Site-directed mutagenesis of human ceruloplasmin:. production of a proteolytically stable protein and structure-activity relationships of type 1 sites.

A fully active recombinant human ceruloplasmin was obtained, and it was mutated to produce a ceruloplasmin stable to proteolysis. The stable ceruloplasmin was further mutated to perturb the environment of copper at the type 1 copper sites in two different domains. The wild type and the mutated ceruloplasmin were produced in the yeast Pichia pastoris and characterized. The mutations R481A, R701A, and K887A were at the proteolytic sites, did not alter the enzymatic activity, and were all necessary to protect ceruloplasmin from degradation. The mutation L329M was at the tricoordinate type 1 site of the domain 2 and was ineffective to induce modifications of the spectroscopic and catalytic properties of ceruloplasmin, supporting the hypothesis that this site is reduced and locked in a rigid frame. In contrast the mutation C1021S at the type 1 site of domain 6 substantially altered the molecular properties of the protein, leaving a small fraction endowed with oxidase activity. This result, while indicating the importance of this site in stabilizing the overall protein structure, suggests that another type 1 site is competent for dioxygen reduction. During the expression of ceruloplasmin, the yeast maintained a high level of Fet3 that was released from membranes of yeast not harboring the ceruloplasmin gene. This indicates that expression of ceruloplasmin induces a state of iron deficiency in yeast because the ferric iron produced in the medium by its ferroxidase activity is not available for the uptake

A hydrodynamic approach to Stark localization

When a free Fermi gas on a lattice is subject to the action of a linear potential it does not drift away, as one would naively expect, but it remains spatially localized. Here we revisit this phenomenon, known as Stark localization, within the recently proposed framework of generalized hydrodynamics. In particular, we consider the dynamics of an initial state in the form of a domain wall and we recover known results for the particle density and the particle current, while we derive analytical predictions for relevant observables such as the entanglement entropy and the full counting statistics. Then, we extend the analysis to generic potentials, highlighting the relationship between the occurrence of localization and the presence of peculiar closed orbits in phase space, arising from the lattice dispersion relation. We also compare our analytical predictions with numerical calculations and with the available results, finding perfect agreement. This approach paves the way for an exact treatment of the interacting case known as Stark many-body localization.Comment: 26 pages, 9 figures. Comments are welcome

Mobile Phones and Outdoor Advertising: Measurable Advertising

Television and newspapers sit at the top of many agency marketing plans, while outdoor advertising stays at the bottom. The reason for this is that it’s difficult to account for who views a billboard, so there is no way of consistently determining the effectiveness of outdoor advertising. As a result, agencies do not consider the medium and allocate their money elsewhere. To change this situation, one needs to create new credible audience measurements for the outdoor marketing industry. Here we propose a new way of performing audience measurements that combines mobile phone location estimations with information freely available on the Internet. We show that it is possible to estimate the number of people who drive or walk by a given area in Greater Boston from location estimations of a large fraction of mobile phone users in the region. We also infer the preferences for social events of the users by combing their location estimations with Internet listings of social events. This makes it possible to profile areas based on their residents’ interests and dynamically change displayed advertising based on those assessments.Engineering and Physical Sciences Research Council (Horizon project

Reconstitution of Ceruloplasmin by the Cu(I)-Glutathione Complex: EVIDENCE FOR A ROLE OF Mg2+ AND ATP

The copper-glutathione complex (Cu(I)-GSH) efficiently acted in vitro as the source of Cu(I) in the reconstitution of apoceruloplasmin. Copper was found to reinstate in the various sites in a multistep process, with metal entry into the protein in a first phase, and a second step involving conformational changes of the protein leading to the recovery of the native structural and functional properties. This latter phase was found to be strongly facilitated by Mg2+ or Ca2+ and by ATP. Both Mg2+ and ATP had to be present for optimal reconstitution. These results may shed some light on the mechanisms governing the biosynthesis of ceruloplasmin in vivo. Cu(I)-GSH was the only complex able to reconstitute ceruloplasmin at neutral pH. Glutathione may thus function to shuttle the metal from the membrane copper pump, as the Wilson disease ATPase, and ceruloplasmin in the secretory compartments of the cell. The finding that ceruloplasmin acquires the native conformation after metal entry through a complex pathway triggered by Mg2+ and ATP suggests that they may act as physiological modulators of this process in vivo

DEMNUni: The imprint of massive neutrinos on the cross-correlation between cosmic voids and CMB lensing

