Impaired Awareness in People with Severe Alcohol-Related Cognitive Deficits Including Korskoff's Syndrome: A Network Analysis.

BACKGROUND: Impaired awareness of one's own functioning is highly common in people with Korsakoff's syndrome (KS). However, it is currently unclear how awareness relates to impairments in daily functioning and quality of life (QoL). METHODS: We assessed how impaired awareness relates to cognitive, behavioral, physical, and social functioning and QoL by applying a network analysis. We used cross-sectional data from 215 patients with KS or other severe alcohol-related cognitive deficits living in Dutch long-term care facilities (LTCFs). RESULTS: Apathy has the most central position in the network. Higher apathy scores relate positively to reduced cognition and to a greater decline in activities of daily living and negatively to social participation and the use of antipsychotic drugs. Impaired awareness is also a central node. It is positively related to a higher perceived QoL, reduced cognition and apathy, and negatively to social participation and length of stay in the LTCF. Mediated through apathy and social participation, impaired awareness is indirectly related to other neuropsychiatric symptoms. CONCLUSIONS: Impaired awareness is closely related to other domains of daily functioning and QoL of people with KS or other severe alcohol-related cognitive deficits living in LTCFs. Apathy plays a central role. Network analysis offers interesting insights to evaluate the interconnection of different symptoms and impairments in brain disorders such as KS.

Penalized estimation of the Gaussian graphical model from data with replicates.

Gaussian graphical models are usually estimated from unreplicated data. The data are, however, likely to comprise signal and noise. These two cannot be deconvoluted from unreplicated data. Pragmatically, the noise is then ignored in practice. We point out the consequences of this practice for the reconstruction of the conditional independence graph of the signal. Replicated data allow for the deconvolution of signal and noise and the reconstruction of former's conditional independence graph. Hereto we present a penalized Expectation-Maximization algorithm. The penalty parameter is chosen to maximize the F-fold cross-validated log-likelihood. Sampling schemes of the folds from replicated data are discussed. By simulation we investigate the effect of replicates on the reconstruction of the signal's conditional independence graph. Moreover, we compare the proposed method to several obvious competitors. In an application we use data from oncogenomic studies with replicates to reconstruct the gene-gene interaction networks, operationalized as conditional independence graphs. This yields a realistic portrait of the effect of ignoring other sources but sampling variation. In addition, it bears implications on the reproducibility of inferred gene-gene interaction networks reported in literature.

Mechanical stress regulates bone regulatory gene expression independent of estrogen and vitamin D deficiency in rats.

Mechanical stress determines bone mass and structure. It is not known whether mechanical loading affects expression of bone regulatory genes in a combined deficiency of estrogen and vitamin D. We studied the effect of mechanical loading on the messenger RNA (mRNA) expression of bone regulatory genes during vitamin D and/or estrogen deficiency. We performed a single bout in vivo axial loading with 14 N peak load, 2 Hz frequency and 360 cycles in right ulnae of nineteen weeks old female control Wistar rats with or without ovariectomy (OVX), vitamin D deficiency and the combination of OVX and vitamin D deficiency (N = 10/group). Total bone RNA was isolated 6 hours after loading, and mRNA expression was detected of Mepe, Fgf23, Dmp1, Phex, Sost, Col1a1, Cyp27b1, Vdr, and Esr1. Serum levels of 25(OH)D, 1,25(OH)2 D and estradiol were also measured at this time point. The effect of loading, vitamin D and estrogen deficiency and their interaction on bone gene expression was tested using a mixed effect model analysis. Mechanical loading significantly increased the mRNA expression of Mepe, and Sost, whereas it decreased the mRNA expression of Fgf23 and Esr1. Mechanical loading showed a significant interaction with vitamin D deficiency with regard to mRNA expression of Vdr and Esr1. Mechanical loading affected gene expression of Mepe, Fgf23, Sost, and Esr1 independently of vitamin D or estrogen, indicating that mechanical loading may affect bone turnover even during vitamin D deficiency and after menopause.

