The NGS technology for the identification of genes associated with the ALS. A systematic review.

BACKGROUND: More than 30 causative genes have been identified in familial and sporadic amyotrophic lateral sclerosis (ALS). The next-generation sequencing (NGS) is a powerful and groundbreaking tool to identify disease-associated variants. Despite documented advantages of NGS, its diagnostic reliability needs to be addressed in order to use this technology for specific routine diagnosis. MATERIAL AND METHODS: Literature database was explored to identify studies comparing NGS and Sanger sequencing for the detection of variants causing ALS. We collected data about patients' characteristics, disease type and duration, NGS and Sanger properties. RESULTS: More than 200 bibliographic references were identified, of which only 14 studies matching our inclusion criteria. Only 2 out of 14 studies compared results of NGS analysis with the Sanger sequencing. Twelve studies screened causative genes associated to ALS using NGS technologies and confirmed the identified variants with Sanger sequencing. Overall, data about more 2,000 patients were analysed. The number of genes that were investigated in each study ranged from 1 to 32, the most frequent being FUS, OPTN, SETX and VCP. NGS identified already known mutations in 21 genes, and new or rare variants in 27 genes. CONCLUSIONS: NGS seems to be a promising tool for the diagnosis of ALS in routine clinical practice. Its advantages are represented by an increased speed and a lowest sequencing cost, but patients' counselling could be problematic due to the discovery of frequent variants of unknown significance.

Energy metabolism and cell motility defect in NK-cells from patients with hepatocellular carcinoma.

Functional rescue of NK-cells in solid tumors represents a central aim for new immunotherapeutic strategies. We have conducted a genomic, phenotypic and functional analysis of circulating NK-cells from patients with HCV-related liver cirrhosis and hepatocellular carcinoma. NK-cells were sorted from patients with HCC or liver cirrhosis and from healthy donors. Comparative mRNA gene expression profiling by whole-human-genome microarrays of sorted NK-cells was followed by phenotypic and functional characterization. To further identify possible mediators of NK-cell dysfunction, an in vitro model using media conditioned with patients' and controls' plasma was set up. Metabolic and cell motility defects were identified at the genomic level. Dysregulated gene expression profile has been translated into reduced cytokine production and degranulation despite a prevalent phenotype of terminally differentiated NK-cells. NKG2D-downregulation, high SMAD2 phosphorylation and other phenotypic and molecular alterations suggested TGF-ß as possible mediator of this dysfunction. Blocking TGF-ß could partially restore functional defects of NK-cells from healthy donors, exposed to TGF-ß rich HCC patients' plasma, suggesting that TGF-ß among other molecules may represent a suitable target for immunotherapeutic intervention aimed at NK-cell functional restoration. By an unbiased approach, we have identified energy metabolism and cell motility defects of circulating NK-cells as main mechanisms responsible for functional NK-cell impairment in patients with hepatocellular carcinoma. This opens the way to test different approaches to restore NK-cell response in these patients.

Counter-regulation of regulatory T cells by autoreactive CD8+ T cells in rheumatoid arthritis.

