Tyrosine phosphorylation modulates peroxiredoxin-2 activity in normal and diseased red cells

Peroxiredoxin-2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells. Prx2 belongs to a well-known family of antioxidants, the peroxiredoxins (Prxs), that are widely expressed in mammalian cells. Prx2 is a typical, homodimeric, 2-Cys Prx that uses two cysteine residues to accomplish the task of detoxifying a vast range of organic peroxides, H2O2, and peroxynitrite. Although progress has been made on functional characterization of Prx2, much still remains to be investigated on Prx2 post-translational changes. Here, we first show that Prx2 is Tyrosine (Tyr) phosphorylated by Syk in red cells exposed to oxidation induced by diamide. We identified Tyr-193 in both recombinant Prx2 and native Prx2 from red cells as a specific target of Syk. Bioinformatic analysis suggests that phosphorylation of Tyr-193 allows Prx2 conformational change that is more favorable for its peroxidase activity. Indeed, Syk-induced Tyr phosphorylation of Prx2 enhances in vitro Prx2 activity, but also contributes to Prx2 translocation to the membrane of red cells exposed to diamide. The biologic importance of Tyr-193 phospho-Prx2 is further supported by data on red cells from a mouse model of humanized sickle cell disease (SCD). SCD is globally distributed, hereditary red cell disorder, characterized by severe red cell oxidation due to the pathologic sickle hemoglobin. SCD red cells show Tyr-phosphorylated Prx2 bound to the membrane and increased Prx2 activity when compared to healthy erythrocytes. Collectively, our data highlight the novel link between redox related signaling and Prx2 function in normal and diseased red cells

Inter-rater agreement of CDC criteria and ASEPSIS score in assessing surgical site infections after cesarean section: a prospective observational study

ObjectiveTo assess and compare the inter-rater agreement of the CDC criteria and the ASEPSIS score in identifying surgical site infections after cesarean section.MethodsProspective observational study including 110 patients subjected to a cesarean section at our institution. Surgical wounds were managed according to standard care and were photographed on the third, seventh, and thirtieth postoperative day or during any evaluation in case of complications. Three expert surgeons reviewed the prospectively gathered data and photographs and classified each wound using CDC criteria and the ASEPSIS score. The inter-rater agreements of CDC criteria and ASEPSIS score were determined with Krippendorff's Alpha with linear weights and compared with a confidence interval approach.ResultsThe weighted α coefficient for CDC criteria was 0.587 (95%CI, 0.411–0.763, p < 0.001, “moderate” agreement according to Altman's interpretation of weighted agreement coefficient), while the weighted α coefficient for the ASEPSIS score was 0.856 (95%CI, 0.733–0.980, p < 0.001, “very good” agreement).ConclusionASEPSIS score presents a “very good” inter-rater agreement for surgical site infections identification after cesarean, resulting in a more objective method than CDC criteria (“moderate” inter-rater agreement). ASEPSIS score could represent an objective tool for managing and monitoring surgical site infections after cesarean section, also by photographic evaluation

The role of clinical and neuroimaging features in the diagnosis of CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common familial cerebral small vessel disease, caused by NOTCH3 gene mutations. The aim of our study was to identify clinical and neuroradiological features which would be useful in identifying which patients presenting with lacunar stroke and TIA are likely to have CADASIL. METHODS: Patients with lacunar stroke or TIA were included in the present study. For each patient, demographic and clinical data were collected. MRI images were centrally analysed for the presence of lacunar infarcts, microbleeds, temporal lobe involvement, global atrophy and white matter hyperintensities. RESULTS: 128 patients (mean age 56.3 ± 12.4 years) were included. A NOTCH3 mutation was found in 12.5% of them. A family history of stroke, the presence of dementia and external capsule lesions on MRI were the only features significantly associated with the diagnosis of CADASIL. Although thalamic, temporal pole gliosis and severe white matter hyperintensities were less specific for CADASIL diagnosis, the combination of a number of these factors together with familial history for stroke result in a higher positive predictive value and specificity. CONCLUSIONS: A careful familial history collection and neuroradiological assessment can identify patients in whom NOTCH3 genetic testing has a higher yield.The Lombardia GENS project has received funding from the Regione Lombardia Government as a Research Independent Project (DGR n°VIII/006128-12/12/2007). Lombardia GENS is an investigator-driven, academic, non-profit consortium and is publicly funded. Hugh Markus is supported by an NIHR Senior Investigator award and his work is supported by the Cambridge University Hospitals NIHR Biomedical Research Centr

Growth response of juvenile dentex (Dentex dentex L.) to varying protein level and protein to lipid ratio in practical diets

