we report hyperspectral RBC imaging in children affected by Autism Spectrum Disorder (ASD) (n = 21) compared to healthy age-matched subjects (n = 20), investigating if statistically significant differences in their HDFM spectra exist, that can comprehensively map a membrane impairment involved in disease. A significant difference concerning one end-member (spectrum 4) was found (P value = 0.0021). A thorough statistical treatment evidenced: i) diagnostic performance by the receiving operators curve (ROC) analysis, with cut-offs and very high predictive values (P value = 0.0008) of spectrum 4 for identifying disease; ii) significant correlations of spectrum 4 with clinical parameters and with the RBC membrane deficit of the omega-3 docosahexaenoic acid (DHA) in ASD patients; iii) by principal component analysis, very high affinity values of spectrum 4 to the factor that combines behavioural parameters and the variable "cc" discriminating cases and controls. These results foresee the use of biophotonic methodologies in ASD diagnostic panels combining with molecular elements for a correct neuronal growth.

Pyrethroid Pesticide Metabolite in Urine and Microelements in Hair of Children Affected by Autism Spectrum Disorders: A Preliminary Investigation.

The number of children affected by Autism Spectrum Disorders (ASD) is dramatically increasing as well as the studies aimed at understanding the risk factors associated with the development of ASD. Since the etiology of ASD is partly genetic and partly environmental, factors (i.e., heavy metals, pesticides) as well as lifestyle seem to have a key role in the development of the disease. ASD and Control (CTR) children, aged 5-12 years, were compared. Gas chromatography coupled with trap mass detector was used to measure the level of 3-PBA, the main pyrethroid metabolite in urine in a group of ASD patients, while optical emission spectrometry analysis was employed to estimate the level of metals and microelements in hair in a different group of ASD children. The presence of 3-PBA in urine seems to be independent of age in ASD children, while a positive correlation between 3-PBA and age was observed in the control group of the same age range. Urine concentration of 3-BPA in ASD children had higher values than in the control group, which were marginally significant (p = 0.054). Mg results were significantly decreased in ASD with respect to controls, while V, S, Zn, and Ca/Mg were marginally increased, without reaching statistical significance. Results of Principal Component (PC) analysis of metals and microelements in hair were not associated with either age or health status. In conclusion, 3-PBA in urine and Mg in hair were changed in ASD children relative to control ones.

Identification of novel plasma glycosylation-associated markers of aging.

The pro- or anti-inflammatory activities of immunoglobulins G (IgGs) are controlled by the structure of the glycan N-linked to Asn297 of their heavy chain. The age-associated low grade inflammation (inflammaging) is associated with increased plasmatic levels of agalactosylated IgGs terminating with N-acetylglucosamine (IgG-G0) whose biogenesis has not been fully explained. Although the biosynthesis of glycans is in general mediated by glycosyltransferases associated with internal cell membranes, the extracellular glycosylation of circulating glycoproteins mediated by plasmatic glycosyltransferases has been recently demonstrated. In this study we have investigated the relationship between plasmatic glycosyltransferases, IgG glycosylation and inflammatory and aging markers. In cohorts of individuals ranging from infancy to centenarians we determined the activity of plasmatic ß4 galactosyltransferase(s) (B4GALTs) and of α2,6-sialyltransferase ST6GAL1, the glycosylation of IgG, the GlycoAge test (a glycosylation-based marker of aging) and the plasma level of inflammatory and liver damage markers. Our results show that: 1) plasmatic B4GALTs activity is a new marker of aging, showing a linear increase throughout the whole age range. 2) plasmatic ST6GAL1 was high only in children and in people above 80, showing a quadratic relationship with age. 3) Neither plasmatic glycosyltransferase correlated with markers of liver damage. 4) plasmatic ST6GAL1 showed a positive association with acute phase proteins in offspring of short lived parents, but not in centenarians or in their offspring. 5) Although the glycosylation of IgGs was not correlated with the level of the two plasmatic glycosyltransferases, it showed progressive age-associated changes consistent with a shift toward a pro-inflammatory glycotype.