Identification of Deregulated Pathways, Key Regulators, and Novel miRNA-mRNA Interactions in HPV-Mediated Transformation.

Next to a persistent infection with high-risk human papillomavirus (HPV), molecular changes are required for the development of cervical cancer. To identify which molecular alterations drive carcinogenesis, we performed a comprehensive and longitudinal molecular characterization of HPV-transformed keratinocyte cell lines. Comparative genomic hybridization, mRNA, and miRNA expression analysis of four HPV-containing keratinocyte cell lines at eight different time points was performed. Data was analyzed using unsupervised hierarchical clustering, integrated longitudinal expression analysis, and pathway enrichment analysis. Biological relevance of identified key regulatory genes was evaluated in vitro and dual-luciferase assays were used to confirm predicted miRNA-mRNA interactions. We show that the acquisition of anchorage independence of HPV-containing keratinocyte cell lines is particularly associated with copy number alterations. Approximately one third of differentially expressed mRNAs and miRNAs was directly attributable to copy number alterations. Focal adhesion, TGF-beta signaling, and mTOR signaling pathways were enriched among these genes. PITX2 was identified as key regulator of TGF-beta signaling and inhibited cell growth in vitro, most likely by inducing cell cycle arrest and apoptosis. Predicted miRNA-mRNA interactions miR-221-3p_BRWD3, miR-221-3p_FOS, and miR-138-5p_PLXNB2 were confirmed in vitro. Integrated longitudinal analysis of our HPV-induced carcinogenesis model pinpointed relevant interconnected molecular changes and crucial signaling pathways in HPV-mediated transformation.

NOX2 Expression Is Increased in Keratinocytes After Burn Injury.

Reepithelialization is crucial for effective wound repair in burn wounds. Reactive oxygen species (ROS) have shown to be important in this. Recent studies suggest that NOX proteins produce ROS in keratinocytes. In the present study, we have studied NOX proteins in burn wounds, including the effect of C1-esterase inhibitor (C1inh) hereon, which is the endogenous inhibitor of complement activity whereof we have shown previously that it also increased the rate of reepithelialization in burn wounds. Skin tissue derived from healthy control Wistar rats (n = 6) were compared with burn-injured rats, with (n = 7) or without C1inh treatment (n = 7). After 14 days, rats were terminated. From the burn-injured rats, the entire wound and nonburned skin from the hind leg, that is, internal control was excised. From the control rats, dorsal skin was excised. In these skin samples, NOX2 and NOX4 were analyzed immunohistochemically. In nonburned rats, NOX2 was found in keratinocytes in both the basal layer and suprabasal layer of the epidermis; and the number of NOX2-positive keratinocytes was 367/mm2 (254-378). In burned rats, the number of NOX2-positive keratinocytes was significantly increased in the newly forming epidermis in the burned area to 1019/mm2 (649-1172), especially in the suprabasal layer, but significantly decreased in remote nonburned skin to 22/mm2 (6-89). C1inh treatment counteracted these changes in epidermal NOX2 expression in burned rats, both in the burned area as in remote nonburned skin. No NOX4 expression was found in the epidermis in none of the groups. NOX2 expression was increased in keratinocytes in newly forming epidermis after burn injury. C1inh, a drug that increases the rate of reepithelialization, counteracted this effect. These results suggest a role for NOX2 in the reepithelialization of burn wounds.

Diagnostic Value of Cerebrospinal Fluid Neurofilament Light Protein in Neurology: A Systematic Review and Meta-analysis.

