An unbiased approach to mapping the signaling network of the pseudorabies virus US3 protein

The US3 serine/threonine protein kinase is conserved among the alphaherpesvirus family and represents an important virulence factor. US3 plays a role in viral nuclear egress, induces dramatic alterations of the cytoskeleton, represses apoptosis, enhances gene expression and modulates the immune response. Although several substrates of US3 have been identified, an unbiased screen to identify US3 phosphorylation targets has not yet been described. Here, we perform a shotgun and phosphoproteomics analysis of cells expressing the US3 protein of pseudorabies virus (PRV) to identify US3 phosphorylation targets in an unbiased way. We identified several cellular proteins that are differentially phosphorylated upon US3 expression and validated the phosphorylation of lamin A/C at serine 404, both in US3-transfected and PRV-infected cells. These results provide new insights into the signaling network of the US3 protein kinase and may serve as a basis for future research into the role of the US3 protein in the viral replication cycle

Development of solvent-casting particulate leaching (SCPL) polymer scaffolds as improved three-dimensional supports to mimic the bone marrow niche

The need for new approaches to investigate ex vivo the causes and effects of tumor and to achieve improved cancer treatments and medical therapies is particularly urgent for malignant pathologies such as lymphomas and leukemias, whose tissue initiator cells interact with the stroma creating a three-dimensional (3D) protective environment that conventional mono- and bi-dimensional (2D) models are not able to simulate realistically. The solvent-casting particulate leaching (SCPL) technique, that is already a standard method to produce polymer-based scaffolds for bone tissue repair, is proposed here to fabricate innovative 3D porous structures to mimic the bone marrow niche in vitro. Two different polymers, namely a rigid polymethyl methacrylate (PMMA) and a flexible polyurethane (PU), were evaluated to the purpose, whereas NaCl, in the form of common salt table, resulted to be an efficient porogen. The adoption of an appropriate polymer-to-salt ratio, experimentally defined as 1:4 for both PMMA and PU, gave place to a rich and interconnected porosity, ranging between 82.1 vol% and 91.3 vol%, and the choice of admixing fine-grained or coarse-grained salt powders allowed to control the final pore size. The mechanical properties under compression load were affected both by the polymer matrix and by the scaffold's architecture, with values of the elastic modulus indicatively varying between 29 kPa and 1283 kPa. Preliminary tests performed with human stromal HS-5 cells co-cultured with leukemic cells allowed us to conclude that stromal cells grown associated to the supports keep their well-known protective and pro-survival effect on cancer cells, indicating that these devices can be very useful to mimic the bone marrow microenvironment and therefore to assess the efficacy of novel therapies in pre-clinical studies

Ankrd2/ARPP is a novel Akt2 specific substrate andregulates myogenic differentiation upon cellular exposure to H(2)O(2).

Activation of Akt-mediated signaling pathways is crucial for survival, differentiation, and regeneration of muscle cells. A proteomic-based search for novel substrates of Akt was therefore undertaken in C(2)C(12) murine muscle cells exploiting protein characterization databases in combination with an anti-phospho-Akt substrate antibody. A Scansite database search predicted Ankrd2 (Ankyrin repeat domain protein 2, also known as ARPP) as a novel substrate of Akt. In vitro and in vivo studies confirmed that Akt phosphorylates Ankrd2 at Ser-99. Moreover, by kinase assay with recombinant Akt1 and Akt2, as well as by single-isoform silencing, we demonstrated that Ankrd2 is a specific substrate of Akt2. Ankrd2 is typically found in skeletal muscle cells, where it mediates the transcriptional response to stress conditions. In an attempt to investigate the physiological implications of Ankrd2 phosphorylation by Akt2, we found that oxidative stress induced by H(2)O(2) triggers this phosphorylation. Moreover, the forced expression of a phosphorylation-defective mutant form of Ankrd2 in C(2)C(12) myoblasts promoted a faster differentiation program, implicating Akt-dependent phosphorylation at Ser-99 in the negative regulation of myogenesis in response to stress conditions

Serum fatty acids and risk of cutaneous melanoma: a population-based case-control study

