TLDc Domain-Containing Genes in Autism Spectrum Disorder: New Players in the Oxidative Stress Response.

Oxidative stress (OS) plays a key role in autism spectrum disorder (ASD), a neurodevelopmental disorder characterized by deficits in social communication, restricted interests, and repetitive behaviors. Recent evidence suggests that the TLDc [Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic] domain is a highly conserved motif present in proteins that are important players in the OS response and in neuroprotection. Human proteins sharing the TLDc domain include OXR1, TLDC1, NCOA7, TBC1D24, and C20ORF118. This study was aimed at understanding whether TLDc domain-containing mRNAs together with specific microRNAs (200b-3p and 32-5p) and long noncoding RNAs (TUG1), known to target TLDc proteins, contributed to regulate the OS response in ASD. Data showed a significant increase in the levels of OXR1 and TLDC1 mRNAs in peripheral blood mononuclear cells (PBMCs) of ASD children compared to their neurotypically developing (NTD) counterparts, along with an increase in TUG1 mRNA expression levels, suggesting its possible role in the regulation of TLDc proteins. A positive correlation between the expression of some TLDc mRNAs and the Childhood Autism Rating Scale (CARS) global score as well as inflammatory gene expression was found. In conclusion, our data suggest a novel biological pathway in the OS response of ASD subjects that deserves further exploration.

Non-Coding RNAs in the Transcriptional Network That Differentiates Skeletal Muscles of Sedentary from Long-Term Endurance- and Resistance-Trained Elderly.

In a previous study, the whole transcriptome of the vastus lateralis muscle from sedentary elderly and from age-matched athletes with an exceptional record of high-intensity, life-long exercise training was compared-the two groups representing the two extremes on a physical activity scale. Exercise training enabled the skeletal muscle to counteract age-related sarcopenia by inducing a wide range of adaptations, sustained by the expression of protein-coding genes involved in energy handling, proteostasis, cytoskeletal organization, inflammation control, and cellular senescence. Building on the previous study, we examined here the network of non-coding RNAs participating in the orchestration of gene expression and identified differentially expressed micro- and long-non-coding RNAs and some of their possible targets and roles. Unsupervised hierarchical clustering analyses of all non-coding RNAs were able to discriminate between sedentary and trained individuals, regardless of the exercise typology. Validated targets of differentially expressed miRNA were grouped by KEGG analysis, which pointed to functional areas involved in cell cycle, cytoskeletal control, longevity, and many signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), which had been shown to be pivotal in the modulation of the effects of high-intensity, life-long exercise training. The analysis of differentially expressed long-non-coding RNAs identified transcriptional networks, involving lncRNAs, miRNAs and mRNAs, affecting processes in line with the beneficial role of exercise training.

Skeletal Muscle Gene Expression in Long-Term Endurance and Resistance Trained Elderly.

Physical exercise is deemed the most efficient way of counteracting the age-related decline of skeletal muscle. Here we report a transcriptional study by next-generation sequencing of vastus lateralis biopsies from elderly with a life-long high-level training practice (n = 9) and from age-matched sedentary subjects (n = 5). Unsupervised mixture distribution analysis was able to correctly categorize trained and untrained subjects, whereas it failed to discriminate between individuals who underwent a prevalent endurance (n = 5) or a prevalent resistance (n = 4) training, thus showing that the training mode was not relevant for sarcopenia prevention. KEGG analysis of transcripts showed that physical exercise affected a high number of metabolic and signaling pathways, in particular those related to energy handling and mitochondrial biogenesis, where AMPK and AKT-mTOR signaling pathways are both active and balance each other, concurring to the establishment of an insulin-sensitive phenotype and to the maintenance of a functional muscle mass. Other pathways affected by exercise training increased the efficiency of the proteostatic mechanisms, consolidated the cytoskeletal organization, lowered the inflammation level, and contrasted cellular senescence. This study on extraordinary individuals who trained at high level for at least thirty years suggests that aging processes and exercise training travel the same paths in the opposite direction.

trans-Double Bond-Containing Liposomes as Potential Carriers for Drug Delivery.