The mechanisms whereby autoreactive T cells escape peripheral tolerance establishing thus autoimmune diseases in humans remain an unresolved question. Here, we demonstrate that autoreactive polyfunctional CD8+ T cells recognizing self-antigens (i.e., vimentin, actin cytoplasmic 1, or non-muscle myosin heavy chain 9 epitopes) with high avidity, counter-regulate Tregs by killing them, in a consistent percentage of rheumatoid arthritis (RA) patients. Indeed, these CD8+ T cells express a phenotype and gene profile of effector (eff) cells and, upon antigen-specific activation, kill Tregs indirectly in an NKG2D-dependent bystander fashion in vitro. This data provides a mechanistic basis for the finding showing that AE-specific (CD107a+) CD8+ T killer cells correlate, directly with the disease activity score, and inversely with the percentage of activated Tregs, in both steady state and follow-up studies in vivo. In addition, multiplex immunofluorescence imaging analyses of inflamed synovial tissues in vivo show that a remarkable number of CD8+ T cells express granzyme-B and selectively contact FOXP3+ Tregs, some of which are in an apoptotic state, validating hence the possibility that CD8+ Teff cells can counteract neighboring Tregs within inflamed tissues, by killing them. Alternatively, the disease activity score of a different subset of patients is correlated with the expansion of a peculiar subpopulation of autoreactive low avidity, partially-activated (pa)CD8+ T cells that, despite they conserve the conventional naïve (N) phenotype, produce high levels of tumor necrosis factor (TNF)-α and exhibit a gene expression signature of a progressive activation state. Tregs directly correlate with the expansion of this autoreactive (low avidity) paCD8+ TN cell subset in vivo, and efficiently control their differentiation rather their proliferation in vitro. Interestingly, autoreactive high avidity CD8+ Teff cells or low avidity paCD8+ TN cells are significantly expanded in RA patients who would become non-responders or patients who would become responders to TNF-α inhibitor therapy, respectively. These data provide evidence of a previously undescribed role of such mechanisms in the progression and therapy of RA.

Immune Gene Expression Profile in Hepatocellular Carcinoma and Surrounding Tissue Predicts Time to Tumor Recurrence.

BACKGROUND: The antitumor immune response may play a major role in the clinical outcome of hepatocellular carcinoma (HCC). We characterized the liver immune microenvironment by direct hybridization of RNA extracted from HCC and nontumorous tissues. METHODS: RNA was extracted from frozen liver tissue samples of HCC (T; n = 30) and nontumorous tissues (NT; n = 33) obtained from 38 patients. Matched samples were available for 25 patients. The immune gene expression profile was analyzed with the nCounter GX Human Immunology v2 system (NanoString Technologies), which detects the expression levels of 579 immune response-related genes simultaneously. RESULTS: Since the immune gene expression profile of T and NT tissues was significantly different, the prognostic relevance of the liver immune microenvironment was evaluated in the T and NT samples separately. Unsupervised clustering detected two main clusters of immune gene expression both in T and in NT liver samples. In both cases, the expression clusters identified groups of patients with a significantly different median time to HCC recurrence (TTR) but similar overall survival. Based on T tissue, two groups with median TTR of 19 and 127 months, respectively, were detected (p < 0.005). Expression of genes related to T-cell activation was associated with longer TTR. The analysis of NT tissue discriminated subsets of patients with median TTR of 22 and 68 months (p < 0.05). In contrast to T tissue, a predominant inflammatory immune environment was associated with shorter TTR. CONCLUSIONS: Immune gene expression profiles predictive of TTR could be identified both in HCC and in adjacent cirrhotic tissues. Longer TTR was associated with overexpression in T tissue and downregulation in NT tissue of the immune response and of inflammation-related genes.

Acute isoproterenol induces anxiety-like behavior in rats and increases plasma content of extracellular vesicles.

Several clinical observations have demonstrated a link between heart rate and anxiety or panic disorders. In these patients, ß-adrenergic receptor function was altered. This prompted us to investigate whether the ß-adrenergic receptor agonist isoproterenol, at a dose that stimulates peripheral ß-adrenergic system but has no effects at the central nervous system, can induce anxiety-like behavior in rats. Moreover, some possible messengers involved in the peripheral to brain communication were investigated. Our results showed that isoproterenol (5 mg kg(-1) i.p.) increased heart rate, evoked anxiety-like behavior, did not result in motor impairments and increased extracellular vesicle content in the blood. Plasma corticosterone level was unmodified as well as vesicular Hsp70 content. Vesicular miR-208 was also unmodified indicating a source of increased extracellular vesicles different from cardiomyocytes. We can hypothesize that peripheral extracellular vesicles might contribute to the ß-adrenergic receptor-evoked anxiety-like behavior, acting as peripheral signals in modulating the mental state.

In vitro effects of cocaine on tunneling nanotube formation and extracellular vesicle release in glioblastoma cell cultures.

