SIRT2 and SIRT3 expression correlates with redox imbalance and advanced clinical stage in patients with multiple myeloma.

OBJECTIVES: Sirtuins comprise seven family elements (SIRT1-7) involved in various cell signalling pathways comprising cancer inhibition and tumorigenesis. The present study aims to evaluate SIRT2 and SIRT3 gene expression and potential redox reactions in patients with multiple myeloma (MM) at onset and its correlation with disease status, extent and presence of organ damage secondary to myeloma. DESIGN & METHODS: Total RNA was extracted from 17 MM patients and 10 controls to assess gene expression using real-time PCR. The NAD+/NADH ratio as well as the levels of glutathione peroxidase (GPx) and hydrogen peroxide (HP) in peripheral blood mononuclear cells (PBMCs) were determined using established biochemical assays. RESULTS: SIRT2 and SIRT3 expression is reduced in MM patients compared to healthy controls. Correlational analysis demonstrated that SIRT2 reduction is associated with advanced clinical stage and with more advanced bone lesions than in the remaining patients. SIRT3 expression is correlated with lytic bone lesions. Biochemical analysis indicated an imbalance of oxidative stress biomarkers with low concentrations of the antioxidant enzyme GPx, low amounts of NAD + and higher concentrations of pro-oxidant enzyme HP in PBMCs of MM patients compared to controls. Moreover, MM patients with bone lesions had lower concentrations of NAD + and GPx in PBMCs than patients without signs of bone disease. In addition, MM patients had higher quantities of intracellular HP than controls. CONCLUSIONS: Our results demonstrate that SIRT2 and SIRT3 are downregulated in MM and that lower concentrations correlate with an advanced stage of disease and redox imbalance. We conclude that SIRT2 and SIRT3 together with oxidative stress biomarkers, may be useful for improved risk stratification of MM patients.

Circulating microRNAs Profile in Patients With Transthyretin Variant Amyloidosis.

Transthyretin variant amyloidosis (ATTRv) is a rare autosomal dominant disease characterized by the accumulation of amyloid in many organs, mostly causing a sensory-motor neuropathy, cardiomyopathy, and dysautonomia. The aim of the study was to report microRNAs (miRNAs) expression profile identified in the blood of ATTRv patients. Ten ATTRv patients, 10 asymptomatic carriers of transthyretin variant (TTRv), 10 patients with Charcot-Marie-Tooth (CMT) disease, and 10 healthy controls were studied. Human Schwann cells cultures were used to study the regulatory effects of miR-150-5p on the expression of cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF). ATTRv patients had 33 miRNAs up-regulated and 48 down-regulated versus healthy controls; 9 miRNAs were up-regulated and 30 down-regulated versus CMT patients; 19 miRNAs were up-regulated and 38 down-regulated versus asymptomatic TTRv carriers. Twelve out of the 19 upregulated miRNAs had a fold increase higher than 100. The validation experiment indicated miR-150-5p as a valuable biomarker to differentiate ATTRv patients from asymptomatic TTRv carriers (AUC: 0.9728; p < 0.0001). Schwann cells culture model demonstrated that miR-150-5p is a powerful negative regulator of CREB, BDNF, and NGF genes. Identification of deregulated miRNAs can help in understanding the complex pathomechamism underlying the development of ATTRv and related multisystemic pathology. Further investigations are needed on the role of circulating miR-150-5p to predict the shift of TTRv carriers from an asymptomatic status to symptoms appearance.

Circulating miRNAs expression as potential biomarkers of mild traumatic brain injury.

TBI is the main cause of death and disability in individuals aged 1-45 in Western countries. One of the main challenges of TBI at present is the lack of specific diagnostic biomarkers, especially for mild TBI (mTBI), which remains currently difficult to value in clinical practice. In this context MiRNAs may be important mediators of the profound molecular and cellular changes that occur after TBI in both the short and the long term. Recently, plasma miRNAs profiling in human TBI, have revealed dynamic temporal regulation of miRNA expression within the cortex. Aim of this study was to select a specific miRNAs panel for mTBI, by focusing the research on the prognostic meaning of miRNAs in the hours following the trauma, in order to be able to use this MIRNAs as potential biomarkers useful for monitoring the follow up of mild TBI. Serum levels of 17 miRNAs were measured by RT-quantitative polymerase chain reaction (qPCR) in 20 patients with mTBI at three different time-points (0 h, 24 h, 48 h) and in 10 controls. For 15 miRNAs we found a significant differences in the comparison among the three time points: for each of these miRNAs the values were greater at baseline and progressively reduced at 24 h and 48 h. These data allow us to consider the miRNAs included in panel as sensitive and specific biomarkers for mTBI, useful in monitoring the post-trauma period.