The use of liposomes has been crucial for investigations in biomimetic chemical biology as a membrane model and in medicinal chemistry for drug delivery. Liposomes are made of phospholipids whose biophysical characteristics strongly depend on the type of fatty acid moiety, where natural unsaturated lipids always have the double bond geometry in the cis configuration. The influence of lipid double bond configuration had not been considered so far with respect to the competence of liposomes in delivery. We were interested in evaluating possible changes in the molecular properties induced by the conversion of the double bond from cis to trans geometry. Here we report on the effects of the addition of trans-phospholipids supplied in different amounts to other liposome constituents (cholesterol, neutral phospholipids and cationic surfactants), on the size, ζ-potential and stability of liposomal formulations and on their ability to encapsulate two dyes such as rhodamine B and fluorescein. From a biotechnological point of view, trans-containing liposomes proved to have different characteristics from those containing the cis analogues, and to influence the incorporation and release of the dyes. These results open new perspectives in the use of the unnatural lipid geometry, for the purpose of changing liposome behavior and/or of obtaining molecular interferences, also in view of synergic effects of cell toxicity, especially in antitumoral strategies.

Dynamic and Systemic Perspective in Autism Spectrum Disorders: A Change of Gaze in Research Opens to A New Landscape of Needs and Solutions.

The first step for a harmonious bio-psycho-social framework in approaching autism spectrum disorders (ASD) is overcoming the conflict between the biological and the psychosocial perspective. Biological research can provide clues for a correct approach to clinical practice, assuming that it would lead to the conceptualization of a pathogenetic paradigm able to account for epidemiologic and clinical findings. The upward trajectory in ASD prevalence and the systemic involvement of other organs besides the brain suggest that the epigenetic paradigm is the most plausible one. The embryo-fetal period is the crucial window of opportunity for keeping neurodevelopment on the right tracks, suggesting that women's health in pregnancy should be a priority. Maladaptive molecular pathways beginning in utero, in particular, a vicious circle between the immune response, oxidative stress/mitochondrial dysfunction, and dysbiosis-impact neurodevelopment and brain functioning across the lifespan and are the basis for progressive multisystemic disorders that account for the substantial health loss and the increased mortality in ASD. Therefore, the biological complexity of ASD and its implications for health requires the enhancement of clinical skills on these topics, to achieve an effective multi-disciplinary healthcare model. Well-balanced training courses could be a promising starting point to make a change.

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.

Myocardial tolerance to ischemia-reperfusion injury, training intensity and cessation.

Training has been shown to induce cardioprotection. The mechanisms involved remain still poorly understood. Aims of the study were to examine the relevance of training intensity on myocardial protection against ischemia/reperfusion (I/R) injury, and to which extent the beneficial effects persist after training cessation in rats. Sprague-Dawley rats trained at either low (60% [Formula: see text]) or high (80% [Formula: see text]) intensity for 10 weeks. An additional group of highly trained rats was detrained for 4 weeks. Untrained rats served as controls. At the end of treatment, rats of all groups were split into two subgroups. In the former, rats underwent left anterior descending artery (LAD) ligature for 30 min, followed by 90-min reperfusion, with subsequent measurement of the infarct size. In the latter, biopsies were taken to measure heat-shock proteins (HSP) 70/72, vascular endothelial growth factor (VEGF) protein levels, and superoxide dismutase (SOD) activity. Training reduced infarct size proportionally to training intensity. With detraining, infarct size increased compared to highly trained rats, maintaining some cardioprotection with respect to controls. Cardioprotection was proportional to training intensity and related to HSP70/72 upregulation and Mn-SOD activity. The relationship with Mn-SOD was lost with detraining. VEGF protein expression was not affected by either training or detraining. Stress proteins and antioxidant defenses might be involved in the beneficial effects of long-term training as a function of training intensity, while HSP70 may be one of the factors accounting for the partial persistence of myocardial protection against I/R injury in detrained rats.

