A Kinetically Controlled Bioconjugation Method for the Synthesis of Radioimmunoconjugates and the Development of a Domain Mapping MS-Workflow for Its Characterization.

Immunoconjugates exploit the high affinity of monoclonal antibodies for a recognized antigen to selectively deliver a cytotoxic payload, such as drugs or radioactive nuclides, at the site of disease. Despite numerous techniques have been recently developed for site-selective bioconjugations of protein structures, reaction of ε-amine group of lysine residues with electrophilic reactants, such as activated esters (NHS), is the main method reported in the literature as it maintains proteins in their native conformation. Since antibodies hold a high number of lysine residues, a heterogeneous mixture of conjugates will be generated, which can result in decreased target affinity. Here, we report an intradomain regioselective bioconjugation between the monoclonal antibody Trastuzumab and the N-hydroxysuccinimide ester of the chelator 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) by a kinetically controlled reaction adding substoichiometric quantities of the activated ester to the mAb working at slightly basic pH. Liquid chromatography-mass spectrometry (LC-MS) analyses were carried out to assess the chelator-antibody ratio (CAR) and the number of chelating moieties linked to the mAb chains. Proteolysis experiments showed four lysine residues mainly involved in bioconjugation (K188 for the light chain and K30, K293, and K417 for the heavy chain), each of which was located in a different domain. Since the displayed intradomain regioselectivity, a domain mapping MS-workflow, based on a selective domain denaturation, was developed to quantify the percentage of chelator linked to each mAb domain. The resulting immunoconjugate mixture showed an average CAR of 0.9. About a third of the heavy chains were found as monoconjugated, whereas conjugation of the chelator in the light chain was negligible. Domain mapping showed the CH3 domain bearing 13% of conjugated DOTA, followed by CH2 and VH respectively bearing 12.5 and 11% of bonded chelator. Bioconjugation was not found in the CH1 domain, whereas for the light chain, only the CL domain was conjugated (6%). Data analysis based on LC-MS quantification of different analytical levels (intact, reduced chains, and domains) provided the immunoconjugate formulation. A mixture of immunoconjugates restricted to 15 species was obtained, and the percentage of each one within the mixture was calculated. In particular, species bearing 1 DOTA with a relative abundance ranging from 4 to 20-fold, in comparison to species bearing 2DOTA, were observed. Pairing of bioconjugation under kinetic control with the developed domain mapping MS-workflow could raise the standard of chemical quality for immunoconjugates obtained with commercially available reactants.

Aß and Tau Interact with Metal Ions, Lipid Membranes and Peptide-Based Amyloid Inhibitors: Are These Common Features Relevant in Alzheimer's Disease?

In the last two decades, the amyloid hypothesis, i.e., the abnormal accumulation of toxic Aß assemblies in the brain, has been considered the mainstream concept sustaining research in Alzheimer's Disease (AD). However, the course of cognitive decline and AD development better correlates with tau accumulation rather than amyloid peptide deposition. Moreover, all clinical trials of amyloid-targeting drug candidates have been unsuccessful, implicitly suggesting that the amyloid hypothesis needs significant amendments. Accumulating evidence supports the existence of a series of potentially dangerous relationships between Aß oligomeric species and tau protein in AD. However, the molecular determinants underlying pathogenic Aß/tau cross interactions are not fully understood. Here, we discuss the common features of Aß and tau molecules, with special emphasis on: (i) the critical role played by metal dyshomeostasis in promoting both Aß and tau aggregation and oxidative stress, in AD; (ii) the effects of lipid membranes on Aß and tau (co)-aggregation at the membrane interface; (iii) the potential of small peptide-based inhibitors of Aß and tau misfolding as therapeutic tools in AD. Although the molecular mechanism underlying the direct Aß/tau interaction remains largely unknown, the arguments discussed in this review may help reinforcing the current view of a synergistic Aß/tau molecular crosstalk in AD and stimulate further research to mechanism elucidation and next-generation AD therapeutics.

Adult Benign, Non-Iatrogenic Bronchoesophageal Fistulae: Systematic Review and Descriptive Analysis of Individual Patient Data.

