Hunting the Carotid Culprit An Intriguing Game:An Intriguing Game

Editoria

Systematic Analysis of the Epidermal Growth Factor Receptor by Mass Spectrometry Reveals Stimulation-dependent Multisite Phosphorylation

Multisite phosphorylation of proteins is a general mechanism for modulation of protein function and molecular interactions. Definition of phosphorylation sites and elucidation of the functional interplay between multiple phosphorylated residues in proteins are, however, a major analytical challenge in current molecular cell biology and proteomic research. In the present study, we used mass spectrometry to determine the major phosphorylated residues of the human epidermal growth factor (EGF) receptor at various well defined cellular conditions. Activation of EGF receptor was achieved by several types of stimulation, i.e. by sodium pervanadate, EGF, and integrin-dependent adhesion. The contribution of cell-matrix adhesion was also determined by activating the EGF receptor by EGF in cells kept in suspension. We developed an analytical strategy that combined miniaturized sample preparation techniques and MALDI tandem mass spectrometry and determined a total of nine phosphorylation sites in the EGF receptor. We discovered one novel phosphorylation site (Ser967) and revealed constitutive phosphorylation of Thr669, Ser967, Ser1002, and Tyr1045 and stimulation-dependent differential phosphorylation of Tyr1068, Tyr1086, Ser1142, Tyr1148, and Tyr1173. The EGF receptor was purified from HeLa cells or ECV304 cells by immunoprecipitation and SDS-PAGE and then digested with trypsin. Phosphopeptides in the range of 0.8-3.7 kDa were recovered by combinations of IMAC, perfusion chromatography, and graphite powder chromatography and subsequently detected and sequenced by MALDI quadrupole time-of-flight tandem mass spectrometry. Two phosphorylation sites were detected in the peptide 1137GSHQISLDNPDYQQDFFPK1155; however, only Tyr1148 was phosphorylated upon EGF treatment; in contrast Ser1142 was only phosphorylated by integrin-dependent adhesion in the absence of EGF treatment, suggesting differential phosphorylation of this region by distinct stimuli. This MALDI MS/MS-based analytical approach demonstrates the feasibility of systematic analysis of signaling molecules by mass spectrometry and provides new insights into the dynamics of receptor signaling processes

Prevalence of hypovitaminosis D and predictors of vitamin D status in Italian healthy adolescents

Background: Vitamin D plays an important role in health promotion during adolescence. Vitamin D deficiency and insufficiency are common in adolescents worldwide. Few data on vitamin D status and risk factors for hypovitaminosis D in Italian adolescents are currently available. Methods. 25-hydroxyvitamin D (25-OH-D) and parathyroid hormone (PTH) levels were evaluated in 427 Italian healthy adolescents (10.0-21.0 years). We used the following cut-off of 25-OH-D to define vitamin D status: deficiency < 50 nmol/L; insufficiency 50-75 nmol/L; sufficiency ≥ 75 nmol/L. Hypovitaminosis D was defined as 25-OH-D levels < 75.0 nmol/L and severe vitamin D deficiency as 25-OH-D levels < 25.0 nmol/L. We evaluated gender, residence, season of blood withdrawal, ethnicity, weight status, sun exposure, use of sunscreens, outdoor physical activity, and history of fractures as predictors of vitamin D status. Results: Enrolled adolescents had a median serum 25-OH-D level of 50.0 nmol/L, range 8.1-174.7, with 82.2% having hypovitaminosis D. Vitamin D deficiency and insufficiency were detected in 49.9% and 32.3% of adolescents, respectively. Among those with deficiency, 38 subjects were severely deficient (38/427, 8.9% of the entire sample). Non-white adolescents had a higher prevalence of severe vitamin D deficiency than white subjects (6/17-35.3% vs 32/410-7.8% respectively, p = 0.002). Logistic regression showed increased risk of hypovitaminosis D as follows: blood withdrawal taken in winter-spring (Odds ratio (OR) 5.64) compared to summer-fall period; overweight-obese adolescents (OR 3.89) compared to subjects with normal body mass index (BMI); low sun exposure (OR 5.94) compared to moderate-good exposure and regular use of sunscreens (OR 5.89) compared to non regular use. Adolescents who performed < 3 hours/week of outdoor exercise had higher prevalence of hypovitaminosis D. Gender, residence, and history of fractures were not associated with vitamin D status. Serum 25-OH-D levels were inversely related to PTH (r = -0.387, p < 0.0001) and BMI-SDS (r = -0.141, p = 0.007). 44/427 (10.3%) adolescents showed secondary hyperparathyroidism. Conclusions: Italian adolescents have high prevalence of vitamin D deficiency and insufficiency. Pediatricians should tackle predictors of vitamin D status, favoring a healthier lifestyle and promoting supplementation in the groups at higher risk of hypovitaminosis D. © 2014 Vierucci et al.; licensee BioMed Central Ltd

Multimodality Imaging in the Diagnostic Work-Up of Endocarditis and Cardiac Implantable Electronic Device (CIED) Infection

Infective endocarditis (IE) is a serious cardiac condition, which includes a wide range of clinical presentations, with varying degrees of severity. The diagnosis is multifactorial and a proper characterization of disease requires the identification of the primary site of infection (usually the cardiac valve) and the search of secondary systemic complications. Early depiction of local complications or distant embolization has a great impact on patient management and prognosis, as it may induce to aggressive antibiotic treatment or, in more advanced cases, cardiac surgery. In this setting, the multimodality imaging has assumed a pivotal role in the clinical decision making and it requires the physician to be aware of the advantages and disadvantages of each imaging technique. Echocardiography is the first imaging test, but it has several limitations. Therefore, the integration with other imaging modalities (computed tomography, magnetic resonance imaging, nuclear imaging) becomes often necessary. Different strategies should be applied depending on whether the infection is suspected or already ascertained, whether located in native or prosthetic valves, in the left or right chambers, or if it involves an implanted cardiac device. In addition, detection of extracardiac IE-related lesions is crucial for a correct management and treatment. The aim of this review is to illustrate strengths and weaknesses of the various methods in the most common clinical scenarios