IMPORTANCE: Neurofilament light protein (NfL) is elevated in cerebrospinal fluid (CSF) of a number of neurological conditions compared with healthy controls (HC) and is a candidate biomarker for neuroaxonal damage. The influence of age and sex is largely unknown, and levels across neurological disorders have not been compared systematically to date. OBJECTIVES: To assess the associations of age, sex, and diagnosis with NfL in CSF (cNfL) and to evaluate its potential in discriminating clinically similar conditions. DATA SOURCES: PubMed was searched for studies published between January 1, 2006, and January 1, 2016, reporting cNfL levels (using the search terms neurofilament light and cerebrospinal fluid) in neurological or psychiatric conditions and/or in HC. STUDY SELECTION: Studies reporting NfL levels measured in lumbar CSF using a commercially available immunoassay, as well as age and sex. DATA EXTRACTION AND SYNTHESIS: Individual-level data were requested from study authors. Generalized linear mixed-effects models were used to estimate the fixed effects of age, sex, and diagnosis on log-transformed NfL levels, with cohort of origin modeled as a random intercept. MAIN OUTCOME AND MEASURE: The cNfL levels adjusted for age and sex across diagnoses. RESULTS: Data were collected for 10 059 individuals (mean [SD] age, 59.7 [18.8] years; 54.1% female). Thirty-five diagnoses were identified, including inflammatory diseases of the central nervous system (n = 2795), dementias and predementia stages (n = 4284), parkinsonian disorders (n = 984), and HC (n = 1332). The cNfL was elevated compared with HC in a majority of neurological conditions studied. Highest levels were observed in cognitively impaired HIV-positive individuals (iHIV), amyotrophic lateral sclerosis, frontotemporal dementia (FTD), and Huntington disease. In 33.3% of diagnoses, including HC, multiple sclerosis, Alzheimer disease (AD), and Parkinson disease (PD), cNfL was higher in men than women. The cNfL increased with age in HC and a majority of neurological conditions, although the association was strongest in HC. The cNfL overlapped in most clinically similar diagnoses except for FTD and iHIV, which segregated from other dementias, and PD, which segregated from atypical parkinsonian syndromes. CONCLUSIONS AND RELEVANCE: These data support the use of cNfL as a biomarker of neuroaxonal damage and indicate that age-specific and sex-specific (and in some cases disease-specific) reference values may be needed. The cNfL has potential to assist the differentiation of FTD from AD and PD from atypical parkinsonian syndromes.

Short-term LPS induces aortic valve thickening in ApoE*3Leiden mice.

BACKGROUND: Recently, it was shown that 12 weeks of lipopolysaccharide (LPS) administration to nonatherosclerotic mice induced thickening of the aortic heart valve (AV). Whether such effects may also occur even earlier is unknown. As most patients with AV stenosis also have atherosclerosis, we studied the short-term effect of LPS on the AVs in an atherosclerotic mouse model. METHODS: ApoE*3Leiden mice, on an atherogenic diet, were injected intraperitoneally with either LPS or phosphate buffered saline (PBS), and sacrificed 2 or 15 days later. AVs were assessed for size, fibrosis, glycosaminoglycans (GAGs), lipids, calcium deposits, iron deposits and inflammatory cells. RESULTS: LPS injection caused an increase in maximal leaflet thickness at 2 days (128.4 µm) compared to PBS-injected mice (67.8 µm; P = 0.007), whereas at 15 days this was not significantly different. LPS injection did not significantly affect average AV thickness on day 2 (37.8 µm), but did significantly increase average AV thickness at day 15 (41.6 µm; P = 0.038) compared to PBS-injected mice (31.7 and 32.3 µm respectively). LPS injection did not affect AV fibrosis, GAGs and lipid content. Furthermore, no calcium deposits were found. Iron deposits, indicative for valve haemorrhage, were observed in one AV of the PBS-injected group (a day 2 mouse; 9.1%) and in five AVs of the LPS-injected group (both day 2- and 15 mice; 29.4%). No significant differences in inflammatory cell infiltration were observed upon LPS injection. CONCLUSION: Short-term LPS apparently has the potential to increase AV thickening and haemorrhage. These results suggest that systemic inflammation can acutely compromise AV structure.

Ridge estimation of network models from time-course omics data.