Cosmic voids are a powerful probe of cosmology and are one of the core observables of upcoming galaxy surveys. The cross-correlations between voids and other large-scale structure tracers such as galaxy clustering and galaxy lensing have been shown to be very sensitive probes of cosmology and among the most promising to probe the nature of gravity and the neutrino mass. However, recent measurements of the void imprint on the lensed Cosmic Microwave Background (CMB) have been shown to be in tension with expectations based on LCDM simulations, hinting to a possibility of non-standard cosmological signatures due to massive neutrinos. In this work we use the DEMNUni cosmological simulations with massive neutrino cosmologies to study the neutrino impact on voids selected in photometric surveys, e.g. via Luminous Red Galaxies, as well as on the void- CMB lensing cross-correlation. We show how the void properties observed in this way (size function, profiles) are affected by the presence of massive neutrinos compared to the neutrino massless case, and show how these can vary as a function of the selection method of the void sample. We comment on the possibility for massive neutrinos to be the source of the aforementioned tension. Finally, we identify the most promising setup to detect signatures of massive neutrinos in the voids-CMB lensing cross-correlation and define a new quantity useful to distinguish among different neutrino masses by comparing future observations against predictions from simulations including massive neutrinos.Comment: 34 pages, 15 figure

Clinical management of severe active ulcerative colitis in the TNF-a inhibitors era

Ulcerative colitis (UC) is a chronic inflammation of the coli mucosa clinically characterized by bloody diarrhea, abdominal pain and other systemic symptoms. The onset, as well as subsequent relapses, may occur with varying degrees of clinical and endoscopic activity and extent of disease. The clinical and endoscopic activity varies from mild to severe, while the extent of disease, without interruption, may involve from the rectum up to the entire colon. The severe form, when not properly and promptly treated, can be life-threatening and may determine various complications requiring urgent surgical treatment. Early recognition of severe forms, their treatment and patient monitoring can reduce morbidity and mortality, and improve surgical outcome. Since the 1950s, systemic corticosteroids have been the first-line treatment in severe active UC. Today, appropriate patient monitoring, and recognition of clinical, radiological and laboratory findings indicative of steroid failure guide the clinician in the use of immunomodulatory drugs or suggest indications for surgery. The aim of our study is to review the more recent data and guidelines that could be useful in clinical practice for the management of severe UC. © Copyright A. Calafiore et al., 2013

Occurrence of Bifidobacteriaceae in human hypochlorhydria stomach

open7noBackground: The human stomach, when healthy, is not a suitable host for microorganisms, but in pathological conditions such as gastritis, when gastric acid secretion is impaired, microbial overgrowth can be observed. Apart from Helicobacter pylori, the composition of microbiota, resident or exogenously introduced during neutral/high pH conditions, has not been investigated thoroughly. Thus, it is possible that Bifidobacteriaceae, important autochthonous and beneficial bacteria of human gastrointestinal microbiota, could over-colonize the stomach of hypochlorhydria patients suffering from autoimmune atrophic gastritis (AAG) or omeprazoletreated (OME) gastritis. This prompted us to characterize the Bifidobacteriaceae in such patients’ gastric microbiota and to study its abnormal colonization. Methods: Samples of gastric juices, and antrum and corpus mucosa from 23 hypochlorhydria patients (13 AAG and 10 OME) and from 10 control volunteers with base-line normochlorhydria, were cultivated in Brain Heart Infusion (BHI) and selective Bifidobacterium-Tryptone-Phytone-Yeast extract (Bif-TPY) media. The isolates were characterized by the fructose-6-phosphate phosphoketolase (F6PPK) test, electrophoresis of cellular proteins, the fermentation test, guanine-cytosine% DNA content, and DNADNA hybridization. Negative F6PPK isolates were characterized by order-specific polymerase chain reaction (PCR). Results: A total of 125 isolates, assigned to the Bifidobacteriaceae family on the basis of their morphology, were obtained from AAG and OME patients, but not from normal subjects. Of these isolates, 55 were assigned to the Bifidobacteriaceae family on the basis of their fructose-6-phosphoketolase (PPK) activity, PPK being the key taxonomic enzyme of this family. The remaining 70 isolates, which were PPK-negative, were attributed to the Actinomycetales order following specific primer PCR analysis. We observed a significantly higher abundance of Bifidobacteriaceae (Bifidobacterium dentium, Scardovia inopinata, and Parascardovia denticolens) in OME group than the AAG group. Furthermore, the Actinomycetales distribution was homogeneous for both hypochlorhydria patient groups. Conclusions: This study suggests that the Bifidobacteriaceae species, typically found in the oral cavity, readily colonizes the hypochlorhydria stomach of OME patients. The clinical relevance and the mechanism underlying this Bifidobacteriaceae presence in OME gastritis requires further functional studies.openPaola Mattarelli; Giovanni Brandi; Carlo Calabrese; Fabio Fornari; Gian Maria Prati; Bruno Biavati; Barbara SgorbatiPaola Mattarelli; Giovanni Brandi; Carlo Calabrese; Fabio Fornari; Gian Maria Prati; Bruno Biavati; Barbara Sgorbat