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.     

Prediction of Arctic plant phenological sensitivity to climate change from historical records

The pace of climate change in the Arctic is dramatic, with temperatures rising at a rate double the global average. The timing of flowering and fruiting (phenology) is often temperature dependent and tends to advance as the climate warms. Herbarium specimens, photographs, and field observations can provide historical phenology records and have been used, on a localised scale, to predict species’ phenological sensitivity to climate change. Conducting similar localised studies in the Canadian Arctic, however, poses a challenge where the collection of herbarium specimens, photographs, and field observations have been temporally and spatially sporadic. We used flowering and seed dispersal times of 23 Arctic species from herbarium specimens, photographs, and field observations collected from across the 2.1 million km² area of Nunavut, Canada, to determine (1) which monthly temperatures influence flowering and seed dispersal times; (2) species’ phenological sensitivity to temperature; and (3) whether flowering or seed dispersal times have advanced over the past 120 years. We tested this at different spatial scales and compared the sensitivity in different regions of Nunavut. Broadly speaking, this research serves as a proof of concept to assess whether phenology–climate change studies using historic data can be conducted at large spatial scales. Flowering times and seed dispersal time were most strongly correlated with June and July temperatures, respectively. Seed dispersal times have advanced at double the rate of flowering times over the past 120 years, reflecting greater late‐summer temperature rises in Nunavut. There is great diversity in the flowering time sensitivity to temperature of Arctic plant species, suggesting climate change implications for Arctic ecological communities, including altered community composition, competition, and pollinator interactions. Intraspecific temperature sensitivity and warming trends varied markedly across Nunavut and could result in greater changes in some parts of Nunavut than in others.     

Respiratory fluoroquinolones for the treatment of community-acquired pneumonia: a meta-analysis of randomized controlled trials

Background

We investigated whether the use of respiratory fluoroquinolones was associated with better clinical outcomes compared with the use of macrolides and β-lactams among adults with pneumonia.

Methods

We searched PubMed, Current Contents, Scopus, EMBASE, ClinicalTrials.gov and Cochrane with no language restrictions. Two reviewers independently extracted data from published trials that compared fluoroquinolones (levofloxacin, moxifloxacin, gemifloxacin) with macrolides or β-lactams or both. A meta-analysis was performed with the clinical outcomes of mortality, treatment success and adverse outcomes.

Results

We included 23 trials in our meta-analysis. There was no difference in mortality among patients who received fluoroquinolones or the comparator antibiotics (OR 0.85, 95% CI 0.65–1.12). Pneumonia resolved in more patients who received fluoroquinolones compared with the comparator antibiotics for the included outcomes in the intention-to-treat population (OR 1.17, 95% CI 1.00–1.36), clinically evaluable population (OR 1.26, 95% CI 1.06–1.50) and the microbiologically assessed population (OR 1.67, 95% CI 1.28–2.20). Fluoroquinolones were more effective than a combination of β-lactam and macrolide (OR 1.39, 95% CI 1.02–1.90). They were also more effective for patients with severe pneumonia (OR 1.84, 95% CI 1.02–3.29), those who required admission to hospital (OR = 1.30, 95% CI 1.04–1.61) and those who required intravenous therapy (OR = 1.44, 15% CI 1.13–1.85). Fluoroquinolones were more effective than β-lactam and macrolide in open-label trials (OR = 1.35, 95% CI 1.08–1.69) but not in blinded randomized controlled trials (OR = 1.13, 95% CI 0.85–1.50).