BACKGROUND: Adult, benign, non-iatrogenic bronchoesophageal fistula (BEF) is a rare condition, which is occasionally described in single case reports. Therefore, little is known about its possible causes, presentation and management. METHODS: A systematic search of the literature in MEDLINE, PubMed Central and EMBASE databases between 1990 and 2020 was carried out to identify all cases of BEF. The initial database search identified 19,452 articles, of which 183 (251 individual patient cases) were included in the final analysis. RESULTS: Main causes of BEF were congenital malformations (97/251, 38.7%) and infections (82/251, 32.7%), while 33/251 (13.1%) fistulae were regarded as idiopathic and 39/251 (15.5%) attributed to other causes. Esophagograpy was the most sensitive method of diagnosis (97.4%) compared with esophagoscopy (78.9%), computed tomography (49.6%) and bronchoscopy (46.0%). Definitive treatment was surgical for 176 patients (70%), endoscopic for 25 (10%) and medical for 37 (14.7%). Compared with congenital BEFs, infective BEFs had shorter median symptom duration and were distributed more proximally over the bronchial tree. Definitive treatment was almost only surgical for congenital BEFs, while infective BEFs were treated also endoscopically (12%) and by medical therapy (38%). Morbidity, treatment failure and recurrence rates were higher for infective BEFs. CONCLUSIONS: BEFs are rare. Symptoms are non-specific and a high index of suspicion is necessary for diagnosis. Patients with infective BEF tend to have a more severe clinical picture than those with congenital BEF. Surgery is the main treatment for patients affected by congenital BEF, while infective BEFs may heal conservatively.

Zinc Interactions with a Soluble Mutated Rat Amylin to Mimic Whole Human Amylin: An Experimental and Simulation Approach to Understand Stoichiometry, Speciation and Coordination of the Metal Complexes.

Islet amyloid polypeptide (IAPP) is a hormone co-secreted with insulin and zinc from pancreatic ß-cells. To overcome the low solubility of human IAPP, we characterized zinc complexes species formed with 1) a mutated form of rat-IAPP(1-37; R18 H) able to mimic the human IAPP, 2) the r-IAPP(1-37) and the IAPP(1-8) fragment. Stoichiometry, speciation and coordination features of zinc(II) complexes were unveiled by ESI-MS, potentiometry and NMR measurements combined with DFT and free-energy simulations. Mononuclear species start to form around pH 6; Zn2+ binds both His18 and N-amino terminus in rat-IAPP(1-37; R18 H). The in silico study allows us to assess not only a structured turn compact domain in r-IAPP(1-37) and r-IAPP(1-37; R18 H) featured by a different free energy barrier for the transition from the compact to elongated conformation upon the coordination of Zn2+ , but also to bring into light a coordination shell further stabilized by noncovalent interactions.

Pilot prospective open, single-arm multicentre study on off-label use of tocilizumab in patients with severe COVID-19.

OBJECTIVES: No agent has yet been proven to be effective for the treatment of patients with severe COVID-19. METHODS: We conducted a pilot prospective open, single-arm multicentre study on off-label use of tocilizumab (TCZ) involving 63 hospitalised adult patients (56 males, age 62.6±12.5) with severe COVID-19. Clinical and laboratory parameters were prospectively collected at baseline, day 1, 2, 7 and 14. No moderate-to-severe adverse events attributable to TCZ were recorded. RESULTS: We observed a significant improvement in the levels of ferritin, C-reactive protein, D-dimer. The ratio of the partial pressure of oxygen (Pa02) to the fraction of inspired oxygen (Fi02) improved (mean±SD Pa02/Fi02 at admission: 152±53; at day 7: 283.73±115.9, at day 14: 302.2±126, p<0.05). The overall mortality was 11%; D-dimer level at baseline, but not IL-6 levels were predictors of mortality. TCZ administration within 6 days from admission in the hospital was associated with an increased likelihood of survival (HR 2.2 95%CI 1.3-6.7, p<0.05). CONCLUSIONS: In hospitalised adult patients with severe COVID-19, TCZ could be a safe option. An improvement in respiratory and laboratory parameters was observed. Future controlled trials in patients with severe illness are urgently needed to confirm the definite benefit with IL-6 target therapy.

