Ribosomal selection of mRNAs with degenerate initiation triplets

To assess the influence of degenerate initiation triplets on mRNA recruitment by ribosomes, five mRNAs identical but for their start codon (AUG, GUG, UUG, AUU and AUA) were offered to a limiting amount of ribosomes, alone or in competition with an identical AUGmRNA bearing a mutation conferring different electrophoretic mobility to the product. Translational efficiency and competitiveness of test mRNAs toward this AUGmRNA were determined quantifying the relative amounts of the electrophoretically separated wt and mutated products synthesized in vitro and found to be influenced to different extents by the nature of their initiation triplet and by parameters such as temperature and nutrient availability in the medium. The behaviors of AUAmRNA, UUGmRNA and AUGmRNA were the same between 20 and 40°C whereas the GUG and AUUmRNAs were less active and competed poorly with the AUGmRNA, especially at low temperature. Nutrient limitation and preferential inhibition by ppGpp severely affected activity and competitiveness of all mRNAs bearing non-AUG starts, the UUGmRNA being the least affected. Overall, our data indicate that beyond these effects exclusively due to the degenerate start codons within an optimized translational initiation region, an important role is played by the context in which the rare start codons are present

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

Elastin-coated biodegradable photopolymer scaffolds for tissue engineering applications

One of the main open issues in modern vascular surgery is the nonbiodegradability of implants used for stent interventions, which can lead to small caliber-related thrombosis and neointimal hyperplasia. Some new, resorbable polymeric materials have been proposed to substitute traditional stainless-steel stents, but so far they were affected by poor mechanical properties and low biocompatibility. In this respect, a new material, polypropylene fumarate (PPF), may be considered as a promising candidate to implement the development of next generation stents, due to its complete biodegradability, and excellent mechanical properties and the ease to be precisely patterned. Besides all these benefits, PPF has not been tested yet for vascular prosthesis, mainly because it proved to be almost inert, while the ability to elicit a specific biological function would be of paramount importance in such critical surgery applications. Here, we propose a biomimetic functionalization process, aimed at obtaining specific bioactivation and thus improved cell-polymer interaction. Porous PPF-based scaffolds produced by deep-UV photocuring were coated by elastin and the functionalized scaffolds were extensively characterized, revealing a stable bound between the protein and the polymer surface. Both 3T3 and HUVEC cell lines were used for in vitro tests displaying an enhancement of cells adhesion and proliferation on the functionalized scaffolds

X chromosome inactivation does not necessarily determine the severity of the phenotype in Rett syndrome patients

Rett syndrome; X chromosome inactivation; MECP2 geneSíndrome de Rett; Inactivación del cromosoma X; Gen MECP2Síndrome de Rett; Inactivació del cromosoma X; Gen MECP2Rett syndrome (RTT) is a severe neurological disorder usually caused by mutations in the MECP2 gene. Since the MECP2 gene is located on the X chromosome, X chromosome inactivation (XCI) could play a role in the wide range of phenotypic variation of RTT patients; however, classical methylation-based protocols to evaluate XCI could not determine whether the preferentially inactivated X chromosome carried the mutant or the wild-type allele. Therefore, we developed an allele-specific methylation-based assay to evaluate methylation at the loci of several recurrent MECP2 mutations. We analyzed the XCI patterns in the blood of 174 RTT patients, but we did not find a clear correlation between XCI and the clinical presentation. We also compared XCI in blood and brain cortex samples of two patients and found differences between XCI patterns in these tissues. However, RTT mainly being a neurological disease complicates the establishment of a correlation between the XCI in blood and the clinical presentation of the patients. Furthermore, we analyzed MECP2 transcript levels and found differences from the expected levels according to XCI. Many factors other than XCI could affect the RTT phenotype, which in combination could influence the clinical presentation of RTT patients to a greater extent than slight variations in the XCI pattern.The work was supported by grants from the Spanish Ministry of Health (Instituto de Salud Carlos III/FEDER, PI15/01159); Crowdfunding program PRECIPITA, from the Spanish Ministry of Health (Fundacion Espanola para la Ciencia y la Tecnologia); the Catalan Association for Rett Syndrome; Fondobiorett and Mi Princesa Rett

Structure of a 30S pre-initiation complex stalled by GE81112 reveals structural parallels in bacterial and eukaryotic protein synthesis initiation pathways

In bacteria, the start site and the reading frame of the messenger RNA are selected by the small ribosomal subunit (30S) when the start codon, typically an AUG, is decoded in the P-site by the initiator tRNA in a process guided and controlled by three initiation factors. This process can be efficiently inhibited by GE81112, a natural tetrapeptide antibiotic that is highly specific toward bacteria. Here GE81112 was used to stabilize the 30S pre-initiation complex and obtain its structure by cryo-electron microscopy. The results obtained reveal the occurrence of changes in both the ribosome conformation and initiator tRNA position that may play a critical role in controlling translational fidelity. Furthermore, the structure highlights similarities with the early steps of initiation in eukaryotes suggesting that shared structural features guide initiation in all kingdoms of life