Time-course omics experiments enable the reconstruction of the dynamics of the cellular regulatory network. Here, we describe the means for this reconstruction and the downstream exploitation of the inferred network. It is assumed that one of the various vector-autoregressive models (VAR) models presented here serves as a reasonably accurate description of the time-course omics data. The models are estimated through ridge penalized likelihood maximization, accompanied by functionality for the determination of optimal penalty paramaters. Prior knowledge on the network topology is accommodated by the estimation procedures. Various routes that translate the fitted models into more tangible implications for the medical researcher are described. The network is inferred from the-nonsparse-ridge estimates through empirical Bayes probabilistic thresholding. The influence of a (trait of a) molecular entity at the current time on those at future time points is assessed by mutual information, impulse response analysis, and path decomposition of the covariance. The presented methodology is applied to the omics data from the p53 signaling pathway during HPV-induced cellular transformation. All methodology is implemented in the ragt2ridges package, freely available from the Comprehensive R Archive Network.

No Plasmatic Proteomic Signature at Clinical Disease Onset Associated With 11 Year Clinical, Cognitive and MRI Outcomes in Relapsing-Remitting Multiple Sclerosis Patients.

Background: The clinical course of relapsing-remitting multiple sclerosis (RRMS) is highly heterogeneous and prognostic biomarkers at time of diagnosis are lacking. Objective: We investigated the predictive value of the plasma proteome at time of diagnosis in RRMS patients. Methods: The plasma proteome was interrogated using a novel aptamer-based proteomics platform, which allows to measure the levels of a predefined set of 1310 proteins. Results: In 67 clinically and radiologically well characterized RRMS patients, we found no association between the plasma proteome at diagnosis and clinical, cognitive or MRI outcomes after 11 years. Conclusions: Proteomics studies on cerebrospinal fluid may be better suited to identify prognostic biomarkers in early RRMS.

Rscreenorm: normalization of CRISPR and siRNA screen data for more reproducible hit selection.

BACKGROUND: Reproducibility of hits from independent CRISPR or siRNA screens is poor. This is partly due to data normalization primarily addressing technical variability within independent screens, and not the technical differences between them. RESULTS: We present "rscreenorm", a method that standardizes the functional data ranges between screens using assay controls, and subsequently performs a piecewise-linear normalization to make data distributions across all screens comparable. In simulation studies, rscreenorm reduces false positives. Using two multiple-cell lines siRNA screens, rscreenorm increased reproducibility between 27 and 62% for hits, and up to 5-fold for non-hits. Using publicly available CRISPR-Cas screen data, application of commonly used median centering yields merely 34% of overlapping hits, in contrast with rscreenorm yielding 84% of overlapping hits. Furthermore, rscreenorm yielded at most 8% discordant results, whilst median-centering yielded as much as 55%. CONCLUSIONS: Rscreenorm yields more consistent results and keeps false positive rates under control, improving reproducibility of genetic screens data analysis from multiple cell lines.

Reconstruction of molecular network evolution from cross-sectional omics data.

Cross-sectional studies may shed light on the evolution of a disease like cancer through the comparison of patient traits among disease stages. This problem is especially challenging when a gene-gene interaction network needs to be reconstructed from omics data, and, in addition, the patients of each stage need not form a homogeneous group. Here, the problem is operationalized as the estimation of stage-wise mixtures of Gaussian graphical models (GGMs) from high-dimensional data. These mixtures are fitted by a (fused) ridge penalized EM algorithm. The fused ridge penalty shrinks GGMs of contiguous stages. The (fused) ridge penalty parameters are chosen through cross-validation. The proposed estimation procedures are shown to be consistent and their performance in other respects is studied in simulation. The down-stream exploitation of the fitted GGMs is outlined. In a data illustration the methodology is employed to identify gene-gene interaction network changes in the transition from normal to cancer prostate tissue.

Prognostic modeling of oral cancer by gene profiles and clinicopathological co-variables.