Effect of training and sudden detraining on the patellar tendon and its enthesis in rats.

BACKGROUND: Different conditions may alter tendon characteristics. Clinical evidence suggests that tendon injuries are more frequent in athletes that change type, intensity and duration of training. Aim of the study was the assessment of training and especially detraining on the patellar tendon (PT) and its enthesis. METHODS: 27 male adult Sprague-Dawley rats were divided into 3 groups: 20 rats were trained on a treadmill for 10 weeks. Of these, 10 rats were euthanized immediately after training (trained group), and 10 were caged without exercise for 4 weeks before being euthanized (de-trained group). The remaining 7 rats were used as controls (untrained rats). PT insertion, structure (collagen fiber organization and proteoglycan, PG, content), PT thickness, enthesis area, and subchondral bone volume at the enthesis were measured by histomorphometry and microtomography. RESULTS: Both PG content and collagen fiber organization were significantly lower in untrained and detrained animals than in trained ones (p < 0.05 and p < 0.0001). In the detrained group, fiber organization and PG content were worse than that of the untrained groups and the untrained group showed a significantly higher score than the detrained group (p < 0.05). In the trained group, the PT was significantly thicker than in untrained group (p < 0.05). No significant differences in the enthesis area and subchondral bone volume among the three groups were seen. CONCLUSIONS: Moderate exercise exerts a protective effect on the PT structure while sudden discontinuation of physical activity has a negative effect on tendons. The present results suggest that after a period of sudden de-training (such as after an injury) physical activity should be restarted with caution and with appropriate rehabilitation programs.

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.

Autism Spectrum Disorder from the Womb to Adulthood: Suggestions for a Paradigm Shift.

The wide spectrum of unique needs and strengths of Autism Spectrum Disorders (ASD) is a challenge for the worldwide healthcare system. With the plethora of information from research, a common thread is required to conceptualize an exhaustive pathogenetic paradigm. The epidemiological and clinical findings in ASD cannot be explained by the traditional linear genetic model, hence the need to move towards a more fluid conception, integrating genetics, environment, and epigenetics as a whole. The embryo-fetal period and the first two years of life (the so-called 'First 1000 Days') are the crucial time window for neurodevelopment. In particular, the interplay and the vicious loop between immune activation, gut dysbiosis, and mitochondrial impairment/oxidative stress significantly affects neurodevelopment during pregnancy and undermines the health of ASD people throughout life. Consequently, the most effective intervention in ASD is expected by primary prevention aimed at pregnancy and at early control of the main effector molecular pathways. We will reason here on a comprehensive and exhaustive pathogenetic paradigm in ASD, viewed not just as a theoretical issue, but as a tool to provide suggestions for effective preventive strategies and personalized, dynamic (from womb to adulthood), systemic, and interdisciplinary healthcare approach.

Modulation of paraoxonase 1 and 3 expression after moderate exercise training in the rat.

Paraoxonases (PONs) are a small family of antioxidant enzymes whose antiatherogenic activity is well known. The aim of the present study was the evaluation of the effects of moderate aerobic training on their expression using a rat model. In order to discriminate between PON1 and PON3 enzymatic activity, we took advantage of some differences in their substrate preferences. PON1 and PON3 enzymatic activities and their protein levels were analyzed in plasma and in liver microsomes, and their mRNA levels in the liver. Exercise training did not affect PON1 expression or enzymatic activity but increased PON3 mRNA, protein levels, and enzymatic activity. Training also induced variations in plasma membrane composition, including an increase in polyunsaturated and a decrease in mono- and di-unsaturated fatty acids. On the other hand, acute exercise inhibited PON activities while increasing PON3 protein content in liver microsomes and reversing the relative composition in mono-, di-, and poly-unsaturated fatty acids, suggesting that physical stress, by altering membrane composition, may impair PON release from liver membranes. In conclusion, we documented, for the first time, the presence of PON3 in rat serum and, notably, found that the upregulation of PON3, rather than PON1, appears to be associated with physical training.