Interaction between Hemin and Prion Peptides: Binding, Oxidative Reactivity and Aggregation.

We investigate the interaction of hemin with four fragments of prion protein (PrP) containing from one to four histidines (PrP106-114, PrP95-114, PrP84-114, PrP76-114) for its potential relevance to prion diseases and possibly traumatic brain injury. The binding properties of hemin-PrP complexes have been evaluated by UV-visible spectrophotometric titration. PrP peptides form a 1:1 adduct with hemin with affinity that increases with the number of histidines and length of the peptide; the following log K1 binding constants have been calculated: 6.48 for PrP76-114, 6.1 for PrP84-114, 4.80 for PrP95-114, whereas for PrP106-114, the interaction is too weak to allow a reliable binding constant calculation. These constants are similar to that of amyloid-ß (Aß) for hemin, and similarly to hemin-Aß, PrP peptides tend to form a six-coordinated low-spin complex. However, the concomitant aggregation of PrP induced by hemin prevents calculation of the K2 binding constant. The turbidimetry analysis of [hemin-PrP76-114] shows that, once aggregated, this complex is scarcely soluble and undergoes precipitation. Finally, a detailed study of the peroxidase-like activity of [hemin-(PrP)] shows a moderate increase of the reactivity with respect to free hemin, but considering the activity over long time, as for neurodegenerative pathologies, it might contribute to neuronal oxidative stress.

Long-Term Monitoring with Fiber Optics Distributed Temperature Sensing at Campi Flegrei: The Campi Flegrei Deep Drilling Project.

Monitoring volcanic phenomena is a key question, for both volcanological research and for civil protection purposes. This is particularly true in densely populated volcanic areas, like the Campi Flegrei caldera, which includes part of the large city of Naples (Italy). Borehole monitoring of volcanoes is the most promising way to improve classical methods of surface monitoring, although not commonly applied yet. Fiber optics technology is the most practical and suitable way to operate in such high temperature and aggressive environmental conditions. In this paper, we describe a fiber optics Distributed Temperature Sensing (DTS) sensor, which has been designed to continuously measure temperature all along a 500 m. deep well drilled in the west side of Naples (Bagnoli area), lying in the Campi Flegrei volcanic area. It has then been installed as part of the international 'Campi Flegrei Deep Drilling Project', and is continuously operating, giving insight on the time variation of temperature along the whole borehole depth. Such continuous monitoring of temperature can in turn indicate volcanic processes linked to magma dynamics and/or to changes in the hydrothermal system. The developed monitoring system, working at bottom temperatures higher than 100 °C, demonstrates the feasibility and effectiveness of using DTS for borehole volcanic monitoring.

Systematic DNA Study for Fabry Disease in the End Stage Renal Disease Patients from a Southern Italy Area.

BACKGROUND/AIMS: Fabry disease (FD) is a lysosomal storage disorder characterized by pervasive renal involvement. However, this disease is underdiagnosed in patient with chronic kidney disease (CKD), including those with end stage renal disease (ESRD), so their investigation represents an unexploited opportunity for early diagnosis of the disease and for its identification in relatives of affected patients. METHODS: We investigated Fabry disease in a clinical and biological database including ESRD patients of unknown cause in a geographical area with 2 million residents. The study was based on state of art GLA gene sequencing and was extended to relatives of affected ESRD patients. RESULTS: Among ESRD patients qualified for enrollment into this study, a previously undiagnosed young man harboring the mutation p.I91T was identified. The study of the proband's family led to the identification of 8 additional cases. In another ESRD male patient, we identified the functional polymorphism p.D313Y. Furthermore, in 55 ESRD patients (24.2%) we found intronic polymorphisms of uncertain functional relevance in the non-coding regions of the GLA gene. CONCLUSION: A comprehensive survey of ESRD patients in a geographical area of 2 million residents identified one undiagnosed case of Fabry disease and led to the identification of 8 additional cases among his relatives. Screening protocols starting from the dialysis population and upstream extended to families of affected individuals may be an effective strategy to maximize the early identification of subjects with Fabry disease.