Accurate staging and outcome prediction is a major problem in clinical management of oral cancer patients, hampering high precision treatment and adjuvant therapy planning. Here, we have built and validated multivariable models that integrate gene signatures with clinical and pathological variables to improve staging and survival prediction of patients with oral squamous cell carcinoma (OSCC). Gene expression profiles from 249 human papillomavirus (HPV)-negative OSCCs were explored to identify a 22-gene lymph node metastasis signature (LNMsig) and a 40-gene overall survival signature (OSsig). To facilitate future clinical implementation and increase performance, these signatures were transferred to quantitative polymerase chain reaction (qPCR) assays and validated in an independent cohort of 125 HPV-negative tumors. When applied in the clinically relevant subgroup of early-stage (cT1-2N0) OSCC, the LNMsig could prevent overtreatment in two-third of the patients. Additionally, the integration of RT-qPCR gene signatures with clinical and pathological variables provided accurate prognostic models for oral cancer, strongly outperforming TNM. Finally, the OSsig gene signature identified a subpopulation of patients, currently considered at low-risk for disease-related survival, who showed an unexpected poor prognosis. These well-validated models will assist in personalizing primary treatment with respect to neck dissection and adjuvant therapies.

4H Leukodystrophy: A Brain Magnetic Resonance Imaging Scoring System.

4H (hypomyelination, hypodontia and hypogonadotropic hypogonadism) leukodystrophy (4H) is an autosomal recessive hypomyelinating white matter (WM) disorder with neurologic, dental, and endocrine abnormalities. The aim of this study was to develop and validate a magnetic resonance imaging (MRI) scoring system for 4H. A scoring system (0-54) was developed to quantify hypomyelination and atrophy of different brain regions. Pons diameter and bicaudate ratio were included as measures of cerebral and brainstem atrophy, and reference values were determined using controls. Five independent raters completed the scoring system in 40 brain MRI scans collected from 36 patients with genetically proven 4H. Interrater reliability (IRR) and correlations between MRI scores, age, gross motor function, gender, and mutated gene were assessed. IRR for total MRI severity was found to be excellent (intraclass correlation coefficient: 0.87; 95% confidence interval: 0.80-0.92) but varied between different items with some (e.g., myelination of the cerebellar WM) showing poor IRR. Atrophy increased with age in contrast to hypomyelination scores. MRI scores (global, hypomyelination, and atrophy scores) significantly correlated with clinical handicap (p < 0.01 for all three items) and differed between the different genotypes. Our 4H MRI scoring system reliably quantifies hypomyelination and atrophy in patients with 4H, and MRI scores reflect clinical disease severity.

The Local and Systemic Inflammatory Response in a Pig Burn Wound Model With a Pivotal Role for Complement.

In patients with burns, a massive inflammatory response is induced which negatively affects the healing process of the burn wound and additionally exerts systemic effects. An important factor herein is the complement system. Here we analyzed the effects of burns on complement and inflammatory cells both locally and systemically after burn in time in a pig burn wound model. In burned pigs, burn wound biopsies and blood were collected up to 60 days after burn. Complement in blood as well as complement and inflammatory cells in the burn wound and several organs were determined. In the blood, C3 was significantly increased after 9 to 60 days, whereas C4 after 21 to 30 days after burn. In the burn wound, C3 levels were significantly increased after 9 days and C4 after 3 days, whereafter both declined after 21 and 9 days, respectively. Neutrophils, macrophages, and lymphocytes were significantly increased in the burn wound after 3 days, all declined after 21 days after burn. In the heart, at 60 days after burn, an increase of neutrophils and macrophages was observed, mainly in the right atrium. In contrast to the heart, the inflammatory cell infiltrates in the lungs, liver, and kidney of burned pigs were lower than in control pigs. In pigs, following burn there is a prolonged increase in complement levels both in the burn wound and the blood and increased inflammatory cell infiltrate in the burn wound and the heart. However, complement levels in the burn wound and in the blood seem not to be correlated in time.