Exposure of alpha2,6-sialylated lactosaminic chains marks apoptotic and necrotic death in different cell types.

Many observations have reported glycosylation changes associated with apoptosis in different biological systems, although none of these has shown any general significance. In this work, we show that in cell lines from different histological origin, (colon, breast, pancreas, and bladder cancer) as well as in normal human and mice neutrophils, apoptosis is accompanied by the exposure of sugar chains recognized by the lectin from Sambucus nigra (SNA), specific for Sia alpha 2,6Gal/GalNAc structures. Also, cells undergoing primary necrosis induced by heat treatment (56 degrees C, 30 min) expose specifically binding sites for SNA. While this modification is recognized also by the lectin from the mushroom Polyporus squamosus, which is highly specific for alpha2,6-sialylated lactosamine, no significant changes were detected in the binding of lectins specific for other carbohydrate structures, such as those from Phaseolus vulgaris, Arachis hypogea, and Maackia amurensis. The binding of SNA to apoptotic/necrotic cells is inhibited by neuraminidase treatment and by alpha2,6-sialylated compounds. In apoptotic, but not in necrotic SW948 cells, SNA reactivity is specifically associated with 65, 69, and 87 kDa glycoproteins. The exposure of SNA-reactive chains by apoptotic/necrotic cells occurs also in cells not expressing sialyltransferases ST6Gal.1 or ST6Gal.2 and is largely independent of the presence of alpha2,6-sialylated lactosaminic chains on the surface of preapoptotic cells. In neutrophils from ST6Gal.1 knock-out mice, the apoptosis-related increase in SNA reactivity is reduced but not abolished. These data demonstrate that apoptosis and primary necrosis induce a specific glycosylation change independent of the cell type and nature of the stimulus.

Gene expression profile of rat left ventricles reveals persisting changes following chronic mild exercise protocol: implications for cardioprotection.

BACKGROUND: Epidemiological studies showed that physical exercise, specifically moderate lifelong training, is protective against cardiovascular morbidity and mortality. Most experimental work has focused into the effects and molecular mechanisms underlying intense, rather than mild exercise, by exploring the acute effect of training. Our study aims at investigating the cardioprotective effect of mild chronic exercise training and the gene expression profile changes at 48 hrs after the exercise cessation. Rats were trained at mild intensity on a treadmill: 25 m/min, 10%incline, 1 h/day, 3 days/week, 10 weeks; about 60% of the maximum aerobic power. By Affymetrix technology, we investigated the gene expression profile induced by exercise training in the left ventricle (LV) of trained (n = 10) and control (n = 10) rats. Cardioprotection was investigated by ischemia/reperfusion experiments (n = 10 trained vs. n = 10 control rats). RESULTS: Mild exercise did not induce cardiac hypertrophy and was cardioprotective as demonstrated by the decreased infarct size (p = 0.02) after ischemia/reperfusion experiments in trained with respect to control rats. Ten genes and 2 gene sets (two pathways) resulted altered in LV of exercised animals with respect to controls. We validated by real-time PCR the increased expression of four genes: similar to C11orf17 protein (RGD1306959), caveolin 3, enolase 3, and hypoxia inducible factor 1 alpha. Moreover, caveolin 3 protein levels were higher in exercised than control rats by immunohistochemistry and Western Blot analysis. Interestingly, the predicted gene similar to C11orf17 protein (RGD1306959) was significantly increased by exercise. This gene has a high homology with the human C11orf17 (alias: protein kinase-A interacting protein 1 or breast cancer associated gene 3). This is the first evidence that this gene is involved in the response to the exercise training. CONCLUSION: Our data indicated that few, but significant, genes characterize the gene expression profile of the rat LV, when examined 48 hrs since the last training section and that mild exercise training determines cardioprotection without the induction of hypertrophy.