Prion Peptides Are Extremely Sensitive to Copper Induced Oxidative Stress.

Copper(II) binding to prion peptides does not prevent Cu redox cycling and formation of reactive oxygen species (ROS) in the presence of reducing agents. The toxic effects of these species are exacerbated in the presence of catecholamines, indicating that dysfunction of catecholamine vesicular sequestration or recovery after synaptic release is a dangerous amplifier of Cu induced oxidative stress. Cu bound to prion peptides including the high affinity site involving histidines adjacent to the octarepeats exhibits marked catalytic activity toward dopamine and 4-methylcatechol. The resulting quinone oxidation products undergo parallel oligomerization and endogenous peptide modification yielding catechol adducts at the histidine binding ligands. These modifications add to the more common oxidation of Met and His residues produced by ROS. Derivatization of Cu-prion peptides is much faster than that undergone by Cu-ß-amyloid and Cu-α-synuclein complexes in the same conditions.

Gene Expression, Oxidative Stress, and Senescence of Primary Coronary Endothelial Cells Exposed to Postprandial Serum of Healthy Adult and Elderly Volunteers after Oven-Cooked Meat Meals.

Epidemiological studies have linked high consumption of meat with major age-related diseases including cardiovascular diseases. Abnormal postprandial increases in plasma lipids after a meat meal have been hypothesized among the pathogenetic mechanisms. However, it is still unknown if the postprandial serum derived after a normal meat meal is able to affect endothelial function, and if the type of meat and the age of the donors are critical factors. Here, we show the effects of postprandial sera derived from healthy adults and elderly volunteers who consumed meat meals on human coronary artery endothelial cell (HCAEC) oxidative stress, gene expression, DNA damage, and cellular senescence. We observed that a single exposure to postprandial serum induces a slight increase in ROS that is associated with modulation of gene expression pathways related to oxidative stress response and metabolism. The postprandial-induced increase in ROS is not associated with a measurable DNA oxidative damage. However, repeated exposure to postprandial serum induces an acceleration of cellular senescence. Taking into account the deleterious role of cellular senescence in age-related vascular diseases, the results suggest a new mechanism by which excessive meat consumption and time spent in postprandial state may affect health status during aging.

Ubiquitin Associates with the N-Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions.

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI-MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF1-14 , which is a short model peptide encompassing the first 14 N-terminal residues of NGF, binds the copper-binding regions of Ub (KD =8.6 10-5 m). Moreover, the peptide undergoes a random coil-polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF1-14 /Ub interactions. Further experiments performed with the full-length NGF confirmed the existence of a copper(II)-dependent association between Ub and NGF and indicated that the N-terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full-length protein.

Synapsin knockdown is associated with decreased neurite outgrowth, functional synaptogenesis impairment, and fast high-frequency neurotransmitter release.

Synapsins (Syns) are an evolutionarily conserved family of synaptic vesicle-associated proteins related to fine tuning of synaptic transmission. Studies with mammals have partially clarified the different roles of Syns; however, the presence of different genes and isoforms and the development of compensatory mechanisms hinder accurate data interpretation. Here, we use a simple in vitro monosynaptic Helix neuron connection, reproducing an in vivo physiological connection as a reliable experimental model to investigate the effects of Syn knockdown. Cells overexpressing an antisense construct against Helix Syn showed a time-dependent decrease of Syn immunostaining, confirming protein loss. At the morphological level, Syn-silenced cells showed a reduction in neurite linear outgrowth and branching and in the size and number of synaptic varicosities. Functionally, Syn-silenced cells presented a reduced ability to form synaptic connections; however, functional chemical synapses showed similar basal excitatory postsynaptic potentials and similar short-term plasticity paradigms. In addition, Syn-silenced cells presented faster neurotransmitter release and decreased postsynaptic response toward the end of long tetanic presynaptic stimulations, probably related to an impairment of the synaptic vesicle trafficking resulting from a different vesicle handling, with an increased readily releasable pool and a compromised reserve pool.