An empirical Bayes approach to network recovery using external knowledge.

Reconstruction of a high-dimensional network may benefit substantially from the inclusion of prior knowledge on the network topology. In the case of gene interaction networks such knowledge may come for instance from pathway repositories like KEGG, or be inferred from data of a pilot study. The Bayesian framework provides a natural means of including such prior knowledge. Based on a Bayesian Simultaneous Equation Model, we develop an appealing Empirical Bayes (EB) procedure that automatically assesses the agreement of the used prior knowledge with the data at hand. We use variational Bayes method for posterior densities approximation and compare its accuracy with that of Gibbs sampling strategy. Our method is computationally fast, and can outperform known competitors. In a simulation study, we show that accurate prior data can greatly improve the reconstruction of the network, but need not harm the reconstruction if wrong. We demonstrate the benefits of the method in an analysis of gene expression data from GEO. In particular, the edges of the recovered network have superior reproducibility (compared to that of competitors) over resampled versions of the data.

Gene Network Reconstruction using Global-Local Shrinkage Priors.

Reconstructing a gene network from high-throughput molecular data is an important but challenging task, as the number of parameters to estimate easily is much larger than the sample size. A conventional remedy is to regularize or penalize the model likelihood. In network models, this is often done locally in the neighbourhood of each node or gene. However, estimation of the many regularization parameters is often difficult and can result in large statistical uncertainties. In this paper we propose to combine local regularization with global shrinkage of the regularization parameters to borrow strength between genes and improve inference. We employ a simple Bayesian model with non-sparse, conjugate priors to facilitate the use of fast variational approximations to posteriors. We discuss empirical Bayes estimation of hyper-parameters of the priors, and propose a novel approach to rank-based posterior thresholding. Using extensive model- and data-based simulations, we demonstrate that the proposed inference strategy outperforms popular (sparse) methods, yields more stable edges, and is more reproducible. The proposed method, termed ShrinkNet, is then applied to Glioblastoma to investigate the interactions between genes associated with patient survival.

Cytotoxic Effects of the Therapeutic Radionuclide Rhenium-188 Combined with Taxanes in Human Prostate Carcinoma Cell Lines.

OBJECTIVE: Rhenium-188-HEDP is an effective radiopharmaceutical for the treatment of painful bone metastases from prostate cancer. The effectiveness of the ß-radiation emitted by 188Re might be enhanced by combination with chemotherapy, using the radiosensitization concept. Therefore, the authors investigated the combined treatment of the taxanes, docetaxel and cabazitaxel, with 188Re in prostate carcinoma cell lines. MATERIALS AND METHODS: The cytotoxic effects of single and combined treatment with taxanes and 188Re were investigated in three human prostate carcinoma cell lines (PC-3, DU 145, and LNCaP), using the colony-forming assay. The half maximal effective concentration (EC50) of all individual agents was determined. The combined treatment was studied at 0.25, 0.5, 1, 2, and 4 times the EC50 of each agent. The interaction was investigated with a regression model. RESULTS: The survival curves showed dose-dependent cell growth inhibition for both the taxanes and 188Re. The regression model showed a good capability of explaining the data. It proved additivity in all combination experiments and confirmed a general trend to a slight subadditive effect. CONCLUSIONS: This proof-of-mechanism study exploring radiosensitization by combining 188Re and taxanes showed no synergism, but significant additivity. This encourages the design of in vivo studies. Future research should explore the potential added value of concomitant treatment of bone metastases with chemotherapy and 188Re-HEDP.

Ridge estimation of the VAR(1) model and its time series chain graph from multivariate time-course omics data.