Interpretation

Fluoroquinolones were associated with higher success of treatment for severe forms of pneumonia; however, a benefit in mortality was not evident. A randomized controlled trial that includes patients with severe pneumonia with or without bacteremia is needed.Community-acquired pneumonia is among the leading reasons for hospital admission¹ and resource consumption.^2,3 It is the most frequent cause of community-acquired infections among patients admitted to intensive care units.⁴ In addition, it is among the leading causes of death worldwide.Physicians must choose an optimal therapeutic regimen that eliminates the infection effectively, minimizes the risk of developing drug resistance and does not compromise the safety of the patient. The combination of β-lactam and macrolide covers the most common possible pathogens involved in the pathogenesis of pneumonia.⁵ More recently, fluoroquinolones with enhanced activity against Streptococcus pneumoniae were introduced in clinical practice. The favourable pharmacokinetic profile of fluoroquinolones allows for once daily administration, often eliminating the need for parenteral treatment. Furthermore, initial treatment with fluoroquinolones was among the predictors of lower treatment failure among patients with pneumonia.⁶In 2007, the Infectious Diseases Society of America and the American Thoracic Society released new guidelines for the management of care for adult patients with community-acquired pneumonia.⁷ In these guidelines, levofloxacin, gemifloxacin and moxifloxacin were reported to be equally effective as the combination of β-lactam and macrolide, and were proposed to be the preferred treatment option for patients who require admission to hospital, as well as for patients with comorbidity who receive treatment as outpatients. In addition to being safe, these fluoroquinolones are more effective against the most common types of bacteria responsible for the development of community-acquired pneumonia.⁷ For example, S. pneumoniae strains are not fully susceptible to ciprofloxacin. On the other hand, trovafloxacin, clinafloxacin, gatifloxacin and other quinolones are not used because of safety concerns or because they are not widely available. The trials that compared fluoroquinolones with other antibiotics regimens for the treatment of pneumonia were designed on the basis of noninferiority (i.e., an antibiotic is equally effective to a comparator), and several were conducted in order to receive approval from the relevant agencies.We sought to examine whether the use of fluoroquinolones was associated with more advantages or disadvantages than the use of macrolides or β-lactams in terms of mortality, resolution of pneumonia and adverse effects.     

Impact of Alginate Overproduction on Attachment and Biofilm Architecture of a Supermucoid Pseudomonas aeruginosa Strain

The supermucoid Pseudomonas aeruginosa strain PDO300Δalg8(pBBR1MCS-5:alg8) showed strongly impaired attachment compared with the respective mucoid or nonmucoid strains and formed a thicker biofilm with large extended mushroom-like microcolonies. Alginate lyase treatment dissolved microcolonies. The data suggested that alginate overproduction impairs attachment but plays a structural role in microcolony formation.Alginate is an important virulence factor for Pseudomonas aeruginosa, and the conversion of nonmucoid strains to alginate-overproducing mucoid strains early after the infection of cystic fibrosis patients is associated with a decline of pulmonary function and survival rate (11, 13). Alginate functions as extracellular matrix material, enabling the formation of differentiated biofilms in which the diffusion of clinical antibiotics is decreased and the embedded cells are protected against human antibacterial defense mechanisms (9, 12). Although alginate is not required for P. aeruginosa biofilm formation (15), previous studies have provided evidence that it plays a role in the formation of thick and three-dimensional biofilms (5, 9). To further investigate the impact of alginate on attachment and biofilm architecture, we used a recently generated supermucoid strain, PDO300Δalg8(pBBR1MCS-5:alg8) (14). This strain showed about 15-fold alginate overproduction compared to alginate-producing mucoid P. aeruginosa. The gene alg8 encodes the proposed catalytic subunit of alginate polymerase and is essential for alginate biosynthesis (14).     

Brain sterol dysregulation in sporadic AD and MCI: relationship to heme oxygenase-1

The objective of this study was to ascertain the impact of aging and Alzheimer's disease (AD) on brain cholesterol (CH), CH precursors, and oxysterol homeostasis. Altered CH metabolism and up-regulation of heme oxygenase-1 (HO-1) are characteristic of AD-affected neural tissues. We recently determined that HO-1 over-expression suppresses total CH levels by augmenting liver X receptor-mediated CH efflux and enhances oxysterol formation in cultured astroglia. Lipids and proteins were extracted from postmortem human frontal cortex derived from subjects with sporadic AD, mild cognitive impairment (MCI), and no cognitive impairment ( n  = 17 per group) enrolled in the Religious Orders Study, an ongoing clinical-pathologic study of aging and AD. ELISA was used to quantify human HO-1 protein expression from brain tissue and gas chromatography–mass spectrometry to quantify total CH, CH precursors, and relevant oxysterols. The relationships of sterol/oxysterol levels to HO-1 protein expression and clinical/demographic variables were determined by multivariable regression and non-parametric statistical analyses. Decreased CH, increased oxysterol and increased CH precursors concentrations in the cortex correlated significantly with HO-1 levels in MCI and AD, but not no cognitive impairment. Specific oxysterols correlated with disease state, increasing neuropathological burden, neuropsychological impairment, and age. A model featuring compensated and de-compensated states of altered sterol homeostasis in MCI and AD is presented based on the current data set and our earlier in vitro work.     