Perspective Biological Markers for Autism Spectrum Disorders: Advantages of the Use of Receiver Operating Characteristic Curves in Evaluating Marker Sensitivity and Specificity.

Autism Spectrum Disorders (ASD) are a heterogeneous group of neurodevelopmental disorders. Recognized causes of ASD include genetic factors, metabolic diseases, toxic and environmental factors, and a combination of these. Available tests fail to recognize genetic abnormalities in about 70% of ASD children, where diagnosis is solely based on behavioral signs and symptoms, which are difficult to evaluate in very young children. Although it is advisable that specific psychotherapeutic and pedagogic interventions are initiated as early as possible, early diagnosis is hampered by the lack of nongenetic specific biological markers. In the past ten years, the scientific literature has reported dozens of neurophysiological and biochemical alterations in ASD children; however no real biomarker has emerged. Such literature is here reviewed in the light of Receiver Operating Characteristic (ROC) analysis, a very valuable statistical tool, which evaluates the sensitivity and the specificity of biomarkers to be used in diagnostic decision making. We also apply ROC analysis to some of our previously published data and discuss the increased diagnostic value of combining more variables in one ROC curve analysis. We also discuss the use of biomarkers as a tool for advancing our understanding of nonsyndromic ASD.

Non-thermal radio frequency and static magnetic fields increase rate of hemoglobin deoxygenation in a cell-free preparation.

The growing body of clinical and experimental data regarding electromagnetic field (EMF) bioeffects and their therapeutic applications has contributed to a better understanding of the underlying mechanisms of action. This study reports that two EMF modalities currently in clinical use, a pulse-modulated radiofrequency (PRF) signal, and a static magnetic field (SMF), applied independently, increased the rate of deoxygenation of human hemoglobin (Hb) in a cell-free assay. Deoxygenation of Hb was initiated using the reducing agent dithiothreitol (DTT) in an assay that allowed the time for deoxygenation to be controlled (from several min to several hours) by adjusting the relative concentrations of DTT and Hb. The time course of Hb deoxygenation was observed using visible light spectroscopy. Exposure for 10-30 min to either PRF or SMF increased the rate of deoxygenation occurring several min to several hours after the end of EMF exposure. The sensitivity and biochemical simplicity of the assay developed here suggest a new research tool that may help to further the understanding of basic biophysical EMF transduction mechanisms. If the results of this study were to be shown to occur at the cellular and tissue level, EMF-enhanced oxygen availability would be one of the mechanisms by which clinically relevant EMF-mediated enhancement of growth and repair processes could occur.

Supplementation of creatine and ribose prevents apoptosis and right ventricle hypertrophy in hypoxic hearts.

BACKGROUND/AIMS: The simultaneous supplementation of creatine and D-ribose has been shown to reduce apoptosis in vitro in non-irreversibly injured cultured ischemic cardiomyocytes through down-regulation of the signaling mechanisms governing adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (Akt). Here, we test the hypothesis that an analogous mechanism exists in vivo when the challenge is chronic exposure to hypoxia. METHODS: Five week-old mice were exposed to an atmosphere containing 10% O2 for 10 days. Mice were gavaged daily with vehicle, creatine, D-ribose or creatine + D-ribose. After sacrifice, myocardial and pulmonary tissue were harvested for structural and biochemical analyses. RESULTS: Hypoxia induced right ventricle hypertrophy and left ventricle apoptosis. Both phenotypes were slightly reduced by either creatine or D-ribose, whereas the simultaneous administration of creatine + D-ribose almost completely reversed the effects of hypoxia. Furthermore, creatine + D-ribose diminished the hypoxia-induced increases in the activity of AMPK, Akt and JNK, but not of ERK. Finally, the hypoxia-induced pulmonary overexpression of endothelin-1 mRNA was markedly reduced by creatine + D-ribose. CONCLUSION: The simultaneous administration of creatine + D-ribose confers additional cardiovascular protection with respect to that observed with either creatine or D-ribose. The mechanism stems from the AMPK and Akt signaling pathways. These findings may form the basis of a paradigm to re-energize non-irreversibly damaged cardiomyocytes, counteracting injury by triggering specific signaling pathways.