Hippo signaling pathway is altered in Duchenne muscular dystrophy.

Hippo signaling pathway is considered a key regulator of tissue homeostasis, cell proliferation, apoptosis and it is involved in cancer development. In skeletal muscle, YAP, a downstream target of the Hippo pathway, is an important player in myoblast proliferation, atrophy/hypertrophy regulation, and in mechano-trasduction, transferring mechanical signals into transcriptional responses. We studied components of Hippo pathway in muscle specimens from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, limb-girdle muscular dystrophy type 2A and type 2B and healthy subjects. Only DMD muscles had decreased YAP1 protein expression, increased LATS1/2 kinase activity, low Survivin mRNA expression and high miR-21 expression. In light of our novel results, a schematic model is postulated: low levels of YOD1 caused by increased inhibition by miR-21 lead to an increase of LATS1/2 activity which in turn augments phosphorylation of YAP. Reduced amount of active YAP, which is also a target of increased miR-21, causes decreased nuclear expression of YAP-mediated target genes. Since it is known that YAP has beneficial roles in promoting tissue repair and regeneration after injury so that its activation may be therapeutically useful, our results suggest that some components of Hippo pathway could become novel therapeutic targets for DMD treatment.

miRNA expression profiling regulates necroptotic cell death in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive types of cancer and is among the leading causes of cancer-related mortality worldwide. Although the dysregulation of microRNAs (miRNAs or miRs) has often been reported in HCC, the precise molecular mechanisms by which miRNAs modulate the process of tumorigenesis and the behavior of cancer cells are not yet clearly understood. In this study, we identified a novel three­miRNA signature, including miR­371-5p, miR­373 and miR­543, that appears to orchestrate programmed cell necrosis in HCC by directly targeting the caspase­8 gene (Casp­8). Our results demonstrated that miR­371-5p, miR­373 and miR­543 were overexpressed in HCC tissues compared with paired adjacent normal tissues. The upregulation of these miRNAs specifically and markedly downregulated the expression of Casp­8, as well as significantly enhanced the Z-VAD/TNF­α-induced necroptosis of HCC cells. By contrast, the selective knockdown of miRNA expression led to a significant increase in Casp­8 levels and a marked reduction in programmed cell necrosis. Intriguingly, the sustained overexpression of Casp­8 reversed the pro­necroptotic effects exerted by miRNA mimics. Finally, a strong inverse association between the level of miR­223 and the expression levels of nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing-3 inflammasome was observed in our HCC specimens. On the whole, the present study revealed a molecular link between the three­miRNA signature, comprising miR­371-5p, miR­373 and miR­543, and the negative necroptotic regulator Casp­8, and presents evidence for its employment as a novel potential diagnostic, prognostic and therapeutic target in HCC.

miRNA regulation of Sirtuin-1 expression in human astrocytoma.

Sirtuins are a family of 7 histone deacetylases largely involved in the regulation of cell proliferation, survival and death. The role of sirtuins in tumorigenesis and cancer progression has been previously studied in certain cancer types. Few studies have investigated sirtuin expression in gliomas, with controversial results. The aim of the present study was to investigate the expression of sirtuin-1 (Sirt-1) in diffuse astrocytoma [low grade astrocytoma (LGA)], anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) and in primary glioma cell lines: PLGAC (primary LGA cells); PAAC (primary AA cells); and PGBMC (primary GBM cells). Tumor samples were obtained from patients who underwent craniotomy for microsurgical tumor resection at the Neurosurgery Unit of the University of Messina between 2011 and 2014. Sirt-1 expression was qualitatively analyzed in 30 human glial tumor samples and 5 non-neoplastic brain tissue (NBT) specimens using immunohistochemistry and western blotting techniques. Sirt-1 expression was quantitatively analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, Sirt-1 expression in primary cell lines was investigated by immunoblotting and RT-qPCR. Sirt-1 expression was downregulated in gliomas compared to NBTs. Sirt-1 levels also varied among different tumor grades, with more evident downregulation in high-grade (P<0.001) than low-grade tumors (P<0.01). These data were confirmed in cell lines, with the exception of upregulation of protein level in the highest malignancy grade cell lines. The present results suggest a role for miRNA-34a, miRNA-132 and miRNA-217 in the epigenetic control of Sirt-1 during gliomagenesis and progression, and demonstrate the different implications of Sirt-1 in human tissues and cell lines. Furthermore, the present results reveal that Sirt-1 may be an intrinsic regulator of tumor progression and the regulation of Sirt-1 involves complex molecular pathways. However, the biological functions of Sirt-1 in gliomagenesis require additional investigation.

