DNA methylation based biomarkers: Practical considerations and applications

A biomarker is a molecular target analyzed in a qualitative or quantitative manner to detect and diagnose the presence of a disease, to predict the outcome and the response to a specific treatment allowing personalized tailoring of patient management. Biomarkers can belong to different types of biochemical molecules such as proteins, DNA, RNA or lipids, whereby protein biomarkers have been the most extensively studied and used, notably in blood-based protein quantification tests or immunohistochemistry. The rise of interest in epigenetic mechanisms has allowed the identification of a new type of biomarker, DNA methylation, which is of great potential for many applications. This stable and heritable covalent modification mostly affects cytosines in the context of a CpG dinucleotide in humans. It can be detected and quantified by a number of technologies including genome-wide screening methods as well as locus- or gene-specific high-resolution analysis in different types of samples such as frozen tissues and FFPE samples, but also in body fluids such as urine, plasma, and serum obtained through non-invasive procedures. In some cases, DNA methylation based biomarkers have proven to be more specific and sensitive than commonly used protein biomarkers, which could clearly justify their use in clinics. However, very few of them are at the moment used in clinics and even less commercial tests are currently available. The objective of this review is to discuss the advantages of DNA methylation as a biomarker, the practical considerations for their development, and their use in disease detection, prediction of outcome or treatment response, through multiple examples mainly focusing on cancer, but also to evoke their potential for complex diseases and prenatal diagnostics.     

The Role of Electron Affinity in Determining Whether Fullerenes Catalyze or Inhibit Photooxidation of Polymers for Solar Cells

Understanding the stability and degradation mechanisms of organic solar materials is critically important to achieving long device lifetimes. Here, an investigation of the photodegradation of polymer:fullerene blend films exposed to ambient conditions for a variety of polymer and fullerene derivative combinations is presented. Despite the wide range in polymer stabilities to photodegradation, the rate of irreversible polymer photobleaching in blend films is found to consistently and dramatically increase with decreasing electron affinity of the fullerene derivative. Furthermore, blends containing fullerenes with the smallest electron affinities photobleached at a faster rate than films of the pure polymer. These observations can be explained by a mechanism where both the polymer and fullerene donate photogenerated electrons to diatomic oxygen to form the superoxide radical anion which degrades the polymer.     

Microtubule stability, Golgi organization, and transport flux require dystonin-a2-MAP1B interaction

Loss of function of dystonin cytoskeletal linker proteins causes neurodegeneration in dystonia musculorum (dt) mutant mice. Although much investigation has focused on understanding dt pathology, the diverse cellular functions of dystonin isoforms remain poorly characterized. In this paper, we highlight novel functions of the dystonin-a2 isoform in mediating microtubule (MT) stability, Golgi organization, and flux through the secretory pathway. Using dystonin mutant mice combined with isoform-specific loss-of-function analysis, we found dystonin-a2 bound to MT-associated protein 1B (MAP1B) in the centrosomal region, where it maintained MT acetylation. In dt neurons, absence of the MAP1B-dystonin-a2 interaction resulted in altered MAP1B perikaryal localization, leading to MT deacetylation and instability. Deacetylated MT accumulation resulted in Golgi fragmentation and prevented anterograde trafficking via motor proteins. Maintenance of MT acetylation through trichostatin A administration or MAP1B overexpression mitigated the observed defect. These cellular aberrations are apparent in prephenotype dorsal root ganglia and primary sensory neurons from dt mice, suggesting they are causal in the disorder.     

HLA-B7-restricted EBV-specific CD8+ T cells are dysregulated in multiple sclerosis

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.     

Partial,selective survival of nitrergic neurons in chagasic megacolon

One frequent chronic syndrome of Chagas’ disease is megacolon, an irreversible dilation of a colonic segment. Extensive enteric neuron loss in the affected segment is regarded as key factor for deficient motility. Here, we assessed the quantitative balance between cholinergic and nitrergic neurons representing the main limbs of excitatory and inhibitory colonic motor innervation, respectively. From surgically removed megacolonic segments of four patients, each three myenteric wholemounts (from non-dilated oral, megacolonic and non-dilated anal parts) was immunohistochemically triple-stained for choline acetyltransferase, neuronal nitric oxide synthase (NOS) and the panneuronal human neuronal protein Hu C/D. Degenerative changes were most pronounced in the megacolonic and anal regions, e.g. bulked, honeycomb-like ganglia with few neurons which were partly enlarged or atrophic or vacuolated. Neuron counts from each 15 ganglia of 12 megacolonic wholemounts were compared with those of 12 age- and region-matched controls. Extensive neuron loss, mainly in megacolonic and anal wholemounts, was obvious. In all three regions derived from megacolonic samples, the proportion of NOS-positive neurons (control: 55%) was significantly increased: in non-dilated oral parts to 61% (p = 0.003), in megacolonic regions to 72% (p < 0.001) and in non-dilated anal regions to 78% (p < 0.001). We suggest the chronic dilation of megacolonic specimens to be due to the preponderance of the nitrergic, inhibitory input to the intestinal muscle. However, the observed neuronal imbalance was not restricted to the dilated regions: the non-dilated anal parts may be innervated by ascending, cholinergic axons emerging from less affected, more anally located regions.     

