Recommendations for accelerating open preprint peer review to improve the culture of science.

Peer review is an important part of the scientific process, but traditional peer review at journals is coming under increased scrutiny for its inefficiency and lack of transparency. As preprints become more widely used and accepted, they raise the possibility of rethinking the peer-review process. Preprints are enabling new forms of peer review that have the potential to be more thorough, inclusive, and collegial than traditional journal peer review, and to thus fundamentally shift the culture of peer review toward constructive collaboration. In this Consensus View, we make a call to action to stakeholders in the community to accelerate the growing momentum of preprint sharing and provide recommendations to empower researchers to provide open and constructive peer review for preprints.

Downstream retraction of preprinted research in the life and medical sciences.

Retractions have been on the rise in the life and clinical sciences in the last decade, likely due to both broader accessibility of published scientific research and increased vigilance on the part of publishers. In this same period, there has been a greater than ten-fold increase in the posting of preprints by researchers in these fields. While this development has significantly accelerated the rate of research dissemination and has benefited early-career researchers eager to show productivity, it has also introduced challenges with respect to provenance tracking, version linking, and, ultimately, back-propagation of events such as corrigenda, expressions of concern, and retractions that occur on the journal-published version. The aim of this study was to understand the extent of this problem among preprint servers that routinely link their preprints to the corollary versions published in journals. To present a snapshot of the current state of downstream retractions of articles preprinted in three large preprint servers (Research Square, bioRxiv, and medRxiv), the DOIs of the journal-published versions linked to preprints were matched to entries in the Retraction Watch database. A total of 30 retractions were identified, representing only 0.01% of all content posted on these servers. Of these, 11 retractions were clearly noted by the preprint servers; however, the existence of a preprint was only acknowledged by the retracting journal in one case. The time from publication to retraction averaged 278 days, notably lower than the average for articles overall (839 days). In 70% of cases, retractions downstream of preprints were due-at least in part-to ethical or procedural misconduct. In 63% of cases, the nature of the retraction suggested that the conclusions were no longer reliable. Over time, the lack of propagation of critical information across the publication life cycle will pose a threat to the scholarly record and to scientific integrity. It is incumbent on preprint servers, publishers, and the systems that connect them to address these issues before their scale becomes untenable.

Differential DNA methylation in umbilical cord blood of infants exposed to low levels of arsenic in utero.

BACKGROUND: There is increasing epidemiologic evidence that arsenic exposure in utero, even at low levels found throughout much of the world, is associated with adverse reproductive outcomes and may contribute to long-term health effects. Animal models, in vitro studies, and human cancer data suggest that arsenic may induce epigenetic alterations, specifically by altering patterns of DNA methylation. OBJECTIVES: In this study we aimed to identify differences in DNA methylation in cord blood samples of infants with in utero, low-level arsenic exposure. METHODS: DNA methylation of cord-blood derived DNA from 134 infants involved in a prospective birth cohort in New Hampshire was profiled using the Illumina Infinium Methylation450K array. In utero arsenic exposure was estimated using maternal urine samples collected at 24-28 weeks gestation. We used a novel cell mixture deconvolution methodology for examining the association between inferred white blood cell mixtures in infant cord blood and in utero arsenic exposure; we also examined the association between methylation at individual CpG loci and arsenic exposure levels. RESULTS: We found an association between urinary inorganic arsenic concentration and the estimated proportion of CD8+ T lymphocytes (1.18; 95% CI: 0.12, 2.23). Among the top 100 CpG loci with the lowest p-values based on their association with urinary arsenic levels, there was a statistically significant enrichment of these loci in CpG islands (p = 0.009). Of those in CpG islands (n = 44), most (75%) exhibited higher methylation levels in the highest exposed group compared with the lowest exposed group. Also, several CpG loci exhibited a linear dose-dependent relationship between methylation and arsenic exposure. CONCLUSIONS: Our findings suggest that in utero exposure to low levels of arsenic may affect the epigenome. Long-term follow-up is planned to determine whether the observed changes are associated with health outcomes.