Analysis of lipid profile in lipid storage myopathy.

Lipid dysmetabolism disease is a condition in which lipids are stored abnormally in organs and tissues throughout the body, causing muscle weakness (myopathy). Usually, the diagnosis of this disease and its characterization goes through dosage of Acyl CoA in plasma accompanied with evidence of droplets of intra-fibrils lipids in the patient muscle biopsy. However, to understand the pathophysiological mechanisms of lipid storage diseases, it is useful to identify the nature of lipids deposited in muscle fiber. In this work fatty acids and triglycerides profile of lipid accumulated in the muscle of people suffering from myopathies syndromes was characterized. In particular, the analyses were carried out on the muscle biopsy of people afflicted by lipid storage myopathy, such as multiple acyl-coenzyme A dehydrogenase deficiency, and neutral lipid storage disease with myopathy, and by the intramitochondrial lipid storage dysfunctions, such as deficiencies of carnitine palmitoyltransferase II enzyme. A single step extraction and derivatization procedure was applied to analyze fatty acids from muscle tissues by gas chromatography with a flame ionization detector and with an electronic impact mass spectrometer. Triglycerides, extracted by using n-hexane, were analyzed by high performance liquid chromatography coupled to mass spectrometer equipped with an atmospheric pressure chemical ionization interface. The most representative fatty acids in all samples were: C16:0 in the 13-24% range, C18:1n9 in the 20-52% range, and C18:2n6 in the 10-25% range. These fatty acids were part of the most representative triglycerides in all samples. The data obtained was statistically elaborated performing a principal component analysis. A satisfactory discrimination was obtained among the different diseases. Using component 1 vs component 3 a 43.3% of total variance was explained. Such results suggest the important role that lipid profile characterization can have in supporting a correct diagnosis.

Telomere shortening is associated to TRF1 and PARP1 overexpression in Duchenne muscular dystrophy.

Telomere shortening is thought to contribute to premature senescence of satellite cells in Duchenne muscular dystrophy (DMD) muscle. Telomeric repeat binding factor-1 (TRF1) and poly (ADP-ribose) polymerase-1 (PARP1) are proteins known to modulate telomerase reverse transcriptase (TERT) activity, which controls telomere elongation. Here we show that an age-dependent telomere shortening occurs in DMD muscles and is associated to overexpression of mRNA and protein levels of TRF1 and PARP1. TERT expression and activity are detectable in normal control muscles and they slightly increase in DMD. This is the first demonstration of TRF1 and PARP1 overexpression in DMD muscles. They can be directly involved in replicative senescence of satellite cells and/or in the pathogenetic cascade through a cross-talk with oxidative stress and inflammatory response. Modulation of these events by TRF1 or PARP1 inhibition might represent a novel strategy for treatment of DMD and other muscular dystrophies.

L-arginine: a new opportunity in the management of clinical derangements in dialysis patients.

L-Arginine is an essential amino acid for infants and growing children, as well as for pregnant women. This amino acid is a substrate for at least 5 enzymes identified in mammals, including arginase, arginine-glycine transaminase, kyotorphine synthase, nitric oxide synthase, and arginine decarboxylase. L-arginine is essential for the synthesis of creatine, urea, polyamines, nitric oxide, and agmatine. Arginine may be considered an essential amino acid in sepsis, and its supplementation could be beneficial in this clinical setting by improving microcirculation and protein anabolism. Rats receiving arginine-supplemented parenteral nutrition showed an increased ability to synthesize acute phase proteins when challenged with sepsis. Finally, L-arginine exerts antihypertensive and antiproliferative effects on vascular smooth muscles. It has been shown to reduce systemic blood pressure in some forms of experimental hypertension. Endothelial dysfunction and reduced nitric oxide bioactivity are associated with increased incidence of cardiovascular diseases. A beneficial effect of acute and chronic L-arginine supplementation on endothelial derived nitric oxide production and endothelial function has been shown. In end-stage renal disease patients, the rate of de novo arginine synthesis seemed to be preserved. Our preliminary data on a group of dialysis patients showed that predialysis arginine levels were stable in a normal range during the dialysis session and that hypertensive patients had lower arginine-citrulline ratio than normotensive patients.