Cross-talk between the octarepeat domain and the fifth binding site of prion protein driven by the interaction of copper(II) with the N-terminus.

Prion diseases are a group of neurodegenerative diseases based on the conformational conversion of the normal form of the prion protein (PrP(C)) to the disease-related scrapie isoform (PrP(Sc)). Copper(II) coordination to PrP(C) has attracted considerable interest for almost 20 years, mainly due to the possibility that such an interaction would be an important event for the physiological function of PrP(C). In this work, we report the copper(II) coordination features of the peptide fragment Ac(PEG11)3PrP(60-114) [Ac = acetyl] as a model for the whole N-terminus of the PrP(C) metal-binding domain. We studied the complexation properties of the peptide by means of potentiometric, UV/Vis, circular dichroism and electrospray ionisation mass spectrometry techniques. The results revealed that the preferred histidyl binding sites largely depend on the pH and copper(II)/peptide ratio. Formation of macrochelate species occurs up to a 2:1 metal/peptide ratio in the physiological pH range and simultaneously involves the histidyl residues present both inside and outside the octarepeat domain. However, at increased copper(II)/peptide ratios amide-bound species form, especially within the octarepeat domain. On the contrary, at basic pH the amide-bound species predominate at any copper/peptide ratio and are formed preferably with the binding sites of His96 and His111, which is similar to the metal-binding-affinity order observed in our previous studies.

Prunus persica var. platycarpa (Tabacchiera Peach): Bioactive Compounds and Antioxidant Activity of Pulp, Peel and Seed Ethanolic Extracts.

A comparative analysis of ethanol extracts from peel, pulp and seed of Prunus persica var. platycarpa (Tabacchiera peach) was done. The total phenol, flavonoid and carotenoid content as well as the antioxidant properties by using different in vitro assays (DPPH, ABTS, FRAP, Fe-chelating, ß-carotene bleaching test) were evaluated. Pulp extract was subjected to liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS). Gallic acid, protocatechuic acid, protocatechualdehyde, chlorogenic acid, p-coumaric acid, and ferulic acid were identified as main constituents. Pulp extract was characterized by the highest total phytonutrients content and exhibited the highest antioxidant activity in all in vitro assays (IC(50) values of 2.2 µg/mL after 60 min of incubation by using ß-carotene bleaching test and 2.9 µg/mL by using Fe-chelating assay). Overall, the obtained results suggest that P. persica var. platycarpa displays a good antioxidant activity and its consumption could be promoted.

Affinity, speciation, and molecular features of copper(II) complexes with a prion tetraoctarepeat domain in aqueous solution: insights into old and new results.

Characterization of the copper(II) complexes formed with the tetraoctarepeat peptide at low and high metal-to-ligand ratios and in a large pH range, would provide a breakthrough in the interpretation of biological relevance of the different metal complexes of copper(II)-tetraoctarepeat system. In the present work, the potentiometric, UV/Vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) studies were carried out on copper(II) complexes with a PEG-ylated derivative of the tetraoctarepeats peptide sequence (Ac-PEG27 -(PHGGGWGQ)4 -NH2 ) and the peptide Ac-(PHGGGWGQ)2 -NH2 . Conjugation of tetraoctarepeat peptide sequence with polyethyleneglycol improved the solubility of the copper(II) complexes. The results enable a straightforward explanation of the conflicting results originated from the underestimation of all metal-ligand equilibria and the ensuing speciation. A complete and reliable speciation is therefore obtained with the released affinity and binding details of the main complexes species formed in aqueous solution. The results contribute to clarify the discrepancies of several studies in which the authors ascribe the redox activity of copper(II)-tetraoctarepeat system considering only the average effects of several coexisting species with very different stoichiometries and binding modes.

Aß8-20 Fragment as an Anti-Fibrillogenic and Neuroprotective Agent: Advancing toward Efficient Alzheimer's Disease Treatment.

