Streptococcus pyogenes Φ1207.3 Is a Temperate Bacteriophage Carrying the Macrolide Resistance Gene Pair mef(A)-msr(D) and Capable of Lysogenizing Different Streptococci

Streptococcus pyogenes prophage phi 1207.3 (formerly Tn1207.3) carries the mef(A)-msr(D) resistance genes, responsible for type M macrolide resistance. To investigate if phi 1207.3 is a functional bacteriophage, we transferred the element from the original S. pyogenes host in a prophage-free and competence-deficient S. pneumoniae strain. Pneumococcal cultures of the phi 1207.3-carrying lysogen were treated with mitomycin C to assess if phi 1207.3 enters the lytic cycle. Mitomycin C induced a limited phage burst and a growth impairment, resulting in early entrance into the stationary phase. To determine if phi 1207.3 is able to produce mature phage particles, we prepared concentrated supernatants recovered from a mitomycin C-induced pneumococcal culture by sequential centrifugation and ultracentrifugation steps. Negative-staining transmission electron microscopy (TEM) of supernatants revealed the presence of phage particles with an icosahedral, electron-dense capsid and a long, noncontractile tail, typical of a siphovirus. Quantification of phi 1207.3 was performed by quantitative PCR (qPCR) and semiquantitatively by TEM. PCR quantified 3.34 x 10(4) and 6.06 x 10(4) excised forms of phage genome per milliliter of supernatant obtained from the untreated and mitomycin C-treated cultures, respectively. By TEM, we estimated 3.02 x 10(3) and 7.68 x 10(3) phage particles per milliliter of supernatant. The phage preparations of phi 1207.3 infected and lysogenized pneumococcal recipient strains at a frequency of 7.5 x 10(-6) lysogens/recipient but did not show sufficient lytic activity to form plaques. Phage lysogenization efficiently occurred after 30 min of contact of the phages with the recipient cells and required a minimum of 10(3) phage particles. © 2023 Santoro et al

The impact of national and international guidelines on newborn care in the nurseries of Piedmont and Aosta Valley, Italy

BACKGROUND: Care procedures for preventing neonatal diseases are carried out according to nurseries' traditions and may be not consistent with the evidence based medicine issues. METHODS: A multi-centric survey was conducted in 2 Regions located in NW Italy (Piedmont and Aosta Valley) in order to collect information on some healthy newborn care procedures. During 2001, a questionnaire was sent to the chief pediatrician in charge to the all 33 nurseries of the region asking the methods used during 2000 as prevention of ophthalmia neonatorum, early and late hemorrhagic disease of newborn, umbilical cord care and recommendations of vitamin D administration. Thereafter, during 2004 the same questionnaire was sent to the 34 chief pediatrician of nurseries to evaluate if the procedures were changed during 2003 according to guidelines. The nurseries care for 32,516 newborns in 2000 and 37,414 in 2003. RESULTS: Aminoglycoside eyes drops as prevention of ophthalmia neonatorum were the first choice in both periods (23 out 33 nurseries in 2000 and 24 out 34 in 2003 p > 0.05; the corresponding figures for newborns were18,984 out 32,516 newborns vs. 28,180 out of 37,414 p < 0.05). The umbilical cord care was carried out with alcohol in 12/33 centers (13,248 newborns) and dry gauze in 3/33 centers (2,130 newborns) in 2000, the corresponding figures in 2003 were 6/34 centers (p > 0.05), (6,380 newborns, p < 0.05) and 12/34 centers (p < 0.05), (18,123 newborns, p < 0.05). The percentage of newborns receiving of i.m. vitamin K. at birth increased during the study period (15,923/32,104 in 2000 vs. 19,684/37,414 in 2003, p < 0.01), but not the number of nurseries (16 in 2000 and 17 in 2003 p > 0.05). The numbers of parents of newborns who receive the recommendations of oral vitamin K during the first months life decreased from 2000 (25,516/30,606) to 2003 (29,808/37,414, p < 0.01) as well as for Vitamin D recommendation (14,582/30,616 in 2000 vs. 11,051/37,414 in 2003, p < 0.01). Oral vitamin K during the first months of life was recommended by 25 nurseries in 2000 and 27 in 2003 (p > 0.05), the corresponding figures for Vitamin D were 15 and 14 (p > 0.05). CONCLUSION: In the present study a large variability of procedures among the nurseries was observed. During the study periods, guidelines and evidence based medicine issues have only partially modified the neonatal care procedures In Piedmont and Aosta Valley nurseries. These observations suggest to implement local forum/consensus conference to standardized procedures as much as possible

Characterization of human frataxin missense variants in cancer tissues

Human frataxin is an iron binding protein involved in the mitochondrial Fe-S clusters assembly, a process fundamental for the functional activity of mitochondrial proteins. Decreased level of frataxin expression is associated with the neurodegenerative disease Friedreich ataxia. Defective function of frataxin may cause defects in mitochondria, leading to increased tumorigenesis. Tumour initiating cells show higher iron uptake, a decrease in iron storage and a reduced Fe-S clusters synthesis and utilization. In this study we selected, from COSMIC database, the somatic human frataxin missense variants found in cancer tissues p.D104G, p.A107V, p.F109L, p.Y123S, p.S161I, p.W173C, p.S181F, and p.S202F to analyze the effect of the single amino acid substitutions on frataxin structure, function and stability. The spectral properties, the thermodynamic and the kinetic stability, as well as the molecular dynamics of the frataxin missense variants found in cancer tissues point to local changes confined to the environment of the mutated residues. The global fold of the variants is not altered by the amino acid substitutions, however some of the variants show a decreased stability and a decreased functional activity in comparison to that of the wild type protein. This article is protected by copyright. All rights reserved