Omics experiments endowed with a time-course design may enable us to uncover the dynamic interplay among genes of cellular processes. Multivariate techniques (like VAR(1) models describing the temporal and contemporaneous relations among variates) that may facilitate this goal are hampered by the high-dimensionality of the resulting data. This is resolved by the presented ridge regularized maximum likelihood estimation procedure for the VAR(1) model. Information on the absence of temporal and contemporaneous relations may be incorporated in this procedure. Its computational efficient implemention is discussed. The estimation procedure is accompanied with an LOOCV scheme to determine the associated penalty parameters. Downstream exploitation of the estimated VAR(1) model is outlined: an empirical Bayes procedure to identify the interesting temporal and contemporaneous relationships, impulse response analysis, mutual information analysis, and covariance decomposition into the (graphical) relations among variates. In a simulation study the presented ridge estimation procedure outperformed a sparse competitor in terms of Frobenius loss of the estimates, while their selection properties are on par. The proposed machinery is illustrated in the reconstruction of the p53 signaling pathway during HPV-induced cellular transformation. The methodology is implemented in the ragt2ridges R-package available from CRAN.

Quantitative MRI in hypomyelinating disorders: Correlation with motor handicap.

OBJECTIVE: To assess the correlation of tissue parameters estimated by quantitative magnetic resonance (MR) techniques and motor handicap in patients with hypomyelination. METHODS: Twenty-eight patients with different causes of hypomyelination (12 males, 16 females; mean age 10 years) and 61 controls (33 males, 28 females; mean age 8 years) were prospectively investigated. We quantified T2 relaxation time, magnetization transfer ratio, fractional anisotropy, mean, axial, and radial diffusivities, and brain metabolites. We performed measurements in the splenium, parietal deep white matter, and corticospinal tracts in the centrum semiovale. We further analyzed diffusion measures using tract-based spatial statistics. We estimated severity of motor handicap by the gross motor function classification system. We evaluated correlation of handicap with MR measures by linear regression analyses. RESULTS: Fractional anisotropy, magnetization transfer ratio, choline, and N-acetylaspartate/creatine ratio were lower and diffusivities, T2 values, and inositol were higher in patients than in controls. Tract-based spatial statistics showed that these changes were widespread for fractional anisotropy (96% of the white matter skeleton), radial (93%) and mean (84%) diffusivity, and less so for axial diffusivity (20%). Correlation with handicap yielded radial diffusivity and N-acetylaspartate/creatine ratio as strongest independent explanatory variables. CONCLUSIONS: Gross motor function classification system grades are in part explained by MR measures. They indicate that mainly lack of myelin and, to a lesser degree, loss of axonal integrity codetermine the degree of motor handicap in patients with hypomyelinating disorders. These MR measures can be used to evaluate strategies that are aimed at promotion of myelination.

Aberrant methylation-mediated silencing of microRNAs contributes to HPV-induced anchorage independence.

Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by high-risk types of the human papillomavirus (hrHPV). During hrHPV-induced malignant transformation keratinocytes become able to grow anchorage independently, a tumorigenic trait at least partly associated with inactivation of tumor suppressor genes. We used hrHPV-containing keratinocytes to investigate the role of DNA methylation-mediated silencing of microRNAs (miRNAs) in the acquisition of anchorage independence.Anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalized keratinocyte cell lines were treated with 2'-deoxy-5-azacytidine (DAC). Genome-wide miRNA expression profiles before and after treatment were compared to identify miRNAs silenced by methylation. Bisulfite sequencing and methylation-specific PCR showed increased methylation of hsa-mir-129-2/-137/-935/-3663/-3665 and -4281 in anchorage independent HPV-transformed keratinocytes and cervical cancer cell lines. Mature miRNAs derived from hsa-mir-129-2/-137/-3663 and -3665 showed functional relevance as they decreased anchorage independence in cervical cancer cell lines. Cervical (pre)cancerous lesions demonstrated increased methylation of hsa-mir-129-2/-935/-3663/-3665 and -4281, underlining the clinical relevance of our findings.In conclusion, methylation-mediated silencing of tumor suppressive miRNAs contributes to acquisition of an anchorage independent phenotype. This study further substantiates the importance of miRNAs during early stages of carcinogenesis and underlines their potential as both disease markers and therapeutic targets.