New Insights into the Hepcidin-Ferroportin Axis and Iron Homeostasis in iPSC-Derived Cardiomyocytes from Friedreich's Ataxia Patient.

Iron homeostasis in the cardiac tissue as well as the involvement of the hepcidin-ferroportin (HAMP-FPN) axis in this process and in cardiac functionality are not fully understood. Imbalance of iron homeostasis occurs in several cardiac diseases, including iron-overload cardiomyopathies such as Friedreich's ataxia (FRDA, OMIM no. 229300), a hereditary neurodegenerative disorder. Exploiting the induced pluripotent stem cells (iPSCs) technology and the iPSC capacity to differentiate into specific cell types, we derived cardiomyocytes of a FRDA patient and of a healthy control subject in order to study the cardiac iron homeostasis and the HAMP-FPN axis. Both CTR and FRDA iPSCs-derived cardiomyocytes express cardiac differentiation markers; in addition, FRDA cardiomyocytes maintain the FRDA-like phenotype. We found that FRDA cardiomyocytes show an increase in the protein expression of HAMP and FPN. Moreover, immunofluorescence analysis revealed for the first time an unexpected nuclear localization of FPN in both CTR and FRDA cardiomyocytes. However, the amount of the nuclear FPN was less in FRDA cardiomyocytes than in controls. These and other data suggest that iron handling and the HAMP-FPN axis regulation in FRDA cardiac cells are hampered and that FPN may have new, still not fully understood, functions. These findings underline the complexity of the cardiac iron homeostasis.

Risk and Protective Environmental Factors Associated with Autism Spectrum Disorder: Evidence-Based Principles and Recommendations.

Autism Spectrum Disorder (ASD) is a complex condition with early childhood onset, characterized by a set of common behavioral features. The etiology of ASD is not yet fully understood; however, it reflects the interaction between genetics and environment. While genetics is now a well-established risk factor, several data support a contribution of the environment as well. This paper summarizes the conclusions of a consensus conference focused on the potential pathogenetic role of environmental factors and on their interactions with genetics. Several environmental factors have been discussed in terms of ASD risk, namely advanced parental age, assisted reproductive technologies, nutritional factors, maternal infections and diseases, environmental chemicals and toxicants, and medications, as well as some other conditions. The analysis focused on their specific impact on three biologically relevant time windows for brain development: the periconception, prenatal, and early postnatal periods. Possible protective factors that might prevent or modify an ASD trajectory have been explored as well. Recommendations for clinicians to reduce ASD risk or its severity have been proposed. Developments in molecular biology and big data approaches, which are able to assess a large number of coexisting factors, are offering new opportunities to disentangle the gene⁻environment interplay that can lead to the development of ASD.

Licofelone, a dual COX/5-LOX inhibitor, induces apoptosis in HCA-7 colon cancer cells through the mitochondrial pathway independently from its ability to affect the arachidonic acid cascade.

Nowadays, no data are available concerning the potential use of dual cyclooxygenase (COX)/5-lipoxygenase (LOX) inhibitors as anticancer agents in colon cancer treatment. Here, we report, for the first time, that the dual COX/5-LOX inhibitor licofelone triggers apoptosis in a dose- and time-dependent manner in HCA-7 colon cancer cells. Induction of apoptosis was related to the recruitment of the intrinsic mitochondrial apoptotic pathway, as shown by loss in mitochondrial membrane potential, cytochrome c release, caspase-9 and 3 activation and poly-(ADP-ribose)polymerase-1 cleavage. Moreover, licofelone induced the cleavage of the full-length p21(Bax) into p18(Bax), a more potent inducer of the apoptotic process than the uncleaved form. Pre-treatment of HCA-7 cells with the pan-caspase inhibitor z-VAD-fmk significantly blocked licofelone-induced apoptosis, confirming that this process occurred primarily in a caspase-dependent pathway. We also present evidences that licofelone was able to affect the arachidonic acid (AA) cascade, as it blocked the activity of 5-LOX and COX enzymes, and it induced, through the phosphorylation of cytoplasmic phospholipase A(2) (cPLA(2)), the release of unesterified AA from HCA-7 membrane phospholipids. However, apoptosis induction was not related to the ability of licofelone to affect the AA cascade, since neither exogenous prostaglandin E(2) and leukotriene B(4) addition, nor pharmacological inhibition of cPLA(2), was able to rescue HCA-7 cells from apoptosis. Even if further studies are needed to clarify the mechanism of licofelone-induced apoptosis, this study suggests that this drug, as well as similar dual COX/5-LOX inhibitors, may represent a novel and promising approach in colon cancer treatment.

