Dual Role of Mitochondrial Porin in Metabolite Transport across the Outer Membrane and Protein Transfer to the Inner Membrane

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Validation of a universal set of primers to study animal-associated microeukaryotic communities

The application of metabarcoding to study animal-associated microeukaryotes has been restricted because the universal barcode used to study microeukaryotic ecology and distribution in the environment, the Small Subunit of the Ribosomal RNA gene (18S rRNA), is also present in the host. As a result, when host-associated microbial eukaryotes are analysed by metabarcoding, the reads tend to be dominated by host sequences. We have done an in silico validation against the SILVA 18S rRNA database of a non-metazoan primer set (primers that are biased against the metazoan 18S rRNA) that recovers only 2.6% of all the metazoan sequences, while recovering most of the other eukaryotes (80.4%). Among metazoans, the non-metazoan primers are predicted to amplify 74% of Porifera sequences, 4% of Ctenophora, and 15% of Cnidaria, while amplifying almost no sequences within Bilateria. In vivo, these non-metazoan primers reduce significantly the animal signal from coral and human samples, and when compared against universal primers provide at worst a 2-fold decrease in the number of metazoan reads and at best a 2800-fold decrease. This easy, inexpensive, and near-universal method for the study of animal-associated microeukaryotes diversity will contribute to a better understanding of the microbiome.     

Human chromosomal bands: nested structure,high-definition map and molecular basis

In this paper, we report investigations on the nested structure, the high-definition mapping, and the molecular basis of the classical Giemsa and Reverse bands in human chromosomes. We found the rules according to which the approximately 3,200 isochores of the human genome are assembled in high (850-band) resolution bands, and the latter in low (400-band) resolution bands, so forming the nested mosaic structure of chromosomes. Moreover, we identified the borders of both sets of chromosomal bands at the DNA sequence level on the basis of our recent map of isochores, which represent the highest-resolution, ultimate bands. Indeed, beyond the 100-kb resolution of the isochore map, the guanine and cytosine (GC) profile of DNA becomes turbulent owing to the contribution of specific sequences such as exons, introns, interspersed repeats, CpG islands, etc. The isochore-based level of definition (100 kb) of chromosomal bands is much higher than the cytogenetic definition level (2-3 Mb). The major conclusions of this work concern the high degree of order found in the structure of chromosomal bands, their mapping at a high definition, and the solution of the long-standing problem of the molecular basis of chromosomal bands, as these could be defined on the basis of compositional DNA properties alone.     

Different functional classes of genes are characterized by different compositional properties

A compositional analysis on a set of human genes classified in several functional classes was performed. We found out that the GC3, i.e. the GC level at the third codon positions, of the genes involved in cellular metabolism was significantly higher than those involved in information storage and processing. Analyses of human/Xenopus ortologous genes showed that: (i) the GC3 increment of the genes involved in cellular metabolism was significantly higher than those involved in information storage and processing; and (ii) a strong correlation between the GC3 and the corresponding GCi, i.e. the GC level of introns, was found in each functional class. The non-randomness of the GC increments favours the selective hypothesis of gene/genome evolution.     

Association between the ACCN1 gene and multiple sclerosis in Central East Sardinia

Multiple genome screens have been performed to identify regions in linkage or association with Multiple Sclerosis (MS, OMIM 126200), but little overlap has been found among them. This may be, in part, due to a low statistical power to detect small genetic effects and to genetic heterogeneity within and among the studied populations. Motivated by these considerations, we studied a very special population, namely that of Nuoro, Sardinia, Italy. This is an isolated, old, and genetically homogeneous population with high prevalence of MS. Our study sample includes both nuclear families and unrelated cases and controls. A multi-stage study design was adopted. In the first stage, microsatellites were typed in the 17q11.2 region, previously independently found to be in linkage with MS. One significant association was found at microsatellite D17S798. Next, a bioinformatic screening of the region surrounding this marker highlighted an interesting candidate MS susceptibility gene: the Amiloride-sensitive Cation Channel Neuronal 1 (ACCN1) gene. In the second stage of the study, we resequenced the exons and the 3' untranslated (UTR) region of ACCN1, and investigated the MS association of Single Nucleotide Polymorphisms (SNPs) identified in that region. For this purpose, we developed a method of analysis where complete, phase-solved, posterior-weighted haplotype assignments are imputed for each study individual from incomplete, multi-locus, genotyping data. The imputed assignments provide an input to a number of proposed procedures for testing association at a microsatellite level or of a sequence of SNPs. These include a Mantel-Haenszel type test based on expected frequencies of pseudocase/pseudocontrol haplotypes, as well as permutation based tests, including a combination of permutation and weighted logistic regression analysis. Application of these methods allowed us to find a significant association between MS and the SNP rs28936 located in the 3' UTR segment of ACCN1 with p = 0.0004 (p = 0.002, after adjusting for multiple testing). This result is in tune with several recent experimental findings which suggest that ACCN1 may play an important role in the pathogenesis of MS.     

