Altered Intracellular Localization of SOD1 in Leukocytes from Patients with Sporadic Amyotrophic Lateral Sclerosis

Several lines of evidence support the hypothesis of a toxic role played by wild type SOD1 (WT-SOD1) in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). In this study we investigated both distribution and expression profile of WT-SOD1 in leukocytes from 19 SALS patients and 17 healthy individuals. Immunofluorescence experiments by confocal microscopy showed that SOD1 accumulates in the nuclear compartment in a group of SALS subjects. These results were also confirmed by western blot carried out on soluble nuclear and cytoplasmic fractions, with increased nuclear SOD1 level (p<0.05). In addition, we observed the presence of cytoplasmic SOD1 aggregates in agreement with an increased amount of the protein recovered by the insoluble fraction. A further confirmation of the overall increased level of SOD1 has been obtained from single cells analysis using flow cytometry as cells from SALS patients showed an higher SOD1 protein content (p<0.05). These findings add further evidence to the hypothesis of an altered WT-SOD1 expression profile in peripheral blood mononuclear cells (PBMCs) from patients with ALS suggesting that WT-SOD1 species with different degrees of solubility could be involved in the pathogenesis of the disease.     

Human monocyte‐derived macrophages spontaneously differentiated in vitro show distinct phenotypes

Tissue macrophages are resident phagocytes that acquire specific phenotypes according to the microenvironment. Morphological and functional heterogeneity has been evidenced in different homeostatic and pathological conditions. Indeed, the nature of macrophage subsets may have either harmful or beneficial functions in disease progression/resolution. Therefore the possibility to pharmacologically manipulate heterogeneity represents a relevant challenge. Since human tissue macrophages are not easily obtained, various in vitro models are currently used that do not adequately reflect the heterogeneity and plasticity of tissue macrophages. We had previously reported that two dominant and distinct macrophage morphotypes co‐exist in the same culture of human monocytes spontaneously differentiated for 7 days in autologous serum. The present study was aimed to the phenotypic characterization of these morphotypes, that is, round‐ and spindle‐shaped. We observed that, besides substantial differences in cytoskeleton architecture, round monocyte‐derived macrophages (MDMs) showed higher lipid content, increased macropinocytosis/efferocytosis capacity, and overexpression of CD163, interleukin (IL)‐10, and transforming growth factor (TGF) β2. Conversely, spindle MDMs exhibited enhanced respiratory burst and higher expression of the chemokine (C‐C motif) ligands 18 and 24 (CCL18 and CCL24). Overall, round MDMs show functional traits reminiscent of the non‐inflammatory and reparative M2 phenotype, whereas spindle MDMs exhibit a pro‐inflammatory profile and express genes driving lymphocyte activation and eosinophil recruitment. MDMs obtained in the culture condition herein described represent a valuable model to disentangle and manipulate the functional heterogeneity of tissue macrophages that has been disclosed in scenarios spanning from inflammatory and wounding responses to atherosclerotic lesions. J. Cell. Physiol. 228: 1464–1472, 2013. © 2012 Wiley Periodicals, Inc.     

Human cytomegalovirus DNA polymerase catalytic subunit pUL54 possesses independently acting nuclear localization and ppUL44 binding motifs

The catalytic subunit of human cytomegalovirus (HCMV) DNA polymerase pUL54 is a 1242-amino-acid protein, whose function, stimulated by the processivity factor, phosphoprotein UL44 (ppUL44), is essential for viral replication. The C-terminal residues (amino acids 1220-1242) of pUL54 have been reported to be sufficient for ppUL44 binding in vitro. Although believed to be important for functioning in the nuclei of infected cells, no data are available on either the interaction of pUL54 with ppUL44 in living mammalian cells or the mechanism of pUL54 nuclear transport and its relationship with that of ppUL44. The present study examines for the first time the nuclear import pathway of pUL54 and its interaction with ppUL44 using dual color, quantitative confocal laser scanning microscopy on live transfected cells and quantitative gel mobility shift assays. We showed that of two nuclear localization signals (NLSs) located at amino acids 1153-1159 (NLSA) and 1222-1227 (NLSB), NLSA is sufficient to confer nuclear localization on green fluorescent protein (GFP) by mediating interaction with importin alpha/beta. We also showed that pUL54 residues 1213-1242 are sufficient to confer ppUL44 binding abilities on GFP and that pUL54 and ppUL44 can be transported to the nucleus as a complex. Our work thus identified distinct sites within the HCMV DNA polymerase, which represent potential therapeutic targets and establishes the molecular basis of UL54 nuclear import.     

Regulated nucleocytoplasmic trafficking of viral gene products: a therapeutic target?

The study of viral proteins and host cell factors that interact with them has represented an invaluable contribution to understanding of the physiology as well as associated pathology of key eukaryotic cell processes such as cell cycle regulation, signal transduction and transformation. Similarly, knowledge of nucleocytoplasmic transport is based largely on pioneering studies performed on viral proteins that enabled the first sequences responsible for the facilitated transport through the nuclear pore to be identified. The study of viral proteins has also enabled the discovery of several nucleocytoplasmic regulatory mechanisms, the best characterized being through phosphorylation. Recent delineation of the mechanisms whereby phosphorylation regulates nuclear import and export of key viral gene products encoded by important human pathogens such as human cytomegalovirus dengue virus and respiratory syncytial virus has implications for the development of antiviral therapeutics. In particular, the development of specific and effective kinase inhibitors makes the idea of blocking viral infection by inhibiting the phosphorylation-dependent regulation of viral gene product nuclear transport a real possibility. Additionally, examination of a chicken anemia virus (CAV) protein able to target selectively into the nucleus of tumor but not normal cells, as specifically regulated by phosphorylation, opens the exciting possibility of cancer cell-specific nuclear targeting. The study of nucleoplasmic transport may thus enable the development not only of new antiviral approaches, but also contribute to anti-cancer strategies.     

