Phylogenetic and Metabolic Tracking of Gut Microbiota during Perinatal Development

The colonization and development of gut microbiota immediately after birth is highly variable and depends on several factors, such as delivery mode and modality of feeding during the first months of life. A cohort of 31 mother and neonate pairs, including 25 at-term caesarean (CS) and 6 vaginally (V) delivered neonates (DNs), were included in this study and 121 meconium/faecal samples were collected at days 1 through 30 following birth. Operational taxonomic units (OTUs) were assessed in 69 stool samples by phylogenetic microarray HITChip and inter- and intra-individual distributions were established by inter-OTUs correlation matrices and OTUs co-occurrence or co-exclusion networks. ¹H-NMR metabolites were determined in 70 stool samples, PCA analysis was performed on 55 CS DNs samples, and metabolome/OTUs co-correlations were assessed in 45 CS samples, providing an integrated map of the early microbiota OTUs-metabolome. A microbiota “core” of OTUs was identified that was independent of delivery mode and lactation stage, suggesting highly specialized communities that act as seminal colonizers of microbial networks. Correlations among OTUs, metabolites, and OTUs-metabolites revealed metabolic profiles associated with early microbial ecological dynamics, maturation of milk components, and host physiology.     

Full Genotyping of a Highly Polymorphic Human Gene Trait by Time-Resolved Fluorescence Resonance Energy Transfer

The ability of detecting the subtle variations occurring, among different individuals, within specific DNA sequences encompassed in highly polymorphic genes discloses new applications in genomics and diagnostics. DQB1 is a gene of the HLA-II DQ locus of the Human Leukocyte Antigens (HLA) system. The polymorphisms of the trait of the DQB1 gene including codons 52–57 modulate the susceptibility to a number of severe pathologies. Moreover, the donor-receiver tissue compatibility in bone marrow transplantations is routinely assessed through crossed genotyping of DQB and DQA. For the above reasons, the development of rapid, reliable and cost-effective typing technologies of DQB1 in general, and more specifically of the codons 52–57, is a relevant although challenging task. Quantitative assessment of the fluorescence resonance energy transfer (FRET) efficiency between chromophores labelling the opposite ends of gene-specific oligonucleotide probes has proven to be a powerful tool to type DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, i.e. time-correlated single-photon counting, which allows working on very diluted template specimens and in the presence of fluorescent contaminants. Here we present a full in-vitro characterization of the fluorescence responses of two probes when hybridized to oligonucleotide mixtures mimicking all the possible genotypes of the codons 52–57 trait of DQB1 (8 homozygous and 28 heterozygous). We show that each genotype can be effectively tagged by the combination of the fluorescence decay constants extrapolated from the data obtained with such probes.     

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.     

Zileuton Improves Memory Deficits,Amyloid and Tau Pathology in a Mouse Model of Alzheimer’s Disease with Plaques and Tangles

The 5-lipoxygenase (5LO) enzyme is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer’s disease (AD), and plays an active role in the development of brain amyloidosis in the APP transgenic mice. In the present paper, we studied the effect of its pharmacological inhibition on the entire AD-like phenotype of a mouse model with plaques and tangles, the 3×Tg mice. Compared with mice receiving placebo, the group treated with zileuton, a specific 5LO inhibitor, manifested a significant improvement of their memory impairments. The same animals had a significant reduction in Aβ levels and deposition, which was secondary to a down-regulation of the γ-secretase pathway. Additionally, while total tau levels were unchanged for both groups, zileuton-treated mice had a significant reduction in its phosphorylation state and insoluble forms, secondary to a decreased activation of the cdk5 kinase. These data establish a functional role for 5LO in the pathogenesis of the full spectrum of the AD-like phenotype and represent the successful completion of the initial step for the preclinical development of 5LO inhibitors as viable therapeutic agents for AD.     