Urine profiling by SELDI-TOF/MS: monitoring of the critical steps in sample collection, handling and analysis

The topic of this study is the impact of several pre-analytical and analytical variables on proteomic profiling of human urine by surface enhanced laser desorption/ionization time of flight-mass spectrometry (SELDI-TOF-MS) in healthy subjects. Urine storage at room temperature caused a progressive degradation of proteins, which was prevented by the addition of protease inhibitors only up to 2 h from the collection. The timing of collection over the day had only a minor impact on protein profile, although influencing the intensity of peaks. Repeated freeze/thaw cycles (up to five) did not affect either the number or the intensity of the peaks. A comparison of the protein profile from eight different healthy individuals showed fairly consistent inter-subject similarities, along with between-subject differences, which were markedly dependent on the sex and the type of ProteinChip array used. The addition of a variety of denaturing agents improved the quality of the spectra with all the chips tested (CM10, Q10 and H50), but not with the copper-coated IMAC-30 chip. Finally, SPA matrix allowed to achieve a better performance of SELDI-TOF/MS spectrum, as compared with CHCA, regardless of the ProteinChip array used and even in the low m/z range (2500-10,000). In conclusion, we suggest that a careful choice of a number of pre-analytical and analytical conditions is required to accomplish and define a unifying protocol for the analysis of human urine by SELDI-TOF/MS, in physiological and in pathological states.     

The strange case of a biofilm-forming strain of Pichia fermentans, which controls Monilinia brown rot on apple but is pathogenic on peach fruit

A biofilm-forming strain of Pichia fermentans proved to be most effective in controlling brown rot on apple fruit when coinoculated into artificial wounds with a phytopathogenic isolate of Monilinia fructicola. Culture filtrates and autoclaved cells had no significant influence on the disease. When sprayed onto the apple fruit surface, this yeast formed a thin biofilm but failed to colonize the underlying tissues. When inoculated into wounds artificially inflicted to peach fruit or when sprayed onto the surface of peach fruit, the same strain showed an unexpected pathogenic behaviour, causing rapid decay of fruit tissues even in the absence of M. fructicola. Both optical and scanning electron microscopy were used to evaluate the pattern of fruit tissue colonization by P. fermentans. While on apple surface and within the apple wound the antagonist retained its yeast-like shape, colonization of peach fruit tissue was always characterized by a transition from budding growth to pseudohyphal growth. These results suggest that pseudohyphal growth plays a major role in governing the potential pathogenicity of P. fermentans, further emphasizing the importance of a thorough risk assessment for the safe use of any novel biocontrol agent.     

Interactions of imidazoline ligands with the active site of purified monoamine oxidase A

The two forms of monoamine oxidase, monoamine oxidase A and monoamine oxidase B, have been associated with imidazoline-binding sites (type 2). Imidazoline ligands saturate the imidazoline-binding sites at nanomolar concentrations, but inhibit monoamine oxidase activity only at micromolar concentrations, suggesting two different binding sites [Ozaita A, Olmos G, Boronat MA, Lizcano JM, Unzeta M & García-Sevilla JA (1997) Br J Pharmacol121, 901-912]. When purified human monoamine oxidase A was used to examine the interaction with the active site, inhibition by guanabenz, 2-(2-benzofuranyl)-2-imidazoline and idazoxan was competitive with kynuramine as substrate, giving K(i) values of 3 microM, 26 microM and 125 microM, respectively. Titration of monoamine oxidase A with imidazoline ligands induced spectral changes that were used to measure the binding affinities for guanabenz (19.3 +/- 3.9 microM) and 2-(2-benzofuranyl)-2-imidazoline (49 +/- 8 microM). Only one type of binding site was detected. Agmatine, a putative endogenous ligand for some imidazoline sites, reduced monoamine oxidase A under anaerobic conditions, indicating that it binds close to the flavin in the active site. Flexible docking studies revealed multiple orientations within the large active site, including orientations close to the flavin that would allow oxidation of agmatine.