Recent findings and technological advances in phosphoproteomics for cells and tissues

Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins – termed phosphoproteomics – strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.     

Genetic analysis of Soil-Borne Cereal Mosaic Virus response in durum wheat: evidence for the role of the major quantitative trait locus QSbm.ubo-2BS and of minor quantitative trait loci

Genetic analysis of Soil-Borne Cereal Mosaic Virus (SBCMV) resistance in durum wheat was carried out using a population of 180 recombinant inbred lines (RILs) obtained from Simeto (susceptible) × Levante (resistant). The RILs were characterized for SBCMV response in the field under severe and uniform SBCMV infection in two growing seasons and genotyped with simple sequence repeat (SSR) and Diversity Arrays Technology^? markers. Transgressive segregation was observed for disease reaction as estimated by symptom severity scores and virus concentration in leaves. Heritability of the disease response was high, with h ² values consistently above 80%. A major quantitative trait locus (QTL) (QSbm.ubo-2BS) in the distal telomeric region of chromosome 2BS accounted for 60–70% of the phenotypic variation for symptom severity, 40–55% for virus concentration and 15–30% for grain yield. The favorable allele was contributed by Levante. Seven additional QTL influenced SBCMV resistance, with the low-susceptibility allele contributed by Levante at five QTL and by Simeto at the remaining two. The meta-QTL analysis carried out using the data from two mapping populations (Simeto × Levante and Meridiano × Claudio) suggests that in both populations SBCMV resistance is likely controlled by QSbm.ubo-2BS. Our results confine QSbm.ubo-2BS to a c. 2-cM-wide interval flanked by SSR markers that are already being used for marker-assisted selection.     

A new role for Drosophila Aurora-A in maintaining chromosome integrity

Chromosoma - Aurora-A is a conserved mitotic kinase overexpressed in many types of cancer. Growing evidence shows that Aurora-A plays a crucial role in DNA damage response (DDR) although this...     

First flowering of wind‐pollinated species with the greatest phenological advances in Europe

Increasing risk of pollinosis (hay fever) is one of the most anticipated consequences of climate change on human health. Wind‐pollinated plants are representative of allergenic species because they include species with the highest capability of causing allergy‐related diseases in humans. Therefore, changes in wind‐pollinated species may reflect impacts of climate change on allergenic plants. In particular, flowering is one of the developmental stages most affected by climate change. This report specifically addresses changes in flowering dates that have occurred during the three decades 1971–2000 as a function of pollination mode and woodiness. The assessment is made using a phenological data set comprising trends of flowering dates of 29 species in 983 locations in Europe. Linear mixed models assessing the statistical significance of trends while adjusting for spatial correlation are used. The main results indicate for the first time that the onset of flowering of wind‐pollinated plants advanced more than for insect‐pollinated plants, while full flowering phases tended to advance less. These novel findings are contrary to the results of Fitter and Fitter (2002) for Oxfordshire, who reported larger advances of insect‐pollinated plants. Onset of flowering and full flowering of insect‐pollinated species are more likely to advance for seasons early in the year; instead, wind‐pollinated plants showed no dependence of trends on the season (first flowering) or a decreased advance of phases that are early in the year (full flowering). The effect of woodiness could not be unambiguously defined, but seems to be of minor importance. The presented findings suggest a lengthening of the flowering period in general, which might lead to an increasing time of exposure to airborne pollen of allergic subjects, with consequent likely increment in severity and incidence of allergic symptoms.     

The Deoxynucleoside Triphosphate Triphosphohydrolase Activity of SAMHD1 Protein Contributes to the Mitochondrial DNA Depletion Associated with Genetic Deficiency of Deoxyguanosine Kinase

The dNTP triphosphohydrolase SAMHD1 is a nuclear antiviral host restriction factor limiting HIV-1 infection in macrophages and a major regulator of dNTP concentrations in human cells. In normal human fibroblasts its expression increases during quiescence, contributing to the small dNTP pool sizes of these cells. Down-regulation of SAMHD1 by siRNA expands all four dNTP pools, with dGTP undergoing the largest relative increase. The deoxyguanosine released by SAMHD1 from dGTP can be phosphorylated inside mitochondria by deoxyguanosine kinase (dGK) or degraded in the cytosol by purine nucleoside phosphorylase. Genetic mutations of dGK cause mitochondrial (mt) DNA depletion in noncycling cells and hepato-cerebral mtDNA depletion syndrome in humans. We studied if SAMHD1 and dGK interact in the regulation of the dGTP pool during quiescence employing dGK-mutated skin fibroblasts derived from three unrelated patients. In the presence of SAMHD1 quiescent mutant fibroblasts manifested mt dNTP pool imbalance and mtDNA depletion. When SAMHD1 was silenced by siRNA transfection the composition of the mt dNTP pool approached that of the controls, and mtDNA copy number increased, compensating the depletion to various degrees in the different mutant fibroblasts. Chemical inhibition of purine nucleoside phosphorylase did not improve deoxyguanosine recycling by dGK in WT cells. We conclude that the activity of SAMHD1 contributes to the pathological phenotype of dGK deficiency. Our results prove the importance of SAMHD1 in the regulation of all dNTP pools and suggest that dGK inside mitochondria has the function of recycling the deoxyguanosine derived from endogenous dGTP degraded by SAMHD1 in the nucleus.     