Background. Some observational studies have suggested that excess dietary intake of polyunsaturated fatty acids such as linoleic acid increases cutaneous melanoma risk. We aimed at examining the association between serum fatty acids and melanoma risk by conducting a population-based case-control study in a northern Italy community. Methods. \ue062e percentage composition of 12 fatty acids was determined in 51 newly diagnosed melanoma patients and 51 age- and sex-matched population controls by extracting total lipids from serum samples using thin layer and gas chromatography. Conditional logistic regression was used to estimate the relative risk of melanoma associated with tertiles of percentage composition of each fatty acid as well as groupings including saturated, monounsaturated, and polyunsaturated fatty acids. Results. We found a slightly increased melanoma risk for stearic and arachidic acids proportion, with and without adjustment for potential confounders. For an n-3 polyunsaturated fatty acid, docosapentaenoic acid, we found a male-specic direct association with melanoma risk. o other associations emerged for the other saturated, monounsaturated, and polyunsaturated fatty acids, individually or grouped by type. Conclusions. These fndings do not suggest a major role of fatty acids, including linoleic acid, on risk of cutaneous melanoma, though their evaluation is limited by the small sample size

Protein kinase B/AKT isoform 2 drives migration of human mesenchymal stem cells.

This study was designed to investigate the migratory behavior of adult human mesenchymal stem cells (MSC) and the underlying mechanism. Cell migration was assessed by transwell, wound healing and time-lapse in vivo motility assays. Pharmacological inhibitors were used to determine the potential mechanism responsible for cell migration and invasion. The tests that were implemented revealed that MSC were fairly migratory. Protein kinase B (AKT) was strongly activated at the basal level. Through our analyses we demonstrated that pharmacological inactivation of AKT2 but not AKT1 significantly decreased cell migration and invasion. Although preliminary, collectively our results indicate that AKT2 activation plays a critical role in enabling MSC migration

Nuclear phospholipase C β1 signaling, epigenetics and treatments in MDS.

Myelodysplastic syndromes (MDS), clonal hematopoietic stem-cell disorders mainly affecting older adult patients, show ineffective hematopoiesis in one or more of the lineages of the bone marrow. Most MDS are characterized by anemia, and a number of cases progresses to acute myeloid leukemia (AML). Indeed, the molecular mechanisms underlying the MDS evolution to AML are still unclear, even though the nuclear signaling elicited by PI-PLCβ1 has been demonstrated to play an important role in the control of the balance between cell cycle progression and apoptosis in MDS cells. Here we review both the role of epigenetic therapy on PI-PLCβ1 promoter and the changes in PI-PLCβ1 expression in MDS patients treated for anemia.Myelodysplastic syndromes (MDS), clonal hematopoietic stem-cell disorders mainly affecting older adult patients, show ineffective hematopoiesis in one or more of the lineages of the bone marrow. Most MDS are characterized by anemia, and a number of cases progresses to acute myeloid leukemia (AML). Indeed, the molecular mechanisms underlying the MDS evolution to AML are still unclear, even though the nuclear signaling elicited by PI-PLCβ1 has been demonstrated to play an important role in the control of the balance between cell cycle progression and apoptosis in MDS cells. Here we review both the role of epigenetic therapy on PI-PLCβ1 promoter and the changes in PI-PLCβ1 expression in MDS patients treated for anemia. © 2012 Elsevier Ltd

Nuclear Nox4 Interaction with Prelamin A is Associated with Nuclear Redox Control of Stem Cell Aging

Mesenchymal stem cells have emerged as an important tool that can be used for tissue regeneration thanks to their easy preparation, differentiation potential and immunomodulatory activity. However, an extensive culture of stem cells in vitro prior to clinical use can lead to oxidative stress that can modulate different stem cells properties, such as self-renewal, proliferation, differentiation and senescence. The aim of this study was to investigate the aging process occurring during in vitro expansion of stem cells, obtained from amniotic fluids (AFSC) at similar gestational age. The analysis of 21 AFSC samples allowed to classify them in groups with different levels of stemness properties. In summary, the expression of pluripotency genes and the proliferation rate were inversely correlated with the content of reactive oxygen species (ROS), DNA damage signs and the onset premature aging markers, including accumulation of prelamin A, the lamin A immature form. Interestingly, a specific source of ROS, the NADPH oxidase isoform 4 (Nox4), can localize into PML nuclear bodies (PML-NB), where it associates to prelamin A. Besides, Nox4 post translational modification, involved in PML-NB localization, is linked to its degradation pathway, as it is also for prelamin A, thus possibly modulating the premature aging phenotype occurrence

