Single-molecule mitochondrial DNA sequencing shows no evidence of CpG methylation in human cells and tissues

Methylation on CpG residues is one of the most important epigenetic modifications of nuclear DNA, regulating gene expression. Methylation of mitochondrial DNA (mtDNA) has been studied using whole genome bisulfite sequencing (WGBS), but recent evidence has uncovered technical issues which introduce a potential bias during methylation quantification. Here, we validate the technical concerns of WGBS, and develop and assess the accuracy of a new protocol for mtDNA nucleotide variant-specific methylation using single-molecule Oxford Nanopore Sequencing (ONS). Our approach circumvents confounders by enriching for full-length molecules over nuclear DNA. Variant calling analysis against showed that 99.5% of homoplasmic mtDNA variants can be reliably identified providing there is adequate sequencing depth. We show that some of the mtDNA methylation signal detected by ONS is due to sequence-specific false positives introduced by the technique. The residual signal was observed across several human primary and cancer cell lines and multiple human tissues, but was always below the error threshold modelled using negative controls. We conclude that there is no evidence for CpG methylation in human mtDNA, thus resolving previous controversies. Additionally, we developed a reliable protocol to study epigenetic modifications of mtDNA at single-molecule and single-base resolution, with potential applications beyond CpG methylation.     

Spontaneous exfoliation of single-walled carbon nanotubes dispersed using a designed amphiphilic peptide

We have observed concentration dependent exfoliation of single-walled carbon nanotubes dispersed in solutions of the synthetic peptide nano-1. As the nanotube concentration is reduced, the bundle diameters tend to decrease before saturating at <2.0 nm for concentrations below 6 x 10(-3) mg/mL. The fraction of individual nanotubes increases with decreasing concentration, saturating at approximately 95% at low concentration. This concentration dependent exfoliation happens even if the dispersions are not sonicated on dilution, albeit over a longer time scale. The populations both of individual nanotubes and of bundles are much higher than expected at high concentrations, indicating the presence of repulsive internanotube interactions stabilizing the dispersions.     

Citrus phylogeny and genetic origin of important species as investigated by molecular markers

Citrus phylogeny was investigated using RAPD, SCAR and cpDNA markers. The genotypes analyzed included 36 accessions belonging to Citrus together with 1 accession from each of the related genera Poncirus, Fortunella, Microcitrus and Eremocitrus. Phylogenetic analysis with 262 RAPDs and 14 SCARs indicated that Fortunella is phylogenetically close to Citrus while the other three related genera are distant from Citrus and from each other. Within Citrus, the separation into two subgenera, Citrus and Papeda, designated by Swingle, was clearly observed except for C. celebica and C. indica. Almost all the accessions belonging to subgenus Citrus fell into three clusters, each including 1 genotype that was considered to be a true species. Different phylogenetic relationships were revealed with cpDNA data. Citrus genotypes were separated into subgenera Archicitrus and Metacitrus, as proposed by Tanaka, while the division of subgenera Citrus and Papeda disappeared. C. medica and C. indica were quite distant from other citrus as well from related genera. C. ichangensis appeared to be the ancestor of the mandarin cluster, including C. tachibana. Lemon and Palestine sweet lime were clustered into the Pummelo cluster led by C. latipes. C. aurantifolia was located in the Micrantha cluster. Furthermore, genetic origin was studied on 17 cultivated citrus genotypes by the same molecular markers, and a hybrid origin was hypothesized for all the tested genotypes. The assumptions are discussed with respect to previous studies; similar results were obtained for the origin of orange and grapefruit. Hybrids of citron and sour orange were assumed for lemon, Palestine sweet lime, bergamot and Volkamer lemon, while a citron × mandarin hybrid was assumed for Rangpur lime and Rough lemon. For Mexican lime our molecular data indicated C. micrantha to be the female parent and C. medica as the male one. Received: 5 October 1999 / Accepted: 3 November 1999     

Plasma and hepatic apoE isoproteins of nonhuman primates. Differences in apoE among humans, apes, and New and Old World monkeys

