Origins and Evolution of the Etruscans’ mtDNA

The Etruscan culture is documented in Etruria, Central Italy, from the 8^th to the 1^st century BC. For more than 2,000 years there has been disagreement on the Etruscans’ biological origins, whether local or in Anatolia. Genetic affinities with both Tuscan and Anatolian populations have been reported, but so far all attempts have failed to fit the Etruscans’ and modern populations in the same genealogy. We extracted and typed the hypervariable region of mitochondrial DNA of 14 individuals buried in two Etruscan necropoleis, analyzing them along with other Etruscan and Medieval samples, and 4,910 contemporary individuals from the Mediterranean basin. Comparing ancient (30 Etruscans, 27 Medieval individuals) and modern DNA sequences (370 Tuscans), with the results of millions of computer simulations, we show that the Etruscans can be considered ancestral, with a high degree of confidence, to the current inhabitants of Casentino and Volterra, but not to the general contemporary population of the former Etruscan homeland. By further considering two Anatolian samples (35 and 123 individuals) we could estimate that the genetic links between Tuscany and Anatolia date back to at least 5,000 years ago, strongly suggesting that the Etruscan culture developed locally, and not as an immediate consequence of immigration from the Eastern Mediterranean shores.     

IL28B Gene Polymorphisms and US Liver Fatty Changes in Patients Who Spontaneously Cleared Hepatitis C Virus Infection

Background

Recent clinical studies have shown that the presence of CC genotype in the rs12979860 region of IL28B gene is associated with an increase in the probability of spontaneous clearance of hepatitis C virus (HCV). Moreover, IL28B polymorphism seems to influence the probability of developing liver steatosis in chronic HCV patients.

Aims

The aims of our clinical study were 1) to verify the distribution of IL28B genotypes (CC, CT or TT) among subjects with spontaneous clearance of HCV infection and 2) to examine the correlation between IL28B polymorphism and hepatic steatosis among these subjects.

Methods and patients

We enrolled 41 subjects with spontaneous resolution of HCV infection (detectable serum anti-HCV but undetectable HCV-RNA) and 134 healthy controls from the same geographical area. The IL28B single-nucleotide polymorphism (SNP) rs12979860 was genotyped by using a Pyrosequencing™ technique. The presence of steatosis was assessed by liver biopsy or ultrasound examination in the 41 study subjects.

Results

CC, CT and TT-genotypes of the SNP rs1979860 were found in 66%, 24% and 10% of the subjects who spontaneously cleared HCV and in 31%, 54% and 15% of controls, respectively (p = 0.0003). Among the study subjects, females with CC-genotype were significantly more represented (p = 0.02). Hepatic steatosis did not correlate with IL28B genotype (p = 0,14) but only with a high body mass index (BMI) value (p = 0.03).

Conclusions

Female subjects carrying IL28B CC-genotype are significantly more represented among Italian patients who spontaneously cleared HCV infection. In addition, among these subjects, the presence of liver steatosis does not correlate with IL28B genotype but is solely related to the occurrence of high BMI. Thus, the association between IL28B polymorphism and steatosis in chronic HCV patients requires the presence of active HCV replication to occur, while in subjects who have cleared the infection, the mechanism(s) inducing liver steatosis are independent from IL28B profile.     

Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.     

Quantitative variations of the isoforms in haptoglobin 1-2 and 2-2 individual phenotypes

Haptoglobin is a hemoglobin-binding protein presenting in humans three distinct phenotypes (Hpt 1-1, Hpt 1-2, or Hpt 2-2). The Hpt 1-2 and Hpt 2-2 phenotypes are in turn represented by populations of isoforms. The relative amounts of the major isoforms of Hpt 1-2 and Hpt 2-2 were found to differ not only in different individuals, but also in the same individual before and after a physical effort. Exercise-dependent changes in the plasma concentrations of ascorbate, urate, alpha-tocopherol, retinol, and glutathione were also observed, but correlations between such changes and those of the amount for any isoform were not found. Samples of Hpt 1-2 or Hpt 2-2 were challenged with oxidants (H(2)O(2) with ferrous ions, spermine-NO, KO(2), and 3-morpholinosydnonimine), but the isoform levels were not altered. Hpt 2-2 isoforms were present in Hpt 1-2, as minor species. Furthermore, different isoforms exhibited different hemoglobin binding abilities. Thus, these parameters should also be taken into consideration in studies correlating Hpt phenotypes prevalence with pathologies or functional differences.     