Alzheimer's disease (AD) is the most common cause of dementia, characterized by a spectrum of symptoms associated with memory loss and cognitive decline with deleterious consequences in everyday life. The lack of specific drugs for the treatment and/or prevention of this pathology makes AD an ever-increasing economic and social emergency. Oligomeric species of amyloid-beta (Aß) are recognized as the primary cause responsible for synaptic dysfunction and neuronal degeneration, playing a crucial role in the onset of the pathology. Several studies have been focusing on the use of small molecules and peptides targeting oligomeric species to prevent Aß aggregation and toxicity. Among them, peptide fragments derived from the primary sequence of Aß have also been used to exploit any eventual recognition abilities toward the full-length Aß parent peptide. Here, we test the Aß8-20 fragment which contains the self-recognizing Lys-Leu-Val-Phe-Phe sequence and lacks Arg 5 and Asp 7 and the main part of the C-terminus, key points involved in the aggregation pathway and stabilization of the fibrillary structure of Aß. In particular, by combining chemical and biological techniques, we show that Aß8-20 does not undergo random coil to ß sheet conformational transition, does not form amyloid fibrils by itself, and is not toxic for neuronal cells. Moreover, we demonstrate that Aß8-20 mainly interacts with the 4-11 region of Aß1-42 and inhibits the formation of toxic oligomeric species and Aß fibrils. Finally, our data show that Aß8-20 protects neuron-like cells from Aß1-42 oligomer toxicity. We propose Aß8-20 as a promising drug candidate for the treatment of AD.

Comparative metagenomics at Solfatara and Pisciarelli hydrothermal systems in Italy reveal that ecological differences across substrates are not ubiquitous.

Introduction: Continental hydrothermal systems (CHSs) are geochemically complex, and they support microbial communities that vary across substrates. However, our understanding of these variations across the complete range of substrates in CHS is limited because many previous studies have focused predominantly on aqueous settings. Methods: Here we used metagenomes in the context of their environmental geochemistry to investigate the ecology of different substrates (i.e., water, mud and fumarolic deposits) from Solfatara and Pisciarelli. Results and Discussion: Results indicate that both locations are lithologically similar with distinct fluid geochemistry. In particular, all substrates from Solfatara have similar chemistry whereas Pisciarelli substrates have varying chemistry; with water and mud from bubbling pools exhibiting high SO4 2- and NH4 + concentrations. Species alpha diversity was found to be different between locations but not across substrates, and pH was shown to be the most important driver of both diversity and microbial community composition. Based on cluster analysis, microbial community structure differed significantly between Pisciarelli substrates but not between Solfatara substrates. Pisciarelli mud pools, were dominated by (hyper)thermophilic archaea, and on average, bacteria dominated Pisciarelli fumarolic deposits and all investigated Solfatara environments. Carbon fixation and sulfur oxidation were the most important metabolic pathways fueled by volcanic outgassing at both locations. Together, results demonstrate that ecological differences across substrates are not a widespread phenomenon but specific to the system. Therefore, this study demonstrates the importance of analyzing different substrates of a CHS to understand the full range of microbial ecology to avoid biased ecological assessments.

Impact of Modern Oven Treatments on Lipid Oxidation and Vitamin E Content of Fillets from Sardine (Sardina pilchardus) at Different Reproductive Cycle Phases.

The beneficial effects of sardine consumption can be related to the presence of bioactive compounds, such as vitamin E and ω3 polyunsaturated fatty acids. In any case, the levels of these compounds in sardine fillet depend on different factors mainly related to the diet and reproductive cycle phase of the fish as well as the technological treatments carried out to cook the fillets. The aim of the present study is two-fold: first, to evaluate changes in the total fatty acid profile, lipid oxidation, and vitamin E content of raw fillets from sardine (Sardina pilchardus) at different reproductive cycle phases (pre-spawning, spawning, and post-spawning); and second, to highlight how these nutritional profiles are affected by three oven treatments (conventional, steam, and sous-vide). For this purpose, raw fish was grouped into pre-spawning, spawning, and post-spawning phases according to the mesenteric fat frequency and the gonadosomatic index evaluation, and submitted to conventional (CO), steam (SO), and sous-vide (SV) baking. The ratio of EPA/DHA and vitamin E increased from post-spawning to pre-spawning, to spawning. Considering the reproductive phases, baking affected the oxidative degree differently: a CO > SO ≥ SV impact was found in the worst scenario (post-spawning), mitigated by vitamin E, to CO ≥ SO > SV in the best scenario (spawning). SV was the best treatment with high values of vitamin E in pre-spawning individuals (110.1 mg/kg). This study shows how vitamin E is correlated to the combined effect of endogenous and exogenous factors.