Targeting SHIP-1 in Myeloid Cells Enhances Trained Immunity and Boosts Response to Infection

beta-Glucan-induced trained immunity in myeloid cells leads to long-term protection against secondary infections. Although previous studies have characterized this phenomenon, strategies to boost trained immunity remain undefined. We found that beta-glucan-trained macrophages from mice with a myeloid-specific deletion of the phosphatase SHIP-1 (LysM Delta SHIP-1) showed enhanced proinflammatory cytokine production in response to lipopolysaccharide. Following beta-glucan training, SHIP-1-deficient macrophages exhibited increased phosphorylation of Akt and mTOR targets, correlating with augmented glycolytic metabolism. Enhanced training in the absence of SHIP-1 relied on histone methylation and acetylation. Trained LysM Delta SHIP-1 mice produced increased amounts of proinflammatory cytokines upon rechallenge in vivo and were better protected against Candida albicans infection compared with control littermates. Pharmacological inhibition of SHIP-1 enhanced trained immunity against Candida infection in mouse macrophages and human peripheral blood mononuclear cells. Our data establish proof of concept for improvement of trained immunity and a strategy to achieve it by targeting SHIP-1.We thank the members of the Immunobiology Lab for useful discussions. We thank the CNIC facilities and personnel, particularly Santiago Rodriguez and Ruben Mota, for their support. P.S.-L. is funded by grant BES-2015-072699 (´´Ayudas para Contratos Predoctorales para la Formacion de Doctores 2015´´) from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO). C.d.F. is supported by the Asociacion Espanola Contra el Cancer (AECC) Foundation as a recipient of an ``Ayuda Fundacion Cientifica AECC a Personal Investigador en Cancer´´ grant. Work in the Sancho laboratory is funded by CNIC and grant SAF2016-79040-R from MINECO, Agencia Estatal de Investigacion, and FEDER (European Fund for Regional Development); grant B2017/BMD-3733 Immunothercan-CM from Comunidad de Madrid; grant RD16/0015/0018-REEM from FIS-Instituto de Salud Carlos III, MINECO, and FEDER; Foundation Acteria; a Constantes y Vitales prize (Atresmedia); Foundation La Marato de TV3 (grant 201723); the European Commission (grant 635122-PROCROP H2020); and the European Research Council (ERC-2016-Consolidator Grant 725091). CNIC is supported by MINECO and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). W.G.K. is an Empire Scholar of the State of New York, the Murphy Family Professor of Children's Oncology Research, and is supported by funds from the Paige Arnold Butterfly Run.S

Experimental study for evaluation of a suitable ground displacement monitoring system: Pilot hole Campi Flegrei Deep Drilling Project case

The paper presents an experimental study carried out in 2012 during the drilling activity for a pilot hole performed in the framework of the Campi Flegrei Deep Drilling Project. A monitoring network has been installed to test and choose a suitable ground deformation system for the subsequent deep drilling of about 3.5 km in the Campi Flegrei Caldera (Italy). We describe the seismic network installed to characterize the structure of the pilot hole area and the ground deformation monitoring system chosen for the small drilling area. Data analysis and results obtained indicate that Total Station is a suitable tool for this case.Published4V. Dinamica dei processi pre-eruttiviN/A or not JC

Therapeutic Effects of the Superoxide Dismutase Mimetic Compound Mn(II)Me2DO2A on Experimental Articular Pain in Rats.

Superoxide anion () is overproduced in joint inflammation, rheumatoid arthritis, and osteoarthritis. Increased production leads to tissue damage, articular degeneration, and pain. In these conditions, the physiological defense against , superoxide dismutases (SOD) are decreased. The complex MnL4 is a potent SOD mimetic, and in this study it was tested in inflammatory and osteoarticular rat pain models. In vivo protocols were approved by the animal Ethical Committee of the University of Florence. Pain was measured by paw pressure and hind limb weight bearing alterations tests. MnL4 (15 mg kg−1) acutely administered, significantly reduced pain induced by carrageenan, complete Freund’s adjuvant (CFA), and sodium monoiodoacetate (MIA). In CFA and MIA protocols, it ameliorated the alteration of postural equilibrium. When administered by osmotic pump in the MIA osteoarthritis, MnL4 reduced pain, articular derangement, plasma TNF alpha levels, and protein carbonylation. The scaffold ring was ineffective. MnL4 (10−7 M) prevented the lipid peroxidation of isolated human chondrocytes when was produced by RAW 264.7. MnL4 behaves as a potent pain reliever in acute inflammatory and chronic articular pain, being its efficacy related to antioxidant property. Therefore MnL4 appears as a novel protective compound potentially suitable for the treatment of joint diseases

Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding

In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state

Von Hippel-Lindau protein is required for optimal alveolar macrophage terminal differentiation, self-renewal, and function

The rapid transit from hypoxia to normoxia in the lung that follows the first breath in newborn mice coincides with alveolar macrophage (AM) differentiation. However, whether sensing of oxygen affects AM maturation and function has not been previously explored. We have generated mice whose AMs show a deficient ability to sense oxygen after birth by deleting Vhl, a negative regulator of HIF transcription factors, in the CD11c compartment (CD11c Delta Vhl mice). VHL-deficient AMs show an immature-like phenotype and an impaired self-renewal capacity in vivo that persists upon culture ex vivo. VHL-deficient phenotype is intrinsic in AMs derived from monocyte precursors in mixed bone marrow chimeras. Moreover, unlike control Vhl(fl/fl), AMs from CD11c Delta Vhl mice do not reverse pulmonary alveolar proteinosis when transplanted into Csf2rb(-/)(-) mice, demonstrating that VHL contributes to AM-mediated surfactant clearance. Thus, our results suggest that optimal AM terminal differentiation, self-renewal, and homeostatic function requires their intact oxygen-sensing capacity