Pulsed electromagnetic fields promote repair of focal articular cartilage defects with engineered osteochondral constructs

Articular cartilage injuries are a common source of joint pain and dysfunction. We hypothesized that pulsed electromagnetic fields (PEMFs) would improve growth and healing of tissue-engineered cartilage grafts in a direction-dependent manner. PEMF stimulation of engineered cartilage constructs was first evaluated in vitro using passaged adult canine chondrocytes embedded in an agarose hydrogel scaffold. PEMF coils oriented parallel to the articular surface induced superior repair stiffness compared to both perpendicular PEMF (p = .026) and control (p = .012). This was correlated with increased glycosaminoglycan deposition in both parallel and perpendicular PEMF orientations compared to control (p = .010 and .028, respectively). Following in vitro optimization, the potential clinical translation of PEMF was evaluated in a preliminary in vivo preclinical adult canine model. Engineered osteochondral constructs (∅ 6 mm × 6 mm thick, devitalized bone base) were cultured to maturity and implanted into focal defects created in the stifle (knee) joint. To assess expedited early repair, animals were assessed after a 3-month recovery period, with microfracture repairs serving as an additional clinical control. In vivo, PEMF led to a greater likelihood of normal chondrocyte (odds ratio [OR]: 2.5, p = .051) and proteoglycan (OR: 5.0, p = .013) histological scores in engineered constructs. Interestingly, engineered constructs outperformed microfracture in clinical scoring, regardless of PEMF treatment (p < .05). Overall, the studies provided evidence that PEMF stimulation enhanced engineered cartilage growth and repair, demonstrating a potential low-cost, low-risk, noninvasive treatment modality for expediting early cartilage repair.     

A novel CD4 T-cell epitope described from one of the cervical cancer patients vaccinated with HPV 16 or 18 E7-pulsed dendritic cells

Previously, safety and immunogenicity of human papillomavirus type 16 (HPV16) or 18 E7-pulsed dendritic cells (DC) vaccinations were demonstrated in a dose-escalation Phase I clinical trial which enrolled ten patients diagnosed with stage IB or IIA cervical cancer (nine HPV 16-positive, one HPV 18-positive). The goal of the study was to define the T-cell epitopes of HPV 16 or 18 E7 protein in these patients in order to develop new strategies for treating HPV-associated malignancies. This was accomplished through establishing T-cell lines by stimulating peripheral blood mononuclear cells with autologous mature DC pulsed with the HPV 16 or 18 E7 protein, examining the T-cell responses using ELISPOT assays, and isolating E7-specific T-cell clones based on IFN-γ secretion. Then, the epitope was characterized in terms of its core sequence and the restriction element. Twelve T-cell lines from eight subjects (seven HPV 16-positive, one HPV 18-positive) were evaluated. Positive T-cell responses were demonstrated in four subjects (all HPV 16-positive). All four were positive for the HPV 16 E7 46-70 (EPDRAHYNIVTFCCKCDSTLRLCVQ) region. T-cell clones specific for the E7 47–70 region were isolated from one of the subjects. Further analyses revealed a novel, naturally processed, CD4 T-cell epitope, E7 58–68 (CCKCDSTLRLC), restricted by the HLA-DR17 molecule. This work was supported by the National Institutes of Health (R21CA094507). An erratum to this article can be found at     

CIN85 regulates the ligand-dependent endocytosis of the IgE receptor: a new molecular mechanism to dampen mast cell function

