Trafficking of Estrella lausannensis in human macrophages.

Estrella lausannensis is a new member of the Chlamydiales order. Like other Chlamydia-related bacteria, it is able to replicate in amoebae and in fish cell lines. A preliminary study investigating the pathogenic potential of Chlamydia-related bacteria found a correlation between antibody response to E. lausannensis and pneumonia in children. To further investigate the pathogenic potential of E. lausannensis, we determined its ability to grow in human macrophages and its intracellular trafficking. The replication in macrophages resulted in viable E. lausannensis; however, it caused a significant cytopathic effect. The intracellular trafficking of E. lausannensis was analyzed by determining the interaction of the Estrella-containing inclusions with various endocytic markers as well as host organelles. The E. lausannensis inclusion escaped the endocytic pathway rapidly avoiding maturation into phagolysosomes by preventing both EEA-1 and LAMP-1 accumulation. Compared to Waddlia chondrophila, another Chlamydia-related bacteria, the recruitment of mitochondria and endoplasmic reticulum was minimal for E. lausannensis inclusions. Estrella lausannensis appears to use a distinct source of nutrients and energy compared to other members of the Chlamydiales order. In conclusion, we hypothesize that E. lausannensis has a restricted growth in human macrophages, due to its reduced capacity to control programmed cell death

A proposito dell\u2019inchiesta della S. Congregazione dell\u2019Indice dei libri proibiti di fine \u2018500

Il paper \u2013 diviso in tre parti \u2013 muove dai risultati della \u201cRicerca sull\u2019inchiesta della Congregazione dell\u2019Indice\u201d che ha preso in esame i codici Vaticani Latini che conservano circa 9500 liste di titoli di libri posseduti dai religiosi italiani alla fine del XVI secolo. Roberto Rusconi sviluppa una panoramica introduttiva sulle biblioteche degli ordini religiosi alla fi ne del Cinquecento. Giovanna Granata sfrutta le molteplici potenzialit\ue0 informative dell\u2019Inchiesta, sia in termini di conoscenza di tali biblioteche, sia rispetto alle modalit\ue0 di circolazione delle diverse edizioni. Rosa Marisa Borraccini prende invece in esame un caso di studio come quello del 'Confessionario' di Girolamo da Palermo per dimostrare l\u2019importanza che un testo oggi quasi del tutto sconosciuto aveva all\u2019epoca della sua produzione. This paper \u2013 divided into three sections \u2013 expands upon the work undertaken in \u201cRicerca sull\u2019inchiesta della Congregazione dell\u2019Indice\u201d, which has examined the Codici Vaticani Latini, a collection which conserves around 9.500 lists of volumes owned by Italian monks and clerics at the end of the sixteenth century. Roberto Rusconi offers an introductory overview of the libraries of religious orders at the end of the sixteenth century. Giovanna Granata analyses the findings of the Inchiesta in terms of actual library holdings and in terms of the circulation of different editions. Rosa Marisa Borraccini examines the case study of the \u201cConfessionario\u201d of Girolamo da Palermo to demonstrate the importance certain texts, although now forgotten and largely ignored, had at the time of their production and circulation

Cryogenic light detectors with enhanced performance for rare events physics

We have developed and tested a new way of coupling bolometric light detectors to scintillating crystal bolometers based upon simply resting the light detector on the crystal surface, held in position only by gravity. This straightforward mounting results in three important improvements: (1) it decreases the amount of non-active materials needed to assemble the detector, (2) it substantially increases the light collection efficiency by minimizing the light losses induced by the mounting structure, and (3) it enhances the thermal signal induced in the light detector thanks to the extremely weak thermal link to the thermal bath. We tested this new technique with a 16 cm$^2$ Ge light detector with thermistor readout sitting on the surface of a large TeO$_2$ bolometer. The light collection efficiency was increased by greater than 50\% compared to previously tested alternative mountings. We obtained a baseline energy resolution on the light detector of 20~eV RMS that, together with increased light collection, enabled us to obtain the best $\alpha$ vs $\beta/\gamma$ discrimination ever obtained with massive TeO$_2$ crystals. At the same time we achieved rise and decay times of 0.8 and 1.6 ms, respectively. This superb performance meets all of the requirements for the CUPID (CUORE Upgrade with Particle IDentification) experiment, which is a 1-ton scintillating bolometer follow up to CUORE.Comment: 6 pages, 4 figure