Methods to account for spatial autocorrelation in the analysis of species distributional data: a review

Species distributional or trait data based on range map (extent‐of‐occurrence) or atlas survey data often display spatial autocorrelation, i.e. locations close to each other exhibit more similar values than those further apart. If this pattern remains present in the residuals of a statistical model based on such data, one of the key assumptions of standard statistical analyses, that residuals are independent and identically distributed (i.i.d), is violated. The violation of the assumption of i.i.d. residuals may bias parameter estimates and can increase type I error rates (falsely rejecting the null hypothesis of no effect). While this is increasingly recognised by researchers analysing species distribution data, there is, to our knowledge, no comprehensive overview of the many available spatial statistical methods to take spatial autocorrelation into account in tests of statistical significance. Here, we describe six different statistical approaches to infer correlates of species’ distributions, for both presence/absence (binary response) and species abundance data (poisson or normally distributed response), while accounting for spatial autocorrelation in model residuals: autocovariate regression; spatial eigenvector mapping; generalised least squares; (conditional and simultaneous) autoregressive models and generalised estimating equations. A comprehensive comparison of the relative merits of these methods is beyond the scope of this paper. To demonstrate each method's implementation, however, we undertook preliminary tests based on simulated data. These preliminary tests verified that most of the spatial modeling techniques we examined showed good type I error control and precise parameter estimates, at least when confronted with simplistic simulated data containing spatial autocorrelation in the errors. However, we found that for presence/absence data the results and conclusions were very variable between the different methods. This is likely due to the low information content of binary maps. Also, in contrast with previous studies, we found that autocovariate methods consistently underestimated the effects of environmental controls of species distributions. Given their widespread use, in particular for the modelling of species presence/absence data (e.g. climate envelope models), we argue that this warrants further study and caution in their use. To aid other ecologists in making use of the methods described, code to implement them in freely available software is provided in an electronic appendix.     

Making choices between rules or between actions

A new study by Klaes et al. in this issue of Neuron shows that the brain can simultaneously apply two rules to the same sensory information in order to specify two parallel potential action goals, which then compete for execution in the sensorimotor system.     

French Experience of 2009 A/H1N1v Influenza in Pregnant Women

Background

The first reports on the pandemic influenza 2009 A/H1N1v from the USA, Mexico, and Australia indicated that this disease was associated with a high mortality in pregnant women. The aim of this study was to describe and compare the characteristics of severe critically ill and non-severe pregnant women with 2009 A/H1N1v-related illness in France.

Methodology/Principal Findings

A national registry was created to screen pregnant women with laboratory-confirmed 2009 A/H1N1v influenza. Three hundred and fifteen patients from 46 French hospitals were included: 40 patients were admitted to intensive care units (severe outcomes), 111 were hospitalized in obstetric or medical wards (moderate outcomes), and 164 were outpatients (mild outcomes). The 2009 A/H1N1v influenza illness occurred during all pregnancy trimesters, but most women (54%), notably the severe patients (70%), were in the third trimester. Among the severe patients, twenty (50%) underwent mechanical ventilation, and eleven (28%) were treated with extracorporeal membrane oxygenation. Three women died from A/H1N1v influenza. We found a strong association between the development of a severe outcome and both co-existing illnesses (adjusted odds ratio [OR], 5.1; 95% confidence interval [CI], 2.2–11.8) and a delay in oseltamivir treatment after the onset of symptoms (>3 or 5 days) (adjusted OR, 4.8; 95% CI, 1.9–12.1 and 61.2, 95% CI; 14.4–261.3, respectively). Among the 140 deliveries after 22 weeks of gestation known to date, 19 neonates (14%) were admitted to a neonatal intensive care unit, mainly for preterm delivery, and two neonates died. None of these neonates developed 2009 A/H1N1v infection.

Conclusions

This series confirms the high incidence of complications in pregnant women infected with pandemic A/H1N1v observed in other countries but depicts a lower overall maternal and neonatal mortality and morbidity than indicated in the USA or Australia. Moreover, our data demonstrate the benefit of early oseltamivir treatment in this specific population.