We have used two-dimensional polyacrylamide gel electrophoresis (PAGE) to study the plasma and hepatic apoE isoproteins of nonhuman primates and have compared them with their human counterparts. We have found that apoE obtained from fresh monkey or ape plasma, as well as nascent apoE synthesized by perfused monkey livers, is composed of several isoproteins that resemble the homozygous (beta) apoE phenotype observed in humans. The nonhuman primate plasma apoE pattern of 90 animals from nine different species consisted of a major isoprotein designated apoE3 and a few minor isoproteins. A group of acidic apoE isoproteins is eliminated after treatment with C. perfringens neuraminidase and has been designated sialo apoE (apoEs). Nonhuman primate liver apoE isoproteins comigrate with their plasma apoE isoprotein counterparts on two-dimensional PAGE, but hepatic apoE is enriched in sialo apoE isoproteins when compared to plasma apoE. The apparent molecular weight of asialo and sialo apoE obtained from Old World monkeys and apes is identical to the molecular weight of the corresponding human isoproteins (E3 = 38K, Es = 38.5-39.5K). However, the apparent molecular weight of apoE isoproteins obtained from New World monkeys is increased by approximately 0.5K (E3 = 38.5K, Es = 39.0-40.0K) as compared to the molecular weight of human and Old World monkey and ape isoproteins. The isoelectric points of apoE3 obtained from Old World monkeys, New World monkeys, chimpanzees, and gibbons are 5.74, 5.76, 5.95, and 5.89, respectively. The entire New or Old World monkey, chimpanzee, and gibbon apoE pattern is shifted by approximately -2.0, -0.5, and -1.0 charges, respectively, relative to the pattern of the corresponding human E3/3 phenotype. The molecular weight difference in apoE observed among New and Old World monkeys, as well as the molecular weight and/or charge differences observed among monkey, ape, and human apoE are consistent with structural changes in the apoE gene which have occurred following the divergence of the different species. The observation of only the homozygous apoE phenotypes in all animals studied suggests that the common apoE genetic polymorphism recently described in humans may not be present in nonhuman primates.     

Effects of dietary fats and cholesterol on liver lipid content and hepatic apolipoprotein A-I, B, and E and LDL receptor mRNA levels in cebus monkeys.

The effects of the long-term administration of the dietary fats coconut oil and corn oil at 31% of calories with or without 0.1% (wt/wt) dietary cholesterol on plasma lipoproteins, apolipoproteins (apo), hepatic lipid content, and hepatic apoA-I, apoB, apoE, and low density lipoprotein (LDL) receptor mRNA abundance were examined in 27 cebus monkeys. Relative to the corn oil-fed animals, no significant differences were noted in any of the parameters of the corn oil plus cholesterol-fed group. In animals fed coconut oil without cholesterol, significantly higher (P less than 0.05) plasma total cholesterol (145%), very low density lipoprotein (VLDL) + LDL (201%) and high density lipoprotein (HDL) (123%) cholesterol, apoA-I (103%), apoB (61%), and liver cholesteryl ester (263%) and triglyceride (325%) levels were noted, with no significant differences in mRNA levels relative to the corn oil only group. In animals fed coconut oil plus cholesterol, all plasma parameters were significantly higher (P less than 0.05), as were hepatic triglyceride (563%) and liver apoA-I (123%) and apoB (87%) mRNA levels relative to the corn oil only group, while hepatic LDL receptor mRNA (-29%) levels were significantly lower (P less than 0.05). Correlation coefficient analyses performed on pooled data demonstrated that liver triglyceride content was positively associated (P less than 0.05) with liver apoA-I and apoB mRNA levels and negatively associated (P less than 0.01) with hepatic LDL receptor mRNA levels. Liver free and esterified cholesterol levels were positively correlated (P less than 0.05) with liver apoE mRNA levels and negatively correlated (P less than 0.025) with liver LDL receptor mRNA levels. Interestingly, while a significant correlation (P less than 0.01) was noted between hepatic apoA-I mRNA abundance and plasma apoA-I levels, no such relationship was observed between liver apoB mRNA and plasma apoB levels, suggesting that the hepatic mRNA of apoA-I, but not that of apoB, is a major determinant of the circulating levels of the respective apolipoprotein. Our data indicate that a diet high in saturated fat and cholesterol may increase the accumulation of triglyceride and cholesterol in the liver, each resulting in the suppression of hepatic LDL receptor mRNA levels. We hypothesize that such elevations in hepatic lipid content differentially alter hepatic apoprotein mRNA levels, with triglyceride increasing hepatic mRNA concentrations for apoA-I and B and cholesterol elevating hepatic apoE mRNA abundance.     

A novel functional crosstalk between DDR1 and the IGF axis and its relevance for breast cancer

ABSTRACT

In the last decades increasing importance has been attributed to the Insulin/Insulin-like Growth Factor signaling (IIGFs) in cancer development, progression and resistance to therapy. In fact, IIGFs is often deregulated in cancer. In particular, the mitogenic insulin receptor isoform A (IR-A) and the insulin-like growth factor receptor (IGF-1R) are frequently overexpressed in cancer together with their cognate ligands IGF-1 and IGF-2. Recently, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis.

Herein, we review recent findings indicating that DDR1 is as a novel modulator of IR and IGF-1R expression and function. DDR1 functionally interacts with IR and IGF-1R and enhances the biological actions of insulin, IGF-1 and IGF-2. Conversely, DDR1 is upregulated by IGF-1, IGF-2 and insulin through the PI3K/AKT/miR-199a-5p circuit. Furthermore, we discuss the role of the non-canonical estrogen receptor GPER1 in the DDR1-IIGFs crosstalk. These data suggest a wider role of DDR1 as a regulator of cell response to hormones, growth factors, and signals coming from the extracellular matrix.     

