Prevalence and follow-up of occult HCV infection in an italian population free of clinically detectable infectious liver disease

Background

Occult hepatitis C virus infection (OCI) is a recently described phenomenon characterized by undetectable levels of HCV-RNA in serum/plasma by current laboratory assays, with identifiable levels in peripheral blood mononuclear cells (PBMCs) and/or liver tissue by molecular tests with enhanced sensitivity. Previous results from our group showed an OCI prevalence of 3.3% in a population unselected for hepatic disease. The present study aimed to evaluate OCI prevalence in a larger cohort of infectious liver disease-free (ILDF) subjects. Clinical follow-up of OCI subjects was performed to investigate the natural history of the infection.

Methods and Findings

439 subjects referred to a Turin Blood Bank for phlebotomy therapy were recruited. They included 314 ILDF subjects, 40 HCV-positive subjects and 85 HBV-positive subjects, of whom 7 were active HBV carriers. Six subjects (4/314 ILDF subjects [1.27%] and 2/7 active HBV carriers [28%]) were positive for HCV-RNA in PBMCs, but negative for serological and virological markers of HCV, indicating OCI. HCV genotypes were determined in the PBMCs of 3/6 OCI subjects two had type 1b; the other had type 2a/2c. OCI subjects were followed up for at least 2 years. After 12 months only one OCI persisted, showing a low HCV viral load (3.73×10¹ UI/ml). By the end of follow-up all OCI subjects were negative for HCV. No seroconversion, alteration of liver enzyme levels, or reduction of liver synthesis occurred during follow-up.

Conclusions

This study demonstrated the existence of OCI in ILDF subjects, and suggested a high OCI prevalence among active HBV carriers. Follow-up suggested that OCI could be transient, with a trend toward the decrease of HCV viral load to levels undetectable by conventional methods after 12–18 months. Confirmation studies with a longer follow-up period are needed for identification of the OCI clearance or recurrence rates, and to characterize the viruses involved.     

Lack of the mitochondrial protein acylglycerol kinase causes Sengers syndrome

Exome sequencing of an individual with congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis, all typical symptoms of Sengers syndrome, discovered two nonsense mutations in the gene encoding mitochondrial acylglycerol kinase (AGK). Mutation screening of AGK in further individuals with congenital cataracts and cardiomyopathy identified numerous loss-of-function mutations in an additional eight families, confirming the causal nature of AGK deficiency in Sengers syndrome. The loss of AGK led to a decrease of the adenine nucleotide translocator in the inner mitochondrial membrane in muscle, consistent with a role of AGK in driving the assembly of the translocator as a result of its effects on phospholipid metabolism in mitochondria.     

Geometric morphometrics throws light on evolution of the subterranean catfish Rhamdiopsis krugi (Teleostei: Siluriformes: Heptapteridae) in eastern Brazil

Rhamdiopsis krugi is a highly specialized troglobitic (exclusively subterranean) catfish from phreatic water bodies of caves located within two separated metasedimentary basins in the region of Chapada Diamantina, Bahia state, Brazil. In order to test the hypothesis of isolation with differentiation of the groups from the Una‐Utinga and Irecê metasedimentary basins, we compared five populations among themselves and with an epigean species of Rhamdiopsis. This was accomplished using geometric morphometrics, a powerful tool for detecting differences in body shape at population and species levels. All studied samples differed significantly from each other, the epigean sample being the most distinct and the Una Basin populations clustering together. Geological and hydrological barriers explain the differences among the subterranean populations. We discuss our results together with the autapomorphies found in R. krugi, which validate its monophyly. These results imply an old age for the R. krugi clade, more than 10 Myr; alternative hypotheses are also presented. We propose a two‐step vertical colonization model of the subterranean habitat through the hyporheic zone by an epigean ancestral, with a progressive acquisition of the autapomorphies characterizing R. krugi. For conservation purposes, the two differentiated sets of populations should be considered and referred to as R. krugi ‘Una morphotype’ and R. krugi ‘Irecê morphotype’. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 114 , 136–151.     

The sexual phase of the diatom <Emphasis Type="Italic">Pseudo-nitzschia multistriata</Emphasis>: cytological and time-lapse cinematography characterization

Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.     

Expression level of heterologous tat genes is crucial for in vivo reconstitution of a functional Tat translocase in Escherichia coli

The Tat system has the remarkable capacity of exporting proteins in folded conformation across the cytoplasmic membrane. The functional Tat translocase from Gram-negative bacteria consists of TatA, TatB and TatC proteins. To gain information about the species specificity of the Tat translocase, we cloned tat genes from Gram-negative pathogens Shigella flexneri 2a str. 301, Vibrio cholerae El Tor N16961, Pseudomonas aeruginosa PAO1, thermophilic Sulfolobus solfataricus P2, Thermus thermophilus HB8 and from three Magnetospirillum species (AMB-1, MS-1 and MSR-1), and assessed the capacity of these Tat systems to restore the Tat-dependent growth defect of Escherichia coli tat mutants. We found that whereas the tat genes from the thermophilic bacterial and archaeal species were not functional in E. coli, other tat genes could all complement the phenotype of the E. coli tat mutants. In addition, a chimera composed of the N-terminus of V. cholerae TatE and C-terminus of M. magneticum TatA was functional. Whereas the expression of the tatABC genes from P. aeruginosa and Magnetospirillum strains must be induced to obtain a functional Tat system, overproduction of the V. cholerae TatABC proteins abolished the complementation. The complementation impairment seemed to be correlated with increasing level of slow-migrating TatC isoforms. In vitro studies showed that slow-migrating TatC isoforms in the purified V. cholerae TatABC complex increased with storage time. Together these results showed that the Tat translocases from the Gram-negative bacteria are generally functional in E. coli and the expression level is crucial for in vivo reconstitution of a functional Tat translocase.     