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAcα1-O-Ser/Thr, where GalNAc is N-acetyl-D-galactosamine) is expressed in many carcinomas, in haemopoietic disorders including the Tn syndrome, and on human immunodeficiency virus (HIV) coat glycoproteins, but is not expressed on normal, differentiated cells because of the expression of a Tn-processing galactosyltransferase. Using Jurkat T leukaemic cells which express high levels of Tn antigen due to deficient Tn galactosylation, we have established the Tn antigen-mediated gene transfer and demonstrate the considerable efficiency of this approach. We used poly(L-lysine) conjugates of the monoclonal antibody 1E3 directed against the Tn antigen to deliver the luciferase and β-galactosidase reporter genes to Jurkat cells by receptor-mediated endocytosis. Addition of unconjugated 1E3 reduced transfection efficiency in a concentration-dependent manner and incubation with free GalNAc abolished DNA transfer completely, indicating that gene delivery is indeed mediated by the Tn antigen. Pre-treatment of Jurkat cells with Vibrio cholerae sialidase, which uncovers additional Tn antigens, resulted in an improvement of gene transfection. Both human and chicken adenovirus particles attached to the DNA/polylysine complex strongly augmented transgene expression. When the β-galactosidase (lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis, up to 60% of the cells were positive in the cytochemical stain using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) as a chromogenic substrate. The efficiency of the transferrin receptor-mediated DNA uptake into Jurkat cells was comparatively low, although these cells were shown to express considerable amounts of transferrin receptor. We show here that a mucin-type carbohydrate antigen mediates highly efficient DNA uptake by endocytosis into Jurkat T cells. This method represents a 50-fold improvement of Jurkat cell transfection efficiency over other physical gene transfer techniques. Specific gene delivery to primary cancer cells exhibiting Tn epitopes may especially be desirable in immunotherapy protocol

Upgrading Monocytes Therapy for Critical Limb Ischemia Patient Treatment: Pre-Clinical and GMP-Validation Aspects

Advanced cell therapy medicinal products (ATMP) are at the forefront of a new range of biopharmaceuticals. The use of ATMP has evolved and increased in the last decades, representing a new approach to treating diseases that are not effectively managed with conventional treatments. The standard worldwide recognized for drug production is the Good Manufacturing Practices (GMP), widely used in the pharma production of synthesized drugs but applying also to ATMP. GMP guidelines are worldwide recognized standards to manufacture medicinal products to guarantee high quality, safety, and efficacy. In this report, we describe the pre-clinical and the GMP upgrade of peripheral blood mononuclear cell (PBMC) preparation, starting from peripheral blood and ending up with a GMP-grade clinical product ready to be used in patients with critical limb ischemia (CLI). We also evaluated production in hypoxic conditions to increase PBMC functional activity and angiogenic potential. Furthermore, we extensively analyzed the storage and transport conditions of the final product as required by the regulatory body for ATMPs. Altogether, results suggest that the whole manufacturing process can be performed for clinical application. Peripheral blood collected by a physician should be transported at room temperature, and PBMCs should be isolated in a clean room within 8 h of venipuncture. Frozen cells can be stored in nitrogen vapors and thawed for up to 12 months. PBMCs resuspended in 5% human albumin solution should be stored and transported at 4 degrees C before injection in patients within 24 h to thawing. Hypoxic conditioning of PBMCs should be implemented for clinical application, as it showed a significant enhancement of PBMC functional activity, in particular with increased adhesion, migration, and oxidative stress resistance. We demonstrated the feasibility and the quality of a GMP-enriched suspension of monocytes as an ATMP, tested in a clean room facility for all aspects related to production in respect of all the GMP criteria that allow its use as an ATMP. We think that these results could ease the way to the clinical application of ATMPs

Crescent and star shapes of members of the Chlamydiales order: impact of fixative methods.