Prevalence of C-reactive protein elevation and time course of normalization in acute pericarditis: implications for the diagnosis, therapy, and prognosis of pericarditis.

BACKGROUND: The role of inflammatory markers is not well defined for either diagnosis or treatment of pericarditis. The aim of this study is to prospectively evaluate the frequency of high-sensitivity C-reactive protein (hs-CRP) elevation in patients with acute pericarditis, its time course of normalization, and the possible importance for diagnosis, therapy, and prognosis. METHODS AND RESULTS: Two hundred consecutive patients with viral or idiopathic acute pericarditis (mean age, 53 ± 15.5 years; 103 men) were studied from August 2005 to August 2007 in 2 Italian referral centers. Hs-CRP was determined at presentation and then every week until normalization. Hs-CRP elevation was recorded in 156 of 200 cases (78%) at presentation. Recognized causes of a negative hs-CRP at presentation were early assessment in 15 of 44 cases (34%) and previous anti-inflammatory therapies in 22 of 44 cases (50%). Hs-CRP normalization was achieved with the following time course: 120 of 200 (60%) at week 1, 170 of 200 (85%) at week 2, 190 of 200 (95%) at week 3, and all cases (100%) at week 4. In multivariable analysis, incomplete response to empirical anti-inflammatory therapy at week 1 (hazard ratio, 2.98; 95% confidence interval, 1.80 to 4.94; P < 0.001), corticosteroid therapy (hazard ratio, 2.80; 95% confidence interval, 1.59 to 4.95; P < 0.001), and the presence of elevated hs-CRP at week 1 (hazard ratio, 2.36; 95% confidence interval, 1.32 to 4.21; P = 0.004) were independent risk factors for recurrence. CONCLUSIONS: Hs-CRP is elevated at the initial presentation in ≈ 3 of 4 cases of acute pericarditis, identifies patients at higher risk of recurrence, and could be used to monitor disease activity and select appropriate therapy length.

MAPK/Erk-dependent phosphorylation of synapsin mediates formation of functional synapses and short-term homosynaptic plasticity.

MAPK/Erk is a protein kinase activated by neurotrophic factors involved in synapse formation and plasticity, which acts at both the nuclear and cytoplasmic level. Synapsin proteins are synaptic-vesicle-associated proteins that are well known to be MAPK/Erk substrates at phylogenetically conserved sites. However, the physiological role of MAPK/Erk-dependent synapsin phosphorylation in regulating synaptic formation and function is poorly understood. Here, we examined whether synapsin acts as a physiological effector of MAPK/Erk in synaptogenesis and plasticity. To this aim, we developed an in vitro model of soma-to-soma paired Helix B2 neurons, that establish bidirectional excitatory synapses. We found that the formation and activity-dependent short-term plasticity of these synapses is dependent on the MAPK/Erk pathway. To address the role of synapsin in this pathway, we generated non-phosphorylatable and pseudo-phosphorylated Helix synapsin mutants at the MAPK/Erk sites. Overexpression experiments revealed that both mutants interfere with presynaptic differentiation, synapsin clustering, and severely impair post-tetanic potentiation, a form of short-term homosynaptic plasticity. Our findings show that MAPK/Erk-dependent synapsin phosphorylation has a dual role both in the establishment of functional synaptic connections and their short-term plasticity, indicating that some of the multiple extranuclear functions of MAPK/Erk in neurons can be mediated by the same multifunctional presynaptic target.