Confirmed association between a single nucleotide polymorphism in the <Emphasis Type="Italic">FTO</Emphasis> gene and obesity-related traits in heavy pigs

We recently showed that a polymorphism in the fat mass and obesity associated (FTO) gene (AM931150: g.276T > G) is associated with fat deposition traits in pigs. To confirm this result, we genotyped this polymorphism in an Italian Duroc population made up by 313 performance tested pigs with known estimated breeding values (EBVs) for average daily gain, back fat thickness (BFT), feed:gain ratio, lean cuts (LC), and visible intermuscular fat (VIF, a measure of intermuscular fat in the hams). In addition, we genotyped 148 commercial heavy pigs for which several fat deposition traits and lean meat percentage were measured. The results of the association analyses confirmed the effect of the FTO mutation on obesity-related traits (VIF, BFT and LC) in the Italian Duroc pigs (P < 0.01) and in the commercial pigs (intramuscular fat content of different muscles, P < 0.05 or P < 0.10; lean meat content, P < 0.05; BFT, P < 0.05; intermuscular fat content in the hams, P < 0.05).     

Impact of drought and increasing temperatures on soil CO2 emissions in a Mediterranean shrubland (gariga)

In arid and semiarid shrubland ecosystems of the Mediterranean basin, soil moisture is a key factor controlling biogeochemical cycles and the release of CO₂ via soil respiration. This is influenced by increasing temperatures. We manipulated the microclimate in a Mediterranean shrubland to increase the soil and air night-time temperatures and to reduce water input from precipitation. The objective was to analyze the extent to which higher temperatures and a drier climate influence soil CO₂ emissions in the short term and on an annual basis. The microclimate was manipulated in field plots (about 25 m²) by covering the vegetation during the night (Warming treatment) and during rain events (Drought treatment). Soil CO₂ effluxes were monitored in the treatments and compared to a control over a 3-year period. Along with soil respiration measurements, we recorded soil temperature at 5 cm depth by a soil temperature probe. The seasonal pattern of soil CO₂ efflux was characterized by higher rates during the wet vegetative season and lower rates during the dry non-vegetative season (summer). The Warming treatment did not change SR fluxes at any sampling date. The Drought treatment decreased soil CO₂ emissions on only three of 10 occasions during 2004. The variation of soil respiration with temperature and soil water content did not differ significantly among the treatments, but was affected by the season. The annual CO₂ emissions were not significantly affected by the treatments. In the semi-arid Mediterranean shrubland, an increase of soil CO₂ efflux in response to a moderate increase of daily minimum temperature is unlikely, whereas less precipitation can strongly affect the soil processes mainly limited by water availability.     

Occurrence and characterization of psychrotolerant hydrocarbon-oxidizing bacteria from surface seawater along the Victoria Land coast (Antarctica)

A total of 253 hydrocarbon-oxidizing bacterial isolates were achieved from eight Antarctic surface seawater samples enriched on diesel oil at 4°C. Isolates were screened by amplified ribosomal DNA restriction analysis prior to 16S rRNA gene sequencing. Sequences were compared to those in available databases using the Basic Local Alignment Search Tool network service to determine their approximate phylogenetic affiliations. The majority of the isolates were affiliated to the Actinobacteria (75.9%) and the Gamma-Proteobacteria (22.9%). The Alpha- and Beta-Proteobacteria represented 0.8 and 0.4% of total isolates, respectively. The Actinobacteria were predominantly allocated to the genera Arthrobacter, Cryobacterium and Rhodococcus. The Gamma-Proteobacteria were mainly found to be related to the genus Pseudomonas. Conversely, the Alpha- and Beta-Proteobacterial isolates shared the highest degree of sequence identity with unclassified bacteria. Differences in the distribution of the detected phylotypes were observed among the analyzed samples. Isolates representing each phylotype were selected for further characterization, including phenotypic assays and screening for the growth ability in the presence of individual hydrocarburic substrates as the sole supplied carbon and energy source. Isolates possessed different patterns of substrate utilization. Aliphatic hydrocarbons supported the growth of a higher number of isolates than aromatics. Results confirm the ability of our Antarctic marine bacteria to utilize hydrocarbons at low temperature and therefore suggesting that isolates with different substrate specificities can act in nature as a consortium in the utilization of complex hydrocarburic mixtures.