Latent profile analysis in frontotemporal lobar degeneration and related disorders: clinical presentation and SPECT functional correlates

Background  

Frontotemporal Lobar Degeneration (FTLD) thus recently renamed, refers to a spectrum of heterogeneous conditions. This same heterogeneity of presentation represents the major methodological limit for the correct evaluation of clinical designation and brain functional correlates. At present, no study has investigated clinical clusters due to specific cognitive and behavioural disturbances beyond current clinical criteria.     

Association of Thyroid Diseases with Primary Extra-Thyroidal Malignancies in Women: Results of a Cross-Sectional Study of 6,386 Patients

We here analyzed the prevalence of extra-thyroidal malignancies (EM) in 6,386 female patients affected by different thyroid disease (TD). At first, an age-matched analysis of EM in all patients was performed. We then evaluated EM prevalence in four TD diagnostic categories: non-nodular TD (n = 2,159); solitary nodule (n = 905); multinodular TD (n = 2,871); differentiated thyroid cancers (n = 451). Finally, patients were grouped based on the absence (n = 3,820) or presence of anti-thyroglobulin (TgAb) and/or anti-thyroperoxidase (TPOAb) (n = 2,369), or anti-Thyroid Stmulating Hormone (TSH) receptor autoantibodies (n = 197). A total of 673 EM were recorded. EM prevalence in TD patients was higher compared to the general population (Odds Ratio, OR 3.21) and the most frequent EM was breast cancer (OR 3.94), followed by colorectal (OR 2.18), melanoma (OR 6.71), hematological (OR 8.57), uterus (OR 2.52), kidney (OR 3.40) and ovary (OR 2.62) neoplasms. Age-matched analysis demonstrated that the risk of EM was maximal at age 0–44 yr (OR 11.28), remaining lower, but significantly higher that in the general population, in the 45–59 and 60–74 year age range. Breast and hematological malignancies showed an increased OR in all TD, while other cancers associated with specific TD. An increased OR for melanoma, breast and hematological malignancies was observed in both TPOAb and/or TgAb autoantibody negative and positive patients, while colorectal, uterus, kidney and ovary cancers showed an increased OR only in thyroid autoantibody negative patients. In conclusions, women affected by both benign and malignant TD, especially at a younger age and in absence of thyroid autoimmunity, have an increased risk of developing primary EM, thus requiring a careful follow-up and surveillance.     

Transforming Growth Factor-β1 Induces Transdifferentiation of Myoblasts into Myofibroblasts via Up-Regulation of Sphingosine Kinase-1/S1P3 Axis

The pleiotropic cytokine transforming growth factor (TGF)-β1 is a key player in the onset of skeletal muscle fibrosis, which hampers tissue repair. However, the molecular mechanisms implicated in TGFβ1-dependent transdifferentiation of myoblasts into myofibroblasts are presently unknown. Here, we show that TGFβ1 up-regulates sphingosine kinase (SK)-1 in C2C12 myoblasts in a Smad-dependent manner, and concomitantly modifies the expression of sphingosine 1-phosphate (S1P) receptors (S1PRs). Notably, pharmacological or short interfering RNA-mediated inhibition of SK1 prevented the induction of fibrotic markers by TGFβ1. Moreover, inhibition of S1P₃, which became the highest expressed S1PR after TGFβ1 challenge, strongly attenuated the profibrotic response to TGFβ1. Furthermore, downstream of S1P₃, Rho/Rho kinase signaling was found critically implicated in the profibrotic action of TGFβ1. Importantly, we demonstrate that SK/S1P axis, known to play a key role in myogenesis via S1P₂, consequently to TGFβ1-dependent S1PR pattern remodeling, becomes responsible for transmitting a profibrotic, antidifferentiating action. This study provides new compelling information on the mechanism by which TGFβ1 gives rise to fibrosis in skeletal muscle, opening new perspectives for its pharmacological treatment. Moreover, it highlights the pleiotropic role of SK/S1P axis in skeletal myoblasts that, depending on the expressed S1PR pattern, seems capable of eliciting multiple, even contrasting biological responses.