The effects of cocaine (150 nM, 300 nM, and 150 µM) on human glioblastoma cell cultures were studied on tunneling nanotube formation (1-h cocaine treatment) and extracellular vesicle release (1-, 3-, and 8-h cocaine treatment). Cocaine significantly increased the number of tunneling nanotubes only at the lowest concentration used. The release of extracellular vesicles (mainly exosomes) into the medium was stimulated by cocaine at each concentration used with a maximum effect at the highest concentration tested (150 µM). Moreover, cocaine (150 nM) significantly increased the number of vesicles with 61-80 nm diameter while at concentrations of 300 nM and 150 µM, and the smaller vesicles (30-40 nm diameter) were significantly increased with a reduction of the larger vesicles (41-60 nm diameter). A time dependence in the release of extracellular vesicles was observed. In view of the proposed role of these novel intercellular communication modes in the glial-neuronal plasticity, it seems possible that they can participate in the processes leading to cocaine addiction. The molecular target/s involved in these cocaine effects could be specific molecular components of plasma membrane lipid rafts and/or cocaine-induced modifications in cytoplasmic lipid composition.

Neural damage biomarkers during open carotid surgery versus endovascular approach.

BACKGROUND: Carotid endarterectomy (CEA) is the gold standard for treating severe carotid artery stenosis, whereas carotid artery stenting (CAS) represents an endovascular alternative. The objective of this study was to assess the potential neural damage following open or endovascular carotid surgery measured by peripheral blood concentration of 3 biomarkers: S100ß, matrix metalloproteinase-9 (MMP-9), and d-dimer. METHODS: Data for this prospective investigation were obtained from the Carotid Markers study (January 2010-2011), which sought to measure the levels of specific biomarkers of neuronal damage and thrombosis on candidates to CEA or CAS presenting at the Department of Vascular Surgery of the Nuovo Ospedale S. Agostino Estense of Modena (Italy) at baseline and at 24 hr after surgery. Relevant medical comorbidities were noted. RESULTS: A total of 113 consecutive patients were enrolled in the study, 41 in the endarterectomy group and 72 in the endovascular group. The baseline levels of the studied biomarkers did not show any statistically significant difference between the groups with the exception of MMP-9, which showed higher concentrations in the endovascular group (median 731 vs. 401, P = 0.0007), while 24 hr after surgery the endarterectomy group featured significantly higher peripheral blood concentrations of MMP-9, S100ß, and d-dimer. Conversely, no significant difference was detected in the endovascular group except the d-dimer level. CONCLUSIONS: Neural damage biomarkers demonstrated a substantial difference between open and endovascular carotid surgery, which, if performed in selected patients, may become a less invasive alternative to CEA.

Information handling by the brain: proposal of a new "paradigm" involving the roamer type of volume transmission and the tunneling nanotube type of wiring transmission.

The current view on the organization of the central nervous system (CNS) is basically anchored to the paradigm describing the brain as formed by networks of neurons interconnected by synapses. Synaptic contacts are a fundamental characteristic for describing CNS operations, but increasing evidence accumulated in the last 30 years pointed to a refinement of this view. A possible overcoming of the classical "neuroscience paradigm" will be here outlined, based on the following hypotheses: (1) the basic morpho-functional unit in the brain is a compartment of tissue (functional module) where different resident cells (not only neurons) work as an integrated unit; (2) in these complex networks, a spectrum of intercellular communication processes is exploited, that can be classified according to a dichotomous criterion: wiring transmission (occurring through physically delimited channels) and volume transmission (exploiting diffusion in the extracellular space); (3) the connections between cells can themselves be described as a network, leading to an information processing occurring at different levels from cell network down to molecular level; (4) recent evidence of the existence of specialized structures (microvesicles and tunneling nanotubes) for intercellular exchange of materials, could allow a further type of polymorphism of the CNS networks based on at least transient changes in cell phenotype. When compared to the classical paradigm, the proposed scheme of cellular organization could allow a strong increase of the degrees of freedom available to the whole system and then of its plasticity. Furthermore, long range coordination and correlation can be more easily accommodated within this framework.