The dentex (Dentex dentex L.) is a fast-growing sparid which represents a possible candidate forMediterranean mariculture. As the basic nutrient requirements of this species are unknown, twofeeding trials were carried out to identify suitable protein and lipid (energy) levels to be used informulating practical diets for dentex. The experiments used groups of 30 specimens (each fishweighing 17 or 20.7 g) which were kept in 12 flow-through 160 1 tanks supplied with seawater at 2 1min-’ (temperature 205 1°C; salinity 33 ppt). An initial 4-week trial was conducted to obtain apreliminary estimate of the optimal protein level. Triplicate groups were fed four practical diets withincreasing levels of protein (44.3,49.3, 55.7 and 58.9% dry matter (DM), over 90% of which wassupplied by white fish meal) and a constant lipid content ( 17.3% DM; mostly supplied by fish lipids)to satiation. An analysis of the nutrient-weight gain relationship indicated that the optimal dietaryprotein level was 49.3% DM. In a second 60 day experiment, duplicate groups of fish were fed sixdiets with three protein levels (44.4,49.5 and 55.8% DM) and two lipid contents ( 12.0 and 17.3%DM) within each protein level, nearly to satiation. Growth performance was lowest in fish given dietscontaining 44.4 or 49.5% protein and 12% lipid and was improved (P<O.O5) either with dietssupplying 55.8% protein (regardless of dietary lipid) or 49.3% protein and 17.4% lipid. The resultsobserved in the second trial suggest that when protein efficiency or retention are considered evendietary levels of 44.3% protein and 17.2% lipid (i.e. 24.3 g protein k.-’ calculated available energy)could be assumed as suitable levels for formulating practical diets for this species, providing highquality fish meal and oil are used

Sustainable Microwave-Assisted Aerobic Oxidation of Tomato Plant Waste into Bioaromatics and Organic Acids

Lignin is the second most abundant polymer in lignocellulosic biomass. A biorefinery approach to lignin valorisation, as an alternative to the use of heating, can produce bioaromatics, and this idea is attracting ever more interest. However, the isolation of lignin for further processing is really quite challenging, making the valorisation of native lignin (i.e., lignin in raw biomass) crucially important. An analysis of vanillin yields in relation to total reaction feedstock weight shows that the economic viability of the process depends on the valorisation of all biomass components. This paper reports a microwave-assisted, catalyst-free aerobic oxidation process for biomass. The protocol yields bioaromatics (mainly vanillin and syringaldehyde, with traces of p-hydroxybenzaldehyde and acetovanillone) and organic acids (38.4 wt % maximum total yield) in only 30 min. Vanillin is the most valuable of the aromatic compounds to be produced by lignin oxidation, accounting for 8.7 wt % of the initial lignin. The use of biomass as the starting material means that no pretreatment is needed for component separation. Furthermore, the use of air as the oxidant and the catalyst-free nature of the protocol render the process environmentally sustainable and scalable, as the energy consumption is counterbalanced by the clean production of high market value products

Grp94 is Tyr-phosphorylated by Fyn in the lumen of the endoplasmic reticulum andtranslocates to Golgi in differentiating myoblasts.

The endoplasmic-reticulum chaperone Grp94 is required for the cell surface export of molecules involved in the native immune response, in mesoderm induction and muscle development, but the signals responsible for Grp94 recruitment are still obscure. Here we show for the first time that Grp94 undergoes Tyr-phosphorylation in differentiating myogenic C2C12 cells. By means of phospho-proteomic and immunoprecipitation analyses, and the use of Src-specific inhibitors we demonstrate that the Src-tyrosine-kinase Fyn becomes active early after induction of C2C12 cell differentiation, in parallel with the recruitment and the Tyr-phosphorylation of Grp94, which peaks at 6-hour differentiation. Grp94 is Tyr-phosphorylated inside the endoplasmic reticulum by a lumenal Fyn, as indicated by fluorescence and electronmicroscopy immunolocalization, co-immunoprecipitation after chemical cross-linking and by treatment of intact endoplasmic-reticulum vesicles with proteinase K. Furthermore, fractionation of cellular membrane compartments and double-immunofluorescence studies showed that Tyr-phosphorylation of Grp94 is necessary for the protein translocation from the endoplasmic reticulum to the Golgi apparatus. These results indicate that Fyn-catalyzed Tyr-phosphorylation of Grp94 is an event required to promote the chaperone export from the endoplasmic reticulum occurring in the early phase of myoblast differentiation

Structural Insights into Complexes of Glucose-Regulated Protein94 (Grp94) with Human Immunoglobulin G. Relevance for Grp94-IgG Complexes that Form In Vivo in Pathological Conditions

While the mechanism by which Grp94 displays its chaperone function with client peptides in the cell has been elucidated extensively, much less is known about the nature and properties of how Grp94 can engage binding to proteins once it is exposed on the cell surface or liberated in the extra-cellular milieu, as occurs in pathological conditions. In this work, we wanted to investigate the molecular aspects and structural characteristics of complexes that Grp94 forms with human IgG, posing the attention on the influence that glycosylation of Grp94 might have on the binding capacity to IgG, and on the identification of sites involved in the binding. To this aim, we employed both native, fully glycosylated and partially glycosylated Grp94, and recombinant, non-glycosylated Grp94, as well as IgG subunits, in different experimental conditions, including the physiological setting of human plasma. Regardless of the species and type, Grp94 engages a similar, highly specific and stable binding with IgG that involves sites located in the N-terminal domain of Grp94 and the hinge region of whole IgG. Grp94 does not form stable complex with Fab, F(ab)2 or Fc. Glycosylation turns out to be an obstacle to the Grp94 binding to IgG, although this negative effect can be counteracted by ATP and spontaneously also disappears in time in a physiological setting of incubation. ATP does not affect at all the binding capacity of non-glycosylated Grp94. However, complexes that native, partially glycosylated Grp94 forms with IgG in the presence of ATP show strikingly different characteristics with respect to those formed in absence of ATP. Results have relevance for the mechanism regulating the formation of stable Grp94-IgG complexes in vivo, in the pathological conditions associated with the extra-cellular location of Grp94