MDCT of the breast.

OBJECTIVE: The purpose of this study was to evaluate retrospectively the accuracy of low-dose MDCT in the differentiation of breast lesions suspected on mammography and sonography. MATERIALS AND METHODS: MDCT was performed on 61 patients with mammographic or sonographic findings suggestive of breast cancer who could not undergo MR mammography. For each lesion, morphologic features, attenuation, and time-attenuation curve pattern were evaluated. The 1-minute cut point of attenuation was analyzed on the images. CT findings were compared with histopathologic results, which were the reference standard. RESULTS: Forty-seven of 61 patients underwent surgery, and the pathologic findings revealed 27 malignant and 20 benign lesions. With CT 25 of 27 malignant lesions and all 20 benign lesions were diagnosed correctly. CT had a sensitivity of 92.6%, specificity of 100%, positive predictive value of 100%, negative predictive value of 90.9%, and accuracy of 95.74%. The cutoff attenuation value, which had the best validity for differentiating malignant and benign lesions, was calculated to be 90 H on the 1-minute images. CONCLUSION: Our results confirm and strengthen the importance of all imaging parameters and not one in particular. Dynamic MDCT can be used in the evaluation of selected patients with suspected breast tumors.

Persistence of regenerative myogenesis in spite of down-regulation of activity-dependent genes in long-term denervated rat muscle.

Contrary to general expectation, in humans, we have recently shown that after complete conus cauda lesion, the lower motoneuron denervated myofibers may survive several years. In adult rats, the sciatectomized muscle progresses in 4-6 months from severe atrophy to a dystrophic stage and undergoes a dramatic weight loss; during this process, myofiber death/regeneration processes maintain a decreasing population of very small, but vital myofibers. At the same time, in vitro electrophysiologic recordings show that denervated fibers can maintain membrane excitability longer than they can retain contractile properties. A certain level of myofiber regeneration seems to have a role in the process, with the early re-expression of embryonic subunits of integrins and acetylcholine receptor subunits. In the present work, using the reliable real-time quantitative PCR, we confirm the long-lasting occurrence of myoblast proliferation-dependent events and their focal nature. In fact, we show here that in sciatectomized muscle, the expression of 12 selected genes was differentially regulated after 3 and 9 month denervation. At both time points, indexes of muscle activity/inactivity and tissue remodeling (proteolysis, energy usage and angiogenic factors) were down-regulated, while indexes of regenerative myogenesis (Myogenin, MyoD, MRF4 and MHCemb) were up-regulated. Immunohistochemistry with anti-MHCemb and anti-NCAM monoclonal antibodies show that such regeneration events were focally distributed. We conclude that myofiber regeneration is a non-compensatory mechanism, which prolongs the chance of reinnervation during long-lasting denervation. It may also contribute to muscle recovery in paraplegic patients, even when rehabilitation strategies based on functional electric stimulation start late after spinal cord injury (SCI).

Oxidative Stress in Autistic Children Alters Erythrocyte Shape in the Absence of Quantitative Protein Alterations and of Loss of Membrane Phospholipid Asymmetry.