A new interpretative paradigm for Conformational Protein Diseases.

Conformational Protein Diseases (CPDs) comprise over forty clinically and pathologically diverse disorders in which specific altered proteins accumulate in cells or tissues of the body. The most studied are Alzheimerß's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, prion diseases, inclusion body myopathy, and the systemic amyloidoses. They are characterised by three dimensional conformational alterations, which are often rich in ß- structure. Proteins in this non-native conformation are highly stable, resistant to degradation, and have an enhanced tendency to aggregate with like protein molecules. The misfolded proteins can impart their anomalous properties to soluble, monomeric proteins with the same amino acid sequence by a process that has been likened to seeded crystallization. However, these potentially pathogenic proteins also have important physiological actions, which have not completely characterized. This opens up the question of what process transforms physiological actions into pathological actions and most intriguing, is why potentially dangerous proteins have been maintained during evolution and are present from yeasts to humans. In the present paper, we introduce the concept of mis-exaptation and of mis-tinkering since they may help in clarifying some of the double edged sword aspects of these proteins. Against this background an original interpretative paradigm for CPDs will be given in the frame of the previously proposed Red Queen Theory of Aging.

Differential sensitivity of A2A and especially D2 receptor trafficking to cocaine compared with lipid rafts in cotransfected CHO cell lines. Novel actions of cocaine independent of the DA transporter.

The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A(2A) and D(2) immunoreactivities in previously characterized A(2A)-D(2) CHO cell lines. Receptor double immunofluorescence staining was performed with D(2) and A(2A) antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D(2) immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A(2A) immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 microM at which concentration the rise of D(2) immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D(2) receptors may primarily increase the insertion of D(2) monomers, homomers and also of a subpopulation of A(2A)-D(2) heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D(2) receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D(2) and A(2A)-D(2) trafficking induced by allosteric actions of cocaine at D(2) receptors may contribute to the alterations of D(2) signaling found in cocaine abusers.

Receptor-receptor interactions: A novel concept in brain integration.

A brief historical presentation of the hypothesis on receptor-receptor interactions as an important integrative mechanism taking place at plasma membrane level is given. Some concepts derived from this integrative mechanism especially the possible assemblage of receptors in receptor mosaics (high-order receptor oligomers) and their relevance for the molecular networks associated with the plasma membrane are discussed. In particular, the Rodbell's disaggregation theory for G-proteins is revisited in the frame of receptor mosaic model. The paper also presents some new indirect evidence on A2A;D2 receptor interactions obtained by means of Atomic Force Microscopy on immunogold preparations of A2A and D2 receptors in CHO cells. These findings support previous data obtained by means of computer-assisted confocal laser microscopy. The allosteric control of G-protein coupled receptors is examined in the light of the new views on allosterism and recent data on a homocysteine analogue capable of modulating D2 receptors are shown. Finally, the hypothesis is introduced on the existence of check-points along the amino acid pathways connecting allosteric and orthosteric binding sites of a receptor and their potential importance for drug development.

Influence of f-MLP, ACTH(1-24) and CRH on in vitro chemotaxis of monocytes from centenarians.