Ligation of the high-affinity receptor for IgE (Fc epsilonRI), constitutively expressed on mast cells and basophils, promotes cell activation and immediate release of allergic mediators. Furthermore, Fc epsilonRI up-regulation on APC from atopic donors is involved in the pathophysiology of allergic diseases. In consideration of the clinical relevance of the IgE receptor, the down-modulation of Fc epsilonRI expression in mast cells may represent a potential target for handling atopic diseases. In an effort to identify new molecular mechanisms involved in attenuating Fc epsilonRI expression and signaling, we focused our attention on CIN85, a scaffold molecule that regulates, in concert with the ubiquitin ligase Cbl, the clathrin-mediated endocytosis of several receptor tyrosine kinases. In the present study, we show that endogenous CIN85 is recruited in Cbl-containing complexes after engagement of the Fc epsilonRI on a mast cell line and drives ligand-induced receptor internalization. By confocal microscopic analysis, we provide evidence that CIN85 directs a more rapid receptor sorting in early endosomes and delivery to a lysosomal compartment. Furthermore, biochemical studies indicate that CIN85 plays a role in reducing the expression of receptor complex. Finally, we demonstrate that CIN85-overexpressing mast cells are dramatically impaired in their ability to degranulate following Ag stimulation, suggesting that the accelerated internalization of activated receptors by perturbing the propagation of Fc epsilonRI signaling may contribute to dampen the functional response. This role of CIN85 could be extended to include other multimeric immune receptors, such as the T and B cell receptors, providing a more general molecular mechanism for attenuating immune responses.     

Image analysis and in vivo imaging as tools for investigation of productivity dynamics in anthocyanin-producing cell cultures of Daucus carota

An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model system to study secondary metabolite production in cell culture at the individual cell level. An approach was set up in which growth and production of anthocyanins were investigated using a combination of biochemical analysis, image (colour) analysis and in vivo imaging. This novel approach was used to segment the culture in different subpopulations and dissect the productive process in the cell culture grown under two different conditions, known to differ mainly for oxygen supply and mixing intensity (volume of 50 ml or 20 ml in 250 ml flasks). The 20 ml batch cultures gave a higher content and yield of anthocyanins, which depended on a complex balance between events that positively or negatively affected anthocyanin production. A model is proposed in which the different ability of cells to respond to environmental stimuli and stress depends on the different amount of anthocyanins accumulated within cells.     

Partial rescue of Rett syndrome by ω-3 polyunsaturated fatty acids (PUFAs) oil

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with ω-3 PUFAs-containing fish oil (DHA: 72.9 ± 8.1 mg/kg b.w./day; EPA: 117.1 ± 13.1 mg/kg b.w./day; total ω-3 PUFAs: 246.0 ± 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F₂-dihomo-isoprostanes, F₃-isoprostanes, F₄-neuroprostanes, and F₂-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the ω-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-012-0285-7) contains supplementary material, which is available to authorized users.     

Interaction between nitric oxide and ethylene in the induction of alternative oxidase in ozone-treated tobacco plants

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain, an alternative pathway that terminates with a single homodimeric protein, the alternative oxidase (AOX). We recorded temporary inhibition of cytochrome capacity respiration and activation of AOX pathway capacity in tobacco plants (Nicotiana tabacum L. cv BelW3) fumigated with ozone (O(3)). The AOX1a gene was used as a molecular probe to investigate its regulation by signal molecules such as hydrogen peroxide, nitric oxide (NO), ethylene (ET), salicylic acid, and jasmonic acid (JA), all of them reported to be involved in the O(3) response. Fumigation leads to accumulation of hydrogen peroxide in mitochondria and early accumulation of NO in leaf tissues. Although ET accumulation was high in leaf tissues 5 h after the start of O(3) fumigation, it declined during the recovery period. There were no differences in the JA and 12-oxo-phytodienoic acid levels of treated and untreated plants. NO, JA, and ET induced AOX1a mRNA accumulation. Using pharmacological inhibition of ET and NO, we demonstrate that both NO- and ET-dependent pathways are required for O(3)-induced up-regulation of AOX1a. However, only NO is indispensable for the activation of AOX1a gene expression.