Quantum-like behavior without quantum physics I

Recently there has been much interest in the possible quantum-like behavior of the human brain in such functions as cognition, the mental lexicon, memory, etc., producing a vast literature. These studies are both empirical and theoretical, the tenets of the theory in question being mainly, and apparently inevitably, those of quantum physics itself, for lack of other arenas in which quantum-like properties are presumed to obtain. However, attempts to explain this behavior on the basis of actual quantum physics going on at the atomic or molecular level within some element of brain or neuronal anatomy (other than the ordinary quantum physics that underlies everything), do not seem to survive much scrutiny. Moreover, it has been found empirically that the usual physics-like Hilbert space model seems not to apply in detail to human cognition in the large. In this paper we lay the groundwork for a theory that might explain the provenance of quantum-like behavior in complex systems whose internal structure is essentially hidden or inaccessible. The approach is via the logic obeyed by these systems which is similar to, but not identical with, the logic obeyed by actual quantum systems. The results reveal certain effects in such systems which, though quantum-like, are not identical to the kinds of quantum effects found in physics. These effects increase with the size of the system.     

Quantum-like behavior without quantum physics II. A quantum-like model of neural network dynamics

In earlier work, we laid out the foundation for explaining the quantum-like behavior of neural systems in the basic kinematic case of clusters of neuron-like units. Here we extend this approach to networks and begin developing a dynamical theory for them. Our approach provides a novel mathematical foundation for neural dynamics and computation which abstracts away from lower-level biophysical details in favor of information-processing features of neural activity. The theory makes predictions concerning such pathologies as schizophrenia, dementias, and epilepsy, for which some evidence has accrued. It also suggests a model of memory retrieval mechanisms. As further proof of principle, we analyze certain energy-like eigenstates of the 13 three-neuron motif classes according to our theory and argue that their quantum-like superpositional nature has a bearing on their observed structural integrity.     

Sulphide oxidation to elemental sulphur in a membrane bioreactor: performance and characterization of the selected microbial sulphur-oxidizing community

In leather tanning industrial areas sulphide management represents a major problem. However, biological sulphide oxidation to sulphur represents a convenient solution to this problem. Elemental sulphur is easy to separate and the process is highly efficient in terms of energy consumption and effluent quality. As the oxidation process is performed by specialized bacteria, selection of an appropriate microbial community is fundamental for obtaining a good yield. Sulphur oxidizing bacteria (SOB) represent a wide-ranging and highly diversified group of microorganisms with the capability of oxidizing reduced sulphur compounds. Therefore, it is useful to select new microbes that are able to perform this process efficiently. For this purpose, an experimental membrane bioreactor for sulphide oxidation was set up, and the selected microbial community was characterized by constructing 16S rRNA gene libraries and subsequent screening of clones. Fluorescence in situ hybridization (FISH) was then used to assess the relative abundance of different bacterial groups. Sulphide oxidation to elemental sulphur proceeded in an efficient (up to 79% conversion) and stable way in the bioreactor. Both analysis of clone libraries and FISH experiments revealed that the dominant operational taxonomic unit (OTU) in the bioreactor was constituted by Gammaproteobacteria belonging to the Halothiobacillaceae family. FISH performed with the specifically designed probe tios_434 demonstrated that this OTU constituted 90.6+/-1.3% of the bacterial community. Smaller fractions were represented by bacteria belonging to the classes Betaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Clostridia, Mollicutes, Sphingobacteria, Bacteroidetes and Chlorobia. Phylogenetic analysis revealed that clone sequences from the dominant OTU formed a stable clade (here called the TIOS44 cluster), within the Halothiobacillaceae family, with sequences from many organisms that have not yet been validly described. The data indicated that bacteria belonging to the TIOS44 cluster were responsible for the oxidation process.     

Oxidative stress in the rat heart,studies on low-level chemiluminescence

Detection of ultraweak chemiluminescence (CL) emission from the surface of the organ is a sensitive and non-disruptive tool to evaluate the oxidative stress in rat heart. Indeed, an increased photon emission rate can be observed when cellular antioxidants such as glutathione or vitamin E are depleted, or when organic hydroperoxides are infused. We used CL recording to demonstrate in rat heart that: (i) different diets may lead to different heart sensitivity to an oxidative stress; and (ii) post-ischaemic reoxygenation induces an oxidative stress. CL emission induced by an oxidative stress is accompanied by an increased release of eicosanoids. However, while non-steroid anti-inflammatory drugs (aspirin, indomethacin and ibuprofen) prevented eicosanoid release, these compounds dramatically enhanced hydroperoxide-dependent CL. The nature of this phenomenon is still obscure, but the increase of steady-state concentration of excited species caused by anti-inflammatory drugs seems to be pathophysiologically relevant, since in all our experimental conditions tissue damage was proportional to CL emission rate.