Polycomb group genes are targets of aberrant DNA methylation in renal cell carcinoma.

The combined effects of genetic and epigenetic aberrations are well recognized as causal in tumorigenesis. Here, we defined profiles of DNA methylation in primary renal cell carcinomas (RCC) and assessed the association of these profiles with the expression of genes required for the establishment and maintenance of epigenetic marks. A bead-based methylation array platform was used to measure methylation of 1,413 CpG loci in ~800 cancer-associated genes and three methylation classes were derived by unsupervised clustering of tumors using recursively partitioned mixture modeling (RPMM). Quantitative RT-PCR was performed on all tumor samples to determine the expression of DNMT1, DNMT3B, VEZF1 and EZH2. Additionally, methylation at LINE-1 and AluYb8 repetitive elements was measured using bisulfite pyrosequencing. Associations between methylation class and tumor stage (p = 0.05), LINE-1 (p < 0.0001) and AluYb8 (p < 0.0001) methylation, as well as EZH2 expression (p < 0.0001) were noted following univariate analyses. A multinomial logistic regression model controlling for potential confounders revealed that AluYb8 (p < 0.003) methylation and EZH2 expression (p < 0.008) were significantly associated with methylation class membership. Because EZH2 is a member of the Polycomb repressive complex 2 (PRC2), we next analyzed the distribution of Polycomb group (PcG) targets among methylation classes derived by clustering the 1,413 array CpG loci using RPMM. PcG target genes were significantly enriched (p < 0.0001) in methylation classes with greater differential methylation between RCC and non-diseased kidney tissue. This work contributes to our understanding of how repressive marks on DNA and chromatin are dysregulated in carcinogenesis, knowledge that might aid the development of therapies or preventive strategies for human malignancies.

Birthweight is associated with DNA promoter methylation of the glucocorticoid receptor in human placenta.

Birthweight has been associated with a number of health outcomes throughout life. Crucial to proper infant growth and development is the placenta, and alterations to placental gene function may reflect differences in the intrauterine environment which functionally contribute to infant growth and may ultimately affect the child's health. To examine if epigenetic alteration to the glucocorticoid receptor (GR) gene was linked to infant growth, we analyzed 480 human placentas for differential methylation of the GR gene exon 1F and examined how this variation in methylation extent was associated with fetal growth. Multivariable linear regression revealed a significant association (p < 0.0001) between differential methylation of the GR gene and large for gestational age (LGA) status. Our work is one of the first to link infant growth as a measure of the intrauterine environment and epigenetic alterations to the GR and suggests that DNA methylation may be a critical determinant of placental function.

Global hypomethylation identifies Loci targeted for hypermethylation in head and neck cancer.

PURPOSE: The human epigenome is profoundly altered in cancers, with a characteristic loss of methylation in repetitive regions and concomitant accumulation of gene promoter methylation. The degree to which these processes are coordinated is unclear so we investigated both in head and neck squamous cell carcinomas. EXPERIMENTAL DESIGN: Global methylation was measured using the luminometric methylation assay (LUMA) and pyrosequencing of LINE-1Hs and AluYb8 repetitive elements in a series of 138 tumors. We also measured methylation of more than 27,000 CpG loci with the Illumina HumanMethylation27 Microarray (n = 91). RESULTS: LINE-1 methylation was significantly associated with LUMA and Infinium loci methylation (Spearman's ρ = 0.52/ρ = 0.56, both P < 0.001) but not that of AluYb8. Methylation of LINE-1, AluYb8, and Infinium loci differed by tumor site (each Kruskal-Wallis, P < 0.05). Also, LINE-1 and LUMA methylation were associated with HPV16 E6 serology (each Mann-Whitney, P < 0.05). Comparing LINE-1 methylation to gene-associated methylation, we identified a distinct subset of CpG loci with significant hypermethylation associated with LINE-1 hypomethylation. An investigation of sequence features for these CpG loci revealed that they were significantly less likely to reside in repetitive elements (Gene Set Enrichment Analysis, P < 0.02), enriched in CpG islands (P < 0.001) and were proximal to transcription factor-binding sites (P < 0.05). We validated the top CpG loci that had significant hypermethylation associated with LINE-1 hypomethylation (at EVI2A, IFRD1, KLHL6, and PTPRCAP) by pyrosequencing independent tumors. CONCLUSIONS: These data indicate that global hypomethylation and gene-specific methylation processes are associated in a sequence-dependent manner, and that clinical characteristics and exposures leading to HNSCC may be influencing these processes.