Members of the Chlamydiales order all share a biphasic lifecycle alternating between small infectious particles, the elementary bodies (EBs) and larger intracellular forms able to replicate, the reticulate bodies. Whereas the classical Chlamydia usually harbours round-shaped EBs, some members of the Chlamydia-related families display crescent and star-shaped morphologies by electron microscopy. To determine the impact of fixative methods on the shape of the bacterial cells, different buffer and fixative combinations were tested on purified EBs of Criblamydia sequanensis, Estrella lausannensis, Parachlamydia acanthamoebae, and Waddlia chondrophila. A linear discriminant analysis was performed on particle metrics extracted from electron microscopy images to recognize crescent, round, star and intermediary forms. Depending on the buffer and fixatives used, a mixture of alternative shapes were observed in varying proportions with stars and crescents being more frequent in C. sequanensis and P. acanthamoebae, respectively. No tested buffer and chemical fixative preserved ideally the round shape of a majority of bacteria and other methods such as deep-freezing and cryofixation should be applied. Although crescent and star shapes could represent a fixation artifact, they certainly point towards a diverse composition and organization of membrane proteins or intracellular structures rather than being a distinct developmental stage

Characterization of a ZnSe scintillating bolometer prototype for neutrinoless double beta decay search

As proposed in the LUCIFER project, ZnSe crystals are attractive materials to realize scintillating bolometers aiming at the search for neutrinoless double beta decay of the promising isotope 82Se. However, the optimization of the ZnSe-based detectors is rather complex and requires a wide-range investigation of the crystal features: optical properties, crystalline quality, scintillation yields and bolometric behaviour. Samples tested up to now show problems in the reproducibility of crucial aspects of the detector performance. In this work, we present the results obtained with a scintillating bolometer operated aboveground at about 25 mK. The detector energy absorber was a single 1 cm3 ZnSe crystal. The good energy resolution of the heat channel (about 14 keV at 1460 keV) and the excellent alpha/beta discrimination capability are very encouraging for a successful realization of the LUCIFER program. The bolometric measurements were completed by optical tests on the crystal (optical transmission and luminescence measurements down to 10 K) and investigation of the crystalline structure. The work here described provides a set of parameters and procedures useful for a complete pre-characterization of ZnSe crystals in view of the realization of highly performing scintillating bolometers

Preclinical and clinical evidence on the approach-avoidance conflict evaluation as an integrative tool for psychopathology

The approach-avoidance conflict (AAC), i.e. the competing tendencies to undertake goal-directed actions or to withdraw from everyday life challenges, stands at the basis of humans' existence defining behavioural and personality domains. Gray's Reinforcement Sensitivity Theory posits that a stable bias toward approach or avoidance represents a psychopathological trait associated with excessive sensitivity to reward or punishment. Optogenetic studies in rodents and imaging studies in humans associated with cross-species AAC paradigms granted new emphasis to the hippocampus as a hub of behavioural inhibition. For instance, recent functional neuroimaging studies show that functional brain activity in the human hippocampus correlates with threat perception and seems to underlie passive avoidance. Therefore, our commentary aims to (i) discuss the inhibitory role of the hippocampus in approach-related behaviours and (ii) promote the integration of functional neuroimaging with cross-species AAC paradigms as a means of diagnostic, therapeutic, follow up and prognosis refinement in psychiatric populations

Capillary electrophoretic and extraction conditions for the analysis of Catha edulis FORKS active principles

A capillary electrophoretic method, which allowed the detection and separation of the active principles of Catha edulis, i.e. cathinone, cathine and phenylpropanolamine, was developed. A suitable internal standard (nicotinamide), which permitted the quantification of the analytes reducing the variability of the migration times due to EOF changes, was identified. The analytical method was validated, assessing linearity, sensitivity and repeatability, showing optimal features for the analysis of the vegetable material. Moreover extraction conditions were investigated to achieve the exhaustion of the plant material in the fastest and most efficient way to meet the requirements of the Court

TeO2_2 bolometers with Cherenkov signal tagging: towards next-generation neutrinoless double beta decay experiments

CUORE, an array of 988 TeO$_2$ bolometers, is about to be one of the most sensitive experiments searching for neutrinoless double-beta decay. Its sensitivity could be further improved by removing the background from $\alpha$ radioactivity. A few years ago it has been pointed out that the signal from $\beta$s can be tagged by detecting the emitted Cherenkov light, which is not produced by $\alpha$s. In this paper we confirm this possibility. For the first time we measured the Cherenkov light emitted by a CUORE crystal, and found it to be 100 eV at the $Q$-value of the decay. To completely reject the $\alpha$ background, we compute that one needs light detectors with baseline noise below 20 eV RMS, a value which is 3-4 times smaller than the average noise of the bolometric light detectors we are using. We point out that an improved light detector technology must be developed to obtain TeO$_2$ bolometric experiments able to probe the inverted hierarchy of neutrino masses.Comment: 5 pages, 4 figures. Added referee correction