Amino acid levels in some brain areas of inducible nitric oxide synthase knock out mouse (iNOS-/-) before and after pentylenetetrazole kindling.

Inducible nitric oxide synthase knock-out (iNOS(-/-)) mice are valid models of investigation for the role of iNOS in patho-physiological conditions. There are no available data concerning neuroactive amino acid levels of iNOS(-/-) mice and their behaviour in response to pentylenetetrazole (PTZ). We found no significant differences in the convulsive dose 50 (CD(50)) between iNOS(-/-) and control (iNOS(+/+)) mice, however, iNOS(-/-) mice reach the kindled status more slowly than control, suggesting that in basal condition the GABA-benzodiazepine inhibitory inputs are unaltered by iNOS mutation. Clear differences between iNOS(+/+) and iNOS(-/-) mice amino acid concentrations were evident both in basal conditions and after kindling. Our results show that aspartate was significantly lower in all brain areas studied except the brain stem whereas glutamate and glutamine were significantly higher in the cortex, hippocampus and brain stem. GABA was slightly and not significantly higher in the cortex, hippocampus and brain stem, whereas taurine was significantly higher in all areas except diencephalon and glycine was significantly lower in the diencephalon and cerebellum. In this context, the inability of iNOS(-/-) mice to increase the NO levels following PTZ administrations indicate that NO might play a pro-epileptogenic role in the genesis and development of some types of epilepsy. Since there is no correlation between neurotransmitter levels and the development of kindling, it is possible to exclude that the difference between the two strains is due to an imbalance between the considered neurotransmitters, and it is then possible that this difference is due to the presence of iNOS, which might be involved in long term plasticity of the brain.

Amino acid levels in some lethargic mouse brain areas before and after pentylenetetrazole kindling.

Genetic animal models have contributed significantly to our understanding of epilepsy causes. Lethargic mice are considered a valid model of absence epilepsy, which have been shown to possess behavioral, electrographic and pharmacological profiles similar to those of humans with absence epilepsies. Single gene mutations that comprise the beta4 subunit of voltage-sensitive Ca2+ channels underlie the spontaneous discharges of the absence, non-convulsive seizures of lethargic mice. There are no available data concerning how the mutant channels actually behave at terminals in response to chemical activation by subconvulsant stimulation with pentylenetetrazole. In this study, we found no significant difference in the convulsive dose 50 between lethargic and control mice. Lethargic mice showed a more rapid development of kindling to pentylenetetrazole than control animals. No significant differences were observed between the groups of mice rechallenged with pentylenetetrazole 30 or 60 days after the end of the chronic treatment. Marked differences in brain amino acid levels were found between the two strains of mice in basal conditions and after kindling. In conclusion, our results indicate that lethargic mice show a range of biochemical and behavioral changes, correlated in particular with a higher susceptibility to develop kindled seizures.

Susceptibility to audiogenic seizure and neurotransmitter amino acid levels in different brain areas of IL-6-deficient mice.

Interleukin-6-deficient (IL-6(-/-)) mice and their normal littermate (WT) were studied to evaluate their susceptibility to seizures induced by electroshock and audiogenic stimuli at different ages. No significant changes in maximal electroshock susceptibility were evidenced between the two strains, while audiogenic seizures (AGS) can be induced only in IL-6(-/-) mice. The effects of age and genetic condition on AGSs were evaluated. The behavioural and electrocortical changes during audiogenic stimulus were observed. In addition, the levels of neurotransmitter amino acids in five brain areas (of both strains) were measured at 60 days of age. Aspartate level significantly increased in the brain stem (BS) and hippocampus (HI), while it decreased in the diencephalon (DE) of IL-6(-/-) mice. Glutamate content significantly decreased in the cerebellum (CB), DE and HI. GABA levels significantly decreased in all the areas studied. Glycine significantly decreased in the BS, CB and DE, while taurine decreased only in the DE. The levels of glutamine significantly decreased in all the areas examined, except in the cortex (CX). The changes of neuroactive amino acid levels, particularly in the BS, might explain the characteristic of high propensity to AGS of IL-6(-/-) mice. The present data support the validity of IL-6(-/-) mice as a novel epileptic model for the study of the pathophysiology and pharmacology of epilepsy.