Isomorphism between Systems of Equivariant Singularities

AbstractIn this article isomorphisms between systems of singularities equivariant under different Lie group actions are investigated and a sufficient condition for two systems to be isomorphic is given. With this sufficiency theorem we show that the system ofO(n)-equivariant singularities in its irreducible representation on Rnis isomorphic to that of one-dimensional Z2-equivariant singularities and the system of[formula]-dimensionalO(n)-equivariant singularities is isomorphic to that ofn-dimensionalSn-equivariant singularities

Aberrant Compartment Formation by HSPB2 Mislocalizes Lamin A and Compromises Nuclear Integrity and Function

Small heat shock proteins (HSPBs) contain intrinsically disordered regions (IDRs), but the functions of these IDRs are still unknown. Here, we report that, in mammalian cells, HSPB2 phase separates to form nuclear compartments with liquid-like properties. We show that phase separation requires the disordered C-terminal domain of HSPB2. We further demonstrate that, in differentiating myoblasts, nuclear HSPB2 compartments sequester lamin A. Increasing the nuclear concentration of HSPB2 causes the formation of aberrant nuclear compartments that mislocalize lamin A and chromatin, with detrimental consequences for nuclear function and integrity. Importantly, phase separation of HSPB2 is regulated by HSPB3, but this ability is lost in two identified HSPB3 mutants that are associated with myopathy. Our results suggest that HSPB2 phase separation is involved in reorganizing the nucleoplasm during myoblast differentiation. Furthermore, these findings support the idea that aberrant HSPB2 phase separation, due to HSPB3 loss-of-function mutations, contributes to myopathy

Clusterin enhances migration and invasion of prostate cancer cells through an isoform-specific Akt2/miR-190/PHLPP1 circuit

During prostate cancer progression cancer cells undergo a variety of molecular alterations that lead to the acquisition of uncontrolled growth properties. One such set of molecular alterations is mediated by the PI3K/Akt signaling pathway. Here, we describe a regulatory system that modulates the phosphoinosited 3-kinase/Akt (PI3K/Akt) pathway downstream of secreted Clusterin (sCLU) in normal and cancer prostate cells. The overexpression of sCLU is very frequent in prostate cancer, and can lead to Akt-activation. This prompted us to investigate how sCLU overexpression influences PI3K/Akt signaling network in a study model represented by human epithelial prostate PNT1A cells stably transfected with sCLU or with empty vector alone. We found that CLU cells show a marked differential phosphorylation of several members of the PI3K/Akt cascade, and in particular of Akt2. Moreover, we found that the phosphatase PHLPP1, known to dephosphorylate Akt2 at S473, is severely downregulated in CLU compared to MOCK cells. We thus investigated whether sCLU alters PHLPP1 protein stability or expression. Our results indicate that sCLU indeed stimulates PHLPP1 degradation by β-TrCP. Interestingly, we further demonstrated that sCLU alters also PHLPP1 through the negative regulator miR-190. Next, because sCLU has been reported to inhibit or to stimulate the aggressive behavior of cancer cells depending on the cell model, we investigated the effects of CLU overexpression or addition of recombinant Clusterin to the medium on cell migration and invasion in PNT1A cell line, which is not expected to display an invasive phenotype, and in the cancer prostate epithelial cell lines LNCaP and PC3. The result was extremely clear: not only CLU overexpression gives PNT1A cells the same behavior of wild-type PC3 cells, but also increases the migration and invasion index of all the above cell models by two to four times, compared to controls. As a confirmation, in the same model silencing of Clusterin abrogates migration of CLU cells. Next, the effect of Akt single-isoform silencing on cell migration was explored. While silencing of Akt1 affected migration only slightly, silencing of Akt2 prevented migration of both MOCK and CLU cells completely. The same result was obtained by pharmacological inhibition of Akt2. All together our results, clearly demonstrate for the first time that Clusterin can switch the low migration phenotype of normal prostate cells towards a high migration phenotype through the modulation of the expression of the PHLPP1 and, in turn, the activity of Akt2