Study of 2ONe(p,γ)21Na reaction at LUNA

openThe synthesis of Ne, Na, Mg, and Al isotopes is connected to the NeNa-MgAl cycles of stellar burning. The entire cycle speed is controlled by the 20Ne(p,gamma)21Na reaction (Qvalue=2431.68 keV) which is the first and slowest reaction of the whole NeNa cycle. At the state of the art, the associated reaction rate uncertainty therefore affects the production of the elements in the NeNa cycle and their yields in various stellar environments. In the relevant temperature range from 0.1 GK to 1 GK, the rate is mainly dominated by the 366 keV resonance, corresponding to the excited state of Ex= 2797.5 keV, and by the direct capture component. The present thesis analyses the direct capture below energies of 400 keV, which has been studied in deep underground at LUNA (Laboratory for Underground Nuclear Astrophysics), located at Gran Sasso National Laboratories in Italy. The reaction has been measured using the intense proton beam delivered by the LUNA 400 kV accelerator and a windowless differentially pumped gas target filled with natural neon at pressure of few mbar. Two high-purity germanium detectors collected the photons produced in the reaction, obtaining detailed gamma spectra. This work will present the experimental details of the campaign and its scientific results, focusing on the 20Ne(p,gamma)21Na cross section at stellar energies and its possible impact on the associated thermonuclear reaction rate

A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants

Abstract Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS). Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). We showed that CCS-D2 forms a stable complex with zinc-bound SOD1 in human cells, that has a twofold stabilizing effect: it both prevents the accumulation of unstructured mutant SOD1 and promotes zinc binding. We further showed that CCS-D2 interacts with apo-SOD1 in vitro, suggesting that in cells CCS stabilizes mutant apo-SOD1 prior to zinc binding. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation

Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells

In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2-3 d, after which the NMR sample is prepared. (1)H and (1)H-(15)N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. Uniform (15)N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. The entire procedure takes 4 d from cell culture seeding to NMR data collection

Protein interaction patterns in different cellular environments are revealed by in-cell NMR

In-cell NMR allows obtaining atomic-level information on biological macromolecules in their physiological environment. Soluble proteins may interact with the cellular environment in different ways: either specifically, with their functional partners, or non-specifically, with other cellular components. Such behaviour often causes the disappearance of the NMR signals. Here we show that by introducing mutations on the human protein profilin 1, used here as a test case, the in-cell NMR signals can be recovered. In human cells both specific and non-specific interactions are present, while in bacterial cells only the effect of non-specific interactions is observed. By comparing the NMR signal recovery pattern in human and bacterial cells, the relative contribution of each type of interaction can be assessed. This strategy allows detecting solution in-cell NMR spectra of soluble proteins without altering their fold, thus extending the applicability of in-cell NMR to a wider range of proteins

Direct structural evidence of protein redox regulation obtained by in-cell NMR

The redox properties of cellular environments are critical to many functional processes, and are strictly controlled in all living organisms. The glutathione-glutathione disulfide (GSH-GSSG) couple is the most abundant intracellular redox couple. A GSH redox potential can be calculated for each cellular compartment, which reflects the redox properties of that environment. This redox potential is often used to predict the redox state of a disulfide-containing protein, based on thermodynamic considerations. However, thiol-disulfide exchange reactions are often catalyzed by specific partners, and the distribution of the redox states of a protein may not correspond to the thermodynamic equilibrium with the GSH pool. Ideally, the protein redox state should be measured directly, bypassing the need to extrapolate from the GSH. Here, by in-cell NMR, we directly observe the redox state of three human proteins, Cox17, Mia40 and SOD1, in the cytoplasm of human and bacterial cells. We compare the observed distributions of redox states with those predicted by the GSH redox potential, and our results partially agree with the predictions. Discrepancies likely arise from the fact that the redox state of SOD1 is controlled by a specific partner, its copper chaperone (CCS), in a pathway which is not linked to the GSH redox potential. In principle, in-cell NMR allows determining whether redox proteins are at the equilibrium with GSH, or they are kinetically regulated. Such approach does not need assumptions on the redox potential of the environment, and provides a way to characterize each redox-regulating pathway separately

TCT-77 Risk and benefits of triple therapy in patients undergoing percutaneous coronary stent implantation requiring chronic oral anticoagulation: a meta-analysis of 12 trials

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In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone

Plasma Renin Concentration in Critically Ill COVID-19 Patients

Investigations of plasma renin concentration as a marker of organ perfusion in several intensive care settings have shown a significant correlation between its increase and a lack of perfusion in critical tissues, especially in septic patients. Castillo et al. proposed that activation of the non-canonical pathway of the renin–angiotensin–aldosterone system could improve cardiovascular homeostasis under COVID-19. During the first wave of COVID-19, we preliminarily enrolled a small cohort of subjects admitted to the Intensive Care Unit with a diagnosis of COVID-19 and acute respiratory distress syndrome. Their plasma renin value was measured in the first 24 h (T0), in the following 72 h (T1), and after one week (T2). In eight patients, we observed a higher plasma renin concentration—patients with difficulty weaning and in non-survivors. This is a preliminary observation. The variation of plasma renin levels in a septic condition is known, but settings such as COVID-19 infection have recently been investigated, showing a correlation with angiotensin-converting enzyme 2 receptor expression and functionality; in the near future, it will be interesting to have more data about its variation and value in COVID-19 patients

Advanced techniques to investigate the internalization mechanism of TiO2 NPs in the roots grown in a biosolid-amended agricultural soil

Plants play an important role in introducing the engineered nanoparticles (ENPs) into the food chain. The pathway of ENPs uptake from soil, their distribution in the edible plant parts, and their impact in the food production are important issues to be investigated. In the present study, Pisum sativum plants were grown at microcosm scale under medium-term TiO2 NPs exposure, to possibly mime environmental conditions in an agricultural soil amended with biosolids from a wastewater treatment plant in Pisa, Italy. TiO2 NPs were applied as pure rutile, pure anatase and a mixture of both crystalline phases in the biosolid amended-soil. Micro-XRF and μ-XANES from ID21 beamline were used for Ti elemental mapping and crystalline phase identification to indicate a relative distribution/localization of TiO2 crystalline phases within a given cross-section of roots, as well as the possible speciation and preferential crystalline phase uptake in the roots. Titanium in roots showed a main localization in the rizoderma, independently of the crystalline phase. Fewer Ti spots were found localized in the cortex or in vessel, however the roots grown in presence of a mixture of both phases showed a main presence of anatase, suggesting a preferential adsorption and translocation of this crystalline form through the roots. Our data indicated also a reduced translocation of Ti to the aerial part of the plant, confirming the chemical analysis of shoots and roots separately, which showed that Ti concentration was about 40 times lower in the upper part than in the below ground tissues. The TiO2 NPs were characterized on the basis of their size and shape by TEM analysis. Moreover, observations on cell ultrastructure of control and of anatase, rutile and mixture of both crystalline phases treated roots were performed. The root cells of plant grown in the presence of all NPs treatments shared the same alterations of ultrastructure: mitochondria with swollen cristae, nuclei with condensed chromatin, and part of the cytoplasm degraded, probably in consequence of an autophagic process. As detected by μ-XRF and μ-XANES, electron dense prismatic or round profiled particles of about 30-40 nm were observed mainly in form of aggregates in the intercellular spaces or crossing the wall of the cells next to rizoderma and in the cortex cells. Furthermore, the anatase treated cells were mostly damaged in respect to control and rutile treated roots, and more frequently internalized NPs were observed in these samples