OBJECTIVE: The lifelong exposure to a variety of stressors activates a plethora of defense mechanisms, including the hypothalamic-pituitary-adrenal axis which releases neuropeptides affecting the immune responses. Here, we report data on the capability of monocytes from young subjects and centenarians to migrate towards chemotactic stimuli (formyl-methionyl-leucyl-phenylalanine, f-MLP; adrenocorticotropic hormone, ACTH, and corticotrophin-releasing hormone, CRH). Plasma levels of ACTH, CRH and cortisol were measured as an index of ongoing stress response. METHODS: Monocyte chemotaxis towards f-MLP (10(-8)M), ACTH(1-24) (10(-14) and 10(-8)M) and CRH (10(-14) and 10(-8)M) was evaluated in vitro in young subjects (n = 8, age range 25-35 years) and centenarians (n = 9, age >100 years) and expressed as chemotactic index. In 9 young subjects and 6 centenarians, plasma levels of cortisol, ACTH and CRH were measured. RESULTS: Monocyte chemotaxis towards f-MLP, ACTH(1-24) and CRH (10(-8)M) was well preserved in centenarians, except when the lowest concentration of CRH was used. CRH, ACTH and cortisol plasma levels were significantly higher in centenarians than in young subjects. CONCLUSIONS: The capability of monocytes from centenarians to respond to chemotactic neuropeptides is well preserved. The decreased responsiveness to the lowest concentration of CRH might be due to downregulation of CRH receptors or to defects in the intracellular signal transduction pathway. The high plasma levels of cortisol, CRH and ACTH in centenarians indicate an activation of the entire stress axis, likely counteracting the systemic inflammatory process occurring with age. This activation fits with the hypothesis that lifelong low-intensity stressors activate ancient, hormetic defense mechanisms, favoring healthy aging and longevity.

Understanding neuronal molecular networks builds on neuronal cellular network architecture.

The central nervous system (CNS) is a nested network at all levels of its organization. In particular, neuronal cellular networks (the neuronal circuits), interconnected to form neuronal systems, are formed by neurons, which operate thanks to their molecular networks. Proteins are the main components of the molecular networks and via protein-protein interactions can be assembled in multimeric complexes, which can work as micro-devices. On this basis, we have introduced the term "fractal logic" to describe networks of networks where at the various levels of the nested organization the same rules (logic) to perform operations are used. If this assumption is true, the description of the information handling at one of the nested levels sheds light on the way in which similar operations are carried out at other levels. This conceptual frame has been used to deduce from some features of neuronal networks the features of the molecular networks as far as modes for inter-node communication and their architecture. It should be noted that these features are such to allow a highly regulated cross-talk between signalling pathways, hence preserving selectivity and privacy. To investigate these aspects, the protein-protein interactions in beta2 Adrenergic Receptor (beta2AR) and Epidermal growth factor receptor (EGFR) signalling pathways have been analysed. The presence of disordered sequences in interacting domains can favour via the "fly-casting mechanism" protein-protein interactions, in addition it favours an induced-fitting rather than a lock-key type of interactions. Thus, by means of a computer assisted analysis the presence of disorder sequences in the main streams of the molecular networks that have beta2AR and EGFR as input proteins leading to MAP kinase activation has been evaluated.

Hyper-homocysteinemia alters amyloid peptide-clusterin interactions and neuroglial network morphology and function in the caudate after intrastriatal injection of amyloid peptides.

Amyloid peptides (Abeta) are fragments of the Amyloid Precursor Protein (APP), an integral membrane protein. Abeta peptides are continuously generated by neurons and non-neuronal cells via sequential cleavage of APP by secretases. In particular, Abeta1-42 is the main component of the senile plaques associated with Alzheimer's disease (AD). Glial cells participate in the uptake of soluble extra-cellular Abeta and in the clearance of this material at localized sites where the Abeta are concentrated. It has been shown that clusterin (Apo J) and apolipoprotein E (ApoE) exert important additive effects in reducing Abeta deposition. In agreement with the fact that homocysteine (Hcy) potentiates Abeta peptide neurotoxicity, and Hcy brain levels increase with age, it has been demonstrated that high plasma levels of Hcy are a risk factor for AD. In the present paper, we used animals subjected to chronic intake of methionine (1 g/kg/day) in the drinking water, since this treatment can increase plasma Hcy levels by 30%. By means of this animal model, interactions between the Abeta beta-sheet rich fibrils and clusterin, have been evaluated in striata of animals after Abeta injection. Furthermore, it has been demonstrated that Abeta peptides are not only signals capable of activating astrocytes but also capable of reducing tyrosine-hydroxylase immunoreactivity in the basal ganglia probably leading to a reduction of volume transmission. These alterations in the neuroglial network morphology and function can, at least in part, explain the enhanced pain threshold observed in the Abeta intra-striatally injected animals.