Iron Release in Erythrocytes and Plasma Non Protein-bound Iron in Hypoxic and Non Hypoxic Newborns

Iron is released in a desferrioxamine (DFO)-chelatable form (DCI) when erythrocytes are challenged by an oxidative stress. In &#103 -thalassemic erythrocytes, both DCI content and release (after aerobic incubation for 24 h) are increased and correlated with the fetal hemoglobin (HbF) levels. Since erythrocytes from newborns have an extremely high content of HbF and are exposed to conditions of oxidative stress, the release of iron in these erythrocytes was investigated. The erythrocyte DCI content was increased in preterm but not in term newborns as compared to adults, while the release was increased in both preterm and term erythrocytes. The level of plasma non protein-bound iron (NPBI), which was not detectable in adults, was much higher in preterm than in term newborns. When term plus preterm newborns were divided in two groups, normoxic and hypoxic, according to cord blood pH, it was found that both iron release and NBPI were markedly higher in the hypoxic newborns compared to normoxic ones. Similar results were also obtained when the preterm and term infants were considered separately on the basis of cord blood pH. Therefore, iron release and NPBI are higher when conditions of hypoxia occur. In fact, when the values for iron release and NPBI were separately plotted against cord blood pH values, significant negative correlations were seen in both cases. NPBI was correlated with iron release seen in all the newborns and a significant part of the released iron could be recovered into the incubation medium at the end of the incubation.     

Prolonged lifespan with enhanced exploratory behavior in mice overexpressing the oxidized nucleoside triphosphatase hMTH1

The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1‐Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1‐Tg mice express high levels of the hMTH1 hydrolase that degrades 8‐oxodGTP and 8‐oxoGTP and excludes 8‐oxoguanine from both DNA and RNA. Compared to wild‐type animals, hMTH1‐overexpressing mice have significantly lower steady‐state levels of 8‐oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age‐dependent accumulation of DNA 8‐oxoguanine that occurs in wild‐type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1‐Tg animals live significantly longer than their wild‐type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1‐Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1‐Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1‐Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids.     

Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD.     

Inhibition of Dual/Mixed Tropic HIV-1 Isolates by CCR5-Inhibitors in Primary Lymphocytes and Macrophages

Background

Dual/mixed-tropic HIV-1 strains are predominant in a significant proportion of patients, though little information is available regarding their replication-capacity and susceptibility against CCR5-antagonists in-vitro. The aim of the study was to analyze the replication-capacity and susceptibility to maraviroc of HIV-1 clinical isolates with different tropism characteristics in primary monocyte-derived-macrophages (MDM), peripheral-blood-mononuclear-cells (PBMC), and CD4⁺T-lymphocytes.

Methods

Twenty-three HIV-1 isolates were phenotipically and genotipically characterized as R5, X4 or dual (discriminated as R5⁺/X4, R5/X4, R5/X4⁺). Phenotypic-tropism was evaluated by multiple-cycles-assay on U87MG-CD4⁺-CCR5⁺−/CXCR4⁺-expressing cells. Genotypic-tropism prediction was obtained using Geno2Pheno-algorithm (false-positive-rate [FPR] = 10%). Replication-capacity and susceptibility to maraviroc were investigated in human-primary MDM, PBMC and CD4⁺T-cells. AMD3100 was used as CXCR4-inhibitor. Infectivity of R5/Dual/X4-viruses in presence/absence of maraviroc was assessed also by total HIV-DNA, quantified by real-time polymerase-chain-reaction.

Results

Among 23 HIV-1 clinical isolates, phenotypic-tropism-assay distinguished 4, 17 and 2 viruses with R5-tropic, dual/mixed-, and X4-tropic characteristics, respectively. Overall, viruses defined as R5⁺/X4-tropic were found with the highest prevalence (10/23, 43.5%). The majority of isolates efficiently replicated in both PBMC and CD4⁺T-cells, regardless of their tropism, while MDM mainly sustained replication of R5- or R5⁺/X4-tropic isolates; strong correlation between viral-replication and genotypic-FPR-values was observed in MDM (rho = 0.710;p-value = 1.4e-4). In all primary cells, maraviroc inhibited viral-replication of isolates not only with pure R5- but also with dual/mixed tropism (mainly R5⁺/X4 and, to a lesser extent R5/X4 and R5/X4⁺). Finally, no main differences by comparing the total HIV-DNA with the p24-production in presence/absence of maraviroc were found.

Conclusions

Maraviroc is effective in-vitro against viruses with dual-characteristics in both MDM and lymphocytes, despite the potential X4-mediated escape. This suggests that the concept of HIV-entry through one of the two coreceptors “separately” may require revision, and that the use of CCR5-antagonists in patients with dual/mixed-tropic viruses may be a therapeutic-option that deserves further investigations in different clinical settings.     

Protozoa and human macrophages infection by Legionella pneumophila environmental strains belonging to different serogroups

Three Legionella pneumophila strains isolated from municipal hot tap water during a multicentric Italian survey and belonging to serogroups 1, 6, 9 and the reference strain Philadelphia-1 were studied to determine the intracellular replication capability and the cytopathogenicity in human monocyte cell line U937 and in an Acanthamoeba polyphaga strain. Our results show that both serogroups 1 and Philadelphia-1 were able to multiply into macrophages inducing cytopathogenicity, while serogroup 6 and ever more serogroup 9 were less efficient in leading to death of the infected macrophages. Both serogroups 1 and 6 displayed a quite good capability of intracellular replication in A. polyphaga, although serogroup 1 was less cytopathogenic than serogroup 6. Serogroup 9, like Philadelphia-1 strain, showed a reduced efficiency of infection and replication and a low cytopathogenicity towards the protozoan. Our study suggests that bacterial pathogenesis is linked to the difference in the virulence expression of L. pneumophila serogroups in both hosts, as demonstrated by the fact that only L. pneumophila serogroup 1 shows the contextual expression of the two virulence traits. Serogroup 6 proves to be a good candidate as pathogen since it shows a good capacity for intracellular replication in protozoan.     

Regulated expression systems for the development of whole-cell biocatalysts expressing oxidative enzymes in a sequential manner

This work reports the preparation of two recombinant strains each containing two enzymatic activities mutually expressed through regulated systems for production of functionalized epoxides in one-pot reactions. One strain was Pseudomonas putida PaW340, containing the gene coding for styrene monooxygenase (SMO) from Pseudomonas fluorescens ST under the auto-inducing Ptou promoter and the TouR regulator of Pseudomonas sp. OX1 and the gene coding for naphthalene dihydrodiol dehydrogenase (NDDH) from P. fluorescens N3 under the Ptac promoter inducible by IPTG. The second strain was Escherichia coli JM109, in which the expression of SMO was under the control of the Pnah promoter and the NahR regulator of P. fluorescens N3 inducible by salicylate, while the gene expressing NDDH was under the control of the Plac promoter inducible by IPTG. SMO and NDDH activities were tested in bioconversion experiments using cinnamyl alcohol as reference substrate. The application that we selected is one example of the sequential use of the two enzymatic activities which require a temporal control of the expression of both genes.