Vaccinia Virus-Mediated Inhibition of Type I Interferon Responses Is a Multifactorial Process Involving the Soluble Type I Interferon Receptor B18 and Intracellular Components

Poxviruses such as virulent vaccinia virus (VACV) strain Western Reserve encode a broad range of immune modulators that interfere with host responses to infection. Upon more than 570 in vitro passages in chicken embryo fibroblasts (CEF), chorioallantois VACV Ankara (CVA) accumulated mutations that resulted in highly attenuated modified vaccinia virus Ankara (MVA). MVA infection of mice and of dendritic cells (DC) induced significant type I interferon (IFN) responses, whereas infection with VACV alone or in combination with MVA did not. These results implied that VACV expressed an IFN inhibitor(s) that was functionally deleted in MVA. To further characterize the IFN inhibitor(s), infection experiments were carried out with CVA strains isolated after 152 (CVA152) and 386 CEF passages (CVA386). Interestingly, neither CVA152 nor CVA386 induced IFN-α, whereas the latter variant did induce IFN-β. This pattern suggested a consecutive loss of inhibitors during MVA attenuation. Similar to supernatants of VACV- and CVA152-infected DC cultures, recombinantly expressed soluble IFN decoy receptor B18, which is encoded in the VACV genome, inhibited MVA-induced IFN-α but not IFN-β. In the same direction, a B18R-deficient VACV variant triggered only IFN-α, confirming B18 as the soluble IFN-α inhibitor. Interestingly, VACV infection inhibited IFN responses induced by a multitude of different stimuli, including oligodeoxynucleotides containing CpG motifs, poly(I:C), and vesicular stomatitis virus. Collectively, the data presented show that VACV-mediated IFN inhibition is a multistep process involving secreted factors such as B18 plus intracellular components that cooperate to efficiently shut off systemic IFN-α and IFN-β responses.     

An effective approach for identification of <Emphasis Type="Italic">in vivo</Emphasis> protein-DNA binding sites from paired-end ChIP-Seq data

Background  

ChIP-Seq, which combines chromatin immunoprecipitation (ChIP) with high-throughput massively parallel sequencing, is increasingly being used for identification of protein-DNA interactions in vivo in the genome. However, to maximize the effectiveness of data analysis of such sequences requires the development of new algorithms that are able to accurately predict DNA-protein binding sites.     

Safety and immunogenicity of a replication-defective adenovirus type 5 HIV vaccine in Ad5-seronegative persons: a randomized clinical trial (HVTN 054)

Background

Individuals without prior immunity to a vaccine vector may be more sensitive to reactions following injection, but may also show optimal immune responses to vaccine antigens. To assess safety and maximal tolerated dose of an adenoviral vaccine vector in volunteers without prior immunity, we evaluated a recombinant replication-defective adenovirus type 5 (rAd5) vaccine expressing HIV-1 Gag, Pol, and multiclade Env proteins, VRC-HIVADV014-00-VP, in a randomized, double-blind, dose-escalation, multicenter trial (HVTN study 054) in HIV-1-seronegative participants without detectable neutralizing antibodies (nAb) to the vector. As secondary outcomes, we also assessed T-cell and antibody responses.

Methodology/Principal Findings

Volunteers received one dose of vaccine at either 10¹⁰ or 10¹¹ adenovector particle units, or placebo. T-cell responses were measured against pools of global potential T-cell epitope peptides. HIV-1 binding and neutralizing antibodies were assessed. Systemic reactogenicity was greater at the higher dose, but the vaccine was well tolerated at both doses. Although no HIV infections occurred, commercial diagnostic assays were positive in 87% of vaccinees one year after vaccination. More than 85% of vaccinees developed HIV-1-specific T-cell responses detected by IFN-γ ELISpot and ICS assays at day 28. T-cell responses were: CD8-biased; evenly distributed across the three HIV-1 antigens; not substantially increased at the higher dose; and detected at similar frequencies one year following injection. The vaccine induced binding antibodies against at least one HIV-1 Env antigen in all recipients.

Conclusions/Significance

This vaccine appeared safe and was highly immunogenic following a single dose in human volunteers without prior nAb against the vector.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00119873","term_id":"NCT00119873"}}NCT00119873