Optimization of left ventricular pacing site plus multipoint pacing improves remodeling and clinical response to cardiac resynchronization therapy at 1 year

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Multipoint pacing by a left ventricular quadripolar lead improves the acute hemodynamic response to CRT compared with conventional biventricular pacing at any site

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Longevity of Replaced ICD/CRT-D

Longevity of Replaced ICD/CRT-D Introduction The longevity of defibrillators (ICD) is extremely important from both a clinical and economic perspective. We studied the reasons for device replacement, the longevity of removed ICD, and the existence of possible factors associated with shorter service life. Methods and Results Consecutive patients who underwent ICD replacement from March 2013 to May 2015 in 36 Italian centers were included in this analysis. Data on replaced devices were collected. A total of 953 patients were included in this analysis. In 813 (85%) patients the reason for replacement was battery depletion, while 88 (9%) devices were removed for clinical reasons and the remaining 52 because of system failure (i.e., lead or ICD generator failure or a safety advisory indication). The median service life was 5.9 years (25th–75th percentile, 4.9–6.9) for single- and dual-chamber ICD and 4.9 years (25th–75th percentile, 4.0–5.7) for CRT-D. On multivariate analysis, the factors CRT-D device, SC/DC ICD generator from Biotronik, percentage of ventricular pacing, and the occurrence of a system failure were positively associated with a replacement procedure. By contrast, the device from Boston Scientific was an independent protective factor against replacement. Considerable differences were seen in battery duration in both ICD and CRT-D. Specifically, Biotronik devices showed the shortest longevity among ICD and Boston Scientific showed the longest longevity among CRT-D (log-rank test, P < 0.001 for pairwise comparisons). Conclusion Several factors were associated with shorter service life of ICD devices: CRT-D, occurrence of system failure and percentage of ventricular pacing. Our results confirmed significant differences among manufacturers

Heterologous Prime-Boost Combinations Highlight the Crucial Role of Adjuvant in Priming the Immune System

The induction and modulation of the immune response to vaccination can be rationally designed by combining different vaccine formulations for priming and boosting. Here, we investigated the impact of heterologous prime-boost approaches on the vaccine-specific cellular and humoral responses specific for a mycobacterial vaccine antigen. C57BL/6 mice were primed with the chimeric vaccine antigen H56 administered alone or with the CAF01 adjuvant, and boosted with H56 alone, or combined with CAF01 or with the squalene-based oil-in-water emulsion adjuvant (o/w squalene). A strong secondary H56-specific CD4+ T cell response was recalled by all the booster vaccine formulations when mice had been primed with H56 and CAF01, but not with H56 alone. The polyfunctional nature of T helper cells was analyzed and visualized with the multidimensional flow cytometry FlowSOM software, implemented as a package of the R environment. A similar cytokine profile was detected in groups primed with H56 + CAF01 and boosted with or without adjuvant, except for some clusters of cells expressing high level of IL-17 together with TNF-α, IL-2, and IFN-γ, that were significantly upregulated only in groups boosted with the adjuvants. On the contrary, the comparison between groups primed with or without the adjuvant showed a completely different clusterization of cells, strengthening the impact of the formulation used for primary immunization on the profiling of responding cells. The presence of the CAF01 adjuvant in the priming formulation deeply affected also the secondary humoral response, especially in groups boosted with H56 alone or o/w squalene. In conclusion, the presence of CAF01 adjuvant in the primary immunization is crucial for promoting primary T and B cell responses that can be efficiently reactivated by booster immunization also performed with antigen alone

Characterization of the Channel Constriction Allowing the Access of the Substrate to the Active Site of Yeast Oxidosqualene Cyclase

In oxidosqualene cyclases (OSCs), an enzyme which has been extensively studied as a target for hypocholesterolemic or antifungal drugs, a lipophilic channel connects the surface of the protein with the active site cavity. Active site and channel are separated by a narrow constriction operating as a mobile gate for the substrate passage. In Saccharomyces cerevisiae OSC, two aminoacidic residues of the channel/constriction apparatus, Ala525 and Glu526, were previously showed as critical for maintaining the enzyme functionality. In this work sixteen novel mutants, each bearing a substitution at or around the channel constrictions, were tested for their enzymatic activity. Modelling studies showed that the most functionality-lowering substitutions deeply alter the H-bond network involving the channel/constriction apparatus. A rotation of Tyr239 is proposed as part of the mechanism permitting the access of the substrate to the active site. The inhibition of OSC by squalene was used as a tool for understanding whether the residues under study are involved in a pre-catalytic selection and docking of the substrate oxidosqualene