Red blood cells (RBCs) from people affected by autism spectrum disorders (ASDs) are a target of oxidative stress. By scanning electron microscopy, we analyzed RBC morphology from 22 ASD children and show here that only 47.5 ± 3.33% of RBC displayed the typical biconcave shape, as opposed to 87.5 ± 1.3% (mean ± SD) of RBC from 21 sex- and age-matched healthy typically developing (TD) controls. Codocytes and star-shaped cells accounted for about 30% of all abnormally shaped ASD erythrocytes. RBC shape alterations were independent of the anticoagulant used (Na2-EDTA or heparin) and of different handling procedures preceding glutaraldehyde fixation, thus suggesting that they were not artefactual. Incubation for 24 h in the presence of antioxidants restored normal morphology in most erythrocytes from ASD patients. By Coomassie staining, as well as Western blotting analysis of relevant proteins playing a key role in the membrane-cytoskeleton organization, we were unable to find differences in RBC ghost composition between ASD and normal subjects. Phosphatidylserine (PS) exposure towards the extracellular membrane domain was examined in both basal and erythroptosis-inducing conditions. No differences were found between ASD and TD samples except when the aminophospholipid translocase was blocked by N-ethylmaleimide, upon which an increased amount of PS was found to face the outer membrane in RBC from ASD. These complex data are discussed in the light of the current understanding of the mode by which oxidative stress might affect erythrocyte shape in ASD and in other pathological conditions.

Advanced glycation endproducts, dityrosine and arginine transporter dysfunction in autism - a source of biomarkers for clinical diagnosis.

Background: Clinical chemistry tests for autism spectrum disorder (ASD) are currently unavailable. The aim of this study was to explore the diagnostic utility of proteotoxic biomarkers in plasma and urine, plasma protein glycation, oxidation, and nitration adducts, and related glycated, oxidized, and nitrated amino acids (free adducts), for the clinical diagnosis of ASD. Methods: Thirty-eight children with ASD (29 male, 9 female; age 7.6 ± 2.0 years) and 31 age-matched healthy controls (23 males, 8 females; 8.6 ± 2.0 years) were recruited for this study. Plasma protein glycation, oxidation, and nitration adducts and amino acid metabolome in plasma and urine were determined by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Machine learning methods were then employed to explore and optimize combinations of analyte data for ASD diagnosis. Results: We found that children with ASD had increased advanced glycation endproducts (AGEs), Nε-carboxymethyl-lysine (CML) and Nω-carboxymethylarginine (CMA), and increased oxidation damage marker, dityrosine (DT), in plasma protein, with respect to healthy controls. We also found that children with ASD had increased CMA free adduct in plasma ultrafiltrate and increased urinary excretion of oxidation free adducts, alpha-aminoadipic semialdehyde and glutamic semialdehyde. From study of renal handling of amino acids, we found that children with ASD had decreased renal clearance of arginine and CMA with respect to healthy controls. Algorithms to discriminate between ASD and healthy controls gave strong diagnostic performance with features: plasma protein AGEs-CML, CMA-and 3-deoxyglucosone-derived hydroimidazolone, and oxidative damage marker, DT. The sensitivity, specificity, and receiver operating characteristic area-under-the-curve were 92%, 84%, and 0.94, respectively. Conclusions: Changes in plasma AGEs were likely indicative of dysfunctional metabolism of dicarbonyl metabolite precursors of AGEs, glyoxal and 3-deoxyglucosone. DT is formed enzymatically by dual oxidase (DUOX); selective increase of DT as an oxidative damage marker implicates increased DUOX activity in ASD possibly linked to impaired gut mucosal immunity. Decreased renal clearance of arginine and CMA in ASD is indicative of increased arginine transporter activity which may be a surrogate marker of disturbance of neuronal availability of amino acids. Data driven combination of these biomarkers perturbed by proteotoxic stress, plasma protein AGEs and DT, gave diagnostic algorithms of high sensitivity and specificity for ASD.