Maternal cigarette smoking during pregnancy is associated with downregulation of miR-16, miR-21, and miR-146a in the placenta.

Maternal cigarette smoking during pregnancy is associated with poor fetal outcome and aberrant miRNA expression is associated with adverse pregnancy outcomes. In 25 human placentas, we analyzed the expression of four candidate miRNA previously implicated in growth and developmental processes: miR-16, miR-21, miR-146a, and miR-182, and used three immortalized placental cell lines to identify if specific components of cigarette smoke were responsible for alterations to miRNA expression. miR-16, miR-21, and miR-146a were significantly downregulated in cigarette smoke-exposed placentas compared to controls. TCL-1 cells exposed to both nicotine and benzo(a)pyrene exhibited significant, dose-dependent downregulation of miR-146a. These results suggest that miR-146a is particularly responsive to exposures, and that smoking may elicit some of its downstream effects through alteration of miRNA expression.

Mature microRNA sequence polymorphism in MIR196A2 is associated with risk and prognosis of head and neck cancer.

PURPOSE: The central role of microRNAs as regulators of translation has been well established, whereas the relationships between genetic variation in microRNAs and disease risk is only beginning to be explored. A polymorphism in the MIR196A2 locus has shown associations with lung, breast, esophageal, and gastric tumors but has not been examined in head and neck cancers, which share similar pathology and etiology to these diseases. EXPERIMENTAL DESIGN: We studied a polymorphism in the mature sequence of MIR196A2 (rs11614913, C/T) in a population-based case-control study (n = 1,039) of head and neck squamous cell carcinoma (HNSCC) to determine if MIR196A2 genotype was associated with disease occurrence and patient survival. RESULTS: Presence of any variant allele was associated with a significantly reduced risk for HNSCC (odds ratio, 0.8; 95% confidence interval, 0.56-0.99). Homozygous variant allele carriers with pharyngeal tumors had significantly reduced survival compared with wild-type and heterozygous cases (hazard ratio, 7.4; 95% confidence interval, 1.9-28.2). Expression analysis in a subset of tumors (n = 83) revealed no significant difference in relative expression of either miR-196a or miR-196a* by MIR196A2 genotype. CONCLUSION: These data demonstrate a role for MIR196A2 genotype in susceptibility and prognosis of HNSCC.

Bisphenol A exposure leads to specific microRNA alterations in placental cells.

Exposure to bisphenol A (BPA) has been observed to alter developmental pathways and cell processes, at least in part, through epigenetic mechanisms. This study sought to investigate the effect of BPA on microRNAs (miRNAs) in human placental cells. miRNA microarray was performed following BPA treatment in three immortalized cytotrophoblast cell lines and the results validated using quantitative real-time PCR. For functional analysis, overexpression constructs were stably transfected into cells that were then assayed for changes in proliferation and response to toxicants. Microarray analysis revealed several miRNAs to be significantly altered in response to BPA treatment in two cell lines. Real-time PCR results confirmed that miR-146a was particularly strongly induced and its overexpression in cells led to slower proliferation as well as higher sensitivity to the DNA damaging agent, bleomycin. Overall, these results suggest that BPA can alter miRNA expression in placental cells, a potentially novel mode of BPA toxicity.