Helical screw sense of peptide molecules: The pentapeptide system (Aib)4/L-Val[L-(αMe)Val] in the crystal state

The crystal-state preferred conformations of six N^α-blocked pentapeptide esters, each containing four helicogenic, achiral α-aminoisobutyric acid (Aib) residues followed by one chiral L -valine (L -Val) or C^α-methyl-L -valine [(αMe)Val] residue at the C-terminus, have been assessed by x-ray diffraction analysis. In all of the compounds the  (Aib)₄ sequence is folded in a regular 3₁₀-helical conformation. In the four pentapeptides characterized by the L -(αMe)Val residue two conformationally distinct molecules occur in the asymmetric unit. Conversely, only one molecule is observed in the asymmetric unit of two pentapeptides with the C-terminal L -Val residue. In the L -Val based peptides the helical screw sense of the  (Aib)₄ sequence is right-handed, whereas in the L  (αMe)Val analogues both right- and left-handed helical screw senses concomitantly occur in the two crystallographically independent molecules. © 1998 John Wiley & Sons, Inc. Biopoly 46: 433–443, 1998     

X-ray structures of new dipeptide taste ligands

The molecular basis of sweet taste was investigated by carrying out the crystal state conformational analysis by X-ray diffraction of the following dipeptide taste igands:N-3,3-dimethylbutyl-aspartyl-phenylalanine methyl ester, I (N-DMB-Asp-Phe-OMe), its sodium salt (N-DMB-Asp-Phe-ONa), II , aspartyl-D -2-aminobutyric acid-(S)-α-ethylbenzylamide, III (Asp-D -Abu-(S)-α-ethylbenzylamide), aspartyl-N′-((2,2,5,5-tetramethylcyclopentanyl)-carbonyl)-(R)-1,1-diamino-ethane, IV (Asp-(R)-gAla-TMCP), and aspartyl-D -valine-(R)-α-methoxymethylbenzyl amide, V (Asp-D -Val-(R)-α-methoxymethylbenzylamide). With the exception of the sodium salt II , all compounds are sweet-tasting, showing in some cases considerable potency enhancement with respect to sucrose. The results of this study confirm the earlier model that an ‘L-shape’ molecular array is essential for eliciting sweet taste for dipeptide-like ligands. In addition, it was established that (i) substitution of the N-terminal group does not inhibit sweet taste, if its zwitterionic character is maintained; (ii) a hydrophobic group located between the stem and the base of the L-shape could be responsible for sweetness potency enhancement, as found in I, III and IV ; in fact, the extraordinary potency of the N-alkylated analogue I would support a model with an additional hydrophobic binding domain above the base of the ‘L’; (iii) removal of the methyl ester at the C-terminus of compound I with the salt formation gives rise to the tasteless compound II ; (iv) for the first time all possible side-chain conformers (g⁻,g⁺andt) for the N-substituted aspartyl residue were observed; and (v) a retro-inverso modification, incorporated at position 2 of the dipeptide chain, confers greater flexibility to the molecule, as demonstrated by the contemporary presence of six conformationally distinct independent molecules in the unit cell and yet sweet taste properties are maintained, as found in IV . © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Oral administration of trans-resveratrol to guinea pigs increases cardiac DT-diaphorase and catalase activities,and protects isolated atria from menadione toxicity

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine. It has antioxidant and antiproliferative activities, and has been shown to induce NAD(P)H:quinone oxidoreductase, also known as DT-diaphorase, in cultured mouse hepatoma cells. DT-diaphorase is a detoxifying enzyme for quinone-containing substances, due to its ability to prevent their one-electron reduction and the consequent generation of reactive oxygen species (ROS). The aim of the present study was to investigate whether oral administration of trans-resveratrol to guinea pigs (60 mg/l in tap water for 16 days, ad libitum) increases cardiac DT-diaphorase and, consequently, reduces the response of isolated atria to 2-methyl-1,4-naphthoquinone (menadione), the positive inotropic effect of which is related to the amount of ROS generated by its cardiac metabolism. In the cardiac tissue of resveratrol-treated animals, DT-diaphorase activity was significantly higher than that measured in control animals, the V(max) of the enzyme reaction being 75.47 +/- 3.87 and 50.73 +/- 0.63 nmoles/mg protein/min, respectively (p < 0.05). Resveratrol administration also significantly increased the activity of cardiac catalase (32.20 +/- 2.39 vs. 25.14 +/- 3.85 units/mg protein in treated and control animals, respectively; p < 0.001). As a consequence, menadione metabolism by the cardiac homogenate obtained from resveratrol-treated animals generated a smaller amount of ROS and, in electrically driven left atria, menadione produced a significantly lower increase in the force of contraction than in atria isolated from control animals. These results indicate that oral administration of resveratrol exerts cardioprotection against ROS-mediated menadione toxicity.