Low density lipoprotein heterogeneity in the cebus monkey

In studies of cebus monkey plasma lipoproteins, we have used an ultracentrifugally generated density gradient to isolate two distinct species of low density lipoproteins (LDL). Compositional analyses revealed that each of the ultracentrifugally isolated fractions was enriched in cholesteryl esters and contained a single apolipoprotein which in terms of its mobility on SDS gels corresponded to apolipoprotein B-100, the major apolipoprotein of human LDL. Hydrodynamic measurements carried out in the analytical ultracentrifuge showed that F1.20 values were 30.0 for LDL1 and 23.5 for LDL2. In a solution of density 1.0069 g/ml, the sedimentation rates were 5.9 and 7.2 S for LDL1 and LDL2, respectively. In addition to sedimentation velocity data, we describe a new approach for using these same data to obtain calculated values for molecular weight. The hydrated densities calculated for the two fractions were 1.033 and 1.045 g/ml and calculated molecular weights were 3.08 million for LDL1 and 2.42 million for LDL2. Hydrated density values were in excellent agreement with those calculated from compositional data. Electron microscopy data showed that LDL1 had a larger mean diameter of 26.7 nm than LDL2 which had a diameter of 19.3 nm. Native gel electrophoretic analyses of the two LDL fractions in 3.5% acrylamide showed that, consistent with its size, LDL1 had slower mobility than LDL2.     

The morphology of Van der Vecth's organ as a tool to measure caste dimorphism in Polistes paper wasps: a comparative approach

Given the centrality of chemical communication in social insects, there are many selective pressures acting on morpho‐functional traits that mediate chemical pheromones. On the last gastral sternite of Polistes females, there is an important exocrine surface secreting chemical pheromone, named Van der Vecht's organ. It is involved in chemical defence of the nest, in rank and nestmate recognition, preventing workers from direct reproduction. Allometric differential growth of phenotypic traits between castes of social insects is generally considered as an indication of incipient physical castes. European Polistes present different nesting strategies and reproductive choices. Here, we carry out a comparison of Van der Vecht's organ size between castes of four European Polistes to provide a general measure of dimorphism. We show that Van der Vecht's organ of Polistes dominula and Polistes nimphus foundresses shows an allometric development being enlarged with respect to workers. Otherwise, no allometries have been highlighted in the other two studied species (i.e. Polistes associus and Polistes biglumis). Therefore, our data show that neither rigid monogyny nor specific nesting habits foster the evolution of true morphological castes in primitively eusocial taxa. Thus, at least two other species of Ezuropean Polistes show real evidence of incipient morphological castes.     

Type-Specific Human Papillomavirus Biological Features: Validated Model-Based Estimates

Infection with high-risk (hr) human papillomavirus (HPV) is considered the necessary cause of cervical cancer. Vaccination against HPV16 and 18 types, which are responsible of about 75% of cervical cancer worldwide, is expected to have a major global impact on cervical cancer occurrence. Valid estimates of the parameters that regulate the natural history of hrHPV infections are crucial to draw reliable projections of the impact of vaccination. We devised a mathematical model to estimate the probability of infection transmission, the rate of clearance, and the patterns of immune response following the clearance of infection of 13 hrHPV types. To test the validity of our estimates, we fitted the same transmission model to two large independent datasets from Italy and Sweden and assessed finding consistency. The two populations, both unvaccinated, differed substantially by sexual behaviour, age distribution, and study setting (screening for cervical cancer or Chlamydia trachomatis infection). Estimated transmission probability of hrHPV types (80% for HPV16, 73%-82% for HPV18, and above 50% for most other types); clearance rates decreasing as a function of time since infection; and partial protection against re-infection with the same hrHPV type (approximately 20% for HPV16 and 50% for the other types) were similar in the two countries. The model could accurately predict the HPV16 prevalence observed in Italy among women who were not infected three years before. In conclusion, our models inform on biological parameters that cannot at the moment be measured directly from any empirical data but are essential to forecast the impact of HPV vaccination programmes.     

Paroxysmal atrial fibrillation triggered by a monomorphic ventricular couplet in a patient with acute coronary syndrome

Atrial fibrillation is a common arrhythmia in patients suffering from acute myocardial infarction, however its pathophysiological mechanisms are not fully understood. We describe the unusual case of a 76-year old woman admitted for non-ST-segment elevation myocardial infarction, who developed multiple episodes of paroxysmal atrial fibrillation triggered by monomorphic ventricular couplets. Beta-blocking and amiodarone therapy resulted efficacious in preventing arrhythmic recurrences. We then discuss the possible arrhythmogenic mechanisms, with special emphasis on the unique electrophysiological, hemodynamic, cellular and anatomical milieu created by acute myocardial ischemia.