Oxidative Stress and Erythrocyte Membrane Alterations in Children with Autism: Correlation with Clinical Features.

It has been suggested that oxidative stress may play a role in the pathogenesis of Autism Spectrum Disorders (ASD), but the literature reports somewhat contradictory results. To further investigate the issue, we evaluated a high number of peripheral oxidative stress parameters, and some related issues such as erythrocyte membrane functional features and lipid composition. Twenty-one autistic children (Au) aged 5 to 12 years, were gender and age-matched with 20 typically developing children (TD). Erythrocyte thiobarbituric acid reactive substances, urinary isoprostane and hexanoyl-lysine adduct levels were elevated in Au, thus confirming the occurrence of an imbalance of the redox status of Au, whilst other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma radical absorbance capacity and carbonyl groups, erythrocyte superoxide dismutase and catalase activities) were unchanged. A very significant reduction of Na(+)/K(+)-ATPase activity (-66%, p<0.0001), a reduction of erythrocyte membrane fluidity and alteration in erythrocyte fatty acid membrane profile (increase in monounsaturated fatty acids, decrease in EPA and DHA-ω3 with a consequent increase in ω6/ω3 ratio) were found in Au compared to TD, without change in membrane sialic acid content. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity. Oxidative stress and erythrocyte membrane alterations may play a role in the pathogenesis of ASD and prompt the development of palliative therapeutic protocols. Moreover, the marked decrease in NKA could be potentially utilized as a peripheral biomarker of ASD.

Oxidative stress in the denervated muscle.

Following experimental hind limb denervation in rats, this study demonstrates that oxidative stress occurs and advances an hypothesis about its origin. In fact: (i) ROS are formed; (ii) membrane lipids are oxidized; (iii) oxidized ion channels and pumps may lead to increased [Ca(2+)](i); all the above mentioned events increase with denervation time. In the denervated muscle, (iv) mRNA abundance of cytoprotective and anti-oxidant proteins (Hsp70, Hsp27, Sod1, Catalase, Gpx1, Gpx4, Gstm1), as well as (v) SOD1 enzymatic activity and HSP70i protein increase; (vi) an unbalance in mitochondrial OXPHOS enzymes occurs, presumably leading to excess mitochondrial ROS production; (vii) increased cPLA2alpha expression (mRNA) and activation (increased [Ca(2+)](i)) may lead to increased hydroperoxides release. Since anti-oxidant defences appear inadequate to counterbalance increased ROS production with increased denervation time, an anti-oxidant therapeutic strategy seems to be advisable in the many medical conditions where the nerve-muscle connection is impaired.

A subpopulation of rat muscle fibers maintains an assessable excitation-contraction coupling mechanism after long-standing denervation despite lost contractility.

To define the time course and potential effects of electrical stimulation on permanently denervated muscle, we evaluated excitation-contraction coupling (ECC) of rat leg muscles during progression to long-term denervation by ultrastructural analysis, specific binding to dihydropyridine receptors, ryanodine receptor 1 (RYR-1), Ca channels and extrusion Ca pumps, gene transcription and translation of Ca-handling proteins, and in vitro mechanical properties and electrophysiological analyses of sarcolemmal passive properties and L-type Ca current (ICa) parameters. We found that in response to long-term denervation: 1) isolated muscle that is unable to twitch in vitro by electrical stimulation has very small myofibers but may show a slow caffeine contracture; 2) only roughly half of the muscle fibers with "voltage-dependent Ca channel activity" are able to contract; 3) the ECC mechanisms are still present and, in part, functional; 4)ECC-related gene expression is upregulated; and 5) at any time point, there are muscle fibers that are more resistant than others to denervation atrophy and disorganization of the ECC apparatus. These results support the hypothesis that prolonged "resting" [Ca] may drive progression of muscle atrophy to degeneration and that electrical stimulation-induced [Ca] modulation may mimic the lost nerve influence, playing a key role in modifying the gene expression of denervated muscle. Hence, these data provide a potential molecular explanation for the muscle recovery that occurs in response to rehabilitation strategies developed based on empirical clinical observations.